Insulin, chemistry, structure, and function of insulin and related hormones: Proceedings of the Second International Insulin Symposium, Aachen, Germany, September 4–7, 1979 9783111646190, 9783110081565

Table of contents :
ACKNOWLEDGEMENTS
Preface
CONTENTS
INTRODUCTION
SECTION I. STRUCTURE OF INSULIN
A Comparison Between the Insulin Molecules in 2-Zinc and 4-Zinc Insulin Crystals
Crystal Structure, Aggregation and Biological Potency of Beef Insulin Cross-Linked at A1 and B29 by Diaminosuberic Acid
Correlation of Structural Details of Insulin in the Crystal and in Solution
SECTION II. PEPTIDE SYNTHESIS
An Assessment of Protecting Group Strategies in Synthesis of Large Peptides
Fully Active Semisynthetic Insulin by Selective Formation of the Disulfide Bridges as an Intermediate Result of the Total Synthesis by Fragment Condensation on Polymer Phase
[A14-Phenylalanine-Insulin]: A Synthetic Analogue
[B25-Tyrosine]B-Chain-S-Sulfonate of Porcine Insulin
The Synthesis of [B25-4-Nitrophenylalanine]-Octapeptide B(23-30) of Porcine Insulin
A Novel Route for the Preparation of Mono-Iodotyrosine and Mono-Iodotyrosine-Containing Peptides by Electrochemical Reduction of Di-Iodo Derivatives
Alternative Approach for Human Proinsulin Synthesis
An Alternative Synthesis of Glucagon by the Method of Solid Phase Condensation of Peptide Fragments
SECTION III. SEMISYNTHESIS AND CHEMICAL MODIFICATION
Progress Towards the Semisynthesis of Preproinsulin
Specific Activation of the Arginine Carboxyl Group of the B-Chain of Bovine Des-Octapeptide-(B23-30)-Insulin
Enzyme-Catalyzed Semisynthesis with Insulin Derivatives
The Relative Reactivity of Insulin Amino Groups as an Indicator of Structural Accessibility and its Use for Synthetic Approaches for Structure-Function Studies
A14-(N-Methylpyridinium) and A14-(2-Nitro-4-Trimethylammoniophenyl) Derivatives of Bovine Insulin
Preparation of Covalent Insulin Dimers
Reactions of Insulin Derivatives with ETAC Reagents
SECTION IV. RADIOACTIVE LABELLING AND SEPARATION TECHNIQUES
The Application of HPLC to the Analysis of Radio- Iodinated Tracers of Glucagon and Insulin
Monoiodoinsulin Specifically Substituted in A14-Tyr or A19-Tyr
Preparation of Homogeneous Insulin Tracers with Monoand Di-Iodo-Desaminotyrosines in Position B1
Insulin and Proteins Labeled by Microwave Discharge Activation of Tritium
Radioactive Labelling (14 C, 3 H) of Insulin and Insulin Derivatives by Reductive Methylation; Isolation of Homogeneous N, N-Di-Methylated Derivatives
Quantitative Determination of Insulin by Gradient Elution HPLC
Some Preliminary Findings in Isotachophoretic Analyses of Insulin
Studies on the Denaturation of Dissolved Insulin
SECTION V. RECEPTORS AND HORMONE-RECEPTOR INTERACTION
125 I-SpHPP-Avidin: A Tool for Hormone Receptor Studies
Chromatographic Properties of the Adipocyte Transport System in Detergent Solution and its Resolution from the Insulin Receptor
Hydrophobic Interaction Chromatography as a Tool in Insulin Receptor Purification
Localization of Insulin-Receptor Complexes on the Surface of Whole Cells by Autoradiography
Insulin Receptors in the Heart Muscle: Evidence for the Negative Cooperativity Binding Model
Insulin Receptors, Insulin Degradation, and Biological Activity
Gammeltoft, S.#Kristensen, L.Ø.
Regulation of Insulin Receptors in Isolated Hepatocytes from Normal and Streptozotocin-Diabetic Rats in the Fed and 48 H Fasted State
Insulin-Receptor Turnover and Down Regulation
Receptor-Mediated Internalisation of Insulin, Glucagon and Growth Hormone in Intact Rat Liver. A Biochemical Study
Kinetics of Insulin Binding to Its Receptors in vivo. Effects of Fasting
Hystricomorph Insulins and Insulin Receptors
SECTION VI. PHOTO-INDUCED HORMONE-RECEPTOR COUPLING
Photoreactive Insulin Derivatives: Preparation and Characterization
Photoreactive Insulin Derivatives: Chemical and Physical Properties
The Preparation and Application of an A1-Substituted Photo-Reactive Insulin Analogue
Photoaffinity Labeling of Insulin Receptor Proteins of Liver Plasma Membranes
