Modern Methods in Protein Chemistry. [Volume 1] Modern methods in protein chemistry: Review articles following the joint meeting of the Nordic Biochemical Societies Damp/Kiel, FR of Germany, September 27–29, 1982 9783111439846, 9783110095142

Table of contents :
Preface
Contents
Protein Studies as Important Tools in Amplifying Different Characterizations of Biologically Active Macromolecules
High Resolution Analytical and Preparative Isoelectric Focusing of Proteins: Principles and Strategy
High Resolution of Complex Protein Solutions by Two- Dimensional Electrophoresis
Some Recent Developments of the Electroimmunochemical Analysis of Membrane Proteins. Application of Zwittergent, Triton x-114 and Western Blotting Technique
Affinity Electrophoresis with Special Reference to the Microheterogeneity of Glycoproteins and Identification of Ligand-Binding Proteins
Principles and Applications of Heterogeneous Enzyme Immunoassays
Immunological Methods for the Detection of Proteins. Application to Proteins Synthesized in Cell Free Systems in Rat Hepatocytes and by E. Coli Cells Transformed with Recombinant DNA
Protein Quantification with Zone Immunoelectrophoresis Assay (ZIA)
High Performance Liquid Chromatography of Peptides and Proteins
Advanced Automatic Microsequencing of Proteins and Peptides
The Current Status of Automated Solid-Phase Sequencing
Microsequence Analysis of Peptides and Proteins
Aminopeptidase M in the Sequence Analysis of Peptides and Proteins
Fast Atom Bombardment Mass Spectrometry. A New Technique for Peptide Sequencing. A Review
Perspectives in the Circular Dichroic Analysis of Protein Main-Chain Conformation
Spin-Labelled Amino Acids, Peptides and Proteins - Synthesis and Application
The Ultrastructure of Macromolecular Complexes Studied with Antibodies
Subject Index
List of Authors

Citation preview

Modem Methods in Protein Chemistry Review Articles

Modern Methods in Protein Chemistry Review Articles following the Joint Meeting of the Nordic Biochemical Societies Damp/Kiel, F R. of Germany, September 27-29,1982 Editor Harald Tschesche

W G DE

Walter de Gruyter • Berlin • New York 1983

Editor Harald Tschesche, Dr. rer. nat. Professor für Biochemie Lehrstuhl für Biochemie Fakultät für Chemie Universität Bielefeld D - 4 8 0 0 Bielefeld, F R. of Germany

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Modem Methods in Protein Chemistry: review articles; following the joint meeting of the Nordic biochem. soc., Damp/Kiel, FR of Germany, September 27 - 29,1982 / ed. Harald Tschesche. Berlin; New York: de Gruyter, 1983 ISBN 3-11-009514-9 NE: Tschesche, Harald [Hrsg.]

Library of Congress Cataloging in Publication Data Modern Methods in Protein Chemistry. Sponsored by Gesellschaft für Biologische Chemie. Bibliography: p. Includes index. I. Proteins—Analysis—Congresses. I. Tschesche, Harald II. Gesellschaft für Biologische Chemie. QP551.M58 1983 547.7'5 83-14009 ISBN 3-11-009514-9

Copyright © 1983 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin - Printed in Germany.

Preface

The intention of this volume is to attempt a survey of the present status in the different fields of analytical methods for the characterization and study of proteins. It is hoped, that the book will serve as a guideline for newcomers as well as for experienced scientists to become acquainted with the recent developments, trends and possibilities of the available analytical tools. The articles review the methodological developments up to the present most advanced applications. The references given should enable the reader to find his orientation among the literature and to adapt the method to his own problems. Most of the subjects have been reviewed during the Joint Meeting of the Nordic Biochemical Societies, Conference A, September 30 and October 1, 1982, Damp/Kiel, FRG. This meeting was kindly organized and supported by the Gesellschaft fur Biologische Chemie and the study group for chemical protein analysis. At this meeting, the general opinion was that there is a trend and demand for micro- and submicroscale analytical procedures in order to facilitate the solution of many interesting biochemical problems. This trend is clearly obvious from the efforts described in this book. It is the aim of the authors to extend knowledge and their analytical experience in order to stimulate progress in the field of protein research. Bielefeld, April 1983 Harald Tschesche

Contents

Protein Studies as Important Tools in Amplifying Different Characterizations of Biologically Active Macromolecules H. Jornvall, N. Kalkkinen, J. Luka, R. Kaiser, M. Carlquist, H. von Bahr-Lindstrom

1

High Resolution Analytical and Preparative Isoelectric Focusing of Proteins: Principles and Strategy B.J. Radola

21

High Resolution of Complex Protein Solutions by TwoDimensional Electrophoresis J. Klose

49

Some Recent Developments of the Electroimmunochemical Analysis of Membrane Proteins. Application of Zwittergent, Triton x-114 and Western Blotting Technique O.J. Bjerrum, K.P. Larsen, M. Wilken

79

Affinity Electrophoresis with Special Reference to the Microheterogeneity of Glycoproteins and Identification of Ligand-Binding Proteins T.C. B0g-Hansen, B. Teisner, J. Hau

125

Principles and Applications of Heterogeneous Enzyme Immunoassays G. Grenner

149

Immunological Methods for the Detection of Proteins. Application to Proteins Synthesized in Cell Free Systems in Rat Hepatocytes and by E. Coli Cells Transformed with Recombinant DNA K. Schneider, W. Northemann, E. Schmelzer, V. Gross, P.C. Heinrich

163

VIII Protein Quantification with Zone Immunoelectrophoresis Assay (ZIA) 0. Vesterberg

187

High Performance Liquid Chromatography of Peptides and Proteins H. Kratzin, C.Y. Yang, H. Götz, F.P. Thinnes, T. Kruse, G. Egert, E. Pauly, S. Kölbel, L. McLaughlin, N. Hilschmann

207

Advanced Automatic Microsequencing of Proteins and Peptides B. Wittmann-Liebold

229

The Current Status of Automated Solid-Phase Sequencing W. Machleidt

267

Microsequence Analysis of Peptides and Proteins K. Beyreuther, B. Bieseler, J. Bovens, R. Dildrop, K. Neifer, K. Stüber, S. Zaiss, R. Ehring, P. Zabel

303

Aminopeptidase M in the Sequence Analysis of Peptides and Proteins K.-D. Jany, J. Czech, G. Pfleiderer

327

Fast Atom Bombardment Mass Spectrometry. A New Technique for Peptide Sequencing. A Review W. Schäfer

337

Perspectives in the Circular Dichroic Analysis of Protein Main-Chain Conformation A. Wollmer, W. Straßburger, U. Glatter

361

IX Spin-Labelled Amino Acids, Peptides and Proteins Synthesis and Application

-

H . R . W e n z e l , H. T s c h e s c h e , E. v o n G o l d a m m e r

T h e U l t r a s t r u c t u r e of M a c r o m o l e c u l a r C o m p l e x e s with Antibodies

385

Studied

G. S t o f f l e r , M . S t o f f l e r - M e i l i c k e

409

S u b j e c t Index

457

L i s t of A u t h o r s

463

PROTEIN STUDIES AS IMPORTANT TOOLS IN AMPLIFYING DIFFERENT TIONS OF BIOLOGICALLY ACTIVE

CHARACTERIZA-

MACROMOLECULES

Hans Jörnvall, Nisse Kalkkinen, Janos Luka, Rudolf Kaiser, Mats Carlquist and Hedvig von Bahr-Lindström Departments of Physiological Chemistry I, Biochemistry II and Tumour Biology, Karolinska Institutet, S-104 01 Stockholm, Sweden, and Department of Biochemistry, University of Helsinki, SF-00170 Helsinki, Finland

Summary

Structures and functions of proteins and their genes require several

dif-

ferent types of investigation for complete characterization. Three kinds of protein study valuable in combined approaches, are discussed: radiosequence analysis and two applications of peptide

analysis.

-- Radiosequence analysis for correlation of proteins and their genes can be extended by use of multi-labelled proteins. This is exemplified by in vivo multi-labelled protein ns72 of Semiiki Forest virus. Results

illus-

trate factors of importance in the selection of labelled residues, and suggest that the spread of specific activities should be low. However, the total amount of label

is less critical, and the amount required is shown

to be smal1. -- Results from selected peptides can give information for subsequent synthesis of oligonucleotide probes intended for hybridizations with corresponding nucleic acid structures. This is exemplified by a cellular, probably transformation-associated protein ("p53^"). Utilization of CNBr-peptides facilitates fragment separation, gives knowledge of several

protein

regions, and ensures information on structures that include residues with little code-degeneracy. This enables subsequent synthesis of probes with few

alternatives.

-- Similarly, protein results for use in subsequent synthesis of peptide replicates to generate specific antibodies can be obtained by limited structural

studies. This has also been demonstrated on the

Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York

above-mentioned

2 cellular protein utilizing results of direct sequence

analysis.

Introduction

Protein sequence studies, X-ray crystallography, enzymology and DNA sequence analyses are well-established methods that combined allow characterization of the structures and functions of proteins in great detail. In relation to the protein studies, one comparatively new aspect concerns radiosequence analyses where use of multi-labelled proteins (1) extends the methodology. Protein studies have also become especially important as steps in new approaches where oligonucleotide probes (f.ex. 2) and antibodies (3) may be produced on the basis of information from limited peptide analyses. These two approaches and radiosequence analysis are discussed in relation to some viral and cellular

proteins.

Methods

In vivo multi-labelling and radiosequence analysis of Semiiki Forest virus protein ns72 was performed by previously described methods (1). Another protein, cellular "p53^" (3) purified as described (4), was treated with CNBr and the peptides obtained were separated by reverse phase high performance liquid chromatography, HPLC (5). Amino acid sequences were determined by manual DABITC (dimethylaminoazobenzene

isothiocyanate)

dations using by-products to assist identifications

(6) degra-

(7), and by liquid-

phase sequencer degradations using polybrene and a 0.1 M peptide program (8).

Radiosequence Analysis

Background

Sequence analysis of biosynthetically labelled proteins is a standard procedure for establishing relationships between proteins and their genes,

3 and for investigating N-terminal protein processing. The protein is labelled with radioactive amino acids of known types during in vivo or in vitro synthesis. After isolation of the product, release of the radioactivity during degradations will residue(s)

identify the original position(s) of the labelled

in the polypeptide chain.

For single types of labelled residues, this approach is fast and sensitive, allowing sequence analysis down to below the picomole scale. This method has therefore also been used in "ordinary" sequence analysis of proteins that could only be obtained in small amounts (cf. 9). However, with increasing sensitivity of non-radioactive protein methods (10-13) and with fast methods for DNA analysis, the main use of radio-sequence

degradations

is not for complete investigations of fully unknown proteins, but for combined investigations of partly known proteins studied also at the DNA level In this way, it is possible to establish positions for initiation of protein synthesis, correct reading frames, and positions of post-translational processing (e.g. removal of signal sequences, cleavage of polyproteins, or glycosylations, cf. 14,15). Most residues have been fully utilized for label

success-

in such studies, and simultaneous degradation of

a cold carrier protein of known structure is standard practice to check yield and sequencer

performance.

In vivo multi-labelled proteins Several

labelled residues can be incorporated into a protein for analysis.

In this way, the positions of more than one type of residue can be determined in each sequencer run, increasing speed and amount of

information

obtained. However, radioactivity alone is then not sufficient for the identification of residues. Instead, ordinary amino acid

phenylthiohydantoin

(PTH) separations are also required, as shown schematically in Fig. 1. Another step in making radiosequence analysis generally applicable is use of in vivo rather than in vitro labelling. This avoids the limitations set by working with cell-free systems but adds the complexities of cellular amino acid transport/metabolism and of isolation of the labelled protein in a radiochemically pure form. A few extensive analyses of proteins multilabelled in vivo have been carried out (9), and one in vivo label of a Semiiki Forest virus protein with seven different types of radioactive amino

In vivo Single labelling x 5

Multiple labelling

^ N^

c

o

^

o

j

In v i t r o

t

a

t

1

o

Sequencer

( D X f f i c o o i b c x i o ( x i o o g t o a i c o t o

HPLC

I Sequencer ( # - s a m p l e )

V

V

V

I

6ODOQ

^^ ^^

I c.p.m. (in 1,9,14)

c.p.m.

• 1 > 1 >1 Separate sequencer + c.p.m. x 4 (for 4 f ) ^

A in 13; • in 1,9,14 •

•

Result X#X0.2%)

Cooor

should

be

avoi-

protein

bind-

the

can

with

po-

detergent

blocking

transfer

layer

sensitivity

when

it b l o c k s

removal

conditions

renature

high

should

excellent

protein

20. As

of

be

the

residu-

obtained

side

effects

to

this

is r e c o m m e n d e d

instead

of

blocking

spotted

p r o t e ins

appears that

not

after

an

with the

treatment

by

performed

blot

(76)).

antibodies the

denaturation

application

because

separation

react with

the

irreversible

exclude

focusing

native

is to r a i s e

likely

In f a c t the

of a n a l y s i s ,

transfered

of

the

with

albumin.

fact

be

tergent

Tween

reaction the

presence

to

SDS-PAGE

gel

0.5%

to

loads

a well-stained

separation

have

In c a s e

the

resolution

antigen

due

Under

needs

of

detergents

with

be d u e

proteins

because

The

after

bovine

proteins paper.

of c h a r g e d

to n i t r o c e l l u l o s e .

ing al

the

Blue.

concentration in the

the

optimal

lyspecific

high

the

antibodies

those

used

is p r o b a b l y

nitrocellulose

fraction the

This

of

the

non-denaturated simple

zone

in p r e s e n c e

Another to

SDS-PAGE

way

this has

technique antigens

can

electrophoresis of

non-ionic

to c i r c u m v e n t

SDS-denatured separated

could

taken

proteins

protein

dethis which

after

the

blotting.

Acknowledgements T h a n k s due to M r s . L e n a B a r o n a n d M i s s Pia H j o r t O l s e n f o r t h e i r s k i l f u l t e c h n i c a l a s s i s t a n c e , a n d to M r s . P. P l o u g f e l t f o r t y p i n g . We a r e g r a t e f u l to D r s . A. G o e n n e , C a l b i o c h e m - B e h r i n g Corp. San D i e g o , C a l i f o r n i a f o r his g e n e r o u s s u p p l y of t h e Z w i t t e r g e n t s a n d M. D a n i e l s e n f o r p r o v i d i n g us w i t h pig i n t e s t i n a l b r u s h b o r d e r . T h e i n v e s t i g a t i o n w a s s u p p o r t e d by the D a n i s h M e d i c a l R e search Council (12-3228), L u n d b e c k f o n d e n and H a r b o e f o n d e n .

120 References la.

Albertsson, (1981 ).

1.

Allen,

2.

Andersson, D.R., Davis, 252, 6617-6627 (1977).

3.

A x e l s e n , N . H . : In H a n d b o o k of I m m u n o p r e c i p i t a t i o n - i n - g e l t e c h n i q u e s , B l a c k w e l l , O x f o r d ; a l s o S c a n d . J. I m m u n o l . 1 7 , Suppl . 10 (in p r e s s ) ( 1 9 8 3 ).

4.

A x e l s e n , N . H . , K r o l l , J . , W e e k e , B . : In A M a n u a l of Q u a n t i tative I m m u n o e l e c t r o p h o r e s i s . Methods and A p p l i c a t i o n s , Univ e r s i t e t s f o r l a g e t , O s l o ; a l s o S c a n d . J. I m m u n o l . 2, S u p p l . 1 , ( 1 973) .

5.

B a r o n , C., T h o m p s o n , 285 (1975).

6.

Bhakdi, A., B h a k d i - L e h n e n , B., phys. Acta 470, 35-44 (1977).

7.

Bhakdi, S., Knüfermann, phys. Acta 2ii> 505-557

H., W a l l a c h , (1975).

8.

Bjerrum,

O.J.:

Biochim.

Biophys.

9.

Bjerrum,

O.J.:

Anal.

10.

B j e r r u m , O . J . ( e d . ) : E l e c t r o i m m u n o c h e m i c a l A n a l y s i s of brane Preoteins, Elsevier Biomedical Press, Amsterdam, York (1983).

11.

B j e r r u m , O . J . : In E l e c t r o i m m u n o c h e m i c a l A n a l y s i s of ne P r o t e i n s ( B j e r r u m , O . J . , e d . ) pp. 3 - 4 4 . E l s e v i e r dical P r e s s , A m s t e r d a m , N e w Y o r k ( 1 9 8 3 ) .

12.

B j e r r u m , O . J . , B h a k d i , S . : In H a n d b o o k of I m m u n o p r e c i p i t a tion-in-qel Techniques (Axelsen, N.H., ed.) B l a c k w e l l , Oxf o r d ; a l s o S c a n d . J. I m m u n o l . 1_Z.> S u p p l . 10 (in p r e s s ) ( 1 9 8 3 )

for

J.C.,

P-Ä., Andersson, Humphries,

T.:

C.:

B.: J. FEBS

J.L.,

Lett.

57,

Carraway,

Biochim.

Biochem.

Chrom.

Acta

90,

D.F.H.:

331-348

J.

Acta

O.J.:

472,

131-141

158-162

K.L.:

Biophys.

Bjerrum,

21_5,

(1975).

Biol. Chem.

332,

276-

Biochim. Biochim.

135-195

BioBio-

(1977).

(1978). MemNew

MembraBiome-

13.

Bjerrum, O.J., Bog-Hansen, T.C.: M e m b r a n e s ( M a d d y , A . H . , e d . ) pp. London (1976).

In B i o c h e m i c a l A n a l y s i s of 378-426, Chapman and Hall,

14.

Bjerrum, O.J., 66-89 (1976).

Biochim.

15.

B j e r r u m , O . J . , G i a n a z z a , E . : In E l e c t r o i m m u n o c h e m i c a l A n a l y s i s of M e m b r a n e P r o t e i n s ( B j e r r u m , O . J . , e d . ) pp. 1 2 5 153. E l s e v i e r Biomedical P r e s s , A m s t e r d a m , New York, (1983).

16.

B j e r r u m , O . J . , Lundahl , P.: 139-143 (1973).

Scand.

17.

Bjerrum, O.J., 69-80 (1974).

Biochim.

B0g-Hansen,

Lundahl,

P.:

T.C.:

J.

Biophys

Immunol.,2, Biophys.

Acta

Acta

455,

Suppl. 342,

1,

121 18.

Bjerrum, Wallach,

O.J., B h a k d i , S., B o g - H a n s e n , T.C., D.F.H.: Biochim. Biophys. Acta 406,

19.

Bjerrum, O.J., Bhakdi, S., Rieneck, M e t h o d s 3, 3 5 5 - 3 6 6 ( 1 9 8 0 ) .

20.

Bjerrum, O.J., Bjerrum, P.J., Larsen, K.P., Norrild, B., B h a k d i , S . : In E l e c t r o i m m u n o c h e m i c a i A n a l y s i s of M e m b r a n e P r o t e i n s ( B j e r r u m , O . J . , e d . ) pp. 1 7 9 - 1 9 8 , E l s e v i e r B i o m e dical P r e s s , A m s t e r d a m , N e w Y o r k ( 1 9 8 3 ) .

21.

B j e r r u m , O . J . , B o g - H a n s e n , T . C . , P l e s n e r , T. , W i l k e n , M. : In L e e t i n s - B i o l o g y , B i o c h e m i s t r y , C l i n i c a l B i o c h e m i s t r y ( B o g - H a n s e n , T . C . , e d . ) V o l . 1, pp. 2 5 9 - 2 6 8 , W a l t e r de G r u y t e r , B e r l i n , New York (1981).

22.

Bjerrum, Biochim.

23.

Bjerrum, O.J., G e r l a c h , J., B o g - H a n s e n , E l e c t r o p h o r e s i s 3, 8 9 - 9 8 ( 1 9 8 2 ) .

24.

B j e r r u m , O.J., H a w k i n s , M., S w a n s o n , P., G r i f f i n , M., Lor a n d , L . : J. S u p r a m o l . S t r u c . C e l l . B i o c h e m . ]_6, 2 8 9 - 3 0 1 (1981 ).

25.

B j e r r u m , O . J . , R a m l a u , J . , B o c k , E . , B 0 g - H a n s e n , T . C . : In T e c h n i q u e s for M e m b r a n e R e c e p t o r C h a r a c t e r i z a t i o n and Pur i f i c a t i o n ( J a c o b s , S . J . , C u a t r e c a s a s , P . , e d s . ) pp. 1 1 6 156, Chapman and Hall, London (1980).

26.

Bonsai 1 , R.W., 280 (1 971 ) .

27.

Bordier,

28.

B r o d b e c k , V . , G e n t i n e t t a , R . , O t t , P . : In M e m b r a n e P r o t e ins. A L a b o r a t o r y M a n u a l ( A z z i , A . , B r o d b e c k , U . , Z a h l e r , P., e d s . ) pp. 8 5 - 9 6 , S p r i n g e r - V e r l a g , B e r l i n ( 1 9 8 1 ) .

29.

Burnette,

30.

B o g - H a n s e n , T . C . : In S o l i d P h a s e B i o c h e m i s t r y - A n a l y t i c a l a n d S y n t h e t i c A s p e c t s ( S c o u t e n , W . H . , e d s . ) J. W i l e y a n d S o n s , I n c . N e w Y o r k (in p r e s s ) ( 1 9 8 3 ) .

31.

B0g-Hansen, T.C., Brogren, S u p p l . 2, 37-51 ( 1 9 7 5 ) .

32.

B0g-Hansen, T.C., Bjerrum, O.J. Ramlau, m u n o l . 4, S u p p l . 2, 1 4 1 - 1 4 7 ( 1 9 7 5 ) .

33.

B 0 g - H a n s e n , T . C . , L o r e n c - K u b i s , I., B j e r r u m , O . J . : In E l e c t r o p h o r e s i s ' 79 ( R a d o l a , B . J . , e d . ) p p . 1 7 3 - 1 9 2 , W. de Gruyter, Berlin (1979).

34.

Calcagno, M., Zagoya, 73, 3 8 6 - 3 9 0 (1976).

J.C.D.,

35.

Chua, N.H., (1979).

F.:

O.J., Lundahl , P., B i o p h y s . A c t a 3JM>

C.:

J.

Hunt, Biol.

W.N. : Anal.

Blomberg,

S.:

K. : J.

Knlifermann, H . , 489-504 (1975).

Biochem.

Hjertén, S., Brogren, 1 73-1 81 (1 975 ).

Biochim.

Chem.

T.C.,

Biophys.

2j[6, 1 604-1 607

Biochem.

1_1_2 , 1 9 5 - 2 0 3

C.-H.:

J.

Scand.

Lozano, Biol.

J. J.:

A.G.:

Chem.

Biophys.

C.-H.:

Hertz,

Acta

249,

J.:

266-

( 1 981 ).

(1 981 ).

Immunol, Scand.

Anal.

25_4,

J.

4, Im-

Biochem.

215-233

36.

Converse, ( 1 975) .

C.A.,

Papermaster,

37.

Dako

38.

D a n i e l s e n , E . M . : In E l e c t r o i m m u n o e h e m i c a 1 A n a l y s i s of M e m brane Proteins ( B j e r r u m , O.J., ed.) pp. 1 5 5 - 1 6 7 , E l s e v i e r B i o m e d i c a i P r e s s , A m s t e r d a m , New Y o r k ( 1 9 8 3 ) .

39.

Dodge, J.T., Mitchel, Biophys. 100, 119-130

40.

Fairbanks, G., Steck, T.L., 10, 2 6 0 6 - 2 6 1 7 (1971).

41.

Förster, Seyler's

M., Kopp, F., R e i n a u e r , H., Z. P h y s i o l . C h e m . 3 6 3 , 1 0 1 0

42.

Gabriel,

0.: M e t h .

43.

Glass, 70-73

43a

G e r l a c h , J . H . : In E l e c t r o i m m u n o c h e m i c a l A n a l y s i s of M e m b r a ne P r o t e i n s ( B j e r r u m , O . J . , e d . ) p p . 2 9 9 - 3 1 7 , E l s e v i e r B i o m e d i c a l P r e s s , A m s t e r d a m , New Y o r k ( 1 9 8 3 ) .

44.

Gerlach, J.H., Bjerrum, 60, 6 5 9 - 6 6 6 (1982).

45.

Goenne,

46.

Grauballe, P.C., J. M e d . V i r o l o g y

47.

Graham, R.C., Lundholm, U., Karnovsky, C y t o c h e m . ]_3, 1 50-1 52 ( 1 962 ).

48.

H e l e n i u s , A. , S i m o n s , 79 ( 1 975 ) .

49.

H a g e n , L., B j e r r u m , O . J . , G o g s t a d , G . O . , K o r s m o , N . O . : B i o c h i m . B i o p h y s . A c t a 701_, 1 - 6 ( 1 982 ).

50.

H a g e n , I., N u r d e n , A . , B j e r r u m , O . J . , J. C l i n . I n v e s t . 6 5 , 7 2 2 - 7 3 1 (1980).

51.

H a r b o e , N . M . G . , I n g i l d , A . : In A H a n d b o o k of I m m u n o p r e c i p i t a t i o n - i n - g e l Techniques ( A x e l s e n , N . H . , e d . ) . B l a c k w e l l , O x f o r d , a l s o S c a n d . J. I m m u n o l . V 7 , S u p p l . 10 (in p r e s s ) 4 (1983).

51a

H e r t z , J . B . , P a r t o n , R . : In E 1 e c t r o i m m u n o e h e m i c a 1 Analysis of M e m b r a n e P r o t e i n s ( B j e r r u m , O . J . , e d . ) pp. 3 7 3 - 3 9 2 , E l s e v i e r B i o m e d i c a l P r e s s , A m s t e r d a m , New York (1983).

52.

H a w k e s , R., N i d a y , 147 ( 1 9 8 2 ) .

53.

H e e g a a r d , N . , C h r i s t e n s e n , U . , B j e r r u m , O . J . : In L e c t i n s Biology. Biochemistry, Clinical Biochemistry (Bog-Hansen, T . C . , S p e n g l e r , G . A . , e d s . ) V o l . 3, p p . 3 8 7 - 3 9 6 . W . de G r u y t e r , B e r l i n , New York (1983).

Immunoglobulins,

Ernst,

Pamphlet

pp.

C., Hanahan, (1963).

R.C.,

R.: Anal.

Rank,

D.J.:

Biochim.

E., G o r d o n ,

Arch.

D.F.H.:

(1975).

Biochem.

Biochemistry

S t a i b , W. : H o p p e (1982). ( 1 971 ).

L.S.:

G.H.:

Biochem.

Vestergaard, B.F., 7, 2 9 - 4 0 ( 1 9 8 1 ) .

469-472

5, C o p e n h a g e n

578-604

Huilica,

O.J.,

K.:

Science ^89,

Wallach,

Enzymol . VI,

W.F., Briggs, (1981).

P.,

D.S.:

Science

Can.

J.

8J7 , 2 8 - 3 8

Meyling, M.J.:

Biophys.

Solum,

J.: Anal.

211,

( 1 978).

A., J.

Biochem.

Genner,

J.:

Histochem.

Acta

415, R.,

N.O.,

Biochem.

29Solum,

Caen,

119,

J.:

142-

123 54.

Hjelmeland, L.M., Nebert, D.W., c h e m . 9_5, 201 - 2 0 8 ( 1 979 ).

55.

Hoek,

56.

K j a e r v i g , M . , I n g i l d , A . : In A H a n d b o o k of I m m u n o p r e c i p i tati o n - i n - g e l T e c h n i q u e s ( A x e l s e n , N . H . , e d . ) B l a c k w e l l , O x f o r d ; a l s o S c a n d . J. I m m u n o l . 1_7, S u p p l . 10 (in p r e s s ) ( 1 9 8 3 ).

57.

Kroll,

58.

Laemmli,

U.K.:

59.

Laurell,

C.-B.:

Anal.

60.

Laurell, (1981).

C.-B.,

McKay,

61.

Lester, R.L., ( 1 974 ) .

62.

L o r a n d , L., Proc. Natl.

63.

Lowry, O.M., Rosenbrough, N.J., Farr, A.L., J. B i o l . C h e m . ]_93 , 2 6 5 - 2 7 5 ( 1 951 ).

64.

Marchesi,

65.

