Food Protein Deterioration. Mechanisms and Functionality 9780841207516, 9780841209954, 0-8412-0751-8

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Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.fw001

Food Protein Deterioration

Mechanisms and Functionality

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.fw001

Food Protein Deterioration Mechanisms and Functionality John P.Cherry,EDITOR

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.fw001

United States Department of Agriculture

Based on a symposium sponsored by the ACS Division of Agricultural and Food Chemistry at the 182nd Meeting of the American Chemical Society, New York, New York, August 23-28, 1981.

ACS SYMPOSIUM SERIES AMERICAN

CHEMICAL

WASHINGTON, D. C.

SOCIETY 1982

206

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.fw001

Library of Congress Cataloging in Publication Data Food protein deterioration. (ACS symposium series, ISSN 0097-6156; 206) Includes bibliographies and index. 1. Proteins—Deterioration—Congresses. I. Cherry, John P., 1941. II. American Chemical Society. Division of Agricultural and Food Chemistry. III. American Chemical Society. National Meeting (182nd: 1981: New York, N.Y.) IV. Series. TP453.P7F66 ISBN 0-8412-0751-8

664 ACSMC8

82-20739 206 1-444 1982

Copyright © 1981 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of thefirstpage of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. P R I N T E D I N THE U N I T E D S T A T E S OF A M E R I C A

Second printing 1982

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.fw001

ACS Symposium Series M. Joan Comstock, Series Editor

Advisory Board David L. Allara

Marvin Margoshes

Robert Baker

Robert Ory

Donald D. Dollberg

Leon Petrakis

Robert E. Feeney

Theodore Provder

Brian M . Harney

Charles N . Satterfield

W. Jeffrey Howe

Dennis Schuetzle

James D. Idol, Jr.

Davis L. Temple, Jr.

Herbert D. Kaesz

Gunter Zweig

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.fw001

FOREWORD T h e A C S SYMPOSIUM SERIES was founded i n 1974 to provide a medium for publishing symposia quickly i n book form. The format of the Series parallels that of the continuing ADVANCES I N CHEMISTRY SERIES except that i n order to save time the papers are not typeset but are reproduced as they are sub­ mitted by the authors i n camera-ready form. Papers are re­ viewed under the supervision of the Editors w i t h the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

PREFACE

Τ

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.pr001

JLHE

THEORIES

AND CONCEPTS

O F FOOD

PROTEIN

DETERIORATION

are

based on the composition, structure, and physicochemical deteriorative changes that occur in proteins during genetic and physiological processes. Endogenous and exogenous enzymatic activities, chemical reactions and modifications, pH changes, salt effects, storage-fungi contamination, and temperature dependency all contribute to making the study of food protein deterioration a vital science. The utilization of proteins as the functionally active constituents in food systems, the processes that control the degree of protein degradation, and the nutritional quality and bioavailability of de­ teriorated and modified proteins are topics that are included in this volume. Proteins undergo denaturation by reaction with hydroperoxides which are formed by lipoxygenase and nonenzymatic oxidation of polyunsaturated fatty acids. Discoloration occurs as a result of the disruption of food sys­ tems by heating and/or blending during processing. Efforts to develop methods to characterize and control these reactions, and the objectionable odors and off-flavors developed during storage, handling, and processing have been fruitful. Temperature also affects food protein deterioration. Below ambient temperature proteins can be affected adversely with notable changes in cellular systems, solubility, and enzymatic activity. These alterations can affect storage time, salt concentration, pH dehydration effects, and the effect of ice on membranes. Evidence has shown that during heat exposure, enzyme-resistant linkages form between polypeptides. Many physicochemi­ cal changes that are caused by heat-induced protein denaturation can result in texturation products having meat-like food properties. In addition, brown-colored beneficial and deleterious products can be formed from carbonylamines which are products of the reaction between the aldehydes of sugars and the amino groups of amino acids. (The latter reaction is the first step in the series of reactions that are collectively known as the Maillard reaction.) These chemical reactions can be pre­ vented by eliminating the aldehyde-containing compounds, or temporarily masking the e-amino group of lysine during processing. The functional properties of foods are influenced by the inherent molecular properties of proteins, the manner in which they interact with other protein and nonprotein constituents, and their behavior in prevailing k

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.pr001

environmental conditions. Most of the reactions that affect food functional­ ity involve disulfide bonds, and the electrostatic and hydrophobic interac­ tions of structure and surface properties of proteins. Chemical modification of these disulfide bonds results in the loss of both structural order and activity. This volume confronts the issue that the nutritional quality of proteins can change as a result of structural modification or deterioration. There may be a small increase or decrease in protein digestibility as the proteins unfold or form aggregates with other protein or nonprotein constituents. Extremes in heat or chemical modification can result in a major loss of bioavailability when essential amino acids form nonreversible molecular complexes. Understanding the mechanisms by which proteins deteriorate during handling, storage, and processing will allow modification and/or control of these processes to protect the inherent functional and nutritional qualities of proteins in raw products. This will then allow for maximum production of quality foods. JOHN P . CHERRY

Eastern Regional Research Center United States Department of Agriculture Philadelphia, PA 19118 June 1982

χ

1 Genetic Mechanisms and Protein Properties with Special Reference to Plant Proteins ASIM ESEN

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

Virginia Polytechnic Institute and State University, Department of Biology, Blacksburg, VA 24061

Continuous intake of high quality dietary protein in adequate amounts is prerequisite for normal growth and development in the young and for maintenance of nitrogen equilibrium in adult humans. The reason for this is that dietary protein provides certain specific (essential) amino acids that cannot be synthesized by man and other monogastric animals as well as indispensable nitrogen required for various physiological functions. Animal cells also cannot store amino acids as they do carbohydrates and lipids, which necessitates continuous protein intake. Malnutrition or undernutrition is a serious problem affecting nearly one half of the world's population. There is no consensus among nutritionists and sociologists on the causes of malnutrition. Some experts (1) attribute it to the lack of good quality protein for human consumption while others (2, 3, 4, 5, 6) to either protein and calorie deficiencies or calorie deficiency alone. Bressani and Elias (7) have recently emphasized the difficulty in identifying a simple nutrient as the causal factor in malnutrition. They concluded that the malnutrition problem is the end result of the interaction of many factors of which low food availability and intake are perhaps the most important. On a worldwide basis, about 90% of the calories and 10% of the protein available for human consumption come directly from plants with the remainder from foods of animal origin (Table i ) . There is a high correlation between income level and the proportion of animal protein in the diet with the prosperous nations and also prosperous social and economic classes in a country receiving their protein primarily from foods of animal origin (8), namely milk, meat, egg, fish and their products. In developed countries large amounts of plant protein are fed to animals for meat production. This is a rather wasteful process from the standpoint of energetics since it takes about

0097-6156/82/0206-0001 $08.75/0 © 1982 American Chemical Society

FOOD P R O T E I N

2

DETERIORATION

e i g h t k g o f p l a n t p r o t e i n t o p r o d u c e one k g o f a n i m a l p r o t e i n . P r e s e n t l y t h e w o r l d ' s p o p u l a t i o n i s a b o u t 4-5 b i l l i o n and i t i s e s t i m a t e d t o r e a c h 6 t o 7 b i l l i o n by t h e t u r n o f t h e c e n t u r y . Therefore, i t i s probable that the d i r e c t consumption o f p l a n t p r o t e i n by humans i s l i k e l y t o i n c r e a s e (9) and a n i m a l p r o t e i n i s t o become somewhat a l u x u r y t h a t o n l y u p p e r e c o n o m i c c l a s s e s and p o p u l a t i o n s o f c e r t a i n d e v e l o p e d c o u n t r i e s c a n a f f o r d on a regular basis.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

Table I .

P r o t e i n s o u r c e s and i n t a k e

(1975-77)

Source Region o r Country World Asia Africa S. A m e r i c a I . â C. A m e r i c a Europe Oceania USA India Zaire

Daily I n t a k e (gm) 69 58 59 66 93 96 96 106 48 36

% Plant 65 79 80 56 39 45 35 32 89 80

% Animal 35 21 20 44 61 55 65 68 11 20

Source: ( 9 6 ) . Regardless of i t s o r i g i n , plant o r animal, a protein s o u r c e i s e x p e c t e d t o meet c e r t a i n r e q u i r e m e n t s i n o r d e r t o be s u i t a b l e f o r food o r feed u s e . First, i t should have a h i g h p r o t e i n content t o p r o v i d e t h e q u a n t i t y o f p r o t e i n needed a t normal i n t a k e l e v e l s . S e c o n d l y , i t s p r o t e i n s s h o u l d be o f h i g h n u t r i t i o n a l q u a l i t y t o p r o v i d e a l l e s s e n t i a l amino a c i d s a t o r above recommended l e v e l s . T h i r d l y , the p r o t e i n s o u r c e must h a v e a c c e p t a b l e o r g a n o l e p t i c p r o p e r t i e s ( e . g . , t a s t e , c o l o r and texture) i n i t s edible state so t h a t i t c a n be i n g e s t e d i n sufficient quantity alone o r i n mixtures with other dietary components. Fourthly, i t s protein should have high digestibility and b i o l o g i c a l value. I n o t h e r words, the p r o t e i n must be r e s i s t a n t t o o r f r e e o f d e t e r i o r a t i v e c h a n g e s and b i o l o g i c a l l y a c t i v e p r o t e i n and n o n p r o t e i n s u b s t a n c e s w h i c h unfavorably a f f e c t the d i g e s t i b i l i t y and b i o l o g i c a l value. Lastly, b u t n o t l e a s t , t h e p r o t e i n s o u r c e and i t s p r o t e i n s should have d e s i r a b l e f u n c t i o n a l p r o p e r t i e s t o l e n d themselves

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

1.

ESEN

Genetic

Mechanisms

and Protein

Properties

3

to p r o c e s s i n g , handling and storage with little o r no d e t e r i o r a t i v e changes. I f a p r o t e i n s o u r c e c a n n o t s a t i s f y one o r more o f t h e s e c r i t e r i a , i t s d e f i c i e n c i e s may b e r e m e d i e d b y genetic o r nongenetic ( e . g . supplementation, improved p r o c e s s i n g and s t o r a g e t e c h n i q u e s ) means. A n i m a l p r o t e i n s meet all o r most o f t h e r e q u i r e m e n t s a s i n g r e d i e n t s w i t h high nutritional q u a l i t y and organoleptic properties. The o n l y g e n e t i c improvement t h a t c a n b e made i n t h e s e p r o t e i n s i s t h a t of quantity b y s e l e c t i n g and developing breeds w i t h high p r o d u c t i o n and f e e d u t i l i z a t i o n c a p a b i l i t y . Futhermore, animal proteins a r e e x c l u s i v e l y s t r u c t u r a l and enzymatic i n nature w i t h w e l l - d e f i n e d m e t a b o l i c a c t i v i t i e s and f u n c t i o n s . Thus any a t t e m p t t o change g e n e t i c a l l y t h e i r n u t r i t i o n a l p r o p e r t i e s and f u n c t i o n a l i t y would be d e l e t e r i o u s o r l e t h a l . However s u c h c o n s t r a i n t s may n o t apply to the s t o r a g e p r o t e i n s o f seeds where t h e r e i s b o t h need and p o t e n t i a l f o r improvement o f p r o t e i n p r o p e r t i e s b y g e n e t i c means. T h e r e f o r e t h i s review's focus w i l l be o n p l a n t p r o t e i n s w i t h s p e c i a l r e f e r e n c e to t h e i r d e f i c i e n c i e s and p o s s i b l e g e n e t i c a p p r o a c h e s t o i m p r o v e them. There are about 80 thousand p l a n t s p e c i e s w i t h an organ o r part s u i t a b l e f o r u s e a s food a n d many o f them may b e a p o t e n t i a l p r o t e i n s o u r c e (10). However, o n l y 12 s p e c i e s a r e the main s t a y o f the w o r l d ' s food supply a c c o u n t i n g f o r about 90% o f t h e h a r v e s t e d p r o d u c t s . Of these, c e r e a l g r a i n s provide some 70% o f t h e w o r l d ' s s u p p l y o f p l a n t p r o t e i n and o v e r 90% o f the c a l o r i e s o f p l a n t o r i g i n . F o r example, r i c e a l o n e i s the m a i n s o u r c e o f p r o t e i n i n A s i a and p r o v i d e s 80% o f t h e c a l o r i e s i n the A s i a n d i e t . I t i s p r o j e c t e d t h a t t h e w o r l d demand f o r c e r e a l s i n t h e y e a r 2000 w i l l b e 2,422 b i l l i o n m e t r i c tons assuming a net p o p u l a t i o n i n c r e a s e r a t e o f 0.7% i n d e v e l o p e d c o u n t r i e s and 2.2% i n d e v e l o p i n g c o u n t r i e s (11 ) . This w i l l r e p r e s e n t a 100$ i n c r e a s e o v e r 1970 p r o d u c t i o n . The n e x t m a j o r source of plant p r o t e i n f o r humans i s legume s e e d s , which account only f o r 18-20$ o f t h e t o t a l p l a n t p r o t e i n produced. E f f o r t s d i r e c t e d t o w a r d s improvement o f q u a n t i t y and p r o p e r t i e s o f p l a n t p r o t e i n s b y g e n e t i c means w o u l d h a v e t o b e c e n t e r e d a r o u n d t h e c o n v e n t i o n a l c r o p p l a n t s s u c h a s c e r e a l s and legumes i n o r d e r to a c h i e v e t a n g i b l e r e s u l t s i n the n e a r f u t u r e . There a r e b a s i c a l l y two r e a s o n s f o r t h i s o b s e r v a t i o n . F i r s t , almost our e n t i r e r e p e r t o i r e o f b a s i c s c i e n t i f i c i n f o r m a t i o n r e l a t i v e to p l a n t g e n e t i c s , b r e e d i n g and b i o c h e m i s t r y i s d e r i v e d f r o m s t u d i e s on t r a d i t i o n a l c r o p p l a n t s ( e . g . , c o r n , wheat, soybean, and others). This basic information c o n s t i t u t e s the most valuable asset f o r further progress through research. Secondly, the t r a d i t i o n a l c r o p p l a n t s have been a p a r t o f s o c i a l , e c o n o m i c and c u l t u r a l l i f e o f m a n k i n d s i n c e t h e d a y s o f prehistoric agriculture; man knows how t o p r o d u c e , process, m a r k e t , and consume them.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

4

FOOD P R O T E I N

DETERIORATION

T h e o r e t i c a l l y speaking, g e n e t i c improvement o f p r o t e i n s w o u l d be a s t r a i g h t f o r w a r d t a s k . P r o t e i n i s a n i m m e d i a t e gene product whose amino a c i d s e q u e n c e ( p r i m a r y structure) is c o l i n e a r t o t h e n u c l e o t i d e s e q u e n c e o f t h e gene e n c o d i n g i t . Thus b o t h t h e s t r u c t u r e and f u n c t i o n o f a p r o t e i n i s u n d e r direct genetic control. From t h e s t a n d p o i n t o f a g e n e t i c i s t , each o f t h e d i f f e r e n t kinds of proteins i s a heritable characteristic, one w h i c h i s c l o s e s t t o t h e gene and one t h a t c a n be d e s c r i b e d w i t h o u t any a m b i g u i t y . This i s i n contrast to many b i o l o g i c a l c h a r a c t e r i s t i c s t h a t g e n e t i c i s t s h a v e t o work w i t h t h a t a r e f a r removed f r o m t h e gene and c a n n o t be o f t e n d e s c r i b e d and m e a s u r e d i n p r e c i s e q u a n t i t a t i v e o r q u a l i t a t i v e terms. One may i d e n t i f y t h r e e m a j o r p r o b l e m s r e l a t i v e to p l a n t p r o t e i n s t h a t a r e p o t e n t i a l l y amenable t o a g e n e t i c s o l u t i o n . These a r e : l o w p r o t e i n c o n t e n t i n c e r e a l s and some l e g u m e s , p o o r p r o t e i n q u a l i t y i n c e r e a l s and l e g u m e s , and t h e p r e s e n c e o f p r o t e i n and n o n p r o t e i n components t h a t a r e e i t h e r t o x i c o r a f f e c t u n f a v o r a b l y n u t r i t i o n a l v a l u e and f u n c t i o n a l i t y o f p l a n t p r o t e i n s and o t h e r e s s e n t i a l n u t r i e n t s . W i t h i n each problem area t h e r e a r e s u b - p r o b l e m s some o f w h i c h a r e amenable t o solution, and some may d e f y a s o l u t i o n i n v i e w o f b i o l o g i c a l and t e c h n i c a l c o n s t r a i n t s . Experimental

Procedures

This chapter will attempt to present a catalogue o f genetic s t r a t e g i e s and a p p r o a c h e s t h a t may be u s e d f o r t h e solution o f t h e above m e n t i o n e d problems r e l a t i v e t o p l a n t protein, and e v a l u a t e t h e p o t e n t i a l and l i m i t a t i o n s o f each approach based on r e p r e s e n t a t i v e e x p e r i m e n t a l data from previous investigations. Finally the potential uses o f r e c o m b i n a n t BNA t e c h n o l o g y and g e n e t i c e n g i n e e e r i n g f o rplant p r o t e i n i m p r o v e m e n t w i l l be b r i e f l y m e n t i o n e d . For additional i n f o r m a t i o n on t o p i c s d i s c u s s e d i n t h i s c h a p t e r and s p e c i f i c methodologies the reader i s referred t o numerous previous r e v i e w s ( 9 , U ) , J_2, V 3 H, V5, 1£» 1_7, U_, 1 9 , 20) and r e s e a r c h and r e v i e w p a p e r s t h a t a p p e a r e d i n t h e p u b l i c a t i o n s o f many p r o c e e d i n g s and s y m p o s i a h e l d o n t h i s s u b j e c t . The g e n e t i c s o l u t i o n t o t h e s e p r o b l e m s w i l l r e l y upon t h e presence o f s u f f i c i e n t g e n e t i c v a r i a t i o n i n t h e e x i s t i n g germ plasm sources. E q u a l l y important i s the a v a i l a b i l i t y of simple, rapid, and i n e x p e n s i v e s c r e e n i n g p r o c e d u r e s t o d e t e c t such v a r i a t i o n . E x t e n s i v e s u r v e y s o f m a j o r c e r e a l s and legumes conducted w i t h i n t h e p a s t two d e c a d e s f o r s p o n t a n e o u s high q u a n t i t y and/or q u a l i t y mutants have r e v e a l e d a g r e a t d e a l o f v a r i a t i o n i n t h e germ p l a s m ( T a b l e I I ) . Moreover, additional v a r i a t i o n has been generated s u c c e s s f u l l y by employing the F

1.

ESEN

Genetic

Mechanisms

and Protein

Properties

v a r i o u s methods o f i n d u c e d m u t a g e n e s i s ( 2 1 , 22, 23, 24). In o r d e r t o make r a p i d p r o g r e s s and j u s t i f y the expenditure o f f i n a n c i a l and t e c h n i c a l resources, high h e r i t a b i l i t y o f the desirable characteristic chosen f o r improvement i s a prerequisite. P r o t e i n c o n t e n t seems t o b e a h i g h l y h e r i t a b l e c h a r a c t e r and shows r e l a t i v e l y a s i m p l e mode o f i n h e r i t a n c e . For example, Johnson et a l . (25) reported heritability e s t i m a t e s a s h i g h a s 83$ f o r p r o t e i n c o n t e n t i n w h e a t .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

Table I I .

Crop

V a r i a t i o n o f p r o t e i n c o n t e n t and l i m i t i n g amino a c i d i n some c e r e a l s and legumes

P r o t e i n {%)

Wheat Rice Corn Barley Sorghum Oats

7-22 5-17 7-17 10-13 7-26 8-22

Soybean Bean Pea Lentil Cowpea Chickpea

35-52 19-31 17-31 20-31 21-34 12-28

Source:

(97,

98,

Improvement o f P r o t e i n

Limiting Amino A c i d L y s i n e (g/16 g Ν) 2.2-4.2 3.1-4.5 2.2-4.0 3.2-4.3 1.8-3.8 3.7-4.8 M e t h i o n i n e (g/16 1.0-1.6 0.5-2.1

0.8-2.3 0.5-1.7

99). Quantity

The p r o t e i n content o f cereal grains and o t h e r starchs t o r i n g c r o p s i s r e l a t i v e l y low. F o r example o n t h e a v e r a g e o n l y 8% o f t h e dry matter i n r i c e , 12$ i n w h e a t , and 10$ i n corn i s p r o t e i n . T h e s e l e v e l s may n o t n e c e s s a r i l y b e too low i n view o f the fact that cereals are the p r i m a r y source o f d i e t a r y energy. I n f a c t i f c e r e a l p r o t e i n s contained a l l the essential amino a c i d s i n required proportions they would a c t u a l l y meet t h e p r o t e i n requirement even f o r i n f a n t s and growing c h i l d r e n , t h e g r o u p s most v u l n e r a b l e to protein malnutrition. Of course, t h i s would be t r u e only i f t h e q u a n t i t y o f c e r e a l consumed was a d e q u a t e t o meet t h e e n t i r e

5

6

FOOD P R O T E I N DETERIORATION

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

c a l o r i e requirement (26). Some n u t r i t i o n i s t s ( e . g . , 6 ) a r g u e t h a t most o f t h e common p l a n t s t a p l e s , w h i c h o f c o u r s e i n c l u d e s cereals, "contain s u f f i c i e n t u t i l i z a b l e p r o t e i n t o meet t h e s a f e l e v e l r e q u i r e m e n t s w i t h o u t any a d d i t i o n s o r s u p p l e m e n t s " . Munck ( 1 9 ) s t a t e s t h a t t h e r e i s no p o i n t f o r i n c r e a s i n g t h e protein quantity i n cereals i f such an i n c r e a s e i s t o be a c h i e v e d a t t h e e x p e n s e o f y i e l d and c a r b o h y d r a t e s . Proponents of t h i s view b e l i e v e that c e r e a l s should remain as energy foods and legumes must be t h e p r i m a r y s o u r c e o f p l a n t p r o t e i n . N e g a t i v e c o r r e l a t i o n b e t w e e n p r o t e i n c o n t e n t and y i e l d . H i g h e r p r o t e i n c o n t e n t h a s a l m o s t a l w a y s b e e n f o u n d t o be n e g a t i v e l y c o r r e l a t e d w i t h g r a i n y i e l d ( 2 7 , 2 8 , 2 9 , 3 0 , 31 )» If this i s i n d e e d a u n i v e r s a l phenomenon, t h e a d v o c a t e s f o r i n c r e a s i n g p r o t e i n content i n c e r e a l s cannot c o n v i n c i n g l y argue t h e i r case before they r e s o l v e t h i s problem. The r e a s o n f o r t h e i n v e r s e r e l a t i o n s h i p b e t w e e n p r o t e i n c o n t e n t and y i e l d h a s been e x p l a i n e d by t h e energy r e q u i r e m e n t s o f v a r i o u s seed b i o m a s s components ( 3 2 , 33). The amount o f t h e p r i m a r y photosynthate, glucose, expended t o p r o d u c e a u n i t mass o f p r o t e i n i s t w i c e a s much a s t h a t needed t o p r o d u c e t h e same u n i t mass o f c a r b o h y d r a t e . To be more s p e c i f i c , 1 g o f g l u c o s e y i e l d s 0.83 g o f c a r b o h y d r a t e and o n l y 0.4 g o f p r o t e i n . If these t h e o r e t i c a l c a l c u l a t i o n s t r u l y r e f l e c t what i s h a p p e n i n g i n the plant i t w o u l d i n d e e d be p o i n t l e s s t o i n c r e a s e p r o t e i n content unless t h e r e i s a premium p a i d f o r h i g h p r o t e i n t o offset yield loss. However, t h e s i t u a t i o n d o e s n o t seem a s hopeless as the s k e p t i c s say i t i s . The n e g a t i v e c o r r e l a t i o n between y i e l d and p r o t e i n d o e s n o t seem t o be t h e r u l e . Johnson e t a l . ( 2 5 , 3 4 ) showed t h a t t h e s i m u l t a n e o u s p r o g r e s s i n g r a i n y i e l d and p r o t e i n c o n t e n t was p o s s i b l e i n wheat. They found t h a t 4 out o f 5 h i g h p r o t e i n s e l e c t i o n s d e r i v e d from t h e h i g h p r o t e i n c u l t i v a r A t l a s s 66 e x c e e d e d a p o p u l a r Nebraska cultivar, Scout 66, both i n y i e l d and p r o t e i n content. L i k e w i s e , new o a t s , rice, and c o r n c u l t i v a r s and e x p e r i m e n t a l lines have been r e p o r t e d t o c o n s i s t e n t l y and s i g n i f i c a n t l y exceed standard c u l t i v a r s i n p r o t e i n content without loss i n y i e l d ( 3 8 , 39, 4 0 ) . These i n c r e a s e s i n p r o t e i n c o n t e n t do n o t seem t o be n e g a t i v e l y c o r r e l a t e d w i t h y i e l d ( 3 8 , 39, 4 0 ) . However, some r e p o r t s i n d i c a t e c o n s i d e r a b l y high negative c o r r e l a t i o n s e s p e c i a l l y i n b e a n s (41 , 4 2 ) . I n soybean, p r o t e i n and o i l contents t e n d t o be n e g a t i v e l y correlated but this r e l a t i o n s h i p may be o f l i t t l e c o n s e q u e n c e now s i n c e t h e o i l i s no l o n g e r t h e more v a l u a b l e p r o d u c t from soybean because t h e value o f p r o t e i n exceeds t h a t o f o i l . Poey e t a l . ( 4 3 ) and R o b b e l e n (10) postulate that the i n v e r s e r e l a t i o n s h i p b e t w e e n y i e l d and p r o t e i n i s a n a r t i f a c t of past s e l e c t i o n p r a c t i c e s which s e l e c t e d f o r o n l y one

1.

ESEN

Genetic

Mechanisms

and Protein

Properties

7

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

c h a r a c t e r at a time, e i t h e r y i e l d or p r o t e i n . I m p l i c i t i n t h i s h y p o t h e s i s i s t h a t i t may be p o s s i b l e t o d e v e l o p h i g h p r o t e i n high yield cultivars by selecting f o r both traits simultaneously. S t r a t e g i e s f o r t h e improvement o f p r o t e i n q u a n t i t y . 1_) Increase y i e l d per u n i t area. I n c r e a s i n g the g r a i n y i e l d per u n i t area w o u l d b e t h e most s t r a i g h t f o r w a r d way t o i n c r e a s e protein production. T h i s w o u l d r e q u i r e t h e d e v e l o p m e n t and use o f h i g h y i e l d c u l t i v a r s , namely, those w i t h h i g h p h o t o s y n t h e t i c efficiency, p e s t r e s i s t a n c e , and h a r v e s t i n d e x . These h i g h y i e l d c u l t i v a r s coupled w i t h better cultural practices, e.g., b e t t e r s e e d bed p r e p a r a t i o n , fertilization, irrigation, weed and pest control, and g r o w t h r e g u l a t o r a p p l i c a t i o n w o u l d further i n c r e a s e the y i e l d p e r u n i t area. One p o t e n t i a l drawback o f t h i s a p p r o a c h i s t h a t i t may d e p r e s s the p r o t e i n percentage below a c c e p t a b l e levels especially i n cereals, assuming t h a t t h e i n v e r s e r e l a t i o n s h i p s between y i e l d and p r o t e i n i s to o p e r a t e . T h i s might l e a d to a p r o t e i n - d e f i c i e n t diet, although adequate i n c a l o r i e s , i n r e g i o n s where c e r e a l s are t h e common s t a p l e . However, t h e development o f h i g h protein-high yield c u l t i v a r s may a l l e v i a t e t h i s possibility. I n c r e a s e d y i e l d p e r u n i t a r e a i s e x p e c t e d t o b e more s u c c e s s f u l i n terms o f p r o t e i n p r o d u c t i o n w i t h legumes t h a n w i t h c e r e a l s . T h i s i s b e c a u s e p r o t e i n c o n t e n t i s l e s s a f f e c t e d b y y i e l d and i s i n h e r e n t l y h i g h i n legumes. 2) Apply a d d i t i o n a l n i t r o g e n - f e r t i l i z e r at or after anthesis. Nitrogen f e r t i l i z a t i o n , especially late applications a t t h e t i m e o f a n t h e s i s , has b e e n shown t o b e r a t h e r e f f e c t i v e i n i n c r e a s i n g the p r o t e i n content i n c e r e a l s . Hucklesby et a l . (44) o b s e r v e d 30-118$ i n c r e a s e i n p r o t e i n p r o d u c t i o n p e r h e c t a r e b y l a t e s p r i n g a p p l i c a t i o n o f n i t r o g e n to w h e a t . T h e i n c r e a s e was due t o b o t h y i e l d and p r o t e i n c o n t e n t . Somewhat s i m i l a r but l e s s d r a m a t i c r e s u l t s were o b t a i n e d w i t h corn by Hageman e t a l . (45_). Zink ( 4 6 ) showed t h a t late nitrogen a p p l i c a t i o n i n c r e a s e d t h e k e r n e l number, w e i g h t , and p e r c e n t p r o t e i n i n c o r n b y 15$ , 1 2 $ , and 3 0 $ , respectively. These r e s u l t s suggested that s o i l nitrogen i s depleted considerably by the vegetative growth p r i o r to anthesis and l a t e Ν a p p l i c a t i o n both provided a d d i t i o n a l reduced nitrogen from roots t o the seed and prolonged the d u r a t i o n o f n i t r o g e n accumulation i n t o seeds. The d e v e l o p m e n t o f h y b r i d s and c u l t i v a r s t h a t w i l l r e s p o n d to nitrogen fertilization can further increase the e f f e c t i v e n e s s o f n i t r o g e n used i n i n c r e a s i n g p r o t e i n content and p r o d u c t i o n . The a p p l i c a t i o n o f a d d i t i o n a l n i t r o g e n a s a method t o increase protein production may n o t b e f e a s i b l e a t a l l i n t h e

8

FOOD P R O T E I N DETERIORATION

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

f u t u r e i n view of high cost of n i t r o g e n f e r t i l i z e r s and t h e i r application. In fact, the author personally believes that future crop plant cultivars will be o n e s t h a t p e r f o r m best u n d e r a d v e r s e e n v i r o n m e n t a l c o n d i t i o n s w i t h minimum c a r e u n l e s s new and c h e a p s o u r c e s o f energy are a v a i l a b l e f o r a g r i c u l t u r a l use. T h e r e f o r e p l a n t g e n e t i c i s t s and b r e e d e r s s h o u l d r e a s s e s s their goals of developing cultivars whose p e r f o r m a n c e i s contingent upon c u l t u r a l practices requiring high inputs of energy. 3) Abolish r a t e - l i m i t i n g steps i n p r o t e i n b i o s y n t h e s i s pathway. C o n v e n t i o n a l wisdom s u g g e s t s t h a t p r o t e i n s y n t h e s i s , l i k e any o t h e r b i o c h e m i c a l p r o c e s s , i s under g e n e t i c c o n t r o l . T h i s c o n t r o l may be e x e r t e d by r e g u l a t i n g t h e q u a n t i t y a n d / o r a c t i v i t y o f any one o r more o f t h e components o f the p r o t e i n s y n t h e s i z i n g m a c h i n e r y , e.g. amino a c i d s , r i b o s o m e s , m e s s e n g e r r i b o n u c l e i c a c i d (RNA), i n i t i a t i o n , e l o n g a t i o n and t e r m i n a t i o n factors, amino a c y l t r a n s f e r RNA synthetases, peptidyl transferase, adenosine triphosphate, guanosine triphosphate, and o t h e r u n i d e n t i f i e d f a c t o r s . F i g u r e 1 shows t h e p a t h w a y o f n i t r o g e n a s s i m i l a t i o n from n i t r a t e to amino a c i d s and p r o t e i n s i n plants (47). A b o l i s h i n g the r a t e - l i m i t i n g steps o f p r o t e i n synthesis p a t h w a y by genetically altering the regulatory components w o u l d be expected to i n c r e a s e the p r o t e i n content. The a v a i l a b i l i t y o f s u b s t r a t e s , amino a c i d s , i s p r o b a b l y t h e most i m p o r t a n t r a t e - l i m i t i n g f a c t o r i n p r o t e i n s y n t h e s i s s i n c e i t a f f e c t s not o n l y the r a t e o f s y n t h e s i s of storage proteins but also that of enzymes and other protein factors that function directly i n nitrogen assimilation. There is considerable evidence f o r the regulation of amino acid b i o s y n t h e s i s by allosteric f e e d b a c k i n h i b i t i o n by the endp r o d u c t amino a c i d s i n p l a n t s (47, 48)• Another l o g i c a l step f o r t h e r e g u l a t i o n o f t h e s y n t h e s i s o f amino a c i d s i s t h o u g h t t o be t h e n i t r a t e r e d u c t a s e r e a c t i o n (45, 47). T h i s stems f r o m the f a c t that i t i s the first enzyme i n t h e nitrogen a s s i m i l a t i o n p a t h w a y , i n d u c i b l e by t h e s u b s t r a t e and r e l a t i v e l y unstable under adverse environmental conditions. The experimental data i n support of t h i s suggestion came f r o m s t u d i e s o f Hageman and h i s a s s o c i a t e s (46, 47, 50) who f o u n d consistently p o s i t i v e c o r r e l a t i o n s between n i t r a t e reductase activity, g r a i n y i e l d and p r o t e i n c o n t e n t . In view of these results, they suggested the use of n i t r a t e reductase a c t i v i t y as a screening tool f o r s e l e c t i n g high protein-high yield genotypes. Most, or a l l , o f the n i t r o g e n r e a c h i n g the seeds i s i n the f o r m o f amino a c i d s c o m i n g f r o m l e a v e s and r o o t s (51) · Amino a c i d s o r i g i n a t i n g i n leaves r e s u l t p r i m a r i l y from p r o t e o l y s i s o f l e a f p r o t e i n s d u r i n g s e n e s c e n c e and a l s o some f r o m new amino acid synthesis coupled to p h o t o s y n t h e s i s . But those coming

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch001

ESEN

Genetic

1

2

Mechanisms

3

and Protein

Glutamate

Glutamine

4

Properties

Glutamate

ocoxo acids

5 P e p s t a t i n OH + HO-j>-0 Ο slow? R

Q

2

D e r i v a t i v e s of n o n - e s s e n t i a l c a r b o x y l group : Pepstatin-COO

+ X

•

Pepstatin-COX ( a c t i v a t e d c a r b o x y l group)

* Pepstatin-COX Figure 7.

+ Z: *

•

Pepstatin-COZ

Possible chemical modifications of pepstatin.

2.

RICHARDSON

Enzymic

Degradation

of

Proteins

47

a c t i v a t e d or otherwise modified to immobilize the i n h i b i t o r or t o couple i t to u n d i g e s t i b l e macromolecules such as carboxymethyl c e l l u l o s e f o r use i n foods. T h i s l a t t e r compound would impart water s o l u b i l i t y to the hydrophobic i n h i b i t o r . Immobilized p e p s t a t i n has been used to i s o l a t e a c i d proteases (58). Pepsin i s known to be i n h i b i t e d by high molar r a t i o s of i t s a c t i v a t i o n peptide (38). When the a c t i v a t i o n peptide i s guanidated, i t s i n h i b i t o r y a c t i v i t y can be more than doubled (Figure 4 ) . The sequence o f t h i s peptide and r e l e v a n t chemical m o d i f i c a t i o n s suggest routes f o r p r e p a r a t i o n of e f f e c t i v e , digestible synthetic inhibitors. T r y p s i n i s i n h i b i t e d by oligomers of homoarginine (n = 10) with a K i approaching 10"" M (53) (Figure 8 ) . T h i s o l i g o m e r i c s u b s t r a t e analog f o r t r y p s i n apparently binds t o the a c t i v e s i t e of t r y p s i n where the geometry f o r r a p i d h y d r o l y s i s i s not adequate. Synthetic peptide i n h i b i t o r s with high a f f i n i t i e s f o r the a c t i v e s i t e of proteases are a l s o p o s s i b l e based on a f f i n i t y l a b e l i n g techniques ( 5 9 ) . There are thus numerous p o s s i b i l i t i e s f o r designing s y n t h e t i c peptide (60) protease i n h i b i t o r s that c o u l d prove u s e f u l i n food systems. Again based on an understanding o f the peptide chemistry o f i n h i b i t o r s , recombinant DNA technology could probably be employed to e v e n t u a l l y produce d e s i r a b l e peptide i n h i b i t o r s u s i n g conventional fermentation technology. Solidphase o l i g o n u c l e o t i d e s y n t h e t i c procedures are p r o g r e s s i n g q u i c k l y and i t i s r a p i d l y becoming p o s s i b l e t o synthesize o l i g o n u c l e o t i d e s , coding f o r a p a r t i c u l a r amino a c i d sequence, f o r i n s e r t i o n i n t o a c l o n i n g v e c t o r (61, 62).

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch002

5

Control of Interactions A few, b r i e f examples from the l i t e r a t u r e should be s u f f i c i e n t to i l l u s t r a t e the p o t e n t i a l of d i r e c t l y managing enzyme-substrate i n t e r a c t i o n s and i n d i r e c t l y c o n t r o l l i n g subsequent product i n t e r a c t i o n s . Since v i r t u a l l y a l l food p r o t e i n s have a net negative charge a t the pH o f most foods, enzymes engineered to be more p o s i t i v e l y or n e g a t i v e l y charged or more hydrophobic may be r e t a i n e d more or l e s s i n a food as d e s i r e d . For example, the amount o f m i l k - c l o t t i n g or l i p o l y t i c enzymes r e t a i n e d i n cheese curd could be governed by charge or f u n c t i o n a l groups on a modified enzyme. I n t e r a c t i o n s of t h i s type might be important i n subsequent r i p e n i n g or aging of the cheese. Holmes e t a l . ( 6 3 ) have demonstrated the d i f f e r e n t i a l r e t e n t i o n of v a r i o u s , p r o t e o l y t i c m i l k - c l o t t i n g enzymes i n cheese curd. M a r s h a l l and Green ( 6 4 ) have s t u d i e d i n some d e t a i l the e f f e c t s of c a t i o n i c substances i n c l u d i n g p r o t e i n s on the rennet c l o t t i n g time o f m i l k . Normal renneting of m i l k i s thought to r e s u l t i n a decreased charge r e p u l s i o n between c a s e i n a t e

American Chemical Society Library 1155 16th S t ft W. Washington, 0. C. 20039

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch002

48

Figure 8. Inhibition of trypsin by polyhomoarginine. (Reproduced, with per­ mission, from Ref. 53. Copyright 1974, Springer-Verlag New York, Inc.)

FOOD PROTEIN

Π,

DETERIORATION

Residues per molecule

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch002

2.

RICHARDSON

Enzymic Degradation

of

Proteins

49

p a r t i c l e s from the enzymic-mediated r e l e a s e of a h y d r o p h i l i c , n e g a t i v e l y charged p e p t i d e . The decreased charge r e p u l s i o n favors c o a g u l a t i o n of the modified caseinate p a r t i c l e s to y i e l d curd. M a r s h a l l and Green (64) observed that as i n c r e a s i n g amounts of c a t i o n i c m a t e r i a l s were added to the milk to pro­ g r e s s i v e l y n e u t r a l i z e the net negative charge on the c a s e i n m i c e l l e s , the r a t e of enzymic-mediated c o a g u l a t i o n of the c a s e i n increased with the reduced net negative charge i n the system. Many o f these observations have been v e r i f i e d and extended by DiGregorio and S i s t o (33, 34) and by M a t t a r e l l a (35), who s t u d i e d the i n t e r a c t i o n s of p o s i t i v e l y charged β-lactoglobu l i n d e r i v a t i v e s with c a s e i n a t e systems. This has r e s u l t e d i n the use of p o s i t i v e l y charged p r o t e i n s as e l e c t r o s t a t i c coagulants of milk which d r a m a t i c a l l y increased the y i e l d of p r o t e i n s i n cheesemaking ( 3 3 , 34). However, i t i s not known what the e f f e c t s would be on the q u a l i t y of r e s u l t a n t cheese. Kang and Kepplinger (65) showed that a d d i t i o n of a c a t i o n i c peptide, p a l m i t o y l - L - l y s y l - L - l y s i n e e t h y l e s t e r , to a pepsin s o l u t i o n before a d d i t i o n t o milk i n h i b i t e d the c l o t t i n g a c t i v i t y of pepsin. On the other hand, when the peptide was added f i r s t to the milk, an enhanced r a t e o f c l o t t i n g was observed. This i s an extreme example of how p r o t e o l y s i s and subsequent product i n t e r a c t i o n s might be r e g u l a t e d e l e c t r o s t a t i c a l l y . C o n t r o l l i n g the p a t t e r n of p r o t e o l y s i s i n p r o t e i n hydrolysates by r e l y i n g on s t e r i c or charge e f f e c t s between p r o t e i n and an immobilized protease i s a d i s t i n c t p o s s i b i l i t y . By analogy, the p a t t e r n s of s t a r c h h y d r o l y s i s are s u b s t a n t i a l l y d i f f e r e n t depending upon whether a s o l u b l e o r immobilized aamylase i s used (66). Conclusions C o n t r o l of endogenous and exogenous enzymic a c t i v i t y i n foods must be extended beyond c u r r e n t , conventional methods t o r e a l i z e advances i n food p r o c e s s i n g and storage. Suggestions i n t h i s review f o r manipulating food enzymes are based on accepted p h y s i c a l , chemical and b i o l o g i c a l p r i n c i p l e s . Among p o s s i b i l i t i e s discussed are: (1) management of enzymic l a t e n c y i n s i t u , ex v i v o and i n v i v o ; ( 2 ) engineering of enzymes using chemical and b i o l o g i c a l techniques; (3) c o n t r o l of enzymes using n a t u r a l and s y n t h e t i c i n h i b i t o r s : and (4) a l t e r a t i o n of e l e c t r o ­ s t a t i c and hydrophobic f o r c e s on exogenous enzymes and w i t h i n foods that may r e g u l a t e enzymic-substrate and post-enzymic product i n t e r a c t i o n s . Although p r o t e o l y t i c enzymes are s p e c i f i ­ c a l l y d i s c u s s e d , many of the concepts can be g e n e r a l i z e d to other f o o d - r e l a t e d enzymes. Future food s c i e n t i s t s may thus be able to exert b e t t e r c o n t r o l over exogenous and a d v e n t i t i o u s enzymes i n foods by designing and engineering the enzymes as w e l l as by using acceptable enzymic i n h i b i t o r s . Powerful techniques such as

50

FOOD PROTEIN

DETERIORATION

those used f o r chemical m o d i f i c a t i o n of enzymes and i n h i b i t o r s , and s o l i d phase s y n t h e s i s of polypeptides and o l i g o n u c l e o t i d e s coupled w i t h emerging recombinant DNA technology can be brought t o b e a r t o e v e n t u a l l y r e a l i z e b e t t e r c o n t r o l of enzymic r e a c t i o n s i n f o o d p r o c e s s i n g a n d i n food d e t e r i o r a t i o n * Regulation o f endogenous enzymes i n foods i s o b v i o u s l y more d i f f i c u l t a n d must a w a i t a b e t t e r understanding of the physiology o f p o s t - h a r v e s t a n d post-mortem processes.

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Acknowledgments T h i s c o n t r i b u t i o n was made p o s s i b l e b y support from the Walter ?. P r i c e Cheese Research I n s t i t u t e a n d f r o m the College of A g r i c u l t u r a l a n d L i f e Sciences, U n i v e r s i t y o f Wisconsin-Madison, Madison, WI 53706.

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Potter, N.N. "Food Science"; AVI Publishing Co., Inc.; Westport, CT, 1968; p 149, 191. Lawrie, R.A. "Meat Science", 3rd E d . ; Pergamon Press: New York, NY, 1979; p 348. Schwimmer, S. "Post-Harvest Biology and Biotechnology"; Hultin, H.O.; Milner, Μ., Eds.; Food and Nutrition Press, Inc.: Westport, CT, 1978; p 317. Tappel, A . L . "The Physiology and Biochemistry of Muscle as a Food"; Briskey, E.J.; Cassens, R . G . ; Trantman, J . C . , Eds.; Univ. Wisconsin Press: Madison, WI, 1966; p 237. Thunell, R.K.; Duersch, J.W.; Ernstrom, C.A. J. Dairy Sci. 1979, 62, 373. Adams, D.M.; Bramley, T.G. J. Dairy S c i . 1981, 64, 1951. Marshall, R . T . ; Marstiller, J.K. J . Dairy Sci. 1981, 64, 1545. Eigel, W.N.; Hofmann, C . J . ; Chibber, B.A.K.; Tomich, J.M.; Keenan, T.W.; Mertz, E.T. Proc. Natl. Acad. Sci. U.S. 1979, 76, 2244. Humbert, G.; Alais, C. J . Dairy Res. 1979, 46, 559. Barry, J . G . ; Donnelly, W.J. J . Dairy Res. 1980, 47, 71. Donnelly, W.J.; Barry, J . G . ; Richardson, T. Biochim. Biophys. Acta 1980, 626, 117. Richardson, T. "Principles of Food Science. I. Food Chemistry"; Fennema, O.R., Ed.; Marcel Dekker, Inc.: New York, NY, 1976; p 285. Canonico, P.G.; Bird, J.W.C. J. Cell Biol. 1970, 45, 321. Pennington, R.J.T. "Proteinases in Mammalian Cells and Tissues"; North-Holland Publ. Co.: New York, NY, 1977; p 528. Dutson, T.R.; Smith, G . C . ; Carpenter, Z.L. J. Food Sci. 1980, 45, 1097.

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20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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Mead, J . F . ; Fulco, A.J. "The Unsaturated and Polyunsatu­ rated Fatty Acids in Health and Disease"; C.C. Thomas: Springfield, IL, 1976; p 122. Richardson, T.; Tappel, A.L. J. Cell Biol. 1962, 13, 43. Bitman, J.; Wrenn, T.R.; Dryden, L . P . ; Edmondson, L . F . ; Yoncoskie, R.A. "Microencapsulation"; Vandegaer, J.E., Ed.; Plenum Press: New York, NY, 1974; p 200. Scott, T.W.; Cook, L.J.; Mills, S.C. J . Am. Oil Chem. Soc. 1971, 48, 358. Snyderman, R.; Goetzl, E.J. Science 1981, 213, 830. Kang, C.K.; Warner, W.D.; Rice, E.E. U.S. Patent 3,818,106, 1974. H i l l , R.D.; Laing, R.R. Nature 1966, 210, 1160. H i l l , R.D.; Laing, R.R. Biochim. Biophys. Acta 1967, 132, 188. Rickert, W. Biochim. Biophys. Acta 1970, 220, 628. Tang, J., Ed. "Acid Proteases: Structure, Function and Biology"; Adv. Exp. Med. Biol., Vol. 95; Plenum Press: New York, NY, 1977; p 355. Kaiser, E.T.; Nakagawa, Y. "Acid Proteases: Structure, Function and Biology"; Tang, J., Ed.; Adv. Exp. Med. Biol., Vol. 95; Plenum Press: New York, NY, 1977; p 159. Chang, T.M.S. "Enzyme Engineering", Vol. 2; Pye, E.K.; Wingard, L . B . , Eds.; Plenum Press: New York, NY, 1973; p 419. May, S.W.; Li, N.N. "Enzyme Engineering", Vol. 2; Pye, E.K.; Wingard, L . B . , Eds.; Plenum Press: New York, NY, 1973; p 77. Magee, E . L . , J r . ; Olson, N.F. J . Dairy Sci. 1981, 64, 600. Magee, E . L . , J r . ; Olson, N.F. J . Dairy Sci. 1981, 64, 611. Magee, E . L . , J r . ; Olson, N.F. J. Dairy Sci. 1981, 64, 616. Malamud, D.; Drysdale, J.W. Anal. Biochem. 1978, 86, 620. DiGregorio, F . ; Sisto, R. U.K. Pat. Appl. 2,052,515, 28 Jan., 1981. DiGregorio, F . ; Sisto, R. J. Dairy Res. 1981, 48, 267. Mattarella, N. Ph.D. Thesis, 1981, University of Wisconsin, Madison, WI 53706. Bovey, E.A.; Yanari, S.S. "The Enzymes", Vol. IV; Boyer, P.D.; Lardy, H.; Myrback, Κ., Eds.; Academic Press: New York, NY, 1960; p 63. Ruenwongsa, P.; Chvlavatnatol, M. "Acid Proteases: Structure, Function and Biology"; Tang, J., Ed.; Adv. Exp. Med. Biol., Vol. 95; Plenum Press: New York, NY, 1977; p 329. Kumar, P.M.H.; Ward, P.H.; Kassell, B. "Acid Proteases: Structure, Function and Biology"; Tang, J., Ed.; Adv. Exp. Med. Biol., Vol. 95; Plenum Press: New York, NY, 1977; p 211.

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44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

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Nishimori, K.; Kawaguchi, Y.; Hidaka, M.; Uozumi, T.; Beppu, T. J . Biochem. 1981, 90, 901. Foltmann, B.; Pedersen, V.B. "Acid Proteases: Structure, Function and Biology"; Tang, J., Ed.; Adv. Exp. Med. B i o l . , Vol. 95; Plenum Press: New York, NY, 1977; p 3. Green, C.; Tibbetts, C. Proc. Natl. Acad. Sci., USA 1980, 77, 2455. Shortle, D.; Koshland, D.; Weinstock, G.M.; Botstein, D. Proc. Natl. Acad. Sci., USA 1980, 77, 5375. Kudo, I.; Leineweber, M.; Raj Bhandary, U.L. Proc. Natl. Acad. Sci., USA 1981, 78, 4753. Woo, S.; Creamer, L.K.; Richardson, T. J . Agric. Food Chem., In Press, 1982. Johansen, J . T . ; O'Hesen, M.; Svendsen, I. Biochim. Biophys. Acta 1967, 139, 211. Mitz, M.A.; Summaria, L . J . Nature 1961, 189, 576. Silman, H.I.; Sela, M. "Poly-α-Amino Acids"; Fasman, G.D., Ed.; Marcel Dekker, Inc.: New York, NY, 1967; p 605. Becker, R. "Polyamino Acids, Polypeptides and Proteins"; Stahmann, M.A., Ed.; Univ. Wisconsin Press: Madison, WI, 1962; p 301. Frensdorf, A.; Sela, M. J. Biochem. 1967, 1, 267. Wade, N. Science 1981, 213, 623. Richardson, M. Food Chem. 1980-81, 6, 235. Fritz, H.; Tschesche, H.; Green, L.J.; Truscheit, L., Eds. "Proteinase Inhibitors"; Proc. 2nd Int. Res. Conf.; Springer-Verlag: New York, NY, 1974; p 311. Rigbi, M.; Elkana, Y.; Segal, N.; Kliger, D.; Schwartz, L. "Proteinase Inhibitors"; Proc. 2nd Int. Res. Conf.; Fritz, H.; Tschesche, H.; Green, L.J.; Truscheit, L . ; Eds.; Springer-Verlag: New York, NY, 1974; p 541. Rich, D.H.; Sun, E . ; Sengh, J . Biochem. Biophys. Res. Commun. 1977, 74, 762. Marciniszyn, J., J r . ; Hartsuck, J . A . ; Tang, J . "Acid Proteases: Structure, Function and Biology"; Tang, J., Ed.; Adv. Exp. Med. B i o l . , Vol. 95; Plenum Press: New York, NY, 1977; p 199. Morrison, R.T.; Boyd, R.N. "Organic Chemistry"; Allyn and Bacon, Inc.: Boston, NY, 1973; p 458. Bruice, T.C.; Benkovic, S.J. "Bioorganic Mechanisms, Vol. II"; W.A. Benjamin, Inc.: New York, NY, 1966; p 3. Kobayashi, H.; Murakami, K. Agric. Biol. Chem. 1978, 42, 2227. Jakoby, W.B.; Wilchek, M. "Methods in Enzymology"; Colowick, S.P.; Kaplan, N.O., Eds.; Academic Press: New York, NY, 1977; p 774. Stewart, J.M.; Young, J.D. "Solid Phase Peptide Synthesis"; W.H. Freeman and Co.: San Francisco, CA, 1969. Duckworth, M.L.; Gait, M.J.; Goilet, P.; Hong, G.F.; Singh, M.; Richards, T.C. Nucleic Acid Res. 1981, 9, 1691.