Characterisation of Insulin Binding Sites in Rat Liver Plasma Membranes Using Photoreactive Insulin Analogues
Permanent Activation of Lipogenesis by Insulin Covalently Bound to Adipocyte Receptors
SECTION VII. STRUCTURE, BINDING, ACTIVITY
The Activation of Adenylate Cyclase by Chemically Modified Forms of Glucagon and its Relationship to Receptor Binding
Structure-Activity Relationships of Insulin-Induced Negative Cooperativity Among Receptor Sites
Properties of a Mutant Insulin Species Causing Human Diabetes
A1-Modified Insulins: Receptor Binding and Biological Activity
Biological Activity of Insulin Analogues Substituted at the Amino Group of B1-Phenylalanine
Functional Role of the N-Terminal Region of the B-Chain of Insulin
Biological Properties of Covalent Insulin Dimers
Biological Activity and Receptor Binding of Six Different Covalent Dimers of Insulin
In vitro and in vivo Effects of Poly-N-Vinylpyrrolidone- Insulin
Study of Insulin Action by Use of Insulin-Dextran Complex: Uptake of Insulin into Subcellular Fractions and Stimulation of Pyruvate Dehydrogenase in Mouse Adipocytes
Studies on the Mechanism of Insulin Action VI: The Interaction of Insulin Fragments with Insulin Receptor
Stimulation of Intracellular H2 02 Production in Rat Epididymal Adipocytes by Insulin, Insulin Fragments, and Other Hormones and Growth Factors with Insulin-Like Activities
Early Events in Insulin Binding and Insulin Action in Fat Cells and Hepatocytes
Role of the Biodegradation Process in the Action of Insulin. A Proposal for a Model Consisting of Sulfhydryl- Disulfide Interchange Reaction and Proteolysis
Insulin Receptors, Receptor Antibodies and the Mechanism of Insulin Action
SECTION VIII. DEGRADATION
Studies on Uptake of [3 H]-Insulin by Erythrocytes
An Insulin-Degrading Enzyme from Human Erythrocytes: Isolation and Characterization
Comparison of 125 I-Insulin and [B1- 3 H-Phenylalanine]- Insulin Degradation by Insulin Protease
The Chemical Characterization of the Products of the Processing of Subcutaneously Injected Insulin
Some Inhibitors of Insulin Degradation
Excretion of Endogenous and Exogenous Insulin by Kidney in Diabetic Ketoacidosis
Incubation of Insulin in Hyperglycemic Blood Generates Insulin Dimers
SECTION IX. IMMUNOLOGY
Genetic Regulation of the Immune Response of Mice to Insulins of Different Species
Genetic Control of the Immune Response to Insulin
Analysis of the Immunogenicity of Three Insulin- Preparations
Studies on the Immunological Properties of Natural and Modified Insulins
Genetic Control of the Immune Response to Insulin: A Clinical Study of Adverse Immunologic Reactions to Insulin
The Induction of Hyperglycemia with Insulin Antibodies to B-Chain Determinants
The Pattern of B-Lymphocyte Specificities for Modified and Unmodified Insulins in the Mouse
Specificity of Rabbit Anti-Insulin Antibody
Radioimmunoassay of Chemically-Modified Insulins - Use and Misuse
Purity of Chromatographycally Purified Insulins Measured by Skin-Reactivity and Insulin-Specific IGE
SECTION X. BIOSYNTHESIS, STORAGE, EVOLUTION
Human Preproinsulin: Primary Structure of the Messenger RNA
Quantification of Proinsulin Biosynthesis by Determination of Specific Radioactivity of [3 H]-Leucine in Pancreatic Islets
Immunoreactive Rat C-Peptides I and II in Glucose Perfusate of Isolated Pancreas
Biosynthesis of Somatostatin in Pancreatic Islets of Wistar Rats
Crystallography of Intracellular Insulin and Glucagon as Revealed by Investigations of Tissues and Models
Pancreatic Hormone Storage Granules: The Role of Metal Ions and Polypeptide Oligomers
On the Molecular Biology of Hagfish Insulin
SECTION XI. INSULIN-RELATED HORMONES
Possible Relationships in the Processing, Storage and Secretion of Some Insulin-Related Peptides
Insulin-Like Growth Factors
Relaxin and Relaxin-Like Peptides in Ovaries of Pregnant Sows and Sharks
Structural Studies on Porcine Relaxins and their Biosynthetic Precursors
PRESENTATIONS NOT INCLUDED IN THIS VOLUME
LIST OF PARTICIPANTS
ABBREVIATIONS
SUBJECT INDEX
AUTHOR INDEX