Nielsen, C.S.: ( 1 975 ) .

Scand.

66.

Nielsen, C.S., ( 1 975 ) .

Bjerrum,

O.J.:

Scand.

67.

Nielsen, C.S., Bjerrum, 496-509 (1977).

O.J.:

Biochim.

68.

Norrild, B., Bjerrum, O.J., c h e m . 8J_, 4 3 2 - 4 4 1 ( 1 977 ) .

69.

N o r r i l d , B . : In A H a n d b o o k of I m m u n o p r e c i p i t a t i o n - i n - g e l T e c h n i q u e s (Axelsen, N.H., ed.) B l a c k w e l l , O x f o r d ; also S c a n d . J. I m m u n o l . V 7 , S u p p l . 10. (in p r e s s ) ( 1 9 8 3 ) .

70.

01 F a r r e l 1 , P . H . :

71.

O w e n , P . , S m y t h , C . J . : In I m m u n o c h e m i s t r y of E n z y m e s a n d t h e i r A n t i b o d i e s ( S a l t o n , M . R . J . , e d . ) pp. 1 4 7 - 2 0 2 , W i l e y a n d S o n s , New Y o r k ( 1 9 7 6 ) .

72.

Parkhouse, R.M.E., 280-281 (1980).

73.

Plesner, T.,Bjerrum, ( 1 980) .

74.

P l e s n e r , T . , W i l k e n , M . , A v n s t r o m , S . : In E l e c t r o i m m u n o c h e m i c a l A n a l y s i s of M e m b r a n e P r o t e i n s ( B j e r r u m , O . J . , e d . ) pp. 4 5 - 5 4 . E l s e v i e r B i o m e d i c a l P r e s s , A m s t e r d a m , New Y o r k ( 1 983) .

A.K.,

Zail,

J.: Meth.

S.S.:

Enzymol.

Nature

Smith,

J.

79,

7-14

Bio-

(1977).

(1981). (1970).

Meth.

J.

A.: Anal.

1_0, 3 5 8 " 3 6 1

Biochem. E.J.:

Acta

52-57

680-685

S.W.:

Meth.

Chim.

73,

227,

Weissmann, Acad. Sci.

V.T.:

Clin.

Chrambach,

(1 9 6 5 )

Enzymol.

Biol.

Chem.

73,

249,

338-339 3395-3405

L.B., Epel, D.L., Bunner-Lorand, U . S. A. 7 3 , 4 4 7 9 - 4 4 8 1 (1976).

Enzymol. J.

28,

Immunol.

Biol.

Lifter, O.J.:

252-254 4,

Suppl. J.

J.,

2,

Scand.

J.

101-105

Biophys. B.F.:

25J), 4 0 0 7 - 4 0 2 1

Choi,

Y.S.:

R.J.:

(1972).

Immunol.

Vestergaard,

Chem.

Randall,

J.:

2,

73-80

Acta

466 ,

Anal

( 1 975 ).

Nature

Immunol.

Bio-

11, —

284 , 341-351

124 75.

Plesner, T., Wilken, M., Bjerrum, O.J., C l i n . L a b . I m m u n o l . 8, 137-141 (1982).

76.

Reinhart, ( 1 982 ) .

77.

S i m o n s , K., H e l e n i u s , A . , M o r e in, B., B a l c o r o v a , J., S h a r p , M . : In N e w D e v e l o p m e n t s w i t h H u m a n a n d V e t e r i n a r y V a c c i n e s ( M i z r a k i , A . , e d . ) P r o g r e s s in C l i n i c a l a n d B i o l o g i c a l Res e a r c h , V o l . 4 7 , pp. 2 1 7 - 2 2 8 , A l a n R. L i s s , New Y o r k (1980).

78.

S j ö s t r ö m , H . , N o r é n , 0 . : In E 1 e c t r o i m m u n o e h e m i c a 1 Analysis of M e m b r a n e P r o t e i n s ( B j e r r u m , O . J . , ed.) pp. 2 4 1 - 2 5 5 , E l s e v i e r B i o m e d i c a l P r e s s , A m s t e r d a m , New York (1983).

79.

S k o v b j e r g , H., Norén, 0., S j ö s t r ö m , Lab. Invest. 38, 723-729 (1978).

H.:

80.

Smith, S.W., (1979).

Chem.

81.

Svendsen, ( 1 973) .

82.

S v e n d s e n , P . J . : In E l e c t r o p h o r e s i s . A S u r v e y o f T e c h n i q u e s and A p p l i c a t i o n s ( D e y l , Z., ed.) P a r t A. T e c h n i q u e s , pp. 349-350, Elsevier, Amsterdam (1979).

83.

Tanford, C., Reynolds, 133-170 (1976).

84.

Tanner, M.J.A., ( 1 976 ) .

85.

Towbin, S c i . U.

86.

U r i e l , J . : In M e t h o d s in I m m u n o l o g y a n d Immunochemistry (Williams, C . A . , C h a s e , M . W . , e d s . ) V o l . 3, pp. 2 9 4 - 3 2 6 , A c a d e m i c Press, New York (1971).

87.

Weeke,

88.

W i l k e n , M., B j e r r u m , O.J., G e i s l e r , C., P l e s n e r , T.: - S e y l e r ' s Z . P h y s i o l . C h e m . 36J3, 1 01 3 - 1 01 4 ( 1 9 8 2 ).

M.P.,

Malamud,

Lester,

P.J.:

D.:

K.L.:

Scand.

Anstee,

J.

J.

Anal.

Biol.

Immunol.

J.A.: D.J.:

Biochem.

2,

Biochim.

Scand.

J.

Clin.

Lab.

Scand. 249,

Biophys.

Biochem.

Invest.

J . ]_53, Proc.

25,

M.M.:

]_2_3,

S u p p l . 1,

H., S t a e h e l i n , T,, G o r d o n , J.: S. A. 7 5 , 4 3 5 0 - 4 3 5 4 (1979).

B.:

Hansen,

J.

229-235

J.

Clin.

3395-3405 69-70

Acta

457 ,

265-270

Natl.

Acad.

269-275

(1970). Hoppe-

AFFINITY

ELECTROPHORESIS

HETEROGENEITY BINDING

T.

C.

SPECIAL

GLYCOPROTEINS

AND

REFERENCE

TO

IDENTIFICATION

THE OF

MICRO-

LIGAND-

PROTEINS

B0g-Hansen

The P r o t e i n Sigurdsgade

B.

OF

WITH

Laboratory, 34, D K - 2 2 o o

University Copenhagen

of N,

Copenhagen Denmark

Teisner

Medical Microbiology, J. B . W i n s l 0 w s v e j 1 9 ,

U n i v e r s i t y of Odense D K - 5 o o o O d e n s e C, D e n m a r k

J . Hau B i o m e d i c a l L a b o r a t o r y , U n i v e r s i t y of J . B. W i n s l e w s v e j 2 1 , D K - 5 o o o O d e n s e

Odense C, D e n m a r k

Introduction

Affinity

electrophoresis

electrophoretic allowed

to

react

electrophoresis to

interact

resis,

23,

after

26).

jugates proach

As

this and can

during is

is in

the

term

which

which or

a system

the

in

which

(6).

used

Affinity

components

before

or

to

describe

components

are

during

are

Immuno-

allowed

electropho-

results

of

the

interaction

quantitatively

by

the

immunoprecipitation

a typical chapter

used

example

describes

lectins. be

commonly

interacting

electrophoresis

non-immunologically,

analytically

tion,

systems

However,

are

of the

are

1igand-macromo1ecu1e interaction

systems

summarized.

Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York

in

between

which

revealed (6,

interacglycocon-

a similar

ap-

126 General The

Principles

analysis

tion

of

liqand

in a g a r o s e .

interaction

Compared

troimmunoprecipitation as p r o t e i n s

tend

to

biological

remain

activity

impaired.

Because

to

the

change

protein, resis.

well

complex

In t h i s in t h e

suited feature

free

migration

which

formation

the

of

their

of

be

gel.

these of e v e n

may it

can

be

utilized

in t h e

protein

reference

identification

of

native

The

be to

include

the

in the

are

not tends

of

the

Immunoelectropho-

that

crossed

Immuno-

m a c r o m o l e c u 1 ar matrix

pore

several

analysis (12,

offers

e-

is

size

li-

especially One

impor-

which

allows

m a c r o m o 1 e c u 1 ar c o m p l e x e s

has

mixtures gel

gel

an

their

complex

macromo1ecu1es. large

large

during

behaviour

by

elecmethod

retain

interactions

studied

agarose

is

very

they

antigenic

of n a t i v e

gels

state

Thus

is i m p o r t a n t

adapted

study

methods, reference

a protein-ligand and

Immunoelectrophoresis

multicomponent dies

in

protein-ligand

connection can

analytical

experiment.

formation

agarose to

tant

Crossed

and

electr•immunoprecipita-

to be a s u p e r i o r

e1ectrophoretic

electrophoresis gands

to o t h e r

seems

1ectroimmunoprecipitation

by

and 34).

unique

(29)

features

characterization The

use

the p o s s i b i l i t y

of

of

antibo-

for

specific

proteins.

Lectins For

developing

extensively

our

(6 - 1 6 ) .

bohydrate-binding blood

cells,

interacting

and

have

"phytohemagg1utinins" Several (2,

3,

reviews 39).

of

Lectins

chromatography 4o).

been as

lectins have

Several

separated types

of

are

They

to at

for

first

properties

and

been

e.g.

carred

"hemagglutinins" in

have for

characterization

have

lectins

cells,

extensively

glycoproteins label

used

or m u l t i v a l e n t

discovered

their used

and

have

divalent

were

and

been

we

agglutinate

referred

they

of p r o t e i n s

trophoretica11y 19,

Lectins

proteins.

systems

appeared

affinity of

elec-

g1ycopeptides

used,

or

plants.

these

(3,

include

127 fluorescein labels, been

or

i s o t h i o c y a n ate antibody

applied

(5).

to

to

led

form

Reactions

Fig.

with

1, F i g .

lectin

tin.

The

the p r o t e i n

the

prior

the

incubation

was

internal

protein

binds

to the

disappears

disappearance

2, or p a r t i a l , of

The

the

same

first

4b)

or w i t h

morphology If by tates

area

the

for

remain result

coupuncoup-

is b o u n d .

Other

mobility

4) or p r o f i l e

plates

(Fig.

lc).

This

(13) m e d i a t e d

unrelated

by

leclectin

that

by

bind

to

and

it

1).

When

that

the

in

a pre-

area.

for

Protein

in

only

could

may

immua

frac-

also

occur

several

lines

la,

Protein

4 - > 4a

(Protein

is u s e d ,

not

reduction

mobility

supernatant

identity

as s e e n

a change

lectin

the

analyzed

does

in

into

(Fig.

in t h e

immuincu-

immobilized was

patterns

split

by

shows

is r e d u c e d

The

dimension

accompanied

lb

1, F i g .

3 indicates

could

after

immobilized

is e i t h e r

or

3.

Crossed

unchanged

be c o m p l e t e ,

Protein

first

Fig.

(Protein

free

lectin

used free

schematically

with

mixing

pattern

Protein

dimension

by

complexes

in t h e

shown

If a p r o t e i n

the

either of

be

are

supernatant

will

the

precipitate

(Protein

nologically At low

may

could

contrast

as l e c t i n partial

and

lectin

different

This

techniques

been

obtained

plate.

performed

from

this protein

in t h e o r y .

5 + x).

control

reference

as s e e n

noprecipitation tion

have

in t h e i r

patterns

lectin

its p r e c i p i t a t e

as an

cipitate

lectins

electrophoresis.

Immunoelectrophoresis.

lectin,

have

or

of g l y c o p r o t e i n s

sample

be u s e d

The

to

with

reaction

with

crossed

enzyme

in b l o t t i n g

lectins

lectin)

precipitation

la b e i n g

after

labelled

labels,

lectin).

of g l y c o p r o t e i n

pattern

Moreover,

(immobilized

noelectrophoretic bation

radioactive

glycoproteins

electrophoresis

Sepharose (free

labels.

identify

In a f f i n i t y

led

labels,

some

as

result

lectin

+

5 ->

precipitate

5).

proteins

and

may

(Protein of

with

may

appear

immunoprecipiin

a pattern

cross-linking

of

of

immu-

proteins.

to g l y c o p r o t e i n

ratios,

the

pattern

is

affected

128 by

the

the

lectin

pattern

concentration,

is l i t t l e

but

changed

above

by

the

further

point1

'saturation

addition

of m o r e

lec-

tin.

Lectins lectin

in i n t e r m e d i a t e into

sis^ w a s

introduced

trophoresis'

(6).

fic a n t i b o d i e s

under

and

on of

interacting

sensitive

troduction, with

the

term

'crossed

is a n a l o g o u s

intermediate

gel,

to

the d e t e c t i o n antibodies

and

used

for

for p r e d i c t i o n

affinity

the

which

for been

incorporating

immuno

and

has

of

Immunoelectrophore-

method

the m e t h o d lectin

in c r o s s e d

antigens

glycoproteins

ments

The p r i n c i p l e

gel

The m e t h o d

in the

cific

many

gels.

an i n t e r m e d i a t e

affino use

of

speci-

is a h i g h l y and

(33).

spe-

identificatiSince

its

identification

of s e p a r a t i o n

chromatography

elec-

in-

of

experi-

of g l y c o p r o t e i n s

(8,

1,11).

Immobilized original tions

lectin

are p o s s i b l e .

or d i m i n u t i o n teristic

for

reasonable

simple

to

complex

tin.

Splitting same

that

compl exes

patterns gel

as

shown

dimension

seen

glycoproteins.

If s h o w s gel.

tins:

Protein

the

lectin,

formation

individual

to some

a limited reactions

(Fig.

Id a n d

may

the

with

of a f f i n i t y

glycoproteins.

with

reac-

are

e).

disappearance

This

free

charac-

it w o u l d would

in

precipitates

give

appear and

lecin

the of

precipitate

in the

changes

features

as

A - > 4a + 4b

is c h a r a c t e r i s t i c

lectin

be

such

reaction

(Protein

le

is

an i m m o b i l i z e d after

The c h a n g e

precipitate

notable

the

glycoproteins,

occur le

5 in F i g .

in of

glycoproteins,

reaction

in F i g .

gel

number

Theoretically

complex

mobility).

reactions

In a d d i t i o n

immobilized of

for

intermediate

containing

after

complex

Fig.

more

of p r e c i p i t a t e s

first

the only

glycoproteins.

profile

ate

to

Characteristic

expect

more

intermediate

added

However,

of p r e c i p i t a t e s

m a c r o m o l e c u 1ar

the

was

experiments.

for

intermedi-

mentioned with

free

with lec-

coprecipitation

129

F i g . 1 . S c h e m a t i c r e p r e s e n t a t i o n of v a r i o u s s y s t e m s of a g a r o s e g e l e l e c t r o p h o r e s i s w i t h a n t i b o d i e s for s t u d i e s of i n t e r a c t i o n b e t w e e n l e c t i n s and g l y c o p r o t e i n s : C r o s s e d I m m u n o e l e c t r o p h o r e s i s ( a - i ) a n d l i n e i m m u n o e l e c t r o p h o r e s i s (j). a - b - c : I n t e r a c t i o n b e f o r e e l e c t r o p h o r e s i s (a: c o n t r o l ) , d - e - f : L e c t i n in the i n t e r m e d i a t e gel (d: c o n t r o l ) , g - h - i : L e c t i n in t h e f i r s t d i m e n s i o n gel (g: c o n t r o l ) , j: L e c t i n in s m a l l w e l l s in an i n t e r m e d i a t e g e l . A n o d e to t h e r i g h t s i d e a n d on t o p . A b b r e v i a t i o n s u s e d : a = a f f i n i t y p r e c i p i t a t e , FL = f r e e l e c t i n , IMM = i m m o b i l i z e d l e c t i n . See the t e x t for f u r t h e r d e t a i l s . M o d i f i e d from (11).

130 Lectins

in

first

ting

lectin

1975

(9) a n d

the

first

first

into

dimension

gels.

the

dimension

first

is a n a l o g o u s

dimension

dimension

terization

and

prediction

of

gel

to

gel

(28).

has

been

outcome

(8,

Fig.

lh a n d

i shows

typical

trophorezed

through

a gel

respectively.

tin

concentration.

action Protein

can

tardation

that

action,

each

a well

Generally, cathodic

Lectins on of

a well the

component

each

with

peak

on

a low

the

very

Krall

and

If t h e r e

a change

of

to

for

dimension

with

and

the

inter-

an

affinity

gel.

In F i g .

to a p p e a r

from

to

a distinct

li the

as a re-

concentration.

respect

lectin

Many inter-

subpopu1 ation

structure. at pH 8 . 6

technique,

conservative

(25).

between

The the

is an i n t e r a c t i o n the p a t t e r n

which

amounts

may

A

lectin

between

a

2 and

3).

modificati-

has

the

of l e c t i n ,

antigen

be

induce

(Figs.

Immunoelectrophoresis.

gel

lectin

3 is l o s t

glycoproteins

Andersen

leclec-

a characteristic

lectin

with

the

position.

reflect

Protein

elec-

free

on

the

sometimes

mobility

reacting

in l i n e

bind

and

representing

intermediate

gel.

lectin,

as

are

and

dependent

cathodically

carbohydrate

of t h e

of u s i n g by

the

charac-

as w e l l

proteins

patterns

first

heterogeneity

defined

in an

antibody

in t h e

3 is s h i f t e d

lectins

introduced

in

in

chromatography

immobilized

not

the p a t t e r n .

immunoelectrophoretic

advantage

in

antibodies

lectins

of u n c h a n g e d

lectin

is d e p e n d e n t

in w e l l s

the

seen

when

is h i g h l y

1 does

with

from

show

shift

with

affinity

reactions

reference

precipitate,

glycoproteins

incorpora-

identification,

of g l y c o p r o t e i n s

containing

Protein

be

Protein

double-peak

for

immunoprecipitation

2 is l o s t

pattern.

of

introduced

of s p e c i f i c

The m e t h o d used

pattern

of t h e p r o t e i n

precipitate

with

The

as a n i n t e r n a l in

was

11).

tin

Alterations

principle gel

use

of l e c t i n

separations

is u s e d

the

quantification the

The

added was

is a d d e d gel

and

a protein

observed

as a

to the and

nega-

131 tive has

deflection been

tographic fast

and

extracts

used

in

for

media

the

(18,

sensitive (Fig.

i

1ine-preeipitate

evaluation 25).

assay

We for

of

binding

use

this

(Fig.

method

screening

lj).

properties

for

The of

routinely

lectins

in

method chromaas

a

plant

4) .

U

MMMm

c

y tt u

F i g . 2. C r o s s e d I m m u n o e l e c t r o p h o r e s i s w i t h l e c t i n s in the first dimension gel. Human serum proteins analyzed with antib o d i e s a g a i n s t h u m a n s e r u m p r o t e i n s ( s t a i n e d for p r o t e i n ) , a: C o n t r o l p l a t e . b: C o n A in the f i r s t d i m e n s i o n g e l . c : L e n t i l a g g l u t i n i n ( L C A ) in t h e f i r s t d i m e n s i o n g e l . d: W h e a t g e r m a g g l u t i n i n ( W G A ) in the f i r s t d i m e n s i o n g e l . A n a f f i n i t y p r e c i p i t a t e is v i s i b l e in t h e f i r s t d i m e n s i o n g e l w i t h all t h r e e l e c t i n s . E v e n t h o u g h c o n A and LCA is i n h i b i t e d by t h e s a m e m o n o s a c c h a r i d e s ( m a n n o s e a n d o t h e r s ) t h e i r r e a c t i o n w i t h g l y c o p r o t e i n s is d i s t i n c t l y d i f f e r e n t ( c o m p a r e b and c). The N - a c e t y 1 - g 1 u c o s a m i n e - b i n d i n g WGA e x h i b i t s a d i s t i n c t l y d i f f e r e n t b e h a v i o u r w i t h a s t r o n g b i n d i n g of o r o s o mucoid.

F i g . 3. As F i g . 2 b u t s t a i n e d for e s t e r a s e a c t i v i t y . Two ester a s e s a r e s e e n : C h o l i n e s t e r a s e (C) i s b o u n d s t r o n g l y i n t h e a f f i n i t y p r e c i p i t a t e by a l l t h r e e l e c t i n s , HDL-associated a r y l e s t e r a s e (A) is s h i f t e d c a t o d i c a l l y by t h e l e c t i n s .

132 Precipitation

It

is

important

cipitation tins.

In

No

to

with

note

there

lectin

interaction

non-binding. 2.

and

No

and

Microheterogeneity

here

are

the

and

distinction

affinity

three

between

immunopre-

precipitation

possible

reactions

with

lec-

between

a

glycoproteins:

with

the

change

Lectin-dependent pitating

and

antibodies

general

multimeric 1.

Patterns

glycoprotein: in

the

immunoprecipitate.

precipitation

binding

non-precipitating ,

of

the

glycoprotein.

glycoprotein:

Disappearance

preci-

of

the

im-

munoprecipitate. 3.

Interaction

without

formation

of

a lectin

non-precipitating , 1ectin-binding the

Characteristically,

may

described The ed

upon be

the

found

glycoproteins lectin

Change

in

as

used

a mixture

are

for of

microheterogeneous

analysis, two

or

a given

three

of

the

and

glycoproforms

above.

microheterogeneity by

glycoprotein.

immunoprecipitate.

depending tein

precipitate:

crossed

classes

can

Immunoelectrophoresis

most with

clearly free

be

distinguish-

lectin

incorpo-

F i g. 4. L i n e i m m u n o e 1 e c t r o p h o r e s is w i t h l e c t i n e x t r a c t s in s m a l l w e l l s for s c r e e n i n g of l e c t i n s , from l e f t s i d e e x t r a c t s of K e n n e d y a r u b i c u n d a , P s i d u m c a t t l e i a w u m , C a s s i a n o d o s a , E r y t h r i n a c r i s t a g a l l i , S c h o t i a a f f r a (T. C . B a g - H a n s e n a n d J. G. G r u d z i n s k a s , in p r e p a r a t i o n ) .

133 rated

into

protein

the

first

structure

In p r i n c i p l e and

the most

use

of b o t h

cipitating

both

dimension

previously

free

and

complete forms.

and

information

Free

lectin

fractions

ding.

lectin

and

non-binding

fractions

form

addition

this,

the

affinity

influences

the

pattern.

lectin

Quantification hods

may

with

free

a basis

lectin

for

Type For

into

sites,

three

Type

forms,

the

intermediate

out

lectin

third

three

Type

different

lectin

The

of

can

(the

simple

calculation

free the

be

will

Type of

the

of

2 (the

in the

the

relative

and

microhete-

required

the

is

with

with-

experiment gel

and

in-

the

intermediate

of e a c h

the

total

amount

of

of

glycoand

experiment).

content

gel.

precipitate amount

experiment)

third

of

without

site

of

are

second

second

2 (the

micro-

constitutes

experiment

area

met-

sites.

intermediate

for

In

the

forms

binding

types

first

experiment),

Type

give

three

enclosed

lec-

Precipitation

lectin

binding

lectin

obtained

first

1 plus

of g l y c o p r o t e i n

gel,

The

present

changes

ij^ v i v o .

experiments The

in the

includes

amount

(11).

these

intermediate

estimate

of

forms.

immobilized

technique.

planimetry

protein

of

The

non-bin-

for

one

By c a r e f u l

glycoprotein

glycoprotein

of m i c r o h e t e r o g e n e i t y

or m o r e

pre-

lectin-

with

a relative the

or

1 - molecules

of e a c h

immobilized experiment

fractions.

0 - molecules

gel

in the

between

Type

two

used

coordinated

between

quantitative

to

be

classes:

with

quantification

cludes

binding

(1).

or n o n - p r e c i p i t a t i n g .

of the

the

glyco-

of a g l y c o p r o t e i n .

of g l y c o p r o t e i n s

vs.

2 - molecules

rogeneity

assess

by

lectin-binding

be p r e c i p i t a t i n g

a classification

glycoproteins binding

to

forms

may

differentiate

of m i c r o h e t e r o g e n e i t y

be u s e d

heterogeneity

lectins

glycoprotein be

of

in d e t a i l

differentiate

tin-binding to

may

may

will

aspects

is o b t a i n e d

will

non-precipitating

Immobilized

These

discussed

immobilized

non-precipitating binding

gel.

were

of e a c h

the A form

134 Table

I.

Orosomucoid

Heterogeneity.

D i s t r i b u t i o n of O r o s o m u c o i d i n t o t h r e e C l a s s e s F o r m s C h a r a c t e r i z e d by t h e i r R e a c t i o n w i t h c o n Dimension

Peak(s ) 3

Source

Normal

serum

8

I 3

(+4)

Peak

43 a )

5 - 3

40

+ +

of M o l e c u l a r A in the F i r s t

2

Peak

3

49

4

55

+ +

1

Ref .

4

41,,42

6

30

7

30

5

* * *

Pregnancy Maternal

serum

23

0 a )

3 - 4 0 Amniotic Chord

Abnormal Prostatic +

fluid

13

15 23

0

22

- 5

45

-

+ +

+

7

85

5

77

7

78

2

44

,42 +

+ + +

7 6

serum cancer

stilboestrol

Post

0

blood

t 0

77 +

b )

10

0 - 0

- 1

9 0 - 1

30

operative c)

cholecystectomy c)

24

Septicaemia

27

Acute

pancrea-

32

titis

C )

Advanced

>11

-

6

: i

a)

4 0 - 3

3 7 - 1 0

39 - 2

3 5 - 8

40

28 - 4

40

48

40

48

cancer

Rheumatoid arthritis c )

13

a)

- 13

a ) Sum of Peak 3 and p e a k 4, R a y n e s f i n d s a f o u r t h p e a k ( 3 o ) . b ) T o t a l a m o u n t of o r o s o m u c o i d , d o u b l e of n o r m a l . c ) T o t a l a m o u n t of o r o s o m u c o i d , four t i m e s n o r m a l . * T. C. B a g - H a n s e n and J. G. G r u d z i n s k a s , in p r e p a r a t i o n .