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Proteins

Matteucci, M.D.; Caruthers, M.H. J . Am. Chem. Soc. 1981, 103, 3185. Holmes, D.G.; Duersch, J.W.; Ernstrom, C.A. J . Dairy Sci. 1977, 60, 862. Marshall, R . J . ; Green, M.L. J . Dairy Res. 1980, 47, 359. Kang, Y.; Kepplinger, J. Unpublished Observations, 1981. Reilly, P.J. "Immobilized Enzymes for Food Processing"; Pitcher, W.H., Jr., Ed.; CRC Press, Inc.: Boca Raton, FL, 1980; p 113. Schröder, E . ; Lübke, K. "The Peptides", Vol. 1; Academic Press: New York, 1965; p 96.

RECEIVED August 30, 1982.

3 Effects of Lipid Oxidation on Proteins of Oilseeds

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch003

ROBERT L. ORY and ALLEN J. ST. ANGELO United States Department of Agriculture, Southern Regional Research Center, Agricultural Research Service, New Orleans, LA 70179

Peanuts (groundnuts) are one of the world's major oilseeds and are grown in tropical and subtropical countries. World pro­ duction of peanuts is annually about 19 million tons (1), with most of them grown in India, China, Africa, and the United States (2). The United States produces about 10% of the world supply and is the only country that consumes most of the crop as whole nut products. Most countries crush peanuts for the edible oil. Peanuts contain 50-55% oil and 27-30% protein. The major proteins of peanuts are the storage globulins, arachin and conarachin, that make up almost 75% of the total proteins. After removal of the oil, the o i l - f r e e peanut meal contains 55-60% protein that can be used to raise the nutritional quality of protein-deficient diets or to provide alternate sources of low-cost functionally useful protein. Nutrition, however, is not simply a matter of supplying individual nutrients. Good nutrition involves groups of nutrients whose functions are closely related, each providing a necessary part of the balanced diet. Storage o i l and protein in oilseeds l i k e soybeans and peanuts are in close proximity and, under normal conditions, no changes occur in either the protein or the o i l , which contains polyunsatu­ rated fatty acids. Peanut o i l contains varying amounts of oleic and linoleic acids; soybean o i l contains these two, plus linolenic acid. It is the latter two fatty acids that can be oxidized to hydroperoxides and their breakdown products ( i . e . : ketones, alde­ hydes , alcohols, etc.) which, in turn, can react with terminal functional groups of amino acids in proteins and enzymes. Such interactions of l i p i d peroxides and the secondary products with proteins can alter the functional and nutritional properties, in addition to affecting their flavor. Lipoxygenase is the principal enzyme that catalyzes oxidation of polyunsaturated fatty acids in oiIseeds. Denatured metalloproteins are the primary nonenzymatic catalysts. Soybean lipoxygenase is possibly the most studied oilseed lipoxygenase. Peanuts also contain the enzyme, about a f i f t h of the a c t i v i t y in

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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FOOD P R O T E I N DETERIORATION

soybeans (3, 4 ) . This enzyme acts on l i n o l e i c and l i n o l e n i c acids to produce hydroperoxides which, in turn, can react with terminal functional groups of amino acids in proteins ( i . e . : -SH, -OH, -C00H, -NH2) to alter t h e i r functional, f l a v o r , and nutritional properties. Hydroperoxides also break down into carbonyl compounds that react readily with these amino acids; especially when catalyzed by metalloproteins. Some effects of protein interactions with l i p i d peroxides and their secondary products and methods for studying them are described.

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Experimental Procedures Interactions of l i p i d peroxides with proteins can be measured by several methods: thin layer chromotography of the amino acids, l i p i d peroxides, and their reaction products (24); dual staining of gel electrophoretic protein patterns for both protein and associated l i p i d (13) ; and flourescence spectroscopy of the extracted proteins (23). Catalysts of Lipid Oxidation Enzymatic Oxidation. Lipoxygenase (E.G. 1.13.1.13) i s the principal enzyme for catalyzing oxidation of polyunsaturated fatty acids in vegetable o i l s . It is present in v i r t u a l l y a l l oilseeds but has been studied more in soybeans than in other oilseeds (3J. This enzyme i s substrate-specific in that i t attacks only c i s , c i s , - l , 4 - pentadiene bonds, such as those in 1inoleic and 1inolenic acids. The primary products are o p t i c a l l y active cis-trans conjugated hydroperoxides (5). Once formed, hydroperoxides can be degraded further into various aldehydes, ketones, and alcohols by enzymatic and/or nonenzymatic c a t a l y s t s . It is these secondary products, plus the intact hydroperoxides that can interact with proteins to lower both nutritional and flavor quality of a food product. Soybean lipoxygenase, because of i t s higher a c i t i v t y , has been the most completely investigated of the seed lipoxygenases (2,8,9). It is not particulate and can be extracted with water. It has a molecular weight of 100,000, consists of a single polypeptide chain, exists in at least two isomeric forms, and has a pH range from 6-9, depending upon the isomer being studied. With l i n o l e i c acid as substrate, i t forms predominantly the C-13 hydroperoxide, rather than the C-9 isomer (3>). Chan and coworkers (10,11) examined methyl esters of the soybean 1ipoxygenase-formed C-9 and C-13 linoleate hydroperoxides after thermal decomposition. The v o l a t i l e degradation products included many of the shorter chain aldehydes that have been implicated in flavor problems of plant food products. Peanut lipoxygenase has not been as well characterized as soybean lipoxygenase but various workers (4,12-17) have identified some s i m i l a r i t i e s in the two enzymes. The p a r t i a l l y purified

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57

Effects

enzyme has a general pH optimum of 6.2 and is rather heat-labile, losing a l l a c t i v i t y at temperatures above 40°C. Sulfhydryl reducing agents (2,3-dimercaptoethanol and d i t h i o t h r e i t o l ) i n h i b i t a c t i v i t y by 55-100%. Sanders et a l . (18) isolated 3 isozymes from raw peanuts; two having a pH optimum of 6.2 and the t h i r d , at pH 8.3. Molecular weight of a l l isomers was 73,000. The alkaline pH enzyme was reported to be CN-tolerant but the acid isozymes were inhibited by NaCN i n their tests. However, work by Siddiqui and Tappel (16) and St. Angelo and Kuck (19) showed no inhibition of peanut 1ipoxygenase by CN when the system was buffered at pH 6.2 to offset the NaCN-induced rise in pH. Increasing concentration of NaCN caused a rise in pH, which then decreased a c t i v i t y of the acid pH isozyme. Nonenzymatic Oxidation. As was shown e a r l i e r by St. Angelo, et a l . (6_,7J, catalysis of l i p i d peroxidation/ degradation by nonenzymatic agents, such as hemeproteins and metals, can induce faster deterioration in a high oil/protein product l i k e peanut butter, than that caused by 1ipoxygenase. Relative rates of l i p i d peroxidation shown in Table I i l l u s t r a t e the faster rates catalyzed by metal ions, Fe and Cu, and the metalloproteins, peroxidase and tyrosinase. Depending upon the types of handling, processing and/or storage conditions, i t is these l a t t e r Table I .

Effect of various catalysts on rates of peroxidation of fatty acids in stored peanut butter

Catalyst None NaCl (0.04 mmole) Cupric acetate (0.02 mmole) FeCI3 (0.02 mmole) Soybean lipoxygenase!/ Tyrosinase Peroxidase Boiled Peroxidase

Peroxide Value (meq/kiT meq. increase 28 days initial 30.0 14.3 11.8 10.3 7.0 6.3 5.4 7.3

31.1 24.9 33.1 36.8 21.0 24.9 27.0 26.6

1.1 10.6 21.3 26.5 14.0 18.6 21.6 19.3

1/ Concn. of enzymes was 0.1% (w:v). catalysts that frequently cause greater deterioration of foods through l i p i d peroxide-protein interactions. Efffects of Lipid Peroxide-Protein Interactions Advanced stages of l i p i d peroxidation cause flavor problems that are well known. Rancidity in a vegetable o i l or oi1containing food w i l l preclude any attempts to consume the food

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because of objectionable odors and off-flavors. These off-flavors may or may not be 1ipid-protein interactions, depending upon the extent of the rancidity. Effects on nutritional value of proteins, however, may not be as obvious without chemical analysis of the products. In general, the protein quality of oilseed proteins i s , with very few exceptions, lower than animal proteins (20). Oilseed proteins have inherent amino acid deficiencies that d i f f e r between seeds. For example, soybeans are low in methionine but have sufficient lysine. Sesame is low in lysine but is high in sulfur-containing amino acids, whereas cottonseed and peanuts are lower in both of these amino acids (20). These deficiencies can be easily improved by blending with other proteins having the complimentary amino acid but, i f large amounts of l i p i d peroxides and their secondary products are present, they can bind to terminal reactive groups of cysteine and methionine or other amino acids (Table II) to inactivate them, i f present in enzymes, or lower nutritional value, i f present in food proteins. Table I I .

Terminal reactive groups of amino acids in proteins Amino Acid ARG, LYS CYS GLU, ASP SER, THR, TYR MET HIS

Reactive Group -NH -SH -C00H -OH -SCH =NH 2

3

Proteins are the most important source of amino acids. Some amino acids can be produced in the body but the essential amino acids must be obtained from the diet as protein, peptides, or free amino acids. Of these, i t is lysine, methionine, and cysteine that are most limiting in oi1 seeds and are the ones that are inactivated by l i p i d peroxides. If peroxidized or autoxidized l i p i d s are ingested, they can also bind to enzymes and inhibit their a c t i v i t y . This was shown by Matsushita (21), who examined the specific interactions of 1inoleic acid hydroperoxide and i t s secondary products with trypsin, pepsin, 1ipase, and RNase. He found a correlation between the incorporation of autoxidized 1ipids into the enzymes and their inactivation caused by resulting damage to the amino acids: aspartic and glutamic acids, threonine, cysteine, methionine, leucine, tyrosine, lysine, and h i s t i d i n e . Methionine, cysteine, h i s t i d i n e , lysine, and tyrosine were the most l a b i l e to hydroperoxide damage, whereas lysine,

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Effects

59

h i s t i d i n e , and methionine were the most susceptible to interac­ tions with the peroxide secondary products ( e . g . : aldehydes, ketones, etc.) Karel, et a l . (22) also showed that peroxidizing methyl 1inoleate could react with proteins and amino acids. They f o l ­ lowed the reaction of peroxidizing 1inoleate with lysozyme by electron spin resonance (ESR). Peroxides did not break disulfide bonds but several reaction products were identified from h i s t i d i n e , methionine, and lysine interactions. In addition to the thorough characterization of ESR signals in peroxide-lysozyme interactions, they also examined ESR spectral characteristics of g e l a t i n , bovine serum albumin, casein, lactalbumin, g l i a d i n , trypsin, several other enzymes and free amino acids. Results were similar to those obtained with lysozyme. Lipid peroxides and/or free radicals can readily abstract hydrogen from -SH groups in proteins but they do not easily break -SS- bonds. As noted e a r l i e r (13), 1ipoxygenase is the prime suspect for catalyzing 1i pid oxidation in raw peanuts but this enzyme is destroyed by roasting temperatures. In cooked or roasted peanut products, l i p i d oxidation is catalyzed by nonenzymic catalysts such as metal loproteins, free Fe and Cu. One of the primary methods for identifying 1ipid-protein interactions in crude protein extracts is polyacrylamide gel elec­ trophoresis, employing dual staining for both protein and 1ipid. Ami do Black and Coomassie Blue are ideal stains for protein, but they do not identify those proteins with associated 1ipid. Sudan stains are the principal stains employed in histochemical analyses for l i p i d s but they are not sensitive enough for oilseed proteins separated by gel electrophoresis. We, therefore, developed a pro­ cedure using Rhodamine 6G or Oi1 Red 0 stain, allowing the gels to soak in the l i p i d s t a i η at 37°c overnight. This produced satisfactory 1ipid-stained gels as shown in Figure 1. Total pro­ teins are stained with ami do black. Lipid-stained gels show only those bands that contain 1ipid material associated with protein. Only three proteins of peanuts appear to bind l i p i d peroxides. Of these, the principal protein that binds 1ipids appears to be the major storage protein, arachin. This general type of 1ipid-protein staining pattern for raw peanuts also appears in roasted peanuts (Figure 2). Peanuts that are roasted for candy or peanut butter manufacture undergo some protein denaturation, which can lower protein s o l u b i l i t y . Because of t h i s , there are fewer protein bands in gel patterns of roasted peanut proteins, but 1i pi d-stai ni ng bands s t i l l appear. Only two major protein bands appear in roasted peanut extracts (compared to three in raw peanuts) and these same two 1ipid-protein complexes are evident in Oi1 Red 0-stained gels of both raw and roasted peanuts. Lipid bands are similar in both raw and roasted peanuts, in that arachin is sti11 the principal protein that binds to l i p i d peroxides. The conarachin fraction of peanut proteins does not appear to bind to the 1ipid peroxides.

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FOOD PROTEIN DETERIORATION

Figure 1. Dual staining of gel electrophoretic patterns of raw peanut proteins with oil red Ο (A), amido black (B), and rhodamine 6G (C). Migration is toward the anode.

Figure 2. Dual staining of gel electrophoretic patterns of roasted peanut proteins with amido black (A and C) and oil red Ο (Β and D). A and Β are freshly roasted peanuts; C and D are rancid roasted peanuts, 1 year old. Migration is toward the anode.

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Thin layer chromatography (TLC) is another method that can be used to identify reactions between peroxidized l i p i d s and amino acids but, because of different s o l u b i l i t i e s of the amino acids and the 1ipids in aqueous and organic solvents, this technique has not been used much. Reactants had to be separated on two or more plates or in 2-phase systems to insure separation of both ma­ t e r i a l s . To avoid the use of several separations and/or 2-phase systems that required an additional development, Kuck, et a l . (24) developed a single phase system for TLC analysis of free amino acids, l i p i d peroxides, and the 1ipid peroxide-ami no acid complex (Figure 3). The developing solvent of petroleum ether: diethyl ether: glacial acetic acid (60:40:1) separated the 1inoleate hydroperoxide-amino acid complex (A), 1inolete hydro­ peroxide (D), oxidized 1inoleic acid (E), and the free ami no acids threonine and lysine (F). Β and C are unknown minor products. Identities of the separated materials on the TLC plates were con­ firmed by infrared spectral analysis and mass spectrometer frag­ mentation patterns. Polyacrylamide gel electrophoresis and TLC therefore, can provide useful information on the effects of l i p i d peroxides binding to proteins and amino acids, but these methods do not identify changes in protein conformation by changes in size and charge of the proteins in the gels. We also employed fluorescence spectroscopy to compare fresh and peroxidized peanut proteins (23). The NaCl-soluble proteins were extracted and scanned. Figure 4 i l l u s t r a t e s fluorescence spectra of salt-soluble proteins from fresh raw peanuts (curve A) and rancid raw peanuts stored 4.5 months at 30°c ( Β ) , plus freshly roasted peanuts (C) and 12-month old roasted peanuts (D). These scans show that both raw and roasted peanuts have some natural fluorescence which can be quenched or decreased by apparent 1ipid peroxide interactions (curves Β and D). Both curves of rancid peanut proteins (B and D) showed some decrease in intensity com­ pared to the fresh samples (A and C). Malonaldehyde is one product of l i p i d peroxidation that fluoresces but this apparent quenching suggests that other nonfluorescing l i p i d peroxide secondary products may be formed in greater amounts than is malonaldehyde. Increasing l i p i d peroxidation in both raw and roasted pea­ nuts can also affect s o l u b i l i t y of salt-soluble proteins (14, 23). Peanuts were ground in a food blender to disrupt the eel 1 s and promote 1ipoxygenase a c t i v i t y on the 1inoleic acid of the storage o i l . Ground samples were stored under separate condi­ tions for 4.5 months: (1) at 4°c in a sealed glass j a r , (2) at 30°C in an open, gauze-covered j a r , and (3) at 30°C in an open, gauze-covered jar with 10% rancid o i l added to enhance 1ipidprotein interactions. Results in Table III i l l u s t r a t e the recoveries of soluble proteins from hexane-deoiled meals prepared from these samples. As absorbance ( l i p i d peroxidation) increased, so did the amounts of salt-soluble proteins extracted from the ground peanuts. Conversely, the amounts of

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FOOD PROTEIN DETERIORATION

Figure 3. Thin layer chromatographic separation of linoleate hydroperoxide, amino acids, and their complexes. Key to spots: 1, pure linoleic acid; 2, oxidized linoleic acid; 3, threonine; and 4, lysine. A-F and the developing solvent are defined in the text.

Lipid Oxidation

Effects

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ORY AND ST. ANGELO

Figure 4. Fluorescence spectra of proteins extracted from fresh and rancid peanuts. Key: A, fresh raw peanuts; B, rancid raw peanuts; C, freshly roasted peanuts; and D, rancid roasted peanuts.

FOOD PROTEIN DETERIORATION

64

salt-extracted residue (oil-and protein-extracted) remaining showed a decrease. The reason for the increased s o l u b i l i t y of proteins after association with l i p i d peroxides is unknown; the amounts of bound l i p i d are not sufficient to account for the increase. Table I I I .

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Sample 1 2 3

Effects of l i p i d peroxidation on protein s o l u b i l i t y in ground-up raw peanuts after 4.5 months storage. (A) I n i t i a l recoveries. % Oil removed

% Deoiled meal

46.10 45.09 48.45

53.90 54.91 51.55

CDHP Units U )

%Protein in meal 46.64 44.47 44.16

0.53 1.21 1.89

(B) Recoveries after NaCl extraction of proteins from deoiled meals.'2)

Sample 1 2 3

%NaClinsoluble 37.48 35.80 34.30

%NaClsoluble 31.60 36.55 39.17

%Total recovered 69.08 72.35 73.47

Protein Content % NaCl% NaClinsoluble soluble 22.99 18.96 17.29

88.48 87.86 88.01

(1) Conjugated diene hydroperoxide units of peroxidation. (2) Values shown are those after dialysis and freeze-drying. Conclusions. Lipid peroxides formed by both enzymatic and nonenzymatic catalysts can react with proteins, very l i k e l y through terminal functional groups of amino acids. These 1ipidprotein interactions can be measured by gel electrophoresis, thin layer chromatography, electron spin resonance, and fluorescence spectroscopy. Lipid peroxide interactions can affect several properties of proteins. If the interactions occur through functional groups of essential amino acids, nutritional value w i l l be lowered. If the binding of l i p i d peroxides with arachin involves lysine, this would be very undesirable since lysine is already low in this protein. If binding affects amino acids in active sites of enzymes, these enzymes would have diminished a c t i v i t y . Peroxidation also appears to decrease fluorescence of oilseed proteins but i t seems to increase their s o l u b i l i t y .

3.

ORY AND ST. ANGELO

Lipid Oxidation Effects

65

LITERATURE CITED 1. 2. 3. 4. 5.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch003

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Lusas, E. W. J . Amer. Oil Chem. Soc., 1979, 56, 425. Anonymous. Southeast Peanut Farmer, 1978, 7(5), 15. Veldink, G. Α.; Vliegenthart, J . F. G; Boldingh, J. Prog. Chem. Fats Other L i p i d s , 1977, 15, 131. St. Angelo, A. J.; Ory, R. L. J . Agr. Food Chem., 1979, 27, 229. P r i v e t t , O. S.; Nickel, C.; Lundberg, W. O.; Boxer, P. D. J. Amer. Oil Chem. Soc., 1955, 32, 505. St. Angelo, A. J.; Ory, R. L. J . Amer. Oil Chem. Soc., 1975, 52, 38. St. Angelo, A. J.; Ory, R. L.; Brown, L. E. Oleagineux, 1973, 28, 351. Axelrod, B. In "Food Related Enzymes"; Whitaker, J. R., Ed., American Chemical Society: Washington, D.C., 1974, p. 324. Tappel, A. L. In "The Enzymes" 2nd ed., v o l . 8; Boyer, P. D., Ed., Academic Press: New York, 1963; p. 275. Chan, H. W. S.; Levett, G. L i p i d s , 1976, 12, 99. Chan, H. W. S.; Prescott, F. A. A. Biochim. Biophys. Acta, 1975, 380, 141. D i l l a r d , M. G.; Hendrick, A. S.; Koch, R. B. J . B i o l . Chem., 1960, 236, 37. St. Angelo, A. J.; Kuck, J. C . ; Ory, R. L. J . Agr. Food Chem., 1979, 27, 229. St. Angelo, A. J.; Ory, R. L. Peanut Sci., 1975, 2, 41. Pattee, H. E . ; Singleton, J. A. J . Amer. Oil Chem. Soc., 1977, 54, 183. S i d d i q i , A. M . ; Tappel, A. L. J . Amer. Oil Chem. Soc., 1957, 34, 529. St. Angelo, A. J.; Ory, R. L. In "Symposium: Seed Proteins"; Inglett, G. Ε., Ed., Avi Publish. Co.: Conn., 1972; p. 284. Sanders, T. H . ; Pattee, H. E . ; Singleton, J. A. L i p i d s , 1975, 10, 681. St. Angelo, A. J.; Kuck, J. C. Lipids 1977, 12, 682. Molina, M. R.; Bressani, R. Dev. Food Sci., 1979, 2, 39. Matsushita, S. J . Agr. Food Chem., 1975, 23, 150. Karel, M . ; Shaich, K . ; Roy, R. B. J . Agr. Food Chem. 1975, 23, 159. St. Angelo, A. J.; Ory, R. L. J. Agr. Food Chem., 1975, 23, 141. Kuck, J. C.; St. Angelo, A. J.; Ory, R. L. Oleagineux, 1978, 33, 507.

RECEIVED May 10,

1982.

4 Discoloration of Proteins by Binding with Phenolic Compounds F. A. BLOUIN, Z. M. ZARINS, and J. P. CHERRY

1

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

United States Department of Agriculture, Southern Regional Research Center, Agricultural Research Service, New Orleans, LA 70179

Phenolic compounds that occur in plant materials have very diverse chemical structures. Some are simple benzene deriva­ t i v e s , l i k e the phenolic acids, p-hydroxybenzoic acid and caffeic acid (Figure 1). Others contain more complex ring systems such as the flavonoid quercetin and the terpenoid gossypol (Figure 1). Still others are extremely complex in structure. The vegetable tannin, procyanidin, i s thought to be an oligomer of flavan-3-ol units (Figure 1). Lignin i s a network type polymer composed of substituted cinnamyl alcohols (Figure 1). Phenolic compounds are found in a l l plant tissues. G r i f f i t h s (1), for example, found gentisic acid in the wood, bark, roots, stems, leaves, flowers, pod w a l l , cotyledons and testa of the cacao tree (Table I). Leucoanthocyanins and epicatechin were also present in most of the parts of this particular plant. The concentration of phenolics in plant materials can be quite high. Lignin represents 20-30% of the dry weight of the woody stems of trees. The dry matter of tea leaves is 40% polyphenols of the tannin type. Chlorogenic acid, one of the com­ monly occurring phenolic esters, is present in fruits at about a 0.1% level whereas in sunflower seed i t s concentration can be as high as 2-3%. In the natural state, the low molecular weight phenolic compounds are generally bound to carbohydrate moieties. For example, one of the commonly occurring flavonoids is rutin (Figure 2). It is a quercetin 3-0-rutinoside; a disaccharide of glucose and rhamnose is bound to the 3-hydroxyl of the C-ring of the quercetin. Even the polymeric polyphenol, 1ignin, i s covalently bound to the hemicellulose components of the cell walls. Phenols sometime occur in the free state but usually in storage tissues such as seeds, in dead tissues such as the heartwood of trees, or in diseased plant tissues. 1

Current address: United States Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118. This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

Figure 1.

p-Hydroxybenzoic acid Quercetin

OAr(Lig)

Gossypol

Structures of various types of phenolic compounds found in plant materials.

Caffeic acid

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

+

+ ++ +

+++ +

+++

+++

+

+

+

+

++

+

Cotyledon

Testa

+

+

++

+

+++

+++

++

++

+

+++

++

+

++++

+

++

+

+

++

+

+

Unhydrolysed extract Leuco(-)-Epianthocyanin catechin

+

,

+++

Pod wall

Flower

Old leaf

Young leaf

Green stem

Roots

++

Bark

Gentisic acid

++

p-Coumaric acid

Heart wood

Caffeic acid

++

Quercetin

Hydrolysed extract

Compound in

j

++++

++

++

Cyanidin glycoside

~

Distribution of Flavonoid Compounds in the Tissues of the Cacao Tree (Treobroma cacao) ( 1 ) ·

Sap wood

Plant part

Table I .

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Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

FOOD P R O T E I N

OH

OH

Figure 2. Structure of rutin.

DETERIORATION

4.

BLOWN ET AL.

Discoloration of Proteins

71

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

Discoloration When plant tissues are disrupted by grinding, heating or blending with other ingredients such as occurs in food processing, the phenolic components usually undergo enzymatic and nonenzymatic reactions that can cause undesirable discoloration in the processed food. Figure 3 i l l u s t r â t e s the effect of several types of plant protein products on the color of biscuits (2). Soybean and peanut flours used at the 20% replacement level do not cause a color problem. Sunflower, a l f a l f a leaf and cottonseed flours, on the other hand, cause a marked discoloration of the b i s c u i t s . In the case of the sunflower seed f l o u r , this discoloration is believed to be due to chlorogenic acid. The coloration caused by the a l f a l f a leaf protein product has been attributed mainly to chlorophyll pigments (3) but polyphenol s are also thought to contribute to the discoloration (4J. Work at the Southern Regional Research Center (SRRC) has shown that flavonoids, gossypol and possibly other phenolic compounds contribute to the color observed when cottonseed flours are used in foods (2). Interactions There are two major types of interactions between proteins and phenolic compounds (5). F i r s t , phenols combine reversibly with proteins by hydrogen bonding. Second, they interact i r r e v e r s i b l y by oxidation and condensation-type reactions. Infrared spectral data indicate that hydrogen bonding between phenols and N-substituted amides is one of the strongest types of H-bonds {§_). Research on col lagen, vegetable tannins and synthetic polymers (j>) has established the importance of the peptide linkage in the formation of Η-bonded complexes between tannins and proteins. The interaction is probably between the peptide oxygen and the hydrogen of the phenol. Condensed tannins are bound to proteins almost independent of pH below pH 7.0 to 8.0. This bonding is thought to involve only un-ionized phenolic hydroxyl groups. Hydrolyzable tannins, on the other hand, are strongly bound to proteins at pH 3.0 to 4.0 but the binding decreases above pH 5.0. In this case, the stronger Η-bonding has been attributed to the un-ionized carboxyl groups and the weaker Η-bonding to the un-ionized hydroxyl groups of the tannins. Oxidation of phenols is the f i r s t step in the irreversible type protein-phenol interaction. This oxidation can be enzymatic (as i l l u s t r a t e d in Figure 4) or nonenzymatic. The primary reac­ tion products are qui nones which are themselves very reactive compounds. These qui nones can then react directly with protein groups or can f i r s t undergo polymerization and/or oxidation-reduc­ tion reactions to produce secondary reaction products which can form covalent bonds with the proteins. The work of Mason and Peterson (7j has shown that sulfhydryl and N-terminal ami no groups are the most reactive. The epsilon-amino group of lysine also

DETERIORATION

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

FOOD PROTEIN

ure 3. Biscuits containing 100% wheat and 20% plant-protein products. (Reproduced from Ref. 2. Copyright 1981, American Chemical Society.)

Figure 4.

SULFHYDRYL. C-AMINO

N - T E R M I N A L AMINO, β

REACTIONS

POLYMERIZATION

POLYPHENOLOXIDASE

• [o]

BONDING

OH

OH

TO PROTEIN GROUPS.

COVALENT

I

Reaction sequences involved in covalent bonding of phenols to proteins.

REACTIONS

REDUCTION

OXIDATION-

0-8ENZ0QUIN0NE

CATECHOL

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

74

FOOD PROTEIN DETERIORATION

reacts with qui nones but more slowly. These qui none-type reactions are most commonly cited as causing discoloration problems in plant food products.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

Cottonseed Flours One of our interests at SRRC is the use of high protein cottonseed flours as edible food products. Glanded cottonseeds contain high concentrations of the phenolic compound gossypol, enclosed in pigment glands. This compound i s toxic to humans but an edible flour can be made by removal of the glands in a process developed at SRRC called the Liquid Cyclone Process (8). Glandless cottonseeds do not contain gossypol and an edible flour is prepared from this material by simple hexane extraction methods. Biscuits prepared using 20% cottonseed flours as a replacement for wheat flour are yellow-brown in color (Figure 3 ) . The biscuit prepared with glanded flour is much darker in color than that containing glandless f l o u r . Our research work was to isolate and identify the pigments responsible for this color problem. Experimental. Cottonseed flour was prepared from glanded seeds at SRRC by the Liquid Cyclone Process of Gardner, et a l . (8). The glanded flour had a proximate composition of 57.3% protein, 32.1% carbohydrate, 1.6% l i p i d , 7.7% ash and 2.3% crude f i b e r . The glandless cottonseed flour was obtained from the Plains Cooperative Oil M i l l , Lubbock, Texas and had a proximate compositon of 56.6% protein, 29.9% carbohydrate, 3.7% l i p i d , 7.5% ash and 2.3% crude f i b e r . Analysis of glanded flour indicated 0.04% free gossypol and 0.15% total gossypol. Free and total gossypol analyses of glandless flour indicated that i t s gossypol content was below the detectable level s of the methods. Extraction with petroleum ether, chloroform, 85% aqueous isopropyl alcohol, water and 10% sodium chloride were conducted at room temperature with s t i r r i n g at a solid to solvent ratio of 1:10. Extraction times were 1 hr for the water and salt solution systems and 24 hr for the other solvents. Solids were separated from extracts either by f i l t r a t i o n or centrifugation and residues were washed with a volume of sol vent equal to the original vol ume used. Insoluble residues were dried in a i r (to remove petroleum ether), in vacuum oven (chloroform) or by freeze drying after removal of alcohol by rotary evaporation or salt by d i a l y s i s . Solvents were removed from the extracts by rotary evaporation and/or freeze drying and salt by d i a l y s i s . Pepsin digestions were conducted on the salt insoluble residues at 37°C for 24 hr at pH 2 using 1:100 ratio of enzyme to substrate. Cellulase treatments were at 45°C for 24 hr at pH 4.5 with an enzyme ratio of 1:50. Digestions with amylase were run at 75°C for 4 hr at pH 6.5 with an enzyme ratio of 1:25.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

4.

BLOWN ET AL.

Discoloration

of

Proteins

75

Insoluble residues were separated from digests by centrifugation, washed with water, neutralized and freeze dried. Digests were also neutralized and freeze dried. Insoluble amylase residues were treated with di methylsulfoxide (DMSO) with s t i r r i n g for 1 hr at 160°C. The insoluble residue was collected on a f i l t e r , washed with 50% methanol , dispersed in water, neutralized and freeze dried. The DMSO extract was poured into a large volume of 50% methanol and stirred for about 1 hr. The precipitated sol ids were separated from the solution by centrifugation, washed with 50% methanol, dispersed in water, neutralized and freeze dried (fraction A ) . The non-precipitable fraction was isolated by rotary evaporation of the methanol-water-DMSO solution (fraction B). Wheat flour biscuits were prepared with 20.0 g wheat or composite f l o u r , 1.0 g baking powder, 0.5 g s a l t , 4.5 g shortening, and 15.0 g f l u i d milk. Biscuits were prepared from plant-protein flours based on 20% replacement for wheat f l o u r . In biscuits prepared with fractions isolated from cottonseed f l o u r s , the quantities used were calculated using the percentages these fractions represented of the original f l o u r , e.g. the salt solution soluble fraction of glanded flour was 35.7% of the original flour and 7.1 % (20 χ .357) replacement for wheat was used to prepare the b i s c u i t . Other methods and procedures are f u l l y described by Blouin et a l . (9,10). Sol vent Extraction Steps. Glandless and glanded cottonseed flours were subjected to a series of five mild extraction steps. Biscuits were prepared with the extracted flours and in most cases with the extracts (Figure 5). The quantity of cottonseed material used in preparation of a biscuit was proportional to the amount that the fraction represented in the original f l o u r . These percentages of the orginal flour are shown in Figure 5 in parentheses. In this way, the preparation of biscuits was used as a guide to determine i f the pigments responsible for the color had been modified or lost and to establish in which fractions they occurred. Figure 5 i l l u s t r a t e s that extraction of glandless flour with petroleum ether and chloroform does not alter the color of b i s ­ c u i t s . After extraction with aqueous alcohol , the yel1ow color is present in the biscuit containing the extract and the brown color is present in the biscuit containing the insoluble flour residue. We were able to establish that the yel1ow is caused by flavonoids present in the flours ( £ ) . Seven flavonol compounds were isolated and identified as glycosides of quercetin and kempf e r o l . No strong interactions between these phenolic components and the cottonseed proteins were evident since they were readily removed by simple room temperature extraction. The pigments responsible for the brown color, however, appear to be strongly bound to the more insoluble flour components. They remain in the

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

FOOD PROTEIN DETERIORATION

Figure 5. Biscuits containing glandless cottonseed flour fractions isolated in five solvent extraction steps. Values in parentheses represent the amount of cottonseed material used in preparation of the biscuit.

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4.

BLOWN ET AL.

Discoloration of Proteins

77

insoluble flour fractions after extractions with water and 10% sodium chloride solution. At this point in the isolation proce­ dure, the brown color causing pigments are contained in a fraction which represents 29% of the orginal f l o u r . Figure 6 shows biscuits prepared from glanded flour frac­ tions obtained by the same five extraction steps. The yellow color due to the flavonoids is in the biscuit containing the aqueous alcohol extract and the brown coloration remains in the insoluble fraction through the salt solution extraction step. Our i n i t i a l assumption about the more intense brown color in glanded flour was that i t was caused by gossypol bound to the cottonseed proteins. The original flour contained 0.1% bound gossypol measured by an analytical method developed by W. Pons and co-workers at SRRC (1JJ. This analytical method assumes gossypol is bound as a Schiff base to the epsilon-amino groups of the lysine in proteins (Figure 7). Although this bonding is not of the type previously described for phenol-quinone-proteiη systems, i t is a type quite common in biological systems (12). In this analytical method, the cottonseed flour is treated with 3-amino-l-propanol and acetic acid in dimethylformamide (DMF) s o l u t i o n . Under these conditions, i t is assumed that the gossypol becomes bound to the aminopropanol as the Schiff base of this low molecular weight com­ pound. This gossypol-aminopropanol derivative can then be washed from the flour and the gossypol content measured spectrophoto­ metrically. This reaction appeared to be a good means to test the role of bound gossypol in the color problem. The flour residues after extraction with salt solution were treated with 2% aminopropanol and 10% acetic acid in DMF solution for 15 min at 90°C. The treated fractions were washed, dried and used in biscuits (Figure 8 ) . Most of the brown color was removed from the glanded flour fraction by this treatment. The color of the biscuit prepared with the glandless flour fraction was not altered by this aminopropanol treatment. These results suggest that bound gossypol is responsible for the intense brown color observed in the glanded cottonseed flour-containing b i s c u i t . Enzyme Digestion Steps. To further concentrate and isolate the brown pigments in glandless and glanded flours, the salt solution insoluble residues were subjected to a series of four sequential enzyme treatments. Biscuits prepared from the soluble and insoluble fractions of glandless flour are shown in Figure 9. Pepsin digestion solubilized about 50% of the salt solution insoluble residue. Treatment of the pepsin residue with a eel 1ulase enzyme, which also contained hemicellulase a c t i v i t y , removed an additional 50-60% from the insoluble f r a c t i o n . This was followed by a second pepsin digestion which removed 20-30% from the eellulase residue. Treatment with amylase solubilized an additional 20-25%. At each step of these treatments, biscuit preparation showed that the brown color-causing pigments remained

FOOD PROTEIN DETERIORATION

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

78

Figure 6.

Biscuits containing glanded cottonseed flour fractions isolated in five solvent extraction steps.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

BLOUIN ET AL.

Discoloration

of

Proteins

CHJT

CH -CH -OH 2

2

NH*

OH CHO

CH C0 H/DMF 5

2

CH-CH£-CH£-OH OH CHO

Figure 7. Reaction of protein-bound gossypol with 3-amino-l-propanol in the presence of acetic acid in DMF solution. (Reproduced, with permission, from Ref. 10. Copyright 1980, Institute of Food Technologists.)

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

FOOD PROTEIN DETERIORATION

Figure 8. Biscuits containing the salt solution insoluble fractions of glanded (top) and glandless (bottom) cottonseed flour before and after treatment with aminopropanol.

BLOUiN ET AL.

Discoloration

of

Proteins

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4.

Figure 9.

Biscuits containing glandless cottonseed flour fractions isolated in four enzyme digestion steps.

81

82

FOOD PROTEIN DETERIORATION

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in the insoluble fractions. The amylase residue represented 2% of the original flour. Glanded cottonseed flour responded in an identical manner when subjected to the same four enzyme treat­ ments. Biscuits prepared with these fractions are shown in Figure 10. The last insoluble fraction in this case was 2.6% of the original f l o u r . Pimethyl sulfoxide Treatment Step. The last step in the iso­ lation procedure was to treat the amylase residues from glanded and glandless flours with DMSO at 160°C for 1 h r . This treatment solubilized 70 to 75% of the residues. For both f l o u r s , the DMSO solution was f i l t e r e d off to isolate the insoluble f r a c t i o n . A second fraction was isolated by precipitation on mixing of the DMSO solution with a large volume of 50% methanol (soluble frac­ tion A ) . A third fraction was obtained by removal of the solvents on a rotary evaporator (soluble fraction B). Biscuits prepared from these DMSO treated fractions, i l l u s ­ trated in Figure 11, showed some coloration i n a l l six b i s c u i t s . However, a marked difference in color d i s t r i b u t i o n is now evident between the two types of f l o u r . For the glanded f l o u r , the intense brown color is clearly present in the DMSO soluble frac­ tion A. With the glandless f l o u r , the major proportion of the brown pigments is in the insoluble f r a c t i o n . Also, a brown color of- similar hue and intensity is observed in the biscuit containing the insoluble fraction of glanded cottonseed f l o u r . This indicated that the pigments responsible for the brown color in glandless flour are also present in glanded f l o u r . Chemical analyses indicated that these insoluble fractions are about 40% carbohydrate (presumably polysaccharide), 30% protein and 5% f a t . As yet, we have no experimental evidence that the brown color in these insoluble fractions is caused by phenolic compounds. Because of the extremely insoluble nature of these residues, how­ ever, we suspect that lignins or condensed tannins are present. The DMSO soluble fraction A of glanded flour is about 70% protein. It also contains about 7% carbohydrates and 8% f a t . The fact that this f r a c t i o n , which contains the intense brown color characteristic of glanded flour, is mainly protein, is in agreement with the assumption that gossypol bound to proteins is responsible for this discoloration. The DMSO soluble fraction A of glandless flour is about 50% protein. This lower proportion of protein i s due to a higher fat content (18%). This high 1i pi d content and the amino acid pro­ f i l e data suggests that the DMSO soluble A fractions are mainly membrane type lipoproteins (13). The DMSO soluble Β fractions appear to be mixtures of pro­ t e i n s , 35 to 50%, and carbohydrates, 14 to 19%. They cause the least amount of brown color in b i s c u i t s . The infrared spectra of these six fractions, shown in Figure 12, mainly confirm the chemical data previously described. The spectra of the DMSO insoluble residues are identical for both

BLOUIN ET AL.

Discoloration

of

Proteins

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

4.

Figure 10.

Biscuits containing glanded cottonseed flour fractions isolated in four enzyme digestion steps.

83

FOOD PROTEIN DETERIORATION

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84

Figure 11. Biscuits containing glanded and glandless flour fractions isolated in DMSO treatment step. Precipitable DMSO soluble fraction is Fraction A and nonprecipitable DMSO soluble fraction is Fraction Β in text.

4.

BLOWN ET AL.

Discoloration

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

4000 30002500 ι ι t

85

of Proteins

2 0 0 0 160014001200 ι i i ι

1000900 ι i

800 ι

700 cm" ι

1

DMSO INSOLUBLE FRACTION

DMSO SOLUBLE FRACTION A

DMSO S O L U B L E FRACTION Β

ι 1 1 1 ι microns 2.5 3.0 3.5 4.0 4.5 Figure 12.

1 5

1 6

1 7

1 8

ι 9

1 10

1 II

1 12

1 13

1 14

1 — r 15 16

IR spectra of DMSO treated fractions of glanded (A) and glandless (B) cottonseed flours.

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86

FOOD PROTEIN DETERIORATION

f l o u r s . The -OH, -CH and -C-0- absorption regions indicate a pre­ dominance of carbohydrate components. For the DMSO soluble A fractions, the -NH and amide I and II absorption regions indicate a predominance of protein components. The -CH absorption region confirms the presence of greater amounts of l i p i d s in the gland­ less f l o u r . There i s , however, no indication of the presence of the intense brown color causing components in the spectrum of the DMSO soluble fraction A of the glanded f l o u r . The spectra of the DMSO soluble Β fractions again confirm the presence of protein and carbohydrate mixtures in these fractions. There are some d i f ­ ferences between these two spectra which are unexplainable at this time. The u l t r a v i o l e t - v i s i b l e spectra of the DMSO soluble fractions, shown in Figure 13, do indicate significant differences between the two types of flours. The concentrations used were 0.2 mg/ml for the four flour fractions and 0.02 mg/ml for the gossypol spectra. The glanded flour fractions exhibited s i g n i f i ­ cantly higher UV-visible absorption than the glandless flour fractions. Even more significant, both of the glanded fractions exhibited absorption in the 375-400 nm region similar to the 379 nm maximum exhibited by free gossypol. These data again suggest that gossypol bound to protein is the cause of the intense brown color in glanded flour-containing b i s c u i t s . Sunflower Seed Flour Chlorogenic acid (Figure 14) i s the phenolic compound, which according to several research groups (14-17), is responsible for the discoloration problem observed when sunflower proteins are used in food systems. The concentration of phenolic compounds in sunflower seed flour is 3.0 to 3.5% (18). Chlorogenic acid and caffeic acid constitute about 70% of these phenolics (18). As an interesting comparison with cottonseed flour studies, the research of M. A. Sabir and co-workers (14,15) on the chlorogenic acidprotein interactions in sunflower is reviewed below. Sabir et a l . (14) extracted proteins from sunflower seed flours with 2.5% sodium chloride at pH 7. They fractionated the salt-extractable proteins on a Sephadex G-200 column and isolated five protein fractions (Figure 15). When the undialyzed extracts (Figure 15a) were used, peak V showed far greater absorption at 280 nm than the high molecular weight fractions. Peak V also exhibited a UV-visible maximum at 328 nm which was not observed in the spectra of the other fractions. If the salt extractable proteins were dialyzed before fractionation, the elution curve shown in Figure 15b was obtained. Peak V exhibited, in this case, an absorption at 280 nm more in proportion to the other peaks. This fraction s t i l l exhibited a second absorption maximum at 328 nm. Chlorogenic acid gives a UV-visible maximum at 328 nm and other phenolic acids and esters absorb between 310 and 335 nm. Presumably, phenolic components were present in fraction V, some

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

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FOOD PROTEIN DETERIORATION

ε c

LU

U

ι

i

m

ELUTION

m

VOLUME

Figure 15. Fractionation of undialyzed (top) and dialyzed (bottom) salt-extractable sunflower proteins on a Sephadex G-200 column with neutral salt solution. (Repro­ duced from Ref. 14. Copyright 1973, American Chemical Society.)

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Discoloration of Proteins

89

of which were removed by d i a l y s i s . The phenolics that remain in fraction V after dialysis are strongly bound to this protein fraction. Sabir and coworkers (15) refractionated fraction V on a Sephadex G-200 column with neutral salt solution and 7 M urea. They reasoned that a strong hydrogen-bonding medium such as 7 M urea would dissociate any phenolics that were hydrogen bonded to the proteins but would not influence any phenolic compounds covalently bound. As shown in Figure 16, they isolated two subfractions, VI and V2. Fraction VI exhibited a maximum at 280 nm characteristic of proteins but no absorption in the 328 nm region (Figure 17). This subfraction represented 68% of the proteins in fraction V. Fraction V2, on the other hand, showed absorption maxima at 280 and 328 nm. Since 7 M urea did not dissociate these bonds, these researchers concluded that chlorogenic acid was covalently bonded to this low molecular weight protein fraction. This fraction represented 4% of the original s a l t extractable proteins. Conclusions Current research on sunflower and cottonseed flours indicates that phenolic compounds contribute to discoloration problems when these plant protein products are used in food systems. In the case of cottonseed flours, flavonoids cause a yel1ow coloration but these phenolics do not interact to any significant extent with the proteins. The phenolic compound gossypol in glanded cottonseed flour is believed to be covalently bonded to membrane type lipoproteins and cause an intense brown color in food products containing this f l o u r . Both glanded and glandless cottonseed flours contain pigments causing 1ight brown color in foods. These pigments are bound to or are part of the most insoluble flour components. Lignin or condensed tannins may be the moieties responsible for this light brown c o l o r . Sunflower seed protein products also cause marked discoloration in food products. Experimental evidence obtained by Sabir et a l . (14, 15) indicates that strong hydrogen bonding and covalent bonding of chlorogenic acid to low molecular weight sunflower proteins occurs and that these interactions are probably responsible for the discoloration problem. If plant protein products are to be used in food systems, discoloration problems must be solved. A knowledge of the nature of the bonding between the color-causing components and the proteins should lead to development of better methods for prevention and elimi nation of these problems.

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90

FOOD PROTEIN DETERIORATION

Vt f ι V™ ι 1— Ο 120 140 160 180 2 0 0 2 2 0 ELUTION V O L U M E ( m l ) f

l

Figure 16. Refractionation of salt-extractable sunflower protein Fraction V on a Sephadex G-200 column with neutral salt solution and 7 M urea. Key: O-O, 280 nm; and Δ - Δ , 328 nm. (Reproduced from Ref. 15. Copyright 1974, Ameri­ can Chemical Society.)

1.0] • ! 0.8

U

yj

ο

ζ 0.6 < m oc ο £ 0.4 CD

1

< 0.2h

; 0.0

340

300 260 220

WAVE LENGTH(nm) Figure 17. UV spectra of Subfractions F , and V of salt-extractable sunflower proteins. (Reproduced from Réf. 15. Copyright 1974, American Chemical Society.) t

4.

BLOWN ET AL.

Discoloration of Proteins

91

Acknowledgement Names of companies or commercial products are given solely for the purpose of providing specific information; t h e i r mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. Literature Cited

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch004

1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

G r i f f i n s , L . Biochem. J., 1958, 70, 120. Blouin, F. A.; Zarins, Ζ. M . ; Cherry, J. P. Color, In: "Protein Functionality in Foods"; Cherry, J. P . , E d . ; Amer. Chem. Soc. Symposium Series 147: Washington, D. C . , 1981; p. 21. Kohler, G. O.; Knuckles, Β. E. Food Technol., 1977, 31(5), 191. Lahiry, N. L.; Satterlee, L. D . ; Hsu, H. M . ; Wallace, G. W. J. Food Sci., 1977, 42, 83. Loomis, W. D.; B a t t a i l e , J. Phytochem., 1966, 5, 423. F l e t t , M. St. C. J. Soc. Dyers Colourists, 1957, 68, 59. Mason, H. S.; Peterson, E. W. Biochim. Biophys. Acta, 1965, 111, 134. Gardner, H. K . , Jr.; Hron, R. J., S r . ; V i x , H. L . E. Cereal Chem., 1976, 53, 549. Blouin, F. A . ; Zarins, Ζ. M . ; Cherry, J. P. J . Food Sci., 1981, 46, 266. Blouin, F. Α.; Cherry, J. P. J. Food Sci., 1980, 45, 953. Pons, W. A., Jr.; Pittman, R. A.; Hoffpauir, C. L . J. Am. Oil Chem. Soc., 1958, 35, 93. Singleton, V. L . Common Plant Phenols Other Than Anthocyanins, Contributions to Coloration and Discoloration, In: "The Chemistry of Plant Pigments"; Chichester, C. A . , Ed; Academic Press: Ν. Υ., 1972; p . 162. Gurr, M. I., James, A. T. "Lipid Biochemistry: An Introduction"; Cornell Univ. Press: Ithaca, Ν. Y., 1972; p. 192. Sabir, M. A.; Sosulski, F. W.; MacKenzie. S. L . J. Agr. Food Chem., 1973, 21, 988. Sabir, M. A.; Sosulski, F. W.; Finlayson, A. J. J. Agr. Food Chem., 1974, 22, 575. Sodini, G . ; Canella, M. J. Agric Food Chem., 1977, 25, 822. Cheftel, C . ; Cuq, J. L.; Pronansal, M . ; Besancon, P. Rev. Francaise des corps Gras, 1976, 23, 9. Sabir, Μ. Α.; Sosulski, F. W.; Kernan, J. A. J. Agr. Food Chem., 1974, 22, 572.

RECEIVED July

16, 1982.