Citation preview

Insulin Chemistry, Structure and Function of Insulin and Related Hormones

Insulin Chemistry, Structure and Function of Insulin and Related Hormones Proceedings of the Second International Insulin Symposium Aachen, Germany, September 4-7,1979 Editors Dietrich Brandenburg • Axel Wollmer

W DE G Walter de Gruyter • Berlin • New York 1980

Editors Dietrich Brandenburg, Dr. rer. nat. H e a d of the Insulin Division Deutsches Wollforschungsinstitut an der Rheinisch-Westfälischen Technischen Hochschule Aachen Veltmanplatz 8 D-5100 Aachen Axel Wollmer, Dr. rer. nat. Professor of Physical Biochemistry Fachgebiet Struktur und Funktion der Proteine Abteilung Physiologische Chemie Rheinisch-Westfälische Technische Hochschule Aachen Melatener Straße 211 D-5100 Aachen

CIP-Kurztltelaufnahme

der Deutschen

Bibliothek

Insulin, chemistry, structure and function of insulin and related hormones: proceedings of 2. Internat. Insulin Symposium, Aachen, Germany, September 4-7,1979 / ed. Dietrich Brandenburg; Axel Wollmer. - Berlin, New York: de Gruyter, 1980. ISBN 3-11-008156-3 NE: Brandenburg, Dietrich [Hrsg.]; International Insulin Symposium

Library of Congress Cataloging in Publication

Data

International Insulin Symposium, 2d, Aachen, 1979. Insulin. Bibliography: p. Includes index. 1. Insulin-Congresses. 2. Peptide-hormonesCongresses. I. Brandenburg, Dietrich, 1932II. Wollmer, Axel, 1935- III. Title. QP572.I5I58 1979 615\365 80-13879

© Copyright 1980 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: Karl Gerike, Berlin. - Binding Lüderitz & Bauer, Buchgewerbe GmbH, Berlin. - Printed in Germany.

T 0

PROFESSOR D O R O T H Y

H O D G K I N

2 N D INTERNATIONAL INSULIN SYMPOSIUM AACHEN 1979

organized DEUTSCHES

in

Fachgebiet Abteilung

by

WOLLFORSCHUNGS INSTITUT

cooperation

Struktur

und

with

Funktion

Physiologische

der

Chemie,

TH

Proteine Aachen

and Sonderforschungsbereich

113

Symposium

Diabetesforschung

Committee

D. B R A N D E N B U R G , W. J.

DANHO FÖHLES

H.-G.

GATTNER

P. K A P L I N

Düsseldorf

Chairman

V.K. B.-E.

NAITHANI

D.

SAUNDERS

RUDOLPH

A.

WOLLMER

A C K N O W L E D G E M E N T S We g r a t e f u l l y

DEUTSCHE MINISTER

acknowledge

the

support

of

FORSCHUNGSGEMEINSCHAFT

FÜR W I S S E N S C H A F T

UND

FORSCHUNG

NORDRHEIN-WESTFALEN

RHEINISCH-WESTFÄLISCHE FACHHOCHSCHULE

BAYER

AG

GRÜNENTHAL LKB

"

CIBA-GEIGY

GmbH

INSTRUMENT

-

DR.

RESEARCH R.

HOECHST

GmbH

LABORATORIES NOVO

TECHNISCHE

AG

AACHEN

STADT

AACHEN

" "

ELI SHARP

NORDISK

»

LILLY

HORMON-CHEMIE MERCK,

»

INSTITUTE

SELLENTIN

HOCHSCHULE

AACHEN

& DOHME

INSULIN

ORGANON/DIOSYNTH

-

GmbH

RESEARCH

LABORATORIUM (AKZO

STADTSPARKASSE

»

Programme

& COMPANY

MÜNCHEN

PHARMA) AACHEN

Committee

P . DE M E Y T S , B r u s s e l s G. DODSON, York R . E . OFFORD, O x f o r d P. SONKSEN, London a n d t h e members o f t h e S y m p o s i u m

J.

GLIEMANN, H. Z A H N , Committee

Aarhus Aachen

C h a i rmen E. M. G. J. E.

A R Q U I L L A , I r v i n e , T. B L U N D E L L , L o n d o n , D.BRANDENBURG, Aachen C Z E C H , P r o v i d e n c e , C . D E H A È N , S e a t t l e , P . DE M E Y T S , B r u s s e l s DODSON, Y o r k , J . GLIEMANN, A a r h u s , C. R. K A H N , B e t h e s d a MEIENHOFER, N u t l e y , R. O F F O R D , O x f o r d , W. R I T T E L , B a s l e RÜDE, M a i n z , P. SÜNKSEN, L o n d o n , H. Z A H N , A a c h e n