135 It

is

the

also

first

possible dimension

determined geneity are

is

plant

material

or

as

Table

I shows

how

(a-1

acid

technique.

forms

the

gel

in

affinity

formation

of

the

pH

shift in

the

have

to

immune an

of

of

is

each

and

component

The

in

there

reason

between

the

fractions

for the

preparation of

concentrations

is

microhetero-

Amazingly

differences

by

pro-

non-homogeneof

divalent

ca-

of

the

a

change

in

is

of

orosomucoid

an

in

strong

phoretic

shi ft.

be

added

to

the

first

first

methyl

antibody-con-

the

dimension

(1,

2o). we

dimen-

glycoprotein

the

specifically

precipitate

or

by

1ectin-binding

precipitate

crossed

affinity

in

the

protein gel

For

prior

release

have

used

from to of

1.5-3o

%

a-D-g1ucopyranoside.

lectins

a lectin

uniformly have

a low

(the

of

the may

glycoproteins. by

in

compared

a gel

veloin

dimension

at

the The

most

pH

8.6

common

interactions the

Higher

and

expressed

first

with

column.

chromatography

in

mobility

Immunoelectrophoresis)

gel

is

migration

electrophoresis

chromatography

binding

and

e1ectrophoretic

distributed

most

retardation

in

the

dimension

electrophoresis

sults

must

measured

a glycoprotein

first

Since

tions

of

release

affinity

between

the

been

forms

Affinity

lectin

the

technique). (the

used.

microheterogeneity

displacer

in

degree

during

which

the

a-D-mannopyra noside

affinity

city

inconsistent

order

the

Determination

by

in

different

precipitate

glycoproteins

The

of

detected

preparations.

lie

quantification

a specific

methyl

of

lectin

extraction

glycoprotein)

precise

taining

amount

forms

manganese.

sion

the

for

The

the

may

microheterogeneity

number

between

giving

by

The

upon

but

used

possibly

lectins,

For

technique.

depends

clear,

tions

quantify

planimetry.

differences

not

cedures, ous

by

forms

great

this

to

interacaffinity

a large

re-

electro-

136 Mathematically, ticular

ligand

the can

relative

be e x p r e s s e d

ents

by

of p r o t e i n s

the

for

retardation

a

par-

coeffici-

^ y2- - i r

R = where

1

(13).

and

r

mension

1

are

o

the

retardation

experiment

is p r e s e n t ,

according

to

ligand

their

differences

the

needs

may

listing

charge

to be p e r f o r m e d

of

with

from an

a

excess

glycoproteins

is i n d e p e n d e n t resulting

densities

method

di-

respectively

where

influences

this

first

be c a l c u l a t e d

coefficient

weights, of

ligand

of a g r o u p

assuming

advantage

in the

In c o n d i t i o n s

retardation

in m o l e c u l a r The

and without

ligand.

concentration,

disregarded. periment

with

distances

coefficient

with

of l i g a n d

{1}

m i qy r a t i o n

electrophoresis

The

single

the

affinity

is

lectin

that

etc. only

of

from can

be

one

ex-

in

the

incorporated

gel. With

several

tion

can

in t h e the

be

first

load

equilibrium tions

calculated (35)

gration

The

5o ng with

concentration

retardation

provided

to

conditions

between

as

the

there

per

retarda-

of

lectin

is i n d e p e n d e n t

is s u r p l u s

analysis

the n o r m a l l y

which

is a s i m p l e

velocity

K is the

in c o m p l e x ,

and

of

the

=

I T mo

constant

relation

did

used

of

not

lectin.

change

lectin

of the

concentra-

(1

+

from

between

a lectin a

may

be

Takeo-Nakamura

the

relative

mi-

i2>

-T>

constant

sites,

glycoprotein

and

concentration:

concentration

of b i n d i n g

the

protein

lectin

dissociation

c is the

concentration mobilily

a retarded

dissociation

TT" mi

where

gel.

amount

of up

concentration-dependent

increasing

(7) .

affinity

plot

the

using

dimension

glycoprotein

A protein

The

experiments, examined

of t h e

of l e c t i n

the

without

lectin

'normality', lectin,

glycoprote-

expressed RmQ

is

R , is t h e

as

the

the mobi-

137 lity

of

the

glycoprotein

mobility

of

internal

standard

albumin The on

or

the

the

lectin such

the

presence

as

plot

complex

component

is

ways

with

met

ferable

using to

involved the

the

difficulty

have

an

kidney

free

of

and in

on

this

non-linear

in

information

determination

of

the

it

is

pre-

of

con

e£

is

al-

pre-

effort

eliminates bound were

lectin. found

plots

(35)

A were

general

dissociation

met

but

the

and

lectins

is

often

saves

glycoprotein

with

an

situati-

not

Takeo-Nakamura

with

complex

is

it

amount

free

the

lectins,

lectins

exact

the

to

albumin,

to

which as

because

and

complex

glycoproteins

Based

the

is

components

However,

immobilized

glycoproteins

p h o s p h o r y 1 ase

trophoretic

lectins.

estimating

between

ligands

Rmc

relation

confined

interacting

lectins

the

e i g e n m o b i 1 ity

serum

(15).

with

preparing

and

in

orosomucoid.

a condition

macrorno 1 e c u 1 ar

immobilized

work

in

Complexes

man

free

lectin

of

implicitly

between

1 e c t r o p h o r e t i c a 11 y i m m o b i l e , priori

of

complex

bromphen o1b1ue-marked

non-reacting

Takeo-Nakamura where

in

glycoprotein

to

for

and

hu-

described

equation

constants

for was

elecde-

rived: 1 - R .

R mo where taken are

is in

as

the

those

(lectin

mo

mobility

used

line

of

the

in

equation

concentration)

^

is

the

(R

the

intercept

on

the

c-"*" i s

The

slope

where

the

of

{2}.

1 - R

R mo

This

(the

{ 3 }

mc

A glycoprotein

standard

the

line

mo

gives the

con

mobility

the

plotted

- R

complex

other

equation

symbols

represents

a

mc

mo

K - R

dissociation

the

is

(R m o (R

mo

-

R m i. ) ^ . R

mc

) ^

and

mc

A glycoprotein of

aqainst

J-"*" a x i s

-K-^.

is R

equation

1 c

when

on

The

con

internal

intercept

mobility

. mc

the

to

The

of

K - R

R

mi

relation

straight

"

con

constant complex.

A glycoprotein

K as In

the

well

as

simple

complex

is

the case

zero

138 (Rmc

= 0),

on of

equation

ligand

Subsequently, f i e d by

the

Horejsi

A prerequisite stant the

is t h a t

gel

with cause tes

the

the

exact

to

allow

.Chromatography A correlation nique

and

there

were

suggested

of

ligands

was

the

affinity

relation

was

milarly

between

veri-

the d i s s o c i a t i o n is not

con-

influenced

suited

large

for

by

work

complexes,

Prediction

found

When

the

be-

aggrega-

of

Affinity

for

affinity

chromatography,

strength,

prediction

the

by

by

three

various and were

above,

not we

of

and

gel

tech-

even

though

temperature

experiments results (13,

of 27).

A fractions

A

of

c o n A in

the

with

A

con

were correcoralpha

first

(4).

chromatography

The

was

si-

Immunoelectrophoresis.

lectins,

we

found

a

correlation

column

affinity

chromatography

bound

strongly

in

predict

use

an a n a l y t i c a l

con with

affinity

affino

glycoproteins

could

pH

experiments

chromatography

separated

with

intermediate

electrophoresis

in the

glycoproteins

as

been

of m a c r o m o l e c u l a r

affinity

separated

study

As m e n t i o n e d

of

forming

in e l e c t r o p h o r e s i s

and

we

has

especially

in t h e

in i o n i c

electrophoresis

trophoresis

lectins.

also

clearly

when

phoresis

column

of A F P

precipitate.

concentraticoncentration.

(22).

complex

chromatographic

(AFP)

gel

In a n o t h e r between

observed

analytical

sponding

forms

The

derivation

is

penetration

to be u s e f u l

dimension

the

gel

{2}.

monomer

(29).

differences

fetoprotein

the

Results

ordinary

Consequently

its

symbols

agarose

• lo7

2

as

determination

mobility

The

pores

and

other

Immunoelectrophoresis

Affino

only

equation

for

equation

calculated

macromolecular

of up

three

be

using

matrix.

free

{3} b e c o m e s

should

were

retarded

binding

interaction prediction

in

on

the

the

line

method

affinity

during

for

the

column

elec-

(27).

Immunoelectropreparation

of

139 Biomedical

Applications

We

the

propose

alternative used

analytical

to

is

when

available

or

in

specific

information

One

step

the

pattern on.

for

and of

This

the

was

ted

major

in

normal

of

five

Qrosomucoid

alpha-1 The

alpha-1

flammation rheumatic the

glycoprotein range

seen

ponents aemia

were

and

severity sponse, of

ins to

of and

with

in

the

disease

generally

proteins

the

disease

the

greatest

to

glycoprotein

highest

carbohydrate

Raynes.

These

of

the

Table

I.

the

each was

in

respon-

be

the

of

pattern

with

acute

phase

outside

a link

acute

that

the com-

septicto

the

phase

re-

in

the

pattern

which

are

the

of

are

after

in-

found

pancreatitis, may

content

forms

(3o).

A non-binding

observed

increases

relates

of

proteins

microheterogeneity Raynes

con

acute

were

=

aseptic

was

pro-

ceru-

associated it

of

orosomucoid

by

concentration

and

There

the

two

and

often

The

size

repor-

phase

(AGP),

pregnancy,

septic

pregnancy

in

the

and

remarkab-

HS-g1ycoprotein

cancer,

and

the

antitrypsin

components

changes

are

acute

surgery.

or

variati-

recently

alpha-1

diagthe

that

the

samples.

for

for

normal

serum

in

spe-

need

has

increased

major

be

establish

their

inflammation

serum

to

conclusion

acute

of

is

the

examined

amounts

may

biological

methodology

and

an

methods.

was

alpha-2

during

that

be

Raynes

metastatic

severe

normal

plasma

variation given

A was

to

proteins,

acid

and

antichymotrypsin

studied show

in

human

serum

chronic and

following

alpha-1

teins

the

in

in

con

wounding,

and

with

circulation.

major

inhibitor

disease

variation

of

= alpha-1

with

surgical

proteins

of

there

these

this

antichymotrypsin,

protease

variation to

by

of

approach

when

diseases

(21)

forms

a study

plasmin,

use

human

approach

amount

cases

offered

in

an

a small

other

serum

constant

teins:

se

of

reported

microheterogeneity ly

methods,

routinely

screening

Immunoelectrophoresis

e1ectrophoretic

only

the

nosis

Affino

preparative

conveniently

cimen

of

the also

five the

pro-

protefastest

a stimulus.

The

orosomucoid

is

of

orosomucoid

140 and

alpha-1

changes

of

lus

which

the

plasma

sylation before The

of

an

sin

has

42)

been

from

of

is

pattern

injury

in

con

of

of

stimurate

the

in

glyco-

antennary

sugars

seen

by

in

compared

by

Raynes

enzymes

(3o)

Raynes

with by

A binding

the

in

subse-

Also

at

the

antichymotryp-

a higher

and

that

the

(3o).

alpha-1

towards

Raynes

whether

from or

the

the

one

changes

whether during

these

blood

con

Wells

et

associated

explanation

may in

be

able

At of

to

A

non-

al.

(41,

with

deduce

carbohydrate

changes

reflect

circulation,

glycoprotein.

any

example

normal of

to

45%

serum

This

in

normalities, as

12

has

A affinity

valuable

alpha

this the

thus

point

a

chan-

microheterothe to

there

clearance reflect

is

no

microheterogeneity

of lead

fetoprotein between

of

has

proportion

32).

con

is

pregnancies

fetal

lower

such

addition

back

by

a

biosynthetic

a change

pancreatitis

change

to

the

reason of

the

glycoproteins.

whereas

of

to

significant

(3o).

explained

acute

of

levels.

alteration

consists

(31,

in

question

eliminate

for

in

glycosylating

reduction

forms,

Another

ly

a

in.synthesis

serum

were

the

found

is

hepatic

plasma

tissue

studied

main

an

to

proteins

function to

of

estrogen

the

lack

HS-g1ycoprotein

result

g1ycosy1 ation

(17).

there

geneity

the

proportion

the

higher The

the

response

attack

in to

responsible into

inflammation

binding With

in

induction

end

or

sequence

the

aIpha-2

a change

secretion

chronic

of

glycoproteins

phase

quent

pattern

causes

changes

acute

ge

a n t i c h y m o t r y p s i n , and the

the been

con to

variants

diagnosing

of

and

33.5

reported

the in

by

fetal

Amniotic

fluid

weeks

pregnancy

A non-reactive to

amniotic

a change serum

fluid

in

across

a

that is

(33). AFP

AFP

(32),

significant-

variants

demonstration

abnormalities

of

form

contain

A non-reactive

fetal

characterized

transudation

15

con

(AFP).

( 2 - 1 o ?£) the

pattern

potentially In

fetal

ab-

compartmentation

exposed

fetal

mem-

141 branes

in t h e p r e s e n c e

of n e u r a l

been

observed

in t h e p a t t e r n

nity

variants

to r e s e m b l e

measurements recently con

of c o n

been

A included

been

found

to

in the

The

affinity

comparison (16, be

24).

of

techniques

fetal

in h e p a t o m a

percentage

of

ra t h a n

fetal

in

C3c

and

LCA sera

have

physiological

role

ses.

as t h e

However,

C3 m o l e c u l e separation we

as w e l l of the

are c u r r e n t l y

procedures analysis

of

of

(36).

and

serum

a shift

has

AFP

A

con

(32,

33).

in a m n i o t i c This than

method the

of the

has

AFP con

and

has

with has

previous

also

lectin

been

used

affinity

A affinity

forms

fetal

sera.

However,

hepatoma

sera,

but

reactive

The

fluid

electrophoresis gel.

affi-

11).

system

among

fluid

reliable

form

is h i g h e r

for

patterns seem the

to LCA

generally

the

in h e p a t o m a

se-

(7). products

been the

complement

as t h e

molecules examining

prior the

third

system

to the

a crossed

of

the

on the

disea-

native

necessitate

specific of con

fac-

patho-

in v a r i o u s

products

usefulness

in a s e r u m

complement

in s t u d i e s

of e p i t o p e s

cleavage

5 shows

C3 m o l e c u l e s

of the

employed

distribution

Fig.

fetal

variants

hepatoma

of s p l i t

C3d,

defects,

affinity

(7,

sera

vary

the

Quantification tor

and

proportion

patterns

of

dimension

simple

electrophoresis

The

similar

affinity

using

first

be m o r e

chromatographic

that

A affinity

performed

tube

of a m n i o t i c

a

measurements A in

these

immunoelectrophoretic

sample

from

a

patient

F i g . 5. C r o s s e d I m m u n o e l e c t r o p h o r e s i s of s e r u m f r o m a p a t i e n t with c o m p l e m e n t a c t i v a t i o n . Anti C3c (lower part) and anti C 3 d ( u p p e r p a r t ) ; c o n A in the f i r s t d i m e n s i o n g e l in b.

142 with

complement

first

dimension

activation

and

gel

precipitation

on

antibody-containing whereas re

the

upper

suggests

acting

with

the

anti

the

lower

the

C3d

con

which

were

sults

are

in

This

C3d

of

in

reacting

addition

reacted

the

in four

differently two

precipitated. of

with

appeared

interaction,

being

figure-

con

forms These

A in

the

re-

direct

Immunoelectrophoresis

described with

e.g.

of

(38,

the

lectins

may

be

molecules, other

principles

as

of

ligands.

potential

hydrophobic

affinity

However, in

been

Immuno-

multitude electropho-

ligands,

metals,

has

a

this

charged

stains,

heparin

in

use

ligands,

dyes,

ligands

ions

used

in

spe-

(11).

As

recent

43). of

heparin

electrophoresis Fig. of

a serum

dimensional

human

serum

a

proteins

in

factor

seen

as

The

proteins

second

gel

B of an

the

the

of

(2o

Using

dimensional

contained

showed

no

system

interaction (anodic

was

is

the

seen

effect

with of

to

that

When

the

antibodies

velocity

shift

added against

mobility.

electrophoretic

Immunoelectrophoresis

it

affi-

been

Immunoelectro-

u/ml)

gel

in

have

antibodies

monospecific

complement

protein

crossed

electrophoretic

increased

which

Heparin H.

plasma

chromatography

results

marked

changed

with

affinity

sample.

gel

the

dimension

was

interaction and

6 shows

first

crossed

no

The

C3d.

ligands

effect

human

gel)

side:

Affinity

has

receptor

cond

upper

form

example

few

(the

anodic

the

lower

molecules

those

In

in

antibodies

antibodies.

from

experiments.

A

The

C3c

immunoprecipitate

utilization

cific

phoresis

A

con

anti

separate

the

Ligands

compared.

no

C3d

to

antibodies as

con

the

anti

used

one

system

The

C3c

be

for

resis

nity

contained

of

pattern.

5 received

and

chapter

years

Fig.

influence

retarded

various

an

of

the

promising

electrophoresis of

in

A can

the

from

quantification

Other

gel con

anti

forms A,

gel

antibodies

gel

molecular with

that

the

of of

heparin

the

total only se-

against heparin

factor

B.

in

migration)

all

143 appeared rose parin when was

in a s h a r p

chromatography. in c r o s s e d analyzed bound

by

tarded

(e.g.

lating

proteins,

Experimental

void

volume

proteins

plasma which

(e.g.

protein

B and

fell

chromatography

Sepharose C4)

showed

into

i.e.

two

A) a n d p r o t e i n s to

the

interaction

Sephawith

III

which

bulk

he-

groups

proteins

antithrombin

relative no

on h e p a r i n

interacting

which and were

of

pregre-

circu-

in e i t h e r

system.

Procedures

following

Our

experimental gel

affinity

factor

The rose

in t h e

By c o n t r a s t

Immunoelectrophoresis

to h e p a r i n

nancy-associated

peak

is a s h o r t

description

procedures

electroimmunoassays

are and

of m a t e r i a l s

essentially the

reader

those

and

methods.

used

is r e f e r r e d

in

aga-

to

F i g . 6. C r o s s e d I m m u n o e l e c t r o p h o r e s i s of s e r u m w i t h a n t i b o dies against serum p r o t e i n s (upper panels) and a n t i b o d i e s a g a i n s t f a c t o r B ( l o w e r p a n e l s ) . H e p a r i n in the f i r s t d i m e n s i o n g e l s m a r k e d H.

144 manuals tion

in e l e c t r o i m m u n o a s s a y s

(8, 9,

lo,

TRIS-barbital 22.4

water

TRIS-barbital 1 part

stock

% agarose

king

bath

ready

for

be

at use

The

solution,

pH 8.6,

tion

solution,

pH 8.6,

4 parts

distilled

(w/v):

1 g agarose,

The g e l C.

again

between of

is b o i l e d

The

gel

after

for

gel.

for

boiling. found

is p e r f o r m e d plate

paper

and

the

wicks

agarose

15 to buffer

(Whatman

employed

the

following

per

wicks

is u s e d :

8 layers

for

the

resis

(2

The

one

bromphenol front second 18

for

the

kept

C, a n d

in it

lectin

first

second

a is

may in

the

C.

vessel 1).

electrophoThe is

connecestablished

Depending

number

of

filter

dimension dimension

on pa-

electrophoelectropho-

V/cm).

first

about

5 layers

(wor-

suitable.

18°

No.

strength V/cm),

di-

0.02:

electroendosmosis

field (10

and

at 4 °

on w a t e r - c o o l e d of

g TRIS,

TRIS-barbital

immobilized

Litex

at a t e m p e r a t u r e

the gel

Low

0.1:

strength

5 minutes

the

resis

instruc-

water.

stored

with

44.3

ionic

100 ml

c a n be

experiments

We h a v e

filter

and

strength

(veronal),

working

apparatus

means

acid

solution,

56°

details

ionic

ml.

electrophoresis

resis

further

to l o o o

advantageous

intermediate

by

stock

solution).

water

for

34).

g 5,5-diethyl-barbituric

stilled

Gel

12,

dimension

electrophoresis

hour,

the

migration

blue

and

serum.

of the

bromphenol

dimension

may

The

blue

is n o r m a l l y

be c h e c k e d

free

albumin

electrophoresis

by

bromphenol

performed

for

a mixture

of

will

in t h e n o r m a l

is p e r f o r m e d

migrate buffer.

overnight

(15

in The to

hours).

Pressing,

washing

precipitated

and

proteins

drying:

It is n e c e s s a r y

in o r d e r

to g e t

low

to

remove

background

non-

staining.

145 This gel

is in

sheet

done

as

order

to

of

sorbant

filter paper

is p l a c e d

nutes

the

0.1 to

for

swell.

be

analyzed, for

5

is

Lectins: they

are

gel

plate.

and

the

gel

for

15 m i n u t e s

plate

may

hot

be

air.

should

be

before

with

then

5

is

The

cold

con-

the

air

in

gel

as

gel

activity

with

mi-

washed

10 m i n u t e s

in

abglass

After

causing

If e n z y m e dried

one

A thick

repeated.

staining

the

of

pressing is

for

over

layers

pressing.

is p r e s s e d

of

activity

gel for

covered many

The

swelling

plate

the

renewed

water

the

gel

is

performed

in

5 g Coomassie

4 5 0 ml

the

450

450

450

to

is and

ml

Coomassie

is

Normally in

aplate is

to

and

Coomassie

Brilliant

We free

ion to

filtered

96%,

1 0 0 ml water.

use

Blue

on

melted

lectins in

an

acid

immunoglobulin

by

salting

anion

agarose

98%

(techni-

may

be

After

rege-

destai-

fraction

from

immunoglobulin out

with

before

ammonium

The

antibo-

casting.

analogously

to

solution

immobilized

or

The

air.

exchanger.

gel

the

use.

hot

The

99%

overnight.

carbon. of

(Hop-

acid

mixing,

destainer

concentrations.

essentially

lectin

acetic

a stream

the

While

left

The

R 250

acetic

before

activated

in

exchange the

treat as

and

cooled

is

Blue

ml

C,

through

we

100

water.

dried

low

purified

added

60°

Brilliant

96%,

distilled

distilled

plate

by

ml

solution

filtration

gel

ethanol

ethanol

antiserum

are

tap

and

heated

day

Antibodies :

fraction

is

5 minutes.

grade),

by

sulfate

paper

the

is

poured

minutes.

grade),

rabbit

on

of

a stream

the

Destaining:

dies

in

placed

paper

solution:

following

the

top

this

& Williams),

nerated

then

on

Staining

for

solution

the

is

Blue.

(technical

ning

1 ) and

enzyme

Staining:

cal

gel

plain

dried

Brilliant

kin

The No.

pressing

then

Staining

well.

(Whatman

another or

is

R 250

fill paper

After

The

stained

water

absorbant

M NaCl

bove.

Distilled

are

plate

tinued

follows:

antibodies, on

whether partic-

146 les.

The

lectins

paration

and

problems

have

of

added

commercial

bility

buffer

trol

to

been

lectins

experiments for

agarose

as

We

an

gel

with

affinity-purified

before

casting.

stability

advise

may

displacer: include

the

be

next

to

(the

water

specific

to

to

test

or

heavy

affinity

cific

displacer

some

pre-

Numerous

and

solubility

the

reproduci-

the

first

totally

or

in

the

and

1.5

an

partially

(over

the

The

A we

to

5%

or in

For

take

the the

have

with

affinity

up

the

inter-

suc-

a-D-glyco-

dissolving

first

or

affini-

used

methyl

gel

the

experigel

electro-

will

gel

bound

con

agarose

overlayered

control

gel.

a-D-mannopyranoside.

been

con-

with

antibody

cathode)

dimension

with

for

washing

the

glycoproteins

containing

electrophore-

lectin

cofactors.

experiments

towards

the

with

repeated

displacer

precipitates, have

by

essential

experiments

gels

methyl

3 times

immobilized

1ectin-containing

into

For

agarose

pyranoside

of

flow

release

precipitate.

cessfully

either

the

displacer gel

produced

For

gel

2 to

Inactive

a specific

endosmosis

mediate

washed

use.

removal

Specific we

are

to

ments

ty

the

used

encountered

preparations.

prior

EDTA-buffer

in

normally

carefully.

Immobilized sis

are

to

3o

dimension

extra spegel

precipitate).

Acknowledgements This

work

Council

was

and

supported

the

Harboe

by

The

Danish

Medical

Research

Foundation.

References

1.

A n d e r s e n , M . M . , H a u , J . , B i a g - H a n s e n , T. C . : In T. C. BiagHansen (ed.) L e c t i n s - B i o 1 o g y , B i o c h e m i s t r y and Clinical B i o c h e m i s t r y . V o l . 2. W . d e G r u y t e r , B e r l i n , p . 7 7 9 (1982).

2.

B a r o n d e s , . S.

3.

B i t t i g e r , H., S c h n e b l i , Wiley, London, (1976).

H.:

Ann.

Rev.

4.

B a y a r d , B . , K e r c k a e r t , J. mun. 95, 7 7 7 - 7 8 4 , (198o).

H.

Biochem., P.:

P.:

5o,

2o7 - 231 , (1981 ) .

Concanavalin Biochem.

A as

Biophys.

a

Tool.

Res.

Com-

147 5.

B j e r r u m , 0. J . : In Protein Chemistry,

H. W.

6.

Bag-Hansen,

Anal.

7.

B o g - H a n s e n , T. C. ( e d . ) L e c t i n s - B i o l o g y , B i o c h e m i s t r y a n d C l i n i c a l B i o c h e m i s t r y . V o l . 1 a n d 2, W . de G r u y t e r , B e r l i n , (1981, 1982).

8.

Bag-Hansen,

T.

9.

Bag-Hansen, I m m u n o l . 4,

T. C . , Suppl.

10.

B a g - H a n s e n , T. C . , B r o g r e n , S u p p l . 2, 1 3 5 - 1 3 9 , ( 1 9 7 5 ) .

C.

11.

B a g - H a n s e n , T. C . , H a u , J . : L i b r a r y , V o l . 18 B , 2 1 9 - 2 5 2

In J o u r n a l (1982).

12.

B a g - H a n s e n , T. C . , L o r e n c - K u b i s , I . , B j e r r u m , 0. J . : In B. J . R a d o l a ( e d . ) E l e c t r o p h o r e s i s ' 7 9 . A d v a n c e d M e t h o d s . B i o c h e m i c a l a n d C l i n i c a l A p p l i c a t i o n s . W. de G r u y t e r , B e r lin, 173-192 ( 198o ) .

13.

Bag-Hansen, M e t h o d s 22,

14.

B o g - H a n s e n , T. C . , S p e n g l e r , G . A . ( e d . ) : Lectins-Biology, B i o c h e m i s t r y a n d C l i n i c a l B i o c h e m i s t r y , V o l . 3, W . d e Gruyter, Berlin (1983).

15.

Bog-Hansen, (198o ) .

16.

B r e b o r o w i c z , J . , B a g - H a n s e n , T. C . : In T. C. B a g - H a n s e n (ed.) L e c t i n s - B i o l o g y , B i o c h e m i s t r y and Clinical Biochem i s t r y , V o l . 2, W. d e G r u y t e r , B e r l i n , 4 4 5 - 4 5 6 (1982).

17.

B o w e n , M . : In T. C. B a g - H a n s e n ( e d . ) L e c t i n s - B i o l o g y , Bioc h e m i s t r y , C l i n i c a l B i o c h e m i s t r y , V o l . 2, W . d e G r u y t e r , Berlin, 4o3-411, (1982).

18.

B r o g r e n , C. H . , B i s a t t i , S . : In T. C. B a g - H a n s e n (ed.) L e c t i n s - B i o l o g y , B i o c h e m i s t r y and Clinical Biochemistry. V o l . 1, W. de G r u y t e r , B e r l i n , 3 7 5 - 3 8 6 , (1981).

19.

Dulaney,

T.

C.:

C.:

Tschesche (ed.) Modern M e t h o d s de G r u y t e r , B e r l i n (1983). Biochem.

Scand.

J.

J.

T.:

C.,

48o-488

Immunol.

Suppl.

B j e r r u m , 0. J . , R a m l a u , 2, 1 4 1 - 1 4 7 , ( 1 9 7 5 ) .

T. C . , P r a h l , P . , 2 9 3 - 3 o 7 , ( 1 9 7 8 ).

T.

56,

Takeo,

Mol.

Cell.

K.:

H.:

Scand. of

(1973).

lo.

J.: J.

press)

Scand.

J.

Immunol.

4,

H.:

Electrophoresis

21,

(In

Chromatography

Lawenstein,

Biochem.,

in

43-62

J.

1,

Immunol.

67-71,

(1979).

2o . F a y e , L . , S a l i e r , J . P . : In T. C. B a g - H a n s e n ( e d . ) L e c t i n s B i o l o g y , B i o c h e m i s t r y a n d C l i n i c a l B i o c h e m i s t r y . V o l . 2, W. de G r u y t e r , B e r l i n , p. 6 o 5 , ( 1 9 8 2 ) . 21.

Hinnérfeldt, D. S t a t a c h o s lin (1983).

F., A l b r e c h t s e n , J., B a g - H a n s e n , ( e d . ) E l e c t r o p h o r e s i s '82, W. de

22.

Horejsi,

V.:

J.

23.

Horejsi, (1979).

V.,

Ticha,

24.

Kerckaert, phys. Acta

Chromatography M.,

178,

Kocourek,

1-13,

J.:

J. P., B a y a r d , B . , B i s e r t e , 576, 99-loB, (1979).

TIBS G.:

T. C . : In G r u y t e r , Ber-ì

(1979b). 4,

1,

N6-N7,

Biochim.

Bio-

148 25 .

Krall, J., (1975) .

Andersen,

26 . N a k a m u r a , S . : Applications. (1966) . 27 .

Cross Igaku

M.

M.:

J.

Electrophoresis. Shoin, Tokyo and

29.

P l u z e k , K. : In T. L e v r i n g S y m p o s i u m . 7 1 1 - 7 1 8 , W. d e

30 .

R a y n e s , J. : B i o m e d i c i n e

Ruoslahti , E 31 . J . C a n c e r 22 S m i t h , C. J . 32 . 1 - 3 2 , ( 1 9 8 o )

34.