5 Seed Protein Deterioration by Storage Fungi 1

JOHN P. CHERRY

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch005

United States Department of Agriculture, Southern Regional Research Center, Agricultural Research Service, New Orleans, LA 70179 Fungi are used to process a variety of seed materials into various fermented food products, such as: 1) miso--a peanut butterlike product prepared by fermenting mixtures of rice and soybeans with Aspergillus oryzae or A. soyae and Saccharomyces rouxii (1-4); 2) shoyu--a liquid food (soya sauce) prepared by fermenting soybeans and rice with A. oryzae or A. flavus and Zygosaccharomyces sp. yeast (5,6,7); 3) tempeh--a material from soybeans fermented with Rhizopus oligosporus (8,9); 4) ang-khak--a rice product fermented with Monasus purpureas and used as a food coloring agent (10); and 5) ontjom--a peanut presscake fermented by Neurospora s i t o p h i l a (4,11). Enhanced nutritive quality and d i g e s t i b i l i t y of these fermented products have been p a r t i a l l y attributed to proteolytic a c t i v i t i e s of the various fungi used in the fermentation processes (12-19). The aging processes of seeds that produce degradative changes leading to seed deterioration are, in many cases, enhanced by microorganisms (20-23). Pathogens produce metabo­ lites that enhance deterioration of their host's c e l l u l a r structures (24-27). Cell membrane disruption allows solute leakage and activation, for example, of autolytic host enzymes such as proteases (27,28). This is in addition to increases in the amounts and kinds of enzymes from the fungi (29,30,31). Biochemical transformations include deletion of some proteins and enzymes, intensification of others, and/or production of new components as evidenced by quantitative and qualitative changes in free amino acids and band patterns in polyacrylamide electrophoretic disc-gels (32-37). This chapter examines the sequence of events i n i t i a t e d by the inoculation of oi1 seeds and their products with selected fungi (A. parasiticus, A. oryzae, A. flavus, R. oligosporus and H. s i t o p h i l a ) , which~leads to the hydrolysis of proteins to small polypeptides and/or aqueous insoluble components, then to free amino acids. Changes in certain enzymes during the infection period that are characterized by gel electrophoretic techniques are also described. 1

Current address: United States Department of Agriculture, Eastern Regional Research Center, Philadelphia,PA19118. This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

94

FOOD PROTEIN DETERIORATION

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Experimental Procedures Aspergillus parasiticus, NRRL A-16,462, A. oryzae, NRRL 1988, nine strains of A. flavus Link ex, N. s i t o p h i l a , NRRL 2884, and R. oligosporus, NRRL 2710, were cultured on potato dextrose agar slants between 24° and 29°C for 4 to 16 days. Fungal spores were collected from the surface of the culture slants with a s t e r i l e solution of 0.005% Span 20. After removal of their skins, the peanut seeds of the c u l t i v a r , Florunner, were soaked in an inoculum of each fungus for 1 min. They were then placed in Petri dishes set in ventilated containers lined with water-saturated absorbent cotton and incubated at 29°C. Control uninoculated seeds were s i m i l a r l y treated, omitting the fungi in the inoculation step. After test periods ranging from 2 to 18 days, duplicate samples of groups of three uninoculated and three of each fungi-infected seeds were collected. They were individually ground in 7 ml of sodium phosphate buffer (pH 7.9; I = 0.01) in a mortar with a pestle, then centrifuged at 43,500 χ g for 30 min to separate soluble and insoluble fractions. Polyacrylamide d i s c - and starch slab-gel electrophoresis were used to indicate the qualitative and semi-quantitative changes that occurred in proteins and/or selected enzymes in the soluble fractions of control and inoculated seeds (30). Al 1 soluble and insoluble samples were then lyophilized, ground into meals, and defatted with diethyl ether. The quantities of proteins in the soluble and insoluble fractions were determined by the macro-Kjeldahl technique (Ν χ 5.41). Defatted whole seed meals were used for determination of free ami no acid content. Measurements for free amino acids were made u t i l i z i n g the procedure of Young et a l . (38). Al 1 tests were conducted twice and labeled as experi­ ments 1 and 2. In instances where there were no s t a t i s t i c a l l y significant differences between experiments, data from both experiments were combined for presentation in the text. Results Quantitative protein changes. Protein quantities decreased in sodium phosphate buffer-soluble fractions of peanut seeds during early stages of A. parasiticus, A. oryzae, IN. sitophila and R. oligosporus contamination; time intervals of fungi growth ranged from 2 to 19 days (Table I ; 32, 34-37). Simultaneously, an increase in protein content occurred in buffer-insoluble fractions. During longer growth periods, fungi grew luxuriantly ( e . g . , A. parasiticus; Figure 1), and most fungi-inoculated seeds showed a continued decrease in solu­ ble proteins with a leveling off at the later stages of growth, while others increased in soluble protein (A. oryzae). The rate of these changes varied significantly between experiments of A. parasiticus-inoculated seeds. The data imply different rates and/or mechanisms of peanut protein hydrolysis by the fungi used in these experiments. The

34 34 34 34 34

63 63 63 63 63

^

I/Averages of 2-4, 7-9, and 11-18 days. 1/Not analyzed.

34

Insoluble

63

Soluble

0

38 45 45 49 36

62

Soluble

2-4

54 51 46 40 53

34

Insoluble

26 45 64 35 31

58

Soluble

7-9

53 47 39 51 54

36

Insoluble

Days Inoculated!/

-11

27 39 68 „

62

Soluble

-

49 48 31

33

Insoluble

11-18

Percentages of protein changes in soluble and insoluble fractions of peanut seeds inoculated with various fungi.

Nom'nfected A. parasiticus: Experiment 1 Experiment 2 A. oryzae N. sitophila R. oligosporus

Treatment

Table I .

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.

ζ3

*

S

£ *

3

m

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96

Figure 1. Typical peanut seeds contaminated with A . parasiticus and then incubated for 0 to 5 days. Included are cross sections of seeds incubated for 5 days to show mold penetration. (Reproduced, with permission, from Ref. 34. Copyright 1974, Physiol. Plant Pathol.j

FOOD PROTEIN DETERIORATION

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5.

CHERRY

Seed Protein

Deterioration

97

organisms are known to exhibit strong and several types of proteolytic a c t i v i t y ( 1 3 - 1 9 , 2 9 » 3 0 , 3 1 , 3 4 , 3 9 ) . The sustained increase in amount of proteins in soluble and insoluble fractions during the later fungi growth stages suggests that there is an accumulation of low molecular weight proteins, peptides and amino acids which have differing molecular properties and s o l u b i l i t i e s in the phosphate buffer. This may be due in part to increased level s of extractable and nonextractable fungal mycelial enzymatic proteins and related metabolites that are present in seed fractions during the later stages of the test periods. A significant contribution to the apparent increase in soluble protein in A. oryzae-contaminated seeds may be due to the accumulation of peanut protein hydrolysates that are not e f f i c i e n t l y u t i l i z e d by this fungus for growth and metabolism. Gel electrophoretic properties. Gel electrophoretic patterns of proteins in buffer-soluble fractions showed that each fungus caused specific changes in these storage components during the growth period (Figures 2, 3, and 4). No protein changes were noted in gel patterns of control seeds. In general, protein patterns of seeds contaminated with the various fungi, when compared to those of the noninoculated control, showed new protein components in region 0-1.0 cm and increased mobility and poor resolution of the major dark staining bands in region 1.0-2.0 cm, as fungi growth progressed. At the same time, bands normally located in region 2.0-3.5 cm disappeared, and a new group of polypeptides appeared in region 3.5-7.0 cm. During later stages of growth, many of the proteins became d i f f i c u l t to distinguish in the lower half of the gel patterns. The changes in protein properties on electrophoretic gel s were more rapid in experiment 1 than experiment 2 with A. parasiticus, as was shown with the quantitative data (cf Table I and Figure 2). With A. oryzae, the major difference between the two experiments was that the smaller molecular weight compounds in region 2.0-6.0 cm seemed to disappear more rapidly in experiment 1 than 2 (Figure 3). The two protein bands in region 0.5-2.0 cm of electrophoretic gels shown in Figures 2, 3, and 4 have been characterized as the major storage globulin, arachin, which is present i n aleurone grains of peanut seeds (40). These bands were not clearly discernable in the gel patterns after day 3; this was especially noted in experiment 1 with A. parasiticus (Figure 2). Evidently, as portions of the arachin components were hydrolyzed by a combination of proteases from the fungi and the peanut seeds, t h e i r sizes, conformations and e l e c t r i c a l charges gradually changed resulting in an increase in electrophoretic mobility. Arachin is most l i k e l y being hydrolyzed to small polypeptide components and free ami no acids.

FOOD PROTEIN DETERIORATION

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98

Figure 2. Polyacrylamide disc-gel electrophoretic patterns of buffer-soluble proteins from A. pMBslticm-inoculated peanut seeds of Experiments 1 and 2 after incubation for 0-18 days. (Reproduced, with permission, from Ref. 35. Copyright 1975, Can. J . Bot.)

Seed Protein

Deterioration

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch005

CHERRY

Figure 3. Polyacrylamide disc-gel electrophoretic patterns of buffer-soluble proteins from A. oryme-inoculated peanut seeds of Experiments 1 and 2 after incubation for 0-18 days. (Reproduced from Ref. 36. Copyright 1976, American Chemical Society.)

FOOD PROTEIN DETERIORATION

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100

Figure 4. Poly aery lamide disc-gel electrophoretic patterns of buffer-soluble proteins from R. oligosporus- and N. sitophilm-inoculated peanut seeds after incubation for 0-7 days. (Reproduced, with permission, from Ref. 32. Copyright 1976, Cereal Chem.)

5.

CHERRY

Seed Protein

Deterioration

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Free amino acids. Analysis of free essential amino acid contents of peanut seeds during a 2- to 7-day period of rapid growth of the various fungi used in this study showed that most of these components increased to levels greater than those observed in uninoculated seeds (Table II ; 32,35,36,37). Similar observations were noted with many of the nonessential amino acids (32,35,36,37). During later stages, many of the free ami no acids decreased in amount, most l i k e l y being used as nutrients by the fungi. These observations coincide with the deterioration of proteins to small molecular weight components and their disappearance from gel electrophoretic patterns. Enzyme analyses. Gel electrophoretic patterns of buffersoluble extracts from fungi-inoculated peanut seeds showed many biochemical transformations in enzymes (29,30,31,34). Examples of selected enzyme patterns of A. flavus-contaminated seeds are shown in Figure 5 (31). Changes included deletion of some enzymes, intensification of others and/or the production of new multiple molecular forms (31). These enzyme systems cause degradation of storage protein peptides (leucine aminopeptidases), hydrolysis of many ester linkages (esterases, acid and alkaline phosphatases) and oxidation reactions (phosphogluconate, alcohol, mal ate and glucose-6-phosphate dehydrogenases). As a result of enzyme changes, other studies suggested increased hormonal interaction and/or oxidation of organic substrates with hydrogen peroxide (peroxidases, oxidases) and decomposition of toxic substances such as hydro­ gen peroxide (catalases) (29,30,34). No doubt, these data on enzyme changes as fungal growth progresses in seeds, make i t d i f f i c u l t to prepare vegetable protein products of known com­ position and s t a b i l i t y during handling, storage and processing. Discussion The observation that proteins, enzymes and amino acid quantities in various preparations of peanut seeds inoculated with A. parasiticus, A. oryzae, A. flavus, R. oligosporus, or N. sitophila are different from those of noninoculated seeds expands presently known information on the effects of saprophytic organisms on plant tissues. For example, gel electrophoretic data show that water-soluble proteins from fungi-inoculated peanut seeds are hydrolyzed to their structural components during various test periods. New multiple molecular forms of enzymes appear, or i f stored in inactive forms, are activated. These data also imply various rates and/or mechanisms of storage protein hydrolysis for the different fungi included in these studies. Other studies have shown that although certain proteolytic enzymes may be common to different fungi, each species has the capacity to produce proteinases endemic to i t s e l f (13-19). Quantitatively, there are decreases and, in some cases, increases, in percentages of proteiη of soluble extracts during

101

0.65b 0.74b 0.92a 0.62b 0.66b

0.51a 0.35a 0.40a 0.29a 0.39a

Methionine

Leucine 0.45c 1.01a 1.10a 0.61 be 0.89ab

Isoleucine 0.48b 0.59ab* 0.69a 0.45b 0.58ab

Arginine 1.34b 1.68b 1.61b 1.66b 2.60a

Lysine 0.44a 0.80a 0.79a 0.59a 0.80a

Phenylalanine 3.47a 1.89b 2.04b 1.81b 2.07b

Time intervals (days) 0.90b 0 0.35c 0.12b 0.40c 0.34c 2.52a 0.37b 2.25a 0.72a 2.05a 0.79b 0.51bc 0.74b 0.58a B: 2 2.22a 0.78a 2.12a 0.89b 0.37ab 0.56b 4 0.83ab 0.87a 2.00a 2.03a 1.23a 7 0.47a 0.76a 0.95a I/Valine, histidine and tryptophan are not l s i t e d because they did not show any s t a t i s t i c a l l y significant changes within either A or B. Values having no common postscript l e t t e r in each amino acid column within A or Β separately are significantly (P < 0.05) different.

A:

Noninfected A. parasiticus A", oryzae R. oligosporus N. sitophila

Threonine

Free Ami no Acids. (nM/100 mg Fat-Free Meal)

Essential free amino acid changes in peanut seeds inoculated for time intervals of 0 to 7 days with various fungi.1/ A: Averages of free amino acid changes within each seed treatment for the entire 7-day test period. B: Free amino acid changes averaged for the five treatments within each time interval of the test period. (37)

Treatments

Table I I .

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Figure 5. Poly aery lamide disc- and starch slab-gel electrophoretic patterns of select enzymes from peanut seeds not inoculated (Controls 1 and 2) and inoculated with nine strains of A.flavus(31).

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104

FOOD PROTEIN DETERIORATION

the test periods, regardless of the fungus used, while at the same time an increase in these constituents occurs in insoluble fractions. These changes are further confirmed by observations showing that the total protein ami no acid composition of the soluble and insoluble fractions are continually changing (32,35,36) and that free amino acid quantities are increasing. Evidently, in the presence of these fungi the major storage proteins of fungi-inoculated seeds are converted to free amino acids and polypeptides of various sizes having different solub i l i t y characteristics. Changes in s o l u b i l i t y of various hydrolyzed products of proteins may also be related to their differential interactions with other degraded constituents stored in peanut seeds ( o i l s , fatty acids, sugars, e t c . ; 33). Previous to these studies on peanut seeds inoculated with different fungi, most research on this subject was on the proximate composition of finished fermented products compared to the nonfermented substrates (18,19,41,42,13). Other studies showed that quantities and proportions of essential ami no acids in certain fermented products were greatly improved over those of raw substrates (12). These improvements were partly attributed to fungal digestion of proteins to t h e i r structural components, which yielded more nutritious food products. Thus, while hydrolyzed protein components in fermenting substrates serve as primary sources of readily available nutrients for fungal metabolism and growth, they in turn have the p o s s i b i l i t y of improving the nutritional and functional properties of ferments as foods or feeds. This chapter showed that techniques normally used to prepare protein extracts from high quality oilseeds w i l l not necessarily produce fractions similar to those from seeds inoculated with A. parasiticus, A. oryzae, A. flavus, R. oligosporus, or N_. s i t o p h i l a . In fact, the resulting extracts w i l l depend on the species of fungus used and the length of the growth period. Since these conditions affect the type and quantity of proteins and ami no acids in various peanut extracts, they should also alter the nutritional and functional properties of peanut protein products to different forms from those of quality seeds. For example, fungus-inoculated peanut seeds have greater quantities of certain essential ami no acids in soluble and insoluble protein fractions than those of noninoculated seeds. In future studies, these factors need to be considered in research to expand u t i l i z a t i o n in foods or feeds of protein isolates or concentrates from various fungi inoculated oilseeds. Conclusions Fungi ( e . g . , A. parasiticus, A. oryzae, A. flavus, R. oligosporus and N. sitophila) growing on oilseeds and their products for various times greatly altered the protein's s o l u b i l i t y and gel electrophoretic properties. In aqueous extracts of inoculated materials, protein quantities changed to

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5.

CHERRY

Seed Protein

Deterioration

levels much lower or higher than those not inoculated with fungi. Simultaneously, the amounts of insoluble proteinaceous compounds changed to quantities greater or lesser than those contained in soluble fractions. Gel electrophoresis of soluble extracts from inoculated seeds showed that proteins were hydro­ lyzed to small molecular-weight components which eventually disappeared as fungal growth progressed. A corresponding increase in quantity of most free ami no acids coincided with the substantial alterations of proteins in both soluble and insoluble fractions. During the interval of protein changes, electrophoretic patterns of inoculated seeds showed that some enzymes were deleted, others were intensified, and/or new molecular forms of these constituents appeared. These data suggested that inoculation of oilseeds and their products with fungi i n i t i a t e d a sequence of events whereby enzyme composition was changed, and proteins were hydrolyzed f i r s t to small polypeptides and/or insoluble components, then to free ami no acids. Acknowledqment Use of a company and/or product named by the U.S. Department of Agriculture does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Shibasaki, K . ; Hesseltine, C. W. J . Biochem. Microbiol. Technol., 1961, 3, 161. Shibasaki, K . ; Hesseltine, C. W. Dev. Ind. M i c r o b i o l . , 1961, 2, 205. Shibasaki, K . ; Hesseltine, C. W. Econ. Bot., 1962, 16, 180. Hesseltine, C. W.; Wang, H. L. Biotechnol. Bioeng., 1967, 9, 275. Dyson, G. M. Pharmeceut., 1928, 121, 375. Lockwood, L. B. Soybean Digest, 1947, 7(12), 10. Yokotsuka, T. Adv. Food Res., 1960, 10, 75. Steinkraus, Κ. H . ; Yap, Β. H . ; Van Buren, J. P . ; Provvidenti, M. I.; Hand, D. B. Food Res., 1960, 25, 777. Djien, K. S.; Hesseltine, C. W. Soybean Digest, 1961, 22(1), 14. Palo, M. A.; Vidal-Adeva, L.; Maceda, L. P h i l i p p . J . Sci., 1961, 89, 1. Gray, W. D. C r i t . Rev. Food Technol., 1970, 1, 225. Hesseltine, C. W. Mycologia, 1965, 57, 149. Plating, S. L.; Cherry, J. P. J . Food Sci., 1979, 44, 1178. Steinkraus, Κ. H . ; Lee, C. Y . ; Buck, P. A. Food Technol., 1965, 19, 1301. Nakadai, T.; Nasuno, S.; Iguchi, N. Agric. B i o l . Chem., 1972, 36, 1481.

105

106

16. 17. 18. 19. 20.

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21. 22.

23. 24. 25. 26. 27.

28. 29.

30.

31. 32. 33. 34. 35. 36. 37. 38. 39.

FOOD PROTEIN DETERIORATION

Nakadai, T.; Nasuno, S.; Iguchi, N. Agric. Biol. Chem., 1972, 37, 2703. Wang, H. L; Vespa, J. P.; Hesseltine, C. W. Appl. Microbiol., 1974, 27, 906. Beuchat, L. R.; Young, C. T.; Cherry, J. P. Can. Inst. Food Sci. Technol. J., 1975, 8, 40. Quinn, M. R.; Beuchat, L. R.; Miller, J.; Young, C. T . ; Worthington, R. E. J . Food Sci., 1975, 40, 470. Barton, L. V. "Seed Preservation and Longevity." Leonard Hill: London, England, 1961; 216 pp. Christensen, C. M. Seed Sci. Technol., 1973, 1, 547. Bothast, R. J . Fungal deterioration and related phenomena in cereals, legumes and oilseeds, In: "Postharvest Biology and Biotechnology"; Hultin, H. O.; Milner, Μ., Eds.; Food and Nutrition Press, Inc.: Westport. Conn., 1978; p. 210. Cherry, J . P. Phytopath., 1982, In press. Harman, G. E.; Granett, A. L. Physiol. Plant Pathol., 1972, 2, 271. Koostra, P. Seed Sci. Technol., 1973, 1, 417. Roberts, Ε. H. Seed Sci. Technol., 1973, 1, 529. Villiers, T. A. Ageing and the longevity of seeds in field conditions, In: "Seed Ecology"; Heydecker, W., Ed.; The Pennsyl. State Univ. Press, Page Bros. (Norwich) Ltd.: Norwich, England, 1973; p. 265. Ryan, C. A. Ann. Rev. Plant Physiol., 1973, 24, 173. Cherry, J . P. Oilseed enzymes as biological indicators for food uses and applications, In: "Enzymes in Food and Beverage Processing"; Ory, R. L . ; St. Angelo, A. J., Eds.; ACS Symposium Series No. 47, American Chemical Society: Washington, D.C., 1977; p. 209. Cherry, J . P. Enzymes as quality indicators in edible plant tissues, In: "Postharvest Biology and Biotechnol­ ogy"; Hultin, H. O.; Milner, Μ., Eds.; Food and Nutrition Press, Inc.: Westport, Conn., 1978; p. 370. Cherry, J . P.; Beuchat, L. R.; Koehler, P. E. J . Agric. Food Chem., 1978, 26, 242. Cherry, J . P.; Beuchat, L. R. Cereal Chem., 1976, 53, 750. Cherry, J . P.; Beuchat, L. R. J . Amer. Oil Chem. Soc., 1976, 53, 551. Cherry, J . P.; Mayne, R. Y.; Ory, L. R. Physiol. Plant Pathol., 1974, 4, 425. Cherry, J . P.; Young, C. T.; Beuchat, L. R. Can. J . Bot., 1975, 53, 2639. Cherry, J . P.; Beuchat, L. R.; Young, C. T. J . Agric. Food Chem., 1976, 24, 79. Cherry, J . P.; Young, C. T.; Beuchat, L. R. Proc. Am. Peanut Res. Educ. Assoc., Inc., 1976, 8, 3. Young, C. T . ; Matlock, R. S.; Mason, M. E.; Waller, G. R. J. Am. Oil Chem. Soc., 1974, 51, 269. Nakadai, T.; Nasumo. S.; Iguchi, N. Agric. Biol. Chem., 1972, 36, 261.

5.

40. 41. 42.

CHERRY

Seed Protein

Basha, S. M. M . ; Cherry, J. P. J . Agric. Food Chem., 1976, 24, 359. Beuchat, L . R.; Worthington, R. E. J . Agric. Food Chem., 1974, 22, 509. van Veen, A. G . ; Graham, D. C. W.; Steinkraus, Ε. H. Cereal S c i . Today, 1968, 13, 96.

RECEIVED September 3, 1982.

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Deterioration

6 Behavior of Proteins at Low Temperatures

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

OWEN FENNEMA University of Wisconsin—Madison, Department of Food Science, Madison, WI 53706

Protein behavior at below ambient temperatures has received l i t t l e attention compared to that of protein behavior at physio­ logical temperatures or above. This is, of course, understand­ able, but nonetheless unfortunate, since proteins are believed by many authorities to be involved in significant, if not substan­ t i a l , ways with the ability or inability of living matter and foods to tolerate a variety of low temperature situations. *The ability of some animals to hibernate and others to acclimate when exposed to a low, nonfreezing environment. *The ability of some plants to "winter harden" and thereby tolerate freezing conditions. *The ability of some fish to avoid freezing at water tempera­ tures normally low enough to cause freezing. *The ability of some microbial cultures and other small biological specimens to survive freezing, frozen storage and thawing. *The inability of humans to tolerate low nonfreezing body temperatures. *The inability of large biological specimens such as whole organs and small, whole animals to survive preservation by freezing. *The inability of plant and animal food tissue to withstand commercial freeze preservation procedures without under­ going detrimental changes, particularly with respect to water holding capacity and texture. In addition, it should be noted that a l l too many researchers pay insufficient attention to the effects of low temperature storage on proteinaceous samples, assuming simply, and often falsely, that the lower the temperature the better. As will be documented in this Chapter, the behavior of proteins at low temperatures often deviates from intuitive expectations. Functional properties, especially enzyme activity, can change nonlinearly as the temperature is lowered and this unexpected behavior becomes even more erratic when cellular systems are involved and/or freezing occurs. Changes in enzyme 0097-6156/82/0206-0109$07.75/0 © 1982 American Chemical Society

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Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

a c t i v i t y r e s u l t i n g f r o m a l o w e r i n g o f t e m p e r a t u r e c a n be r e v e r s i b l e , p a r t i a l l y r e v e r s i b l e o r i r r e v e r s i b l e d e p e n d i n g on f a c t o r s s u c h a s t h e k i n d o f enzyme, t h e n a t u r e o f t h e s a m p l e and the t i m e - t e m p e r a t u r e scheme e m p l o y e d . From what h a s a l r e a d y b e e n s a i d , a r e d u c t i o n i n t e m p e r a t u r e a l s o w o u l d be e x p e c t e d t o c a u s e s i g n i f i c a n t i n t r a - and i n t e r m o l e c u l a r c h a n g e s i n p r o t e i n s , and t h e s e a l t e r a t i o n s do i n f a c t o c c u r . The b e h a v i o r o f p r o t e i n s i n n o n c e l l u l a r and c e l l u l a r s y s t e m s w i l l be c o n s i d e r e d a t t e m p e r a t u r e s c o v e r i n g above and b e l o w f r e e z i n g c o n d i t i o n s . A t t e n t i o n w i l l be g i v e n t o b o t h c h a n g e s i n p r o t e i n f u n c t i o n a l i t y ( m a i n l y enzyme a c t i v i t y ) and c h a n g e s i n protein structure. Experimental Procedures One o b v i o u s r e a s o n f o r t h e l i m i t e d amount o f r e s e a r c h b e i n g g e n e r a t e d on p r o t e i n s a t l o w t e m p e r a t u r e s r e l a t e s t o t h e d i f f i c u l t i e s of conducting t h i s k i n d of r e s e a r c h , p a r t i c u l a r l y i n the p r e s e n c e o f i c e . O p t i c a l p r o c e d u r e s t h a t a r e r e l i e d on so h e a v i l y i n u n f r o z e n l i q u i d s a m p l e s a r e g e n e r a l l y u s e l e s s when an i c e phase i s p r e s e n t . F u r t h e r m o r e , g r o s s nonhomogeneity e x i s t s i n f r o z e n s a m p l e s and t h i s n o n h o m o g e n e i t y c h a n g e s w i t h f r o z e n storage. Procedures to a c c u r a t e l y assess the c h a r a c t e r i s t i c s of s a m p l e s o f t h i s n a t u r e s i m p l y do n o t e x i s t . T h u s , many r e s e a r c h e r s e v a l u a t e t h e e f f e c t s o f f r e e z i n g and f r o z e n s t o r a g e a f t e r t h a w i n g . F u r t h e r m o r e , a l l t o o many i n v e s t i g a t o r s f a i l t o d e s i g n t h e i r s t u d i e s so t h a t t h e e f f e c t s o f f r e e z i n g r a t e c a n be i s o l a t e d from the e f f e c t s of f r e e z i n g depth ( n a d i r ) . One a p p r o a c h t o s t u d y i n g e n z y m e - c a t a l y z e d r e a c t i o n s a t v e r y l o w t e m p e r a t u r e s (down t o a b o u t -100°C) i n v o l v e s t h e u s e o f aqueous-organic s o l v e n t s w i t h f r e e z i n g p o i n t s s u f f i c i e n t l y low t o a v o i d i c e f o r m a t i o n (_1_, 2). T h i s t e c h n i q u e was d e v e l o p e d p r i m a r i l y t o s t u d y r e a c t i o n i n t e r m e d i a t e s and h a s p r o v e n r e a s o n a b l y successful. Some c o n c e r n e x i s t s , h o w e v e r , t h a t h i g h c o n c e n t r a t i o n s of o r g a n i c s o l v e n t s might i n f l u e n c e r e a c t i o n pathways. F a c t o r s I n f l u e n c i n g Enzyme A c t i v i t y a t Low

Temperatures

I n t h i s s e c t i o n , enzyme a c t i v i t y i n s i m p l e , n o n c e l l u l a r s y s t e m s w i l l be c o n s i d e r e d f i r s t and t h i s w i l l be f o l l o w e d by d i s c u s s i o n o f enzyme a c t i v i t y i n c e l l u l a r s y s t e m s .

a

Enzymes i n n o n c e l l u l a r , s i m p l e s y s t e m s a t l o w t e m p e r a t u r e s . F a c t o r s c o n s i d e r e d t o be o f s i g n i f i c a n c e i n g o v e r n i n g t h e a c t i v i t y o f enzymes a t l o w t e m p e r a t u r e s i n c l u d e n a t u r e o f t h e enzyme, t e m p e r a t u r e d e p e n d e n c e , c o o l i n g - f r e e z i n g p r o c e d u r e s , and c o m p o s i t i o n o f t h e medium. 1) N a t u r e o f t h e enzyme. I t i s w e l l known t h a t enzymes d i f f e r g r e a t l y i n t h e i r r e s p o n s e t o a v a r i e t y o f c o n d i t i o n s so i t s h o u l d be no s u r p r i s e t h a t m a r k e d d i f f e r e n c e s i n t h e b e h a v i o r o f

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v a r i o u s enzymes are observed during c o o l i n g and f r e e z i n g . Some enzymes, f o r example, e x h i b i t great t o l e r a n c e t o freeze-thaw treatments, whereas others do not (3, 4_» 5); and some e x h i b i t s i g n i f i c a n t a c t i v i t y i n p a r t i a l l y f r o z e n systems (e.g. oxidases and l i p a s e s ) whereas others do not (e.g. some p r o t e i n a s e s ) (6-10). Based l a r g e l y on a review of the Japanese and Russian l i t e r a t u r e , L o z i n a - L o z i n s k i i (11) concluded that f r e e z i n g causes greater changes i n f i b r i l l a r p r o t e i n s than i n g l o b u l a r p r o t e i n s . The r a m i f i c a t i o n s of t h i s c o n c l u s i o n , o f course, i n v o l v e more than j u s t enzyme a c t i v i t y . 2) Temperature dependence. Temperature i s , of course, a f a c t o r of primary importance i n determining enzyme a c t i v i t y and r e a c t i o n r a t e s . The r e l a t i o n s h i p between the r e a c t i o n r a t e constant and absolute temperature i s expressed by the w e l l known Arrhenius equation (k = se~^ ' ) , and i t i s normally expected that Arrhenius p l o t s ( l o g k v s . 1/T) w i l l be l i n e a r over reasonably small (20-30°C) temperature spans. T h i s e x p e c t a t i o n i s not normally met, however, f o r enzyme suspensions a t low or even mode r a t e l y low temperatures (12, 13, 14). For example, n o n l i n e a r Arrhenius p l o t s have been observed a t s l i g h t l y above-freezing temperatures f o r pyruvate carboxylase (15), glutamate carboxylase (16), a c e t y l CoA-carboxylase (17), pyruvate orthophosphate d i k i n a s e (18), l i p a s e (10) and f o r phosphatase and peroxidase (9). The behavior of l i p a s e i s shown i n Figure 1. I t has been suggested that these data conform e q u a l l y w e l l to a smooth curve and that more acceptable i n t e r p r e t a t i o n s are p o s s i b l e i n t h i s form (14). I t a l s o should be emphasized that the p l o t i n F i g u r e 1 i s obtained both i n the presence and absence o f i c e . Thus, the independent e f f e c t of decreasing temperature can cause enzymec a t a l y z e d r e a c t i o n s i n simple systems t o depart i n a s i g n i f i c a n t negative f a s h i o n from the Arrhenius equation. I t should not be assumed, however, that the e f f e c t s o f temperature lowering and the e f f e c t s of temperature lowering p l u s the formation of i c e always produce Arrhenius p l o t s that a r e superimposable. F r e e z i n g introduces many more c o m p l e x i t i e s (changes i n pH, i o n i c s t r e n g t h , s o l u t e c o n c e n t r a t i o n , e t c . ) than temperature lowering alone and the e f f e c t s of f r e e z i n g are t h e r e f o r e not always c o n s i s t e n t with the p a t t e r n i n F i g u r e 1. Although r a r e , f r e e z i n g can, f o r example, a c t u a l l y cause an i n c r e a s e i n the r a t e constant f o r some enzyme-catalyzed r e a c t i o n s (19, 20). The general p a t t e r n of a negative d e v i a t i o n from the Arrhenius r e l a t i o n s h i p as temperature i s lowered (with o r without f r e e z i n g ) probably i n v o l v e s conformational changes i n the enzyme and/or an a s s o c i a t i o n - d i s s o c i a t i o n type t r a n s i t i o n that leads to enzyme i n a c t i v i t y . In t h i s case, one of the assumptions underl y i n g the a p p l i c a b i l i t y t o the Arrhenius r e l a t i o n s h i p i s v i o l a t e d and l i n e a r i t y would not be expected. A second property of enzymes that i s sometimes temperature dependent i s t h e i r a f f i n i t y f o r s u b s t r a t e . Under a p p r o p r i a t e

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Figure 1. Log rate of hydrolysis (i.e., log reciprocal of time required for 1% hydrolysis) of tributyrin by lipase as influenced by temperature. (Reproduced, with permission, from Ref. 10. Copyright 1942, Institute of Food Technologists.)

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

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circumstances, enzyme-substrate a f f i n i t y i s i n d i c a t e d by the m a g n i t u d e o f t h e M i c h a e l i s c o n s t a n t (Km), and r e p o r t s o f a s t r o n g t e m p e r a t u r e dependence o f t h i s v a l u e h a v e b e e n o b s e r v e d f o r s e v e r a l enzymes i n c l u d i n g l a c t a t e d e h y d r o g e n a s e s (21, 2 2 ) , p h o s p h o p h e n o l p y r u v a t e c a r b o x y l a s e ( 2 3 ) a n d p h e n y l a l a n i n e ammonia l y a s e ( 2 4 ) . The r e l a t i o n s h i p b e t w e e n Km and t e m p e r a t u r e f o r l a c t a t e d e h y d r o g e n a s e s f r o m v a r i o u s s o u r c e s i s shown i n F i g u r e 2. I n t h e s e e x a m p l e s , maximum a p p a r e n t e n z y m e - s u b s t r a t e a f f i n i t y (minimum Km) c o r r e s p o n d s v e r y c l o s e l y t o t h e h a b i t a t o r a c c l i m a t i o n t e m p e r a t u r e o f t h e h o s t f r o m w h i c h t h e enzyme i s d e r i v e d . A r e v e r s i b l e change i n c o n f o r m a t i o n n e a r t h e enzyme a c t i v e s i t e c o u l d be r e s p o n s i b l e f o r t h i s b e h a v i o r ( 2 3 ) . S i n c e s u b s t r a t e c o n c e n t r a t i o n s i n c e l l s a r e n o r m a l l y l o w ( u s u a l l y 1 mM o r l e s s ) and s e l d o m , i f e v e r , a t t a i n s a t u r a t i o n l e v e l s f o r most enzymes, t h e t e m p e r a t u r e dependence o f Km v a l u e s i n l i v i n g systems i s c o n s i d e r e d o f f a r g r e a t e r importance than t h e temperat u r e dependence o f V values (22). S t i l l a n o t h e r t e m p e r a t u r e - d e p e n d e n t p r o p e r t y o f enzymec a t a l y z e d r e a c t i o n s i s worthy of mention. T h i s concerns t h e u l t i m a t e extent t o which these r e a c t i o n s proceed. I t i s commonly observed t h a t t h e u l t i m a t e a c c u m u l a t i o n o f r e a c t i o n p r o d u c t s tends to decrease as the s u b f r e e z i n g temperature i s decreased. F o r example, t h i s r e s u l t has been observed f o r d e n a t u r a t i o n o f c h y m o t r y p s i n o g e n (25) and f o r r e a c t i o n s c a t a l y z e d b y l i p a s e (26), i n v e r t a s e ( 2 7 ) a n d l i p o x y g e n a s e (28), w i t h d a t a i l l u s t r a t i n g t h e l a t t e r example a p p e a r i n g i n F i g u r e 3. P o s s i b l e e x p l a n a t i o n s f o r t h i s behavior include; ^ R e v e r s i b l e c h a n g e s i n enzyme a c t i v i t y stemming f r o m r e v e r s i b l e c h a n g e s i n enzyme c o n f o r m a t i o n a n d / o r a s s o c i a tion. These changes c o u l d o c c u r because o f t h e d i r e c t e f f e c t s o f temperature o r the i n d i r e c t e f f e c t s o f f r e e z i n g (pH c h a n g e , i n c r e a s e i n i o n i c s t r e n g t h , e t c . ) . ^Restraints on d i f f u s i o n of substrate, reaction products a n d / o r enzyme. T h i s c o u l d o c c u r b e c a u s e o f e n t r a p m e n t i n i c e and/or slow d i f f u s i o n i n t h e c o n c e n t r a t e d , h i g h v i s c o s i t y , u n f r o z e n phase. I n l i q u i d systems, v i s c o s i t y i s known t o d e c r e a s e a s a f u n c t i o n o f 1/T, b u t t h e c o n s e q u e n c e s on enzyme a c t i v i t y , e v e n a t v e r y l o w t e m p e r a t u r e s , i s n o t c o n s i d e r e d i m p o r t a n t ( 2 9 ) . However, once i c e f o r m a t i o n occurs, v i s c o s i t y increases abruptly to a very high l e v e l and t h e e f f e c t o n enzyme a c t i v i t y , a l t h o u g h n o t w e l l known, i s l i k e l y t o be s u b s t a n t i a l . 3) C o o l i n g a n d f r e e z i n g p r o c e d u r e s . C o o l i n g and f r e e z i n g p r o c e d u r e s , e s p e c i a l l y t h e l a t t e r , c a n have pronounced e f f e c t s o n enzyme a c t i v i t y . F a c t o r s such as r a t e s o f c o o l i n g , f r e e z i n g and w a r m i n g , t e m p e r a t u r e n a d i r , and t h e t i m e and t e m p e r a t u r e o f f r o z e n s t o r a g e a l l a r e s i g n i f i c a n t (3, 8^, 30, 3 1 ) . I n e v a l u a t i n g t h e s e e f f e c t s , one s h o u l d be aware t h a t most i n v e s t i g a t o r s have m e a s u r e d enzyme a c t i v i t y f o l l o w i n g t h a w i n g r a t h e r t h a n d u r i n g f r o z e n s t o r a g e , and h a v e c o n f o u n d e d t h e e f f e c t s o f f r e e z i n g r a t e and t e m p e r a t u r e n a d i r . m a x

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20 LUNGFISH M - L D H ( 2 7 - 3 0 ° ) /

TUNA M-LDH C~I5°)

lEMATOMUS

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M-LDH (-2°)

06h

10

20

40

30

TEMPERATURE (°C)

Figure 2. Temperature dependence of Michaelis constant (K ) of pyruvate in reactions catalyzed by lactate dehydrogenases. Temperatures indicate average tissue temperatures. (Reproduced, with permission, from Ref. 21. Copyright 1968, Pergamon Press.) m

ο

200

400 MINUTES

I

π ο—ό

600

Figure 3. Effect of sub free zing storage temperatures on the ultimate accumulation of oxidation products of linolenic acid. (All samples were rapidly frozen to —78.5 °C, then stored at temperatures indicated.) Each value is a mean of four deter­ minations. (Reproduced, with permission, from Ref. 28. Copyright 1980, Academic Press.)

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4) Composition of the sample. Enzyme p u r i t y and concent r a t i o n , pH o f the medium, the kind and q u a n t i t y o f e l e c t r o l y t e s present and the absence or presence o f p r o t e c t i v e agents (such as p r o t e i n s , g l y c e r o l , sugars, dimethyl s u l f o x i d e , etc.) can g r e a t l y i n f l u e n c e enzyme a c t i v i t y at low temperatures. For example, r e t e n t i o n of a c t i v i t y o f l a c t a t e dehydrogenase i n a simple s o l u t i o n f o l l o w i n g thawing i s favored when enzyme concentration i s high, s a l t concentration i s low, c h l o r i d e s r a t h e r than phosphates are present, other p r o t e i n s or g l y c e r o l are present, and pH i s a t the upper end o f the range 6.0-7.8 (31, 32). During f r e e z i n g , the concentration of s o l u t e s i n the unfrozen phase always increases and, as a r e s u l t , p r o p e r t i e s such as pH (31, 33, 34), i o n i c s t r e n g t h , surface t e n s i o n , concentration of d i s s o l v e d gases and v i s c o s i t y undergo marked changes. A l l o f these p r o p e r t i e s have important i n f l u e n c e s on enzyme a c t i v i t y . Enzymes i n c e l l u l a r systems a t low temperatures. Enzyme a c t i v i t y i n c e l l u l a r systems i s i n f l u e n c e d by a l l of the f a c t o r s p r e v i o u s l y discussed, with a d d i t i o n a l complications a r i s i n g because of the c e l l u l a r s t r u c t u r e . Two complications of p a r t i c u l a r importance i n v o l v e the behavior o f enzymes during c h i l l i n g i n j u r y of p l a n t s , and d e r e a l i z a t i o n of enzymes during f r e e z i n g of both p l a n t and animal t i s s u e s . It has long been recognized that p l a n t s of t r o p i c a l and s u b t r o p i c a l o r i g i n s undergo detrimental p h y s i o l o g i c a l changes, known as " c h i l l i n g i n j u r y , " when exposed to low nonfreezing temperatures. Two occurrences a s s o c i a t e d w i t h c h i l l i n g i n j u r y are 1) a l t e r a t i o n of membrane s t r u c t u r e i n o r g a n e l l e s (35, 36) and 2) d i s c o n t i n u i t i e s i n , o r a t l e a s t a sharp change i n the curvature o f , Arrhenius p l o t s f o r r e a c t i o n s c a t a l y z e d by membranebound enzymes. These d i s c o n t i n u i t i e s g e n e r a l l y c o i n c i d e w e l l with the maximum temperature f o r the onset o f membrane damage and c h i l l i n g i n j u r y (35, 3 7 ) . The l a t t e r occurrence e x e m p l i f i e s p r o t e i n behavior that i s apparently unique t o c e l l u l a r systems and i s worthy o f f u r t h e r c o n s i d e r a t i o n . Numerous enzymes, t y p i c a l l y nonsoluble (e.g. a s s o c i a t e d with membrane systems or s t a r c h grains) and from c h i l l i n g - s e n s i t i v e p l a n t s , e x h i b i t n o n l i n e a r Arrhenius p l o t s at above-freezing temperatures. An example i n v o l v i n g cytochrome oxidase a c t i v i t y i n i n t a c t mitochondria from tomato i s shown i n Figure 4. The most a t t r a c t i v e explanation f o r t h i s behavior i s that the enzyme i s a s s o c i a t e d with a l i p i d , and i t i s temperature-induced changes i n t h i s a s s o c i a t i o n , perhaps changes i n f l u i d i t y o f the l i p i d and/ or changes i n the geometry of the l i p i d molecules, that produce a conformational change i n the enzyme and r e v e r s i b l e i n a c t i v a t i o n . Although most r e p o r t s of the anamolous behavior mentioned above i n v o l v e enzymes that are attached t o membranes or other c e l l u l a r p a r t i c l e s , attachment i s not a p r e r e q u i s i t e f o r t h i s behavior. Enzymes experimentally detached from membranes o f

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Figure 4. Arrhenius plot of cytochrome oxidase activity in mitochondria. (Reproduced, with permission, from Ref. 40. Copyright 1979, Academic Press.)

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c h i l l i n g - s e n s i t i v e p l a n t s w i l l e x h i b i t t h i s b e h a v i o r (39) and t h e r e i s a t l e a s t one r e p o r t o f a s o l u b l e enzyme, p h o s p h o p h e n o l p y r u v a t e c a r b o x y l a s e from tomato ( 2 3 ) , t h a t e x h i b i t s t h i s behavior. S i n c e t r e a t m e n t o f t h e enzyme w i t h a d e t e r g e n t , o r some o t h e r s u b s t a n c e t h a t w i l l remove l i p i d s , w i l l o f t e n ( b u t n o t a l w a y s ) c a u s e t h e enzyme t o y i e l d l i n e a r A r r h e n i u s p l o t s , i t i s t h e l i p i d - p r o t e i n i n t e r a c t i o n , and p e r h a p s t h e n a t u r e o f t h e l i p i d i t s e l f , that i s b e l i e v e d r e s p o n s i b l e f o r the n o n l i n e a r A r r h e n i u s p l o t s ( 3 8 , 39, 4 0 ) . A l t h o u g h most i n v e s t i g a t o r s b e l i e v e t h a t p r o t e i n s a r e n o t d i r e c t l y i n v o l v e d i n c h i l l i n g i n j u r y (41) t h i s v i e w i s n o t universally held (23). The b e h a v i o r o f enzymes d u r i n g f r e e z e - p r e s e r v a t i o n c a n v a r y g r e a t l y d e p e n d i n g o n w h e t h e r t h e enzyme i s l o c a t e d i n a c e l l u l a r o r n o n c e l l u l a r e n v i r o n m e n t . Many e n z y m e - c a t a l y z e d r e a c t i o n s i n c e l l u l a r s y s t e m s a c t u a l l y i n c r e a s e i n r a t e d u r i n g f r e e z i n g . Examp l e s i n c l u d e enzyme-catalyzed d e g r a d a t i o n of glycogen and/or a c c u m u l a t i o n of l a c t a t e i n f r o g , f i s h , beef or p o u l t r y muscle ( 4 2 - 4 6 ) ; enzyme-catalyzed degradation of h i g h energy phosphates i n f i s h , b e e f and p o u l t r y m u s c l e ( 4 2 , 4 7 - 5 2 ) ; e n z y m e - c a t a l y z e d h y d r o l y s i s o f p h o s p h o l i p i d s i n cod m u s c l e ( 5 3 ) ; e n z y m i c d e c o m p o s i t i o n o f p e r o x i d e s i n r a p i d l y f r o z e n p o t a t o e s and i n s l o w l y f r o z e n p e a s (54); and o x i d a t i o n o f L - a s c o r b i c a c i d i n r o s e h i p s (55) , s t r a w b e r r i e s ( 3 0 ) and B r u s s e l s s p r o u t s ( 5 6 ) . In n o n c e l l u l a r systems, t h i s b e h a v i o r i s v e r y uncommon, and has b e e n o b s e r v e d o n l y when the sample i s e x t r e m e l y d i l u t e p r i o r t o f r e e z i n g . The i n c r e a s e i n s o l u t e c o n c e n t r a t i o n t h a t o c c u r s i n t h e u n f r o z e n phase d u r i n g f r e e z i n g i s o f t e n suggested a s b e i n g responsible f o r instances o f freeze-induced increases i n reaction rates. For enzyme-catalyzed r e a c t i o n s i n c e l l u l a r systems, t h i s e x p l a n a t i o n i s i n a p p r o p r i a t e because: * E n h a n c e d r e a c t i o n r a t e s i n f r o z e n c e l l u l a r s y s t e m s o f t e n do n o t d e c r e a s e upon t h a w i n g ( 5 7 , 58, 5 9 ) . T h i s would not be so i f f r e e z e - i n d u c e d c o n c e n t r a t i o n s o f s o l u t e s w e r e t h e cause s i n c e thawing would r e v e r s e the c o n c e n t r a t i o n effect. *Enzyme-catalyzed r e a c t i o n s i n n o n c e l l u l a r systems r a r e l y a c c e l e r a t e d u r i n g f r e e z i n g , even though the freeze-concent r a t i o n phenomena i s f u l l y o p e r a t i v e . The most r e a s o n a b l e e x p l a n a t i o n f o r t h e f r e e z e - i n d u c e d r a t e enhancement o f e n z y m e - c a t a l y z e d r e a c t i o n s i n c e l l u l a r s y s t e m s i n v o l v e s membrane damage. I t i s w e l l known t h a t f r e e z i n g c a n d i s r u p t membranes and t h e r e b y c a u s e r e l e a s e o f enzymes. Documented i n s t a n c e s i n c l u d e r e l e a s e o f c y t o c h r o m e o x i d a s e f r o m m i t o c h o n d r i a i n b e e f and t r o u t m u s c l e and i n c h i c k e n l i v e r ( 6 0 ) ; r e l e a s e of a c i d l i p a s e s from lysosomes from rainbow t r o u t (61); and r e l e a s e o f m a l a t e and g l u t a m a t e d e h y d r o g e n a s e s , g l u t a m a t e pyruvate transaminase, glutamate or a l o a c e t a t e transaminase, a c o n i t a s e and f u m a r a s e f r o m m i t o c h o n d r i a o f b o v i n e and p o r c i n e muscle ( 6 2 ) . I t i s a l s o reasonable to expect that not o n l y

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enzymes would be d e l o c a l i z e d by f r e e z i n g , but a l s o substrates and other c o n s t i t u e n t s that i n f l u e n c e r a t e s of enzyme r e a c t i o n s ( 8 ) . F i n a l l y , i t should be noted that c h i l l i n g , or f r e e z i n g and frozen storage, can a l s o a l t e r the b i n d i n g s t a t e s ( i o n i c a l l y bound v s . s o l u b l e ) , and the r e l a t i v e concentrations of isoenzymes i n c e l l u l a r systems ( 5 , 63, 64).

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

E f f e c t of Low Temperature on P r o t e i n

Structure

Although i t i s commonly b e l i e v e d that p r o t e i n s become more s t a b l e as the temperature i s lowered, i t has already been shown that many p r o t e i n s e x h i b i t i n s t a b i l i t y , as measured by p a r t i a l l o s s of f u n c t i o n a l i t y , at low, e s p e c i a l l y s u b f r e e z i n g , tempera­ tures. In t h i s s e c t i o n , a t t e n t i o n w i l l be d i r e c t e d to molecular changes that p r o t e i n s can undergo at low temperatures. These are important because they are no doubt r e s p o n s i b l e f o r observed a l t e r a t i o n s i n p r o t e i n f u n c t i o n a l i t y a t low temperatures. C l a s s i f i c a t i o n of molecular changes i n p r o t e i n s a t low temperatures. 1) D i s s o c i a t i o n of oligomers. According to Taborsky (65) and L o z i n a - L o z i n s k i i (11), d i s s o c i a t i o n of p r o t e i n oligomers i n t o subunits i s a primary response o f enzymes to f r e e z i n g . Examples of p r o t e i n s behaving i n t h i s manner i n c l u d e l a c t a t e dehydrogenase (66) , α-chymotrypsin (67) , glutamate dehydrogenase (68), an apoprotein of high-density serum l i p o p r o t e i n s (69), glucose-6-phosphate dehydrogenase (70) , pyruvate orthophosphate d i k i n a s e (18), albumins and g l o b u l i n s (71) and deoxyribonucleop r o t e i n s (72). % 2) Rearrangement of subunits w i t h i n oligomers. An example of t h i s kind i n v o l v e s l a c t a t e dehydrogenase ( 3 ) . 3) Aggregation. Low temperature a s s o c i a t i o n (a s p e c i f i c form o f aggregation u s u a l l y i n v o l v i n g subunits of oligomers and s p e c i f i c bonding s i t e s ) of p r o t e i n subunits i s comparatively r a r e . One example i n v o l v e s a t r a n s i t i o n of monomers to trimers i n a membrane proteinase from Streptococcus l a c t i s (73). Fink (2) a l s o has found that a s s o c i a t i o n of enzyme subunits can occur at low temperatures i n aqueous-organic s o l v e n t s . G e l a t i o n of egg yolk i s r o u t i n e l y observed during f r e e z i n g and thawing (74, 75). Low temperature aggregation has a l s o been reported f o r 17B-hydroxysteroid dehydrogenase (76) , urease (77) and myosin (78). Casein m i c e l l e s i n milk can a l s o aggregate during frozen storage. In the e a r l y stages, a f l o c c u l e n t p r e c i p i t a t e forms, and i n the more advanced stages, a g e l (79). 4) Changes i n conformation. Reports of conformâtional changes i n p r o t e i n s at low temperatures are reasonably abundant (11, 12, 80). Examples i n c l u d e p h o s v i t i n (81) , actomyosin (82), myosin Β (83), v i r i o n s of southern bean mosaic v i r u s (84), snake venom L-amino oxidase (85) and numerous instances of denaturation c i t e d by Brandts (12).