Preface More than f i f t y years a f t e r i t s discovery, twenty-five years a f t e r the determination of i t s amino acid sequence and ten

years a f t e r the e l u c i -

dation of i t s three-dimensional s t r u c t u r e , i n s u l i n has s t i l l

not l o s t

its

f a s c i n a t i o n . The d r i v i n g force for the continued e f f o r t s to study t h i s hormone l i e s mainly in the s t i l l

unsolved problem of diabetes, which ranks

among the most wide-spread diseases of our time. In addition i n s u l i n continues to play an important part in many f i e l d s of science, and often takes a leading role as a model or even nearly a symbol. Undoubtedly t h i s molecule can be regarded as a f i n e example of i n t e r d i s c i p l i n a r y

coopera-

tion and the i n t e r r e l a t i o n between basic and applied research. This again became impressively apparent during the 2nd International I n s u l i n Symposium in Aachen. The f i r s t Symposium had been held in 1973 on the occasion of the 10th anniversary of the Aachen i n s u l i n s y n t h e s i s . Both Symposia were linked to a Congress of the International Diabetes Federation ( B r u s s e l s and Vienna sis fell

r e s p . ) . Whilst in those large meetings empha-

rather on the manifold aspects of the d i s e a s e , the Aachen Sym-

posia were designed to concentrate on the hormone. In 1973 i n s u l i n chemistry was treated s u c c e s s f u l l y in a s p e c i a l i z e d meeting which a l s o managed to present some of the various connections with other d i s c i p l i n e s . Our proposal to i n t e n s i f y and adequately complement these r e l a t i o n s in a Second Symposium had an extremely p o s i t i v e echo. We were therefore del i g h t e d to welcome to Aachen about 180 colleagues a c t i v e l y working in the f i e l d s of chemistry, biochemistry, b i o l o g y , c r y s t a l l o g r a p h y , immunology and medicine in 16 countries. Their 35 lectures and 61 poster present a t i o n s make up

the body of t h i s book. Since a l l of the communications

contained new experimental r e s u l t s , these Proceedings present an up-todate state of the a r t . Despite previous good i n t e n t i o n s , i t proved imposs i b l e to paraphrase the l i v e l y d i s c u s s i o n s throughout the Symposium adequately within t h i s framework; they properly deserve an edition of t h e i r own. "Wir s o l l t e n wieder ein Insulinsymposium machen"; with these words, and a promise of a s s i s t a n c e from a l l members of the Deutsches Wollforschungs-

X institut, Professor Zahn set this Symposium in motion late in 1977. We wish to thank him for his encouragement and help. Indeed, without the active involvement of everybody at the Institute, especially in the Insulin Division, the Symposium would have never materialized. We thank all

concerned.

We most gratefully appreciate the co-operation of our colleagues on the Symposium and the Programme Committees, and the Chairmen of the Sessions. Our sincere thanks are due to our colleagues from all over the world, whose participation and contributions made the Symposium a success. Once again we would like to gratefully acknowledge the generous support by the institutions and individuals listed above. We wish to express our warm thanks to our Symposium Secretary Mrs. Pamela Kaplirf, and also to Dr. B.-E. Rudolph and Dr. D. Saunders for their invaluable help in the preparation of this volume. Lastly, we are indebted to Walter de Gruyter & Co. for agreeing to publish the Proceedings, and to Dr. Weber for overseeing its production. Aachen, January

1980.

Dietrich Brandenburg Axel

Wollmer

CONTENTS

1

INTRODUCTION

SECTION I.

STRUCTURE OF INSULIN

A Comparison Between the Insulin Molecules in 2-Zinc and 4-Zinc Insulin Crystals E.J. Dodson, G.G. Dodson, C.D. Reynolds and D. Vallely . . Crystal Structure, Aggregation and Biological Potency of Beef Insulin Cross-Linked at A1 and B29 by Diaminosuberic Acid G.G. Dodson, S. Cut field, E. Hoenjet, A. Wollmer and D. Brandenburg

9

17

Correlation of Structural Details of Insulin in the Crystal and in Solution A. Vollmer, W. Straßburger, E. Eoenjet, U. Glatter, J. Fleischhauer, D.A. Meroola, R.A.G. de Graaf, E.J. Dodson, G.G. Dodson, D.G. Smith, D. Brandenburg and W. Danho . . . 27

SECTION II.

PEPTIDE SYNTHESIS

An Assessment of Protecting Group Strategies in Synthesis of Large Peptides J. Meienhofer

39

Fully Active Semisynthetic Insulin by Selective Formation of the Disulfide Bridges as an Intermediate Result of the Total Synthesis by Fragment Condensation on Polymer Phase C. Birr, R. Pipkorn, E.-G. Gattner, R. Renner and H.-U. Earing

51

[A14-Phenylalanine-Insulin]: A Synthetic Analogue W. Danho, A. Sasaki, E. Büllesbach, E.-G. Gattner, A. Wollmer

59

[B25-Tyrosine]B-Chain-S-Sulfonate of Porcine Insulin R. Knorr, W. Danho, E. Büllesbach, E.-G. Gattner and E. Zahn

67

The Synthesis of [B25-4-Nitrophenylalanine]-Octapeptide B(23-30) of Porcine Insulin M. Casaretto, W. Danho, E.-G. Gattner and H. Zahn

73

XII A Novel Route for the Preparation of Mono-Iodotyrosine and Mono-Iodotyrosine-Containing Peptides by Electrochemical Reduction of Di-Iodo Derivatives H.-G. Gattner and W. Danho

77

Alternative Approach for Human Proinsulin Synthesis N. Yanaihara, M. Sakagami and C. Yanaihara

81

An Alternative Synthesis of Glucagon by the Method of Solid Phase Condensation of Peptide Fragments Y-T. Rung, D-Y. Zhu, X-D. Qiu, X-W. Yuan, L-T. Ke and W. Wei

91

SECTION III.