Methods

9,

141-146,

Its P r i n c i p l e and Elsevier, Amsterdam,

N i l s s o n , M . , B a g - H a n s e n , T. C . : In H . P e e t e r s ( e d . ) P r o t i d e s of the B i o l o g i c a l E l u i d s , P r o c e e d i n g s of the X X V I I C o l l o q u i m , Vol. 27, P e r g a m o n P r e s s , O x f o r d , (1979).

28 . P i a t t , H. S . , S e w e l l , B. M . , Clin. Chim. Acta 46, 419-429

33 .

Immunol.

S m i t h , C. J . Brit. Med. J

Eeldman, (1973).

T.

Souhami , R . L . :

(ed.) Xth I n t e r n a t i o n a l Seaweed Gruyter, Berlin, (1981).

36,

77-86

(1982).

, E n g v a l l , E. P e k k a l a , 515-520 , (1978 ) . C.:

Kelleher.

A.,

Seppälä,

M.:

Int,

Biochem,

Biophys.

Acta

6o5,

K e l l e h e r , P . C . , B e l a n g e r , L. , D a l l a i r e , L. : 1, 9 2 0 - 9 2 1 , (1979).

S v e n d s e n , P. J . : In D e y l , Z . , E v e r a e r t s E. M . , P r u s i k , Z., S v e n d s e n , P. J. ( e d s . ) , Electrophoresis A S u r v e y of T e c h n i q u e s a n d A p p l i c a t i o n s , P a r t A : T e c h n i q u e s . J. C h r o m a t . L i b r a r y , V o l . 18 A , E l s e v i e r S c i e n t i f i c P u b l i s h i n g C o . , A m s t e r d a m , 13 3 - 1 5 4 , ( 1 9 7 9 ) .

33 .

Takeo, K., (1972).

36 .

Teisner, B., Hau, Svehag , S.-E.:(To

Nakamura,

S.:

Arch.

Biochem.

J., B r a n d s l u n d , be p u b l i s h e d ) .

I.,

Biophys.

Bag-Hansen,

37 . T e i s n e r , B . , B r a n d s l u n d , I . , H a u , J . , S v e h a g , P a t h . M i c r o b i o l . I m m u n o l . S c a n d . (In p r e s s ) . 38 . T e i s n e r , A c t a (In

B., Davey, press).

39.

Tobiska, (1964).

J.:

40 .

Turkova, J.: A f f i n i t y Library (1978).

41 .

W e l l s , C . , C o o p e r , E . ( G l a s s , R. M . , Clin. Chem. Acta lo9, 59, (1981).

Die

M.

W.,

Grudzinskas,

Phytohamagg1utine.

J.

G.:

1-7 ,

153,

T. C . ,

S.-E, Clin.

Acta Chim.

Akademie-Ver1ag-Ber1in,

Chromatography.

J.

Chromatography

Bag-Hansen.

T.

C.:

4 2 . W e l l s , C . , C o o p e r , E . , B a g - H a n s e n , T. C . : In T. C . B a g Hansen (ed.) L e c t i n s - B i o 1 o g y , Biochemistry and Clinical B i o c h e m i s t r y . V o l . 1. W. de G r u y t e r , B e r l i n , 3 3 9 - 3 4 6 (1981). 43 . W e s t e r g a a r d , G.: P l a c e n t a

J. G . , (1983)

Teisner, H a u , 3., (In p r e s s ) .

B .

Grudzinskas,

J.

PRINCIPLES AND APPLICATIONS OF HETEROGENEOUS ENZYME IMMUNOASSAYS

Gerd Grenner Research Laboratories, Behringwerke AG D-3550 Marburg

Introduction The l a b e l l i n g of antigens and antibodies with radioactive substances in s e n s i t i v e immunoassays has been the method of choice for many years. In 1971, two research groups (1, 2) independently described immunoassays following conventional separation techniques but using enzymes f o r l a b e l l i n g instead of radioactive isotopes (heterogeneous enzyme immunoa s s a y s ) . One year l a t e r an enzyme immunoassay not requiring a separation step (3) was reported (homogeneous enzyme immunoassay). The heterogeneous ®

method has been called ELISA (enzyme-linked immunosorbent a s s a y ) . EMIT (enzyme multiplied immunoassay technique) i s used f o r homogeneous procedures. Enzyme immunoassay has been extensively reviewed (4-8). The f i r s t part of t h i s paper deals with the p r i n c i p l e s of heterogeneous enzyme immunoassays with special reference to protein determinations. In a second part some developments of ELISAs f o r protein determinations from our own laboratory are described.

Assay p r i n c i p l e s D i f f e r e n t assay types f o r the determination of antigens and antibodies have been developed. The main p r i n c i p l e s for antigen assays are shown in f i g . 1.

Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York

150 Competitive enzyme immunoassay

O.D.

O - E

o

antigen concentration

Immuno enzymometrlcal assay

-o

O > E

Sandwich assay •P k

o

>

E

= solid phase F i g u r e 1:

0 =

A s s a y principles

antigen

for antigen

- Q = antibody

E = enzyme

determinations

In t h e c o m p e t i t i v e type the enzyme-label led a n t i g e n competes w i t h the sample antigen for the binding sites of a n t i b o d i e s bound to a solid (9). A f t e r phase s e p a r a t i o n

the free or the bound phase is d e t e r m i n e d . T h e e n z y m e activity is proportional

phase

(see b e l o w ) the e n z y m a t i c a c t i v i t y in e i t h e r

to t h e s a m p l e a n t i g e n

inversely

concentration.

T h e sample antigen inhibits the binding of an e n z y m e - l a b e l l e d a n t i b o d y to a s o l i d - p h a s e bound a n t i g e n in the i m m u n o e n z y m o m e t r i c t i t i v e and a sequential

procedure is

assay

(10). A c o m p e -

possible.

The s a n d w i c h assay is r e s t r i c t e d to antigens w i t h h i g h e r m o l e c u l a r

weight,

s i n c e it requires antigens b e a r i n g at least two b i n d i n g sites for antibody. T h e sample a n t i g e n is reacted w i t h excess s o l i d - p h a s e a n t i b o d i e s .

After

s e p a r a t i o n , e n z y m e - l a b e l l e d a n t i b o d i e s are added to c o m b i n e w i t h the remaining antigenic determinants

(11). T h e o n e - s t e p v e r s i o n o f the

assay is c h a r a c t e r i z e d by the s i m u l t a n e o u s

reaction of the sample

sandwich antigen

w i t h the s o l i d - p h a s e and the labelled a n t i b o d y . This assay t y p e is m a i n l y a p p l i c a t e d w h e n monoclonal

a n t i b o d i e s a r e used

(12).

151 Phase separation A f t e r completion of the immunochemical reaction steps a separation of the immune complexes from the excess reagents i s required. Most of the c l a s s i cal methods of phase separation employed in radioimmunoassays, e.g. precip i t a t i o n by s a l t , a l c o h o l , polyethylene g l y c o l , second antibody etc. were found to be inconvenient in enzyme immunoassay due to problems with determination of enzyme a c t i v i t y in sediments. Therefore solid-phase matrices are most commonly used f o r separation (8). In table 1 some solid-phase materials are l i s t e d . Table 1:

Solid-phase matrices for phase separation

Material

Immobilisation of antigen

Separation by

Cel 1 ulose Agarose Latex

covalent linkage (e.g. BrCN a c t i v a t i o n )

centrifugation

magnetic f i e l d

magnetic p a r t i c l e s p l a s t i c tubes, bead, d i s c s (polystyrene, PVC, s i l i c o n rubber)

physical

paper d i s c s

covalent linkage

adsorption

décantation or aspiration

Antigens and antibodies can e a s i l y becovalentlylinked to c e l l u l o s e , agarose or latex p a r t i c l e s but c e n t r i f u g a t i o n steps and long washing cycles are required. The separation of magnetic p a r t i c l e s i s quite simple but f o r complete separation washing i s also necessary. Most widely used i s the physical adsorption of antigens and antibodies to p l a s t i c materials (tubes, m i c r o t i t e r p l a t e s , macro beads e t c . ) , mainly made from polystyrene (8). Phase separation i s achieved by simple decantation or a s p i r a t i o n . Because of the plane surface washing i s not complicated by d i f f u s i o n problems. Very e f f i c i e n t phase separation i s possible by adding and d i r e c t l y a s p i r a t i n g a s u i t a b l e washing s o l u t i o n containing a detergent.

152

The mechanism of adsorption of proteins to p l a s t i c surfaces i s not complet e l y understood. Both electronic charge differences between p l a s t i c s u r face and protein and hydrophobic i n t e r a c t i o n s may be responsible f o r the binding. Therefore d i f f e r e n t proteins do not bind with the same efficiency to the same s o l i d phase. The binding i s not t o t a l l y i r r e v e r s i b l e (13) but f o r most a p p l i c a t i o n s the release of proteins from the s o l i d phase during the incubations can be neglected. No general rules e x i s t for highly e f f i c i e n t and reproducible coating. Good solid-phase bio-reactants r e s u l t in most cases from a time consuming t r i a l and error process. Polystyrene m i c r o t i t e r p l a t e s and beads e s p e c i a l l y made f o r ELISA a p p l i c a t i o n s are a v a i l a b l e from d i f f e r e n t manufacturers.

Marker enzymes Enzymes for l a b e l l i n g in enzyme immunoassays must f u l l f i l l the following c r i t e r i a : As the enzyme i s used as an amplifier only those with very high s p e c i f i c a c t i v i t y can be used. Simple and reproducible procedures for the covalent coupling to antigens or antibodies must be p o s s i b l e . Another requirement i s an excellent s t a b i l i t y of the enzymatic a c t i v i t y during the assay procedure and during storage of the reagent. The enzyme of choice should be absent in the t e s t sample and should not be influenced in i t s a c t i v i t y by sample components. In

heterogeneous enzyme immunoassays horse-radish peroxidase

(E.C.

1.11.1.7), a l k a l i n e phosphatase (E.C. 3 . 1 . 3 . 1 ) , 6-galactosidase (E.C. 3.2.1.23) and glucose oxidase (E.C. 1.1.3.4) are most often used (8). No one enzyme i s i d e a l l y s u i t e d , the choice depending on the requirements of an p a r t i c u l a r assay, e.g. high s e n s i t i v i t y , stable s u b s t r a t e , simple l i n k i n g procedure etc.

153

Conjugates Conjugates are obtained by covalent linkage of the antigen or the antibody to the marker enzyme. The ideal conjugation procedure does not reduce the biological a c t i v i t i e s

(enzymatic a c t i v i t y and immunoreactivity) and has a

high y i e l d of a very stable product. No c r o s s - l i n k i n g between the same proteins (enzyme-enzyme conjugates) should occur. The reaction should be s t o i c h i m e t r i c a l and give conjugates of small s i z e (no polymerisation). None of the e x i s t i n g methods can achieve a l l these c r i t e r i a .

G1utaraldehyde method G1utaraldehyde has been widely a c r o s s - l i n k e r of proteins. In the one-step method (14) enzyme and antigen or antibody are mixed and glutaraldehyde i s added. Conjugates have very high molecular weight and are heterogeneous. Extensive homopolymerisation of the antibody has been described in the case of peroxidase-antibody conjugates (15). Immunoreactivity i s considerably diminished. Despite these drawbacks the method has been very often used because of i t s

simplicity.

The two-step method i s limited to peroxidase-conjugates

(15). Peroxidase i s

reacted with an excess of the aldehyde, the unreacted aldehyde i s removed by gel f i l t r a t i o n and the activated enzyme i s added to the protein to produce the conjugate. Homogeneous populations of conjugates are obtainable, but with low y i e l d .

Periodate method (16) The carbohydrate moiety of peroxidase can be oxidized to aldehyde groups. F i g . 2 shows the procedure in d e t a i l . After blocking the small number of free amino groups and p a r t i a l oxidation of the carbohydrates, the excess of reagents i s removed by d i a l y s i s or gel f i l t r a t i o n . The activated peroxidase i s then reacted with the protein followed by borohydride reduction of the S c h i f f bases. The method has a high y i e l d and the b i o l o g i c a l

154 NO 2

/' "V-NH—/ V - N02

( POD )

Figure 2:

\==/

Periodate method

activities are sufficiently retained. The degree of polymerization can be controlled by the concentration of periodate and the conditions

(pH, dura-

tion) of the coupling step. Glucose oxidase can be coupled using the same procedure

(17).

Diamaleimide

procedure

N,N'-0-phenylendimaleimide

has been used in a one-step procedure to couple

[3-galactosidase to proteins via the sulfhydryl and the protein

(18). In a two-step method

groups of both the enzyme

m-maleimidobenzoyl-N-hydroxy-

succinimidate acetylates the free aminogroups of the protein, the maleimide function is then reacted with sulfhydryl

groups of the enzyme.

N-Succinimidyl-3(2-pyridylthio)propionate method

In this two-step method (fig. 3)

(19)

N-succinimidyl-3(2-pyridylthio)propionate

(SPDP) is reacted firstly with free aminogroups of the protein. In the

155

Figure 3:

N-succinimidyl-3(2-pyridylthio)propionate

procedure

second step the activated d i s u l f i d e groups reacts with the s u l f h y d r y l groups of the enzyme to form a s t a b l e d i s u l f i d e bridge. The reagent can be used to introduce SH-groups to antigens and enzymes.

P u r i f i c a t i o n of conjugates P u r i f i c a t i o n of conjugates has been achieved by gel f i l t r a t i o n (14), ion exchange chromatography ( 2 0 ) , density gradient c e n t r i f u g a t i o n (21) and a f f i n i t y chromatography (22). In the case of antibody-enzyme conjugates simple gel f i l t r a t i o n i s s u f f i c i e n t , although the p u r i f i e d conjugate may contain free antibody. P u r i f i c a t i o n of antigen-enzyme conjugates i s

inmost

cases morecomplicated, i f high immunoreactivity i s necessary. The coupling procedure may have dramatic effects on the r e a c t i v i t y of antigens, e.g. i f the linkage i s via a functional group at or near to an antigenic determinant.

156 Applications Heterogeneous enzyme immunoassays have been described f o r the detection and quantitation of numerous parameters during the l a s t ten years. In the f o l lowing some appl i c a t i o n s for the determinations of c l i n i c a l l y relevant prot e i n s are described.

Alpha-fetoprotein

(AFP)

A1 pha-fetoprotein, a glycoprotein with a molecular weight of 70,000 Dalton, i s synthesized in the yolk sac and in the fetal l i v e r (23). Being a fetal protein i t proved useful in monitoring pregnancy, e s p e c i a l l y in screening for neural tube defects (24). Diagnosis and follow-up of l i v e r carcinoma i s another use for AFP determination (25). Both a p p l i c a t i o n s require very s e n s i t i v e assays which must be able to measure l e s s

than

10 pg/1. To achieve t h i s requirement, we selected the sandwich assay p r i n c i p l e for the development of an enzyme immunoassay (26). Fig. 4 shows the procedure in d e t a i l . 2 ml-polystyrene tubes are used for s o l i d - p h a s e , coated with antibodies s p e c i f i c f o r AFP. The sample (serum or plasma) i s d i l u t e d 1+ 10. After reaching the equilibrium of the f i r s t r e a c t i o n , the tube i s emptied by a s p i r a t i o n of the sample and then f i l l e d with phosphate-buffered saline, followed by immediate a s p i r a t i o n . This washing process i s repeated. This separation step i s necessary to remove excess, unbound antigen otherwise would react with the antibody in the conjugate preventing a reaction with the solid-phase bound antigen. This would r e s u l t in a low s i g n a l at very high concentrations of the analyte ("high-dose hook e f f e c t " ) (27). The addition of the conjugate i s followed by a s u f f i c i e n t incubation time and the second washing. This second phase separation i s c r i t i c a l , as a l l remaining excess conjugates molecules w i l l contribute to the solid-phase enzyme a c t i v i t y g i v i n g f a l s e l y elevated r e s u l t s of the antigen concent r a t i o n . The chromogen/substrate s o l u t i o n i s incubated for 30 min. Diluted acid i s then added to stop the reaction by destroying the enzymatic a c t i v i t y . The colour i n t e n s i t y of the s o l u t i o n i s measured in a photometer.

157 solid-phase antibodies (anti-AFP from sheep)

antibodies peroxidaseconjugate (anti-AFP from rabbit)

anti gen (AFP)

—





€

V . pipette incubate diluted sample* wash

substrate (o-phenylendiaminedihydrochloride, H2O2)

incubate wash 3*2 ml

200 pi

oo o o o oo

«»ti

oo o o o oo

€*|

pipette substrate/ chromogen

incubate

pipette stopping solution

photometry

200 pi

1/2"

1000 pi

492 nm

•diluted 1:10 with incubation medium

Figure 4:

Assay procedure of the enzyme immunoassay f o r AFP determination

F i g . 5 shows a typical c a l i b r a t i o n curve for the AFP assay. As in most immunoassays the nonlinear r e l a t i o n s h i p between antigen concentration and s i g n a l necessiates the use of a multipoint c a l i b r a t i o n . We use f i v e d i f f e r e n t c a l i b r a t o r concentrations to cover a range from 3 to 300 IU/ml (1 IU AFP i s approx. 1.6 ng).

2,0- A/.92r

1.0-

0,3-

0,1-

0.03-

-1— 10

—r30

AFP

Figure 5:

C a l i b r a t i o n curve of the AFP enzyme immunoassay

100

300

C o n c e n t r a t i o n (I.U./ml)

158 Lower concentrations can be measured d i l u t i n g the sample 1 + 1 . In t h i s case we determined a detection l i m i t of 0.4 Mg/1- The assay has a good precision. Intraassay c o e f f i c i e n t of v a r i a t i o n i s in the range of 4 - 6 %, and day to day c o e f f i c i e n t s of v a r i a t i o n are between 6 and 10 % (26).

Pancreatic

lipase

Pancreatic l i p a s e ( t r i a c y l g l y c e r o l l i p a s e , E.C. 3 . 1 . 1 . 3 ) i s an important marker in pancreatic d i s e a s e s . The glycoprotein has a molecular weight of 46,000 to 48,000 Dalton (28). The routine methods in the c l i n i c a l

labora-

tory measure i t s enzymatic a c t i v i t y . To overcome the problems of low s p e c i f i c i t y or low s e n s i t i v i t y of most of these methods, we developed a procedure measuring the protein concentration of the enzyme immunochemically

(29).

This enzyme immunoassay follows exactly the same protocol as the AFP t e s t . The measuring range i s 3 to 300 pg/l- Results from c l i n i c a l samples i n d i cate the usefulness of the assay in the diagnosis and monitoring of chronic

pancreatitis.

B2"microglobulin Determinations of B^-microglobulin, a protein with a molecular weight of 11,800 Dalton, are requested in kidney diseases and can be useful in the monitoring of some tumours (30). The c l i n i c a l l y relevant concentration range from 1 to 25 mg/1 would require an extremely high d i l u t i o n

(e.g.

1 + 1000) of the sample for a sandwich assay procedure. Therefore we decided to apply a competitive procedure ( f i g . 6). P u r i f i e d ^ - m i c r o g l o b u l i n conjugated to peroxidase i s used. Diluted sample (e.g. 1 +25 for serum samples) and antigen-enzyme conjugate are incubated in an antibody-coated p l a s t i c tube. After a 2 h incubation and a s i n g l e washing step, the solid-phase linked enzyme a c t i v i t y i s determined as in the other assays.

159 o OIo

f<
H fl) >H r-H

u

in IN

oo

r-v o O Osi o o o o o o o S) N ^ ^ CVi

+ + +

o o

co

n T—

n

o

O

o

• 0) c •H IH 3 C •H z 1—1 LU 1— o Di

CL

c •H E 3 -Q i—1
+-> 00 IO i—i — ta "Cl E o c to c c O o s- ai a; •— I IO a) •E i ai 00 s_ IO IO -O uo o c o . +-> +-> ai c ai un o "O sz a; ai S• to 0) ai +-> 3 +-> en .—t s- c c CT" O—i 1 0) • r-4 >, ai Í - ta LL. CO 3 E 00 00 cx en 4O SO 00

00

00

o t= aj • í - •rt c .—. +-I O m e o +-> —i > o S - CM C a . . to C • I—( CTIi—1 -CT 4- ai LL. co o +-> to c • t/l C : - H cn • I—to • .—! t/1 •^H 0 0 L I S-.+-I

UJ cj

);

m/e

no 120

indicates Phe at the amino end of the peptide chain and this is confirmed by the ion m/e 796 (3)

Interpretation:

( =

943-147(Phe)).

a) W i t h the a s s u m p t i o n

Phe

amino end of the peptide one can establish the with h e l p of ACO ions: Phe = 148 ( ^ 9 9 ) , Phe-Val-Pro = 344

457

(A 113), P h e - V a l - P r o - I l e - P h e = 604

(a97),

ions AA, AlkC, AC and

the above sequence. Signals m/e ( = 340 -101

sequence

Phe-Val-Pro-Ile

722

=

(A 147).

ACO

for

the

sequence - ions m/e 796, 697, 487, 474 and 621 [Thr]) and m/e 239

the

(not found), Phe-Val =

247

Calculating

at

(

=

above confirm

621

+

101

[Thr]) allow to add Thr

to the sequence. The sequence at the c a r b o x y l e n d of the peptide chain - T y r , G l y - remains (4)

unclear.

The FAB-MS of this neutral peptide shows some ACO

ions,

and ions of type AA and AC but no ion of type AlkC.

128

y r —- - Ala — -H 2 N -—T Tyr Q b

(181)

c d

(164) 1 136

- - Leu —- OH Gly —- - Phe — 309

(252)

(456)

¡392:

(321

(264)

(117)

407

336

279

132

235 1 207

292

439 1 411

MG 569 M - H 570

552

H2N — A l a - - Tyr - - Gly — P Phe h e — Leu —•OH MG569 a ]89) (252) 309 (256) M+H 570 1454) (321; b (264) (117) c (499) 336 279 132 d

(72)

235

I

207

292

439

I

411

552

353 Example 4 (1)

A m i n o acid a n a l y s i s : A l a , G l y , Leu, P h e , T y r , MG

(2)

I n s p e c t i o n of the m a s s s p e c t r u m : M + H 570; and 107 p r o v e the p r e s e n c e of Phe 86), Phe

(m/e 120) and Tyr

on the amino

(3)

and

(m/e acids

end of the p e p t i d e . D i f f e r e n c e s

411, 336 — >

the p r e s e n c e of ACO and AI

308, 235 — >

Interpretation:

In

several

for

candidates

this

207,

the

205

to

increments

of

has

of

the

peptide

construct

the

peptide

M+H

570

allows

to

all

amino

552.

Now

acids

present

f r a g m e n t 439 = 5 5 2 - 1 1 3

(Leu/Ile)

e s t a b l i s h e s Leu to

at

of

peptide;

the

carboxylside

increments not

(57-Gly, 7 1 - A l a , 147-Phe

verified

Proceeding

the

in

sequence

by

fragments

the

same

in

way

for

all are

mass

can

possible no

good

Ala-Tyr-Gly-Phe-Leu,

sequence

spectrum.

one

the the

the amino

masses

finds

Tyr-Ala-Gly-Phe-Leu.

arguments because

are

establish

Calculating

fragmentations

the

against

the

end.

good But

sequence

ion m/e 136 c a n n o t

t a k e n as a proof for Tyr at the amino

be

other

163-Tyr)

G l y - P h e - L e u . The s e q u e n c e of

a g r e e m e n t w i t h the there

the

and

the

one

end A l a , Tyr r e m a i n s u n c l e a r .

28

177)

one

the h e a v i e s t ACO ion to 570 - 18 =

the

^

—>

where

aminoend

it seems a d v a n t a g e o u s

of

ions.

situation

b e g i n n i n g w i t h the c a r b o x y l g r o u p . testing

91

Leu

(439 — >

calculate

Tyr.

m/e

(m/e 136) may be amino

indicate

chain

ions

569

be

354 1 1 2 1 20

h2n — Arg - - Pro - -Pro -- Gly - - Phe --Sér- -Pro 174

Q b c d

-- Phe -- Arg -

X25> S7? iés?) 903 421 — 3Q7 ¿z. ¿35 m 160 827 710 653 sai (5061 419 322 175 1157) 1254) (3511 14061 15551 1642] (739) 18661 11042) I I t 1 858 1014 MG 1059

—

ja

M. H 1060

167 ] 30

1 0-

1060 1 061

1 062

1 058 1000 90-4 960

800 2 0 -NO V - S 1 1 6 : 5 8

BRRDYKIN1N

850

950

g 7 3

ggg

1 0 1 5

1000

355 Example

5

B r a d i k i n i n ; this w a s one of the first

studied with (1)

Amino acid analysis: A r g 2 , G l y , P r o 3 , P h e 2 , S e r

(2)

I n s p e c t i o n of the mass s p e c t r u m : two p a i r s of s i g n a l s w i t h a observed,

t h e r e f o r e one

has

no

Only ACO

remember

indicating

AC

the p r e s e n c e

that a m i n o - a m i d

the c a r b o x y l s i d e

and

of

three

ions

1059. Only

A

of

chance

s e q u e n c e w i t h h e l p of ACO ions. A 15

MG

M + H 1060.

difference

with

Concerning

to

a

28

are

find

the

few

dubletts

ions

are

found.

prolin

one

should

(AA) are not p o s s i b l e

and A l k y l - c a r b o x y l - i o n s

not

p o s s i b l e on the a m i n o s i d e of p r o l i n ; also m i g r a t i o n

of

ion is

sometimes

F r a g m e n t m/e 120 c o u l d be r e s p o n s i b l e amino

end but all c a l c u l a t i o n s

acid met with

not

for

Phe

found. at

s t a r t i n g w i t h this

the amino

failure.

Interpretation:

A favourable

s e q u e n c e may be c o m p a r i n g acids

(AlkC)

on

are

a p r o t o n to the f r a g m e n t

(3)

peptides

FAB-MS.

present

in

the

s e a r c h for the

integral peptide

peptide

i n c r e m e n t s of with

amino

differences

p r o m i n e n t p e a k s of the m a s s s p e c t r u m s t a r t i n g w i t h molecular

ion: 1060 - 1043 = 17

(OH?)

(result n e g . ) , 1060 - 995 = 101

(Thr,

acid

is m i s s i n g

(Pro), 710 - 653 = 57

(Pro),

807

(Gly). This r e s u l t .... or

=

61

amino 155

-

847

=

58

-

710

=

97

inciates

the

Gly-Pro-Pro-Arg.

(653 - 1_5) tell us that

the

former

f r a g m e n t s are those of type A C and t h e r e f o r e

the

above

sequence

expected

(710 - 15) and 638

....

=

this

905

(result n e g . ) , 905

905 - 807 = 98

sequence Arg-Pro-Pro-Gly Ions 695

but

in the p e p t i d e ) , 1060 -

(Arg), 905 - 858 = 47 (result neg.)

1060 - 999

of the

is

indeed

Arg-Pro-Pro-Gly

there are no f r a g m e n t s of type AC and A l k C

As for

356 the A r g - P r o - and the P r o - P r o - b o n d .

Proceeding

further

in the same w a y w i t h the a m i n o - c a r b o x y l - i o n m/e 653 the a l k y l - c a r b o x y - i o n

638

(

15)

reveals

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe, the

carboxyl

possible

end

of

fragments

the

the

Arg

for

Calculating

all

ions for the p r o p o s e d

s o m e more p r o o f s for

the

sequence

leaving only

peptide. postulated

and

sequence

and

shows

well

known

sequence. As

Barber

and

biological

coworkers

a c t i v i t y of

have

demonstrated

bradykinin

is

not

s i g n i f i c a n t l y d u r i n g F A B - M S , those a n a l y s e s considered

to be

conditions with

carried

out

under

"could

non

r e s p e c t to b i o l o g i c a l

the

diminished be

destructive

activity"

^^.