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119

D i s c u s s i o n of changes i n p r o t e i n s t r u c t u r e a t low temperat u r e . The types o f changes c i t e d above can be best understood i f two premises, that appear to have general a p p l i c a b i l i t y to these s i t u a t i o n s , are accepted. F i r s t , i t i s g e n e r a l l y agreed that the t e r t i a r y n a t i v e s t r u c t u r e o f p r o t e i n i s maintained by s e v e r a l types of i n t e r a c t i o n s , namely hydrophobic, hydrogen-bonding, e l e c t r o s t a t i c and van der Waals f o r c e s (86). Among these, hydrophobic i n t e r a c t i o n s are regarded as being o f primary importance i n most p r o t e i n s (65, 80, 87) and the s t r e n g t h o f these i n t e r a c t i o n s i s weakened by lowering the temperature. Therefore, lowering the temperature w i l l tend to produce s t r u c t u r a l changes i n those p r o t e i n molecules that are dependent on hydrophobic i n t e r a c t i o n s f o r maintenance o f n a t i v e s t r u c t u r e (65, 87). I t should a l s o be recognized that t h i s statement a p p l i e s e q u a l l y w e l l t o both a s s o c i a t i o n - d i s s o c i a t i o n t r a n s i t i o n s and t o changes i n conformation (88) . Although t h i s c l e a r l y a p p l i e s to the s i t u a t i o n s being considered here, i t should nevert h e l e s s be recognized that the simple r e l a t i o n s h i p between hydrophobic i n t e r a c t i o n s and temperature i s not capable o f accounting f o r a l l p r o t e i n s t r u c t u r a l changes that are observed a t low temperatures. For example, f a c t o r s other than temperature have profound e f f e c t s on the s t r u c t u r e o f p r o t e i n s , and some o f these f a c t o r s (pH, i o n i c s t r e n g t h , s u r f a c e t e n s i o n , p r o t e i n concentrat i o n , c o n c e n t r a t i o n o f nonprotein s o l u t e s ) change s u b s t a n t i a l l y during f r e e z i n g . Second, a thermodynamic approach t o the molecular behavior of p r o t e i n s a t low temperatures provides i n f o r m a t i o n o f great importance and must t h e r e f o r e occupy a p o s i t i o n of c e n t r a l importance i n t h i s d i s c u s s i o n . A l l of the p r o t e i n a l t e r a t i o n s mentioned p r e v i o u s l y i n v o l v e the t r a n s f e r of some p o r t i o n o f a p r o t e i n molecule from an aqueous environment to an e s s e n t i a l l y organic environment (to the i n t e r i o r o f a f o l d e d p r o t e i n ) or the r e v e r s e . D i s s o c i a t i o n and u n f o l d i n g i n v o l v e i n c r e a s e d exposure of p r o t e i n s u r f a c e t o an aqueous environment (increased p r o t e i n water i n t e r a c t i o n ) . Whether d i s s o c i a t i o n and/or u n f o l d i n g w i l l a c t u a l l y occur depends s o l e l y on the change i n t o t a l f r e e energy. I f the change i n f r e e energy f o r i n c r e a s e d i n t e r a c t i o n with water i s n e g a t i v e , the d i s s o c i a t i o n - u n f o l d i n g type t r a n s i t i o n s w i l l occur spontaneously, but nothing w i l l be known about r a t e . I f the change i n f r e e energy o f i n c r e a s e d water i n t e r a c t i o n i s p o s i t i v e , the a s s o c i a t i o n - f o l d i n g - a g g r e g a t i o n type t r a n s i t i o n s w i l l occur spontaneously, but again, nothing w i l l be known about r a t e . These statements, of course, apply to the o v e r a l l behavior of the sample, not to i n d i v i d u a l molecules. Furthermore, changes i n temperature can a l t e r whether the t r a n s i t i o n s j u s t mentioned w i l l r e s u l t i n negative or p o s i t i v e changes i n f r e e energy, so at one temperature, i n c r e a s e d water i n t e r a c t i o n may be favored and at another temperature, the reverse may be t r u e . It i s now i n s t r u c t i v e t o examine some q u a n t i t a t i v e data f o r thermodynamic p r o p e r t i e s of g l o b u l a r p r o t e i n s exposed to low

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temperatures. At the o u t s e t , i t should be emphasized that data i s not abundant s i n c e the thermodynamic p r e r e q u i s i t e of r e a c t i o n r e v e r s i b i l i t y s e v e r e l y l i m i t s the c o n d i t i o n s that can be used experimentally. Furthermore, the data presented here were developed on the assumption that the t r a n s i t i o n s under c o n s i d e r a t i o n (associâtionjdissociation; n a t i v e ^ e n a t u r e d ) i n v o l v e only two s t a t e s . Reaction i n t e r m e d i a t e s , i f they occur, must not be s t a b l e under the c o n d i t i o n s used. T h i s assumption i s considered reasonable f o r many p r o t e i n s , i n c l u d i n g those considered here, but not n e c e s s a r i l y f o r a l l p r o t e i n s (12, 8 9 , 9 0 , 91). Shown i n Figure 5 i s the temperature dependence of the standard f r e e energy (AG° or AF°) of denaturation f o r chymotrypsinogen at three d i f f e r e n t pH v a l u e s . Conditions were such that a s s o c i a t i o n d i d not occur and observations t h e r e f o r e p e r t a i n to n a t i v e j u n f o l d e d t r a n s i t i o n s of monomers. Since accurate values of AG° could be d i r e c t l y determined only w i t h i n the range of ±2,000 c a l o r i e s , the remainder of the data were c a l c u l a t e d from a four-term power s e r i e s i n v o l v i n g absolute temperature (92). The data i n Figure 5 c l e a r l y i n d i c a t e that AG° i s not a l i n e a r f u n c t i o n of temperature as had been f r e q u e n t l y assumed p r i o r to p u b l i c a t i o n of these data. Instead, the r e l a t i o n s h i p i s p a r a b o l i c i n nature, with a temperature of maximum s t a b i l i t y ( T ) occurr i n g at about 10-12°C, i . e . r a i s i n g or lowering the temperature from T can r e s u l t i n denaturation ( u n f o l d i n g ) . Thus, low temperature denaturation i s c l e a r l y p o s s i b l e . Lowering the pH of the sample causes the e n t i r e curve to s h i f t to a more negative value of AG° and thereby reduce the temperature at which a value of AG° = 0 ( f o r the high-temperature l e g of the graph) i s a t t a i n e d . According to Brandts (12), the e s s e n t i a l f e a t u r e s of these data are undoubtedly t y p i c a l of denaturation r e a c t i o n s f o r most p r o t e i n s . The data i n F i g u r e s 6 and 7 provide c l e a r evidence that the c o n c l u s i o n s drawn from Figure 5 are v a l i d . The data i n F i g u r e 6 r e l a t e to chymotrypsin at pH 1.47 and i t i s evident that t h i s p r o t e i n does, i n f a c t , e x h i b i t maximum s t a b i l i t y at about 12°C, with increased denaturation o c c u r r i n g at higher or lower temperatures. T f o r chymotrypsin i s t h e r e f o r e almost i d e n t i c a l to max chymotrypsinogen i n Figure 5. Figure 7 i s a van't Hoff p l o t ( l o g e q u i l i b r i u m constant, Κ = unfolded/native v s . 1/T) f o r β-lactoglobulin A showing T at about 35°C (90). A s u f f i c i e n t number of other i n v e s t i g a t o r s have reported i n c i d e n c e s of low temperature denaturation to v e r i f y that t h i s behavior i s a reasonably common occurrence (12, 25). From the slopes of the curves i n Figure 7 i t i s p o s s i b l e to c a l c u l a t e the enthalpy of t r a n s i t i o n from the n a t i v e to the denatured s t a t e , assuming a two-state process at a l l temperatures. From such c a l c u l a t i o n s , i t i s found that the u n f o l d i n g r e a c t i o n f o r β-lactoglobulin A i s exothermic below 35°C and endothermic above t h i s temperature (90). C a l c u l a t e d thermodynamic parameters f o r the a-chymotrypsinom a x

m a x

m a x

T

f o r

m a x

FENNEMA

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121

Temperatures

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6.

pH 3.0, 0.01 M Cl

J 0

1 10

I 20

I 30

TEMPERATURE

I 40

I 50

X60

Figure 5. Temperature dependence of the free energy of denaturation of chymotrypsinogen at different pH values and ionic strengths. (Reproduced from Ref. 92. Copyright 1964, American Chemical Society.)

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

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j

Ο

ι

10

ι

20

i

30

T E M P E R A T U R E (°)

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I

40

DETERIORATION

Ι­

50

Figure 6. Temperature dependence of the extinction coefficient at 293 nm for chymotrypsin at pH 1.47. The dashed lines at the top and bottom of the plot indicate the extinction coefficients for the denatured and native states, respectively, (Reproduced, with permission, from Ref. 12. Copyright 1967, Academic Press.)

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

FENNEMA

Behavior

of Proteins at Low

Temperatures

σ* ο

3.5 1/Tx Figure

7.

unfolded^ ^

Van't Hoff

3.6

10 ( ° K ~ )

plots (logarithm of

3

1

the equilibrium constant, Κ

reciprocal of absolute temperature) for β-lactoglobulin

=

A in urea at

various concentrations. (Reproduced from Ref. 90. Copyright 1968, American Chemical Society.)

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gen data (native^unfolded) i n F i g u r e 5 (pH 3.0) are shown i n Figure 8 ( 9 2 ) . Values f o r ΔΗ° and AS° are h i g h l y temperature dependent, both e x h i b i t i n g l a r g e p o s i t i v e values at 65°C, decreasing to values o f zero at about 10°C and becoming negative below 10°C. The change i n heat c a p a c i t y (ACp) between the n a t i v e and unfolded forms a t a given temperature i s l a r g e and p o s i t i v e f o r a l l temperatures i n F i g u r e 8. The l a r g e p o s i t i v e v a l u e s of ACp are r e s p o n s i b l e f o r the temperature dependency of both

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

d(AH) ΔΗ° (dT

= ACp) and AS° (AS = IC 2 S&dT) 1 and f o r the occurrence of a temperature of maximum p r o t e i n s t a b i l i t y (Figures 5 and 7) (88, 90, 92, 93). Although ACp i s temperature dependent i n Figure 8, t h i s i s not e s p e c i a l l y important nor i s t h i s c h a r a c t e r i s t i c true f o r a l l d e n a t u r a t i o n reactions ( 8 8 ) . The l a r g e heat c a p a c i t y values apparently a r i s e from exposure of a p o l a r groups to water d u r i n g the d e n a t u r a t i o n p r o c e s s . In water, a p o l a r groups e x h i b i t a p a r t i a l molal heat c a p a c i t y that i s about three times greater than t h e i r heat c a p a c i t y i n an organic environment, such as i n the i n t e r i o r of the n a t i v e p r o t e i n . Consequently, c o n s i d e r a b l e energy must be expended during temperature i n c r e a s e s to p a r t i a l l y d i s r u p t the s t r u c t u r a l water e x i s t i n g around apolar groups at low temperature ( 2 5 ) . The general importance of the heat c a p a c i t y e f f e c t at atmospheric pressure was emphasized by Edelhock and Osborne (80) i n t h e i r statement that ". . .most, i f not a l l , p r o t e i n r e a c t i o n s i n which nonpolar groups are exposed to water, i . e . , d e n a t u r a t i o n , l i g a n d d i s s o c i a t i o n , the d i s s o c i a t i o n of macromolecules or microscopic s t r u c t u r e s composed of a l a r g e number of s u b u n i t s , are c o n t r o l l e d , i n l a r g e p a r t , by a p o s i t i v e heat c a p a c i t y change which appears to increase w i t h temperature." D i s s o c i a t i o n of o l i g o m e r i c p r o t e i n s i n t o subunits at low temperatures probably occurs more e a s i l y than simple u n f o l d i n g s i n c e the b i n d i n g f o r c e s i n v o l v e d i n d i s s o c i a t i o n are not as strong as those i n v o l v e d i n u n f o l d i n g . In F i g u r e 9 the tempera­ ture dependence o f heats o f d i s s o c i a t i o n ( e n t h a l p i e s , ΔΗ°) are shown f o r glutamate dehydrogenase at pH 7 (68), α-chymotrypsin at pH 4.12 (67) and apo A - I I p r o t e i n a t pH 7.4 from h i g h - d e n s i t y serum l i p o p r o t e i n (69) . The e n t h a l p i e s f o r glutamate dehydro­ genase and α-chymotrypsin are negative (exothermic) a t low temperatures and p o s i t i v e (endothermic) a t high temperatures. The enthalpy of d i s s o c i a t i o n f o r carboxymethylated (Cm) apo A - I I i s negative below, and p o s i t i v e above, 23°C, and c o n t r a r y to glutamate dehydrogenase and α-chymotrypsin, which y i e l d g l o b u l a r molecules upon d i s s o c i a t i o n , Cm apo A - I I d i s s o c i a t e s i n t o molecules that are randomly c o i l e d . Thus, d i s s o c i a t i o n i n t h i s instance i s accompanied by major changes i n secondary and tertiary structure. T

FENNEMA

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6.

of Proteins at Low

Temperatures

TEMPERATURE ( ° ) Figure 8. Temperature dependence of enthalpy (àH°), entropy (AS°), and heat capacity (àC ) pertaining to denaturation of chymotrypsinogen. (Reproduced from Ref, 92, Copyright 1964, American Chemical Society.) p

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20 TEMP. (°C)

40

60

Figure 9. Temperature dependence of heats of dissociation for a-chymotrypsin (O) (61), glutamate dehydrogenase (A) (68), and reduced and carboxymethylated apo A-II protein (•) (69). (Reproduced, with permission, from Ref. 80. Copyright 1976, Academic Press.)

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6.

FENNEMA

Behavior

of Proteins at Low

Temperatures

127

As mentioned a t the beginning o f t h i s s e c t i o n , examples do e x i s t f o r both a s s o c i a t i o n and d i s s o c i a t i o n type r e a c t i o n s i n p r o t e i n s exposed t o low temperatures, and the type of t r a n s i t i o n favored w i l l be that which e x h i b i t s a decrease i n f r e e energy. From the expression AG = ΔΗ - TAS (constant Τ and P) i t i s evident that AG w i l l become more negative as AH becomes more negative (exothermic) or AS becomes more p o s i t i v e (greater d i s o r d e r or g r e a t e r p r o b a b i l i t y of e x i s t e n c e ) . S i t u a t i o n s do e x i s t where AH i s negative and AS i s p o s i t i v e , but i t i s more common f o r both terms t o have the same s i g n (94). Based on the data presented, the thermodynamic p r o p e r t i e s of u n f o l d i n g and d i s s o c i a t i o n r e a c t i o n s f o r p r o t e i n s can be summarized as shown i n Table I . The two assumptions mentioned e a r l i e r apply to these data. Since these assumptions are con­ s i d e r e d v a l i d f o r many p r o t e i n s (12), these data, and the a s s o c i a t e d behavior d e p i c t e d i n F i g u r e s 5-7, can be considered reasonably t y p i c a l . Omitted from Table I are the a s s o c i a t i o n type t r a n s i t i o n s that some p r o t e i n s undergo a t low temperatures. In these i n s t a n c e s , i n t e r a c t i o n s ( e l e c t r o s t a t i c , hydrogen bonding, d i s u l f i d e interchange) other than hydrophobic may p l a y d e c i s i v e r o l e s , or hydrophobic i n t e r a c t i o n s may be i n f l u e n c e d s i g n i f i c a n t ­ l y by f a c t o r s other than temperature (pH, i o n i c s t r e n g t h , e t c . ) . Thus, the data i n Table I are*an o v e r s i m p l i f i c a t i o n , but they n e v e r t h e l e s s are b e l i e v e d to r e p r e s e n t , reasonably w e l l , the behavior of many p r o t e i n s a t low temperatures. Beginning w i t h a d i s c u s s i o n o f the data at intermediate t o high temperatures (row 1 o f Table I ) , i t i s evident that the e n t h a l p i e s and e n t r o p i e s of t r a n s i t i o n to the u n d i s s o c i a t e d or unfolded s t a t e are both p o s i t i v e . E i t h e r o f these t r a n s i t i o n s are favored (-AG) when the temperature i s high enough and AS i s l a r g e enough so that TAS > AH. The entropy term, i n a l l i n s t a n c e s i n Table I i s composed o f two components, one i n v o l v i n g p r o t e i n s t r u c t u r e and the other water s t r u c t u r e . U n f o l d i n g o r d i s s o c i a ­ t i o n would i n v o l v e a p o s i t i v e change i n entropy f o r the p r o t e i n and a negative change i n entropy f o r water, the l a t t e r o c c u r r i n g because groups exposed t o water during u n f o l d i n g or d i s s o c i a t i o n are predominantly hydrophobic, thus promoting i n c r e a s e d water s t r u c t u r e (88). As the temperature i s r a i s e d i n the r e g i o n above the temperature o f maximum s t a b i l i t y ( T ) f o r the p r o t e i n , water e x h i b i t s a reduced tendency t o a s s o c i a t e i n an ordered manner around hydrophobic groups, and more hydrophobic groups become exposed. Thus, the change i n p r o t e i n s t r u c t u r e becomes an i n c r e a s i n g l y dominant component of AS, and AS assumes a l a r g e r p o s i t i v e v a l u e , and AG a l a r g e r negative v a l u e , as the tempera­ ture i s r a i s e d above T . At T , the values of AH and AS both decrease to near zero (row 2, Table I and F i g u r e 8 ) . Since AG assumes the l a r g e s t p o s s i b l e p o s i t i v e value a t t h i s temperature, AH and AS cannot both have values o f zero. Small changes i n e i t h e r AH o r AS near m a x

m a x

m a x

128

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T a b l e I . Thermodynamic s o f p r o t e i n u n f o l d i n g o r d i s s o c i a t i o n when h y d r o p h o b i c i n t e r a c t i o n s a r e o f m a j o r i m p o r t a n c e

Thermodynamic d a t a Temperature AG Sufficiently to cause disordering

ACp

2

AH

AS

|TAS/AH|

+

+

>1

@55°C

-0

small -

>1

~+2 @10°C

-

>1

H-1.5 @-10 C

-

|ΔΗ|, w i t h t h e AS t e r m b e i n g d o m i n a t e d b y t h e change i n w a t e r s t r u c t u r e . A t s t i l l l o w e r t e m p e r a t u r e s (row 4» T a b l e I ) AG p r e s u m a b l y becomes n e g a t i v e ( l o g Κ > 0» where AG = -RT I n K ; F i g u r e 7), i n w h i c h c a s e t h e a b s o l u t e v a l u e o f AH(-) must e x c e e d t h e a b s o l u t e v a l u e o f TAS ( - ) . m a x

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m a x

Conclusions P o s s i b l e adverse e f f e c t s o f low temperatures on p r o t e i n s i n c l u d e d e r e a l i z a t i o n o f p r o t e i n s i n c e l l u l a r s y s t e m s and a l o s s of p r o t e i n s o l u b i l i t y and f u n c t i o n a l i t y . Factors influencing the d e g r e e o f damage d u r i n g f r e e z i n g i n c l u d e t h e t y p e o f p r o t e i n , the c o m p l e x i t y o f t h e s y s t e m ( c e l l u l a r v s . n o n c e l l u l a r ) , s t o r a g e t e m p e r a t u r e ( r e l a t e d t o w a t e r a c t i v i t y ) , s t o r a g e t i m e , and t h e c o n d i t i o n s o f f r e e z i n g and t h a w i n g . P r o t e i n s a t l o w t e m p e r a t u r e may u n d e r g o c h a n g e s i n c o n f o r m a t i o n a n d t h e d e g r e e o f a s s o c i a t i o n , w i t h t h e degree o f r e v e r s i b i l i t y depending on t h e c o n d i t i o n s . C a u s a t i v e f a c t o r s i n f l u e n c i n g p r o t e i n damage d u r i n g f r e e z i n g i n c l u d e c h a n g e s i n s a l t c o n c e n t r a t i o n , c h a n g e s i n pH, m e c h a n i c a l e f f e c t s o f i c e o n membranes and d e h y d r a t i o n e f f e c t s . D i f f e r e n t p r o t e i n s d i f f e r g r e a t l y i n t h e i r responses t o low t e m p e r a t u r e s , and t h e b e h a v i o r o f a s i n g l e p r o t e i n a t a g i v e n l o w t e m p e r a t u r e c a n be u n u s u a l and d i f f i c u l t t o p r e d i c t . The l a t t e r i s t r u e because l o w t e m p e r a t u r e s c a n have a d i r e c t i n f l u e n c e o n p r o t e i n s t r u c t u r e and f u n c t i o n a l i t y , a n d b e c a u s e low t e m p e r a t u r e s , e s p e c i a l l y s u b f r e e z i n g t e m p e r a t u r e s , c a n i n d u c e d r a m a t i c changes i n t h e p r o t e i n ' s environment. A proper perspec­ t i v e i s perhaps best achieved by r e c o g n i z i n g that a great d e a l r e m a i n s t o b e l e a r n e d a b o u t t h e muçh-studied b e h a v i o r o f p r o t e i n s a t a m b i e n t t o h i g h t e m p e r a t u r e s , a n d t h a t f a r more r e m a i n s t o b e l e a r n e d about t h e l i t t l e - s t u d i e d b e h a v i o r o f p r o t e i n s a t l o w temperatures. Acknowledgments R e s e a r c h s u p p o r t e d by t h e C o l l e g e o f A g r i c u l t u r a l and L i f e Sciences, U n i v e r s i t y of Wisconsin-Madison.

FOOD

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DETERIORATION

Literature Cited 1. 2. 3. 4.

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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28.

Douzou, P. Adv. Enzymol., 1980, 51, 1. Fink, A. L . In "Proteins at Low Temperatures"; Fennema, O., Ed.; ACS Advances in Chem. Series: Washington, D . C . , 1979; p. 35. Chilson, O. P.; Costello, L. A.; Kaplan, N. D. Fed. Proc., 1965, 24 (2), Suppl. 15, S-55. Duke, S. H.; Koukkari, W. L.; Soulen, T. B. Physiol. Plant., 1975, 34, 8. Krasnuk, M.; Jung, G. A.; Witham, F. H. Cryobiology, 1976, 13, 375. Balls, A. K.; Tucker, T. W. Ind. Eng. Chem., 1938, 30, 415. Balls, A. K.; Lineweaver, H. Food Res., 1938, 3, 57. Fennema, O. In "Dry Biological Systems"; Crowe, J . H.; Clegg, J . S., Eds.; Academic Press: New York, 1978; p. 297. Maier, V. P.; Tappel, A. L.; Volman, D. H. J . Amer. Chem. Soc., 1955, 77, 1278. Sizer, I. W.; Josephson, E. S. Food Res., 1942, 7, 201. Lozina-Lozinskii, L. K. "Studies in Cryobiology"; John Wiley & Sons: New York, 1974; p. 202, 208, 209. Brandts, J . F. In "Thermobiology"; Rose, Α. Η., Ed.; Academic Press: New York, 1967; p. 25. Han, M. H. J . Theor. Biol., 1972, 35, 543. Kavanau, J . L. J . Gen. Physiol., 1950, 34, 193. Scrutton, M. C.; Utter, M. F. J . Biol. Chem., 1965, 240, 1. Shukuya, R.; Schwert, G. J . Biol. Chem., 1960, 235, 1658. Numa, S.; Ringelmann, E. Biochem. Ζ., 1965, 343, 258. Shirahashi, K.; Hayakawa, S.; Sugiyama, T. Plant Physiol., 1978, 62, 826. Grant, Ν. H.; Alburn, H. E. Nature (London), 1966, 212, 194. Tong, M-M.; Pincock, R. E. Biochemistry, 1969, 8, 908. Hochachka, P. W.; Somero, G. N. Comp. Biochem. Physiol., 1968, 27, 659. Somero, G. N. Amer. Naturalist, 1969, 103, 517. Graham, D.; Hockley, D. G.; Patterson, B. D. In "Low Temperature Stress in Crop Plants"; Lyons, J . M.; Graham, D.; Raison, J . Κ., Eds.; Academic Press: New York, 1979; p. 453. Uritani, I. In "Postharvest Biology and Biotechnology"; Hultin, H. O.; Milner, Μ., Eds.; Food and Nutrition Press: Westport, CT, 1978; p. 136. Brandts, J . F . ; Fu, J.; Nordin, J . H. In "The Frozen Cell"; Wolstenholme, G. E. W.; O'Connor, Μ., Eds.; J . and A. Churchill: London, 1970; p. 189. Parducci, L. G.; Fennema, O. Cryobiology, 1978, 15, 199. Parducci, L. G.; Fennema, O. Cryobiology, 1979, 16, 578. Fennema, O.; Sung, J . C. Cryobiology, 1980, 17, 500.

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37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

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Douzou, P. Mol. Cell. Biochem., 1973, 1, 15. Fennema, O. In "Water Relations in Foods"; Duckworth, R. Β., Ed.; Academic Press; New York, 1975; p. 397. Soliman, F. S.; van den Berg, L. Cryobiology, 1971, 8, 73. Greiff, D.; Kelly, R. T. Cryobiology, 1966, 2, 335. van den Berg, L. Arch. Biochem. Biophys., 1959, 84, 305. van den Berg, L . ; Rose, D. Arch. Biochem. Biophys., 1959, 81, 319. Raison, J. K. J . Bioenerg., 1973, 4, 285. Raison, J. K.; Chapman, E. A. Austr. J. Plant Physiol., 1976, 3, 291. Zeylemaker, W. P.; Jansen, H.; Veeger, C.; Slater, E. C. Biochim. Biophys. Acta., 1971, 242, 14. Downton, W. J. S.; Hawker, J . S. Phytochemistry, 1975, 14, 1259. Duke, S. H.; Schrader, L. E . ; Miller, M. G. Plant Physiol., 1977, 60, 716. Waring, A.; Glatz, P. In "Low Temperature Stress in Crop Plants"; Lyons, J . M.; Graham, D.; Raison, J. Κ., Eds.; Academic Press: New York, 1979; p. 365. Lyons, J . M.; Graham, D.; Raison, J . Κ., Eds. "Low Temperature Stress in Crop Plants"; Academic Press: New York, 1979. Behnke, J . R.; Fennema, O.; Cassens, R. G. J . Agric. Food Chem., 1973, 21, 5. Sharp, J . G. Proc. Roy. Soc., 1934, Ser B, 114, 506. Sharp, J . G. Biochem. J., 1935, 29, 850. Smith, E. C. Proc. Roy. Soc., 1929, Ser B, 105, 198. Tomlinson, N.; Jonas, R. Ε. E . ; Geiger, S. E. J . Fish. Res. Bd. Can., 1963, 20, 1145. Bito, M.; Amano, K. Bull. Tokai Reg. Fish. Res. Lab., 1962, 32, 149. (As cited by Tomlinson et al., 1963, Ref. #46.) Partmann, W. Z. Ernahr. Wiss., 1961, 2, 70. Partmann, W. J . Food Sci., 1963, 28, 15. Saito, T.; Arai, K. Bull. Jap. Soc. Scient. Fish., 1957, 22, 569. (As cited by Tomlinson et al., 1963, Ref. #46.) Saito, T.; Arai, K. Bull. Jap. Soc. Scient. Fish., 1957, 23, 265. (As cited by Tomlinson et al., 1963, Ref. #46.) Saito, T.; Arai, D. Arch. Biochem. Biophys., 1958, 73, 315. Lovern, J . A.; Olley, J . J . Food Sci., 1962, 27, 551. Kiermeier, F. Biochem. Ζ., 1949, 319, 463. Mapson, L. W.; Tomalin, A. W. J . Sci. Food Agric., 1958, 9, 424. Suhonen, I. J . Sci. Agric. Soc. Finland, 1967, 39, 99. Fishbein, W. N.; Stowell, R. E. Cryobiology, 1966, 6, 227. Rhodes, D. N. J . Sci. Food Agric., 1961, 12, 224. Tappel, A. L. In "Cryobiology"; Meryman, H. T . , Ed.; Academic Press: New York, 1966; p. 163. Barbagli, C.; Crescenzi, G. S. J . Food Sci., 1981, 46, 491.

132

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66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82.

83. 84. 85. 86. 87. 88. 89. 90.

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DETERIORATION

Geromel, E. J.; Montgomery, M. W. J . Food Sci., 1980, 45, 412. Hamm, R. In "Proteins at Low Temperatures"; Fennema, O., Ed.; ACS Advances in Chem. Series: Washington, D.C., 1979; p. 191. Gkinis, A. M.; Fennema, O. R. J . Food Sci., 1978, 43, 527. Gusta, L. V.; Weiser, C. J. Plant Physiol., 1972, 49, 91. Taborsky, G. In "Proteins at Low Temperatures"; Fennema, O., Ed.; ACS Advances in Chem. Series: Washington, D.C., 1979; p. 1. Markert, C. L. Science., 1963, 140, 1329. Aune, Κ. C.; Goldsmith, L. C.; Timasheff, S. Ν. Biochemistry, 1971, 10, 1617. Reisler, Ε.; Eisenberg, Η. Biochemistry, 1971, 10, 2659. Osborne, J. C.; Palumbo, G.; Brewer, Η. B.; Edelhoch, H. Biochemistry, 1975, 14, 3741. Kirkman, Η. N.; Hendrickson, Ε. M. J. Biol. Chem., 1962, 237, 2371. Pallavicini, C. Sci. Tecnol. Degli Alimenti., 1973, 3, 35. Khenokh, M. A.; Pershina, V. P.; Lapinskaya, Ε. M. Tsitologiya., 1966, 8, 769-772; In Russian (as cited by Lozina-Lozinskii, 1974, Ref. #11). Cowman, R. A.; Speck, M. L. Cryobiology, 1969, 5, 291. Moran, T. Proc. Roy. Soc., 1925, Ser Β, 98, 436. Thomas, A. W.; Bailey, M. I. Ind. Eng. Chem., 1933, 6, 669. Jarabak, J.; Seeds, J r . , A. E . ; Talalay, P. Biochemistry, 1966, 5, 1269. Hofstee, B. F. J . J . Gen. Physiol., 1948, 32, 339. Connell, J . J . Nature, 1959, 183, 664. Saito, Z . ; Niki, R.; Hashimoto, Y. J . Fac. Agr. Hokkaido Univ., 1963, 53, 200. Edelhoch, H.; Osborne, Jr., J . C. Adv. Protein Chem., 1976, 30, 183. Taborsky, G. J . Biol. Chem., 1970, 24, 1054. Kenokh, M. A.; Aleksandrova, V. N. "Reaktsiya Kletok i ikh belkovykh Komponentov na ekstremal'nye vozdeistviya"; Leningrad, 1966; p. 27. (As cited by Lozina-Lozinskii, 1974, Ref. #11.) Hanafusa, N. Contr. Inst. Low Temp. Sci., 1972, Ser Β (17), 1. Sehgal, O. P.; Das, P. D. Virology, 1975, 64, 180. Curti, B.; Massey, V.; Zmudka, M. J . Biol. Chem., 1968, 243, 2306. Wynn, C. H. "The Structure and Function of Enzymes"; Edward Arnold: London, 1973; p. 18. Oakenfull, D.; Fenwick, D. E. Aust. J . Chem., 1977, 30, 741. Tanford, C. Adv. Protein Chem., 1968, 23, 121. Eaglund, D. In "Water--A Comprehensive Treatise"; Vol. 4; Franks, F . , Ed.; Plenum Press: New York, 1975; p. 305. Pace, N. C.; Tanford, C. Biochemistry, 1968, 7, 198.

6.

91. 92. 93. 94.

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of Proteins at Low

Temperatures

133

Privalov, P. L . ; Khechinashvili, N. N. J . Mol. Biol., 1974, 86, 665. Brandts, J . F. J . Amer. Chem. Soc., 1964, 86, 4291. Brandts, J . F. J . Amer. Chem. Soc., 1964, 86, 4302. Mahan, Β. H. "Elementary Chemical Thermodynamics"; W. A. Benjamin: New York, 1963.

RECEIVED May 17,

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Behavior

1982.

7 Structural Changes and Metabolism of Proteins Following Heat Denaturation J. N. NEUCERE and J. P. CHERRY

1

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch007

United States Department of Agriculture, Southern Regional Research Center, Agricultural Research Service, New Orleans, LA 70179

Heat is perhaps the primary factor that enhances the safety, nutritional quality, and palatability of most foods. Mild or moderate temperatures can be beneficial in achieving these goals, whereas, extreme temperatures often are deleterious in terms of biological value and toxicity. The complex reactions that occur during extreme heat treatments, which usually involve simple and intricate molecules and ions, modify the physicochemical properties and metabolic efficiency of proteins. A key concept in protein chemistry is the effect heat has on active centers or sites within these molecules. These centers, involving any number of functional groups, are defined as the regions of space where the binding of other molecules and ions occur. Some of the most reactive sites in proteins are amine, sulfhydryl, tyrosyl, and imidizole groups. Heat-induced changes in any or all of these functional groups are directly related to physicochemical modifications such as solubility, enzymatic activity, antigenic reactivity, electrophoretic migration and association-dissociation constraints. Anti-nutritional parameters and toxicological aspects of products from protein heated in the presence of carbohydrates are well recognized from the many studies via the Amadori and Maillard reactions. The formation of diverse isopeptides induced by heat are also critical factors that can adversely affect the biological value of proteins. Measuring toxicity or harmful effects, for example, of such complicated reaction products is not a simple matter. Adversity could involve either a modified intact protein or the presence of intricate amino acids and peptides that have formed. This chapter concentrates on several empirical views of structural changes and anti-nutritional aspects of proteins under various conditions of heat treatments. Experimental Procedures The reader is referred to the fol 1 owing publications on techniques of gel electrophoresis Q), immunochemistry (j2), Current address: United States Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118. 1

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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functionality (3) and nutrition (4). Studies on the determination of multiple forms of enzymes and t h e i r use as biological indicators for food uses and applications were reviewed by Cherry ( ! , £ ) . Whole seeds, f u l l - f a t and fat-free meal s, concentrates and/or isolates are used in research to determine the effects of moist and dry heat on protein properties. For example, peanut kernels are moist-heated in water in a temperature-control 1ed steam retort kept at various temperatures for different time intervals (7). After moist heat treatment, the seals are lyophilized and then ground into a meal for protein analyses. Dry heat treatments may be conducted by placing seeds in a forced-draft oven at various temperatures and time intervals ( £ ) ; purified proteins such as peanut α-arachin are s i m i l a r l y heat-treated (9). Seeds can be made to imbibe varying amounts of water to 40% and then dry heated (8). Microwave heating of seeds containing only t h e i r innate moisture or after they have been placed in water is conducted at operational frequencies as high as 2,480 MHz (10). Evidence of Protein Structural Changes Induced by Heat S o l u b i l i t y . Perhaps the most obvious evidence of conforma­ tional changes i n protein structure are the alterations in protein s o l u b i l i t y . In general, protein s o l u b i l i t y decreases with time and temperature of heat treatment; and considerable variations i n the degree of change exist among proteins. Interactions with several functional groups ( e . g . , SH- or tyrosyl-residues) become more prevalent fol 1 owing unfolding of protein chains, resulting in diverse and complicated precipitation reactions. The exact mechanisms leading to precipitation are most d i f f i c u l t to study after heat treatments because the process is usually i r r e v e r s i b l e . It is well known that the interactions of water molecules with ionic and polar groups of proteins strongly inf1uence the folded conformations of proteins (11). Consequently, moist heat usually has a more complex effect on the s o l u b i l i t y properties of pro­ teins than does dry heat, and is strongly influenced by ionic strength and pH. An example of s o l u b i l i t y changes of peanut proteins induced by heat is shown in Figure 1. In this study (8), seeds were al 1 owed to imbibe d i s t i l l e d water for 16 hr at 25°C to a final moisture content of 40% and were heated for 1 hr from 110 to 155°C. Other seed samples were heated on an "as is" basis (5% moisture) under identical conditions. S o l u b i l i t y of total pro­ tein decreased almost 1inearly with temperature for the dry-heated seeds. For the wet-heated samples, the relationship was quite d i f f e r e n t ; a plot of s o l u b i l i t y versus temperature showed a sigmoid-1ike curve with a minimum at 120°C decreasing sharply after 145°C. Compared to the 110°C wet- and dry-heated samples, the control had an intermediate degree of s o l u b i l i t y . This information implies that the i n i t i a l steps of denaturation i n the

7.

NEUCERE AND CHERRY

Heat Denaturation

of

Proteins

140

150

137

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40.0H

35.0

g 30.0h

— 25.0 Ο Ζ Ο Ο 20.0h Ζ UJ

Ο

15.0

01 CL 10.0

5.0

UN

IÏÔ

120

130

160

TEMP. °C Figure 1. Relative protein solubilities of intact peanuts, after moist (O) and dry (Φ) heat. Triangles indicate native seed or control. (Reproduced, from Ref. 9. Copyright 1974, American Chemical Society.)

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imbibed seed involve a gradual change of the water-protein system within the seed between 110 and 120°C. Beyond this point, a more complex water-protein system appears to develop possibly involving protein fragments and other macromolecules. At low moisture con­ tent, a continuous change in the water-protein system seems to occur. Cherry et - 9 5 0 h u ζ tu

ζ ο u

A\% 25 HOLD-

JL

A0%

Lm

, - ι — — r5 5r

55 HEATING PERIOD

92.5 92.5 •HOLD-

25

-COOLING PERIOD

TEMPERATURE C O Figure 14.

Viscograph consistency patterns of 10, 11, and 12% suspensions of SP-isolate at pH 8.5.

Figure 15.

Texturized cottonseed SP.

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184

FOOD PROTEIN DETERIORATION

Addition of 1% or more of NSP-isolate to a 12% SP-isolate suspension at pH 5·5 hindered the texturing process (Figure 16). Non-storage proteins or any number of non-protein constituents in these products could be interferring with the texturization mechanism of the SP-isolate. Figure 17 shows a comparison of the consistency changes during slow heat-stir treatment of 18% suspensions of SP-isolate without and with added 1% NSP-isolate to those occurring i n suspensions of the HgO-HzO-concentrate and the a i r - c l a s s i f i e d fine concentrate at pH 5.5. The H2Q-H2Û-concentrate was r e l a t i v e l y free of NSP. It contained mainly protein bodies with SP and any carbohydrate moitiés not extracted with water. The a i r - c l a s s i f i e d fraction contained maninly NSP, protein bodies with their SP contents and carbohydrates, and yielded a dual-phase system of a l i q u i d and beads of agglomerated protein bodies upon treatment. However, the H2Û-H2Û-concentrate increased in consistency during the cooling period. The final pudding-like consistency may have been due to structural changes in SP content and especially i n the fibrous and pectinaceous components under conditions of the slow heat-stir treatment. On the other hand, the changes in the l a t t e r ' s consistency may be the result of the slow heating rate that permitted some protei η denaturation before heat-setting of protei ns into the fibrous networks of the final product. Consistency Changes During Rapid Heat-Stir Processing Twenty percent SP-isolate and 16% CI suspensions at various pHs of 3.0 to 10.0 were subjected to a rapid heat-stir (20 min to raise the temperature of a s t i r r i n g protein suspension from 25°C to 95-98°C) to determine t h e i r t e x t u r a b i l i t y by a method other than that used with the C. W. Brabender Visco-Amylo-Graph (see Experimental Procedures). Table II compares the results of these experiments. Texturization of SP-isolate by the viscograph method only occurred in suspensions at pH's 5.0, 5.5 and 8.5. The rapid heat-stir method produced texturized products in a l l suspensions at pH's 5.0 to 9.0, and gelled products at pH's 3.0, 4.0 and 10.0. Gels made at pH's 3 and 10 were dark colored and translucent. At pH 4.0, they were light cream-colored and opaque. The gels were self-sustaining, i . e . , they maintained t h e i r shape at room temperatures. No gelled products were produced with the slow heat-stir process. The rapid heat-stir method converted pH 3.0, 4.0 and 10.0 suspensions of CI to gelled products. Like the suspensions of the slow heat-stir process, those at the other pH values became only s l i g h t l y thickened pourable suspensions. Addition of 0.3% NaCl to the CI suspensions caused those at pHs between 4.0 and 9.0 to texturize during the rapid heat-stir process. Possibly, the salt reduced the s o l u b i l i t y of NSP or another component(s) and thus reduced interference with the texturization properties of SP. Figure 18 shows the textural

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(15, Pub-

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s o l i d state peptide synthesis (67-70), the amino groups (nucleophiles) of a candidate protein are reacted with an activated carboxyl of the amino acid to be attached. The resultant bond is either a peptide (for alpha-ami no nucleophile) or an isopeptide (epsilon-amino nucleophile). The N-hydroxy-succi namide esters of tert-butoxycarbonyl-Lamino acids (boc), both hydrophobic (glycine, alanine, methionine, N-acetyl-methionine) and hydrophilie (asparagine, asparatic acid), have been found to be quite effective in covalent attachment to casein (54). The chemistry employed in this case yields peptidylproteins only with non poly-peptidy1-derivatives. The boc-groups were removed by anhydrous trifluoroacetic acid. The urethane type boc-group offers complete protection against racemization. Since casein lacks an ordered tertiary structure or conformational complexity, changes in physical properties are neither expected nor observed. Although the s o l u b i l i t y and viscosity properties of casein did not change appreciably, in vitro enzyme digestion of modified casein decreased as the size of the attached amino acid increased. Methionine and tryptophan can be covalently linked to soy protein by carbodiimide condensation reaction up to as high as 5.9% and 10.7%, respectively. The reaction involves activation of protein carboxyls by 1-ethyl-3-(3-dimethyl-amino propyl) carbodiimide (EDC) and condensation thereof with the amino acid of choice. Factors such as protein concentration, EDC concentrat i o n , activation time, and reaction time were found to determine the degree of covalent binding. This offers a way to control the f o r t i f i c a t i o n of proteins of low nutritive value by covalent attachment of essential ami no acids. Although there may be potential health hazards, the excess carbodiimide and the corresponding urea can be easily removed by working with d i l u t e acids or water. Non-Covalent Interactions Chemical modification of food proteins can also be accomplished by the manipulation of their non-covalent interactions with themselves, carbohydrates, and l i p i d s in formulated foods (72, 73). For instance, 1ipid-protein interactions (74-76) have been described to originate from electrostatic attraction, salt bridging (divalent metals, i . e . , calcium), and cooperative effects of salt-bridging and electrostatic attractions. In addition to electrostatic forces and salt bridges, other proteinprotein interactions such as hydrogen bonding, disulfide bonds, and van der Waal s interactions can play a significant role in s t a b i l i z i n g metal complexes. On the other hand, the glycoproteins are covalently bonded protein and carbohydrate complexes

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FOOD PROTEIN DETERIORATION

(22, 77, 78). The Amadori-rearrangement mechanism of the food products is a good example of the involvement of covalent bonds (77) between proteins and carbohydrates. If the protein, carbohydrate, and l i p i d constituents in a food system were structurally defined and their environment (pH, solutes, solvents) were accurately known, an assessment of the contribution of various interaction forces could be made (79-81). It is possible to safely predict the functional property of the protein under investigation once the type and the magnitude of such interactions are known. The determination of the secondary structures of a protein molecule from the knowledge of amino acid sequence (79) along with the methods of indirect evaluation of the conformation (80), surface area, and hydrophobicity relationships (81) are the techniques u t i l i z e d for the prediction of the physical properties. It must be emphasized that a l l physical properties of matter are a reflection of molecular structure, e.g., a protein of known amino acid sequence would give net charge and therefore a defin­ able standard chemical potential and s o l u b i l i t y in an environment of known temperature and ionic strength. This knowledge permits estimation of both s o l u b i l i t y and thermal s t a b i l i t y . Likewise, a good assessment of the surface active properties of a protein can be determined from the knowledge of i t s hydrophobicity, a b i l i t y to hydrogen-bond and i o n i c , e l e c t r o s t a t i c , and ion-dipole interac­ tions. Furthermore, while emulsifying capacity depends on the shape, charge, hydrophobicity, hydration of polar groups, and neutrality of dipoles, the emulsion s t a b i l i t y depends on the magnitude of these interactions (88). Once a food protein is defined, i t s non-covalent interactions can be manipulated both in kind and degree in formulated foods containing carbohydrates and lipids. It is an easier task, both economically and technically, to change the environment of food proteins to induce changes in t h e i r physical properties than to change the proteins for a given environment. The molecular basis for the functionality of pro­ teins resides in the structure, interactions, and their a b i l i t y to interact with other food ingredients. This is true for any protein, modified or unmodified. In accordance with this concept, the i n i t i a l studies on proteins in coscervate systems (86, 87) should be followed up. This author strongly believes that an iη-depth understanding of the non-covalent interactions of proteins in food systems can be of great help in formulating safe food products. Protein Salts As amphoteric molecules, proteins can bind both anions and cations. Binding with small ions in particular is very important in several foods. If the anions of the protei η molecule can selectively bind metal ions, i t attains a net negative charge, îtç nH increases, and i t s i s o e l e c t r i c point shifts to an even

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more acidic pH i f the binding occurs preferentially around the isoelectric point. The converse is true for cation binding. Some ions, e.g., t r i c h l o r a c e t i c acid, form soluble s a l t s , and some combine to form coordination complexes with proteins. The metal complexes and salts of proteins do exhibit different properties in formulated foods. A recent study compares calcium and sodium binding a b i l i t y of soy protein, gluten protein, leaf protein concentrate, and blood serum albumin (46). The combined a b i l i t y of various fractions of soy and leaf protein for calcium binding is approximately 55.0 and 50.0 mg/g, respectively, although t h e i r s o l u b i l i t i e s at pH 11.0 are 85% and 15%, respec­ t i v e l y . The gluten and blood proteins bind only 25.0 and 10.0 mg/g of calcium under similar conditions, but blood protein isolate is far more soluble at pH 11.0 (92%) than gluten protein (45%). Ion binding by proteins can be either a non-specific (pH and ligand concentration dependent) or a specific (defined stoichiometry) phenomenon which, in general, is a case of multi­ ple equilibrium wherein the small molecules bind to an aggregat­ ing or dissociating protein system in which the monomer co-exists with the polymer and either can bind to the small molecule (82). Other ligands such as chlorogenic acid (33), caffeic acid and isochlorogenic acid (83), flavor compounds (84) and tannins (85) are known to bind to proteins. The forces for binding may be ionic (36), covalent (53), and/or hydrophobic (84, 85). The precise knowledge of ligand-protein interactions in terms of s p e c i f i c i t y , e q u i l i b r i a , and reaction conditions can be employed to create more functional and stable food systems. For instance, removal of chlorogenic acid improves the biological value and protein efficiency ratio of leaf protei η concentrate (33). The binding and release of flavor components to proteins improves food acceptability (84), and removal of tannins and lignins from vegetable and cereal proteins renders them b i o l o g i c a l l y more available. Food protein systems should be designed wherein desired interactions are promoted in place of those that are undesired. Although production of protei η salts of different bulk and solution properties is well understood (82), knowledge of ligand-protein interactions due to hydrophobic forces is poor. It is l i k e l y , however, that ligand-protein interactions result from non-covalent interactions, including metal-binding, and constitute chemistry that is not understood. Nutritive Value and Toxicity of Chemical by Chemically Modified Food Proteins An obvious barrier against the use of chemically modified food proteins is biological in nature. The modified protein products must have acceptable flavor, improved nutritive value, and undetectable t o x i c i t y . Most food proteins that are modified may contain other functional groups either as an integral part of their structure or as impurities. The concomitant modification

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of such groups may increase the concentration of toxic substances. These concerns have not been sufficiently answered by prior research and, of necessity, require extensive evaluation and experimentation. The results from a few studies (11, 26, 34, 71) impact rather negatively on the protein modification technology. For example, determinations of protein efficiency ratio and toxicological t r i a l s (1) suggest that acetylated and succinylated casein and whey proteins cannot be regarded as suitable ingredi­ ents for acid foods despite demonstratable improvement in their s o l u b i l i t y and heat s t a b i l i t y around pH 4.0. Groninger and M i l l e r (26) investigated acetylated and succinylated rnyofibrillar fish protein with respect to 1) ami no acid composition, 2) pro­ tein efficiency r a t i o , 3) rates of enzyme digestion, and 4) the u t i l i z a t i o n of C-1abelled derivatives by rats, and reported a reduction in protein efficiency r a t i o , only partial u t i l i z a t i o n of protein-bound acetyl-lysine, and metabolic unavailability of both C - l a b e l l e d succinyl- and acetyl-lysine. They concluded that acetyl- and succinyl-derivatives are only p a r t i a l l y u t i l i z e d and that the level of u t i l i z a t i o n depends on the type of acylgroup. Yet another succinylated protein, casein, has been reported to resist tryptic (but not chymotryptic) hydrolysis (34). Even the chymotrypsin a c t i v i t y is inhibited by N-tosyllysine methyl ester. Neither carboxypeptidase A nor Β release N-succinyllysine from t-butoxy-carbonyl-N-succi nyllysi ne. Obviously the succinyllysyl bonds are not hydrolyzed by gastric and pancreatic proteases. 14

14

Conclusions A few potential edible proteins have been covalently derivatized by acylation and reductive alkylation. The modified proteins have been found to exhibit different hydration and sur­ factancy properties. However, without the detailed knowledge of the primary structure of the proteins under investigation, the control of derivatization reactions and the physical properties of the product are s t i l 1 a matter of considerable speculation. Although some evidence as to the change in net charge, conforma­ t i o n , molecular weight, polydispersity, and the propensity to associate and/or dissociate has been found, there has been very 1 i t t l e work done on the actual food formulations based on covalently modified food proteins. A number of modified food proteins including acylated-casein and myofibrillar fish protein have been found to be resistant to complete digestion by the pancreatic enzymes. The major process c r i t e r i a such as t o x i c i t y of reagents, cost of manufacturing, and purification have not been identified. Considerations, such as t o x i c i t y of the product and its in vivo digestion products, physiological and pharmacological compatibility, flavor and odor, and overal1 aesthetics and consumer acceptance, have not received

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due research inputs. The questions regarding regulatory clear­ ance and test method development, although raised a few years ago, have not been answered. Although the benefit of fundamental knowledge for preparing functional proteins are known, addition of ami no acids to a deficient protein, blocking free amino groups, and thereby preventing undesirable chemical reactions in processed foods such as formation of lysinoalanine and lanthionine, is questionable at this time on account of t o x i c i t y , cost, and demonstratable reduction in nutritive value. The nature and extent of non-covalent interactions of food protein can be quantified on the basis of primary structure, and solution, and surface behavior. This particular concept deserves careful study. The precise knowledge of both non-covalent and covalent interactions between major food components (starch, pro­ t e i n , and l i p i d s ) along with the knowledge of the chemistry of protein salts and protein surfactant complexes, can f a c i l i t a t e food formulation. This concept needs more rigorous definition at the molecular level in terms of interactions between major and mi nor food components of a formulated food system. The anticipa­ ted research should include study of food proteins in coscervate systems. The covalently modified food proteins may have special s i g n i ­ ficance i f they are to be identified as highly functional at low level s of incorporation. This w i l l overcome any objections to their food use on the grounds of lower nutritive value, t o x i c i t y , and cost. Acknowledgements The author sincerely thanks the R&D staff members of Krause M i l l i n g Company for their review and constructive c r i t i c i s m of t h i s a r t i c l e . A special thank you is given to the senior manage­ ment of Krause M i l l i n g Company for t h e i r permission to write this chapter. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

Barman, B.G.; Hansen, J.R.; Mossey, A.R. J . Agric. Food Chem., 1973, 25, 638. Belikow, W.M.; Besrukow, M.G.; Walnowa, A.I. Die Nahrung., 1975, 19, 65. Bigelo, C.C. Theoret. Biol., 1967, 16, 187. Brinegar, A.C.; Kinsella, J . E . J . Agric. Food Chem., 1980, 28, 818. Βjarson, J.; Carpenter, K.J. Br. J. Nutr., 1967, 23, 859. Brekke, C . J . ; Eisele, T.A. Food Technol., 1981, 35, 231. Chen, L . F . ; Richardson, T.; Amundson, C.H. J . Milk Food Technol., 1975, 38, 89. Child, E.A.; Parks, K.K. J . Food Sci., 1976, 41, 713.

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RECEIVED June 11, 1982.

12 Protein Structure and Functional Properties: Emulsification and Flavor Binding Effects JOHN E. KINSELLA

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

Cornell University, Institute of Food Science, Ithaca, NY 14853

Proteins are the p r i n c i p a l structural and functional compo­ nents of many food systems; e.g., meat, cheese, gelatin, egg white and many cereal products. In addition, proteins are being used increasingly to fabricate and facilitate the engineering of new foods such as protein beverages and extruded foods. These and other applications depend upon the physicochemical properties of protein ingredients, c o l l e c t i v e l y referred to as the functional properties. Proteins per se as dry powders, have very limited appeal to potential users or consumers. To facilitate their use in foods and their conversion to desirable ingredients they must possess appropriate functional properties following interactions with other food components; e.g., water, carbohydrates or l i p i d s , during processing. Functional properties of proteins are those physicochemical properties of proteins which affect their behavior i n food systems during preparation, processing, storage, and con­ sumption, and contribute to the quality and organoleptic a t t r i b u ­ tes of food systems (1). Generally, n u t r i t i o n a l properties are not included in this category. Several typical classes of func­ tional properties are shown i n Table I . There are numerous examples of functional properties i n t r a ­ d i t i o n a l foods; e . g . , v i s c o e l a s t i c i t y of wheat gluten, texture, succulence and color of myofibrillar proteins i n meats, curd for­ mation of caseins, and whippability of egg white proteins. Different food applications require different characteristics; e . g . , i n beverages, proteins should be soluble; i n comminuted meats, they should have emulsion s t a b i l i z i n g and g e l l i n g proper­ ties ; and i n whipped toppings they must s t a b i l i z e the foam. The type of functional properties required i n a protein or a protein mix varies with the particular food system i n question. Thus, i n meat systems, water binding, s o l u b i l i t y , emulsifying a c t i v i t y , adhesiveness, swelling, viscosity and gelation are typical proper­ ties of proteins that determine their usefulness and impact on the f i n a l quality. These c r i t e r i a are c r i t i c a l , and as the number of processed and fabricated foods increase, greater reliance w i l l be placed on the consistent performance of ingredients i n specific food formulations. 0097-6156/82/0206-0301 $07.50/0 © 1982 American Chemical Society

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Table I.