SEMISYNTHESIS AND CHEMICAL MODIFICATION

Progress Towards the Semisynthesis of Preproinsulin V.K. Naithani, E.E. Büllesbach, H. Zahn, J. Shield, R. Chance and U.A. Root

99

Specific Activation of the Arginine Carboxyl Group of the B-Chain of Bovine Des-Octapeptide-(B23-30)-Insulin E. Canova-Davis and F.H. Carpenter

107

Enzyme-Catalyzed Semisynthesis with Insulin Derivatives H.-G. Gattner, W. Danho and V.K. Naithani

117

The Relative Reactivity of Insulin Amino Groups as an Indicator of Structural Accessibility and its Use for Synthetic Approaches for Structure-Function Studies Ü.-J. Friesen

125

A14-(N-Methylpyridiniüm) and A14-(2-Nitro-4-Trimethylammoniophenyl) Derivatives of Bovine Insulin S.E. Drewes, B.R.D. Easter, H.M. Robinson and H.E.M. Magojo

135

Preparation of Covalent Insulin Dimers A. Schüttler and D. Brandenburg

143

Reactions of Insulin Derivatives with ETAC Reagents R.G. Lawton

151

SECTION IV.

RADIOACTIVE LABELLING AND SEPARATION TECHNIQUES

The Application of HPLC to the Analysis of RadioIodinated Tracers of Glucagon and Insulin J. Markussen and U.D. Larsen

161

Monoiodoinsulin Specifically Substituted in A14-Tyr or A1 9-Tyr B. Hansen, S. Linde, 0. Sonne and J. Gliemann

169

XIII Preparation of Homogeneous Insulin Tracers with Monoand Di-Iodo-Desaminotyrosines in Position B1 S. Bahrami and D. Brandenburg

177

Insulin and Proteins Labeled by Microwave Discharge Activation of Tritium H. Tschesche, W. Behr and R. Wick 14 3 Radioactive Labelling ( C, H) of Insulin and Insulin Derivatives by Reductive Methylation; Isolation of Homogeneous N, N-Di-Methylated Derivatives J. Uschkoreit, D. Brandenburg and H.-J. Friesen Quantitative Determination of Insulin by Gradient Elution HPLC M.E.F. Biemond, W.A. Sipman, J. Olivie

201

Some Preliminary Findings in Isotachophoretic Analyses of Insulin A. Baldesten

207

Studies on the Denaturation of Dissolved Insulin H. Thurow

215

SECTION V.

185

191

RECEPTORS AND HORMONE-RECEPTOR INTERACTION

125

I-SpHPP-Avidin: A Tool for Hormone Receptor Studies F.M. Finn and K. Hofmann

225

Chromatographic Properties of the Adipocyte Transport System in Detergent Solution and its Resolution from the Insulin Receptor M.P. Czech, C. Carter-Su, and P.F. Pilch

233

Hydrophobic Interaction Chromatography as a Tool in Insulin Receptor Purification L. Kuehn, H. Meyer and H. Reinauer

243

Localization of Insulin-Receptor Complexes on the Surface of Whole Cells by Autoradiography E. Junger, C. Giesen, M. Semmler, H. Reinauer and L. Bachmann

251

Insulin Receptors in the Heart Muscle: Negative Cooperativity Binding Model J. Eckel and H. Reinauer

259

Evidence for the

Insulin Receptors, Insulin Degradation, and Biological Activity 0. Sonne and J. Gliemann

263

Regulation of Insulin Receptors in Isolated Hepatocytes from Normal and Streptozotocin-Diabetic Rats in the Fed and 48 H Fasted State S. Gammeltoft, L.0. Kristensen, M. Folke and L.Sestoft . . 271

XIV Insulin-Receptor Turnover and Down Regulation S. Terris and D.F. Steiner

277

Receptor-Mediated Internalisation of Insulin, Glucagon and Growth Hormone in Intact Rat Liver. A Biochemical Study B. Desbuquois and M.-C. Postel-Vinay

285

Kinetics of Insulin Binding to Its Receptors in vivo. Effects of Fasting. J.C. Sodoyez, F. Sodoyez-Goffaux and Y.M. Thiry-Moris.