Conclusion

FAB-MS represents of p e p t i d e s without

an i m p o r t a n t a d v a n c e

for several

derivatization

reasons: peptides by

chemical

n e c e s s a r y to have the p e p t i d e Nevertheless derivatization g r e a t h e l p for

Molecular high

by

volatile

be

enough

for

are EI-MS.

is s o m e t i m e s n e c e s s a r y and/or

integral

compounds. mellitin

observed

The

ions. Lys and

increment

and

molecular

Molecular

(128) are

amino

reactions

ions

for

insulin / ^ - c h a i n ion

b e t w e e n G l n - A s n and G l u / A s p . F A B / M S d e t e c t novel

measured

which

ions are of r e l a t i v e l y h i g h i n t e n s i t y ,

molecular

analysis

of Gin

easily

acetylation.

somastotatin, 211

chemical

can

methods,

i d e n t i f i c a t i o n of s e q u e n c e

w h i c h both have the same distinguished

in s e q u e n c e

allows has

a c i d s w h i c h are d i f f i c u l t

with

glucagon, have

the the

even

been

distinction potential

to d e t e c t

that m i g h t c h a n g e the u n k n o w n

to

after

structure.

357 A t the p r e s e n t

state

of

development

e a s i l y d e t e c t e d , also w i t h small fragment clear

molecular

a m o u n t s of

ions are not a l w a y s of s u f f i c i e n t i n f l u e n c e d by

the

peptide

are But

intensity

cut s e q u e n c e d e t e r m i n a t i o n . M o r e o v e r

greatly

ions

substance. fragmentation

structure

and

d e t a i l e d s t u d i e s are n e c e s s a r y to find out how has to be h a n d l e d conditions

before

have

fragmentation.

to

be

ms

analysis

tuned

to

and

get

the m e a s u r e m e n t

Further

is

from

peptide of

important

each

the r e m a i n i n g

e a s i l y be r e i s o l a t e d and p u r i f i e d

is

operating

optimum

The a m o u n t of p e p t i d e a v a i l a b l e

a

further

the how

an

in this r e s p e c t . As o n l y a small a m o u n t of destroyed during

for

sample peptide

is can

glycerol.

technical development, perhaps with

more

efficient

22)

ionization

techniques

', should e n h a n c e

the

sensitivity;

the m e t h o d s e e m s to be s u i t e d for L C - M S c o u p l i n g high

field

magnet

technology

opens

the

and the new

possibility

to

a n a l y z e m o l e c u l e s of MW > 2000. Literature 1) B i e m a n n , K. in B i o c h e m i c a l Spectrometry,

Ed.

A p p l i c a t i o n s of

Waller,

G.R.,

W i l e y - I n t e r - S c i e n c e , N e w York 1972 p

Mass Chapter

405

-

428

15, and

1982, p 469 - 525. 2) M o r r i s , H . R . , N a t u r e 286, 447 3) A p r i n o , P . J . and M c L a f f e r t y , of O r g a n i c S t r u c t u r e s

by

A c a d e m i c P r e s s , N e w York

(1980). in N a c h o d . Physical

Determination Methods,

Vol.

1976.

4) B e c k e y , H . D . , H o f f m a n n , G . , M a u r e r , K . H . and

Winkler,

6,

358 H . U . , A d v . M a s s S p e c t r o m . j>, 626

(1971).

5) W i n k l e r , H.U. and B e c k e y , H . D . , B i o c h e m . B i o p h y s . C o m m u n . £ 6 , 391

6) M a c f a r l a n e , R . D . and T o r g e r s o n , D . F . , Science 920

Res.

(1970). 191

(1976).

7) B u r l i n g a m e , A . L . , D e l l , A. and R u s s e l l , D . H . , C h e m . 54., 363R

Anal.

(1982) .

8) B e n n i n g h o v e n , A., S i c h t e r m a n n , W . , Org. M a s s S p e c t r o m . 12,

595

(1977).

9) B a r b e r , M., B o r d o l i , R . S . , S e d g w i c k , R.D. and A . N . , J. C h e m . Soc. C h e m . C o m m u n .

1981,

325

10) D e v i e n n e , F.M. and R o u s t a n , I., Org. M a s s 17 173

Tyler,

Spectrom.

(1982) .

11) F r a n k s , I. and G h a n d e r , A . M . , V a c u u m 2A, 489 (1979) . 12) M a r t i n , S.A., C o s t e l l o , C.E. and B i e m a n n , K., C h e m . 54^, 2362

Anal.

(1982) .

13) W i l l i a m s , D . H . , B r a d l e y , C . V . , S a n t i k a r n , S. and B o j e s e n , G., B i o c h e m . J. 201, 105

(1982).

14) B a r b e r , M., B o r d o l i , R . S . , S e d g w i c k , R.D., T y l e r , and

Whalley,

E.T.,

Biomed.

Mass.

Spectrom.

A.N. 8^

337

(1981) . 15) B a r b e r , M., B o r d o l i , R . S . , G a r n e r , G . V . , G o r d o n ,

D.B.,

359 S e d g w i c k , R . D . , T e t l e r , L.W. and T y l e r , A . N . , J. 197, 401

Biochem.

(1981).

16) K ö n i g , W . A . , A y d i n , M. S c h u l z e , V . , R a p p , U., H o h n , M. P e s c h , R. and K a i i k h e w i t c h , S p e c t r o m . 46, 403

V.N.,

Int.

J.

of

(1983).

17) R i n e h a r t J r . , K . L . , G a u d i s o , L.A., M o o r e , M . L . , R . C . and C a r t e r Cook J r . , I. J. A m e r . C h e m . 6517

Mass.

Pandy,

Soc.

103,

(1981).

18) M o r r i s , H . R . , P a n i c o , M. B a r b e r , M., B o r d o l i ,

R.S.,

S e d g w i c k , R.D. and T y l e r , A . N . , B i o c h e m . B i o p h y s . C o m m u n 101, 623

Res.

(1983).

19) W i l l i a m s , D . H . , B r a d l e y , C., B o j e s e n , G . , S a n t i k a r n , and T a y l o r , L . C . E . ,

J.

Amer.

Chem.

Soc.

103,

S. 5700

(1981). 20) K a m b a r a , H., Org. M a s s . S p e c t r o m . r7, 29

(1982).

21) B a r b e r , M., B o r d o l i , R . S . , S e d g w i c k , R . D . , T y l e r , Garner, G.V., Gordon, D.B.,

Tetler,

R . C . , B i o m e d . M a s s . S p e c t r o m . 9, 265

L.W.

and

Hider,

(1982).

22) A b e r t h , W . , S t r a u b , K.M. and B u r l i n g a m e , A . L . , C h e m . 54, 2029

A.N.,

Anal.

(1982).

23) W i e l a n d , F., H e i t z e r , R. and S c h ä f e r , W. , s u b m i t t e d P r o c . N a t . A c a d . Sei.

1983.

to

PERSPECTIVES MAIN-CHAIN

Axel

IN T H E

CIRCULAR

DICHROIC

ANALYSIS

OF

PROTEIN

CONFORMATION

Wollmer,

Wolfgang

Straßburger,

and

Uwe

Glatter

L e h r - und F o r s c h u n g s g e b i e t S t r u k t u r und F u n k t i o n der Abteilung Physiologische Chemie Rheinisch-Westfälische Technische Hochschule Aachen Schneebergweg 211, D-5100 Aachen

Proteine

I n t r o d u c t i on

In a v o l u m e chapter

on

on m o d e r n circular

As

a method,

CD

spectroscopy

well

CD

protein

tion

to

most

routinely

which,

however,

troscopy protein tions,

is

an

and

Where

in

7)

this

would

also

by

the

It

may

a more The

the

widely

find

the

frame

of

instrumentation, The

rather

critical

to

chapter for

is

workers

applying

CD

to

be

there

article

even

results

Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York

the

contem-

much

atten-

is

now

al-

structure As

CD

for

is

circular

the

however,

familiar

of

new

spec-

studying of

a

condi-

certain

context.

specializing

of

a

place

nor

in

range

ecological

this or

gives

a wide

of

its

of

is o n e used

technique

spectroscopy

interpretation

assumed

this

not,

it

secondary

used,

landscape

going

in

out

it r e c e n t ,

experiment.

under

reviewed

the

of

by

well

exceed

is

most

analysis

determination

convenient

solution

one

that

volume

Sequence

it

envisage

reader

present

verified

is

currently

prediction

and

protein slightly

nor

sense

treating

dichroism. is m e a n t

be

in

appear

for

chapter

far

phenomenon,

who

to

conformation

justification

is

has

the

The

by

adequate

may

techniques

sequencing. followed

chemical

chemical, in

physical

research.

protein

of

dichroism neither

is m o d e r n

established

porary

lar

is

methods

with

dichroism niche?

to

outline

theory meant in

of

for

other

themselves reported

by

absorption

(1-

It the

circuexperts.

fields, or

want

others. spectro-

362 ai .e +J

!

ai t/1 o 1/1 T - E t E O Sd

iaj "O

LO CM • O r-H

co

1 CL so c l/l o J D •.IO - P

aj sz +-> c

10 S-P 1 -C (_> " O - P O) c e Q . IO •!«1X1 ï

u a)

O) û£

CO CO CO PO

o

o

o

o

—t E c —i

o

o

o

o

LO 1—1

LO LO LO LO LO

"3-

0) .¡r 4->

CTÌ CO LO LO LO O

r-H r - t H

.-H

SO +-> IO i— "O o E

Í. io ai >> x> CD +J ra o -a c i—i co ci o -1— +-> •1— "O c o o

-p c aj E rj s-p 00 c

CM CM CM CM

rtHH

t—1 t—t r—( «-H

C M H H H r H

so 1

c — i

o o

-Q IO

o

sai s-

+-> 10 o

ü

t/1 Saj +J aj E O s+J o ai O00

aj o c IO E So M— s_ ai a.

1— 1 — C O C O O O H

o

co IO

4o

LO CO LO • CO i—1 o o

— ,

-o

t/l

o. o

CM oo CTI I—I

o o LO 1 •n

S0) •P ai E -rs10 1— o o. o í. +-> o cu O. 00

o O t/i (0 o •"3 C_)

N re

o lo

lo o co o rH N

o o o o PO < í

1 O -P L0

>

1 c •r-O O

ai a)

o IO

>

» >

10 u

1

¿í iIO s

u cu o. oo

363 scopy ism.

in g e n e r a l

given

enough

treatment

the

far

chain

by

the only

l i s t of

lection cited

i s , of

important

CD

and

tion

Lakewood,

of

puter

assistance)

identical

the

re-

with

proteins

secondary cover

and

the

se-

literature

the m a j o r i t y

Jersey,

ago.

under

of

same

modern cell.

CD

on and

the

(in

conditions basis

of

in

of

idea

manufac-

of

field,

the

per-

Table I

the a b s e n c e

comparison

the Asso-

moderniza-

to be

indicated.

cannot

Spec-

the A v i v

i ns t r u m e n t s

for

the m a n u f a c t u r e r s

an

in the

noise

the d i f f e r e n t fair

ceased

to g i v e

Japan

Devision

specialized

spectrometers (rms)

the

Recently

which

In o r d e r

square

from

Jobin-Yvon

USA,

samples

specifications

Pockels

and

New

as a p r a g m a t i c

In all

be d e a l t

complete

available France.

root mean

the

presently

Tokyo,

standard

the

exactly

can

at-

latter

Aspects

Longjumeau,

years

lists

used

The

been

CD. The

to

main-

indirectly.

spectropolarimeters

several

formance

of

are

S.A.,

of C a r y

tured

being

hopefully

Experimental

Company,

Instruments ciates,

least

or

spectrometers

spectrometers

troscopic

will

is r e s t r i c t e d

and

individual

from

not

performance.

structure

side-chain

subjective.

however, at

of

dichro-

sometimes

a u t h o r s .have, a l w a y s

of

is far

course,

dichroism

structures

level

are

better

secondary

the

specific

a t the

papers

Instrumental Modern

of

of c i r c u l a r

that

procure

hence,

although

references

therein,

that

circular

and,

idea

touched

potentialities

to d e t a i l s

adequately

a rough

are

and

protein

conformation

lates

CD

of

ultraviolet

tracted

to h a v e

aspects

attention

The

The

and

Experimental

of

com-

Measurements should since

be the

be n o r m a l i z e d

to

conditions.

instruments The main

hygroscopic

nature

and

or a m m o n i u m

dideuterium

photoelastic

disadvantages optical

of

modulators the

imperfection

phosphate

crystal,

replace

latter

lie

of

potassium

its

the

coating

in

the the

with

364 transparent voltages the

foil

and

electrodes,

low m o d u l a t i o n

low f r e q u e n c y

ty of flat

circular

level. time

Operation

to

Higher

square-wave

polarization.

baseline.

ceptability

lamp

IV,V

in

turn

methods

The a n a l y s i s

of

are

protein

wavelength

limit

of

Vacuum u l t r a v i o l e t research yet.

a superior

modulators in

speed

have a

lower

shorter

noise

response

IV,V

0.5

Stopped

in detail was

flow

by B a y l e y

shown

to b e n e f i t

CD s p e c t r o m e t e r s

( A > 185

were d e v e l o p e d

not commercially

involving

the

in

outlined

in

subshort-

nm). several

available

m a g n e s i u m and c a l c i u m

is

Mark

(8).

standard

and m o d u l a t o r s ,

ms)

CD

beyond

but are

sus-

( J - 5 0 0 < 8 nm/s,

studies.

CD s p e c t r o m e t e r s

quali-

reduced

the m e a s u r e m e n t s

construction,

polarizers

scanning

hand,

of

laboratories,

Their

oride

means

conformation

from e x t e n s i o n

yields

0 . 2 5 ms, Mark

discussed

stantially

operating

and a somewhat

J-500

kinetic

high

On the o t h e r

kHz r e s u l t s

course,

constant high

the

Photoelastic 20-50

of

and f a s t

as

voltage

instabilities

allows

< 40 nm/s)

and r e l a t e d

at

frequency,

(minimum time

which

as well

frequencies.

as flu-

references

9-12. Circular

dichroic

Depending

on t h e

difference AA

(i.e.

should the

in

due to =

M

signal or

preference

underlying

unfortunate

a;

displayed

polarized

the

since choice exists

however,

definition

in

it

for

a lot

M

molecular

weight

1

optical

c

concentration

pathlength

[g-mol_1J [dm] [g-cm~3]

the

The It

9.

AA

reflects literamay

appear

ellipticity

of c o n f u s i o n

traditional -1

the

absorption,

directly

though.

[deg • cm 2 • d e c i mol

R

as

ellipticity,

the m e a s u r e m e n t .

by the o p p o s i t e rotation

is

circularly

a common f o r m a l i s m

the o p t i c a l its

the

ellipticity.

dichroism),

be g i v e n

that

and

and r i g h t

process

dominated

attractive 9 and

left

clearly

is

instrument

the c i r c u l a r

physical

ture

absorption

]

units.

is

365

Ae

[103-cm2-mol -1]

=

1

optical

pathlength

c

concentration

[cm] [mol-l"1]

[0] = 3300•Ae Main-chain

conformations

of p o l y p e p t i d e s

and proteins are

pared on the basis of the m o l a r c o n c e n t r a t i o n mophores which

is c a l c u l a t e d

of p e p t i d e

from the mean m o l e c u l a r w e i g h t of

c o n s t i t u e n t r e s i d u e s . For c o m p a r i s o n s with r e s p e c t to c h a i n or p r o s t h e t i c c h r o m o p h o r e s the a n a l y t i c a l empirical

data are lacking

value

hardly e x c e e d

Factors affecting Calibration.

accuracy

of

reported

from d i f f e r e n t

calibration.

the CD

test s o l u t i o n s

involving

(III)

(CSA) as a s t a n d a r d . A of (+)-tris

17 i n s t r u m e n t s

(13 J a s c o , all

2 Jouan

up to 30% at 220 and 490 nm d e p e n d i n g calibrating

(13). This o b s e r v a t i o n

(or third) w a v e l e n g t h

r e q u i r e m e n t of a c o n t i n u o u s additional

calibration

longer w a v e l e n g t h s

(later

Jobinwhich

deviations in

calibration

at

investiga-

it c a n n o t m e e t the

calibration

f u n c t i o n . For

in the far u.v. is a d v i s a b l e

for p r o t e i n s with c e r t a i n

of

the risk

in the range under

that r i s k ; h o w e v e r ,

D-pantolac-

on the type and age demonstrates

at a single w a v e l e n g t h . A d d i t i o n a l

tion may reduce

scale

compara-

over the w o r l d

c a l i b r a t e d with CSA at 290 nm, r e v e a l e d

the m o d u l a t o r

rathattri-

(ethylene-

iodide m o n o h y d r a t e , CSA, and

Y v o n ) , 1 C a r y , and 1 n o n - c o m m e r c i a l )

a second

laboratories

less

This is c o m m o n l y a c h i e v e d at 290 nm with

tive study on identical

were all

will

( A A ) or e l l i p t i c i t y

acid

tone

by an

d i f f e r e n c e s , w h i c h are m o s t l y

(+)-camphor-10-sulfonic cobalt

If

measurement.

to the sample. H o w e v e r ,

diamine)

introduced

CD spectra of the same p r o t e i n under more or

the same c o n d i t i o n s

requires

the error

side-

is i n a d e q u a t e .

cent.

er o f t e n show d i s q u i e t i n g buted

this basis

(mean residue w e i g h t , MRW s 115 g/mol)

10 per

comchro-

basic proteins

(and

at

prosthetic

g r o u p s ) . This could be a c h i e v e d w i t h D - p a n t o l a c t o n e at 220 a wavelength

nearly

coincident with

the n-ir* band of the a -

nm,

366 helix; nm

or a d d i t i o n a l l y

(14),

a wavelength

larized |0

9 2 9 0. 5

/

| =

carefully

purified

its

hygroscopic

of

the

concentration

reliable

puted with ORD

[ e ] 2 9 0 .5

However,

since

terest

that

lution

can

the

normal

used

226.

rule out

ranges

calibrated Protein errors

at

can

itself

mounts

of

of CD

and from

analysis

and

1.1.

pendent the

The on

protein

sated

in

the

field

it is of

in-

inconsistencies

of

the

sensi -

the

spectrometer

used

for

differences

is

increases be are

cent

so-

in the

Increases

by a d e c r e a s e

Very

known with

of

sample.

substantial

to be a

optically

three

a-

active

proteins

seem

are

of

the

prove

composition to and

for

of 0 . 9 ,

fractions

in g - s t r u c t u r e

or

difficulty.

a factor

structural

De-

problem

decreasing

by a n a l y s i s

resulting in h e l i x

be

in c o n c e n t r a t i o n

derived

secondary

actual

a related

with

should

in c o n c e n t r a t i o n .

they

error

below)

were

the

factors.

multiplication

analyzed.

(15).

CSA

(see

II. T h e y

the a b s o l u t e

for m a i n l y

standardized

the

water,

conformation

after

its

The com-

of

Contaminants,

adherent

the

spectra

from

as

absorbance

concentration

of a 10 p e r

three

desirable.

CD of CSA

spectrometers

uncertainties

exponentially

Table

the

spectrum

related

consequences from

highly

because

determination

(14);

The

visible

problematic

spectropolarimeters,

setting

protein

or e v e n

on

(isotropic)

same

of m a t e r i a l .

inactive,

po-

0.77.

instrument

originate which

192.5

perpendicularly

an a c c u r a t e

transform

eventual

the

the

at

or

concentration

termination in

of

-

instead

9

290.5 / E285 =

CSA

2.33-10-\

of O R D

0.068

to

solutions

is b a s e d

number

A E 2 9 o . 5 / E 2 85 = In o r d e r

the

remains makes

dispersion)

2 70

that

the be

(14)

Kronig-Kramers

[M] 3 0 6 "

exceeds

with

with

a-helix:

This

of CSA

rotatory

vastly

tivity

CSA

[M] 3 0 6 - [M]270 = /

the

nature.

calibration

the

(optical

A e 2 9 0. 5 /

of

also

2.00.

of

most

preferably,

coinciding

77-7t* t r a n s i t i o n

1 9 2.5

Even

and

be

1.0, deof

compen-

vice

versa.

367

Table

II:

Dependence of C i r c u l a r D i c h r o i c S e c o n d a r y D e t e r m i n a t i o n * on E r r o r i n C o n c e n t r a t i o n error i n concentration

protein

helix [«1 84

-10% myoglobin

pancreatic trypsin inhibitor

ribonuclease A

Structure

g-structure m

B-turn [%1

unordered m

0

3

13

± 0

79

0

5

16

+10%

70

10

4

16

-10%

31

27

4

38

± 0

28

33

3

36

+10%

24

40

2

34

-10%

26

34

15

25

± 0

23

40

13

24

+10%

20

46

11

23

*)Least-squares a n a l y s i s with the reference data of Chang et a l . under the c o n s t r a i n t s Ef. = 1 and f . > 0. i If

the f r a c t i o n s

low)

their

are

error

is

not c o n s t r a i n e d directly

to

sum to u n i t y

proportional

to

(38)

(see

the e r r o r

be-

in

con-

centration. There

still

is

another

important

experimental

parameter:

of a p r o t e i n

should

dence: wide

changes

range

libria. should teins

in

are

be n o t i c e d

of d i f f e r e n t in

a defined

state

lengths

are

solvent.

of

concentration

depen-

of

concentration

over a

of a s s o c i a t i o n / d i s s o c i a t i o n reflect

derivatives

an

investigation

a study

equi-

quaternary

structure.

It

studies

homologous

pro-

of

of

one

protein

structure,

are

reasonable

concentration

from lyg/ml

5 cm to is

as

which

are

only

for

association.

protein

extends

lationship the

of

CD s p e c t r o s c o p i c

comparative

a quaternary

The m e a s u r a b l e u.v.

that

to c o n c e n t r a t i o n

variation

CD c a n a l s o

organized

far

include

CD w i t h

indicative

Hence,

or

Every

aspect

to

range

10 mg/ml;

for

studies

corresponding

10 pm. The p a t h l e n g t h / c o n c e n t r a t i o n

substantially

determined

by t h e

in

the

pathre-

transparency

of

368 The

less

small

pathlengths

tions. can

transparent

Phosphate

be u s e d

down

to

small ror.

185

and,

the m o r e

consequently,

buffers

are

high

and

0.5 mm

nm.

are

suitable

Fluorides

cells,

Nominal

optical

pathlengths

absorption

one

should

be

to r e s o r t

as

at

neutral

may

with

rotation

Tris

wavelengths salts.

be a s o u r c e

checked

to

concentra-

transparency;

pathlength

pathlength

(16), or o p t i c a l

has

protein

of a c c e p t a b l e

at 0.02 mol/1

sample

infrared

a solvent,

For

of

er-

standards

by

(17), or

inter-

is p a i d

to

ferometri cally. Artefacts.

In g e n e r a l

possibility important corded

same

for

the

come

a cell

cell

that

tests

the

absorption.

optically

sample

are

such

absence

as

not mean

are

that

the most

baseline compound

reof

from

Artefacts

liposomes

in t h i s

of a r t e f a c t s

does

The

the

on o r i e n t e d ,

films,

treated

of

not deviate

solvent.

in m e a s u r e m e n t s not

One

inactive

should

a transparent

in p u b l i c a t i o n s

collection

In r e s e a r c h any

its

proved lows

by

the

that

may

be-

scattering or

membrane

article). normally

they were

The not

fact men-

a matter

solvent

processing

the

always

than

of

improvements

be

operated

under

optimum

detectabi1ity

at

of a s i g n a l

at fixed w a v e l e n g t h

the

technique"

cell are

"step

to be w i t h d r a w n position.

referred

graphically

peak-to-peak

(18): from

noise

the and

instrumentationlimits

The

and can

of

Without

the

its

per-

computer

reproducibili-

be c o n s i d e r a b l y

A special

the

Measurements

to a i r p a t h . as

in the

conditions.

recording

identical

obtained

will

rather

assistance ty of

and

-irrespective

technique

formance

the

the

samples

(which

for

isotropic an

attention

of a r t e f a c t s .

course.

Data

an

as

concern

reflecting

tioned

is

holding

holding

preparations

insufficient

occurrence

absorbance

a primary

and/or

of

the

artefacts

for

the

of

cell

holder*

beam

and

re-inserted

of b o t h

the

sample

height

difference is r e f e r r e d

im-

and

of

the

signal

between

the

centres

to as a s t e p .

At

alin the

is of

signal/

369 noise

(s/n)

ducible

ratios

with

Modern

commercial

digital

computer

provement,

as

data

CD

facilities.

takes

into

play.

of

sample

the

kind

same the time

These

time

can

described has

separation

proportional baseline

of

to

$ 1 m°/day;

data.

The m a i n

Mark

in the

lies

of m a g n i t u d e .

tween

the

operator for

of

IV,V

of c o l l e c t i n g

order

the

cell

and

with

and

small

and

(Cary

order

increased

experiment

which

the

is

the

manual

within

is

saves

m°/day;

rather

remains

the

like

affect over

of p r o c e s s i n g

of

however,

61 ^ 2

will

come

in

baseline

assistance

measurements,

Solvent scanning

the d a t a w h i c h the

may

instabilities

+ +

IO O ^ CCL CCL + + +

+ + + + +

+ + + + + C O

cytochrome c

+

+ +

+ + +

+ +

+

+

+ +

+

+

+

triosephosphate i somerase

subtil i si n Novo

+

hemog1 obi n

+

prea1bumi n

glyceraldehyde-3-P-DH

+

dehydrogenase

+

f1 avodoxi n

+

alcohol

¡3

carbonic anhydrase B

CO

thermolysin

+

tryps in i nhi b i tor

+ +

ribonuclease S

+

parva 1bumi n

+

insulin

+

nuclease

1981

-H h»

el as tase

1980

Hennessey S Siegel et al.(40) !johnson*(41)

+ •*•

concanava1 i n A

ko m N N Ö «icCL

ca rboxypepti dase A

r\

+

a-chymotrypsi n

+

-*- +

adenylate kinase

1979

Bo loti na et al.(39)

+

subtil i si n BPN'

+

lactate dehydrogenase,

1978

1974

+• +

papa i n

Chang et al.(38)

Chen et al.(37)

+

lysozyme

— +

ribonuc1 ease A

to in

myoglobi n

Saxena S Mitläufer (35) 1971

Table

protei n

374

I V : P r o t e i n s w i t h Kn wn X - R a y S t r u c t u r e U s e d f o r E x t r a c t i o n o f CD R e f e r e n c e S p e c t r a f o r S e e o n dary Structures

+ +

375 real

from

the

tain margin extreme

in

the

by d i f f e r e n t

et al.(38)

the

(42) of

criteria

utilized

Bolotina

which

were

g-bends ments

were

are

Microfiche stereo

(AMSOM

pictures

necessarily use al.

of

for

leaving

a few

of

the

the

Hennessey's

a

cer-

examples same

for

protein

& Greer

of

analysis

crystal-

and,

for

g-turns,

and

Glockner

bonds

the

criteria

were

helices two

and

central

criteria

(44)

reference such

which

as

(41)

of

assign-

Structure

for

helices

on and

discrepancies

data

the

were

g-structure, residues

Johnson's

Classification the

procedure

Levitt

the

Provencher

and

H-bond

in

by

o f M a c r o m o 1 e c u 1ar

g-bends.

reflected

given

structures

defining Only

(43)) p l u s

for

the

Hydrogen

the A t l a s

of a s t a n d a r d i z e d

rithms

and

call

automatic

applied

will

for

algo-

by S i e g e l

et

(40).

Execution The

actual

search* by a the

for

linear

the

analysis the

usually f,>0 1

to

of

number

of

their

and/or

*)Computer

consists

in a

fit of

the

of

reference

the

spectrum

least-mean-squares

uninterpretable

out with

the

computer-mediated under

spectra

investigation according

to

deviation:

solutions

following

calculations

obvious

are

constraints:

n i f. = 1 i=l 1

unconstrained

viation

optimum

exclude

carried

and

normally

combination

criterion

In o r d e r

In

be

set.

(39)

on

thus

gives

fractions

e t al „ ( 3 4 )

priority.

counted.

based

the

solved

same

et al. given

types, IV

interpretations

on

of Lewis

the

Table

authors.

relied

1 o g r a p h e r s who a c t u a l l y on

structural

subjectivity.

differences

structure Chang

theoretical

for

analyses, sum

nature

programs

from

negative unity

are

signs

of

fractions

indicative

of

reference

spectra.

are

available

upon

request.

of

and

de-

inadequate

376 Least-mean-squares curacy

fitting

is based on the a s s u m p t i o n

is limited by random e r r o r s .

uncertainties

and s h o r t c o m i n g s

h o w e v e r , c o n s i s t e n t rather portant.