T y p i c a l f u n c t i o n a l p r o p e r t i e s performed by p r o t e i n s i n food systems.

Functional Property

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

Solubility Water a b s o r p t i o n and b i n d i n g Viscosity Gelation Cohesion-adhesion

Elasticity

Emulsification Fat

absorption

Flavor-binding Foaming

Mode o f A c t i o n Protein solvation Hydrogen-bonding o f w a t e r , Entrapment o f water T h i c k e n i n g , Water b i n d i n g Protein matrix formation and s e t t i n g P r o t e i n a c t s as adhesive material Hydrophobic bonding i n gluten, Disulfide links i n gels F o r m a t i o n and s t a b i l i z a t i o n o f f a t emulsions Binding of free f a t A d s o r p t i o n , entrapment, release Form s t a b l e f i l m s t o e n t r a p gas

Food S y s t e m Example Beverages Meats, Sausages, B r e a d s , Cakes Soups, G r a v i e s Meats, Curds, Cheese Meats, Sausages, Baked g o o d s , Pasta products Meats, Bakery

Sausages, Bologna, Soup, Cakes Meats, Sausages, Donuts Simulated meats, Bakery, e t c . Whipped t o p p i n g s , Chiffon desserts, Angel cakes

Though t h e i m p o r t a n c e o f f u n c t i o n a l p r o p e r t i e s have b e e n r e c o g n i z e d b y commodity s p e c i a l i s t s f o r many y e a r s , t h e w i d e s p r e a d awareness o f t h e i r i m p o r t a n c e t o food s c i e n c e and t e c h n o l o g y i s r e l a t i v e l y recent. T h i s h a s been a c c e l e r a t e d by t h e i n c r e a s e d e m p h a s i s o n f o o d p r o c e s s i n g , m a n u f a c t u r i n g , a n d f o r m u l a t i o n . The u n i v e r s a l attempts t o u t i l i z e l e s s expensive sources o f p r o t e i n s t o f a b r i c a t e new f o o d a n a l o g s , t o s i m u l a t e t r a d i t i o n a l f o o d s , t o e x t e n d t r a d i t i o n a l f o o d s , a n d t o d e v e l o p new f u n c t i o n a l i n g r e d i e n t s have a c c e n t u a t e d t h e need f o r i n f o r m a t i o n on f u n c t i o n a l properties of proteins. Factors A f f e c t i n g Functional Properties S e v e r a l f a c t o r s and p r o c e s s i n g s t e p s a f f e c t t h e f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s . Thus, t h e p r o t e i n source c a n a f f e c t functionality. I n t h e c a s e o f meat t h e age o f t h e a n i m a l , w h i c h i s r e l a t e d to the q u a n t i t y o f connective t i s s u e , can markedly a f f e c t t h e f u n c t i o n a l p r o p e r t i e s . The p r o g r e s s i v e m o d i f i c a t i o n of t h e c o l l a g e n molecules v i a c r o s s l i n k i n g i n c r e a s e s t h e toughness

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

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KINSELLA

Emulsification

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Flavor

Binding

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of meat. In the case of egg white there i s a p r o g r e s s i v e t h i n ning of the egg white with storage time. The g l u t e n i n content v a r i e s immensely with the d i f f e r e n t v a r i e t i e s of wheat, and t h i s a f f e c t s the f u n c t i o n a l p r o p e r t i e s o f f l o u r s prepared from these cereals. Both i n t r i n s i c and a p p l i e d f a c t o r s i n f l u e n c e the observed f u n c t i o n a l p r o p e r t i e s of p r o t e i n s . The inherent molecular p r o p e r t i e s of the p r o t e i n per se ( s i z e , shape, conformation, whether n a t i v e o r denatured), the methods and c o n d i t i o n s o f i s o l a t i o n ( r e f i n i n g , d r y i n g , s t o r a g e ) , the degree of p u r i f i c a t i o n , (processing a l t e r a t i o n s ) and m o d i f i c a t i o n by p h y s i c a l (heat), chemical ( d e r i v a t i z a t i o n , h y d r o l y s i s ) or enzymatic processes, a l l i n f l u e n c e performance i n food systems. The methods employed i n determining a p a r t i c u l a r f u n c t i o n a l property, the composition of the assay system (model o r food), the p r o t e i n concentration, the pH, temperature, i o n c o n c e n t r a t i o n and composition, o x i d a t i o n / r e duction p o t e n t i a l , the type of equipment/machinery used (geometry, c o n f i g u r a t i o n ) , energy i n p u t , temperature c o n t r o l s o f the system and the method of measurement can a l l a f f e c t the observed prope r t y (1-6). Thus, m u l t i p l e i n t e r a c t i o n s are i n v o l v e d , and f o r the d e r i v a t i o n of u s e f u l data a l l experimental v a r i a b l e s should be defined and c o n t r o l l e d . F a i l u r e to do so renders the data redundant f o r most researchers and v i t i a t e s v a l i d comparisons of data from d i f f e r e n t l a b o r a t o r i e s . The l i t e r a t u r e i s r e p l e t e with papers on the f u n c t i o n a l prope r t i e s of p r o t e i n s prepared by d i f f e r e n t methods as measured by a v a r i e t y of methods under d i f f e r i n g c o n d i t i o n s ( 6 ) . The tendency has been f o r each i n v e s t i g a t o r to devise methods and/or c o n d i t i o n s to s u i t a p a r t i c u l a r s i t u a t i o n with l i m i t e d concern f o r the v a l i d i t y , comparative v a l u e , o r experimental design to f a c i l i t a t e e l u c i d a t i o n of the b a s i s of the p h y s i c a l property being s t u d i e d . Thus, much o f the data i n the l i t e r a t u r e are of very l i m i t e d value and d e f i n i t e l y cannot be used i n the systematic t a b u l a t i o n of p h y s i c a l p r o p e r t i e s i n a standardized format. This s i t u a t i o n r e f l e c t s and i s f u r t h e r complicated by the heterogeneous mixture of p r o t e i n s being s t u d i e d ; the very complex s e r i e s of i n t e r a c t i o n s which govern expression o f a p a r t i c u l a r f u n c t i o n a l property (and which are not amenable to easy measurement); the m u l t i p l e i n t e r a c t i n g f a c t o r s which impact on the aggregate e f f e c t and the l a c k of appropriate m e a s u r i n g devices which can a c c u r a t e l y quant i f y the r e s u l t a n t of these i n t e r a c t i o n s as a f u n c t i o n o f i n herent p r o p e r t i e s , i n t e r a c t i o n s per se and environmental f a c t o r s . Because of the complexity of food systems, the heterogeneity of the p r o t e i n s and the v a r i e t y o f f u n c t i o n s r e q u i r e d i n the d i f f e r e n t food systems f o r which they are used, i t has been d i f f i c u l t to standardize t e s t s f o r measuring f u n c t i o n a l p r o p e r t i e s (1,6). I t i s impossible to g e n e r a l i z e concerning the a v a i l a b l e information and d i f f i c u l t to e x t r a p o l a t e data from one system to p r e d i c t the behavior o f that p a r t i c u l a r p r o t e i n i n other systems. Measurements, i n order to have more general a p p l i c a b i l i t y ,

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must be based upon the fundamental p r o p e r t i e s of the system. In order to p r e d i c t the behavior of p r o t e i n s i t i s necessary to determine t h e i r b a s i c composition and physicochemical p r o p e r t i e s under s p e c i f i e d c o n d i t i o n s . However, to date, there are few methods that can be v a l i d l y or u n i v e r s a l l y used to p r e d i c t the behavior of p r o t e i n s i n food systems. In measuring the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s , i d e a l l y , e q u i l i b r i u m c o n d i t i o n s should be a t t a i n e d so that thermodynamic c r i t e r i a are met and so that the p a r t i c u l a r measurement r e f l e c t s the p h y s i c a l p r o p e r t i e s of the p r o t e i n i n question. For many f u n c t i o n a l p r o p e r t i e s a number of o p e r a t i o n a l d e f i n i t i o n s have been used to d e f i n e those part i c u l a r p r o p e r t i e s . Each has i t s advantages and disadvantages, and, of course because thermodynamic c r i t e r i a are r a r e l y a t t a i n e d , thermodynamic a n a l y s i s cannot e a s i l y be a p p l i e d (7). E v a l u a t i o n of a p a r t i c u l a r property or a p p l i c a t i o n i n food systems u s u a l l y i n c l u d e s t e s t s i n model systems; i . e . , systems that mimic the food system i n a l i m i t e d way and that are s e l e c t e d to h i g h l i g h t the performance of that p a r t i c u l a r f u n c t i o n . U l t i m a t e l y , of course, a l l f u n c t i o n a l a t t r i b u t e s should be evaluated i n food systems ( 6 ) . The method of e x t r a c t i o n and r e f i n i n g can have a marked e f f e c t on the f u n c t i o n a l p r o p e r t i e s o f i s o l a t e d p r o t e i n s . This depends upon the solvent used, the degree o f heating, the presence or absence of a l k a l i , pH, i o n s , mode of p r e c i p i t a t i o n and d r y i n g , and c o n d i t i o n s of storage (1-8). Temperature markedly a f f e c t s f u n c t i o n a l p r o p e r t i e s ; e.g., high temperatures which i n duce denaturation may impair f u n c t i o n a l p r o p e r t i e s . Time-temperature r e l a t i o n s h i p s used i n d r y i n g markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s ; e.g., spray versus drum d r y i n g a f f e c t s the whipping p r o p e r t i e s of egg white (9) . The exposure o f p r o t e i n s to d i f f e r ent ions during p r e p a r a t i o n can a f f e c t f u n c t i o n a l p r o p e r t i e s (10). Moisture l e v e l s and storage temperature can a f f e c t i n t e r a c t i o n s during storage and thereby impact on f u n c t i o n a l p r o p e r t i e s . The presence of r e a c t i v e components such as unsaturated f a t t y a c i d s and reducing sugars can r e s u l t i n chemical i n t e r a c t i o n s and thereby markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s . To f a c i l i t a t e the development of p r o t e i n s with p a r t i c u l a r f u n c t i o n a l p r o p e r t i e s i t i s f i r s t necessary to define which physicochemical p r o p e r t i e s are most important. To achieve t h i s i t i s very necessary to develop good techniques f o r measuring funct i o n a l p r o p e r t i e s and d e v i s i n g methods which e l u c i d a t e r e l a t i o n ships between s t r u c t u r e and f u n c t i o n . L i m i t e d advances have been made i n t h i s area though i n t e r e s t has i n c r e a s e d . Obviously, i n approaching t h i s challenge one should s e l e c t a w e l l c h a r a c t e r i z e d p r o t e i n and use i t i n a model system wherein f u n c t i o n a l p r o t e i n s are the major a c t i v e reagents ; e.g., protein-based foams, meat systems, cheese curds, e t c . When such r e l a t i o n s h i p s are understood i t may be f e a s i b l e to modify inexpensive p r o t e i n s and impart the necessary f u n c t i o n a l p r o p e r t i e s . For t h i s the s t r u c t u r a l features of the p r o t e i n , molecular p r o p e r t i e s , and

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i n t e r a c t i o n s r e q u i r e d f o r p a r t i c u l a r f u n c t i o n s need to be understood.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

P r o t e i n Structure and F u n c t i o n a l

Relationships

The s t r u c t u r e and chemical p r o p e r t i e s of food components determine t h e i r behavior and u l t i m a t e l y the a c c e p t a b i l i t y o f many foods. One challenge to the modern food s c i e n t i s t i s to define the chemical and/or p h y s i c a l a t t r i b u t e s that are r e s p o n s i b l e f o r these d e s i r a b l e p r o p e r t i e s and apply t h i s knowledge toward the development of f u n c t i o n a l i n g r e d i e n t s and the f a b r i c a t i o n of new foods. The amino a c i d composition, the sequence of amino a c i d s , the degree of m o d i f i c a t i o n of the nascent polypeptides during synthes i s , the manner i n which secondary and t e r t i a r y f o l d i n g occurs and p o s s i b l e a s s o c i a t i o n s between polypeptides a l l a f f e c t the f i n a l s t r u c t u r e and p h y s i c a l p r o p e r t i e s of d i f f e r e n t food prot e i n s . These s t r u c t u r e s i n turn vary i n t h e i r response to environmental f a c t o r s (temperature, pH, i o n i c s t r e n g t h ) , which a l s o a f f e c t f u n c t i o n a l p r o p e r t i e s (Table I I ) . Table I I .

S t r u c t u r e of polypeptides and i n t e r a c t i o n s that i n f l u e n c e f u n c t i o n a l p r o p e r t i e s of food p r o t e i n s .

Amino a c i d composition (major groups) Amino a c i d sequence (segments/polypeptides) Secondary/tertiary conformation (compact/coil) Surface charge, hydrophobicity, p o l a r i t y S i z e , shape (topography) Quaternary s t r u c t u r e s Secondary i n t e r a c t i o n s ( i n t r a - and i n t e r - p e p t i d e ) Hydrogen bonding, i o n i c , Van der Waals, hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n s . D i s u l f i d e / s u l f h y d r y l content Environmental c o n d i t i o n s (pH, 0/R s t a t u s , s a l t s , temperature) Amino A c i d s . The r e l a t i v e amounts o f d i f f e r e n t amino a c i d s and t h e i r d i s p o s i t i o n i n the polypeptide chain a f f e c t f u n c t i o n a l p r o p e r t i e s . Because hydrophobic e f f e c t s are important f o r c e s determining the p h y s i c a l behavior of p r o t e i n s the content and d i s p o s i t i o n of apolar amino a c i d s ( l e u c i n e , v a l i n e , i s o l e u c i n e , a l a nine, p r o l i n e , phenylalanine, tryptophan, t y r o s i n e and methionine) s i g n i f i c a n t l y a f f e c t the s t r u c t u r e and f u n c t i o n of p r o t e i n s (11-13). The shape of p r o t e i n molecules r e f l e c t the i n t e r n a l i z a t i o n of the maximum number o f apolar amino a c i d s , and the subun i t s of many o l i g o m e r i c p r o t e i n s a s s o c i a t e v i a hydrophobic i n t e r a c t i o n s (14). The content of apolar amino a c i d s (which range from 25-35% f o r most p r o t e i n s ) a f f e c t conformation, h y d r a t i o n , s o l u b i l i t y , denaturation and g e l a t i o n p r o p e r t i e s . Shimada and Matushita (15) showed that p r o t e i n s ; e.g., soybean, bovine serum

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FOOD

PROTEIN

DETERIORATION

a l b u m i n , c o n a l b u m i n , c o n t a i n i n g f r o m 2 6 - 3 1 % a p o l a r amino a c i d s formed g e l s upon h e a t i n g , w h e r e a s p r o t e i n s w i t h >31%, c o a g u l a t e d . K a t o and N a k a i (16) showed a c l o s e r e l a t i o n s h i p b e t w e e n t h e h y ­ d r o p h o b i c i t y o f p r o t e i n s and t h e i r e m u l s i f y i n g p r o p e r t i e s . The p r e s e n c e o f c h a r g e d amino a c i d s e n h a n c e s e l e c t r o s t a t i c i n t e r a c t i o n s which are s i g n i f i c a n t i n s t a b i l i z i n g globular pro­ t e i n s and i n w a t e r b i n d i n g ; e . g . , a s p a r t i c and g l u t a m i c a c i d b i n d 6-7 m o l e c u l e s w a t e r p e r c h a r g e d r e s i d u e ( 1 7 ) . This i s important f o r many f u n c t i o n a l p r o p e r t i e s ( h y d r a t i o n , s o l u b i l i t y , g e l a t i o n , surfactancy). P o l a r amino g r o u p s a r e c h e m i c a l l y r e a c t i v e and e n ­ gage i n h y d r o g e n - b o n d i n g , w h i c h i n f l u e n c e s c o n f o r m a t i o n o f a h e l i x and 3-sheet s t r u c t u r e s . T h e s e may be m o d i f i e d ; e . g . , a c ­ y l a t e d , t o manipulate the p h y s i c a l p r o p e r t i e s o f food p r o t e i n s (18)· The h y d r o p h i l i c amino a c i d s on t h e s u r f a c e o f g l o b u l a r proteins impart molecular f l e x i b i l i t y (19). Hence, p r o t e i n s w i t h a l a r g e p r o p o r t i o n o f h y d r o p h i l i c r e s i d u e s may be good f u n c t i o n a l p r o t e i n s ; e.g., egg w h i t e p r o t e i n s . C y s t e i n e and c y s t i n e s i g n i f i c a n t l y a f f e c t s t r u c t u r e and f u n c ­ tion of proteins. S u l f h y d r y l g r o u p s may be o x i d i z e d t o f o r m d i ­ s u l f i d e bonds ( i n t r a - and i n t e r m o l e c u l a r ) , t h i o l s a n d d i s u l f i d e s can u n d e r g o i n t e r c h a n g e r e a c t i o n s w h i c h m a r k e d l y a f f e c t s t r u c t u r e and f u n c t i o n ; e . g . , g l u t e n f o r m a t i o n ( 2 0 ) . Reduction of d i s u l ­ f i d e bonds can s i g n i f i c a n t l y a f f e c t m o l e c u l a r p r o p e r t i e s such as t h e u n f o l d i n g o f g l u t e n i n artel enhance t h e d i g e s t i b i l i t y o f s o y 11S. D i s u l f i d e c r o s s l i n k s are important i n s t a b i l i z i n g the t e r t i a r y structure of proteins. The f o r m a t i o n o f t h e B - l a c t o globulin/κ-casein d i s u l f i d e l i n k e d c o m p l e x e s and i m p r o v e s b a k i n g p r o p e r t i e s o f m i l k powders ( 2 1 ) . G e n e r a l l y , e x c e p t i n c a s e s where a p a r t i c u l a r t y p e o f amino a c i d p r e d o m i n a t e s , k n o w l e d g e o f amino a c i d c o m p o s i t i o n i s o f l i m i t e d value i n p r e d i c t i n g t e r t i a r y s t r u c t u r e or p h y s i c a l proper­ t i e s o f p r o t e i n s ; e . g . , α-lactalbumin and l y s o z y m e h a v e v e r y s i m i ­ l a r amino a c i d c o m p o s i t i o n b u t d i f f e r m a r k e d l y i n p r o p e r t i e s ; t h e mere r e p l a c e m e n t o f g l u t a m i n e w i t h v a l i n e c h a n g e s t h e c o n f o r m a ­ t i o n and p r o p e r t i e s o f h e m o g l o b i n , and c a s e i n v a r i a n t s w i t h m i n o r d i f f e r e n c e s i n amino a c i d s show s m a l l d i f f e r e n c e s i n h e a t s t a ­ bility. Protein Structure. The p a r t i c u l a r s e q u e n c e o f amino a c i d s i n a p r o t e i n d e t e r m i n e s i t s s t r u c t u r e , c o n f o r m a t i o n and p r o p e r ­ ties. Knowledge o f p r o t e i n s t r u c t u r e and t h e s t a b i l i t y o f d i f f e r ­ ent c o n f o r m a t i o n s i s p e r t i n e n t t o u n d e r s t a n d i n g f u n c t i o n a l prop­ e r t i e s o f f o o d p r o t e i n s ( T a b l e I I ) . The s t r u c t u r e o f p r o t e i n i s c a t e g o r i z e d as s e c o n d a r y , t e r t i a r y , and q u a t e r n a r y , a c c o r d i n g t o the s p a t i a l arrangement o f t h e p o l y p e p t i d e c h a i n s . I n a n aqueous environment primary polypeptides tend t o c o i l i n a c h a r a c t e r i s t i c f a s h i o n t o form l o c a l i z e d secondary s t r u c t u r e s ; i . e . , α - h e l i x o r B - p l e a t e d s h e e t . The d r i v i n g f o r c e f o r t h i s f o l d i n g i s h y d r o ­ p h o b i c i n o r i g i n due t o t h e f a v o r a b l e change i n f r e e e n e r g y o f s o l v e n t upon f o l d i n g o f t h e p o l y p e p t i d e ( 1 1 - 1 3 ) . The s e c o n d a r y s t r u c t u r e s a r e s t a b i l i z e d by hydrogen b o n d i n g .

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The t e r t i a r y s t r u c t u r e r e f e r s to the p r e f e r r e d three-dimen­ s i o n a l arrangement of the f o l d e d polypeptide chains. In a t y p i c a l t e r t i a r y s t r u c t u r e the polypeptides are t i g h t l y f o l d e d to give a compact molecule i n which most of the p o l a r groups of the amino a c i d s are l o c a t e d on the outer s u r f a c e and are hydrated. Most o f the apolar groups are i n t e r n a l i n the hydrophobic region from which water i s e s s e n t i a l l y excluded (a few Η-bonded water mole­ cules may remain). The p r o l i n e residues are l o c a t e d at the bends or folds i n the polypeptide chain where i s o l e u c i n e , s e r i n e and charged amino a c i d residues (α-helix d i s r u p t i n g molecules) a l s o tend to be l o c a t e d . These f o l d regions l a c k h e l i c a l s t r u c ­ ture tending to be random (11, 12). The gross conformation; i . e . , t e r t i a r y s t r u c t u r e of most g l o b u l a r p r o t e i n s tend to be s i m i l a r i n general o r g a n i z a t i o n . The degree of f o l d i n g and r e l a t i v e pro­ p o r t i o n of α-helix, 3-sheet o r random c o i l v a r i e s immensely among p r o t e i n s . Some p r o t e i n s are l o o s e l y f o l d e d and the t e r t i a r y s t r u c t u r e i s s t a b i l i z e d by d i s u l f i d e bonds, others are compactly f o l d e d with many S-S bonds (lysozyme, g l y c i n i n ) . Cytochrome C has no α-helix and many residues are i n the extended 3-conformat i o n . The polypeptides i n f i b r o u s p r o t e i n s ( c o l l a g e n , myosin, f i b r i n o g e n ) are mostly α - h e l i x , myoglobin has mostly 70% α-helix; however other g l o b u l a r p r o t e i n s serum albumin, lysozyme, r i b o n u c l e a s e , are made up o f f o l d e d polypeptides with extensive regions of unordered s t r u c t u r e and p r o t e i n s l i k e c a s e i n s , e l a s t i n which possess very l i t t l e order i n manner of polypeptide f o l d i n g . The compactness and extent of i n t e r p e p t i d e i n t e r a c t i o n s or bonding markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s (22). Both e n t r o p i e and e n t h a l p i c f a c t o r s c o n t r i b u t e to the f o l d ­ ing of p r o t e i n s , but i t i s g e n e r a l l y f e l t that the dominant d r i v i n g f o r c e i s the negative entropy e f f e c t caused by the ex­ posure of polypeptides to the aqueous solvent phase (10, 11). This r e s u l t s i n the unfavorable s t r u c t u r i n g of the water i n the immediate v i c i n i t y of the hydrophobic peptide m o i e t i e s . Conse­ quently, f o l d i n g of hydrophobic regions out of the water i n t o the p r o t e i n i n t e r i o r i n c r e a s e s the entropy of the p r e v i o u s l y s t r u c ­ tured water. Though the enthalpy of t h i s process i s u s u a l l y s l i g h t l y p o s i t i v e and t h e r e f o r e unfavorable, the l a r g e p o s i t i v e entropy g r e a t l y favors f o l d i n g . Hydrophobic i n t e r a c t i o n s are n o n s p e c i f i c because they r e s u l t from solvent p r o p e r t i e s r a t h e r than a t t r a c t i v e bonding between s p e c i f i c groups. Once apolar groups are forced w i t h i n a c r i t i c a l d i s t a n c e , Van der Waals forces, e l e c t r o s t a t i c i n t e r a c t i o n s , and other i n t e r a c t i o n s may c o n t r i b u t e to s t a b i l i z a t i o n of s t r u c t u r e (11, 13, 23). Peturbat i o n of e n t r o p i e r e l a t i o n s h i p s can a l t e r f u n c t i o n a l p r o p e r t i e s of p r o t e i n s (10). Many polypeptides with molecular weights exceeding 50,000 daltons tend to a s s o c i a t e and form quaternary s t r u c t u r e s . From the amino a c i d composition of known p r o t e i n s , i t has been ob­ served that p r o t e i n s c o n t a i n i n g more than 28 mole percent o f

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p a r t i c u l a r amino a c i d s ( v a l i n e , p r o l i n e , l e u c i n e , i s o l e u c i n e and p h e n y l a l a n i n e ) t e n d t o s e l f - a s s o c i a t e (14). T h u s , t h e α a n d β c h a i n s o f hemoglobin a s s o c i a t e , whereas m y o g l o b i n , w h i c h has l e s s t h a n 28% a p o l a r amino a c i d s , does n o t s e l f - a s s o c i a t e . An e x p l a n ­ a t i o n f o r t h i s phenomenon i s t h a t b e l o w a c r i t i c a l f r a c t i o n o f h y d r o p h o b i c r e s i d u e s i t i s p o s s i b l e t o b u r y a l l o f t h e s e com­ p l e t e l y w i t h i n t h e m o l e c u l e . When t h i s f r a c t i o n i s e x c e e d e d t h e f o l d i n g o f m o l e c u l e c a n n o t accomodate t h e s e i n t e r n a l l y and some a r e e x p o s e d on t h e s u r f a c e . When enough s u c h r e s i d u e s a p p e a r on the s u r f a c e h y d r o p h o b i c p a t c h e s w i l l be c r e a t e d , a n d t h e s e c a n o n l y be p r o t e c t e d o r removed f r o m t h e p o l a r medium by s e l f - a s s o ­ ciation. I n many i n s t a n c e s f o r c e s o t h e r t h a n h y d r o p h o b i c i n t e r ­ actions are involved i n maintaining multi-subunit structures. S e v e r a l m a j o r f o o d p r o t e i n s show q u a t e r n a r y s t r u c t u r e . C a s e i n s a s s o c i a t e s t r o n g l y v i a h y d r o p h o b i c f o r c e s ; t h e a c i d i c and b a s i c s u b u n i t s o f s o y U S and t h e component p o l y p e p t i d e s o f g l u t e n i n a r e d i s u l f i d e l i n k e d , and t h e s u b u n i t s o f m y o s i n a s s o c i a t e v i a h y d r o p h o b i c and e l e c t r o s t a t i c i n t e r a c t i o n s . These s t r u c t u r e s markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s o f these p r o t e i n s . The f o o d s c i e n t i s t i s f a c e d w i t h t h e s i t u a t i o n w h e r e i n t h e p r o t e i n s w i t h w h i c h he i s w o r k i n g p o s s e s s t h e t e r t i a r y a n d q u a t e r ­ n a r y s t r u c t u r e s and c o n f o r m a t i o n t h a t p r e s u m a b l y have e v o l v e d t o p e r f o r m s p e c i f i c b i o l o g i c a l f u n c t i o n s and were n o t n e c e s s a r i l y d e s i g n e d f o r p a r t i c u l a r f o o d a p p l i c a t i o n s . I n most i n s t a n c e s t h e p h y s i c a l p r o p e r t i e s o f p a r t i c u l a r food p r o t e i n s have d i c t a t e d t h e manner i n w h i c h t h e y a r e u s e d o r consumed. T h u s , t h e p e c u l i a r nature o f m i l k p r o t e i n s w h i c h under a p p r o p r i a t e c o n d i t i o n s form curds l e d t o the development o f cheese. I n the case o f soy pro­ t e i n s , t h e i r a b i l i t y t o c o a g u l a t e i n t h e p r e s e n c e o f c a l c i u m and h e a t i n g t o form t o f u f a c i l i t a t e d t h e i r p r e s e r v a t i o n , improved t h e i r d i g e s t i b i l i t y and q u a l i t y a t t r i b u t e s . The p a r t i c u l a r com­ p o s i t i o n , s t r u c t u r e and c o n f o r m a t i o n o f e g g w h i t e p r o t e i n made i t the p r e m i e r w h i p p i n g p r o t e i n and l e d t o t h e e v o l u t i o n o f foamb a s e d f o o d s and b a k e r y p r o d u c t s . The f i b r o u s s t r u c t u r e o f t h e a c t i n and m y o s i n i n meats i s r e s p o n s i b l e f o r t h e t e x t u r e o f m u s c l e f o o d s , and t h e p r o d u c t i o n o f l e a v e n e d b r e a d s r e f l e c t t h e u n i q u e v i s c o e l a s t i c p r o p e r t i e s o f wheat g l u t e n . The consumer h a s been c o n d i t i o n e d t o t h e s e f o o d s , h e n c e much e f f o r t w i l l be r e ­ q u i r e d i n t r y i n g to s i m u l a t e these p r o p e r t i e s u s i n g other pro­ t e i n i n g r e d i e n t s . I n order t o m a n i p u l a t e these p r o t e i n s and/or s i m u l a t e them w i t h o t h e r f u n c t i o n a l p r o t e i n s , i t i s i m p o r t a n t t o u n d e r s t a n d t h e i r s t r u c t u r e and p h y s i c a l p r o p e r t i e s and t o e l u c i ­ d a t e t h e r e l a t i o n s h i p b e t w e e n s t r u c t u r e and s p e c i f i c f u n c t i o n a l properties. Non-Covalent Forces S t a b i l i z i n g P r o t e i n S t r u c t u r e . Because f u n c t i o n a l p r o p e r t i e s a r e r e l a t e d t o s t r u c t u r e and s i n c e changes i n s t r u c t u r e o f food p r o t e i n s f r e q u e n t l y a r e important i n a spe­ c i f i c f u n c t i o n ( s u r f a c e d e n a t u r a t i o n o f egg w h i t e d u r i n g w h i p p i n g o r t h e u n r a v e l i n g o f g l u t e n p r o t e i n s d u r i n g dough d e v e l o p m e n t ) ,

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i t i s important to consider the forces r e s p o n s i b l e f o r the n a t i v e s t r u c t u r e of p r o t e i n s and f a c t o r s which e f f e c t these i n r e l a t i o n to t h e i r f u n c t i o n i n the n a t i v e o r denatured s t a t e . The forces i n v o l v e d i n s t a b i l i z i n g the t e r t i a r y s t r u c t u r e of p r o t e i n s may i n c l u d e hydrogen bonding, Van der Walls f o r c e s , d i pole and e l e c t r o s t a t i c i n t e r a c t i o n s , and hydrophobic a s s o c i a t i o n s . Covalent d i s u l f i d e bonds are important i n s e v e r a l p r o t e i n s . The nature and importance o f these i n t e r a c t i o n s are described i n Chapter 13_ of t h i s volume (10). The s t a b i l i t y of the quaternary s t r u c t u r e o f p r o t e i n s i s governed by the same general c l a s s e s o f s t a b i l i z i n g i n t e r a c t i o n s , p a r t i c u l a r l y hydrophobic i n t e r a c t i o n s , i o n c r o s s b r i d g i n g (Ca-casein) and d i s u l f i d e c r o s s l i n k i n g ; e.g., a c i d i c and b a s i c subunits of 11S p r o t e i n s and g l u t e n i n subunits. Manipulation of these f o r c e s to a l t e r the p h y s i c a l p r o p e r t i e s o f these p r o t e i n s provides an approach f o r improving f u n c t i o n a l p r o p e r t i e s of some food p r o t e i n s . Surface P r o p e r t i e s / E m u l s i f y i n g A c t i v i t y . Surface a c t i v i t y c l o s e l y r e f l e c t s the s t r u c t u r a l features of p r o t e i n s and these a f f e c t the usefulness of p r o t e i n s i n emulsions. In protein-based emulsions the p r o t e i n f u n c t i o n s by forming an i n t e r f a c i a l f i l m . The p h y s i c a l p r o p e r t i e s of t h i s f i l m matrix and i t s surface c h a r a c t e r i s t i c s determine i t s c a p a c i t y to form and s t a b i l i z e emul­ sions . Several researchers have studied the f i l m forming proper­ t i e s of p r o t e i n s , the p r o p e r t i e s of p r o t e i n f i l m s and t r i e d to c o r r e l a t e these with e m u l s i f y i n g p r o p e r t i e s (22, 24-28)· During the formation of an emulsion under i d e a l c o n d i t i o n s , the s o l u b l e p r o t e i n d i f f u s e s to and concentrates a t the o i l - w a t e r i n t e r f a c e once the i n t e r f a c i a l e l e c t r o s t a t i c b a r r i e r i s overcome. The pro­ t e i n , depending upon i t s s t r u c t u r a l s t a b i l i t y (compactness, f l e x ­ i b i l i t y , charge, d i s u l f i d e bonds, hydrophobicity) tends to unfold to e s t a b l i s h a new thermodynamic e q u i l i b r i u m . The hydrophobic segments or loops o r i e n t i n the apolar o i l phase while the p o l a r and charged segments tend to occupy the aqueous phase. The forma­ t i o n of an i n t e r f a c i a l f i l m occurs i n s e q u e n t i a l stages, a l l o f which are i n f l u e n c e d by the molecular p r o p e r t i e s of the p r o t e i n . The i n i t i a l d i f f u s i o n of n a t i v e molecules to the i n t e r f a c e i s concentration dependent and v a r i e s with the p r o t e i n (24). The newly a r r i v e d molecule must penetrate the i n t e r f a c e and u n f o l d . The r a t e and extent of these events i s p r o t e i n dependent and i s a f f e c t e d by the surface pressure and c o m p r e s s i b i l i t y of the p r o t e i n f i l m already formed (25). F i n a l l y , rearrangement o f ad­ sorbed surface denatured p r o t e i n molecules occurs to a t t a i n the lowest free energy s t a t e . S o l u b i l i t y of p r o t e i n i s an important p r e r e q u i s i t e f o r f i l m formation because r a p i d migration to and adsorption a t the i n t e r ­ face i s c r i t i c a l . This i s p a r t i c u l a r l y r e l e v a n t i n f l u i d emul­ s i o n s . D i f f e r e n t p r o t e i n s r e q u i r e d i f f e r e n t times to e q u i l i b r a t e and form a f i l m . These events are r a p i d f o r l o o s e , f l e x i b l e pro­ t e i n s l i k e β-casein, intermediate f o r g l o b u l i n s (BSA) and slow

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f o r p r o t e i n s w i t h a r i g i d t e r t i a r y s t r u c t u r e l i k e lysozyme o r soy I I S (24) · Graham and P h i l l i p s (24) r e l a t e d t h e p h y s i c a l p r o p e r t i e s o f p r o t e i n f i l m s made w i t h d i f f e r e n t p r o t e i n s t o e m u l s i o n f o r m a t i o n and s t a b i l i t y . They o b s e r v e d t h a t d i f f e r e n t p r o t e i n s behave d i f f e r e n t l y a t i n t e r f a c e s ; e.g., a t low c o n c e n t r a t i o n s , 3 - c a s e i n s p r e a d s o u t a t t h e i n t e r f a c e . As t h e s u r f a c e c o n c e n t r a t i o n p r o g r e s s i v e l y i n c r e a s e s l o o p i n g of the a p o l a r r e g i o n s i n t o the o i l phase o c c u r s . Subsequent compression of these loops occurs to y i e l d a condensed f i l m . Further increases i n protein c o n c e n t r a t i o n does n o t i n c r e a s e f i l m p r e s s u r e o r r e d u c e s u r f a c e t e n s i o n t h o u g h t h e t h i c k n e s s i n c r e a s e s due t o a d s o r p t i o n o f m u l t i layers. Lysozyme m o l e c u l e s do n o t u n f o l d as r e a d i l y as 3 - c a s e i n and take l o n g e r to form a f i l m . The f i l m i s composed o f l a y e r s o f minimally unfolded molecules. B o v i n e serum a l b u m i n b e h a v e s i n an i n t e r m e d i a t e f a s h i o n ; i . e . , i t u n f o l d s t o a l i m i t e d e x t e n t presumably because the d i s u l f i d e bonds p r e v e n t complete l o s s o f i t s t e r t i a r y s t r u c t u r e . B e c a u s e i t p o s s e s s e s a s i g n i f i c a n t numb e r o f h y d r o p h o b i c d o m a i n s b o v i n e serum a l b u m i n i s q u i t e s u r f a c e active. I t r e a d i l y l o c a t e s a t an i n t e r f a c e and u n f o l d s t o p e r m i t p o l a r and a p o l a r segments t o p r o t r u d e i n t o t h e aqueous and l i p i d phases r e s p e c t i v e l y w h i l e the b u l k of the m o l e c u l e occupies the interface. A d j a c e n t m o l e c u l e s may a s s o c i a t e v i a h y d r o p h o b i c and e l e c t r o s t a t i c i n t e r a c t i o n s to form a cohesive film.. I n model s y s t e m s t h e i n t e r f a c i a l c o n c e n t r a t i o n o f p r o t e i n and t h e t h i c k n e s s o f t h e i n t e r f a c i a l f i l m a r e a f f e c t e d by t h e t y p e and c o n c e n t r a t i o n o f p r o t e i n i n s o l u t i o n (22, 2 4 ) . B o v i n e serum a l b u m i n i s more e f f e c t i v e i n s t a b i l i z i n g e m u l s i o n s t h a n 3 - c a s e i n o r l y s o z y m e . I t forms a s t r o n g e r f i l m a t lower p r o t e i n l e v e l s , r e f l e c t i n g a g r e a t e r number o f i n t e r m o l e c u l a r i n t e r a c t i o n s . In the case of food emulsions the r a t e of i n t e r f a c i a l f i l m f o r m a t i o n and t h e t y p e and p r o p e r t i e s o f t h e f i l m f o r m e d a r e g r e a t l y a f f e c t e d by t h e m e c h a n i c s o f e m u l s i o n f o r m a t i o n ; i . e . , s h e a r i n g , m i x i n g , d e n a t u r a t i o n , o x i d a t i o n , and t h i s , p e r h a p s , has more i m p a c t on e m u l s i o n f o r m a t i o n t h a n t h e p r o p e r t i e s o f t h e p r o t e i n p e r se (j4, 29). However, i t i s i m p o r t a n t t o r e c o g n i z e t h a t t h e p h y s i c a l and r h e o l o g i c a l p r o p e r t i e s o f t h e f i l m , o n c e f o r m e d , i s c r i t i c a l i n governing emulsion s t a b i l i t y . Globular proteins w i t h s u f f i c i e n t f l e x i b l e d o m a i n s can r a p i d l y f o r m f i l m s and d e press i n t e r f a c i a l t e n s i o n . Furthermore, they r e t a i n r e s i d u a l t e r t i a r y s t r u c t u r e which f a c i l i t a t e s extensive molecular i n t e r a c t i o n s ( e l e c t r o s t a t i c , h y d r o p h o b i c , d i s u l f i d e , Van d e r W a a l s forces). These c o n t r i b u t e t o t h e r h e o l o g i c a l p r o p e r t i e s ( s h e a r r e s i s t a n c e , v i s c o e l a s t i c i t y , d i l a t a t i o n a l modulus, i n c o m p r e s s i b i l i t y ) o f the f i l m w h i c h enhance f i l m s t a b i l i t y . However, t h e i r i n d i v i d u a l c o n t r i b u t i o n s to emulsion s t a b i l i t y remains u n c l e a r . G e n e r a l l y the s t a b i l i t y of p r o t e i n based emulsions are i n f l u e n c e d by t h e p r o p e r t i e s o f t h e i n t e r f a c i a l f i l m m a t e r i a l s and t h e v i s c o s i t y o f t h e c o n t i n u o u s p h a s e ( T a b l e I I I ) .

12.

KINSELLA

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Table I I I .

Emulsification

and

Flavor

Binding

311

Factors a f f e c t i n g formation and s t a b i l i t y of p r o t e i n based emulsions.

PROTEIN I n t r i n s i c p h y s i c a l p r o p e r t i e s of the p r o t e i n : s i z e , shape, compactness, surface charge, hydrophobicity, solubility, f l e x i b i l i t y , d i s u l f i d e / t h i o l groups PROPERTIES OF ADSORBED PROTEIN FILM Thickness; r h e o l o g i c a l p r o p e r t i e s ( v i s c o s i t y , cohesiveness, e l a s t i c i t y ) ; net charge and d i s t r i b u t i o n ; degree of hydrat i o n ( h y d r o p h i l i c , p o l a r , charged groups) ENVIRONMENTAL FACTORS pH, i o n s , temperature PROCESSING PARAMETERS Shear f o r c e s , temperature, o i l composition, d r o p l e t s i z e , viscosity CONTINUOUS PHASE Viscosity In a packed emulsion the p o l y h e d r a l o i l d r o p l e t s are separated by an aqueous l a m e l l a r l a y e r . The v i s c o s i t y of t h i s l a y e r determines the r a t e a t which adjacent globules can approach each other. E l e c t r o s t a t i c and s t e r i c f a c t o r s r e t a r d the approach and coalescence of f a t d r o p l e t s . The i n t e g r i t y of the p r o t e i n f i l m and i t s s t a b i l i t y to shock (thermal, a g i t a t i o n , aging) i s governed by i t s p h y s i c a l and r h e o l o g i c a l p r o p e r t i e s (thickness, v i s c o s i t y , f l e x i b i l i t y and cohesiveness). Thinning of the f i l m r e s u l t s i n weak segments that may upon shock, rupture and coal e s c e . Hence, the r e s t o r a t i v e nature of the p r o t e i n f i l m i t s e l a s t i c i t y , d i l a t a t i o n a l modulus, shear v i s c o s i t y determines i t s i n t r i n s i c s t a b i l i t y (24-30). Coalescence of o i l d r o p l e t s r e s u l t i n g from the breakdown of the aqueous l a m e l l a i s a major f a c t o r i n emulsion i n s t a b i l i t y . Graham and P h i l i p s (24) concluded that the d i s j o i n i n g pressure s t a b i l i z i n g the l a m e l l a i s mostly a f f e c t e d by e l e c t r o s t a t i c and s t e r i c f a c t o r s c o n t r i b u t e d by the p r o t e i n s i n the f i l m . Thus, while c a p i l l a r y s u c t i o n , Van der Waals f o r c e s , and g r a v i t y tend to cause t h i n n i n g o f the aqueous l a m e l l a these are counteracted by e l e c t r o s t a t i c and s t e r i c f a c t o r s c o n t r i b u t e d by the surface l a y e r s of the adjacent p r o t e i n f i l m s . Thus, the surface charge, the s i z e , nature of the degree of h y d r a t i o n o f exposed groups a l l impair the approach and coalescence o f adjacent f i l m s . For the formation of a s t a b l e emulsion the i n t e r f a c i a l p r o t e i n f i l m should be as t h i c k as p o s s i b l e with good cohesive and r h e o l o g i c a l p r o p e r t i e s , be w e l l hydrated, contain exposed p o l a r and charged groups, and possess a net negative charge (24, 26). Current Research;

Experimental Procedures

Much current research

i s concerned with the determination o f

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312

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DETERIORATION

the e m u l s i f y i n g p r o p e r t i e s o f food p r o t e i n s . A wide d i v e r s i t y o f m o d e l s y s t e m s and d i f f e r e n t c o n d i t i o n s have b e e n u s e d t o d e t e r ­ mine e m u l s i f i c a t i o n p r o p e r t i e s o f numerous p r o t e i n s , b u t u s e f u l c o m p a r i s o n s o f methods and r e s u l t s a r e d i f f i c u l t (1, 4_, 6^, 3 1 ) . E m u l s i f y i n g a c t i v i t y r e f l e c t s the a b i l i t y of the p r o t e i n to a i d e m u l s i o n f o r m a t i o n and t o s t a b i l i z e t h e n e w l y c r e a t e d e m u l s i o n . E m u l s i f y i n g a c t i v i t y c a n be e a s i l y m e a s u r e d by m a k i n g an e m u l s i o n and d e t e r m i n i n g t h e p a r t i c l e s i z e d i s t r i b u t i o n o f t h e d i s p e r s e d phase by m i c r o s c o p y o r s p e c t r o t u r b i d i t y . I n each procedure the a v e r a g e d i a m e t e r o f t h e d i s p e r s e d p h a s e i s d e t e r m i n e d , and f r o m t h e s e d a t a t h e i n t e r f a c i a l a r e a c a n be c a l c u l a t e d . The s p e c t r o ­ t u r b i d i t y method i s s i m p l e , r a p i d and t h e o r e t i c a l l y sound and p r o v i d e s i n f o r m a t i o n a b o u t t h e a v e r a g e d i a m e t e r and p a r t i c l e s i z e distribution. The method i s a p p l i c a b l e t o e m u l s i o n s w i t h a v e r a g e p a r t i c l e s i z e ; i . e . d i a m e t e r s b e t w e e n 0.2-8 μ ( 2 6 , 32, 33). The o p t i c a l density of d i l u t e d emulsions i s d i r e c t l y r e l a t e d to the i n t e r f a c i a l area; i . e . , the s u r f a c e area of a l l the d r o p l e t s , f o r c o a r s e emulsions (See T a b l e I ? ) ( 3 4 ) . S e v e r a l t y p e s o f b l e n d e r s and h o m o g e n i z e r s and many s i z e s and s h a p e s o f c o n t a i n e r s have b e e n u s e d i n e m u l s i o n p r e p a r a t i o n ( 4 , 2 9 ) . These i n s t r u m e n t s v a r y i n t h e i r a b i l i t y t o f o r m an emulsion; i . e . , the p a r t i c l e s i z e d i s t r i b u t i o n of the o i l drop­ l e t s v a r y and f r e q u e n t l y i n g r e d i e n t f a c t o r s a f f e c t i n g e m u l s i f y i n g a c t i v i t y a r e o v e r r i d d e n by t h e c h a r a c t e r i s t i c s o f t h e equipment used. The J a n k e - K u n k e l h o m o g e n i z e r h a s c o n s i s t e n t l y p r o v e d t o be the b e s t f o r p r o d u c i n g model e m u l s i o n s i n o u r l a b o r a t o r y (29). U s i n g model s y s t e m s and t h e p r o c e d u r e s d e s c r i b e d by P e a r c e and K i n s e l l a ( 3 4 ) and W a n i s k a , e t a l . ( 2 9 ) some o f t h e s t r u c t u r a l f a c t o r s and i n t e r a c t i o n s a f f e c t i n g e m u l s i o n f o r m a t i o n and s t a ­ b i l i t y are being studied. Electrostatic Effects. The i m p o r t a n c e o f e l e c t r o s t a t i c i n ­ t e r a c t i o n s on t h e f o r m a t i o n and s t a b i l i z a t i o n o f p r o t e i n - b a s e d e m u l s i o n s i s r e f l e c t e d by t h e e f f e c t s o f c h a r g e a l t e r a t i o n v i a p r o t e i n m o d i f i c a t i o n , pH v a r i a t i o n , and i o n c o n c e n t r a t i o n o n e m u l s i o n f o r m a t i o n and s t a b i l i t y ( 2 4 , 2 7 , 28, 29, 3 1 , 5 2 ) .

T a b l e IV.

E m u l s i f y i n g a c t i v i t y o f s u c c i n y l a t e d y e a s t proteins.. 2 -1 Emulsifying A c t i v i t y (M .g ) pH 6.5 pH 8

Modification Yeast Yeast Yeast Yeast

protein protein protein protein

(unmodified) (24% s u c c i n y l a t e d ) (62% succinylated) (88% s u c c i n y l a t e d )

From P e a r c e and K i n s e l l a

(34).

8 no 262 322

59

204 332 341

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Binding

S u c c i n y l a t i o n of r e l a t i v e l y i n s o l u b l e p r o t e i n s markedly improves the e m u l s i f y i n g c a p a c i t y of p l a n t p r o t e i n s (35). Yeast p r o t e i n i n which an i n c r e a s i n g number of l y s i n e residues were s u c c i n y l a t e d showed a progressive increase i n emulsion a c t i v i t y (Table IV). This i s a t t r i b u t e d to enhanced s o l u b i l i t y and expanded molecular s t r u c t u r e which enhanced emulsion formation. However, e l e c t r o s t a t i c r e p u l s i o n between the n e g a t i v e l y charged, e m u l s i f i e d d r o p l e t s enhanced s t a b i l i t y . Waniska, e_t a l . (29) studied the e f f e c t s of pH and s t r u c t u r a l m o d i f i c a t i o n on the e m u l s i f y i n g p r o p e r t i e s of p r o t e i n s i n c l u d i n g bovine serum albumin which possesses e x c e l l e n t e m u l s i f y ing a c t i v i t y (EA). The EÂ of bovine serum albumin (BSA) was two to t h r e e f o l d b e t t e r than other food p r o t e i n s (Table V) r e f l e c t i n g the favorable molecular c h a r a c t e r i s t i c s of BSA; i . e . , molecular s i z e , a b i l i t y to unfold to a l i m i t e d extent and allow polypept i d e s to r e o r i e n t at the i n t e r f a c e , surface hydrophobicity, d i s u l f i d e l i n k a g e s which maintained s u f f i c i e n t t e r t i a r y s t r u c t u r e and a balance of charged groups (24, 25)· Table V.