. . 293

Hystricomorph Insulins and Insulin Receptors N.R. Lazarus, K. O'Connor, R.W.J. Neville, P. Goodwin, R. Horuk and D. Stone

SECTION VI.

301

PHOTO-INDUCED HORMONE-RECEPTOR COUPLING

Photoreactive Insulin Derivatives: Preparation and Characterization P. Thamm, D. Saunders and D. Brandenburg

309'

Photoreactive Insulin Derivatives: Chemical and Physical Properties D. Saunders, P. Thamm and D. Brandenburg

317

The Preparation and Application of an A1-Substituted Photo-Reactive Insulin Analogue A.R. Rees and M.R. Whittle

327

Photoaffinity Labeling of Insulin Receptor Proteins of Liver Plasma Membranes C.C. Yip, C.W.-T. Yeung and M.L. Moule

337

Characterisation of Insulin Binding Sites in Rat Liver Plasma Membranes Using Photoreactive Insulin Analogues M.H. Wisher, P. Thamm, D. Saunders, P.H. Sonksen and D. Brandenburg

345

Permanent Activation of Lipogenesis by Insulin Covalently Bound to Adipocyte Receptors C. Diaaonesou, P. Thamm, D.J. Saunders and D. Brandenburg. 353

SECTION VII.

STRUCTURE, BINDING, ACTIVITY

The Activation of Adenylate Cyclase by Chemically Modified Forms of Glucagon and its Relationship to Receptor Binding R.M. Epand

363

Structure-Activity Relationships of Insulin-Induced Negative Cooperativity Among Receptor Sites M. Piron, M. Michiels-Place, M. Waelbroeok, P. Ve Meyts, A. Schüttler and D. Brandenburg

371

XV Properties of a Mutant Insulin Species Causing Human Diabetes J.M. Olefsky, M. Saekow, M. Kobayashi, O.G. Kolterman, H. Tager, B. Given, D. Baldwin, M. Mako, J. Markese, A.M. Hubenstein and H. Poucher

393

A1-Modified Insulins: Receptor Binding and Biological Activity P. Rösen, M. Simon, H. Reinauer, D.Brandenburg, Ü.-J. Friesen and C. Diaconescu. . . .

403

Biological Activity of Insulin Analogues Substituted at the Amino Group of B1-Phenylalanine R. Geiger, R. Obermeier, V. Teetz, R. Uhmann, H.D. Summ, H. Neubauer, K. Geisen and G. Regitz

409

Functional Role of the N-Terminal Region of the B-Chain of Insulin C. W.-T. Yeung, M.L. Moule and C.C. Sip

417

Biological Properties of Covalent Insulin Dimers K.P. Willey, M.A. Tatnell, R.H. Jones, A. Schüttler and D. Brandenburg

425

Biological Activity and Receptor Binding of Six Different Covalent Dimers of Insulin K. Schlüter, K.-G. Petersen, A. Schüttler, D. Brandenburg and L . Kerp 4 33 In vitro and in vivo Effects of Poly-N-VinylpyrrolidoneInsulin H.J. Ko lb, R. Renner, B.U. von Specht and K.D. Hepp. . . .439 Study of Insulin Action by Use of Insulin-Dextran Complex: Uptake of Insulin into Subcellular Fractions and Stimulation of Pyruvate Dehydrogenase in Mouse Adipocytes Y. Sakamoto and T. Kuzuya 447 Studies on the Mechanism of Insulin Action VI: The Interaction of Insulin Fragments with Insulin Receptor Y-M.Feng, J-L. Gu, X-T. Zhang, Z-X. Lu, W-J.Xu, J-H Zhu.

.455

Stimulation of Intracellular 1^02 Production in Rat Epididymal Adipocytes by Insulin, Insulin Fragments, and Other Hormones and Growth Factors with Insulin-Like Activities C. de Haën, D.B. Muchmore and S.A. Little

461

Early Events in Insulin Binding and Insulin Action in Fat Cells and Hepatocytes H. Häring, D.G. Brocks, E.A. Siess and W. Kemmler 469 Role of the Biodegradation Process in the Action of Insulin. A Proposal for a Model Consisting of SulfhydrylDisulfide Interchange Reaction and Proteolysis P.T. Varandani 475

XVI Insulin Receptors, Receptor Antibodies and the Mechanism of Insulin Action C.R. Kahn, C. Grunfeld, G.L. King, E. Van Obberghen and K.L. Baird

DEGRADATION 3 Studies on Uptake of [ H]-Insulin by Erythrocytes W. Behr and H. Tsohesahe

483

SECTION VIII.