Furthermore,

that ac-

remaining

parameters

than random errors are more can still

for curve fitting

(45) p r e s e n t e d a m e t h o d e m p l o y i n g

spectral was

of the s t a n d a r d

very poor p a r a m e t e r s

good fits. As an a l t e r n a t i v e berg

In view of the

Baker and

integrals

over

data instead of the data t h e m s e l v e s w h i c h ,

later shown to be e q u i v a l e n t

im-

yield Isen-

the

however,

to a l e a s t - s q u a r e s

fit

(38,

46). Alternative Extraction tures

approaches of s t a n d a r d

is not the only

tion e m b o d i e d reference

spectra for the single s e c o n d a r y possibility

for u t i l i z i n g

informa-

in the c i r c u l a r d i c h r o i s m and X-ray s t r u c t u r e

proteins.

Provencher

and G l o c k n e r

s p e c t r u m d i r e c t l y as a linear c o m b i n a t i o n spectra

the

struc-

|X(A)| of m r e f e r e n c e

of

(42) a n a l y z e a CD

of the

original

proteins,

m [9(A)]= I Y , [ X ( A ) ] J j=l J The f r a c t i o n s

f^ of s e c o n d a r y

the known c o m p o s i t i o n s

structure

of the r e f e r e n c e

can be d e t e r m i n e d proteins F^^

from

as

m f,= I Yy .F. . i j=1 J U Because of the high number m of c o e f f i c i e n t s y the squares a n a l y s i s propriate

needs s t a b i l i z a t i o n .

11 m m i n j ^ O G U , ) ] - ^ Hennessey "basis

(41)

calculated

Y

= 1

is a c h i e v e d by ap-

term: , 21 ( V i ) |

the use of

orthogonal

from the CD spectra of suffice

inal CD s p e c t r a of the r e f e r e n c e independent

+

suggested

Five basis spectra

there are five

m

2

Yj[X(Xk)].)

and J o h n s o n

spectra"

proteins.

This

choice of a in a r e g u l a r i z i n g

least-

reference

to re c o n s t r u c t

proteins.

the

orig-

Correspondingly,

"superstructures",

each

consisting

377 of a s p e c i f i c are

capable

reference

under

How many

of

ambiguity curve

on

how

On

are.

co, Woody,

and

supercoiling t w i s t of

of

calculations.

stance

a and

whereas The

in b r o a d e r 310

helix

diversity

of

types. Attempts have

been made

spectra

of

type

conformational each of

type

the

occur of

as m i r r o r

their

CD

I, II a n d

which ones

the

into

these III

hand

for

should

the

in the

it will of

the

case depend

single

work

as

be

of

Tino-

(49-56). related

length

of s t r a n d s

or

and

the

by q u a n t u m

mechanical

categories

have

not

account

(56)

on

closely

such

should

types

Woody

finally

depend

helical

be r e d u c e d

in

reasonably class,

for

a be in-

distinguished, by C h a n g

et al.

terms

spectral

using

of

synthetic

predicted

be r a t h e r

that

(38).

models the

similar.

specific

the o t h e r

using

an overall

representation

similar

Brahms

found

very

complication

images

with

in a s t r u c t u r e

contributions.

Thus

Chang

and

preferred

to c o n s i d e r

part

the

unordered

form.

the

by

g-turns leading et al.

data

for

using

were

reference

Two

one

g-turns of

uncon-

importance

number

generally

can

above).

analyses,

types

In

are

to c o n f i r m

(11). An additional

the

predictable

is t a k e n

will

theoretical

greatest

and which

g-turns

(see

the

indicate

helices

of b e n d ,

three

of

classes.

length

the

for

spectra.

particularly other

of f e a t u r e s

are

will

to be d i s c e r n e d

combined

of

structures

by an

characteristics

is of

or

sheets

which

structures

basis

number will

or d i s s i m i l a r i t i e s

helices

These

the

elements

the

aspect

the e f f e c t s

pleated

On

spectral

Schellman

and

determined

techniques,

this

similarities

geometries,

chance

the

X-ray

secondary

with

the

procedures. For

are

structures,

categories

hand

of e v a l u a t i o n

different

of

structural

the o n e

fitting

Spectral

of

secondary

original

analysis

structural

categories

the

fractions

investigation

categories

discernible? of

The

least-squares

Resolution

of e i g h t

to r e c o n s t r u c t

proteins.

a protein strained

combination

&

Brahms

is

that

to

cancellation

(38)

cancelling

they

counted residues

may "net" as

378

special features

Q--U CI o n o "5 C i -h •-h -t> O-h o 0) in a o OO -s fD < -S-S T c 01 c+ Q. 3 ui n oi 3 3 o o fD' -s -s c -s fD — o w a c o CL CL » -s on 3 3 fD fD -s -! Ol Ol C c+ C fD fD -S —Q. CL 0) • 3 fD 3• 01 3 Qio 01 -J Q-•• s

Ol 1—-a (0 o i/i •— I -C •— i o +-> (/i O) CL) >i cle: i— cn rO -a C "O QJ C c: +-> to o i— cu CL>o t 3 i/i •— o 4-> a. 4- (O OE CTI Sc (/I o •1 — a) 4— > S- c 1— 3 o o 00 CD fD fD fD >TOfD ' I r+ I- XI t —' L &f> D I c+ r fD I a. 3" fD fD

authors 1969 Greenfield & Fasman (23) 1971 Rosenkranz & Scholtan (24) 1971 Saxena & Wetlaufer (35) 1971 Chen & Yang (36) 1972 Chen et a l . (47) 1974 Chen et a l .

(37)

1978 Chang et a l .

(38)

1979 Brahms & Brahms (48) 1979 Bolotina et a l . (39) 1980 Siegel et a l .

(40)

1980 Brahms & Brahms (11) 1981 Provencher & Glöckner (42) 1981 Hennessey & Johnson (41)

379 Inclusion

of

the

178-195

portance

for

cernible

structural

analytical

the m e a s u r e m e n t s sized

by B r a h m s

show minima

nm.

Surprisingly of

this

tiation

baseline

the

(41).

is

Brahms

to

the

Their

do

of

is a l s o

of

emphaspec-

168 a n d

not affected

in the

im-

dis-

reference

between

were

data

great

number

0-turn

intersections

& Brahms

be of

of an e x t e n s i o n

ultraviolet

48).

g-turn categories

190

by

Hennessey/John-

not describe

employed

of

Tests

the

on

of

a study

of

vacuum

analytical

formation the

procedure analysis the

the

of one

permits

from

the m e t h o d

and

removed

was

15 w a s

actually

of

various

differen&

Johnson

a special

used

eigen-

of

noticed

that

carried

X-ray

the

The

the

Permutation of

out:

structure

at a time.

to a n a l y z e

separated.

deviation

been

known

characterization

X-ray

the

have

protein

methods

in-

spectrum

of

this

quality

of

the

and

correlation

coefficient

structural

composition

and

structure.

numerical

The

performances

analysis

are

that

of

compared

three

in

VI. be

coefficient category

to

frequency

the

approaches

sey

& Johnson

et al.

of

secondary

lation in the

data

with

the

It m a y tural

degree

by H e n n e s s e y

16 p r o t e i n s

of r m s

resulting

different

ultraviolet

remaining

protein

in t e r m s

manifest Table

on

one

highest

in

approaches

reliability

spectrum,

V. T h e

is a t t e m p t e d

achieved

evaluation.

a collective CD

differentiation

in T a b l e

8 categories

method

Accuracy

From

structural

given

with

(41) w h o vector

for

vacuum (11,

i.e. value

shortest wavelength

of t h e

analyses

of

the

The

found

kind.

A survey

and

into and

the

son a n a l y s i s

specificity,

categories.

& Brahms

tra

omission

nm r a n g e w a s

ratio

the

other.

(41)

Provencher have seems

of r m s

varies This

of o c c u r r e n c e

of

(38) w h i c h

the

strongly

of

and

certain

from

deviation one

is d u e the

to

advantages

comparatively

weak

correstruc-

large

single

Glockner

and

secondary

differences

categories.

(42) over

and

of

Both

Hennes-

t h a t of

Chang

in q u a n t i f y i n g

g-

380 Table

VI:

Root-Mean-Square Deviations and Correlation Coeffic i e n t s b e t w e e n CD E s t i m a t e s a n d X - R a y V a l u e s f o r the F r a c t i o n s of S e c o n d a r y S t r u c t u r e s in 16 P r o t e i n s rms

secondary structure

Chang et al.(38)

d e v i a t i o n [Xj

Provencher & Glbckner(42)

11

hel i x

5

anti para 11 el B-structure p a r a 11 el 8-s tructure

21

6

8-turn

15

10

r e m a i nder

15

11

The

the

how

spectral

structure

(see T a b l e

range and

can

nm, whereas the

facile

to

bounds

they

produced

studied

been

200

nm.

nm - w e r e

Notably,

categories,

0.25

10

0 46

0.49

0.72

while the

for

none

helix

of

and

non-ideality limitations

for

of

the

on

the

of

It b e c a m e

above

the

16

of

apparent

proteins

fraction

between

inves-

restrictions

of

only

that

helical

210

and

240

deny

the

significance

of

g-structure

and

random

This

fact

that

because

the

of

affected

large

waveerror

Hennessey

at

178,

first

&

184,

- by

"other"

truncations

coil.

shorter

the

fit.

cut-offs

$-sheet and

Johnson 190,

cut-off

and at

(irregular)

caused

substantial

the

neglected

chromophores

of m a i n - c h a i n for

the

side

create

dichroic

complications

transitions

and

In n e a r l y

they

(1). due

the

dif-

analysis

originate

chains

contributions because

geometry

circular

Additional

peptide

cystinyl

of CD a n a l y s i s

to

analysis.

(40)

side-chain

conformation.

are

should

respond

the

fractions.

and

and

that one

the

ficulties

overlap

I) s u g g e s t s

the

the

Diversity

of a r o m a t i c

towards

of d a t a

from

protein

problems

the e s t i m a t e s

those

0.51

in a p r e l i m i n a r y

Interferences

ods

0.31

of n o i s e

discarded

the e f f e c t

190

mophores

- 0 31

from data

for

had

in the

8

0.95 0.66

statisties

to be d u e

data

of

0. 94

estimation

resulting

length

in

e t al.

be o b t a i n e d

seems

changes

0 25

results

utilized

unfavorable

fractions

(41)

Hennessey & Johnson(41)

0.96

7

increase

by S i e g e l

accurate

finding

(38)

0 85

8

the a n a l y t i c a l

in a s t u d y an

steep

ultraviolet

tigate

coefficient

Provencher & G18ckner(42)

10

structure. far

correlation Chang e t al

Hennessey & Johnson(41)

absorption all

meth-

to n o n - p e p t i d e

chro-

are a s s u m e d

to be

small.

381 While as

contributions

a "background"

in r e f e r e n c e known

X-ray

spectra ed

will,

data

structure,

from model

to c o r r e c t

sentatives.

for

between positive density

of

lation has

of

proteins tional out

for

they

the

(61).

than

handle

and

being

Spectra

from

the of

of

unexpectedly

detailed

a

also

u.v.

near

increase

two excep(58,

tyrosyl

the of

con-

destructive

is e x c e p t i o n a l deserve

sometimes

insights

two

side

dichroic

a truly

59)

with-

case

interaction two

iso-

spectrum of

u.v.

In o n e

in-

high

Clearcut

smoothly

features

protein

generate

there were

that

chromophores

unusual

least

in a

to

spectra

circular

not analyzing

Their

repre-

simultaneously

case

indication

side-chain

far

affected.

success

is an

at

(57),

(6).

in the

abolished

attempt-

chromophores, nm

the

because

in the o t h e r

relative

attention. to a t t a i n

far

and

structure

from

normal.

ticular

in the

reference

interactions

should

in the

o u t to o r i g i n a t e

The

222

For

with

spectral

expected

contributions

included

(11)

occurs

tyrosyl

hand,

present

lysyl-phenylalanine, as

Though

cancellation (1).

with

acids

possible

both

analysis

interference

other

be

corrections

around

be

proteins

& Brahms

always

be q u a n t i f i e d

(58, 60),

formational

the

influenced

turned

tryptophyl chains

be

secondary

phenomena

er

could

phenomena could

on

or

are

at wavelengths

been

of

case

bands.

phenylalanyl

always

other,

using

amino

peptide

compensatory

ever

the

Brahms

applying

geometry,

side-chain

hardly

which

the

of a r o m a t i c s ,

the

glutamyl-tryptophan

of a r o m a t i c

and

less,

spectra

contributions

and

their

or or

the

is n o t

recommended

effects

chances

from

this

such

low CD of

peptide

dependent

in o n e w a y

polypeptides.

a high content

displaying

the

They

may, more

derived

glutamyl-tyrosine, when

which

rathpar-

offer

into

a

a

struc-

ture . This tural

article

restricted

composition.

function used.

is

of CD

One

has

to d e t e r m i n a t i o n

Although

this

spectroscopy, to

realize

principle.

However,

it a p p e a r s

in o u t l i n e s

from

it is

that the

that

cannot the

be

of s e c o n d a r y considered

one m o s t

growing

l i s t of

solved

architecture

best

extensively

it is an a p p r o x i m a t e

protein

the

struc-

is

method

in

structures achieved

382 with

a limited

(62). make

(though

Utilization the

method

of

more

large)

these

set

of

basic

back-bone

for

CD a n a l y s i s

will

reading

text

structures

and more

patterns

reliable.

Acknowledgements We a r e to ing

Mrs.

indepted Renate

to

Dr.

Kehren

a manuscript

Derek

Saunders

who m u s t e r e d

ready

for

the

for

care

the

required

for

and typ-

reproduction.

References 1.

S e a r s , D. & Beychok, S . : i n P h y s i c a l P r i n c i p l e s and Techniques of P r o t e i n C h e m i s t r y , P a r t C, Leach, S . , E d . , Academic P r e s s , New Y o r k , p. 445 (1973).

2.

A d l e r , A . J . , G r e e n f i e l d , N . , Fasman, G.D.: i n Methods i n Enzymology, H i r s , C.H.W., E d . , V o l . 27, Academic P r e s s , New Y o r k , p. 675 (1973).

3.

Woody, R.W.: J . Polym. S e i . , Macromol. Rev. 12, 181 (1977).

4.

Bewley, T . A . & Yang, J . T . : i n Hormonal P r o t e i n s and P e p t i d e s , V o l . Academic P r e s s , New Y o r k , p. 175 (1980).

5.

Simons, E . R . :

6.

Strickland,

7.

Kahn, P . C . : i n Methods i n Enzymology, H i r s , C.H.W., E d . , V o l . 61, Academic P r e s s , New Y o r k , p. 339 (1979).

9,

i n CRC Handbook o f B i o c h e m i s t r y , p. 63 (1980).

E . H . : i n CRC C r i t i c a l

Reviews i n B i o c h e m i s t r y ,

8.

B a y l e y , P . M . : Prog. B i o p h y s . molec. B i o l .

9.

Johnson, W.C.: Rev. S e i .

p. 113 (1974).

37, 149 (1981).

Instrum. 42, 1283 (1971).

10. Brahms, S. & Brahms, J . : P r o c . Nat. Acad. S e i . USA 74, 3208 (1977). 11. Brahms, S. & Brahms, J . : J . Mol. B i o l .

138, 149 (1980).

12. Düben, J . A . & Bush, C . A . : A n a l . Chem. 52, 635 (1980). 13. Tuzimura, K . , Konno, T . , Meguro, H . , Hatano, M., Murakami, T . , wabara, K . , S a i t o , K . , Kondo, Y . , S u z u k i , T . M . : A n a l y t . Biochem. 81, 167 (1977). 14. Chen, G.C. & Yang, J . T . : A n a l y t . L e t t e r s 10, 1195 (1977). 15. Cassini, J . Y . & Yang, J . T . : B i o c h e m i s t r y 8, 1947 ( 1 9 6 9 ) . 16. Bree, A. & Lyons, L . E . : J . Chem. S o c . , 2658 (1956). 17. Samejima, T. & Yang, J . T . : B i o c h e m i s t r y

613 (1964).

Kashi-

383 18. Wood, S.P., Blundel1, T.L., Wollmer, A., Lazarus, N.R., Neville, R.W.J.: Eur. J. Biochem. 55, 531 (1975). 19. Tinoco, I. & Cantor, C.R.: Methods Biochem. Anal. 18, 81 (1970). 20. Savitsky, A. & Golau, M.J.E.: Anal. Chem. 36, 1627 (1964). 21. Bush, C.A.: Anal. Chem. 46, 890 (1974). 22. Glatter, U., Straßburger, W., Wollmer, A.: Biophys. Struct. Mech. 7, 258 (1981). 23. Greenfield, N. & Fasman, G.D.: Biochemistry 8, 4108 (1969). 24. Rosenkranz, H. & Scholtan, W.: Hoppe-Seyler's Z. Physiol. Chem. 352, 896 (1971). 25. Li, L.K. & Spector, A.: J. Amer. Chem. Soc. 91, 220 (1969). 26. Quadrifoglio, F. & Urry, D.W.: J. Amer. Chem. Soc. 90, 2760 (1968). 27. Urry, D.W., Long, M.M., Ohnishi, T., Jacobs, M.: Biochem. Biophys. Res. Comm. 61, 1427 (1974). 28. Brahms, S., Brahms, J., Spach, G., Brack, A.: Biochemistry 74, 3208 (1977). 29. Kawai, M. & Fasman, G.: J. Amer. Chem. Soc. 100, 3630 (1978). 30. Bush, C.A., Sarkar, S.K., Kopple, K.D.: Biochemistry 17, 4951 (1978). 31. Ananthanarayanan, V.S. & Shyamasundar, N.; Biochem. Biophys. Res. Comm.'102, 295 (1981). 32. Gierasch, L.M., Deber, C.M., Madison, V., Niu, C.-H., Blout, E.R.; Biochemistry 20, 4730 (1981). 33. Venkatachalam, C.M.: Biopolymers 6, 1425 (1968). 34. Lewis, P.N., Momany, F.A., Scheraga, H.A.: Biochim. Biophys. Acta 303, 221 (1973). 35. Saxena, V.P. & Wetlaufer, D.B.: Proc. Nat. Acad. Sei. USA 68, 969 (1971). 36. Chen, Y.-H. & Yang, J.T.: Biochem. Biophys. Res. Comm. 44, 1285 (1971). 37. Chen, Y.-H., Yang, J.T., Chau, K.H.: Biochemistry 13, 3350 (1974). 38. Chang, C.T., Wu, C.-S.C., Yang, J.T.: Analyt. Biochem. 91, 13 (1978). 39. Bolotina, J.A., Chekhow, V.O., Lugauskas, V.Yu.: Internat. J. Quant. Chem. 16, 819 (1979). 40. Siegel, J.B., Steinmetz, W.E., Long, G.L.: Analyt. Biochem. 104, 160 (1980). 41. Hennessey, J.P. & Johnson, W.C.Jr.: Biochemistry 20, 1085 (1981). 42. Provencher, S.W. & Glöckner, J.: Biochemistry 20, 33 (1981). 43. AMS0M, Feldman, R.J. Ed., Tracor Jitco Inc., 1776 East Jefferson Street, Rockville, Md. 20852, USA. 44. Levitt, M. & Greer, J.: J. Mol. Biol. 114, 181 (1977).

384 45. Baker, C.C. & Isenberg, J . : Biochemistry 15, 629 (1976). 46. W i l l i c k , G. & Zuker, M.: Biophys. Chem. 7, 223 (1977). 47. Chen, Y.-H., Yang, J . T . , Martinez, H.M.: Biochemistry

4120 (1972).

48. Brahms, S. & Brahms, J.G.: J. Chim. Phys. 76, 841 (1979). 49. Woody, R.W. & Tinoco, J . : J. Chem. Phys. 46, 4927 (1967). 50. Woody, R.W.: J. Chem. Phys. 49, 4797 (1968). 51. Woody, R.W.: Biopolymers 8, 669 (1969). 52. Johnson, W.C. & Tinoco, J . J r . : Biopolymers ]_, I I I

(1969).

53. Pysh, E . S . : J. Chem. Phys. 52, 4723 (1970). 54. Vournakis, J . N . , Yan, J . F . , Scheraga, H.A.: Biopolymers 6, 1531 (1968). 55. Madison, 0. & Schellman, J . : Biopolymers 11, 1041 (1972). 56. Woody, R.W.: in Peptides, Polypeptides and P r o t e i n s , Blout, E.R. et a l . , Eds., John Wiley & Sons I n c . , New York, p. 338 (1974). 57. Woody, R.W.: Biopolymers 17, 1451 (1978). 58. Wollmer, A. & Buse, G.: FEBS Letters 16, 307 (1971). 59. Straßburger, W., G l a t t e r , U., Wollmer, A . , Fleischhauer, J . , Mersola, D.A., Blundel1, T . L . , Glover, I . , P i t t s , J . E . , T i c k l e , I . J . , Wood. S . P . : FEBS Letters 139, 295 (1982). 60. Wollmer, A.: paper presented at the Advanced Study I n s t i t u t e on ORD and CD at T i r r e n i a , P i s a , I t a l y , Sept. 5-18 (1971). 61. Wollmer, A., Straßburger, W., P i t t s , J . E . : to be published. 62. Schulz, G.E.: Angew. Chem. 93, 143 (1981).

Note a d d e d

in

An a r t i c l e

on " e x p e r i m e n t a l

lyzing after It

has

proof:

CD s p e c t r a the d e a d l i n e still

of

errors

proteins"

for

this

been p o s s i b l e

and t h e i r

appeared

in

effect

the

on

ana-

literature

chapter. to a t

least

Hennessey, J . P . , J r . and Johnson, W.C., J r . Analyt. Biochem. 125, 177 (1982).

give

the

reference:

SPIN-LABELLED AMINO ACIDS, PEPTIDES AND PROTEINS - SYNTHESIS AND APPLICATION

Herbert R. Wenzel and Harald Tschesche U n i v e r s i t ä t B i e l e f e l d , Fakultät für Chemie, D-4800 B i e l e f e l d 1

Eberhard von Goldammer R u h r - U n i v e r s i t ä t , I n s t i t u t für B i o p h y s i k , D-4630 Bochum 1

Contents Introduction 1.

Spin-Label Modification Reagents Dinitrofluorobenzene d e r i v a t i v e s Acylating agents Imidoesters Alkyl a l k a n e t h i o l s u l f o n a t e s C r o s s - l i n k i n g reagents I s o t o p i c a l l y labelled reagents

2.

S p i n - L a b e l l i n g of Amino Acids and Peptides Amino group s p i n - l a b e l l e d amino acids Carboxyl group s p i n - l a b e l l e d amino acids Side chain s p i n - l a b e l l e d amino acids N-Terminal s p i n - l a b e l l e d peptides C-Terminal s p i n - l a b e l l e d peptides Side chain s p i n - l a b e l l e d peptides

3.

Minimum Perturbation Spin-Label Amino Acids Nitronyl n i t r o x i d e amino acids Pyrrolidine-oxyl derivatives Pyrroline-oxyl d e r i v a t i v e s Piperidine-oxyl d e r i v a t i v e s Peptides with integrated s p i n - l a b e l s References

Introduction Since i t s introduction as a reporter group technique by McConnell almost twenty years ago, s p i n - l a b e l l i n g has become a well established powerful tool to study structure and function of biological systems on the molecular

Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York

386 level. The method requires a stable paramagnetic nitroxide group to be placed in the system of interest either by covalent attachment label') or by introduction as a »suitably tailored radical

('spin-

('spin-probe')

which may interact with the system through hydrophobic, ionic, or hydrogen bonds. As the electron spin resonance of the nitroxide moiety is sensitive to orientation, molecular motion, and electric and magnetic environment, it potentially offers the ability to study these features in the biological Thus, many biochemical spin-label

and biophysical

system.

problems have been the subject of

experiments, e.g. molecular architecture of membranes, orienta-

tion or tumbling of macromolecules, structural

or conformational

proteins upon ligand binding, protein-protein interactions,

changes of

interconversion

among molecular species, distances between distinct sites in a macromolecule, rates and mechanisms of denaturation, active site geometry of enzymes, and rates of reactions catalyzed by them. The rapid development and the broad scope of spin-labelling are well

docu-

mented by three books entirely devoted to the various aspects of this method (1-3). Important advances have recently been achieved and can certainly be expected for the future in many respects: new instrumental ches such as the use of the saturation transfer ESR spectroscopic

approa-

technique

(4), improvement of computer programs for the simulation of ESR spectra, and the development of new nitroxide spin-labels, new methods o f attachment of these labels to molecules of interest, and new chemistry of the nitroxide grouping as it pertains to the spin-label method. Up to now a great variety of biologically important compounds have been spin-labelled including m e m brane components (5-8), nucleic acids (9-11), coenzymes (12-14), and carbohydrates (15,16). Since the very beginning of the technique, proteins and especially enzymes have ranked among the most prominent targets of spinlabelling. The vast literature which has accumulated up to about 1978 is thoroughly discussed in numerous reviews, wherein detailed synthetic

proce-

dures for spin-labels and representative examples for their covalent attachment to proteins can be found (1-3, 17-34). The focus of this present paper is threefold. The first chapter will some additions to the arsenal of spin-label

protein modification

present

reagents

made during the last four years. A review on the use of spin-labelled amino acid derivatives and peptides as spin-probes and methods for their

synthesis

387 will

follow in the second chapter. Finally, the third chapter will

discuss

the synthesis of amino acid analogues containing a nitroxide moiety as integral

part and their potential

applications.

1. Spin-Label Modification Reagents (Chart 1)

Dinitrofluorobenzene The spin-label

derivatives

reagents I, II and III combine in a single rather hydro-

phobic molecule the paramagnetic nitroxide function, a means to attach the radical

to proteins by an aromatic nucleophilic substitution re-

action, and a chromophoric group which facilitates quantitating the extent of reaction. Reagent I has been mentioned in an early review article (17) and is now commercially available

(Syva, Palo Alto, USA),

the syntheses of reagents II (35, 36) and III (37) have been described recently. Histone HI from calf thymus (38), human serum albumin

(36)

and phosphorylase b (37) were the targets of labelling. A general drawback of the aryl fluorides is their low specificity. They not only react with amino groups but also with sulfhydryl

and

imidazole

groups and with the phenolic hydroxyl of tyrosine. Modification of the latter three groups can be reversed, however, by thiolysis (37, 39). Moreover, the different substitution products can be distinguished

by

their ultraviolet spectra (36, 39).

Acylating

agents

The three acid chlorides of the pyrrolidine series IV (40, 41), the pyrroline series V (22, 38, 41, 42), and the piperidine series VI (41) respectively have been standard reagents e.g. for the spin-labelling of steroids. They may, however, also be suitable for the labelling of rather stable peptides and proteins, since the hydroxyl

groups of serine,

threonine and tyrosine can be esterified by these agents in the presence o f trifluoroacetic acid without affecting amino groups

(38).

The N-hydroxysuccinimide derivatives VII (available from Serva, Heidelberg) and VIII are convenient reagents to label

proteins at their amino

groups. VII was first used to attach a nitroxide moiety to the a-amino

388 Chart 1. Spin-label

reagents for protein modification

NO 2 OH/~Y-

N H - / > N O 11

2

F

NO 2 x

O — N \ — O — — N O ILL

C—CI

c-ci

2

F 0

iv

"N I. 0 0.

Y/ - \. Oii QR-N^ Y - C H 2 - C - 0 - N viii

0

H 3 CO

0

K I

¿7 I.

IX

0

^

^

XVCH

x

2

- S - S - CH 3 XI

NH-C- U CH 2 I xiii

ICH2-C-NH

CH2-NH-C-CH2I

D3C

CD 3

Q

O=-15N I. 0

XII

D3C

CD 3

0

XIV

group of valyl-tRNA (43), recently l a b e l l i n g of snake neurotoxins 45) and of a t r y p s i n i n h i b i t o r (Kunitz) d e r i v a t i v e in which a l l

(44,

e-amino

groups had been guanidinated (Wenzel, H.R. unpublished) was achieved with V I I and V I I I . The p y r r o l i d i n e d e r i v a t i v e I X was prepared in the course of developing a reagent which could be used to s e l e c t i v e l y attach a n i t r o x i d e group to t y r o s i n e residues of proteins (46). I t seems to be more stable than the corresponding pyrrol ine d e r i v a t i v e prepared e a r l i e r (47). Reagent IX has recently served to spin-label

histone HI from c a l f thymus (38)

and both the nucleosome core p a r t i c l e s and histone core extracted from chicken erythrocytes

(48).