The r e l a t i o n s h i p between the pH and r e l a t i v e emsusifyi n g c a p a c i t i e s of v a r i o u s p r o t e i n s .

pH of solution

BSA

3 4 5 6 7 8 9 10

0.20 0.60 0.70 0.73 0.77 0.80 0.82 0.68

Absorbance (550 nm) of d i l u t e d emulsion soy Oval3S-Lg i s o l a t e bumin c a s e i n R-BSA S-BSA 0.15 0.53 0.56 0.51 0.48 0.47 0.42 0.30

0.01 0.03 0.60 1.20 1.40 1.23 1.24 1.20

0.18 0.25 0.34 0.31 0.32 0.33

-

-

0.01 0.10 0.30 0.30 0.35 _

-

0.26 0.25 0.21 0.21 0.22 0.26

-

0.02 0.01

-

0.07 0.08 0.08 0.10

soy lis 0.20 0.38 0.42 0.47 0.45 0.43 0.24 0.22

The e m u l s i f y i n g c a p a c i t y was determined by t u r b i d i m e t r y (29, 34); BSA - bovine serum albumin; R-BSA - a l l d i s u l f i d e s reduced; S-BSA - s u c c i n y l a t e d BSA; 3 - L g - B - l a c t o g l o b u l i n and U S = soy glycinin. Between pH 3 and 4 there was a s i g n i f i c a n t i n c r e a s e i n emuls i f y i n g a c t i v i t y of BSA. At pH 4 BSA undergoes an a c i d induced molecular expansion, presumably caused by e l e c t r o s t a t i c r e p u l s i o n , which r e s u l t s i n d i s r u p t i o n of some hydrophobic i n t e r a c t i o n s (36). This f a c i l i t a t e d emulsion formation. The EA of BSA p r o g r e s s i v e l y increased between pH 4 and 9 i n d i c a t i n g that as net charge i n creased the a b i l i t y to form a f i l m was enhanced. At pH 9.0 BSA undergoes an a l k a l i n e expansion (36) which may i n v o l v e some d i s u l f i d e cleavage and d i s r u p t i o n of hydrophobic a t t r a c t i o n s (37). The EA of BSA a b r u p t l y decreased a t ρH 9.0 r e f l e c t i n g the a l t e r e d t e r t i a r y s t r u c t u r e as d i s u l f i d e bonds were broken. These data

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FOOD P R O T E I N

DETERIORATION

r e f l e c t the importance o f a c e r t a i n degree o f s t r u c t u r a l s t a b i l i t y ( t e r t i a r y s t r u c t u r e ) f o r optimum e m u l s i o n f o r m a t i o n . Above pH 9, EA d e c r e a s e d ; t h i s may r e f l e c t t h e a l k a l i i n d u c e d h y d r o l y s i s o f some d i s u l f i d e bonds and p o s s i b l y some c r o s s l i n k i n g following ^-elimination reactions. The i m p o r t a n c e o f t e r t i a r y and s e c o n d a r y s t r u c t u r e i s r e ­ v e a l e d by t h e a l m o s t c o m p l e t e e l i m i n a t i o n o f EA f o l l o w i n g t r e a t ­ ment o f BSA w i t h u r e a ( 8 M ) . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e s u g g e s t i o n t h a t w h i l e d e n a t u r e d BSA may o c c u p y t h e i n t e r f a c e the l a c k o f o r d e r e d s t r u c t u r e p r e v e n t s t h e f o r m a t i o n o f a c o h e s i v e continuous f i l m . T h i s was c o r r o b o r a t e d by t h e EA o f BSA i n w h i c h a l l t h e 17 d i s u l f i d e bonds w e r e r e d u c e d b y d i t h i o t h r e i t o l (0.01M). T h e r e was a s i g n i f i c a n t r e d u c t i o n i n EA, e s p e c i a l l y above pH 5.0. T h u s , t h e t e r t i a r y c o n f o r m a t i o n s t a b i l i z e d by d i s u l f i d e bonds a r e a p p a r e n t l y i m p o r t a n t i n t h e f o r m a t i o n o f a s t a b l e c o h e s i v e mem­ brane around the o i l d r o p l e t s . The r e d u c e d BSA was more s e n s i t i v e t o pH i n d i c a t i n g g r e a t e r s e n s i t i v i t y t o e l e c t r o s t a t i c r e p u l s i o n s . S u c c i n y l a t i o n , which s i g n i f i c a n t l y increased the net negative c h a r g e and c a u s e s m o l e c u l a r e x p a n s i o n m a r k e d l y i m p r o v e d t h e EA o f BSA b e t w e e n pH 5 and 1 0 . The maximum was o b s e r v e d a t pH 6 and subsequently decreased, p o s s i b l y because the n e t n e g a t i v i t y be­ came e x c e s s i v e and i m p a i r e d f o r m a t i o n o f a c o h e s i v e f i l m . Pre­ sumably s u c c i n y l a t i o n f a c i l i t a t e d m o l e c u l a r u n f o l d i n g a t t h e i n ­ t e r f a c e , enhanced e l e c t r o s t a t i c r e p u l s i o n between e m u l s i f i e d d r o p l e t s and t h e c h a r g e d g r o u p s , b e i n g h i g h l y h y d r a t e d , may h a v e i n c r e a s e d the v i s c o s i t y o f t h e l a m e l l a r phase thereby r e t a r d i n g coalescence. M e t h y l a t i o n o f t h e ε-amino g r o u p s o f l y s i n e w h i c h u n l i k e s u c c i n y l a t i o n d i d n o t i n t r o d u c e a n e g a t i v e charge, b u t reduced t h e n e t p o s i t i v e c h a r g e b e l o w ρH 8.5, d i d n o t s i g n i f i c a n t l y a l t e r EA o f BSA. These o b s e r v a t i o n s i n d i c a t e t h a t i n d i v i d u a l e f f e c t s ; i . e . , n e t c h a r g e , a r e m o d i f i e d by m o l e c u l a r s t r u c t u r e and f l e x i ­ b i l i t y , and optimum f u n c t i o n a l i t y i s o b t a i n e d o n l y when t h e s e a r e a t an optimum b a l a n c e . I o n s a t r e l a t i v e l y l o w c o n c e n t r a t i o n s ( 0 . 1 - 0.3 M) i n f l u ­ ence e l e c t r o s t a t i c e f f e c t s i n e m u l s i o n s . Thus, t h e o i l / w a t e r i n t e r f a c e possesses a c o n c e n t r a t i o n o f charges which tend t o r e ­ p e l approaching charged p r o t e i n s . T h i s r e s u l t s i n a reduced r a t e o f p r o t e i n a d s o r p t i o n , and u n f o l d i n g , t h u s i n h i b i t i n g t h e f o r m a ­ t i o n o f a n i n t e r f a c i a l f i l m ( 2 5 ) . A t a p a r t i c u l a r pH, t h e n e t c h a r g e o n t h e p r o t e i n may r e s u l t i n e x c e s s i v e i n t e r m o l e c u l a r r e ­ p u l s i o n and i m p a i r t h e i n t e r m o l e c u l a r a s s o c i a t i o n s n e c e s s a r y f o r the f o r m a t i o n o f a c o h e s i v e f i l m . S a l t s a t low c o n c e n t r a t i o n s f u n c t i o n by c o u n t e r a c t i n g s u c h e l e c t r o s t a t i c e f f e c t s v i a c o u n t e r i o n b i n d i n g , t h e r e b y p e r m i t t i n g a d s o r p t i o n and f i l m f o r m a t i o n . F u r t h e r m o r e , t h i s weakens c o u l o m b i c i n t e r a c t i o n s , e s p e c i a l l y i n aqueous s o l u t i o n s w h i c h i n t u r n p e r m i t s more e x t e n s i v e h y d r o p h o ­ b i c i n t e r a c t i o n s between p r o t e i n s a t t h e i n t e r f a c e . Waniska e t a l . ( 2 9 ) o b s e r v e d t h a t b o t h c h l o r i d e and s u l f a t e a n i o n s a t c o n ­ c e n t r a t i o n s o f z e r o t o 0.1 M m a r k e d l y e n h a n c e d t h e e m u l s i f y i n g

12.

KINSELLA

Emulsification

and Flavor

315

Binding

a c t i v i t y of BSA. C h l o r i d e enhanced EA s l i g h t l y up to 0.6 M, but the ' s a l t i n g i n ' e f f e c t o f sodium c h l o r i d e reduced the r a t e o f ab­ s o r p t i o n at the i n t e r f a c e a t s a l t concentrations above 0.6 M. S u l f a t e , a water s t r u c t u r e promoting s a l t ( ' s a l t i n g o u t ) favored the t r a n s f e r of BSA to the i n t e r f a c e a t maximum r a t e s a t concen­ t r a t i o n s from 0.1 to 1M. Thus, ions i n a d d i t i o n to i n f l u e n c i n g coulombic i n t e r a c t i o n s , may a l s o a f f e c t conformational s t a b i l i t y of p r o t e i n s , e s p e c i a l l y at higher concentrations v i a the chaotrop­ i c e f f e c t s (10). P r o t e i n s with d i f f e r e n t molecular s i z e s and s t r u c t u r e s showed markedly d i f f e r e n t EA under i d e n t i c a l c o n d i t i o n s . Soy 11S p r o t e i n and a r a c h i n are o l i g o m e r i c p r o t e i n s , around 330,000 d a l tons, with over 40 d i s u l f i d e c r o s s l i n k s and very l i t t l e (~12%) h e l i c a l s t r u c t u r e . These are s t r u c t u r a l l y very s t a b l e p r o t e i n s yet U S had a much greater EA compared to a r a c h i n . This obser­ v a t i o n i s being f u r t h e r explored. 3-Casein i s a h i g h l y amphip a t h i c molecule that i s surface a c t i v e and r a p i d l y forms f i l m s :nd f a c i l i t a t e s e m u l s i f i c a t i o n , but because f i l m s o f β-casein are weak, emulsion s t a b i l i t y i s poor. Ovalbumin i s reasonably hydro­ phobic, but tends to denature e a s i l y and coagulate r e s u l t i n g i n weak i n t e r f a c i a l f i l m s .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

1

H y d r o p h i l i c E f f e c t s . The h y d r o p h i l i c character o f glycopro­ t e i n s may improve t h e i r surface a c t i v i t y . Conceivably the carbo­ hydrate moieties because o f t h e i r h y d r a t i o n provide s t e r i c s t a ­ b i l i z a t i o n i n emulsions. To study the e f f e c t of h y d r o p h i l i c groups we c o v a l e n t l y a c y l a t e d d i f f e r e n t carbohydrate groups to 3 - l a c t o g l o b u l i n which contains 16 f r e e amino groups and 25 c a r ­ boxyl groups a v a i l a b l e f o r m o d i f i c a t i o n . 3-Lactoglobulin has a low i n t r i n s i c v i s c o s i t y (0.034 dl/g) r e f l e c t i n g i t s g l o b u l a r na­ ture (38). I t has an average hydrophobicity of 1060 compared to 1000 and 980 f o r BSA and ovalbumin, r e s p e c t i v e l y (39). 3-Lacto­ g l o b u l i n i s reasonably surface a c t i v e ; i . e . surface tension = 60, i n t e r f a c i a l tension 11.0 and e m u l s i f y i n g a c t i v i t y index 151; bovine serum albumin has corresponding values of 58, 10.3 and 166, r e s p e c t i v e l y (16). A c t i v a t e d c y c l i c carbonate d e r i v a t i v e s of maltose and 3c y c l o d e x t r i n were l i n k e d to 3 - l a c t o g l o b u l i n v i a the f r e e amino groups on the p r o t e i n (R-NH-CO-maltose). Glucosamine was a t ­ tached to 3 - l a c t o g l o b u l i n f o l l o w i n g carbodiimide a c t i v a t i o n o f the free carboxyl groups (R-CO-NH-glucose)· The number o f groups attached to the 3 - l a c t o g l o b u l i n was v a r i e d by manipulating r e ­ a c t i o n c o n d i t i o n s (27). From s t u d i e s of the molecular p r o p e r t i e s ( v i s c o s i t y , c i r c u ­ l a r dichroism, fluorescence and UV d i f f e r e n c e spectroscopy) g l y c o s y l a t i o n of 3 - l a c t o g l o b u l i n with maltose caused greater hy­ d r a t i o n , molecular expansion and e l o n g a t i o n as the number of c a r ­ bohydrate residues added was i n c r e a s e d . Fluorescence and UV s p e c t r a together with r a t e s of p r o t e o l y s i s i n d i c a t e d a more open t e r t i a r y s t r u c t u r e and some d i s o r d e r i n g of the molecule ; i . e . ,

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

316

FOOD PROTEIN

DETERIORATION

r e d u c e d amount o f α h e l i x . As more m a l t o s e g r o u p s were added t h e s u r f a c e h y d r o p h o b i c i t y was d e c r e a s e d and n e t n e g a t i v e c h a r g e was increased. M o d i f i c a t i o n w i t h glucosamine caused l i t t l e c o n f o r ­ m a t i o n a l changes e x c e p t a t h i g h l e v e l s of m o d i f i c a t i o n ; i . e . , when 22 r e s i d u e s were g l y c o s y l a t e d some l o s s o f s e c o n d a r y s t r u c ­ t u r e became e v i d e n t (27). Some s u r f a c e a c t i v e p r o p e r t i e s o f t h e g l y c o s y l a t e d 3 - l a c t o ­ g l o b u l i n were s t u d i e d t o d e t e r m i n e i f g l y c o s y l a t i o n a f f e c t e d these. Because g l y c o s y l a t i o n , e s p e c i a l l y w i t h m a l t o s e , caused a more expanded s t r u c t u r e t h e g a i n i n f r e e e n e r g y d u r i n g p r o t e i n a d s o r p t i o n s h o u l d be l o w e r and t h e r a t e o f a d s o r p t i o n a t an o i l w a t e r i n t e r f a c e s h o u l d be l e s s . However, g l y c o s y l a t i o n may a l s o f a c i l i t a t e u n f o l d i n g and r e o r i e n t a t i o n a t t h e i n t e r f a c e and a l t e r s u r f a c e a r e a o c c u p i e d by e a c h m o l e c u l e . I n i t i a l l y the p r o p e r t i e s o f f i l m s o f 3 - l a c t o g l o b u l i n and m o d i f i e d 3 - l a c t o g l o b u l i n were studied. The s o l u b l e g l o b u l a r 3 - l a c t o g l o b u l i n (Lg) a d s o r b e d r a p i d l y and p a r t l y u n f o l d e d t o f o r m a f a i r l y t i g h t l y p a c k e d c o n d e n s e d film. Maximum s u r f a c e p r e s s u r e o f 3-Lg was o b s e r v e d a t pH 5.0 s l i g h t l y b e l o w t h e i s o e l e c t r i c p o i n t ( 5 . 2 5 ) o f 3-Lg. T h u s , when n e t c h a r g e was c l o s e t o z e r o more p r o t e i n was a d s o r b e d b e c a u s e 3 - l a c t o g l o b u l i n had a more compact s t r u c t u r e . As pH was i n ­ c r e a s e d t h e e l e c t r o n e g a t i v e c h a r g e c a u s e d r e p u l s i o n and p r o b a b l y f e w e r p r o t e i n segments o c c u p i e d t h e i n t e r f a c e . S y s t e m a t i c s t u ­ d i e s of f i l m s r e v e a l e d t h a t g l y c o s y l a t i o n of 3 - l a c t o l o b u l i n w i t h m a l t o s e , g l u c o s a m i n e o r c y c l o d e x t r i n r e d u c e d the r a t e s o f a d ­ s o r p t i o n and r e a r r a n g e m e n t a t t h e i n t e r f a c e . T h i s was a t t r i b u t e d to l o w e r f r e e energy g a i n , the i n c r e a s e d v i s c o s i t y , the s t e r i c h i n d r a n c e by c a r b o h y d r a t e s , t h e s t a b i l i z i n g e f f e c t o f t h e c a r ­ bohydrate moiety a g a i n s t u n f o l d i n g , a l t e r a t i o n of net charge, and r e d u c e d s u r f a c e h y d r o p h o b i c i t y o f t h e m o d i f i e d 3-lactoglobu­ l i n (27)· Though g l y c o s y l a t i o n r e s u l t e d i n a r e d u c t i o n o f e l e c t r o s t a t i c r e p u l s i o n the c a r b o h y d r a t e s a p p a r e n t l y r e s t r i c t e d c l o s e m o l e c u l a r p a c k i n g i n t h e i n t e r f a c i a l f i l m , i n h i b i t e d un­ f o l d i n g o f t h e h y d r o p h o b i c r e g i o n s t o t h e a p o l a r p h a s e and s i g n i f i c a n t l y i n c r e a s e d t h e amount o f w o r k r e q u i r e d f o r e a c h m o l e c u l e t o p e n e t r a t e the i n t e r f a c e . The g e n e r a l c o n c l u s i o n o f t h e s e s t u d i e s were t h a t w h i l e g l y c o s y l a t i o n e n h a n c e d t h e h y d r o p h i l i c c h a r a c t e r o f 3-Lg i t c o n ­ c u r r e n t l y d e c r e a s e d t h e number o f i o n i z a b l e g r o u p s and p e r h a p s more i m p o r t a n t l y , i t r e s t r i c t e d t h e e x p r e s s i o n o f t h e h y d r o ­ p h o b i c p r o p e r t i e s a t the i n t e r f a c e t h e r e b y r e d u c i n g the s u r f a c e a c t i v i t y o f 3 - l a c t o g l o b u l i n as i n d i c a t e d by p r o p e r t i e s o f i n t e r facial films. B e c a u s e t h e h y d r o p h i l i c g l y c o s y 1 - s u b s t i t u e n t s w o u l d be e x ­ p e c t e d t o e n h a n c e e m u l s i o n f o r m a t i o n and s t a b i l i t y v i a h y d r a t i o n and s t e r i c e f f e c t s , the e m u l s i f y i n g p r o p e r t i e s o f g l y c o s y l a t e d 3 - l a c t o g l o b u l i n were e x a m i n e d ( T a b l e V I ) .

12.

KINSELLA

Table VI.

Protein Preparation

Emulsification

and

Flavor

317

Binding

E f f e c t s of g l y c o s y l a t i o n of 3 - l a c t o g l o b u l i n on e m u l s i f y i n g p r o p e r t i e s a t d i f f e r e n t pH v a l u e s . Emulsion A c t i v i t y (OD

3

1:10 D i l u t i o n s )

Stability O i l Separation

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

pH

3L Β C D Ε F G

5 0.20 0.24 0.23 0.21 0.25 0.26 0.12

6.5 0.23 0.30 0.29 0.26 0.21 0.32 0.10

(%)

pH 7.5 0.26 0.40 0.31 0.26 0.34 0.29 0.23

5 9.0 7.5 6.8 8.0 9.5 12.3 9.9

6.5 13.0 8.2 11.5 11.4 12.8 7.9 10.0

7.5 11.6 6.1 13.0 10.8 8.5 9.8 9.8

Emulsions were prepared with 0.5% p r o t e i n and measured as r e ­ ported by Waniska et_ a l . (29). Legend: 3-Lactoglobulin (3Lg) with amino or carboxyl groups modified with JB 7 m a l t o s y l ; £ U m a l t o s y l ; I) 3 3 - c y c l o d e x t r i n ; _E 6 glucosamine ; JF 16 glucosamine and G_ 12 glucosamineoctaosyl residues, respectively. I s o e l e c t r i c p o i n t s 3Lg 5.2; Β = 4.8; C = 4.3; D = 4.3; G = 5.5.

G l y c o s y l a t i o n o f 3 - l a c t o g l o b u l i n improved emulsion formation and s t a b i l i t y . This was i n c o n t r a s t to i t s general e f f e c t s on the p r o p e r t i e s of i n t e r f a c i a l f i l m s . However, the energy input i n emulsion p r e p a r a t i o n may have overcome some of the energy b a r r i e r s encountered i n the f i l m s t u d i e s where e q u i l i b r i u m c o n d i ­ t i o n s p r e v a i l e d . O v e r a l l the data are c o n s i s t e n t with the sug­ g e s t i o n that the increased h y d r o p h i l i c nature of the p r o t e i n en­ hances emulsion p r o p e r t i e s by s t a b i l i z i n g the aqueous i n t e r f a c e through enhanced h y d r a t i o n and perhaps s t e r i c hindrance prevent­ i n g coalescence of the o i l droplets· However, d e r i v a t i z a t i o n a l t e r e d e l e c t r o s t a t i c and hydrophobic i n t e r a c t i o n s w i t h i n and between the polypeptides and these may have impeded attainment of maximum e m u l s i f y i n g p r o p e r t i e s . E m u l s i f y i n g a c t i v i t y increased with increased pH i n d i c a t i n g that as the net e l e c t r o n e g a t i v i t y i n c r e a s e d e l e c t r o s t a t i c r e p u l ­ s i o n f a c i l i t a t e d formation and s t a b i l i z a t i o n of a greater number of o i l d r o p l e t s . The number and s i z e o f h y d r o p h i l i c groups i n ­ troduced a f f e c t e d both EA and emulsion s t a b i l i t y . The attachment of, seven maltose groups i n t o 3 - l a c t o g l o b u l i n was s u p e r i o r to 11 groups i n improving e m u l s i f y i n g p r o p e r t i e s . Conceivably the lower number of maltose groups provided l e s s s t e r i c hindrance to i o n i c and hydrophobic i n t e r a c t i o n s between the polypeptides i n the i n t e r f a c e . The 3 - c y c l o d e x t r i n had l i t t l e impact on emulsi­ f y i n g p r o p e r t i e s . The e m u l s i f y i n g p r o p e r t i e s of 3 - l a c t o g l o b u l i n , modified with glucosamine r e s i d u e s , were g e n e r a l l y improved.

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

318

FOOD P R O T E I N DETERIORATION

Near the i s o e l e c t r i c range of the d e r i v a t i z e d p r o t e i n , emulsion formation was good. P r o t e i n s have a low net charge i n t h i s pH range and maximum hydrophobic i n t e r a c t i o n s are p o s s i b l e (40). Therefore, i t i s p o s s i b l e that small glucose d e r i v a t i v e s (even when 16 carboxyl groups were d e r i v a t i z e d ) d i d not completely impede hydrophobic i n t e r a c t i o n s at t h i s pH. D e r i v a t i z a t i o n with a l a r g e amino sugar, i . e . , glycosamineoctaose, impaired emulsion formation, but s t a b i l i t y of the emulsion was good. These data i n d i c a t e the importance of net charge, h y d r o p h i l i c i n t e r a c t i o n s and s t e r i c e f f e c t s i n emulsion formation and emulsion s t a b i l i z a t i o n and confirm the p o s i t i v e e f f e c t s of hydrop h i l i c groups. However, l a r g e , bulky h y d r o p h i l i c residues may i n t e r f e r e with hydrophobic i n t e r a c t i o n s causing excessive s t e r i c hindrance and a c t u a l l y reduce e m u l s i f y i n g p r o p e r t i e s . Hydrophobic E f f e c t s . The i o n i c , h y d r o p h i l i c and hydrophobic c h a r a c t e r i s t i c s of p r o t e i n a f f e c t t h e i r surface a c t i v i t y and functional properties. In an emulsion one s i d e ' of the p r o t e i n f i l m , i . e . , that exposed to the o i l phase, should be prepondera n t l y hydrophobic. The hydrophobicity i n f l u e n c e s adsorption and o r i e n t a t i o n of p r o t e i n at the i n t e r f a c e (22). The s i d e of the f i l m that i s exposed to the continuous aqueous phase has most of the p o l a r and charged groups exposed. Hydrophobic i n t e r a c t i o n s s t a b i l i z e the conformation of p r o t e i n s i n the n a t i v e s t a t e i n s o l u t i o n . At an i n t e r f a c e the normal thermodynamic e q u i l i b r i u m i s disturbed. The apolar segments of the polypeptides tend to u n f o l d , r e o r i e n t and occupy the l e s s p o l a r o i l phase. T h i s phenomenon depends upon the amphipathic a t t r i b u t e s of the p r o t e i n , a property required f o r surface a c t i v i t y . The hydrophobicity of p r o t e i n s i s r e l a t e d to t h e i r contents of apolar amino a c i d s . The r e l a t i v e h y d r o p h o b i c i t i e s of these; i . e . , t r p , i l e u , t y r , phe, pro, l e u , v a l , l y s , meth, cys/2, a l a , arg are 3.00, 2.95, 2.85, 2.65, 2.60, 2.40, 1.7, 1.5, 1.3, 1.0, 0.75, 0.75 K c a l / r e s i d u e r e s p e c t i v e l y (13). The average hydrophob i c i t y (HQ) r e p r e s e n t i n g the t o t a l hydrophobicity d i v i d e d by the t o t a l number of residues was c a l c u l a t e d f o r s e v e r a l p r o t e i n s by Bigelow (39). P r o t e i n s have HQ values ranging from 1000-12000 c a l / r e s i d u e (Table V I I ) . While general r e l a t i o n s h i p s are apparent, i t should be remembered that the d i s p o s i t i o n of the apolar residues (sequence and whether l o c a t e d i n t e r n a l l y , on the s u r f a c e , or on f l e x i b l e segments) a f f e c t p h y s i c a l p r o p e r t i e s more than the average hydrophobic amino a c i d content. L e s l i e (41) r e l a t e d the strong NMR s i g n a l s of the apolar residues i n soy p r o t e i n s to the e m u l s i f y i n g p r o p e r t i e s and Kato and Nakai (16) reported good c o r r e l a t i o n s between molecular hydrophobicity and s u r f a c t a n t p r o p e r t i e s . They s u c c e s s f u l l y q u a n t i f i e d e f f e c t i v e hydrophobicity; i . e . , true surface hydrop h o b i c i t y , using cii>-parinaric a c i d , an e x c e l l e n t f l u o r e s c e n t probe f o r measuring hydrophobicity of food p r o t e i n s . Proteins were reacted with c i s - p a r i n a r i c a c i d . The conjugates were 1

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Binding

e x c i t e d a t 325 nm and fluorescence i n t e n s i t y measured at 420 nm. The i n i t i a l slope ( S ) obtained by p l o t t i n g fluorescence against p r o t e i n c o n c e n t r a t i o n i n d i c a t e s s u r f a c e hydrophobicity. The energy t r a n s f e r e f f i c i e n c y of conjugates a t zero p r o t e i n concen­ t r a t i o n (T ) also r e f l e c t hydrophobicity. These two values were h i g h l y c o r r e l a t e d and corresponded to data obtained using hydro­ phobic chromatography (42). 0

Table V I I .

The average hydrophobicity of some food p r o t e i n s .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

MW 1 0 Tropomyosin Ovomucoid Collagen ( s o l u b l e c a l f ) Lysozyme Myosin Actin Gliadin Conalbumin Myoglobin Ovalbumin BSA α-Lactalbumin α-Casein 3-Lactoglobulin Zein

-

27 80 15 500 57 120 87 17 46 65 16

-17 50

3

HQ

NPS

870 920 960 970 1020 1050 1080 1080 1090 1110 1120 1150 1200 1230 1310

0.20 0.25 0.19 0.26 0.28 0.31

-

0.31 0.32 0.34 0.32 0.34 0.38 0.37

-

Ρ 1.94 1.50 0.89 1.18 1.43 1.15

-

1.30 1.12 0.92 1.22 1.11 1.27 0.96

-

Charge 0.47 0.30 0.16 0.16 0.35 0.27

-

0.32 0.34 0.24 0.33 0.28 0.24 0.28

-

MW = mol. weight; HQ = t o t a l hydrophobicity d i v i d e d by t o t a l number of r e s i d u e s ; NPS « sum o f Trp, l i e , T y r , Phe, Pro, Leu, and V a l residues as a f r a c t i o n of t o t a l residues ; Ρ = p o l a r i t y r a t i o roughly a l l p o l a r s i d e c h a i n s / a l l nonpolar s i d e chains and charge i s sum of Asp, G l u , H i s , L y s , Arg as f r a c t i o n of t o t a l amino a c i d s . From Bigelow (39).

Bovine serum albumin, κ-casein and 3 - l a c t o g l o b u l i n showed high S values (Table VIII) r e f l e c t i n g the b i n d i n g o f c i s - p a r i n a r i c a c i d to t h e i r hydrophobic r e g i o n s . The surface hydropho­ b i c i t y showed strong c o r r e l a t i o n s w i t h surface tension, i n t e r f a c i a l t e n s i o n and e m u l s i f y i n g a c t i v i t y o f these p r o t e i n s . This was f u r t h e r demonstrated by s t u d i e s i n d i c a t i n g that the p r o g r e s s i v e thermal denaturation o f lysozyme increased surface hydrophobicity and the EA was c o n c u r r e n t l y enhanced (3)· These data i n d i c a t e that hydrophobic i n t e r a c t i o n s are important, par­ t i c u l a r l y f o r the i n t e r m o l e c u l a r i n t e r a c t i o n s and a s s o c i a t i o n s which enhance the cohesiveness w i t h i n the p r o t e i n f i l m s surround­ i n g f a t d r o p l e t s i n emulsions. The above s e c t i o n s demonstrate some r e l a t i o n s h i p s between the molecular p r o p e r t i e s of food p r o t e i n s and t h e i r emulsion p r o p e r t i e s . However, much research i s needed to determine the optimum q u a n t i t a t i v e r e l a t i o n s h i p s between i n d i v i d u a l molecular Q

320

FOOD PROTEIN

DETERIORATION

p r o p e r t i e s ( c h a r g e , h y d r o p h o b i c i t y and h y d r o p h i l i c i t y ) , e n v i r o n m e n t a l p a r a m e t e r s , and p a r t i c u l a r t y p e s o f f o o d e m u l s i o n s .

Table V I I I .

Relative Hydrophobicity (fluorescence i n t e n s i t y , S ) S u r f a c e T e n s i o n , I n t e r f a c i a l T e n s i o n and E m u l s i f y i n g A c t i v i t y Index Values of Various P r o t e i n s . 0

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

S

0

Bovine serum albumin 1400 k-Casein 1300 3-lactoglobulin 750 Trypsin 90 Ovalbumin 60 Conalbumin 70 Lysozyme 100

Surface Tension (Dynes/cm)

57.9 54.1 59.8 64.1 61.1 63.7 64.0 From K a t o and

P r o t e i n Ligand

Interactions - Flavor

Interfacial Tension (Dynes/cm)

Emulsifying Activity Index (m g 1)

10.3 9.5 11.0 12.0 11.6 12.1 11.2

166 185 151 93 57 105 55

Nakai

2

(16)

Binding

P r o t e i n s b e i n g s u r f a c e a c t i v e can b i n d l i p i d s , f a t t y a c i d s , a l d e h y d e s , k e t o n e s , t a n n i n s , c h l o r o g e n i c a c i d , e t c . , and a d v e r s e l y a f f e c t f u n c t i o n a l and n u t r i t i o n a l p r o p e r t i e s ( 4 3 , 4 4 ) . The b i n d i n g o f low amounts o f s u r f a c e a c t i v e compounds and l i p i d s t o f o o d p r o t e i n s may a l t e r t h e i r s t a b i l i t y and t h e r m a l p r o p e r t i e s . Thus, t h e b i n d i n g o f l a u r i c a c i d t o egg w h i t e p r o t e i n s e n h a n c e s t h e i r t h e r m a l s t a b i l i t y (45) and low m o l e c u l a r w e i g h t a l c o h o l s o r c a r b o n y l s a t l o w c o n c e n t r a t i o n s s t a b i l i z e b o v i n e serum a l b u m i n (46). The b i n d i n g o f f l a v o r s t o f o o d components, e s p e c i a l l y p r o t e i n s i s o f p a r t i c u l a r i m p o r t a n c e , and i t i s a p r o b l e m t h a t has b e e n d r a m a t i z e d as f o o d t e c h n o l o g i s t s h a v e t r i e d t o f a b r i c a t e a n a l o g s and new f o o d s f r o m n o v e l p r o t e i n s ( 4 7 ) . More f u n d a m e n t a l i n f o r m a t i o n concerning the nature of f l a v o r - p r o t e i n i n t e r a c t i o n i s needed to h e l p s o l v e problems o f o f f - f l a v o r b i n d i n g , to a i d the d e v e l o p m e n t o f more s u c c e s s f u l f l a v o r i n g p r o c e d u r e s and u n d e r s t a n d t h e mechanism and t h e r m o d y n a m i c s o f f l a v o r r e l e a s e when f o o d i s chewed. Because the p e r c e i v e d f l a v o r i s important i n d e t e r m i n i n g f o o d a c c e p t a b i l i t y , t h e phenomenon o f f l a v o r b i n d i n g and r e l e a s e i s extremely s i g n i f i c a n t . The o f f - f l a v o r s t h a t become a s s o c i a t e d w i t h p r o t e i n s l i m i t s t h e i r a p p l i c a t i o n . T h i s i s e x e m p l i f i e d by many soy p r o t e i n p r e p a r a t i o n s w h i c h , t h o u g h p o s s e s s i n g good

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

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Binding

f u n c t i o n a l p r o p e r t i e s , have l i m i t e d use because of the impact o f bound o f f - f l a v o r s (48). To develop e f f e c t i v e methods f o r the r e ­ moval o f o f f - f l a v o r s we have been studying f l a v o r - p r o t e i n i n t e r ­ a c t i o n s i n simple model systems composed of soy p r o t e i n s and carbonyl compounds and determining b i n d i n g using e q u i l i b r i u m d i a l y s i s (49). Binding isotherms i n d i c a t e d a p r o g r e s s i v e i n c r e a s e i n b i n d ­ i n g o f carbonyls with t h e i r c o n c e n t r a t i o n . There were four binding s i t e s per 100,000 molecular weight soy p r o t e i n f o r 2heptanone, 2-octanone and 2-nonanone, r e s p e c t i v e l y . The b i n d i n g a f f i n i t i e s i n c r e a s e d with chain length of the carbonyls i n d i c a t ­ i n g that hydrophobic i n t e r a c t i o n s are i n v o l v e d (Table I X ) . There was a t h r e e f o l d i n c r e a s e i n Keq f o r each increment of methylene group (-CH^) i n the chain l e n g t h o f the carbonyl which c o r r e ­ sponded to a change i n f r e e energy of -600 c a l o r i e s / C H 2 group. S i m i l a r r e s u l t s were obtained with bovine serum albumin except that i n the case of soy p r o t e i n and BSA the Keq f o r nonanone was 930 and 1800 M~l r e s p e c t i v e l y . This r e f l e c t s the d i f f e r e n c e i n s t r u c t u r e of these p r o t e i n s and the greater surface hydropho­ b i c i t y i n BSA which possesses s i x b i n d i n g s i t e s f o r these c a r ­ bonyls (50). Table

IX.

Thermodynamic constants f o r i n t e r a c t i o n s of carbonyls with soy p r o t e i n s i n model systems. K

Ligand

2-Nonanone 2-Nonanone 2-Nonanone 2-Nonanone

AG

η

native native native native native

25 25 25 25 25

4 4 4 4 4

310 930 541 1094

-2.781 -3.395 -4.045 -3.725 -4.141

(succinylated) native native

25 25 5

2 4 2

850 930 2000

-3.992 -4.045 -4.221

25

4

1240

-4.215

7S 25 (0.03M S a l t ) 25 (0.5M S a l t ) 25

4

930

4.040

-8

290

-

-

2-Heptanone 2-0ctanone 2-Nonanone 5-Nonanone 1-Nonanal 2-Nonanone 2-Nonanone 2-Nonanone

eq

Temp °C

Protein

(heated

IIS IIS

90°C)

1

(M" )

no

(Kcal/mole)

-

These data s t r o n g l y support hydrophobic b i n d i n g and the AG value f o r the b i n d i n g of each C H 2 group, i . e . , -550 and -600 cal/-CH« group f o r BSA and soy p r o t e i n c l o s e l y approximate the -540 c a l / m o l e - C H 2 observed f o r the t r a n s f e r of a C H 2 group from water to an apolar s o l v e n t (51). Further evidence of hydrophobic i n t e r a c t i o n i s obtained by comparing the b i n d i n g of nonanol, 2-nonanone and 5-nonanone to soy p r o t e i n (Table I X ) . The b i n d i n g

322

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c o n s t a n t Κ d e c r e a s e d as t h e k e t o g r o u p was moved t o w a r d t h e m i d d l e o f t h e c h a i n ; i . e . , Keq was 1094, 930 and 541 M" for t h e s e t h r e e c a r b o n y l s r e f l e c t i n g t h e s t e r i c h i n d r a n c e c a u s e d by t h e k e t o g r o u p , e s p e c i a l l y when l o c a t e d n e a r t h e c e n t e r o f t h e hydrocarbon chain. The b i n d i n g a f f i n i t y o f s u c c i n y l a t e d soy p r o ­ t e i n f o r 2-nonanone was s l i g h t l y r e d u c e d compared t o t h e n a t i v e p r o t e i n , and t h e number o f b i n d i n g s i t e s was r e d u c e d t o two r e ­ f l e c t i n g m a j o r c o n f o r m a t i o n a l changes i n t h e s o y p r o t e i n i n t h e n e i g h b o r h o o d of the b i n d i n g s i t e s . Though t h e two b i n d i n g s i t e s have a l m o s t t h e same i n t r i n s i c b i n d i n g c o n s t a n t as t h e n a t i v e soy p r o t e i n , t h e b i n d i n g c a p a c i t y ( m o l e s l i g a n d bound p e r mole p r o t e i n ) o f t h e s u c c i n y l a t e d s o y was d e c r e a s e d . T h e r e f o r e , suc­ c i n y l a t i o n d e c r e a s e d f l a v o r b i n d i n g and i m p r o v e d t h e f l a v o r o f s u c c i n y l a t e d soy p r o t e i n as n o t e d p r e v i o u s l y ( 5 2 ) . A t l o w t e m p e r a t u r e (5°C) t h e number o f b i n d i n g s i t e s was r e d u c e d t o two, b u t t h e s e had s i g n i f i c a n t l y g r e a t e r b i n d i n g c o n ­ s t a n t s r e f l e c t i n g changes i n the c o n f o r m a t i o n o f soy p r o t e i n a t low t e m p e r a t u r e s . The b i n d i n g c o n s t a n t was s i g n i f i c a n t l y i n ­ c r e a s e d (30%) f o l l o w i n g h e a t i n g o f soy (90°C, 1 h r ) , b u t t h e r e were s t i l l f o u r b i n d i n g s i t e s . The e n h a n c e d b i n d i n g upon h e a t i n g corroborates the d a t a o f A r a i ejt a l . (53) and i n d i c a t e s t h a t t h e t h e r m a l p r o c e s s i n g o f soy p r o t e i n may e x a c e r b a t e t h e p r o b l e m o f o f f - f l a v o r b i n d i n g by i n c r e a s i n g t h e b i n d i n g a f f i n i t y o f t h e soy p r o t e i n f o r c a r b o n y l s . The e f f e c t s o f h e a t i n g o f s o y p r o t e i n on l i g a n d b i n d i n g seemed i n c o n s i s t e n t w i t h t h e k n o w l e d g e t h a t soy U S d i s s o c i a t e s , and t h e r e f o r e h e a t i n g s h o u l d i n c r e a s e the number o f b i n d i n g s i t e s . T h e r e f o r e , we s t u d i e d t h e b i n d i n g o f c a r b o n y l s t o U S and 7S, t h e m a j o r p r o t e i n components o f s o y b e a n . S i g n i f i c a n t l y , t h e r e was n e g l i g i b l e b i n d i n g o f nonanone t o t h e 11S f r a c t i o n , w h e r e a s t h e b i n d i n g t o 7S was c o m p a r a b l e t o t h a t f o r w h o l e soy p r o t e i n . This s e l e c t i v e i n t e r a c t i o n o b v i o u s l y r e f l e c t s the d i f f e r e n c e s i n the m o l e c u l a r s t r u c t u r e , c o n f o r m a t i o n and s u r f a c e p r o p e r t i e s o f t h e s e two p r o t e i n s . The 7S p r o t e i n (17,500 d a l t o n s ) i s c o m p r i s e d o f t h r e e s u b u n i t s o f d i f f e r e n t s i z e s and amino a c i d c o m p o s i t i o n ( 5 4 ) . The U S p r o t e i n (320,000 d a l t o n s ) c o n t a i n s s i x a c i d i c and s i x basic subunits (55). The s p a t i a l a r r a n g e m e n t o f t h e s u b u n i t s i n t h e s e two p r o t e i n s may be s u c h t h a t t h e 7S p r o t e i n has h y d r o ­ phobic r e g i o n s which are a c c e s s i b l e f o r l i g a n d b i n d i n g , whereas i n t h e c a s e o f U S p r o t e i n s u c h h y d r o p h o b i c r e g i o n s may be b u r i e d i n s i d e t h e p r o t e i n o r be a t t h e p o i n t s o f c o n t a c t o r a s s o c i a t i o n b e t w e e n s u b u n i t s , and h e n c e may n o t be a c c e s s i b l e t o t h e l i g a n d . B e c a u s e the weak i n t e r a c t i o n b e t w e e n U S and 2-nonanone may r e f l e c t i t s unique q u a t e r n a r y s t r u c t u r e , then changes i n t h i s s t r u c t u r e may a f f e c t t h e b i n d i n g a f f i n i t y f o r 2-nonanone. The o l i g o m e r i c s t r u c t u r e o f soy 11S i s i n f l u e n c e d by i o n i c s t r e n g t h ; i . e . , a t 0.5 M i o n i c s t r e n g t h t h e p r o t e i n has a s e d i m e n t a t i o n c o e f f i c i e n t o f U S , b e l o w 0.1 M i o n i c s t r e n g t h i t d i s s o c i a t e s i n t o ' h a l f U S ' u n i t s ( 5 6 ) . A t the i o n i c s t r e n g t h u s e d i n o u r i n i t i a l s t u d y t h e U S was i n h a l f 11S f o r m and p e r h a p s had a

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

1

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

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Binding

323

r e l a t i v e l y high surface charge which impaired b i n d i n g . A t higher i o n i c (0.5 M) s t r e n g t h when the e l e c t r o s t a t i c r e p u l s i o n i s weakened, the i n t a c t U S i s formed, and t h i s i s accompanied by a marked increase i n nonanone b i n d i n g . This o b s e r v a t i o n i s s i g n i f i c a n t because i f the U S e x i s t s i n an environment of low i o n i c strength i n the soybean seed or i f low i o n i c s t r e n g t h prev a i l s during m i l l i n g of the seed, the 11S f r a c t i o n which i s ^35% of soy p r o t e i n , should possess l e s s o f f - f l a v o r s . Because hydrophobic i n t e r a c t i o n s are the predominant f o r c e s i n v o l v e d i n b i n d i n g of apolar organic l i g a n d s to p r o t e i n i t may be f e a s i b l e to remove o f f - f l a v o r s by c h a o t r o p i c agents which weaken hydrophobic i n t e r a c t i o n s (57). Thus, we determined the e f f e c t s o f i n c r e a s i n g urea concentrations on f l a v o r b i n d i n g c h a r a c t e r i s t i c s of soy p r o t e i n . There was a p r o g r e s s i v e decrease i n b i n d i n g a f f i n i t y f o r nonanone as urea was increased from 0 to 4.5 M. I t i s known that the denaturing a c t i o n of urea i n v o l v e s a hydrophobic mechanism that favors exposure of nonpolar residues i n the p r o t e i n to s o l v e n t environment (58). As these conformat i o n a l changes occurred i n the soy p r o t e i n s the b i n d i n g a f f i n i t y concomitantly decreased. At low concentrations of urea the b i n d i n g a f f i n i t y of soy f o r nonanone was decreased s i g n i f i c a n t l y ; i . e . , by ^50 and 70% a t 1.5 and 3.0 M urea, r e s p e c t i v e l y . This observation may have p r a c t i c a l i m p l i c a t i o n s . Thus, r e v e r s i b l e d i s s o c i a t i o n o f soy p r o t e i n subunits; e.g., using urea or other d i s s o c i a t i n g agent may f a c i l i t a t e the removal o f o f f - f l a v o r s such as carbonyls, because b i n d i n g i s an e q u i l i b r i u m process. Thus, u l t r a f i l t r a t i o n of d i l u t e urea s o l u t i o n s of undenatured soy p r o t e i n s may be u s e f u l f o r the p r e p a r a t i o n of bland soy protein. Conclusion The f u n c t i o n a l p r o p e r t i e s of p r o t e i n s r e f l e c t the inherent molecular p r o p e r t i e s of the p r o t e i n s ( s i z e , shape, conformation, molecular f l e x i b i l i t y , s u s c e p t a b i l i t y to d e n a t u r a t i o n ) , the manner i n which they i n t e r a c t with other food c o n s t i t u e n t s and how they i n t e r a c t with the p r e v a i l i n g environmental c o n d i t i o n s . Thus numerous i n t r i n s i c and e x t r i n s i c f a c t o r s a c t i n g i n concert d e t e r mine the f i n a l e f f e c t . T h i s s i t u a t i o n makes i t extremely c h a l l e n g i n g to the food s c i e n t i s t to develop standard methods based on the physicochemical behavior of p r o t e i n i n a simple system. Because of the complexity o f the systems i n v o l v e d , i t i s obvious that the conventional e m p i r i c a l methods f o r comparing the funct i o n a l p r o p e r t i e s of p r o t e i n s w i l l continue to be used. Recogn i z i n g t h i s , food s c i e n t i s t s should s t r i v e to standardize the methods as much as p o s s i b l e so that published data can be v a l i d l y used to compare the r e l a t i v e f u n c t i o n a l i t y of d i f f e r e n t p r o t e i n preparations. There i s a c o n t i n u i n g need f o r dedicated b a s i c research (Table X) d i r e c t e d toward the e l u c i d a t i o n of the physicochemical

324

FOOD PROTEIN DETERIORATION

bases o f s p e c i f i c f u n c t i o n a l p r o p e r t i e s ; e.g., g e l a t i o n , s u r f a c e a c t i v i t y , f i l m formation. W h i l e i t may n o t be p o s s i b l e t o d e ­ f i n e each f u n c t i o n a l property i n mathematical terms, the knowl­ edge g a i n e d f r o m f u n d a m e n t a l r e s e a r c h w i l l p r o v i d e a c l e a r e r u n d e r s t a n d i n g o f t h e p h y s i c a l and c h e m i c a l r e l a t i o n s h i p s i n v o l v e d . This i n f o r m a t i o n w i l l enable t h e food s c i e n t i s t t o s e l e c t t h e most a p p r o p r i a t e p r o t e i n f o r a s p e c i f i c f u n c t i o n a l p r o p e r t y o r a p p l i c a t i o n and a l s o e n a b l e t h e s c i e n t i s t t o m o d i f y t h e p r o t e i n s to o p t i m i z e d e s i r a b l e p h y s i c a l p r o p e r t i e s .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

T a b l e X.

Some r e s e a r c h properties.

needs i n t h e a r e a o f f u n c t i o n a l

S t a n d a r d i z i n g the d e f i n i t i o n o f f u n c t i o n a l p r o p e r t i e s D e v e l o p m e n t o f s t a n d a r d i z e d methods b a s e d o n p h y s i c a l p r o p e r t i e s R e l a t i n g f u n c t i o n a l p r o p e r t i e s t o s t r u c t u r a l f e a t u r e s and secondary i n t e r a c t i o n s M a n i p u l a t i o n o f p r o t e i n s t r u c t u r e by c h e m i c a l o r p h y s i c a l alterations T e s t i n g i n food systems Acknowledgments The a u t h o r g r a t e f u l l y a c k n o w l e d g e s t h e s u p p o r t and d i s ­ c u s s i o n s o f D r s . J . S h e t t y , S. Damodaran, a n d R. W a n i s k a , some o f whose d a t a a r e p r e s e n t e d i n t h i s p a p e r . F i n a n c i a l s u p p o r t f r o m t h e N a t i o n a l S c i e n c e F o u n d a t i o n G r a n t #CPE 80-18394 i s g r a t e f u l l y acknowledged.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

Kinsella, J . E. CRC Crit. Rev. Food Sci. Nutr., 1976, 7, 219. Cherry, J . P., Ed. "Protein Functionality in Foods", American Chemical Society Symp. Series #147: Washington, DC, 1981. Hermansson, A. M. In "Problems in Human Nutrition"; Porter, J. & Rolls, Β., Eds., Acad. Press, New York, 1973; p. 407. Tornberg, E . ; Hermansson, A. M. J . Food Sci., 1977, 42, 468; 1978, 43, 1553. Pour-El, Α., Ed. "Functionality and Protein Structure"; American Chemical Society Symp. Series #92: Washington, DC, 1980. Pour-El, A. In "Protein Functionality in Foods"; Cherry, J. P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981; p. 1. Shen, J . In "Protein Functionality in Foods"; Cherry, J . P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981. Smith, Α.; Circle, A. J . "Soybeans: Chemistry and Technology"; Avi Publ. Co.: Westport, CT, 1978.

KINSELLA

9. 10. 11. 12.

Galyean, R.; Cotterill, O. J . Food Sci., 1979, 44, 1365. Damodaran, S.; Kinsella, J . E. This volume Chap. 13. Kauzman, P. Advances Protein Chem., 1959, 14, 1. Anfinsen, C.; Scheraga, H. Adv. Protein Chem., 1975, 29, 205. Tanford, C. "The Hydrophobic Effect"; Wiley, J., Ed., New York, 1973. Van Holde, N. In "Food Proteins"; Whitaker, J . & Tannenbaum, S., Eds.; Avi Publ. Co.; Westport, CT, 1971; p. 1. Shimada, K.; Matushita, S. J . Agr. Food Chem., 1980, 28, 413. Kato, A.; Nakai, S. Can. Inst. Food Sci. & Technol. J., press, 1981. Bull, H.; Breese, K. Arch. Biochem. Biophys., 1968, 128, 488. Feeney, R.; Whitaker, J., Eds.; "Food Proteins: Improvement Through Chemical and Enzymatic Modification"; Advances in Chem. Series 160, American Chemical Society: Washington, DC, 1977. Williams, R. J . Biol. Rev., 1979, 54, 389. Ewart, J . J . Sci. Food Agr., 1972, 23, 687. Morr, C. J . Dairy Sci., 1975, 58, 977. Phillips, M. C. Food Technol., 1981, 35, 50. Brandts, J . In "Structure and Stability of Macromolecules"; Timasheff, S. & Fasman, G., Eds., Marcel-Dekker: New York, 1969, Chap. 3. Graham, D. E . ; Phillips, M. C. In "Theory and Practice of Emulsion Technology"; Smith, A. L., Ed., Acad. Press: New York, 1976; p. 75. MacRitchie, F. Adv. In Protein Chem., 1978, 32, 283. Halling, P. J . Crit. Rev. Food Sci. and Nutr., 1981, 12, 155. Waniska, R.; Kinsella, J . E. Unpubl. research, 1982. Tornberg, E. In "Functionality and Protein Structure"; Pour-El, Α., Ed.; American Chemical Society Symp. Series #92: Washington, DC, 1976, p. 105. Waniska, R.; Shetty, J.; Kinsella, J . E. J . Agr. Food Chem., 1981, 29, 826. Kinsella, J . E. Food Chem. (Lond.) 1982 (press). McWatters, K; Cherry, J . P. In "Protein Functionality in Foods"; Cherry, J . P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981, p. 217. Mulder, H.; Walstra, P. "The Milk Fat Globule: Emulsion Science as Applied to Milk Products"; Pub. Commonwealth Agr. Bureau: Bucks, England, 1974. Walstra, P.; Ourtwijn, H.; deGroaf, J . Neth. Milk Dairy J., 1969, 23, 12. Pearce, K.; Kinsella, J . E. J . Agr. Food Chem., 1976, 26, 716.

13. 14. 15.

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16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

Emulsification

and

Flavor

Binding

325

12.

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35. Kinsella, J. E.; Shetty, J. In "Functionality and Protein Structure"; Pour-El., Ed.; American Chemical Society Symp. Series #92: Washington, DC, 1979, p. 37. 36. Leonard, W.; Foster, J. J. Biol. Chem., 1961, 236, 2262. 37. Aoki, K.; Murata, M.; Hiramatus, K. Anal. Biochem., 1974, 59, 16. 38. McKenzie, H. "Milk Proteins: Chemistry and Technology"; Acad. Press: New York, 1971; p. 257. 39. Bigelow, C. J. Theor. Biol., 1967, 16, 187. 40. Kuntz, L . ; Kauzmann, W. Adv. Protein Chem., 1974, 28, 239. 41. Leslie, R. B. J. Am. Oil Chem. Soc., 1976, 56, 290. 42. Kato, A.; Makai, S. Biochem. Biophys. Acta., 1980, 624, 13. 43. Kinsella, J. E. In "Criteria of Food Acceptance"; Solms, J. & Hall, R., Eds.; Forster Verlog A. G.: Zurich, Switzerland, 1981, p. 140. 44. Blouin, F.; Zarins, Z.; Cherry, J. In "Protein Functionality in Foods"; Cherry, J. P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981, p. 21. 45. Hegg, P. O.; Lofquist, B. J. Food Sci., 1974, 39, 1231. 46. Damodaran, S.; Kinsella, J. E. J. Biol. Chem., 1980, 255, 8503. 47. Kinsella, J. E. Crit. Rev. Food Sci. Nutr., 1978, 10, 147. 48. Kinsella, J. E. J. Am. Oil Chem. Soc., 1979, 56, 242. 49. Damodaran, S.; Kinsella, J. E. J. Agr. Food Chem., 1981, 29, 1253. 50. Damodaran, S.; Kinsella, J. E. J. Agr. Food Chem., 1980, 28, 567. 51. Abraham, M. J. Am. Chem. Soc., 1980, 102, 5910. 52. Franzen, K.; Kinsella, J. E. J. Agr. Food Chem., 1976, 24 914. 53. Arai, S.; Noguchi, M.; Kato, H.; Fujimaki, M. Agr. Biol. Chem., 1970, 34, 1420 54. Thanh, V.; Shibasaki, K. J. Agr. Food Chem., 1978, 26, 692. 55. Badley, R.; Atkinson, D.; Oldani, D.; Green, J.; Stubbs, J. Biochem. Biophys. Acta., 1975, 412, 216. 56. Koshiyama, L. Int. J. Peptide & Protein Res., 1972, 4, 167. 57. Damodaran, S.; Kinsella, J. E. J. Biol. Chem., 1981, 256, 3394. 58. Wetlaufer, D.; Malik, S.; Stoller, L . ; Coffin, R. J. Am. Chem. Soc., 1964, 86, 508. RECEIVED June

1, 1982.