An Insulin-Degrading Enzyme from Human Erythrocytes: Isolation and Characterization H.J. Kolb and E. Standi 125 3 Comparison of I-Insulin and [B1- H-Phenylalanine]Insulin Degradation by Insulin Protease W.C. Duckworth and P.A. Ualban

495 501

509

The Chemical Characterization of the Products of the Processing of Subcutaneously Injected Insulin J.G. Davies, R.E. Offord, P.A. Halban and M. Berger. . . . 517 Some Inhibitors of Insulin Degradation R.E. Offord, J. Philippe, S.M. Hoare, P.A. Halban and M. Berger Excretion of Endogenous and Exogenous Insulin by Kidney in Diabetic Ketoacidosis H.S. Sacks, F.B. Stenz and A.E. Kitabohi

531

Incubation of Insulin in Hyperglycemic Blood Generates Insulin Dimers T.R. Csorba, M.M. Cannon, M. Ungar and N.S. Track

539

SECTION IX.

525

IMMUNOLOGY

Genetic Regulation of the Immune Response of Mice to Insulins of Different Species K. Keek and M. Momayezi

551

Genetic Control of the Immune Response to Insulin A.S. Rosenthal, J.W. Thomas, J. Schroer, W. Danho, E. Büllesbach and J. Föhles

559

Analysis of the Immunogenicity of Three InsulinPreparations U. Kiesel, F.K. Jansen, D. Brandenburg and B.-G. Gattner. Studies on the Immunological Properties of Natural and Modified Insulins H.P. Neubauer and H.H. Schöne

. 569

575

Genetic Control of the Immune Response to Insulin: A Clinical Study of Adverse Immunologic Reactions to Insulin A.S. Rosenthal, J. Galloway

J.T.

Blake,

C.R. Kahn,

D. Mann

and

•

.

585

The Induction of Hyperglycemia with Insulin Antibodies to B-Chain Determinants E.R. Arquilla,

J.M.

Kelso,

I.Y.

Tamai

and M.D.

Roth.

. . . 593

The Pattern of B-Lymphocyte Specificities for Modified and Unmodified Insulins in the Mouse U. Kiesel

and Hj-G. Gattner

Specificity of Rabbit Anti-Insulin Antibody

K. Keck, K. Jäger, H.-G. Gattner

R. Geiger,

D. Brandenburg

and

603

611

Radioimmunoassay of Chemically-Modified Insulins Use and Misuse

J.H. Thomas, D.I. D. Brandenburg

Dron,

R.Ü. Jones,

P.H.

Sönksen

and

Purity of Chromatographycally Purified Insulins Measured by Skin-Reactivity and Insulin-Specific IGE H.-G.

Velaovsky,

SECTION X.

E. Mäser

D.V.

Primary Structure of the Messenger

Goeddel,

637

A. Gray and I. Sures

Quantification of Proinsulin Biosynthesis by Determination of Specific Radioactivity of [^H]-Leucine in Pancreatic Islets

S. Schmidt,

D. Schröder,

H. Jahr

and H. Zühlke

Immunoreactive Rat C-Peptides I and II in Glucose Perfusate of Isolated Pancreas

C. Yanaihara, T. Keneko

J. Ozaki,

N. Nishida,

N. Yanaihara

and

Biosynthesis of Somatostatin in Pancreatic Islets of Wistar Rats S. Schmidt,

H. Zühlke,

D. Schröder,

M. Ziegler

and H. Jahr

Crystallography of Intracellular Insulin and Glucagon as Revealed by Investigations of Tissues and Models

R.ü.

Lange

Pancreatic Hormone Storage Granules: Ions and Polypeptide Oligomers

J.E. T.L.

627

BIOSYNTHESIS, STORAGE, EVOLUTION

Human Preproinsulin: RNA

A. Ullrich,

and K. Federlin

619

Pitts, S.P. Blundell

Wood,

R. Horuk,

643

651

659

665

The Role of Metal

S. Bedarkav

and

673

XVIII On the Molecular Biology of Hagfish Insulin S.O. Emdin and S. Falkmer

SECTION XI.

683

INSULIN-RELATED HORMONES

Possible Relationships in the Processing, Storage and Secretion of Some Insulin^Related Peptides G.G. Dodson, N. Isaaas, R.A. Bradshaw and H.D. Hi all . . . 695 Insulin-Like Growth Factors R.E. Humbel, R. Andres, C. Ernest, G. Haselbacher, E. Rinderknecht, E.R. Froesch, J. Schwander, H. Walter and J. Zapf

703

Relaxin and Relaxin-Like Peptides in Ovaries of Pregnant Sows and Sharks C. Schwabe

709

Structural Studies on Porcine Relaxins and their Biosynthetic Precursors H.D. Niall, M. John, R. James, S. Kwok, R. Mercado, G. Bryant-Greenwood, R.A. Bradshaw, M. Gast and I. Boime

. 719

PRESENTATIONS NOT INCLUDED IN THIS VOLUME

72 7

LIST OF PARTICIPANTS

729

ABBREVIATIONS

735

SUBJECT INDEX AUTHOR INDEX

739 749

INTRODUCTION

The complex network of information from a multidisciplinary meeting on so complex a subject as insulin cannot be presented in the linear course of a book without compromises.