Imidoesters The imidoester X ( a v a i l a b l e from Pierce, Rockford, USA) was recently syn thesized and used to spin-label cytochrome c in order to study the inter actions of cytochrome c with l i p i d and protein preparations ( 4 9 ) . The spin-label reagent shares the advantages of other imidoesters (50) such as high water s o l u b i l i t y , l y s i n e s p e c i f i c i t y , rapid modification at moderate temperature and pH, and retention of p o s i t i v e charge at the reaction s i t e . Alkyl al kanethiol sul fonates In an e f f o r t to render the advantages of alkyl a l k a n e t h i o l s u l f o n a t e s as r e v e r s i b l e t h i o l - b l o c k i n g agents (51) accessible to the spin-label

tech-

nique, compound XI was prepared (52). I t s unique s p e c i f i c i t y was demonstrated with the thiol proteinase papain, which was r a p i d l y and s t o i c h i o m e t r i c a l l y inhibited r e s u l t i n g from the formation of a mixed d i s u l f i d e between the active s i t e cysteine residue and the methyl pyrrol ine-oxyl moiety of X I . The radical was r a p i d l y and completely released from the protein by d i t h i o t h r e i t o l . Thus the spin-label

i s useful not only as a

s p e c i f i c conformational reporter group for t h i o l s i t e structures but a l s o as a s e n s i t i v e t h i o l t i t r a t i n g agent.

390 C r o s s - l i n k i n g reagents I f one i s interested in a f a i t h f u l r e f l e c t i o n of the motion of a protein by the ESR spectrum of a covalently attached s p i n - l a b e l , i t must be ascertained that the label i s r i g i d l y bound, that i t undergoes

little

or no motion r e l a t i v e to the protein structure (53). I t i s obvious that a radical anchored to several s i t e s would most l i k e l y meet t h i s r e q u i r e ment . With t h i s in mind n i t r o x i d e X I I of the p y r r o l i d i n e s e r i e s with two functional iodoacetamido groups was synthesized (54). I t appears to be useful for c r o s s - l i n k i n g of cysteine residues which are not more than 1.3 nm d i s t a n t from each other; other potential reaction partners are the side chains of h i s t i d i n e and l y s i n e . Very recently, progress in the development of several new hetero- and homobifunctional

n i t r o x i d e s has been reported (55, 56).

I s o t o p i c a l l y l a b e l l e d reagents Synthesis of i s o t o p i c a l l y labelled spin-label modification reagents has been carried out for two purposes. The use of radioactive reagents such as the iodo[2-

14 C]acetamide d e r i v a -

t i v e X I I I (57) allows c a l c u l a t i o n of the extent of protein l a b e l l i n g d i r e c t l y from the r a d i o a c t i v i t y incorporated and determination of the s i t e s of reaction by peptide mapping methods. Recently the maleimide d e r i v a t i v e XIV with deuterium and nitrogen-15 subs t i t u t i o n s in the n i t r o x i d e part was synthesized (58, 59) and used to label glyceraldehyde-3-phosphate dehydrogenase (60). Compared with conventional unlabelled reagents, XIV showed a considerable gain in s e n s i t i v i t y and a marked sharpening of spectral features. These improvements are due to a reduction in the spectral linewidths r e s u l t i n g from the r e l a t i v e l y weak i n t e r a c t i o n s of the unpaired electron with the deuterium nuclei and to spectral s i m p l i f i c a t i o n because of the reduction 14 In the15 number of nuclear manifolds from three to two in replacing N with N.

391 2. S p i n - L a b e l l i n g of Amino Acids and Peptides (Charts 2 and 3) Considering the extensive l i t e r a t u r e on s p i n - l a b e l l e d p r o t e i n s , papers dealing with the l a b e l l i n g of peptides and amino acids have been rather scarce. Amino acids or d e r i v a t i v e s thereof have served as model compounds to test properties of protein modification reagents. Moreover, s p i n - l a b e l l e d amino acid d e r i v a t i v e s or small peptides have been used as spin-probes. The synthesis of labelled peptides s t a r t s either from s u i t a b l y protected peptides using n i t r o x i d e reagents, part of which was presented in the previous chapter, or from s p i n - l a b e l l e d amino acid d e r i v a t i v e s , which are elongated by standard coupling procedures. Amino group s p i n - l a b e l l e d amino acids Acylation of amino acid esters to y i e l d the highly v e r s a t i l e

spin-label-

led d e r i v a t i v e s XX can be accomplished by several synthetic routes. A key s t a r t i n g compound i s

3-carboxy-2,2,5,5-tetramethylpyrroline-l-oxyl

XV (22). Either the free carboxylic acid in the presence of d i c y l o h e x y l carbodiimide and 1-hydroxybenzotriazole (61) or s u i t a b l e active d e r i v a t i v e s such as active e s t e r s , e.g. V I I (43, 61), mixed anhydrides (61 63) or the corresponding azide (61, 63) acylate amino acid e s t e r s . Compound XXb was also prepared by reaction of ethyl glycinate and reagent XVI (64, 65). A l k a l i n e h y d r o l y s i s of XX leads to N-acylamino acids X X I , which can a l s o be obtained in good y i e l d by reacting the acid azide of XV with the free amino a c i d s . Compounds XXI can be converted to the active esters X X I I with N-hydroxysuccinimide/dicyclohexylcarbodiimide and further to the azides X X I I I with sodium azide in aqueous acetone. An a l t e r n a t i v e pathway to X X I I I leads from the esters XX or the active esters X X I I via the acid hydrazides XXIV. The general procedures mentioned above and c h a r a c t e r i z a t i o n of the c o r responding individual compounds of chart 2 can be found in the l i t e r a ture (61). The isocyanate X V I I , prepared from the azide of XV by Curtius

reaction(63),

392 Chart 2. Spin-labelling of amino acids

COOH

0

N = C = 0

>d< O

xv

i

0

XVI

xvii

„

XVIII

SL1 — C O — G l y — O C H 3

xxa

SL1—CO—Gl y — OC2H5

xxb

SL1—CO—His — O C H 3

xxc

S L 1 — C O — G l y

xxia

S L 1 — C O — A l a

xxi b

SL1—CO — P h e

xxic

SL1—CO—Gly — O S u

XXlla

SL1 — C O — G l y — N 3

xxilla

S L 1 — C O — A l a — O S u

xxiib

S L 1 - C O - P h e - N

xxnib

SL1—CO — P h e — O S u

xxnc

SL1 — C O — G l y — N H N H 2

xxiva

Phe—NHNH2

xxivb

SL1 — C O —

SL1 — N H — C O — G l y — O C H 3

xxva

SL1 — N H — C O — T r p — O C H 3

xxvb

3

0 /7~\ II (' V - 0 - P - G l y - 0 C \ = / | 0-SL3

2

H

xxviia

" \ = J

° " P ~

A l a _ 0 C H

3 xxvi I b

0-SL3 S L 3 — 0 — C O — P h e

xxvia

SL3—0—CO—Trp

xxvib

SL3—0—CO—Gly—OC2H5

xxvic

5

0 0 - P - L e u - 0 C 0-SL3

2

H

5

xxviic

393 Chart 2.

(Continued)

N0

2

NO 2

SL3-NH

Glu

XXVIIIb

XXVII la

NH

2

Gly—NH—SL3

XXX

XXIX

H

2

N - ( C H

2

)

n

xxxi

- C O - N H - S L 2

Thr — N H — S L 2 I

n = 1,5,10

xxxiia-c

P h e — N H — S L 2

XXXIIIa

xxxnib

Bu*

A c — L y s — N H

CH 2 -O-S-CH 3

Tyr—OC2H5 I CH2 I SL1 xxxvi

2

O2N.

8

NH-SL3 0

N0

XXXIV

Abbreviations: - SL1,

Ac, acetyl; Bu

T

; - SL2,

2

XXXV

'» " SL3,

, tert-butyl; OSu, N-hydroxysuccinimide

ester.

—

;

394 adds to amino acid esters to y i e l d a-ureido acid esters XXV (61). The alkoxycarbonyl azide X V I I I was designed to label amino groups.

It

proved to be a convenient reagent for the preparation of s p i n - l a b e l l e d amino a c i d s , e.g. XXVIa, b and t h e i r e s t e r s , e.g. XXVIc under conditions generally used in N-protection with tert-butyloxycarbonyl

azide (66).

N-Labelling procedures for amino acids or d e r i v a t i v e s thereof, which, in addition to the n i t r o x i d e moiety, introduce a rather bulky group, make use of reagents XIX leading to the phosphorylated s p i n - l a b e l l e d amino acid esters XXVIIa-c (67) or reagent I I y i e l d i n g compounds X X V I I I a and XXVII lb

(36)

(68-70).

Carboxyl group s p i n - l a b e l l e d amino acids Amide bond formation using amino n i t r o x i d e s XXIX (22, 71) or XXX (22) has been found to be the method of choice for carboxyl group l a b e l l i n g of amino acid d e r i v a t i v e s . N-protecting groups have to be chosen which can be s p l i t o f f again without destruction of the n i t r o x i d e group. The glycine d e r i v a t i v e XXXI was prepared from XXIX and t r i f l u o r o a c e t y l glycine anhydride, followed by a l k a l i n e deprotection (72). The t r i f l u o r o acetyl protecting group was also s u c c e s s f u l l y used in the s y n t h e s i s of the s p i n - l a b e l l e d amino acids XXXIIa-c ( 7 3 ) . Coupling of N - t r i f l u o r o a c e t y l amino acids and XXX was achieved by dicyclohexylcarbodiimide,

deprotection

by ammonia. 2-Nitrophenyl sul fenyl amino acid N-hydroxysuccinimidesters were reacted with XXX to y i e l d after mild deprotection r a d i c a l s X X X I I I a and b ( 7 4 ) . Cleavage of the 2-nitrophenylsulfenyl

group was carried out in methanol/

acetic acid with rhodanide and 2-methylindole without i n v o l v i n g the nitroxide group. Side chain s p i n - l a b e l l e d amino acids Derivatives of l y s i n e and t y r o s i n e have been l a b e l l e d at t h e i r side c h a i n s . The spin-label reagent used in the preparation of XXXV was compound I I ( 3 6 ) , XXXVI was synthesized by reacting t y r o s i n e ethyl ester with the spin-label

s u l f o n i c ester XXXIV (75).

395 N-Terminal s p i n - l a b e l l e d peptides Peptides l a b e l l e d at t h e i r amino group have been prepared using two s t r a t e g i e s : The s y n t h e s i s s t a r t s either from active d e r i v a t i v e s of N - l a b e l l e d amino acids or from the complete peptide, which i s s u i t a b l y C-protected i f necessary. S p i n - l a b e l l e d N-acylpeptide esters such as XXXIXa or b were obtained by coupling amino acid esters to N-hydroxysuccinimide esters X X I I , to azides X X I I I or to acylamino acids XXI in the presence of dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (61). The second strategy was followed using d i f f e r e n t spin-label

reagents.

Dipeptide XL, exerting a strong binding to the proteinase papain (76), was prepared by dicyclohexylcarbodiimide-coupling of Phe-Leu-OCH^ and c a r b o x y l i c acid XV followed by a l k a l i n e ester h y d r o l y s i s

(77).

Application of the N-hydroxysuccinimide ester procedure using V I I I or V I I yielded the labelled tetrapeptide ester XLI (78) and the oxidized g l u t a thion d e r i v a t i v e X L I I with two n i t r o x i d e groups (Wenzel, H.R. unpublished). The ESR spectrum of X L I I revealed a strong i n t e r a c t i o n between the two paramagnetic centers. The s p i n - l a b e l l e d p e n i c i l l i n X L I I I was synthesized by the mixed carbonic anhydride method using the mixed anhydride from XXXVII (79) and e t h y l chloroformate (80). Reagent X X X V I I I was employed in the synthesis of compound XLIV, a s p i n l a b e l l e d analogue of pepstatin used to probe the active s i t e of porcine pepsin (81). C-Terminal s p i n - l a b e l l e d peptides Again, elongation of carboxyl group s p i n - l a b e l l e d amino acids and l a b e l l i n g of s u i t a b l y protected peptides have been employed. N-Hydroxysuccinimide-coupling led from X X X I l i b to dipeptide XLVI, a z i d e coupling with XXX to t r i p e p t i d e X L V I I . In both cases the amino group which should not react was intermediately protected with the 2 - n i t r o p h e n y l sulfenyl group (74). XLVI and XLVII were used as substrate-analogous

in-

h i b i t o r s for leucine aminopeptidase (74, 82). Amine XXIX and dicyclohexylcarbodiimide were used to synthesize the s p i n l a b e l l e d pepstatin X L V I I I (81) and, with intermediate N-terminal

trifluoro-

396 Chart 3. S p i n - l a b e l l i n g of peptides

COOH

N=C=S

SL3-CH

2

XXXlXa

SL1—CO—Gly—His—OCH3

xxxixb

SL1—CO—Phe —Leu

XXXVIII

XXXVII

SL1—CO—Gly—Gly—OC2H5

-C0-Gly-Gly-Leu-Gly-0C

H

XLI

5

r

SL3-CO-NH

S H - C O - G l u L

2

XL

Cys-Gly I S XLir

S I |-Cys—Gly

XLII

COOH

SL3 —NH—C—Val—Sta—Ala—Iaa

SLI-CO-Glu

Thr—Phe—NH—SL2

xliv

xlvi

But Thr—Phe—Phe—NH—SL2

xlvii

But

Iva—Val—Val—Sta—Ala—Sta—NH—SL3 Gly—Gly—Leu—Gly—NH—SL3

XLVHI

XLIX

SL3—CH2—CO—Gly—Gly—Leu—Gly—NH—SL3

Gly—Phe—NH—SL3

n

L

lie—Val—NH—SL3

HI

397 Chart 3. (Continued)

0

rA 0

CH 2 -COOH I

NH-C-CH2I

LV

LI 11

COOH /—( 0

LVI

Cys—Tyr—NH2 I

S-CH2-CO-NH-SL2

LIV

acetylation, the tetrapeptide XLIX (78). The latter could be converted to the biradical L by the amino group-labelling procedures discussed above (78). The spin-labelled dipeptide LI, capable of binding to the hormone-binding site of neurophysin (83-86), was synthesized by a route which circumvents problems associated with nitroxide instability under standard conditions of peptide deblocking (86): The N-carbobenzoxy derivative of GlyPhe was coupled with 4-amino-2,2,6,6-tetramethylpiperidine XLV using dicyclohexyl carbodiimide / 1-hydroxybenzotriazole. The carbobenzoxy group was removed by catalytic hydrogénation and replaced by the citraconyl group. After oxidation of the piperidine to the nitroxide, the citraconyl group could be split off at pH 3 to yield LI without destroying the paramagnetic center. The same route was followed from N-carbobenzoxy-Ile-Val to the spin-labelled dipeptide L U

(Wenzel, H.R. unpublished). LII was found to bind

398 Chart 3.

(Continued) LVII

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

a

SL3-CH 2 -C0-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

b

SL3-CH 2 -CO-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg CH 2 -C0-SL3

c

SL2 -CO-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg CH 2 -SL2

d

SL3-CH 2 -CO-Arg-Pro-Pro-Gly-Tyr-Gly-Pro-Phe-Arg CH 2 -C0-SL3

e

SL3-CH 2 -C0-Arg-Pro-Pro-Gly-Phe-Gly-Pro-Tyr-Arg CH 2 -C0-SL3

f

Boc-Arg-Pro-Pro-Gly-Tyr-Gly-Pro-Tyr-Arg CH 2 -C0-SL3 CH 2 -C0-SL3

g

Boc-Gly-Pro-Pro-Gly-Tyr-Gly-Pro-Tyr-Arg CH2-C 0-SL3 CH 2 -CO-SL3

h

SL3-CH 2 -CO-Pro-Pro-Gly-Tyr-Gly-Pro-Phe-Arg

1

CH 2 -C0-SL3 SL3-CH 2 -C0-Pro-Pro-Gly-Phe-Gly-Pro-Tyr-Arg CH 2 -CO-SL3

j

Boc-Pro-Pro-Gly-Tyr-Gly-Pro-Tyr-Arg CH 2 -C0-SL3 CH 2 -C0-SL3

k

Boc-Tyr-Gly-Pro-Tyr-Arg CH 2 -C0-SL3 CH 2 -C0-SL3

i

Abbreviations:

- SL1,

"T

Boc, t e r t - b u t y l o x y c a r b o n y l ; B u \ Iva, i s o v a l e r y l ; Sta,

statine.

; - SL2,

—/*T

; - SL3, — /

t e r t - b u t y l ; I a a , i soamyl amide ;

N-0

;

399 strongly to the complex of trypsinogen and bovine trypsin inhibitor

(Ku-

nitz). This corroborates the proposed mechanism of trypsinogen activation (87, 88).

Side chain spin-labelled The standard spin-label

peptides reagent LI 11 (89) with high affinity for sulfhydryl

groups was used to prepare the labelled dipeptide amide LIV (86), which found application along with LI in studies on neurophysin complexes (83 86). Bradykinin LVIIa is a pharmacologically highly active nonapeptide

(90),

which has often served as model to study peptide conformations in solution. A series of mono spin-labelled, LVIIb, and bis-spin-labelled,

LVIIc-1,

bradykinins or bradykinin analogues have been prepared (91-93). The syntheses were carried out using activated derivatives of the carboxylic acids LV (94) or LVI (22), such as 4-nitrophenyl mide ester, 2,4,5-trichlorophenyl symmetrical

ester, N-hydroxysuccini-

ester, pentachlorophenyl

anhydride (95). The 4-nitrophenyl

ester and

ester proved to be most

satisfactory especially for labelling the hydroxyl

groups of serine or

tyrosine residues. On the basis of distance measurements between the paramagnetic centers of the biradicals in vitrified solutions it was proposed that conformers having a bent or pseudocyclic structure with closely adjacent N - and C-termini predominate in the conformational

equi-

librium of bradykinin (93). This hypothesis has, however, been questioned as a result of

13

C nuclear magnetic resonance studies

3. Minimum Perturbation Spin-Label

(96).

Amino Acids (Chart 4)

A continuing concern in the study of biological

systems by the spin-label

technique has been the extent to which the system under investigation is perturbed by the steric bulk of the nitroxide moiety. This is a crucial question especially when a nitroxide group is attached to a rather small molecule like a membrane component or a peptide. In an effort to minimize the perturbation of the nitroxide group on the labelled molecule, azethoxyl

spin-labelled lipids have recently been in-

400 Chart 4. Minimum s t e r i c perturbation spin-label amino acids

H 2 N ^ ^COOH CH

H2N

COOH

H

2

N ^

/COOH

CH I

I

CH2

CH2 O ^Nr +JW N O

I. 0

f Y -

LIX

LVIII

LX

C-NH HNL

X -

"N' I. 0

H2Nv/COOH "N' I.

0

LXII

H

H 2 C0H

C=N

2

N \

LX I I I

/COOH

CH

LXVI

COOBIR

LXVI I

LXVIII

LXIX

401 Chart 4. (Continued)

Ac—NNAla—Pro—Phe

LXXa

Ac—NNAla—Phe

LXXb

Ac—NNAla—Phe—Phe

LXXc

NNAla—Pro—Phe

LXXd

Abbreviations: Ac, a c e t y l ; Bu1*, tert-butyl ; NNAla, nitronyl n i t r o x i d e amino acid L V I I I . troduced (97 - 99), which have three of the f i v e atoms of the p y r r o l i d i n e ring integrated into t h e i r hydrocarbon chains. A straightforward t r a n s f e r of t h i s concept of 'minimum s t e r i c perturbation spin-labels'

to amino acids would aim at analogues which contain a

stable radical as integral part of t h e i r side c h a i n s . E f f o r t s in t h i s d i r e c t i o n w i l l be presented in the following s e c t i o n s . Nitronyl n i t r o x i d e amino acids Amino acid L V I I I , i t s acetyl and carbobenzoxy d e r i v a t i v e s and i t s a c e t y l a ted ethyl ester were prepared s t a r t i n g from N- or C-protected L - a s p a r t i c (J-semi aldehyde (100). The nitronyl n i t r o x i d e amino acid i s an e f f e c t i v e analogue of h i s t i d i n e . I t approximates the s i z e and shape of the natural amino acid and p a r a l l e l s the ion-dipole interactions observed in h i s t i d i n e d e r i v a t i v e s (100). Drawbacks in comparison with simple n i t r o x i d e s are the reduced s t a b i l i t y of nitronyl n i t r o x i d e s and t h e i r more complex ESR spectra due to s p l i t t i n g by the two r i n g nitrogens and the p-methylene protons Pyrrolidine-oxyl

derivatives

The only representative of p y r r o l i d i n e - d e r i v e d spin-label amino acids

is

p-amino acid LIX (101). I t shares a high structural r i g i d i t y with prol i n e and may become i n t e r e s t i n g in c r o s s - l i n k i n g studies or as ligand for metal

ions.

402

Pyrroline-oxyl

derivatives

Derivatives of the spin-label a-amino acid LX have been described very recently (102). S t a r t i n g from the mesylate XXXIV or the corresponding bromomethyl compound ( 7 5 ) , three d i f f e r e n t preparative methods were s u c c e s s f u l l y employed. Because of i t s structural resemblance to the natural aromatic amino a c i d s , LX i s a very promising paramagnetic amino acid synthon for studies of peptides and proteins. Piperidine-oxyl

derivatives

S t a r t i n g from ketone LXI the f i r s t member of a homologous s e r i e s of p i p e r i dine-oxyl-derived a-amino acids LXI11 can e a s i l y be prepared in two steps (101). LXI i s f i r s t converted by conventional methods (103) to the s p i r o hydantoin L X I I , which i s then hydrolyzed to y i e l d LXI11. This spin-label amino acid was used in proton NMR r e l a x a t i o n studies y i e l ding structural and dynamical parameters such as the number and l i f e t i m e of water protons in the f i r s t hydration sphere around the paramagnetic center (104). Moreover, a s e r i e s of complexes between t r a n s i t i o n metal ions and LXI11 were prepared and studied by d i f f e r e n t techniques 105, 106). Recently the ESR spectra of LXI11 and the N-acetyl

(101,

derivative

were studied as a function of pH. The pH-dependence found both for the i s o t r o p i c hyperfine s p l i t t i n g and the g value should enable these compounds to be used as pH-indicators in model and b i o l o g i c a l

systems

(107). Due to i t s r i g i d structure and the lack of an a-hydrogen, radical

LXIII

would probably not make a promising s u b s t i t u t e of natural amino acids in peptides or proteins. The next homologue, amino acid LXVI, i s c e r t a i n l y a more s u i t a b l e candidate for such an approach. I t possesses an a-hydrogen atom and shares the p-branching of i t s side chain with v a l i n e and i s o l e u cine. The synthesis of LXVI has been carried out using standard reactions (Wenzel, H.R. unpublished). Treatment of ketone LXI with tosylmethyl isocyanide in the presence of base (108) yielded the n i t r i l e LXIV (79). A l k a l i n e h y d r o l y s i s gave the corresponding carboxylic acid ( 7 9 ) , which

403 was reduced by lithium aluminum hydride to the primary alcohol Pyridinium dichromate in methylene chloride

LXV

(109).

(110) allowed the oxidation

of LXV to the corresponding a-ldehyde, which had previously been prepared by a more laborious procedure (111). Conversion to the hydantoin

followed

by alkaline hydrolysis yielded the amino acid LXVI. The homologous amino acid LXIX closely approaches the requirements for an ideal spin-label

reporter group, as its structure and dimensions

favourably compare with those of the natural amino acids and tyrosine

phenylalanine

(112).

The last steps of its synthesis (Wenzel, H.R. unpublished) parallel

those

of the two amino acids described above. A suitable intermediate was the unsaturated ester LXVII, which could be prepared from ketone LXI and tert-butyl

trimethylsilylacetate

(113). Reduction of the ester group

with lithium aluminum hydride and subsequent catalytic hydrogenation of the exo double bond yielded the primary alcohol

LXVI11, whose

synthesis

involving a Wittig reaction was mentioned in a short note (114). Pyridinium dichromate oxidation to the aldehyde, hydantoin formation and alkaline hydrolysis led to the amino acid LXIX.

Peptides with integrated

spin-labels

The paramagnetic analogues LXXa-d of angiotensin fragments have been prepared by solid-phase methods using suitably N-protected derivatives of the nitronyl

nitroxide amino acid LVIII (115). The ESR spectra of these

peptides changed with pH in a way which indicated the existence of an ion-dipole bond between the phenylalanine carboxylate group and the nitronyl nitroxide ring system. Solid-phase peptide synthesis also yielded the dipeptide LXXI with the piperidine-oxyl

amino acid LXIII incorporated

(107).

It can certainly be expected that this short list will soon be substantially enlarged. Incorporation of minimum steric perturbation amino acids into a peptide or protein can be achieved by total

spin-label synthesis

(116, 117) or by semisynthetic methods using naturally occurring

peptide

fragments (118). Situated at a well chosen site of a peptide chain the spin-label

could

'report the news in its environment and not make the

news (1)', as it is to be postulated from an ideal reporter group.

404 The literature has been reviewed up to June 1982. Acknowl edgement Our own spin-label work has been supported by the Deutsche Forschungsgemeinschaft .

References 1.

Berliner, L.J., ed.: Spin Labeling Theory and Applications, Academic Press, New York 1976.

2.

Berliner, L.J., ed.: Spin Labeling II Academic Press, New York 1979.

3.

Likhtenshtein, G.I.: Spin Labeling Wiley-Interscience, New York 1976.

4.

Hyde, J.S.: Meth. Enzymol. 49, 480-511 (1978).

5.

Jost, P., Waggoner, A.S., Griffith, O.H.: in Structure and Function of Biological Membranes, Rothfield, L.I., ed., Academic Press, New York 1971, pp. 83-144.

6.

Keith, A.D., Sharnoff, M., Cohn, G.E. : Biochim. Biophys. Acta 300, 379-419 (1973).

7.

Gaffney, B.J.: Meth. Enzymol. 32, 161-197 (1974).

8.

Cafiso, D.S., Hubbell, W.L.: Ann. Rev. Biophys. Bioeng. 10, 217-244 (1981).

9.

Caspary, W.J., Greene, J.J., Stempel, L.M., Ts'o, P.O.P.: Nucleic Acids Res. 3, 847-861 (1976).

10.

Dugas, H.: Acc. Chem. Res. 10, 47-54 (1977).

11.

Kamzolova, S.G., Postnikova, G.B.: Quart. Rev. Biophys. 14, 223-288 (1981),

12.

Wenzel, H.R., Pfleiderer, G., Trommer, W.E., Paschenda, K., Redhardt Biochim. Biophys. Acta 452, 292-301 (1976).

Theory and Applications,

Methods in Molecular Biology,

13.

Sinha, B.K., Chignell, C.F.: Meth. Enzymol. 62, 295-308 (1979).

14.

Misharin, A.Yu., Polyanovsky, O.L., Timofeev, V.P.: Meth. Enzymol. 62, 495-510 (1979).

15.

Darcy, R., McGeeney, K.F.: Experientia 32, 1129-1131 (1976).

16.

Adam, M.J., Hall, L.D.: Carbohydrate Res. 68, C17-C20 (1979).

17.

Hamilton, C.L., McConnell, H.M.: in Structural Chemistry and Molecul Biology, Rich, A., Davidson, N., eds., W.H. Freeman and Co., San Francisco 1968, pp. 115-149.

405 18.

Ingham, J.D.: J. Macromol . Sei.-Revs. Macromol. Chem. C2, 279-302 (1968).

19.

Griffith, O.H., Waggoner, A.S.: Acc. Chem. Res. 2, 17-24 (1969).

20.

Feher, G.: Electron Paramagnetic Resonance with Applications to Selected Problems in Biology, Gordon and Breach, New York 1970.

21.

McConnell, H.M., McFarland, B.G.: Quart. Rev. Biophys. 3, 91-136 (1970).

22.

Rozantsev, E.G.: Free Nitroxyl Radicals, Plenum Press, New York 1970.

23.

Jost, P., Griffith, O.H.: Meth. Pharmacol. 2, 223-276 (1972).