13 Effects of Ions on Protein Conformation and Functionality SRINIVASAN DAMODARAN and JOHN E. KINSELLA

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

Cornell University, Institute of Food Science, Ithaca, NY 14853

The unique three-dimensional structure of a protein molecule is the resultant of various attractive and repulsive interactions of the protein chain within itself and with the surrounding sol­ vent. The various types of interactions and forces which con­ tribute to the overall stability of the native structure of a protein may be broadly categorized as hydrogen bonding, electro­ static and hydrophobic interactions. For the protein to assume a particular ordered structure, the favorable free energy change from the above interactions in the ordered form should be more than the unfavorable free energy increase resulting from the configurational entropy of the chain. In other words, the favorable change in the free energy for the stability of the folded native protein may be expressed as Equation 1: ΔG = ΔG + ΔG + ΔG n

h

e

ф

TΔS

congifuraoitn

where ΔG , ΔG and ΔG are the free energy contribution from hydrogen bonding, electrostatic and hydrophobic interactions, respectively, and ΔS is the configurational entropy of the pro­ tein chain and Τ is the temperature. For many proteins the net free energy favoring the formation and stability of the native structure is only about 10-20 Kcal/ mole (1). Hence any perturbation leading to decrease in the free energy of stabilization even by few kilocalories would profoundly affect the structural stability of the native protein and hence its function. Such changes may be brought about by agents like heat, organic solvents and chaotropic salts. The major emphasis of this chapter will be to discuss the effect of ionic solutes on protein conformation and function, and to elucidate the mecha­ nisms of these interactions. Before venturing into this i t may be appropriate to understand the nature and magnitude of some of the major forces responsible for the stability of protein struc­ ture, viz. hydrogen bonds, electrostatic and hydrophobic inter­ actions . h

e

ф

0097-6156/82/0206-O327$09.00/0 © 1982 American Chemical Society

FOOD P R O T E I N

328 Major Forces i n P r o t e i n Structure

DETERIORATION

Stability

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

Hydrogen bonds. The existence and importance of hydrogen bonds i n p r o t e i n s i s w e l l documented. In f a c t , the α-helical and the 3-sheet s t r u c t u r e s i n p r o t e i n s are based on the formation of hydrogen bonds between the peptide groups. However, there has been considerable disagreement about the degree of s t a b i l i z a t i o n they provide to the p r o t e i n s t r u c t u r e . The reason f o r t h i s i s the f a c t that i n aqueous s o l u t i o n s , the solvent i t s e l f can com­ pete with both the acceptor and donor groups i n p r o t e i n s f o r hydrogen bond formation. In other words, i f the formation of hydrogen bonds i n aqueous s o l u t i o n i s expressed as Equation 2: protein-Η···Η 0 + Η 0··«H-protein 2

2

p r o t e i n - p r o t e i n + Η^0··«Η^Ο then, i n order to form i n t r a p e p t i d e hydrogen bonds the change i n the free energy of the above r e a c t i o n should favor the forward r e a c t i o n . With N-methylacetamide as a model compound, which i s comparable to groups i n p r o t e i n s , i t has been shown that the change i n the free energy f o r the formation of hydrogen bonds between N-me thy lace tamide molecules i s +0.75 Kcal/mole (2). But the data f o r h e l i x - c o i l t r a n s i t i o n i n s y n t h e t i c polypeptides shows that the f r e e energy c o n t r i b u t e d by the hydrogen bonds f o r the s t a b i l i t y of p r o t e i n s i s only about -0.15 Kcal/mole residue (3). One can, t h e r e f o r e , assume that the energy contributed by hydrogen bonds to the o v e r a l l s t a b i l i t y of p r o t e i n s t r u c t u r e i s very s m a l l . I t should be borne i n mind that the hydrogen bond i s p r i m a r i l y i o n i c i n character due to the d i p o l e moment of the pep­ t i d e group. Although the hydrogen bonds give s t a b i l i t y to the ah e l i c a l s t r u c t u r e , the d r i v i n g force f o r the formation of hydro­ gen bonds i s the hydrophobic i n t e r a c t i o n between the s i d e c h a i n groups ( 4 ) . Conversely, any p e r t u r b a t i o n which causes d e s t a b i l i z a t i o n of these hydrophobic i n t e r a c t i o n s i n the p r o t e i n would a l s o d e s t a b i l i z e the hydrogen bonds i n t h i s v i c i n i t y . E l e c t r o s t a t i c i n t e r a c t i o n s . The presence of charged groups l i k e glutamic, a s p a r t i c , l y s i n e and a r g i n i n e residues i n p r o t e i n s form the b a s i s of e l e c t r o s t a t i c i n t e r a c t i o n s . But l i t t l e i s known about the thermodynamic c o n t r i b u t i o n they provide f o r the o v e r a l l s t a b i l i t y of the p r o t e i n s t r u c t u r e . Near the i s o e l e c t r i c pH the e l e c t r o s t a t i c i n t e r a c t i o n between the o p p o s i t e l y charged groups may be s i g n i f i c a n t and may provide some s t a b i l i z a t i o n energy to the p r o t e i n s t r u c t u r e (5). Above or below the i s o e l e c t r i c pH range the p r o t e i n w i l l have a net negative or p o s i t i v e charge and hence r e p u l s i v e i n t e r a c t i o n s w i l l e x i s t w i t h i n the p r o t e i n . But binding of counter ions may decrease these r e p u l s i v e f o r c e s and provide s t a b i l i t y to the p r o t e i n s t r u c t u r e . Since most of the charged groups are present on the surface of the p r o t e i n , the charge r e p u l s i o n or a t t r a c t i o n between these groups may be minimal

13.

D A M O D A R A N

A N D

KINSELLA

Ion Effects on

Proteins

329

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because of the high d i e l e c t r i c constant of the surrounding s o l vent. But the magnitude of such i n t e r a c t i o n s may become s i g n i f i cant i f these charges are p a r t i a l l y or completely b u r i e d i n s i d e the p r o t e i n where the d i e l e c t r i c constant i s lower. In such cases, each of the b u r i e d charges w i l l d e s t a b i l i z e the n a t i v e p r o t e i n s t r u c t u r e to the order of 20 Kcal/mole ( 6 ) . In the p r e s ence of an o p p o s i t e l y charged residue i n the i n t e r i o r of the prot e i n , a s a l t bridge may be formed which w i l l provide c o n s i d e r a b l e energy f o r s t a b i l i z i n g p r o t e i n s t r u c t u r e . Hydrophobic i n t e r a c t i o n s . I t i s recognized that the major force r e s p o n s i b l e f o r the s t a b i l i t y of p r o t e i n s t r u c t u r e i s the hydrophobic i n t e r a c t i o n between the nonpolar s i d e c h a i n s i n prot e i n s . The d r i v i n g force f o r such i n t e r a c t i o n s a r i s e not from the inherent a t t r a c t i o n o f nonpolar s i d e c h a i n s f o r each other, but i n the e n e r g e t i c a l l y unfavorable e f f e c t they have on the s t r u c t u r e of the water molecules around them. The b a s i s f o r t h i s fundamental concept i s the thermodynamic data concerning the t r a n s f e r o f v a r i o u s nonpolar compounds from apolar s o l v e n t s to water (7-10)· When a hydrocarbon i s t r a n s f e r r e d from a nonpolar solvent to water, the changes i n volume and enthalpy are negative and there i s a very large negative excess entropy change over the entropy of i d e a l mixing. T h i s leads to a l a r g e p o s i t i v e change i n f r e e energy and hence, low s o l u b i l i t y (11). These abnormal changes are considered to be the r e s u l t of an o r d e r i n g o f water molecules around the hydrocarbon chain forming c l a t h r a t e or cagel i k e s t r u c t u r e s . The above mentioned n o n - i d e a l thermodynamic behavior of hydrocarbon s o l u t i o n s i s due to the formation of these c l a t h r a t e s t r u c t u r e s and to the o r i e n t a t i o n a l s p e c i f i c i t y of the water molecules i n these c l a t h r a t e s t r u c t u r e s compared to that i n the f r e e bulk water i n the absence of nonpolar s o l u t e s (12). The requirements on the o r i e n t a t i o n a l s p e c i f i c i t y of water molecules f o r the formation of c l a t h r a t e - l i k e s t r u c t u r e s around hydrocarbons i s more s p e c i f i c (Figure 1; 12). F i g u r e 1A r e p r e sents the water p a i r geometry i n i c e or i d e a l i z e d bulk water. This i s the g e o m e t r i c a l l y p o s s i b l e o r i e n t a t i o n of two hydrogen bonded water molecules which permit the maximum entropy p o s s i b l e . The s t r u c t u r e shown i n Figure IB represents the water-water o r i e n t a t i o n i n c l a t h r a t e hydrates. The d i f f e r e n c e between these two o r i e n t a t i o n s i s that i n the second c o n f i g u r a t i o n one of the water molecules i s r o t a t e d through a t e t r a h e d r a l angle on the hydrogen bond. T h i s s t r u c t u r e w i l l have a higher energy s t a t e because of the closeness of the hydrogen atoms and the lone p a i r s of e l e c t r o n s which w i l l enhance the r e p u l s i v e i n t e r a c t i o n (12, 13). This would e x p l a i n the p o s i t i v e standard f r e e energy change observed f o r the t r a n s f e r o f hydrocarbons i n t o aqueous s o l u t i o n . Furthermore, such o r d e r i n g of the water molecules around the hydrocarbon would r e s t r i c t t h e i r freedom of r o t a t i o n a l and t r a n s l a t i o n a l motions and thus decrease the entropy of the system (14)· Such a s t a t e would be h i g h l y unstable and would tend to go back

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DETERIORATION

Figure 1. Mutual orientation of two water molecules in ice (A) (and liquid water), and in clathrate hydrates (B). Dots indicate the direction of lone electron pair orbitals, small circles refer to hydrogen atoms and big circles refer to oxygen atoms. Configuration Β is obtained from A by rotating one water molecule through a tetrahedral angle about the axis of the hydrogen bond, (Reproduced, with permis­ sion, from Ref. 12, Copyright 1975, Academic Press Inc. (London) Ltd.)

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

13.

DAMODARAN

A N D KINSELLA

Ion Effects

on

Proteins

331

to the o r i g i n a l s t a t e o f higher entropy. To do so i t i s o b l i g a tory f o r the system to expel the hydrocarbon from the s o l u t i o n . This i s p a r t i a l l y accomplished by f o r c i n g two hydrophobic molecules together, which allows formation o f a s i n g l e sphere of hyd r a t i o n around them with r e l e a s e o f some o f the water molecules to t h e i r e n t r o p i c a l l y f a v o r a b l e s t a t e (Figure 2 ) . Such grouping of hydrophobic molecules i n aqueous s o l u t i o n i s known as hydrophobic i n t e r a c t i o n . In other words, i t i s the i n t e r a c t i o n between the hydrophobic molecules f o r c e d by the thermodynamically unfavorable s t r u c t u r a l s t a t e of water i n the presence of these molecules. Since p r o t e i n s c o n t a i n such nonpolar r e s i d u e s , the hydrophobic i n t e r a c t i o n between these r e s i d u e s would r e s u l t i n the formation of hydrophobic regions which would impose a p a r t i c u l a r three dimensional conformation on the p r o t e i n . From the s o l u b i l i t y measurements of amino a c i d s i n water and i n e t h a n o l , Nozaki and Tanford (15) c a l c u l a t e d the f r e e energy o f t r a n s f e r of amino a c i d r e s i d u e s from water to e t h a n o l . Assuming that the t r a n s f e r from water t o ethanol i s comparable to t r a n s f e r of hydrophobic r e s i d u e s i n p r o t e i n from water environment to a nonpolar r e g i o n i n the p r o t e i n the data obtained by Nozaki and Tanford (15) suggest that on an average the hydrophobic f r e e energy c o n t r i b u t i o n f o r the p r o t e i n f o l d i n g and s t a b i l i t y by each hydrophobic r e s i d u e i s about -2 Kcal/mole r e s i d u e . Since many p r o t e i n s c o n t a i n about 4 0 to 60% content o f hydrophobic amino a c i d r e s i d u e s , i t may be deduced that the major f o r c e r e s p o n s i b l e f o r the s t a b i l i t y of the p r o t e i n conformation i s the hydrophobic interaction. As a c o r o l l a r y , i f the entropy d r i v e n hydrophobic f r e e energy i s r e s p o n s i b l e f o r the n a t i v e s t r u c t u r e of the p r o t e i n , then i t should be p o s s i b l e to manipulate the s t r u c t u r e o f the p r o t e i n by d e c r e a s i n g t h i s hydrophobic f r e e energy by changing the s o l vent c o n d i t i o n s . Such changes can be induced by c h a o t r o p i c i o n s . Before attempting t o study the i o n i c e f f e c t s on hydrophobic i n t e r a c t i o n , and hence on the p r o t e i n conformation and f u n c t i o n , i t i s p e r t i n e n t to understand the e f f e c t s of ions on the s t r u c ture of water. The type of i n t e r a c t i o n between an i o n and water i n s o l u t i o n i s the strong i o n - d i p o l e compared to the weak d i p o l e induced d i p o l e i n t e r a c t i o n i n hydrocarbon s o l u t i o n s . Such i o n d i p o l e i n t e r a c t i o n would c e r t a i n l y d i s r u p t the r e g u l a r t e t r a h e d r a l l y hydrogen bonded s t r u c t u r e of water while concomitantly imposing a new k i n d o f order around each i o n . A d e t a i l e d d i s cussion on the s t r u c t u r e of water w i l l not be presented s i n c e t h i s i s done elsewhere (13, 16). The o r i e n t a t i o n of water molecules around an i o n depends on the type o f i o n ; i . e . c a t i o n o r anion. In the h y d r a t i o n s h e l l o f a c a t i o n the hydrogen atoms of water are d i r e c t e d out, whereas with the anion they are d i r e c t e d i n (Figure 3)· T h i s q u a l i t a t i v e d i f f e r e n c e i n the o r i e n t a t i o n o f the water molecules around ions i t s e l f profoundly a f f e c t s the p o l a r i t y o f the bulk water (17) and i n t h i s respect the anions decrease the p o l a r i t y o f water to a

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332

FOOD

PROTEIN

DETERIORATION

Free water Figure 2,

Figure 3.

Scheme of water structure induced association of hydrocarbons.

Scheme of orientation of water molecules in the hydration spheres of cations and anions.

DAMODARAN

13.

A N D KINSELLA

Ion Effects

on

Proteins

333

greater extent than c a t i o n s . The degree to which the water s t r u c t u r e i s a f f e c t e d depends on the s i z e and charge d e n s i t y of the anion. In general f o r anions, the extent of s t r u c t u r e breaking e f f e c t on water p r o g r e s s i v e l y f o l l o w s the order F~* < CHLCOO"" < S0 < C I " < Br"" < I"" < N0 ~ < ClO^-SCN"" < C l CCOO"; t h i s s e r i e s i s known as the l y o t r o p i c o r c h a o t r o p i c s e r i e s (18). One of the thermodynamic m a n i f e s t a t i o n s o f the e f f e c t o f ions on water s t r u c t u r e i s the p a r t i a l molar entropy (Table I ) . For monovalent i o n s , a p r o g r e s s i v e i n c r e a s e i n entropy accompanies with i n c r e a s i n g i o n i c r a d i u s , suggesting that the entropy gain may a r i s e p r i m a r i l y from the s t e r i c hindrance to the format i o n o f the water framework. =

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

4

3

Table I . Anion

E f f e c t s of anions on water s t r u c t u r e . * S.0(M)

a

S^ h y d r a t i o n

1

F~~ ciBr~ I~ SCN"

-2.3 13.5 19.7 25.3 36.0

P a r t i a l molar entropy of aqueous ions; of h y d r a t i o n . *Taken from H a t e f i and Hanstein (18).

-31.2 -18.2 -14.4 -10.1 - 8.1 p a r t i a l molar entropy

I t may a l s o be noted that with decreasing entropy of h y d r a t i o n , the p a r t i a l molar entropy o f the s o l u t i o n a l s o decreases. T h i s shows that the entropy of i o n i c s o l u t i o n s i s c l o s e l y r e l a t e d to the o r i e n t a t i o n and to magnitude of water molecules i n the hydrogen sphere o f the i o n s . Small ions with greater charge d e n s i t y on the surface tend to have the l e a s t e f f e c t on the entropy of bulk water, whereas large ions with lower charge d e n s i t y on the surface tend to break the hydrogen bonded s t r u c t u r e of water. Reviews on the e f f e c t of ions on the p h y s i c a l p r o p e r t i e s of water were presented by von Hippel and S c h l e i c h (16) and Dandliker and De Saussure (19). I t i s reasonable a t t h i s p o i n t to question what e f f e c t the chaotropic ions would have on the hydrophobic i n t e r a c t i o n s i n p r o t e i n s . Since the hydrophobic i n t e r a c t i o n i s i n t i m a t e l y r e l a t e d to the s t r u c t u r e of water, c h a o t r o p i c ions should have profound e f f e c t s on the s t r e n g t h o f the hydrophobic i n t e r a c t i o n . As explained i n the previous s e c t i o n s , the conformation of a p r o t e i n i n aqueous s o l u t i o n i s mainly induced by the c h a r a c t e r i s t i c s of the s o l v e n t such as water. Although the grouping of the nonpolar s i d e c h a i n s i n t o a p r o t e i n ' s hydrophobic r e g i o n i n v o l v e s a decrease i n the conformational entropy, the high gain i n the e n t r o py of the s o l v e n t i n the process o v e r r i d e s the decrease i n the

334

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Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

c o n f o r m a t i o n a l e n t r o p y and t h u s t h e r m o d y n a m i c a l l y s t a b i l i z e s t h e f o l d e d n a t i v e s t r u c t u r e ( 5 ) . I f t h e h y d r o g e n bonded s t r u c t u r e o f w a t e r i s a l t e r e d by c h a o t r o p i c i o n s , t h e d r i v i n g f o r c e f o r t h e h y d r o p h o b i c i n t e r a c t i o n s w i t h i n and between p r o t e i n m o l e c u l e s ( a s s o c i a t i o n and p o l y m e r i z a t i o n r e a c t i o n s ) w i l l d e c r e a s e a n d hence would d e s t a b i l i z e t h e i r s t r u c t u r e s . E f f e c t o f s a l t s on t h e s o l u b i l i t y o f h y d r o c a r b o n s a n d m o d e l peptides. One p o s s i b l e a p p r o a c h t o u n d e r s t a n d i n g t h e e f f e c t o f s a l t s on p r o t e i n c o n f o r m a t i o n and f u n c t i o n i s t o s t u d y t h e s o l u ­ b i l i t y o f a p p r o p r i a t e m o d e l compounds i n s a l t s o l u t i o n s . The c l a s s i c a l s t u d y was on t h e s o l u b i l i t y o f a m o d e l p e p t i d e , a c e t y l t e t r a g l y c y l e t h y l e s t e r (ATGEE), i n s a l t s o l u t i o n s ( 2 0 ) , T h i s s t u d y showed t h a t t h e l o g a r i t h m o f t h e a c t i v i t y c o e f f i c i e n t ( l o g S /S) o f ATGEE i n s a l t s o l u t i o n s e x h i b i t e d a l i n e a r r e l a ­ tionship with the s a l t concentration. T h a t i s E q u a t i o n 3: 0

Log

S /S 0

=

K C S

S

where S i s t h e s o l u b i l i t y o f ATGEE i n w a t e r , S i s t h e s o l u b i l i t y i n a s a l t s o l u t i o n , C i s t h e m o l a r c o n c e n t r a t i o n o f t h e added s a l t and K i s t h e s a l t i n g - o u t c o n s t a n t . Here i t s h o u l d be men­ t i o n e d t h a t a n i n c r e a s e i n t h e s o l u b i l i t y o f ATGEE i n a s a l t s o l u ­ t i o n corresponds t o a decrease i n the a c t i v i t y c o e f f i c i e n t (S /S). I o n s s u c h a s I " * , C 1 0 ^ " , SCN~ a n d C I 3 C C O O " e x h i b i t e d n e g a t i v e K v a l u e s , w h i l e S 0 ^ , ^PO^" a n d c i t r a t e h a d p o s i t i v e K values. C h l o r i d e a n d b r o m i d e e x h i b i t e d n e i t h e r s a l t i n g - i n nor s a l t i n g - o u t p r o p e r t i e s ; i . e . , t h e i r K v a l u e s were a l m o s t z e r o . I n a s i m i l a r a p p r o a c h S c h r i e r a n d S c h r i e r (21) s t u d i e d t h e s o l u b i l i t i e s o f N - m e t h y l a c e t a m i d e and N - m e t h y l p r o p i o n a m i d e i n various s a l t s o l u t i o n s . While theorder of effectiveness of v a r i ­ ous n e u t r a l s a l t s a s a c t i v i t y c o e f f i c i e n t a f f e c t o r s were t h e same as t h a t o f ATGEE, t h e l e v e l a t w h i c h Κ c h a n g e s f r o m a p o s i t i v e t o a n e g a t i v e v a l u e f o r t h e s a l t s was d i f f e r e n t . F u r t h e r m o r e , i n c r e a s e i n t h e c h a i n l e n g t h o f t h e amide i n c r e a s e d t h e s a l t i n g out c o e f f i c i e n t i n d i c a t i n g t h a t t h e e f f e c t o f i o n s on t h e s o l u ­ b i l i t y b e h a v i o r o f t h e s e m o d e l compounds was m a i n l y on t h e n o n p o l a r m o i e t i e s i n t h e s e m o d e l compounds. The s a l t i n g - o u t c o n ­ s t a n t f o r N - m e t h y l a c e t a m i d e was 0.099 i n N a C l a n d -0.023 i n NaSCN. The change i n t h e f r e e e n e r g y f o r t r a n s f e r o f one mole o f N-methy l a c e t a m i d e f r o m w a t e r t o 1 M s a l t s o l u t i o n c a n be c a l c u l a t e d from the K v a l u e s by E q u a t i o n 4 ( 2 2 ) : Q

g

g

Q

g

=

g

g

g

AG

= 2.3 RTK C tr s s where C i s t h e m o l a r c o n c e n t r a t i o n o f t h e s a l t , R i s t h e g a s c o n s t a n t a n d Τ i s t h e t e m p e r a t u r e . A t 25°C, t h e f r e e e n e r g y o f t r a n s f e r o f N-methylacetamide from water t o 1M NaCl i s u n f a v o r ­ a b l e t o t h e m a g n i t u d e o f a b o u t +133 c a l / m o l w h e r e a s t h e t r a n s f e r t o 1 M NaSCN i s f a v o r a b l e t o t h e o r d e r o f -30.9 c a l / m o l e . The e f f e c t s o f n e u t r a l s a l t s o n s o l u b i l i t y o f many a p o l a r g

13.

DAMODARAN A N D

KINSELLA

Ion Effects

on

335

Proteins

s o l u t e s such as amides, benzene, purines and p y r i m i d i n e s , e t c . , i n aqueous s o l u t i o n s were reported (22, 23). These s t u d i e s with simple compounds r e v e a l that the n e u t r a l s a l t s a f f e c t both the p o l a r and apolar moieties i n these compounds and c e r t a i n ions e x h i b i t p o s i t i v e e f f e c t s while other ions have negative e f f e c t s on s o l u b i l i t y . In general the s a l t i n g - o u t e f f e c t of the anions and c a t i o n s f o l l o w the order =

S0, > C l ~ > Br" > NO ~ > CIO," > SCN~ > C l C C 0 0 " 4 3 4 3 + + + + 2+ 2+ 2+ NH > Κ ,Na > L i > Mg > Ca > Ba

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

o

Since p r o t e i n s have both p o l a r and apolar groups, more com­ parable to the above model compound, the s a l t i n g - i n or s a l t i n g out e f f e c t o f i o n s on these groups would have profound a f f e c t on the conformational s t a b i l i t y o f p r o t e i n s . Since the hydrophobic i n t e r a c t i o n between the apolar residues i s the major s t a b i l i z i n g force of the n a t i v e conformation o f the p r o t e i n , the ions which s a l t - o u t the hydrocarbons should enhance these hydrophobic i n t e r ­ a c t i o n s i n the p r o t e i n and provide more s t a b i l i z i n g energy. In the presence of ions which e x h i b i t s a l t i n g - i n e f f e c t on hydro­ carbons , the hydrophobic i n t e r a c t i o n s i n the p r o t e i n would be de­ creased and hence d e s t a b i l i z e the p r o t e i n s t r u c t u r e . The simplest way to study the e f f e c t s o f ions on the confor­ mational s t a b i l i t y o f p r o t e i n s i s t o f o l l o w the change i n α-heli­ c a l content o f a p r o t e i n as a f u n c t i o n o f temperature a t d i f f e r ­ ent s a l t concentrations. von Hippel and Wong (24) s t u d i e d the e f f e c t s of CaCl2 on the melting temperature o f c o l l a g e n at ρΗ 7.0. The melting temperature, Τ , which i s defined as the tem­ perature at which the t r a n s i t i o n from h e l i c a l conformation to random c o i l i s 50% complete, decreased l i n e a r l y with increase i n CaCl2 c o n c e n t r a t i o n . In a s i m i l a r approach von H i p p e l and Wong (25) s t u d i e d the temperature t r a n s i t i o n o f ribonuclease as a f u n c t i o n o f s a l t c o n c e n t r a t i o n . The p l o t s o f T versus s a l t con­ c e n t r a t i o n f o r many s a l t s e x h i b i t e d l i n e a r r e l a t i o n s h i p s . In general the r e l a t i o n s h i p between T and the s a l t concentration may be f u n c t i o n a l l y expressed as Equation 5 (16): m

m

Τ

m

= Τ ° + ΚC m ss

where T ° i s the t r a n s i t i o n temperature i n the absence o f s a l t , C i s the molar c o n c e n t r a t i o n o f the s a l t and K i s the molar e f f e c t i v e n e s s of the s a l t i n p e r t u r b i n g T . In the study with r i b o n u c l e a s e , PO^", S0^ and C l ~ e x h i b i t e d p o s i t i v e K values i n d i c a t i n g that these ions s t a b i l i z e d the p r o t e i n . Br and SCN" e x h i b i t e d negative K values i n d i c a t i n g that they d e s t a b i l i z e d the p r o t e i n s t r u c t u r e . The r e l a t i v e e f f e c t i v e n e s s o f both anions and c a t i o n s i n p e r t u r b i n g the s t a b i l i t y o f r i b o n u c l e a s e followed the same chaotropic or l y o t r o p i c order observed with model com­ pounds. One can assume that the observed e f f e c t s o f ions on the m

g

s

m

=

g

g

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336

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s t a b i l i t y o f p r o t e i n s c a n n o t be due t o s i m p l e n o n s p e c i f i c i n t e r a c t i o n s w i t h t h e i r charged groups. As mentioned i n t h e p r e v i o u s s e c t i o n , t h e f r e e e n e r g y c o n t r i b u t e d by t h e e l e c t r o s t a t i c i n t e r a c t i o n s t o t h e o v e r a l l s t a b i l i t y o f p r o t e i n i s v e r y s m a l l and hence cannot a c c o u n t f o r t h e magnitude o f t h e s a l t e f f e c t on p r o t e i n c o n f o r m a t i o n . I n most p r o t e i n s t h e e l e c t r o s t a t i c i n t e r a c t i o n s a r e e s s e n t i a l l y s u p p r e s s e d a t about 0.1-0.15 M s a l t c o n c e n t r a t i o n (26). H e n c e , t h e l y o t r o p i c e f f e c t o f i o n s o n t h e p r o t e i n conformation, which i s observed a t r e l a t i v e l y higher conc e n t r a t i o n s , may be due t o t h e i r e f f e c t on t h e h y d r o p h o b i c i n t e r a c t i o n s mediated v i a changes i n t h e w a t e r s t r u c t u r e . I f t h e c o n f o r m a t i o n a l changes i n p r o t e i n i n t h e presence o f v a r i o u s i o n s a r e due t o s t a b i l i z a t i o n o r d e s t a b i l i z a t i o n o f t h e h y d r o p h o b i c r e g i o n s t h a t a r e mediated v i a changes i n t h e w a t e r s t r u c t u r e , t h e n s u c h c h a n g e s w i l l be r e f l e c t e d i n t h e b i n d i n g a b i l i t y o f h y d r o p h o b i c l i g a n d s t o t h e p r o t e i n . Damodaran and K i n s e l l a (27) s t u d i e d t h e e f f e c t o f a n i o n s o n t h e b i n d i n g o f 2nonanone t o b o v i n e serum a l b u m i n . B i n d i n g o f 2-nonanone t o b o v i n e serum a l b u m i n e x h i b i t e d p o s i t i v e c o o p e r a t i v i t y w h i c h was i n t e r p r e t e d as b e i n g due t o i n i t i a l b i n d i n g i n d u c e d c o n f o r m a t i n a l changes i n t h e p r o t e i n w h i c h i n c r e a s e d t h e b i n d i n g a f f i n i t i e s a t t h e s u b s e q u e n t b i n d i n g s i t e s (27). I n o t h e r w o r d s , t h e p o s i t i v e c o o p e r a t i v i t y may be due t o s t a b i l i z a t i o n o f t h e h y d r o p h o b i c b i n d i n g s i t e s i n b o v i n e serum a l b u m i n upon i n i t i a l b i n d ing. As a c o r o l l a r y , i f t h e e x h i b i t i o n o f p o s i t i v e c o o p e r a t i v i t y i s due t o b i n d i n g i n d u c e d s t a b i l i z a t i o n o f t h e b i n d i n g s i t e s , then i n the presence o f s t r u c t u r e s t a b i l i z i n g o r d e s t a b i l i z i n g a g e n t s t h e d e g r e e o f s u c h s t a b i l i z a t i o n s h o u l d be i n c r e a s e d o r decreased, r e s p e c t i v e l y . The b i n d i n g i s o t h e r m s f o r t h e i n t e r a c t i o n o f 2-nonanone w i t h b o v i n e serum a l b u m i n i n t h e p r e s e n c e o f v a r i o u s a n i o n s i s shown i n F i g u r e 4. I n t h e p r e s e n c e o f C l " * , Br"", SCN" and C I 3 C C O O " , t h e c u r v e s a r e s i g m o i d a l , w h e r e a s i n t h e p r e s e n c e o f F " and S0^ , t h e y a r e h y p e r b o l i c . When t h e same d a t a a r e p r e s e n t e d i n t h e f o r m of Scatchard p l o t s , the b i n d i n g e x h i b i t e d p o s i t i v e c o o p e r a t i v i t y i n t h e p r e s e n c e o f a l l a n i o n s e x c e p t F~ ( F i g u r e 5 ) . The i n i t i a l p o s i t i v e slopes a t low molal r a t i o s o f binding v a r i e d d i s t i n c t l y w i t h the type o f a n i o n . The e f f e c t o f a n i o n s i n i n c r e a s i n g t h e i n i t i a l p o s i t i v e slope followed the c l a s s i c a l Hofmeister s e r i e s o r c h a o t r o p i c s e r i e s (18) ; i . e . , F" > S O ^ > CI"" > B r " > SCN" > CI3CCOO"". These i o n s , i n t h e same o r d e r , have b e e n known t o a f f e c t t h e w a t e r s t r u c t u r e (18) as w e l l as s t a b i l i t y o f g l o b u l a r p r o t e i n s (16). Apart from changing the s l o p e s o f t h e Scatchard p l o t s , these ions a l s o induced a s h i f t i n the p o s i t i o n o f the maximum i n t h e S c a t c h a r d p l o t away f r o m t h e o r d i n a t e . A s h i f t away f r o m t h e o r d i n a t e may r e f l e c t i n c r e a s e d c o o p e r a t i v i t y i n binding. I n t h i s r e s p e c t , t h e r e s u l t s show t h a t t h e b i n d i n g o f 2-nonanone t o b o v i n e serum a l b u m i n i s h i g h l y c o o p e r a t i v e i n t h e p r e s e n c e o f Cl^CCOO" and SCN" i n c o n t r a s t t o i t s b e h a v i o r i n t h e p r e s e n c e o f SO, and F". =

=

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13.

Figure 4. Binding isotherms for the interaction of 2-nonanone to bovine^ serum albumin (BSA) in the presence of various anions at 0.15 M ionic strength, ν is the number of moles of 2-nonanone bound per mole of BSA and [L] is the free 2-non­ anone concentration. (Reproduced from Ref. 27.)

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The above changes i n the b i n d i n g behavior o f 2-nonanone to bovine serum albumin i n the presence of v a r i o u s anions may be i n t i m a t e l y r e l a t e d to changes i n the p r o t e i n s t r u c t u r e . I n other words, the i n i t i a l p o s i t i v e slopes (Figure 5 ) , which may be cons i d e r e d to be p r o p o r t i o n a l to the net s t a b i l i z a t i o n o f the hydrophobic s i t e s upon l i g a n d b i n d i n g , are the r e s u l t o f two opposing f o r c e s ; i . e . , the d e s t a b i l i z i n g e f f e c t o f anions and the s t a b i l i z i n g e f f e c t o f 2-nonanone upon b i n d i n g . Assuming that the s t a b i l i z a t i o n energy provided by 2-nonanone b i n d i n g i s constant, the i n c r e a s e i n the d e s t a b i l i z i n g energy provided by d i f f e r e n t anions would o b v i o u s l y decrease the i n i t i a l p o s i t i v e slope i n the order of the l y o t r o p i c s e r i e s . If the d e s t a b i l i z a t i o n / s t a b i l i z a t i o n o f the p r o t e i n s t r u c ture by anions i s v i a changes i n the water s t r u c t u r e , then one would expect a c o r r e l a t i o n between the changes i n the b i n d i n g parameters and the p a r t i a l molar entropy o f the s o l u t i o n s o f v a r ious anions. F i g u r e 6 shows the c o r r e l a t i o n between the f r e e energy of i n t e r a c t i o n , AG , (27), which i s c a l c u l a t e d from the i n i t i a l p o s i t i v e s l o p e s , t i e H i l l c o e f f i c i e n t (28) which i n d i cates the degree o f cooperative changes, and the p a r t i a l molar entropy o f s o l u t i o n . I t i s obvious that as the p a r t i a l molar entropy o f the s o l u t i o n i s increased (which i n d i c a t e s increased d i s o r d e r l i n e s s of the water s t r u c t u r e ) , the hydrophobic s i d e chains i n the b i n d i n g s i t e s would be d i s s o c i a t e d mainly by a decrease i n the hydrophobic i n t e r a c t i o n between them. In such an environment, the b i n d i n g induced s t a b i l i z a t i o n o f the b i n d i n g s i t e s w i l l be r e l a t i v e l y unfavorable. This i s r e f l e c t e d by an increase i n the f r e e energy change (more p o s i t i v e ) with i n c r e a s e i n the p a r t i a l molar entropy o f the s o l u t i o n . However, the coo p e r a t i v i t y o f 2-nonanone b i n d i n g to bovine serum albumin i n creases with i n c r e a s e i n the p a r t i a l molar entropy o f the s o l u t i o n as revealed from the H i l l c o e f f i c i e n t . In the presence o f F~ i o n which has s a l t i n g - o u t e f f e c t on hydrocarbons, i t i s p o s s i ble that the s t r u c t u r a l s t a t e o f the b i n d i n g s i t e s would be compact so that the i n i t i a l b i n d i n g o f 2-nonanone would not r e s u l t i n f u r t h e r s t a b i l i z a t i o n o f the b i n d i n g s i t e s . In the presence of CI"*, Br"" and SCN" which have p o s i t i v e p a r t i a l molar entropy values (Table I; 18), the b i n d i n g s i t e s may have some degree of freedom f o r f u r t h e r strengthening o f these regions upon 2-nonanone b i n d i n g . T h i s i s r e f l e c t e d i n the c o o p e r a t i v i t y o f b i n d i n g . I t may be pointed out that the above b i n d i n g s t u d i e s with the various ions were done a t 0.15 M i o n i c s t r e n g t h . The r e s u l t s show that even a t t h i s i o n i c s t r e n g t h the anions e x h i b i t l y o t r o pic e f f e c t s on the hydrophobic f o r c e s i n the p r o t e i n , and such e f f e c t s f o l l o w the same Hofmeister s e r i e s as one would expect a t much higher s a l t c o n c e n t r a t i o n s . This i s f u r t h e r confirmed from the s o l u b i l i t y s t u d i e s o f 2-nonanone a t 0 to 0.15 M concentration of NaCl and NaSCN (Figure 7). Even a t t h i s low c o n c e n t r a t i o n , NaCl tends to s a l t - o u t 2-nonanone whereas NaSCN tends to s a l t - i n the hydrocarbon. This i n d i c a t e s that even a t t h i s low

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Figure 6. Relationship between partial molar entropy of solutions of various anions and the free energy of interaction, aG (O - O), and the Hill coefficient (• - •). (Reproduced from Ref. 27.) int

C10^, I " > B r " > CI"". S i n c e a p p a r e n t l y no e l e c t r o s t a t i c i n t e r a c t i o n s are i n v o l v e d i n the p o l y m e r i z a t i o n o f s i c k l e hemoglobin, the s a l t i n g - i n e f f e c t by s a l t s c a n n o t be due t o e l e c t r o s t a t i c s h i e l d ing effects. Even i f e l e c t r o s t a t i c i n t e r a c t i o n s a r e i n v o l v e d , a s o l u t i o n a b o u t 0.15 M i o n i c s t r e n g t h s h o u l d be s u f f i c i e n t t o s u p p r e s s t h e s e i n t e r a c t i o n s ( 2 6 ) . The o b s e r v e d s a l t i n g - i n e f f e c t e v e n a t c o n c e n t r a t i o n s a s h i g h as 1 M i n d i c a t e s t h a t t h e

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Salting-in effects of anions on the solubility of deoxysickle hemoglobin. (Reproduced from Ref. 36.)

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s o l u b i l i z a t i o n o f s i c k l e h e m o g l o b i n was due t o d e c r e a s e d h y d r o p h o b i c i n t e r a c t i o n s between s i c k l e hemoblobin m o l e c u l e s . This i s incompatible w i t h the n o t i o n that the increase i n surface t e n s i o n o f the s o l u t i o n should always r e s u l t i n s a l t i n g - o u t o f the p r o t e i n (33). A l s o , i t i s g e n e r a l l y b e l i e v e d t h a t t h e e f f e c t of i o n s on the h y d r o p h o b i c i n t e r a c t i o n s o c c u r o n l y a t h i g h e r s a l t c o n c e n t r a t i o n s (16)· The d a t a on s i c k l e h e m o b l o b i n and o u r r e ­ s u l t s on t h e s o l u b i l i t y ( F i g u r e 7 ) and b i n d i n g o f 2-nonanone t o b o v i n e serum a l b u m i n ( 2 7 ) i n d i c a t e t h a t t h e l y o t r o p i c e f f e c t o f i o n s on h y d r o p h o b i c i n t e r a c t i o n s c a n o c c u r a t r e l a t i v e l y l o w concentrations. The argument t h a t t h e d e c r e a s e i n e l e c t r o s t a t i c interactions at low s a l t c o n c e n t r a t i o n s i s r e s p o n s i b l e f o r the s a l t i n g - i n e f f e c t (34, 35) may be v a l i d f o r s i m p l e g l o b u l a r p r o t e i n s . B u t , complex p r o t e i n systems, l i k e p r o t e i n s h a v i n g m u l t i s u b u n i t s o r s y s t e m s h a v i n g more t h a n one f r a c t i o n , may behave d i f f e r e n t l y a t low s a l t c o n c e n t r a t i o n s . F o r example, as the s a l t c o n c e n t r a t i o n i s i n c r e a s e d t o a b o u t 0.2 M i o n i c s t r e n g t h , t h e s o l u b i l i t y o f s o y p r o t e i n d e c r e a s e s , and t h e n i n c r e a s e s a t h i g h e r c o n c e n t r a t i o n s ( F i g u r e 12; 38, 39). I n t h i s r e s p e c t b o t h N a C l and C a C l exhib­ i t e d t h e same e f f e c t s ( 3 9 ) . S i m i l a r r e s u l t s were o b t a i n e d w i t h s o l u b i l i t y o f wheat g l u t e n i n v a r i o u s s a l t s o l u t i o n s (40). T h u s , i f the e l e c t r o s t a t i c e f f e c t a t low s a l t c o n c e n t r a t i o n has s a l t i n g i n e f f e c t s , t h e n how d o e s t h e s o l u b i l i t y o f s o y p r o t e i n d e c r e a s e at low s a l t c o n c e n t r a t i o n . T h i s may be due t o c o m p l e x i n t e r ­ a c t i o n s i n v o l v i n g b o t h e l e c t r o s t a t i c and h y d r o p h o b i c f o r c e s b e ­ tween t h e s u b u n i t s and d i f f e r e n t p r o t e i n f r a c t i o n s ( g l y c i n i n and c o n g l y c i n i n ) i n soy. Although the e l e c t r o s t a t i c s h i e l d i n g of the c h a r g e d g r o u p s by s a l t s may i n c r e a s e p r o t e i n s o l u b i l i t y , t h e d e ­ c r e a s e i n t h e e l e c t r o s t a t i c r e p u l s i o n b e t w e e n p r o t e i n s may e n ­ hance t h e h y d r o p h o b i c i n t e r a c t i o n b e t w e e n t h e i r n o n p o l a r s u r f a c e s and l e a d t o f o r m a t i o n o f a g g r e g a t e s . This i s f u r t h e r supported by t h e f a c t t h a t t h e o b s e r v e d s o l u b i l i t y i s minimum a t a b o u t 0.Ι­ Ο.15 M i o n i c s t r e n g t h ( F i g u r e 1 2 ) , a t w h i c h t h e e l e c t r o s t a t i c i n t e r a c t i o n i n p r o t e i n s i s s u p p r e s s e d ( 2 6 ) . The d a t a a l s o s u p ­ p o r t s o u r c o n t e n t i o n t h a t even a t low c o n c e n t r a t i o n s t h e i o n s h a v e l y o t r o p i c e f f e c t s on t h e h y d r o p h o b i c i n t e r a c t i o n s . I f t h e i o n i c strength i s s o l e l y responsible f o r the decrease i n the s o l ­ u b i l i t y a t low s a l t c o n c e n t r a t i o n s , then the s o l u b i l i t y l e v e l a t 0.15 M i o n i c s t r e n g t h s h o u l d be t h e same i r r e s p e c t i v e o f t h e n a ­ t u r e o f t h e i o n . B u t t h e d a t a ( F i g u r e 12) show t h a t t h e minimum s o l u b i l i t y l e v e l a t 0.15 M i o n i c s t r e n g t h v a r i e s w i t h t h e n a t u r e o f t h e a n i o n and t h e i n c r e a s e i n s o l u b i l i t y a t t h i s i o n i c s t r e n g t h a p p a r e n t l y f o l l o w s t h e l y o t r o p i c s e r i e s ; i . e . , Cl"*= B r " < N O 3 < I"". T h i s c l e a r l y i n d i c a t e s t h a t e v e n a t t h i s l o w c o n c e n ­ t r a t i o n t h e a n i o n s e x e r t l y o t r o p i c e f f e c t s on t h e h y d r o p h o b i c i n ­ t e r a c t i o n s . B u t , the magnitude o f t h i s l y o t r o p i c ( s o l u b i l i z i n g ) e f f e c t a t t h i s l o w s a l t c o n c e n t r a t i o n may be s m a l l e r t h a n t h e magnitude o f t h e f a v o r a b l e h y d r o p h o b i c i n t e r a c t i o n between mole­ c u l e s and h e n c e r e s u l t i n i n s o l u b i l i t y . As t h e s a l t c o n c e n t r a t i o n 2

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i s i n c r e a s e d , t h e m a g n i t u d e o f t h e s e h y d r o p h o b i c i n t e r a c t i o n s may be e i t h e r i n c r e a s e d o r d e c r e a s e d d e p e n d i n g on t h e l y o t r o p i c n a ­ t u r e o f t h e i o n and t h u s r e s u l t i n d e c r e a s e d o r i n c r e a s e d s o l u ­ b i l i t y , respectively. To s u p p o r t t h e above a r g u m e n t , we s t u d i e d t h e b i n d i n g o f 8 - a n i l i n o n a p h t h a l e n e - 1 - s u l f o n a t e (ANS) t o s o y p r o t e i n i n t h e presence of v a r i o u s s a l t s . The r a t i o n a l e i n t h i s method i s t h a t when ANS i s h y d r o p h o b i c a l l y bound t o s o y p r o t e i n , i t s quantum y i e l d of fluorescence i s increased. Thus by m o n i t o r i n g t h e f l u o r e s c e n c e o f ANS i n a s o y p r o t e i n - A N S s y s t e m , i t i s p o s s i b l e t o measure t h e c h a n g e s i n t h e h y d r o p h o b i c i n t e r a c t i o n a s a function of salt concentration. The e f f e c t s o f s a l t s on t h e f l u o r e s c e n c e i n t e n s i t y o f ANS bound t o s o y p r o t e i n i s shown i n F i g u r e 1 3 . The ANS c o n c e n t r a ­ t i o n was 1,54 χ 1 0 " ^ M and t h e p r o t e i n c o n c e n t r a t i o n was 0,0027%. A t t h i s p r o t e i n c o n c e n t r a t i o n no p r e c i p i t a t i o n was o b s e r v e d i n s a l t s o l u t i o n s a t t h e c o n c e n t r a t i o n range r e p o r t e d here. In the presence o f a l l the s a l t s s t u d i e d , the fluorescence i n t e n s i t y of ANS i n c r e a s e d up t o a b o u t 0.15 M s a l t c o n c e n t r a t i o n . The i n ­ crease i n the f l u o r e s c e n c e i n t e n s i t y a t t h i s low i o n i c s t r e n g t h i s due t o an i n c r e a s e i n t h e h y d r o p h o b i c i n t e r a c t i o n b e t w e e n ANS and s o y p r o t e i n . The f l u o r e s c e n c e i n t e n s i t y u n d e r g o e s a t r a n s i ­ t i o n a t a b o u t 0.15 M s a l t c o n c e n t r a t i o n and above 0.15 M t h e f l u o r e s c e n c e i n t e n s i t y e i t h e r i n c r e a s e s or decreases depending on t h e l y o t r o p i c e f f e c t o f t h e i o n on h y d r o p h o b i c i n t e r a c t i o n s . I n t h i s r e s p e c t S 0 ^ and F~ f u r t h e r i n c r e a s e d t h e f l u o r e s c e n c e , i n d i c a t i n g i n c r e a s e d h y d r o p h o b i c i n t e r a c t i o n b e t w e e n ANS and s o y p r o t e i n , and Br"", I"* and SCN~ i o n s d e c r e a s e t h e f l u o r e s c e n c e intensity. These r e s u l t s c l e a r l y d e m o n s t r a t e t h a t t h e o b s e r v e d d e c r e a s e i n t h e s o l u b i l i t y o f s o y p r o t e i n ( F i g u r e 11) a t l o w s a l t c o n c e n t r a t i o n s i s due t o enhancement o f h y d r o p h o b i c i n t e r a c t i o n s between p r o t e i n m o l e c u l e s . I n o t h e r w o r d s , t h e s a l t i n g - i n and s a l t i n g - o u t p r o f i l e o f p r o t e i n s by s a l t s a t v a r i o u s c o n c e n t r a ­ t i o n s i s the n e t r e s u l t o f the maximization or m i n i m i z a t i o n o f e l e c t r o s t a t i c and h y d r o p h o b i c f o r c e s , a t d i f f e r e n t s a l t c o n c e n ­ trations. I t w o u l d a l s o depend on t h e r a t i o o f n o n p o l a r s u r f a c e t o c h a r g e d e n s i t y on t h e s u r f a c e o f t h e p r o t e i n . I f t h e p r o t e i n has l a r g e r n o n p o l a r s u r f a c e a r e a s t h e d e c r e a s e i n t h e e l e c t r o ­ s t a t i c i n t e r a c t i o n a t l o w s a l t c o n c e n t r a t i o n s w o u l d promote h y ­ drophobic aggregation of such p r o t e i n s r e s u l t i n g i n decreased solubility. At higher s a l t c o n c e n t r a t i o n s , f u r t h e r decrease or i n c r e a s e i n s o l u b i l i t y depends on t h e l y o t r o p i c n a t u r e o f t h e ion. I n soy p r o t e i n , S 0 ^ f u r t h e r decreases the s o l u b i l i t y whereas o t h e r anions i n c r e a s e the s o l u b i l i t y i n the o r d e r o f C I " < B r " < NO3" < I*" ( F i g u r e 1 2 ) . On t h e o t h e r h a n d , i f t h e h y d r o p h o b i c s u r f a c e a r e a on t h e p r o t e i n i s l e s s and t h e c h a r g e d e n s i t y i s h i g h , t h e p r o t e i n w i l l be s a l t e d - i n a t l o w e r s a l t c o n c e n t r a t i o n s and a t h i g h e r s a l t c o n c e n t r a t i o n s t h e s o l u b i l i t y w o u l d f o l l o w t h e l y o t r o p i c e f f e c t o f d i f f e r e n t i o n s . Such com­ p l e x i n t e r p l a y o f d i f f e r e n t f o r c e s may be t h e r e a s o n f o r t h e =

=

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.(>

•4

Salt

-B

1-0

( one

Figure 13. Effect of sodium salts on the binding of 8-anilinonaphthalene-l-sulfo­ nate (ANS) to soybean protein in 30 mM tris-HCl buffer, pH 8.0. Protein and ANS concentrations were 0.04% and 1.5 χ 10 M , respectively. ANS binding was monitored as the increase in fluorescence intensity at 485 nm with excitation at 360 nm. 6

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observed d i f f e r e n c e s i n the s o l u b i l i t y b e h a v i o r of v a r i o u s p r o t e i n s i n s a l t s o l u t i o n s (36, 38, 4 0 , 41).

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Mechanisms o f S a l t E f f e c t on P r o t e i n

Structure

The e f f e c t s o f i o n s on p r o t e i n c o n f o r m a t i o n and f u n c t i o n may be d i v i d e d i n t o two c a t e g o r i e s : First, their electrostatic i n t e r a c t i o n w i t h t h e c h a r g e d g r o u p s and p o l a r g r o u p s ( e . g . , amide g r o u p s ) i n p r o t e i n s , and s e c o n d , t h e i r e f f e c t on h y d r o p h o b i c f o r c e s v i a i n f l u e n c e on t h e s t r u c t u r e o f w a t e r . From t h e f o r e g o i n g arguments t h e e l e c t r o s t a t i c s h i e l d i n g e f f e c t w h i c h r e a c h e s a maximum a t 0.15 M i o n i c s t r e n g t h may n o t have g r e a t i n f l u e n c e on t h e c o n f o r m a t i o n a l t r a n s i t i o n s i n p r o t e i n s t h a t a r e more p r o n o u n c e d a t h i g h s a l t c o n c e n t r a t i o n s . The s a l t may i n t e r a c t w i t h the p e p t i d e groups i n p r o t e i n s (42, 4 3 ) . Mandelkern e t a l . (44) have s u g g e s t e d t h a t s a l t i n t e r a c t i o n w i t h t h e p e p t i d e g r o u p s may a l t e r t h e p a r t i a l d o u b l e bond c h a r a c t e r o f t h e p e p t i d e bond. T h i s p e r m i t s f r e e r o t a t i o n o f the p e p t i d e backbone w h i c h m i g h t l e a d t o c o n f o r m a t i o n a l changes i n p r o t e i n s . A l t h o u g h the p o s s i b i l i t y o f s u c h i n t e r a c t i o n s b e t w e e n s a l t s and p e p t i d e g r o u p s e x i s t , i t has b e e n shown t h a t t h e h y d r a t i o n o f t h e i o n s g r e a t l y p r e v e n t s f o r m a t i o n o f s u c h c o m p l e x e s ( 4 5 ) . Hence i n aqueous s o l u t i o n s , c o n c e n t r a t i o n s a t which the ions induce c o n f o r m a t i o n a l changes i n p r o t e i n s , i t i s u n l i k e l y t h a t t h e f o r m a t i o n o f t h e c o m p l e x b e t w e e n i o n s and p e p t i d e g r o u p s i s t h e m a j o r c a u s e f o r t h e c o n f o r m a t i o n a l changes i n p r o t e i n s . Although i t i s thought t h a t s u c h a mechanism e x i s t s ( 4 3 ) , 46, 47) t h e r e a r e no d i r e c t evidence to support t h i s h y p o t h e s i s . The c o n f o r m a t i o n a l c h a n g e s i n p r o t e i n s i n t h e p r e s e n c e o f i o n s may be m a i n l y v i a i o n i c e f f e c t s on w a t e r s t r u c t u r e (16, 19). As m e n t i o n e d e a r l i e r , t h e n a t i v e s t r u c t u r e o f a p r o t e i n i s i m p o s e d by t h e s t a t e o f t h e w a t e r s t r u c t u r e b e c a u s e o f i t s t h e r m o dynamically unfavorable i n t e r a c t i o n with nonpolar sidechains. The c h a n g e s i n t h e h y d r o g e n bonded s t r u c t u r e o f b u l k w a t e r i n t h e p r e s e n c e o f s a l t s , due t o i o n - d i p o l e i n t e r a c t i o n , w o u l d a l t e r t h e d e g r e e o f h y d r a t i o n as w e l l as t h e o r i e n t a t i o n o f t h e w a t e r m o l e c u l e s around the n o n p o l a r s i d e c h a i n s . I n t h i s r e s p e c t , the i o n s w h i c h enhance t h e h y d r o g e n bonded s t r u c t u r e o f t h e b u l k w a t e r ( e . g . , F", S0^ ) p r e s u m a b l y h a v e t h e t e n d e n c y t o i n c r e a s e the degree of h y d r a t i o n of the n o n p o l a r groups i n p r o t e i n s . The i o n s w h i c h i n c r e a s e t h e e n t r o p y o f t h e b u l k w a t e r ( e . g . , B r " , I"", C10^~, SCN") tend to decrease the h y d r a t i o n o f the hydrophobic s i d e c h a i n s . As m e n t i o n e d e a r l i e r , t h e d r i v i n g f o r c e f o r t h e h y d r o p h o b i c i n t e r a c t i o n s between the n o n p o l a r m o l e c u l e s i s the e n t r o p i c a l l y u n f a v o r a b l e water-water o r i e n t a t i o n i n the h y d r a t i o n s p h e r e o f t h e h y d r o c a r b o n ( F i g u r e I B ) . Such r e l a t i v e o r i e n t a t i o n o f the water m o l e c u l e s around the hydrocarbon e x e r t s an i n t e r f a c i a l t e n s i o n o f a b o u t 51 e r g s / c m ^ o f w a t e r - h y d r o c a r b o n c o n t a c t a r e a (48). I n o t h e r w o r d s , s e p a r a t i o n o f 1 cm^ a r e a o f w a t e r - h y d r o c a r b o n i n t e r f a c e t o f o r m 1 cm^ o f w a t e r - w a t e r and =

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z

1 cm o f hydrocarbon-hydrocarbon i n t e r f a c e s w i l l be favored by about -102 e r g s . I t i s t h i s f r e e energy which i s r e s p o n s i b l e f o r the formation o f hydrophobic regions i n p r o t e i n s . The ions which s t a b i l i z e or d e s t a b i l i z e these hydrophobic regions may do so by i n c r e a s i n g o r d e c r e a s i n g the i n t e r f a c i a l tension, r e s p e c t i v e l y . I f we a s s i g n an i n t e r f a c i a l t e n s i o n o f 51 ergs/cm f o r the waterwater o r i e n t a t i o n i n the h y d r a t i o n s h e l l as shown i n Figure IB, (13), the ions which i n c r e a s e the i n t e r f a c i a l t e n s i o n above 51 ergs/cm^ may do so by a l t e r i n g the water-water geometry i n the h y d r a t i o n s h e l l i n such a way that the r e p u l s i o n between the lone p a i r o f e l e c t r o n s and the protons i s i n c r e a s e d . Conversely, the ions which decrease the i n t e r f a c i a l tension below 51 ergs/cm^ may do so by changing the water-water geometry such that the r e p u l s i v e i n t e r a c t i o n s between the lone p a i r o f e l e c t r o n s and the hydrogen atoms are decreased. However, such changes i n the water-water o r i e n t a t i o n i n the h y d r a t i o n s h e l l o f hydrocarbon by ions i s only through the changes i n the bulk water s t r u c t u r e , which can be d e f i n e d i n terms o f the p a r t i a l molar entropy. One can e x p e r i m e n t a l l y determine the decrease i n the i n t e r f a c i a l tension between water and hydrocarbon i n the presence of v a r i o u s i o n s . From these e s t i m a t i o n s and assuming that 51 ergs/cm corresponds to water-water o r i e n t a t i o n as shown i n Figure IB (12, 13), i t may be p o s s i b l e to c a l c u l a t e the r e l a t i v e water-water o r i e n t a t i o n around the hydrocarbon with a s t a t i s t i c a l - m e c h a n i c a l approach f o r v a r i o u s i n t e r f a c i a l tensions i n the presence of d i f f e r e n t i o n s . T h i s would provide an understanding of the r e l a t i o n s h i p s between the p a r t i a l molar entropy, i n t e r f a c i a l t e n s i o n and the hydrophobic e f f e c t and the i n f l u e n c e o f these e f f e c t s on p r o t e i n conformation and f u n c t i o n . Conclusion The primary e f f e c t of i o n s on p r o t e i n s t r u c t u r e a t low conc e n t r a t i o n i s t h e i r e l e c t r o s t a t i c i n t e r a c t i o n with the counter charges on the p r o t e i n . Such charge n e u t r a l i z a t i o n may induce d i s s o c i a t i o n o f the subunits i n o l i g o m e r i c p r o t e i n s which are h e l d together by e l e c t r o s t a t i c i n t e r a c t i o n s . At s u f f i c i e n t l y higher c o n c e n t r a t i o n s the ions e i t h e r weaken or strengthen the hydrophobic i n t e r a c t i o n s i n p r o t e i n s , depending on the nature o f the i o n . Such e f f e c t s on hydrophobic i n t e r a c t i o n s are mediated v i a changes i n the water s t r u c t u r e i n the presence of i o n s . However, some of the evidences suggest that even a t low concentrat i o n s (0.15 M) anions a f f e c t hydrophobic i n t e r a c t i o n s . The e f f e c t i v e n e s s of v a r i o u s anions i n d e s t a b i l i z i n g hydrophobic i n t e r a c t i o n s f o l l o w the Hofmeister s e r i e s . The changes i n e l e c t r o s t a t i c and hydrophobic f o r c e s i n p r o t e i n s by ions induce c o n f o r mational changes i n p r o t e i n s and hence a f f e c t t h e i r f u n c t i o n a l behavior. Systematic i n v e s t i g a t i o n of such e f f e c t s may provide b e t t e r understanding of the f a c t o r s a f f e c t i n g the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s i n food systems.

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Acknowledgment Support from the N a t i o n a l S c i e n c e F o u n d a t i o n Grant //CPE 80-18394 i s g r a t e f u l l y a c k n o w l e d g e d .

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2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Pain, R. H. In "Characterization of Protein Conformation and Function"; Franks, F . , Ed.; Symposium Press: London, 1978, p. 19. Klotz, I. M.; Franzen, J . S. J . Am. Chem. Soc., 1962, 84, 3461. Schachman, H. K. Cold Spring Harbor Symp. Quant. Biol., 1963, 28, 409. Ross, R. D.; Subramanian, S. Biochemistry, 1981, 20, 3096. Tanford, C. "Physical Chemistry of Macromolecules"; Wiley: New York, 1961; p. 480. Lumry, R.; Biltonen, R. In "Structure and Stability of Biological Macromolecules"; Timasheff, S. N. & Fasman, G. D., Eds.; Marcel-Dekker, Inc.: New York, 1969; p. 106. Kauzmann, W. Adv. Protein Chem., 1959, 14, 1. Nemethy, G.; Scheraga, H. A. J . Chem. Phys., 1962, 36, 3382. Nemethy, G.; Scheraga, H. A. J . Chem. Phys., 1962, 36, 3401. Miller, K. W.; Hildebrand, J . H. J . Am. Chem. Soc., 1968, 90, 3001. Frank, H. S.; Evans, M. W. J . Chem. Phys., 1945, 13, 507. Franks, F. In "Water Relations of Foods"; Duckworth, R. Β . , Eds.; Academic Press: New York, 1975; p. 3. Stillinger, F. H. Science, 1980, 209, 451. Lewin, S. "Displacement of Water and Its Control of Biochemical Reactions"; Academic Press: New York, 1974. Nozaki, Y.; Tanford, C. J . Biol. Chem., 1971, 276, 2211. von Hippel, P. H.; Schleich, T. In "Structure and Stability of Biological Macromolecules"; Vol. 2; Timasheff, S. N. & Farman, G. D., Eds.; Marcel-Dekker: New York, 1969; p. 417. Greyson, J . J . Phys. Chem., 1967, 71, 2210. Hatefi, Y . ; Hanstein, W. G. Proc. Natl. Acad. S c i . , 1969, 62, 1129. Dandliker, W. B.; De Saussure, V. A. In "The Chemistry of Biosurfaces"; Hair, M. L., Ed.; Marcel-Dekkar, Inc.: New York, 1971; p. 1. Robinson, D. R.; Jencks, W. P. J . Am. Chem. Soc., 1965, 87, 2470. Schrier, Ε. E.; Schrier, E. G. J. Phys. Chem., 1967, 71, 1951. Hamabata, A.; Chang, S.; von Hippel, P. H. Biochemistry, 1973, 12, 1271. Robinson, D. R.; Grant, M. E. J. Biol. Chem., 1966, 241, 4030. von Hippel, P. H.; Wong, Κ. Y. Biochemistry, 1963, 2, 1387.

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28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

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von Hippel, P. H.; Wong, Κ. Y. J. Biol. Chem., 1965, 240, 3909. Eagland, D. In "Water Relations of Foods"; Duckworth, R. B., Ed.; Academic Press: New York, 1975; p. 73. Damodaran, S.; Kinsella, J. E. J . Biol. Chem., 1980, 255, 8503. Dahlquist, F. W. Methods in Enzymology, 1978, 48, 270. Damodaran, S.; Kinsella, J. E. J . Biol. Chem., 1981, 256, 3394. Puett, D.; Garmon, R.; Ciferri, A. Nature, 1966, 211, 1294. Kendrew, J . C. Science, 1963, 139, 1259. Dixon, M.; Webb, E. C. Adv. Protein Chem., 1961, 16, 197. Melander, W.; Horvath, C. Arch . Biochem. Biophys., 1977, 183, 200. Czok, R.; Bucher, T. Adv. Protein Chem., 1960, 15, 315. Dixon, V. P.; Webb, E. C. Adv. Protein Chem., 1961, 16, 197. Poillon, W. N.; Bertles, J. F. J . Biol. Chem., 1979, 254, 3462. Benesch, R.; Benesch, R. Nature, 1981, 289, 637. Shen, J . L. In "Protein Functionality in Foods"; Cherry, J. J. P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981; p. 89. van Megan, W. H. J . Agric. Food Chem., 1974, 22, 126. Preston, K. R. Cereal Chem., 1981,58,317. Sawyer, W. H.; Puckridge, J. J. Biol. Chem., 1973, 248, 2429. Bello, J . ; Bello, H. R. Nature, 1961, 190, 440. Jencks, W. P. Federation Proceedings, 1965, 24, S-50. Mandelkern, L . ; Halpin, J . C.; Posner, A. S. J. Am. Chem. Soc., 1962, 84, 1383. Diorio, A. F.; Lippincott, E.; Mandelkern, L. Nature, 1962, 195, 1296 Gordon, J . Α.; Jencks, W. P. Biochemistry, 1963, 2, 47. Herskovits, T. T. Biochemistry, 1963, 2, 335. Tanford, C. Proc. Natl. Acad. Sci., 1979, 76, 4175.

RECEIVED June1,1982.

14 Effect of Disulfide Bond Modification on the Structure and Activities of Enzyme Inhibitors MENDEL FRIEDMAN, OK-KOO K. GROSJEAN, and JAMES C. ZAHNLEY

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

United States Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Berkeley, CA 94710

Enzyme (trypsin, chymotrypsin, α-amylase, carboxypeptidase, etc.) inhibitors appear in many agricultural products (1-4) such as legumes (4), corn (5), wheat (6), and potatoes (7,8). Soybeans and other legumes containing active trypsin inhibi­ tors depress growth in rats compared to analogous feeding of inhibitor-free soybeans. Growth inhibition and its accompanying pancreatic hypertrophy are presumably partly due to the antitryptic activity of the inhibitor(s) (4, 9-13). Since naturally occurring protease inhibitors such as trypsin and chymotrypsin inhibitors are proteins that require their own structural integrity in order to inactivate proteolytic enzymes by complex formation (14-19), any alteration of the inhibitor's structure will inactivate it if interaction with the enzyme is thereby prevented. A reasonable strategy is to cleave the inhibitor's disulfide bonds, which often control protein structure (20). Reduced trypsin inhibitors are expected to be inactive. In addition to the cited primary benefit, i.e., inactivation of protease inhibitors, reductive cleavage of disulfide bonds of both inhibitors and structural proteins in legumes by, for example, cysteine or other thiols, should also offer an important secondary benefit, namely, improvement in the digestibility of the treated proteins, because disulfide cleavage is expected to decrease crosslinking thus permitting more rapid digestion. In this chapter we examine effects of 1) reductive S-pyridylethylation and S-quinolylethylation of disulfide bonds on analytical, structural, and inhibitory aspects of enzyme inhibitors; and, 2) cooperative and synergistic effects of heat and thiols in inactivating soybean and lima bean inhibitors.

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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FOOD P R O T E I N DETERIORATION

Procedures

Enzymes, i n h i b i t o r s , and s u b s t r a t e s were o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n o r Sigma C h e m i c a l Co. Soy f l o u r was a g i f t o f A r c h e r D a n i e l s M i d l a n d C o r p o r a t i o n (Decatur, ID. L i m a bean f l o u r was p r e p a r e d f r o m raw beans p u r c h a s e d i n a l o c a l s t o r e . 2 - V i n y I p y r i d i n e was o b t a i ned f r o m A l d r i c h C h e m i c a l Co. and was r e d i s t i I Ied b e f o r e u s e . 2 - V i n y l q u i n o l i n e was f r o m Eastman Kodak and was v a c u u m - d i s t i l l e d b e f o r e u s e . T r i b u t y I phosph î ne was o b t a i ned f r o m A I d r i c h C h e m i c a l Co. and was s t o r e d under n i t r o g e n . S - 6 - ( 2 - P y r i d y l e t h y I ) - L - c y s t e i ne (2-PEC) was s y n t h e s i z e d a s d e s c r i b e d by F r i e d m a n and Noma (21 ) and S-Ê?( 2 - q u i n o l y l e t h y I ) - L - c y s t e i n e (2-QEC) a c c o r d i n g t o t h e p r o c e d u r e o f K r u I I e t a_[. ( 2 2 ) . O t h e r c h e m i c a l s were r e a g e n t g r a d e . Instrumental• A b s o r p t i o n s p e c t r a were r e c o r d e d on a C a r y 15 s p e c t r o p h o t o m e t e r ; and t r y p s i η was a s s a y e d by a b s o r b a n c e measured w i t h a Beckman DB s p e c t r o p h o t o m e t e r . A R a d i o m e t e r pHM 26 m e t e r and Beckman 39030 thiη-probe c o m b i n a t i o n e l e c t r o d e were used f o r most pH m e a s u r e m e n t s . Τitrimetric ( p H - s t a t ) e x p e r i ­ ments were c a r r i e d o u t u s i n g a R a d i o m e t e r TTT1b T i t r a t o r , SBR 2c T i t r i g r a p h , S B U l a s y r i n g e b u r e t t e , and a TTA31 m i c r o t i t r a t i o n a s s e m b l y , e q u i p p e d w i t h a G2222c g l a s s e l e c t r o d e and a K4112 c a l o m e l e l e c t r o d e . C o n s t a n t t e m p e r a t u r e was ma i n t a i ned w i t h a Haake Model F E c i r c u l a t o r and a t h e r m o s t a t t i n g j a c k e t ( R a d i o m e t e r V 5 2 6 ) . Thermal d e n a t u r a t i o n measurements were made w i t h a DuPont Model 990 d i f f e r e n t i a l s c a n n i n g c a l o r i m e t e r ( D S C ) , a t a h e a t i n g r a t e o f 10 °/m i η and a n o m i n a l s e n s i t i v i t y o f 100 y e a I s " · C a l i b r a t i o n and o p e r a t i o n o f t h e DSC and t h e i n t e r p r e t a t i o n o f r e s u I t s have been d e s c r i bed ( 2 3 , 2 4 , 2 5 ) • F I u o r e s c e n c e s p e c t r a were r e c o r d e d on a T u r n e r Model 210 s p e c t r o f I u o r o m e t e r . S o l u t i o n s . B u f f e r s were p r e p a r e d a t room t e m p e r a t u r e by a d d i n g HCI o r NaOH t o a d j u s t t h e pH t o t h e d e s i r e d v a l u e . D i s t i I l e d , de i o n i z e d w a t e r was u s e d . Un I e s s o t h e r w i s e i n d i c a t e d , n a t i v e and S - p y r i d y I e t h y I a t e d t r y p s i n i n h i b i t o r s were d i s s o l v e d i n 0.05 M g l y c i n e a d j u s t e d w i t h 1 H_ HCI t o pH 3.0. T r y p s i n was d i s s o l v e d i n 2 mM HCI/20 mM C a C U , and k e p t a t 0-4°C. BenzoylD L - a r g i n i n e p - n i t r o a n i I i d e (ΒΑΡΝΑ) s t o c k s o l u t i o n s (0.1 M) were made w i t h d i m e t h y l s u I f o x i d e (DMSO), and N - a c e t y I - L - t y r o s î ne e t h y I e s t e r (ATEE) s t o c k s o l u t i o n s ( 0 . 2 M) were made w i t h 100$ ethanol· Mod i f i c a t i o n o f i n h i b i t o r s . 200 mg o f K u n i t z s o y b e a n t r y p s i n i n h i b i t o r ( S T I ) was d i s s o l v e d i n 10 ml o f pH 7.6 T r i s - 8 M u r e a b u f f e r . To t h i s s o l u t i o n were t h e n added 10 ml e t h a n o l , 0.2 ml t r i - n - b u t y I p h o s p h i ne and 0.2 ml o f 2 - v i n y I p y r i d i n e . N i t r o g e n was bubbIed i n f o r one m i n u t e . The r e a c t i o n m i x t u r e was s h a k e n and

14.

FRIEDMAN ET AL.

Disulfide

Bond

Modification

361

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

5 ml s a m p l e s were removed a t v a r i o u s t i m e s . The s a m p l e s were d i a l y z e d a b o u t t w o days i n w a t e r - 2% m e t h a n o l , and l y o p h i l i z e d . The p r o c e d u r e w i t h o v o m u c o i d (OMI) was i d e n t i c a l t o t h a t d e s c r i b e d f o r S T I . R e d u c t i v e a l k y l a t i o n w i t h l i m a bean p r o t e a s e î n h i b i t o r (LB I) was c a r r i e d o u t w i t h 50 mg o f i nh i b i t o r , 5 ml pH 7.6-8M u r e a b u f f e r , 5 ml o f e t h a n o l , 0.05 ml o f n-Bu3P, and 0.05 ml 2-vînyI p y r i d i n e . C o n t r o l e x p e r i m e n t s were done i n pH 7.6 b u f f e r w i t h o u t u r e a and i n pH 7.6 b u f f e r w i t h o u t 11-BU3P and 2 - v i n y I p y r i d i n e . A s i m i l a r p r o c e d u r e was used t o t r a n s f o r m h a l f - c y s t i n e r e s i d u e s i n STI t o 2-QEC s i d e c h a i n s ( 2 6 ) . Reaction with t h i o l s . The f o l l o w i n g p r o c e d u r e w i t h s o y f l o u r i l l u s t r a t e s t h e g e n e r a l method used t o i n a c t i v a t e STI (18). Soy f l o u r (300 mg) and N - a c e t y I - L - c y s t e i ne ( Ν Α Ο (67.4 mg, 0.4 m i I I i mo I e s ) i n 30 ml o f 0.5 M T r i s b u f f e r were ρ laced i n a water bath a t a spec i f ied temperature f o r 1 h r . The r e a c t i o n m i x t u r e was t h e n c o o l e d i n i c e - w a t e r . Portions (0.05 m l ) o f t h e s u s p e n s i o n were d i I u t e d t o 0.5 ml w i t h pH 8.2, 0.05 M T r i s b u f f e r . C o n t r o l e x p e r i m e n t s w i t h o u t ΝAC were c a r r i e d out concurrent Iy. P u r e ST I was s t u d i e d s i m i I a r I y e x c e p t t h a t d i I u t i o n s were 1:300 w i t h t h e pH 8.2 T r i s b u f f e r . T h e r e m a i n i n g t r y p s î n i nh i b îtory a c t i v i t y was t h e n a s s a y e d a s d e s c r îbed b e l o w . A l I e x p e r i m e n t s were c a r r i e d o u t i n d u p I i c a t e . The r e p r o d u c i b i I i t y i s e s t i m a t e d t o be ± 3% o r b e t t e r . Ami no a c i d ana l y s i s . Ami no a c i d ana I y s e s o f a I i q u o t s o f h y d r o l y z e d w e i g h e d samp l e s d i s s o l v e d i n pH 2.2 b u f f e r were c a r r i e d o u t w i t h a s i n g l e - c o l u m n Durrum Amino A c i d A n a l y z e r , Model D500 under t h e f o I l o w i ng c o n d i t i o n s : s i n g I e - c o l u m n i o n e x c h a n g e c h r o m a t o g r a p h i c method ( Γ7 ); R e s i n , Durrum DC-4A, c i t r a t e b u f f e r pH, 3.25, 4.25, 7.90; P h o t o m e t e r , 590 and 440 nm; Column, 1.75 mm χ 48 cm; a n a l y s i s t i m e , 105 m i n u t e s .

Norleucine was used as an internal standard (Figures 1-6). Extent of reaction was deduced from the amount of 2-PEC found. U l t r a v i o l e t s p e c t r o s c o p y o f 2-PEC and 2 - p y r i d y I e t h y I a t e d (2-PE) i n h i b i t o r s . E x t e n t o f r e a c t i o n o f 2 - v i n y I p y r i d ine w i t h t h e i nh i b i t o r s was a I s o d e t e r m i ned f r o m t h e d i f f e r e n c e between ^263 ^ equ i m o l a r c o n c e n t r a t i o n s o f t h e mod i f i e d and n a t i ve i n h i b i t o r s , u s i n g ε f o r 2-PEC o f 7070 a t pH 3.0. The v a l u e of ε a t pH 1, 7200, was s I i g h t I y h i g h e r t h a n p r e v i o u s I y f o u n d in 6 Ν HCI ( 2 j _ ) . To d e t e r m i n e ε f o r 2-PEC, a 2.7 mM s t o c k s o l u t i o n i n 2 mM HCI was made and d i I u t e d 1:20 i n a p p r o p r i a t e buffers. D u p I i c a t e d i l u t i o n s i n 0.05 M g l y c i n e / H C I (pH 3.0) gave ε va Iues c o n s î s t e n t w i t h i η 1$. S p e c t r a f o r 2 - p y r i d y I e t h y I a t e d i n h i b i t o r s were a I s o a n a l y z e d s i m u l t a n e o u s l y f o r c o n c e n t r a t i o n s o f 2-PE s i d e cha i ns and p r o t e i η by us i ng a two-wave I e n g t h two-component ana Iys i s (27)· One wave I e n g t h was 263 nm, t h e a b s o r p t i o n Ο Γ

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

362 food protein deterioration

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

FRIEDMAN E T A L . Disulfide Bond Modification

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

364 FOOD PROTEIN DETERIORATION

Asp

Ser

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

FRIEDMAN E T AL.

-J 10

ι 20

Disulfide

I 30

ι 40

Bond

Modification

ι 50

ι 60

ι 70

I 80

TIME (MINUTES) Figure 4.

A mino acid analysis of

2-PE-LBI.

L 90

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

366 FOOD PROTEIN DETERIORATION

Asp

Disulfide

Bond

Modification

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

FRIEDMAN ET AL.

Figure 6.

A mino acid analysis of

2-PE-OML

368

FOOD PROTEIN DETERIORATION e

e

maximum f o r 2-PEC a t pH 3, s i n c e P E C / p r o t e i η i s maximal a t t h i s wavelength. The s e c o n d w a v e l e n g t h , a t w h i c h e f i / pFÇ * maximal o r n e a r l y s o , was 285 nm f o r PE-OMI, 280 nm f o r P t - L B I , o r 289 nm f o r P E - S T I . V a l u e s o f e f j n and ερ^ς f o r w a v e l e n g t h s f o r w h i c h ε i s n o t l i s t e d were c a l c u Y a f e a f r o m s p e c t r a u s i n g t h e formuI a e

D

D

ε

o

x (

max

o

r

A

280

o

e

s

n

e

=

χ

A

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

r

r

e

m

a

Cor ε

x

2 8 0

)

)

Two s i m u l t a n e o u s e q u a t i o n s o f t h e f o r m : A

ε

£

C

= p r o t e i η χ V o t e in + P E C

were s o l v e d i n e a c h

χ PEC

case.

F o r e x a m p l e , e q u a t i o n s f o r PE-STI w e r e : A

A

263 289

=

=

6 S T I

e

263

STI

x

289

The ε v a l u e s λ ( nm) 263 280 285 289

x

C

C

STI STI

+

e

+

€

PEC

x

C

PEC

=

1 4

'

9 0 0

C

STI

+

7 0 7 0

C

PEC

=

1 2

'

6 0 0

C

STI

+

3

263

PEC

x

289

9

5

C

C

PEC

PEC

used a r e t a b u l a t e d below: £

ε

0Μ! 10,850 14,890 12,860

ε

PEC 7070 1460 700 395

STI 14,900 20,300 .

.

ε

LB 1 2480 2390

12,600

U l t r a v i o l e t s p e c t r o s c o p y o f 2-qu i n o I y I e t h y I - S T I

(2-QE-STI).

S i n c e 2-QE-STI was I n s o l u b l e i n nondenaturÏng aqueous s o l v e n t s , i t was f i r s t d i s s o l v e d i n f o r m a m i d e a t 5-15 mg/ml a n d t h e n d i l u t e d i n aqueous s o l u t i o n s . A l i q u o f s o f s t o c k s o l u t i o n s o f 2-QEC o r 2-QE-STI were d i l u t e d t o A < 1.0. D i l u e n t s w e r e : 0.1 and 0.2 M H C I ; 0.1 N H S 0 : 0 . T and 0.2 M a c e t i c a c i d ; 0.2 M g l y c i n e — H C I , pH 3.0; 0.2 M a c e t a t e b u f f e r s , pH 3.6 and pH 5.0; 0.2 M 3 - ( N - m o r p h o l i n o ) p r o p a n e s u i f o n i c a c i d (MOPS)—NaOH b u f f e r , pH 7.0; 0.2 M g l y c i n e — N a O H b u f f e r , pH 9.0. S p e c t r a were r e c o r d e d a g a i n s t sol vent bIanks. 2

4

F I u o r e s c e n c e s p e c t r o s c o p y . 2-QE-STI s t o c k s o l u t i o n s were norma I I y d i l u t e d i n w a t e r o r 0.1 U H S 0 * t o A (1-cm I i g h t p a t h ) o f 0.04 o r l e s s , and t h e pH was a d j u s t e d a s needed w i t h 1 H H S 0 * and 1 Ν o r 4 Ν NaOH. F l u o r e s c e n c e was e x c i t e d a t 316 nm and 25°C. Y i e l d s were d e t e r m i n e d f r o m a r e a s o f t h e e m i s s i o n 2

2

14.

FRIEDMAN ET AL.

Disulfide

Bond

369

Modification

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

s p e c t r a , and i n t e n s i t i e s were measured a s n e t peak h e i g h t s a t maximum e m i s s i o n . T r y p s i η i nh i b i t o r assay. Many u n i t s a r e used t o d e f i n e t h e i n h i b i t o r y a c t i v i t y of protease i n h i b i t o r s (28). The f o l l o w i n g method, based on t h e d e f i n e d s t o i c h i o m e t r y o f enzyme, i n h i b i t o r , and s u b s t r a t e , was a d a p t e d from Mi k o I a and S u o l i n n a ( 2 9 ) . I n h i b i t i o n o f t r y p s i n a c t i v i t y was measured a t pH 8.2 and 37°C, w i t h ΒΑΡΝΑ a s s u b s t r a t e . I n h i b i t o r was i n c u b a t e d w i t h 25 yg o f t w i c e - c r y s t a l l i z e d b o v i n e t r y p s i n i n a t o t a l volume o f 0.75 ml f o r 5 min a t 37°C. ΒΑΡΝΑ (30 mg i n 1.00 ml o f DMSO) was d i l u t e d t o 100 ml w i t h 0.05 M T r i s - H C I , pH 8.2, c o n t a i n i n g 0.02 M CaCI . The d i l u t e d ΒΑΡΝΑ was k e p t a t 37°C and u s e d w i t h i n 1 h r . To s t a r t t h e r e a c t i o n , 3.0 ml o f s u b s t r a t e - b u f f e r m i x t u r e was added r a p i d I y t o t h e t r y p s i n - i n h i b i t o r m i x t u r e . The r e a c ­ t i o n was s t o p p e d a f t e r 5 o r 10 min by a d d i n g 0.50 ml o f 30% acetic acid. 2

D e t e r m i n a t i o n o f a c t i v e t r y p s i η. The c o n c e n t r a t i o n o f a c t i ve t r y p s i η was d e t e r m i ned by a c t i ve s i t e t i t r a t i o n w i t h p_-n i t r o p h e n y I p_ -guan i d i n o b e n z o a t e (NPGB), e s s e n t i a I I y a c c o r d i ng t o C h a s e and Shaw (30). A b s o r b a n c e a t 410 nm was f o l lowed i n a C a r y 15 s p e c t r o p h o t o m e t e r , w i t h t h e 0.1 a b s o r b a n c e s I i dew i r e . NPGB (0.01 M) was d i s s o l v e d i n d i methyIformam i d e - a c e t o n i t r i I e (1:4). T r y p s i n was d i s s o l v e d i n 2 mM HCI/0.02 M C a C I a t 5-6 mg/ml, t h e n d i I u t e d 1:10 i n t h e same s o l v e n t . Each c u v e t t e c o n t a i n e d 0.05-0.09 M Na b a r b i t a l b u f f e r (pH 8.35 o r 8.39 a t 23°C), 0.02 M C a C I o , 0.1 M K C I . T r y p s i n (3-5 ηmo I o f a c t i v e enzyme) was added t o t h e samp l e c u v e t t e a s 0.20 ml o f t h e 1:10 d i I u t i o n o r 25 y g o f t h e s t o c k s o l u t i o n . A f t e r t h e b a s e I i ne was d e t e r m i ned, 5 yl_ o f NPGB was added t o each c u v e t t e . TotaI volume was 1.00 m l . I η o u r i n s t r u m e n t , 1 A ^ Q = 61.1 mol o f p_-n i t r o p h e n o l a t e (31 ). The t r y p s i n used was 61 % a ο enzyme, based on i t s p r o t e i η c o n t e n t a s d e t e r m i ned by ab?'* je at 280 nm and an actî ve s i t e t i t r a t i o n . !

2

Calculations. The f o l l o w i n g samp l e c a l c u l a t i o n was used t o e s t i m a t e t h e t r y p s i n i n h i b i t o r a c t i ν i t y p e r mg o f a c t i v e enzyme, where A^Q

= a b s o r b a n c e measured a t 410 nm = mol a r e x t î n c t i o n c o e f f i c i e n t V = r a t e o f ΒΑΡΝΑ h y d r o l y s i s by t r y p s i η i n t h e a b s e n c e o f inhibitor V. = r a t e o f ΒΑΡΝΑ h y d r o l y s i s by t r y p s i n î η t h e p r e s e n c e o f inhib itor TU = t r y p s i n un i t , def i ned a s t h e amount o f t r y p s i η t h a t c a t a I y z e s t h e h y d r o l y s i s o f 1 μ mol o f ΒΑΡΝΑ p e r min a t 37°C and pH 8.2 TIU = t r y p s i n î nh i b i t o r un i t , def i ned a s r e d u c t i o n i n a c t i v i t y o f t r y p s î η by o n e t r y p s i η u n i t ( T U ) . ε

Q

370

FOOD PROTEIN DETERIORATION

Usi ng ε^ f o r p_-n i t r o a n î I i ne = 8800 1/mol-cm ( 3 2 ) , and a t o t a l volume o f 4.25 ml , 1 TU c o r r e s p o n d s t o an i n c r e a s e i n A ^ Q of 2.07 m i n " . We f o u n d t r y p s i n a c t i v i t y o f 2.51 TU p e r mg o f a c t i v e enzyme ( s e e s a m p l e c a l c u l a t i o n ) . U s i n g m o l e c u l a r w e i g h t s o f 23,400 f o r t r y p s i n and 21,500 f o r S T I , we c a l c u l a t e d 2.92 T I U p e r mg o f ST I. F o r t r y p s i n o n l y , 0.358 A ^ i / 5 m i n ; R a t e , V = 0.0716/min; f o r t r y p s i n and i n h i b i t o r , 0.389 A ^ Q / 1 0 m i n . 1

Q

Q

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

ν V

ν i

v

AJIQ (no inh.)

A,

1

"

(inh.) T j

_ 0.358 5

0

= 0.0716 - 0.0389 = 0.0327 A

4

1

min"

0

1

f ^ I -

i

0.389 10

1

S i n c e 2.07 A min" i s equivalent t o 1 t r y p s i n amount o f i nhιbι t o r p r e s e n t i s : 4

the

"

n

unît ( T U ) ,

0.0158 TIU

F o r p u r e ST I t h e i n h i b i t o r c o n t e n t o f t h e samp l e u s e d i s : 0.0158 T I U _ , 2.92 TIU/mg STI ' A

5

1 Λ

4 1

X

S i n c e 1 mg o f a c t i v e

MrfuXg the

_ m

9

trypsin

=

trypsin

-3

1 0

6

'

2

9

x

1 0

S T I

=

5

*

4 1

^

S T I

= 2.51 TU, 0.0158 T I U r e p r e s e n t s "

3

m

9

a

c

t

i

v

e

t r

yP

s i n

inhibited in

p u r e ST I samp l e .

If i n h i b i t o r s o t h e r t h a n STI a r e a l s o p r e s e n t ( a s i s a p p a r e n t l y t h e c a s e i n most ρ I a n t f o o d s ) , a more p r e c i s e e s t i m a t e i s : TIU i n samp l e wt. o f s a m p l e

T

"

T

l

n

/

,

U

/

, M

9

(

O

R

» 9

K

The f o I l o w i ng a v e r a g e v a Iues d e m o n s t r a t e t h e r e p r o d u c i b i I i t y o f t h e a s s a y . P u r e c o m m e r c i a l ST I i nh î b i t e d 1.23 ± 0.03 mg o f a c t i v e t r y p s i n p e r mg STI ( e i g h t r e p I i c a t e s ) . (Th î s v a Iue i s h i gher t h a n un i t y b e c a u s e t h e r a t i o o f mol ecu I a r w e i g h t s o f t r y p s i n t o STI i s g r e a t e r t h a n o n e and b e c a u s e o f p o s s i b I e mi nor c o n t r i b u t i o n s o f l o w e r mo I e c u l a r w e i g h t i n h i b i t o r s t o t h e t o t a I enzyme i nh î b i t o r y p r o c e s s . ) F o r u n t r e a t e d soy f l o u r , t h e a v e r a g e f o r f o u r d e t e r m i n a t i o n s was f o u n d t o be 109.51 ± 1.52 T I U o r 43.63 ± 0.605 mg t r y p s i n i n h i b i t e d p e r gram of f l o u r . S i m i l a r l y , t h e t r y p s i n i n h i b i t o r a s s a y f o r LB I gave t h e f o l l o w i ng a v e r a g e va Iues f o r f o u r d e t e r m i n a t i o n s : 2.76 ± 0.041 TIU/mg c o m m e r c i a l LB I and 51.33 ± 0.994 T l U / g Iima bean f I o u r .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

14.

FRIEDMAN ET AL.

Disulfide

Bond

371

Modification

Chymotrypsin I n h i b i t i o n assay* I n h i b i t i o n of chymotrypsin was d e t e r m i n e d t i t r i m e t r i c a l I y , w i t h 0.01 M ATEE a s s u b s t r a t e ; t h e p r o c e d u r e was a d a p t e d f r o m t h a t o f W i l c o x ( 3 3 ) . Inhibitor was i n c u b a t e d w i t h c h y m o t r y p s i n (25 μ I o f 0.44 mg/ml s o l u t i o n ) a t room t e m p e r a t u r e , t h e n e q u i l i b r a t e d a t 25°C, i n 1.9 ml o f 5 mM T r i s / H C I , 0.1 M i n C a C U , pH 8.0. A t l e a s t 10 min was a l l o w e d f o r enzyme and i n h i b i t o r t o r e a c t . After the baseline u p t a k e o f t i t r a n t (0.1 Ν NaOH) was r e c o r d e d , 100 μΙ o f 0.2 M ATEE was i n j e c t e d i n t o t h e s o l u t i o n . I n i t i a l r a t e s were d e t e r ­ mined f r o m t h e l i n e a r p o r t i o n o f t h e p l o t o f base u p t a k e a g a i n s t t i m e . C h a r t speed was 2 cm/min. M e a s u r e m e n t s o f t h e n o n e n z y ­ m a t i c breakdown o f ATEE and t h e e f f e c t o f o r d e r o f a d d i n g ATEE and c h y m o t r y p s i n ( n o i n h i b i t o r ) were a l s o made and used t o c o r r e c t assays of i n h i b i t i o n . P r o t e o l y t i c d i g e s t i o n assay* T r e a t e d s o y f l o u r was d i a ­ l y z e d f o r t h r e e d a y s a g a i n s t d i s t i l l e d w a t e r and l y o p h i l i z e d . Samples were d i s s o l v e d o r d i s p e r s e d (5 mg/ml) i n 0.05 M KCI w i t h s t i r r i n g and a d j u s t e d t o pH 8.5. T o d e t e r m i n e d i g e s t i b i l i t y by t r y p s i n , a I i q u o t s (5 mg o f p r o t e i n ) were p i p e t t e d i n t o p l a s t i c t i t r a t i o n v e s s e l s ( R a d i o m e t e r V 524) and d i l u t e d t o 4.0 ml w i t h 0.05 M K C I . S a m p l e s were i n c u b a t e d under N t o m i n i m i z e C 0 absorption. In a t y p i c a l r u n , t h e s u b s t r a t e was b r o u g h t t o pH 8.5 and 37°C, and t h e b a s a l i ne r a t e o f u p t a k e o f t i t r a n t ( 0 . 0 2 NaOH) was d e t e r m i n e d . T h e n , 20 μΙ_ o f b o v i n e t r y p s i n (5 mg/ml in 2 mM HCI-0.02 M C a C I ) was a d d e d . F u r t h e r a d d i t i o n s o f enzyme were made when t h e u p t a k e o f a l k a l i became v e r y s l o w . When t h e r e a c t i o n was e s s e n t i a l l y c o m p l e t e , t h e n e t u p t a k e was d e t e r m i n e d . 2

2

2

S i n c e l i b e r a t i o n o f h y d r o g e n i o n s by p r o t e o l y s i s i s incom­ p l e t e a t pH v a l u e s where some new α-amîno g r o u p s a r e i o n i z e d ( 3 4 ) , c a l c u l a t i o n o f t h e number o f p e p t i d e bonds c l e a v e d r e q u i r e s a v a l u e f o r t h e a v e r a g e pK o f t h e n e w l y formed p e p t i d e α - a m i n o groups. I f we assume a pK o f 7.5 ( 3 5 ) , f r e e h y d r o g e n i o n s w i l l be p r o d u c e d i n 90% o f t h e p e p t i d e bond c l e a v a g e s a t an a s s a y pH 8.5. To c a l c u l a t e t h e a c t u a l a v e r a g e number o f p e p t i d e bonds c l e a v e d p e r c h a i n c o r r e c t e d f o r i n c o m p l e t e l i b e r a t i o n o f hydrogen i o n s , we a l s o assumed an a v e r a g e m o l e c u l a r w e i g h t o f 23,000 f o r the soy p r o t e i n ( 3 6 ) . Q

ResuIts The

2 - P E - d e r i v a t i v e s o f a l I t h r e e i n h i b i t o r s showed

absorp­

tion maxima at 263-264 nm i n acidic (pH 3.0) solution (Figures 7-10), coinciding with that f o r free 2-PEC. Reaction was rapid in buffers containing 8 M urea, especially i n t h e cases of STI and chicken 0ΜΙ. A l l 4 half-cystine groups of STI and 12 of 18 half"cystine groups o f 0ΜΙ reacted within 30 min under these conditions, based on an increase i n A (Figure 8 and Table I ) . As expected, LBI reacted more slowly (figure 10 and Table I ) . Only 5 to 6 of the 14 half-cystines, on the average, reacted

PROTEIN

ALKYLATED

REDUCED

PROTEIN

REDUCED

PROTEIN

9

3

TRIBUT Y L PHOSPHINE

4

9

2

HCI

S

2

oc

it

ϊ

Figure 7.

(2-PEC)

3

2

HYDROLYSIS

S-PYRIDYL-ETHYLATION

1 REDUCTION

Reductive S-pyridylethylation of a protein.

?

CH -S„

S-/Q —(2-PYRIDYLETHYL)—L —CYSTEINE

2 Ν

α » ,

HOOC-CH-CH -S-CH -CH - \ 2

Ν

(? - V I N Y L P Y R I D I N E )

9

CH =CH

4

F?-SH + HS-R-SH + ( n - C H ) - P = 0

r

P _S-S-f?-SH + ( n - C H ) - P + H 0

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

ο

S ο

Η W

w

Η m

SO Ο

to

FRIEDMAN ET AL.

Disulfide

Bond

373

Modification

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

14.

240

260

280

300

320

340

360

W A V E L E N G T H (nm) Figure 8. UV spectra of trypsin inhibitors and their 2-PEC derivatives at pH 3.0. Lyophilized proteins were dissolved in 0.1 M KCl by adjusting the pH to 3.2 at 7.8 mg/mL (native) or 3.1-3.7 mg/mL (modified). Aliquots were diluted to 0.5 mg/mL (2.3 χ 10 M) in 0.05 M glycine/HCl (pH 3.0). Key: a, native STI; and b, modi­ fied STI. 5

FOOD PROTEIN DETERIORATION

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

374

ob

ι

240

ι

ι

260

ι

1

280

ι

Γ ι

300

I 320

I

l340

WAVELENGTH (nm) Figure 9. UV absorption spectra of chicken ovomucoid inhibitors (OMl). Protein samples were dissolved and diluted to 0.25 mg/mL. Key: a, untreated OMl: 8.2 X 1CT M; and b, treated OMl: 7.6 X 10 M. 6

e

14.

FRIEDMAN ET AL.

Disulfide

Bond

375

Modification

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

d

240

260

280

300

320

340

W A V E L E N G T H (nm) Figure 10. The UV absorption spectra of lima bean trypsin inhibitors (LBI) is shown. Control samples in the presence or absence of urea gave almost identical spectra; therefore, only one is shown. Key to protein concentrations (w/w): a, untreated, 2.28 X 10 M; controls (urea), 2.36 χ 10 M; b, treated 30 min without urea, 2.17 X 10 M ; c, treated 30 min with urea, 2.10 X 10 M; and d, treated 240 min, 2.48 χ 10~ M . s

5

s

s

5

FOOD PROTEIN DETERIORATION

376

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

Table

I.

Protein

2-PE-STI

2-PEC Cha I f - c y s t i n e ) c o n t e n t s o f r e d u c e d a l k y l a t e d S T I , LB I , and OMl d e t e r m i n e d by amino a c i d a n a l y s i s and u l t r a v i o l e t s p e c t r o s c o p y .

Time o f Reduction (min )

2-PE-OMl

2-PE-LB 1

Amino a c i d

analysis

4.0 4.6 4.4

30 60 120 240 Mean ±

M o l e s 2-PEC/mole p r o t e i η UV

1

spectra

4.372

4.38^ 4.47*

4.24 S.D.

30 60 240 30 no u r e a 30 8M u r e a 240

4.3±0.3

z

4.361.10

11.4 12.3 15.3

13.27* 13.63^ 14.89

7.4

2.0 , 5.6^ 7.1

z

3

3

M o l e c u l a r w e i g h t s = STI = 21,500; OMl = 29,600; LB I = 9 0 0 0 . D e t e r m i n e d by s i m u l t a n e o u s e q u a t i o n s ( s e e t e x t ) . D e t e r m i n e d by d i r e c t s u b t r a c t i o n o f 2-PE LB I and LB I a b s o r b a n c e s a t 263 nm ( p e a k max imum f o r 2 - P E C ) .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

14.

FRIEDMAN ET AL.

Disulfide

Bond

Modification

377

w i t h i n 30 min i n t h e p r e s e n c e o f u r e a . Even a f t e r 2 4 0 m i n , o n l y a b o u t 7 o f 14 had r e a c t e d . O m i t t i n g u r e a from t h e r e a c t i o n medium s l o w e d t h e a l k y l a t i o n o f LB I , s u g g e s t i n g t h a t t h e r a t e of r e a c t i o n and p o s s i b l y t h e s e l e c t i v i t y f o r d i f f e r e n t d i s u l f i d e b r i d g e s c o u l d be m o d i f i e d by a p p r o p r i a t e c h o i c e o f r e a c t i o n conditions. T a b l e II shows t h e e f f e c t o f e x t e n t o f r e d u c t i o n and a l k y l a t i o n on t r y p s i n i n h i b i t i o n . E s s e n t i a l l y complete loss o f a c t i v i t y o f LB I was o b s e r v e d when 7 ha I f - c y s t i nes p e r m o l e c u l e , on t h e a v e r a g e , were m o d i f i e d . T h i s r e s u l t i s c o n s i s t e n t w i t h t h e f i n d i n g s o f F e r d i n a n d e t a l . ( 3 7 ) . L o s s o f a c t i v i t y was d i s p r o p o r t i o n a t e t o t h e e x t e n t o f m o d i f i c a t i o n a t lower l e v e l s . When an a v e r a g e o f 1 d i s u l f i d e b r i d g e p e r LB I m o l e c u l e was l o s t , a p p r o x i m a t e l y 2/3 o f t h e i n h i b i t o r y a c t i v i t y was l o s t . When m o d i f i c a t i o n was a l m o s t 40% com ρ I e t e ( a v e r a g e o f 3 d i s u l f i d e b r i d g e p e r LBI m o l e c u l e ) a l m o s t 90% o f t h e i n h i b i t o r y a c t i v i t y was l o s t . The c o n t r o l s show t h a t l i t t l e o r no i n a c t i v a t i o n o f i n h i b i t o r was due t o t h e p r o c e d u r e i t s e l f and t h a t , under t h e s e c o n d i t i o n s , the e f f e c t o f urea i s r e v e r s i b l e . E s s e n t i a l l y c o m p l e t e r e d u c t i o n and a l k y l a t i o n o f STI and OMl a b o l i s h e d t h e i r t r y p s i n i n h i b i t o r y a c t i v i t i e s , a s shown in T a b l e I I . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h o s e o b t a i n e d with other reducing agents. S i n c e some v a r i a n t s o f LBI a l s o i n h i b i t c h y m o t r y p s i n ( 3 8 ) , t h e e f f e c t o f m o d i f i c a t i o n on i n h i b i t o r y a c t i v i t y a g a i n s t c h y m o t r y p s i n was a l s o d e t e r m i n e d t o s e e w h e t h e r t h e s e n s i t i v i t y o f a n t i c h y m o t r y p s i n a c t i v i t y t o r e d u c t i o n and a l k y l a t i o n d i f f e r e d from t h a t o f t r y p s i n i n h i b i t i o n . A s shown i n T a b l e I I , t h e l o s s e s o f i n h i b i t o r y a c t i v i t y a g a i n s t b o t h enzymes were similar. L e s s t h a n 30% o f t h e LBI c o n s i s t e d o f v a r i a n t s t h a t i n h i b i t chymotryps i n . F r e e 2-PEC (0.01 M) i n h i b i t e d c h y m o t r y p s i n w e a k l y ( 1 3 ? ) a t a mol r a t i o o f - 5 χ 1 0 . ΡΕ-OMl (0.8 nmol) d i d n o t i n h i b i t 0.4 nmol o f c h y m o t r y p s i n (Mol r a t i o o f 2-PEC m o i e t y t o enzyme 30-40). T h u s , t h e 2-PEC d o e s n o t a p p e a r t o b i n d e f f e c t i v e l y t o chymotrypsiη· Where I i t t I e o r no i n h i b i t o r y a c t i v i t y i s found , f r e e t r y p s in o r c h y m o t r y p s in c a n a t t a c k t h e i n h i b i t o r , i f s i t e s o f potentiaI cleavage are accessi ble. T h u s , t h e mod i f i e d i n h i b i t o r s may we I I show i n c r e a s e d s u s c e p t i b i I i t y t o p r o t e o l y s i s . T a b l e I I I shows t h a t t h e 2 - P E - i n h i b i t o r s a r e a t t a c k e d more r e a d iI y by t r y p s i n o r c h y m o t r y p s in t h a n a r e t h e n a t i v e i n h i b i t o r s , even t h o s e t h a t d o n o t i n h i b i t c h y m o t r y p s i η. Digestion was i n c o m p l e t e , h o w e v e r , under t h e cond i t i o n s u s e d . (Both pr imary and s e c o n d a r y s p e c i f i c i t i e s o f c h y m o t r y p s i η were c o u n t e d in deducing t h e p o t e n t i al s i tes.) I η t h e c a s e o f LB I , s u s c e p t i ­ b i I i t y t o d i g e s t i o n by t r y p s i η o r c h y m o t r y p s i η was n o t i n c r e a s e d u n t i I t h r e e o r more d i su If i d e br i d g e s were b r o k e n . These r e s u I t s and t h o s e i n T a b l e I I show t h a t i n h i b i t o r y a c t i ν i t i e s o f LB I a r e more e a s iI y d e s t r o y e d by t h i s mod i f i c a t i o n p r o c e d u r e t h a n a r e the s t r u c t u r a l elements r e s p o n s i b l e f o r r e s i stance t o p r o t e o l y s i s .

FOOD PROTEIN DETERIORATION

378

Table

II.

E f f e c t o f m o d i f i c a t i o n on i n h i b i t o r y a g a i n s t t r y p s i n and c h y m o t r y p s i n .

Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch014

Reductive treatment ;

Time (min ) Inhibitor

STI 2-PE-STI

LBI LBI-controls 2-PE-LBI

1

% Initial specific inhibitory a c t i v i t y agai nst :

Tryps î η

100

100 0

30^ 30 240

14 40 51

100 92 32 10