24.

Smith, I.C.P.: in Biological Applications of Electron Spin Resonance, Swartz, H.M., Bolton, J.R., Borg, D.C., eds., Wiley-Interscience, New York 1972, pp. 483-539.

25.

Dugas, H.: Can. J. Spectrosc. 18, 110-118 (1973).

26.

Dwek, R.A.: Nuclear Magnetic Resonance in Biochemistry: Applications to Enzyme Systems, Clarendon Press, Oxford 1973.

27.

Kalmanson, A.E., Grigoryan, G.L.: in Experimental Methods in Biophysical Chemistry, Nicolau, C., ed., J. Wiley & Sons, London 1973, pp. 589-612.

28.

Lassmann, G.: in ESR und andere spektroskopische Methoden in Biologie und Medizin, Wyard, S.J., ed., Akademie-Verlag, Berlin 1973, pp. 381-403.

29.

Berliner, L.J.: Prog. Bioorg. Chem. 3, 1-80 (1974).

30.

Axel, F.S.: Biophys. Struct. Mechanism 2, 181-218 (1976).

31.

Smith, I.C.P., Schreier-Muccillo, S., Marsh, D.: in Free Radicals in Biology, Vol. 1, Pryor, W.A., ed., Academic Press, New York 1976, pp. 149-197.

32.

Berliner, L.J.: Meth. Enzymol. 49, 418-480 (1978).

33.

Jost, P.C., Griffith, O.H.: Meth. Enzymol. 49, 369-418 (1978).

34.

Keana, J.F.W.: Chem. Rev. 78, 37-64 (1978).

35.

Hsia, J.C.: Ph.D. Thesis, University of Hawaii (1969).

36.

Gerig, J.T., Reinheimer, J.D., Robinson, R.H.: Biochim. Biophys. Acta 579, 409-420 (1979).

37.

Cazianis, C.T., Sotiroudis, T.G., Evangelopoulos, A.E.: Biochim. Biophys. Acta 621, 117-129 (1980).

38.

Lawrence, J.-J., Berne, L., Ouvrier-Buffet, J.L., Piette, L.H.: Eur. J. Biochem. 107, 263-269 (1980).

39.

Shaltiel, S.: Biochem. Biophys. Res. Commun. 29, 178-183 (1967).

40.

Benson, W.R., Maienthal, M., Yang, G.C., Sheinin, E.B., Chung, C.W.: J. Med. Chem. 20, 1308-1312 (1977).

41.

Defaye, G., Basset, M., Chambaz, E.M.: Bull. Soc. Chim. Fr. 1978 II, 471-473.

42.

Cor.vaja, C., Giacometti, G., Kopple, K.D., Ziauddin: J. Am. Chem. Soc. 92, 3919-3924 (1970).

406 43.

Hoffman, B.M., Schofield, P., Rich, A.: Proc. Natl. Acad. Sci. USA 62, 1195-1202 (1969).

44.

Tsetlin, V.I., Karlsson, E., Arseniev, A.S., Utkin, Yu.N., Surin, A.M., Pashkov, V.S., Pluzhnikov, K.A., Ivanov, V.T., Bystrov, V.F., Ovchinnikov, Yu. A.: FEBS Lett. 106, 47-52 (1979).

45.

Ellena, J.F., McNamee, M.G.: FEBS Lett. 110, 301-304 (1980).

46.

Adackaparayil, M., Smith, J.H.: J. Org. Chem. 42, 1655-1656 (1977).

47.

Barratt, M.D., Dodd, G.H., Chapman, D.: Biochim. Biophys. Acta 194, 600-602 (1969).

48.

Chan, D.C.F., Piette, L.H.: Biochim. Biophys. Acta 623, 32-45 (1980).

49.

Mehlhorn, R., Swanson, M., Packer, L., Smith, P.: Arch. Biochem. Biophys. 204, 471-476 (1980).

50.

Hunter, M.J., Ludwig, M.L.: Meth. Enzymol. 25, 585-596 (1972).

51.

Kenyon, G.L., Bruice, T.W.: Meth. Enzymol. 47^, 407-430 (1977).

52.

Berliner, L.J., Grunwald, J., Hankovszky, H.O., Hideg, K.: Anal. Biochem. 119, 450-455 (1982).

53.

McCalley, R.C., Shimshick, E.J., McConnell, H.M.: Chem. Phys. Lett. 13_, 115-119 (1972).

54.

Wenzel, H.R., Becker, G., von Goldammer, E.: Chem. Ber. Ill, 2453-2454 (1978).

55.

Hankovszky, H.O., Hideg, K., Lex, L., KulcsSr, G., Halasz, H.A.: Can. J. Chem. 60, 1432-1438 (1982).

56.

Keana, J.F.W., Hideg, K., Birrell , G.B., Hankovszky, O.H., Ferguson, G., Parvez, M.: Can. J. Chem. 60, 1439-1447 (1982).

57.

Price, N.C.: FEBS Lett. 36, 351-354 (1973).

58.

Venkataramu, S.D., Pearson, D.E., Beth, A.H., Perkins, R.C., Park, C.R., Park, J.H.: J. Labelled Compd. 18, 371-383 (1979).

59.

Beth, A.H., Venkataramu, S.D., Balasubramanian, K., Dalton, L.R., Robinson, B.H., Pearson, D.E., Park, C.R., Park, J.H.: Proc. Natl. Acad. Sci. USA 78, 967-971 (1981).

60.

Beth, A.H., Balasubramanian, K., Wilder, R.T., Venkataramu, S.D., Robinson, B.H., Dalton, L.R., Pearson, D.E., Park, J.H.: Proc. Natl. Acad. Sci. USA 78, 4955-4959 (1981).

61.

Hideg, K., Lex, L., Hankovszky, H.O., Tigyi, J.: Synthesis 1978, 914-917.

62.

Griffith, O.H., Keana, J.F.W., Noall , D.L., Ivey, J.L.: Biochim. Biophys. Acta 148, 583-585 (1967).

63.

Hankovszky, H.O., Hideg, K., Tigyi, J.: Acta Chim. Acad. Sci. Hung. 98, 339-348 (1978).

64.

Golding, B.T., Ioannou, P.V., O'Brien, M.M.: Synthesis 1975, 462-463.

65.

Alcock, N.W., Golding, B.T., Ioannou, P.V., Sawyer, J.F.: Tetrahedron 33, 2969-2980 (1977).

407 66.

Hankovszky, H.O., Hideg, K . , L e x , L . , T i g y i , S y n t h e s i s 1979, 530-531.

J.:

67.

S o s n o v s k y , G., Konieczny, M.: S y n t h e s i s 1976, 537-539.

68.

S o l t i s , B . J . , H s i a , J . C . : J . B i o l . Chem. 253, 3029-3034

(1978).

69.

S o l t i s , B . J . , H s i a , J . C . : J. B i o l . Chem. 253, 4266-4269

(1978).

70.

H s i a , J . C . , E r , S . S . , Tan, C . T . , T i n k e r , D.O.: J . B i o l . Chem. 257, 1724-1729 ( 1 9 8 2 ) .

71.

Rosen, G.M.: J . Med. Chem. 17_, 358-360

72.

Misharin, A.Yu., Vinokurov, L.M., Polyanovskii, O.L.: I z v . Akad. Nauk SSSR, S e r . Khim. 1974, 1897-1898.

73.

P i r r w i t z , J . , Damerau, W.: Z. Chem. J j s 401-402

74.

F i t t k a u , S . , Damerau, W.: J . P r a k t . Chem. 322, 1032-1038

75.

Hankovszky, H.O., Hideg, K . , Lex, L . : S y n t h e s i s 1980, 914-916.

76.

K a l k , A . , de Jong, A . S . H . , B a r r a t t , M.D.: FEBS L e t t . 60, 58-61 ( 1 9 7 5 ) .

77.

K a l k , A . : Ph.D. T h e s i s , R i j k s u n i v e r s i t e i t , Groningen, Netherlands

78.

Maksimova, L . A . , G r i g o r y a n , G . L . , Rozantsev, E . G . : I z v . Akad. Nauk S S S R , S e r . Khim. 1975, 945-948.

79.

Rauckman, E . J . , Rosen, G.M., Abou-Donia, M . B . : J. Org. Chem. 564-565 ( 1 9 7 6 ) .

80.

Goldberg, J . S . , Rauckman, E . J . , Rosen, G.M.: Biochem. B i o p h y s . Res. Commun. 79, 198-202 ( 1 9 7 7 ) .

81.

Schmidt, P . G . , B e r n a t o w i c z , M . S . , R i c h , D . H . : B i o c h e m i s t r y 2^, 1830-1835 ( 1 9 8 2 ) .

82.

F i t t k a u , S . , Kämmerer, G., Damerau, W.: Ophthalmic Res. U , 381-385 ( 1 9 7 9 ) .

83.

Lundt, S . L . , B r e s l o w , E . : J. Phys. Chem. 80, 1123-1126

84.

Lord, S . T . , B r e s l o w , E . : Biochem. B i o p h y s . Res. Commun. 8 0 , 63-70

85.

Lord, S . T . , B r e s l o w , E . : I n t . J . Peptide P r o t e i n Res. U ,

(1974).

(1976). (1980).

(1975).

(1976). 71-77

(1978).

(1979).

86.

Lord, S . T . , B r e s l o w , E. : B i o c h e m i s t r y lj}, 5593-5602

87.

Bode, W., Huber, R . : FEBS L e t t . 68, 231-236

(1980).

88.

Bode, W.: J . Mol. B i o l . 127, 357-374

89.

Ogawa, S . , McConnell , H.M.: Proc. N a t l . Acad. S e i . USA 58, 19-26

90.

R e g o l i , D . , Barabe, J . : Pharmacol. Rev. 32, 1 - 4 6 ( 1 9 8 0 ) .

91.

F i l a t o v a , M . P . , Reissmann, S . , Ravdel , G . A . , I v a n o v , V . T . , G r i g o r y a n , G . L . , S h a p i r o , A . B . : i n Peptides 1972, Hanson, H . , Jakubke, H . - D . , e d s . , North Holland Publ. C o . , Amsterdam 1973, pp. 333-340.

92.

Reissmann, S . , F i l a t o v a , M . P . , Reutova, T . O . , A r o l d , H . , I v a n o v , J . P r a k t . Chem. 318, 429-440 ( 1 9 7 6 ) .

93.

F i l a t o v a , M . P . , Reissmann, S . , Reutova, T . O . , I v a n o v , V . T . , G r i g o r y a n , G . L . , S h a p i r o , A . M . , R o z a n t s e v , E . G . : B i o o r g . Khim. 3, 1181-1189 ( 1 9 7 7 ) .

(1976).

(1979). (1967).

V.T.:

408 94.

Shapiro, A.B., Baimagambetov, K., Gol'dfel'd, M.G., Rozantsev, E.G.: Zh. Org. Khim. 8, 2263-2269 (1972).

95.

Reißmann, S., Arold, H., Filatova, M.P., Reutova, T.O., Ivanov, V.T.: Z. Chem. 15, 399-400 (1975).

96.

London, R.E., Stewart, J.M., Cann, J.R., Matwiyoff, N.A.: Biochemistry 1_7, 2270-2277 (1978).

97.

Lee, T.D., Birrell, G.B., Keana, J.F.W.: J. Am. Chem. Soc. 100, 1618-1619 (1978).

98.

Lee, T.D., Keana, J.F.W.: J. Org. Chem. 43, 4226-4231 (1978).

99.

Lee, T.D., Birrell, G.B., Bjorkman, P.J., Keana, J.F.W.: Biochim. Biophys. Acta 550, 369-383 (1979).

100.

Weinkam, R.J., Jorgensen, E.C.: J. Am. Chem. Soc. 93, 7028-7033 (1971).

101.

Rassat, A., Rey, P.: Bull. Soc. Chim. Fr. 1967, 815-817.

102.

Lex, L., Hideg, K., Hankovszky, H.O.: Can. J. Chem. 60, 1448-1451

(1982).

103.

Bucherer, H.T., Lieb, V.A.: J. Prakt. Chem. 141, 5-43 (1934).

104.

v. Goldammer, E., Kreysch, W., Wenzel, H.: J. Solution Chem. 2 , 197-204 (1978).

105.

Jahr, D., Rebhan, K.H., Schwarzhans, K.E., Wiedemann, J.: Z. Naturforsch. 28b, 55-62 (1973).

106.

Terzian, G., Cormons, A., Asso, M., Benl'ian, D.: J. Chim. Phys. 73, 146-148 (1976).

107.

Nakaie, C.R., Goissis, G., Schreier, S., Paiva, A.C.M.: Brazilian J. Med. Biol. Res. 14, 173-180 (1981).

108.

Oldenziel, O.H., van Leusen, D., van Leusen, A.M.: J. Org. Chem. 42, 3114-3118 (1977).

109.

Rauckman, E.J., Rosen, G.M.: Synth. Commun. 6, 325-329 (1976).

110.

Corey, E.J., Schmidt, G.: Tetrahedron Lett. 1979, 399-402.

111.

Schlude, H.: Tetrahedron ^9, 4007-4011 (1973).

112.

Morrisett, J.D.: in Spin Labeling Theory and Applications, Berliner, L.J., ed., Academic Press, New York 1976, pp. 273-338.

113.

Rosen, G.M., Rauckman, E.J.: Org. Prep. Proc. Int. 10, 17-20 (1978).

114.

Szykula, J., Zabza, A.: Experientia 36, 149-150 (1980).

115.

Weinkam, R.J., Jorgensen, E.C.: J. Am. Chem. Soc. 93, 7033-7038 (1971).

116.

Gross, E., Meienhofer, J., eds.: The Peptides, Analysis, Synthesis, Biology, Vol. 1, Major Methods of Peptide Bond Formation, Academic Press, New York 1979.

117.

Gross, E., Meienhofer, J., eds.: The Peptides, Analysis, Synthesis, Biology, Vol. 2, Special Methods in Peptide Synthesis, Part A, Academic Press, New York 1980.

118.

Offord, R.E.: Semisynthetic Proteins, John Wiley & Sons Ltd., Chichester 1980.

THE ULTRASTRUCTURE OF MACROMOLECULAR COMPLEXES STUDIED WITH ANTIBODIES

Georg Stöffler and Marina Stoffler-Meilicke Max-Planck-Institut für Molekulare Genetik, Abt. Wittmann, Berlin-Dahlem, Germany

I. Introduction The translation of mRNA into protein occurs on ribosomes with the participation of macromolecules such as tRNAs and protein synthesis factors. The factors play important roles during the three main steps of the cycle of protein synthesis, namely initiation, elongation and termination. Ribosomes are nucleoprotein particles which consist of two subunits of unequal size. The most extensively studied species of ribosomes is that of Escherichia coli. One of the main topics on which research into bacterial ribosome structure has recently concentrated was directed towards the description of the shape of ribosomal particles and on studies as to the spatial arrangement of the various ribosomal components, i.e. ribosomal topography (1-6). Another main objective from the very beginning was to relate the three-dimensional distribution of ribosomal proteins and rRNAs to ribosomal function. The small subunit recognizes the initiation site on mRNA with the participation of three initiation factors and initiator tRNA. It is responsible for the binding of aminoacyl tRNAs and for the translational fidelity of messenger reading. The large subunit binds the acceptor stem of aminoacyl tRNAs, catalyses peptide bond formation and participates in translocation and chain termination.

Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York

410

The localization of functional domains can be approached by relating the three-dimensional distribution of ribosomal proteins and of rRNA to known biochemical information on the functional contribution of individual ribosomal components. Such attempts to localize functional domains have been published (1,7) . The unraveling of the structure and insight into the details of the function of E^ coli is derived from a variety of technologies: the isolation, purification and chemical and physical characterization of the proteins of E^ coli ribosomes (8), the structure determination of the ribosomal RNAs (9), the reconstitution of active ribosomal subunits from their molecular components, i.e., from RNA and protein (4,5). The topography of ribosomal components has been studied by immuno electron microscopy, by crosslinking experiments, neutron scattering studies and by enery transfer (1-7, 9). Since 1968 a concentrated effort has been undertaken to use antibodies in the analysis and function of ribosomes. It occurred to us fifteen years ago that the immunochemical methods that had been used to explore the structure of enzymes might well be applied to more complex assemblies and perhaps even to organelles. For this reason we began to raise antibodies against Escherichia coli ribosomes, ribosomal subunits and then, as they became available, against each of the 53 individual ribosomal proteins. The purpose of this article is to show the contribution which immunochemical experiments have made to the knowledge of the structure and function of ribosomes (reviewed in 1, 2, 7, 10-14). Most of the experiments have been performed with antisera to Escherichia coli ribosomes, some antisera were raised against

411

ribosomes from other bacterial species (e.g.. Bacillus stearothermophilus, Bacillus subtilis), from plants (cytoplasmic and chloroplast), from chicken and from mammals (preferentially rat). Bacterial and plant ribosomes and their subunits are strongly immunogenic in mice, rabbits and sheep, more immunogenic than mixtures of extracted ribosomal proteins. Immunisation with ribosome particle leads to the formation of precipitating antibodies directed primarly against the protein; formation of precipitating antibodies to RNA is an exceptional occurrence (1,10). Ribosomal proteins from chicken liver and rat liver are less immunogenic than bacterial ribosomal proteins (11-13); reasonable antisera can be obtained by immunizing sheep, rabbit or swine (11-14). Only few attempts have so far been made to use monoclonal antibodies (15, 16); they may however become very useful for several applications in the near future. Most of the immunological techniques applied for the elucidation of ribosome structure and function may be applied in other research fields for the study of other complex structures, such as multienzyme complexes or for the study of a variety of enzymes and proteins in cell membranes and organelles.

II. Molecular Composition and Shape of the E. coli Ribosome Before discussing immunochemical methods it is necessary to describe the components which build up the ribosome as well as the shape of ribosomes and of ribosomal subunits. Molecular composition Bacterial ribosomes sediment with a sedimentation co-

412

32 Proteins

21 Proteins

[ Z M.W. 460.000]

IXM.W350.000]

Figure 1: Molecular composition of E^ coli ribosomes (modified from reference 17) efficient of 70S. By lowering the magnesium ion concentration, 70S ribosomes dissociate into two subunits of unequal size, the small 30S subunit and the large 50S subunit. The 30S subunit consits of one 16S RNA molecule (1542 nucleotides) and 21 proteins which are numbered in E^ coli S1-S21. The 50S subunit contains two RNA molecules, the small 5S RNA (120 nucleotides) and the large 23S RNA (2904 nucleotides) and 32 proteins, designated L1-L34 (Fig. 1). The rRNA molecules represent two-thirds of the mass of a ribosome. Sequence studies have provided us with the complete primary structures of the RNA and protein moieties of E^ coli ribosomes (for review see 8, 9).

413

Mica

1. Carbon film

\

5 y y v v

f

9

3

i

h

J jt

1

2

\\

Sedimentation

Figure 6: Antibody-ribosome complex formation after centrifugation of 50S subunits with increasing antibody concentrations (anti-L11) in sucrose gradients: (a,e) 50S subunits; (b-d) 50S subunits + a 30 to 170fold excess of IgG; (f-h) 50S subunits + a 3 to 9fold molar excess of specific IgG which had been purified by affinity chromatography (36). Peak 1 = 50S-IgG-50S complexes; Peak 2 = 50S subunits; Peak 3 = IgG. Methods which employ sedimentation select for the binding of those antibodies which have an affinity sufficiently high to survive centrifugation. Antibodies to proteins S9 and S12 did not form such Complexes, although these antibodies reacted with 30S subunits under the equilibrium conditions that prevail in double antibody precipitation assays. On the other hand, antibodies to protein S1 removed the antigen from the 30S particle (unpublished observation).

421

IV. Protein Topography in Ribosomal Subunits The principle of immuno electron microscopy is to bind a purified IgG-antibody, specific to a single ribosomal protein, to the appropriate ribosomal subunit; the bivalent antibody dimerizes two subunits which can then be examined under the electron microscope. The location of the bound antibody on the subunit surface can be determined and the position of an antigenic determinant of a particular protein can thus be directly made visible. The dimeric immunocomplexes obtained by density gradient centrifugation (Fig. 6, peak 1) are directly used for specimen preparation. The 30S subunit Figure 7 shows examples of electron micrographs of small subunits reacted with protein-specific antibodies. Images of subunits connected by an IgG molecule are easily recognized, and the location of the antibody attachment site can readily be described in two dimensions. For example, antibodies against proteins S10 and S13 bind at the head of the small subunit (Fig. 7a,b), anti-S11 and anti-S18 bind at the large lobe (Fig. 7e,f) and anti-S17 binds near the lower pole (Fig. 7h). In order to determine the three-dimensional location of a given protein it is necessary to locate the antibody attachment site to subunits seen in different orientations. This proceedure is exemplified in Figure 3 for the location of protein S15. Determination of the antibody binding site on all four projections (Fig. 8) led to the three-dimensional location as shown in Figure 11. Thus 15 of the 21 ribosomal proteins have been localized in three dimensions on the surface of the 30S subunit of E^ coli at distinct sites (Fig. 11).

422

423

jy. -

mm -

•1 fUSP''

è

w m Figure 8: General field and selected electron micrographs of 30S subunits, reacted with anti-S15. The interpretative diagrams relate to the micrograph to their left and illustrate antibody binding to the different 30S projections.

Figure 7: Electron micrographs of 30S subunits, reacted with anti-S13 (a), anti-S10 (b), anti-S5 (c), anti-S4 (d), anti-S11 (e) , anti-S18 (f), anti-S6 (g) and anti-S17 (h) . »-30S-IgG-30S complexes, 30S-IgG-30S complexes, in which two antibodies are simultaneously bound.

424

425

Protein S8 has been mapped in previous studies by Tischendorf et al. (31) and by Lake (32). The specificity of the antibody was not sufficiently proven by either group (see below). It is, however, very likely from other studies (protein-protein crosslinking and neutron scattering) that protein S8 is located at the one-third/two-thirds partition. The 30S proteins are localized in distinctive domains (Fig. 11). There are two domains on the head of the subunit, one of them comprises proteins S3, S10 and S14. The second domain comprises proteins S7, S13 and S19. There is evidence that protein S9 is also located at the head of the 30S subunit. Four proteins are located on the larger lobe, whereas three proteins, viz. S4, S5 and S16 are located at or below the small lobe (Fig. 11). Proteins S12 and S20 are, according to preliminary data, also in this region. The antigenic sites of two proteins, namely S15 and S17, have been mapped at the body of the 30S subunit. The distribution of the proteins in domains agrees well with other studies on protein topography, e.g. protein-protein crosslinking (34) and neutron scattering (35). The 50S subunit Altogether, 14 of the 32 ribosomal proteins of the 50S subunit have been mapped in comprehensive studies. Figure 9 shows general fields of electron micrographs obtained from 50S subunits which had been incubated with protein-specific antibodies. Since 50S subunits are predominantly observed in the crown-view, a two-dimensional localization of the antibody binding site on this projection is easily achieved: Figure 9: Electron micrographs of 50S subunits, reacted with anti-L1 (a), anti-L18 (b), anti-L11 (c), anti-L7/L12 (d), anti-L19 (e), anti-L17 (f), anti-L9 (g) and anti-L23 (h). Arrows indicate characteristic 50S-IgG-50S complexes.

426

For example, anti-L1 binds to the broad lateral protuberance (Fig. 9a), anti-L18 to the central protuberance (Fig. 9b), and anti-L7/L12 to the rod-like appendage (Fig. 9d). For an unambiguous three-dimensional localization it is necessary to determine the antibody binding site also on the kidney projection. This necessity is illustrated by the example in Fig. 10: The binding sites for antibodies against L17 and L19 are indistinguishable from each other when only the crown-view is considered (first row), but they are clearly seen to be different in the kidney-view (second row) Complexes, in which a crown form is connected with a kidney projection prove that (because of the identity of the two Fab-arms of one IgG molecule) the two sites observed on the two-dimensional electron micrographs correspond to a single

a-L17

a-L19

50 n m

Figure 10: Selected electron micrographs and interpretative diagrams of 50S subunits, reacted with anti-L17 (left) and anti-L19 (right).

427

.«ns- -»^

f ;

..

.

¿(MMMfct i )

m

\ *

ML 9

ilt 10

'

6

H

k

7/

\>

•

^

%

19

Figure 11: Three-dimensional models of the 30S (a) and the 50S subunit (b) of E^ coli with the locations of the centers of the antibody binding sites for individual proteins. site in the three-dimensional structure (Fig. 11). Thus the binding site on both crown- and kidney-views has been determined for antibodies specific for 14 proteins of the 50S subunit, the resulting three-dimensional locations of these proteins are given in Figure 11. As in the 30S subunit, the proteins are clustered in domains. The central protuberance contains antigenic determinants of proteins L18 and L25, both of which bind independently to 5S RNA. This data is in agreement with the location of the 3'-end of 5S RNA (see Chapter V). Protein L27 is, according to Lake (36) , also located on this protuberance. Another domain is found on the broad lateral protuberance and comprises proteins L1 and L9. The stalk contains antigenic determinants of L7/L12, whereas the protuberance from which the stalk originates contains proteins L6, L10 and L11. A

428

determinant of protein L19 was found slightly separated from the latter domain (Fig. 11). Another domain is on the concave surface of the 50S subunit, below proteins L1 and L9 and comprises determinants of proteins L23 and L29. Protein L17 has been mapped on a unique position. The domain structure as found by immuno electron microscopy also agrees in principle with other topographical studies as described above (4-6) . Determination of antibody specificity It is a general working principle in immunochemistry that it should always be assumed that an antigen which is pure by biochemical criteria, is not pure immunologically. The consequences of this assumption have been drawn by most investigators working with antisera against pure ribosomal proteins. Double

immunodiffusion, quantitative immunopreci-

pitation, Immunoelectrophoresis etc. have been used for assessing the purity of the various antisera. The purity, as established by these criteria, was sufficient for most applications. Impure antisera have also been purified by immunoaffinity chromatography (37). When studying the reaction of antibodies with ribosomal particles it is essential to prove the specificity of that particular interaction, since a small population of contaminating antibodies which escaped detection with the above mentioned techniques may be very reactive with determinants on the intact particle. Therefore, even for "pure" antisera, it cannot a priori be assumed that the antibodies react only with the antigen protein and no other protein in situ. Several methods have so far been applied for testing antibody specificity, none of which was sufficient to prove the specificity of the antibodies used for immuno electron microscopy (1, 7, 22, 30, 31, 36, 37).

429 We have recently developed a series of control experiments which clearly eliminate effects of contaminating and crossreacting antibody (38). Dimeric immunocomplexes must be completely abolishable by preincubation of the antibody with stoichiometric amounts of the antigen protein. An example of such an adsorption experiment is shown in Figure 12a. This experiment clearly shows that the determinant to which antiLi 1 binds, is contained in protein L11. The experiment does, however, not exclude that the reactive determinant on the ribosome is present on another protein which has the same determinant. It is possible that some antibody to protein L11 may bind to protein L16, since both proteins contain a common hexapeptide (Thr-Phe-Val-Thr-Lys-Thr). Such cases of crossreactivity may frequently occur, since common tetrapeptides have been found with a high frequency (reviewed in 8) and a tetrapeptide is the minimal size for an antigenic determinant. A second control is thus necessary. The formation of dimeric immuno-complexes should also be inhibitable with a mixture of all ribosomal proteins (Fig. 12b), but not with total proteins lacking the protein to be mapped (Fig. 12c). Such mixtures can be obtained in various ways. (1) The most easiest way is the use of mutants which lack a single protein; a mutant lacking protein L11 exists (39). (2) A mixture of single ribosomal proteins, omitting the protein in question, can be prepared. (3) Total ribosomal protein can be passed over an immunoaffinity column to which an antibody against the particular protein has been bound (to be published). It is clear that this second type of control experiments is also necessary when monoclonal antibodies are being used. The mapping data for the proteins shown in Figure 11 have all been performed with the complete set of these control experiments.

430

50 S • anti - L 11 • ljig SP L11

• 2¿ig SP L11

•5/ig SP L11

• 25 Jig TP 70 WT

.50/ig TP 70 WT

•100¿ig TP 70 WT

• 25(¿g TP 70 AM 68

.50/ig TP 70 AM 66

•100 /ig TP 70 AM 66