It was

decided to organize the wealth of material in 11 sections.

In

Sections I - IV the hormone is the central subject, with the main emphasis on its chemistry.

In Sections V - VII the re-

ceptor comes into play, and attention is focussed on the nature and effect of its interaction with the hormone. Section VIII deals with receptor-unrelated insulin degradation.

Insu-

lin immunology forms the relatively self-consistent Section IX.

Section X is the last on pancreatic hormones, and relates

to different aspects of biosynthesis.

The final section re-

flects the present state of research on insulin-like growth factors and relaxin. Section I Insulin is one of the few proteins for which the crystal structure has been refined to the present limits of perfection. Furthermore, X-ray analyses of so many crystal forms, species variants, natural and synthetic homologues, are not available for any other protein.

The existence of insulin in different

crystal states suggests that a dynamic component is necessary to the understanding of this molecule.

This very aspect also

emerges when the compatibility of crystal and solution structure is tested with quantum chemical approximations.

Confor-

mational rearrangements may well be part of the insulin/receptor interaction. Section II The total synthesis 0'f large molecules like proinsulin remains, in spite of all advances in synthesis and purification techniques, a difficult and often extremely cumbersome task, and the two-chain hormone insulin still is an undiminished challenge for peptide chemists.

A key role has to be attributed

to protecting groups, and current strategies and ideas on

2 their organisation are reviewed.

With respect to the prepar-

ation of homogeneous insulins, peptide synthesis in solution has been the method of choice since 1963.

Its highlight was

the elegant total synthesis with unequivocal formation of the disulfide bonds by Rittel and his colleagues in 1974.

The

syntheses reported at the Symposium followed the strategy of building up one chain and combining it with the natural partner chain.

Fragment condensation in solution was used to syn-

thesize modified A- and B-chains, and also to assemble the whole sequence of human proinsulin.

Peptides with unnatural

amino acids, such as nitrophenylalanine, as a photo-activatable group (see also S.VI) and mono-iodo-tyrosine for the synthesis of defined iodo-insulins (see also S. IV),have been prepared. Advances in Merrifield synthesis are reported. Modified procedures involving fragment condensation on a solid phase yielded an insulin A-chain with differential thiol protection and glucagon. Section

III

Semisynthesis and chemical modification of insulin have reached a high level of expertise (see also S. IV,VI,VII).

These

techniques have made possible the unequivocal synthesis of photochemically-labelled insulin derivatives (see S.VI) as well as a number of analogues and derivatives (see S.VII) including radioactively-labelled insulins (S.IV).

The semi-

synthesis of preproinsulin extends this approach to the largest molecule dealt with in this Symposium.

Carboxyl pro-

tection is the main problem in semisynthetic modification of the C-terminus of the B-chain. help to overcome obstacles.

Enzymatic peptide coupling may

Methodological investigations are

described as well as the special case of trypsin-catalyzed couplings to prepare analogues, and human from porcine insulin. However the method seems not to be generally applicable. Since most insulin modifications are based on reactions at the amino groups, their relative reactivity is discussed in detail.

The

small number of defined tyrosyl derivatives has been increased. The preparation of insulin dimer's with crosslinks between

3 amino groups has now been systematically explored (see also S. VII) and insulin has also been used as a model for crosslinking with alkylating reagents. Section

IV

The reliability of receptor binding studies and many biological and medical investigations depends on well-characterized tracer hormones. However the tracer properties of many iodinated insulins have been questioned. The distribution of iodine after labelling of insulin and glucagon can be rapidly analyzed by high pressure liquid chromatography. A14- and A19monoiodoinsulin of high specific activity have been separated and further characterized (see also S. V).

Other procedures

14

to label insulin with iodine, tritium and

C are being tested.

In the second part of this section the applications of HPLC and isotachoelectrophoresis to insulin are investigated, as well as solubility problems encountered in insulin administration by the artificial pancreas. Section

V

The 11 papers in Section V range from biochemical studies on receptor purification to in vivo binding, and some evolutionary aspects (see also S. XI).

Avidin has been chemically

modified and iodinated and shown to maintain its affinity for receptor-bound, biotin-labelled hormones.

The insulin recept-

or and D-glucose transport system of adipocytes were demonstrated to be distinct membrane components.

Four investi-

gations deal with receptor distribution, the characterization of the heart-muscle receptor, differences in binding and degradation by target and non-target cells, and receptor regulation in experimental diabetes.

It appears that a signal

different to insulin binding is necessary for modulation of receptor numbers.

After administration of insulin, glucagon

and growth hormone in rats it was shown that the hormones are rapidly bound and internalized.

A discussion on the proper-

ties of the receptors as well as the insulins of hystricomorph rodents concludes this chapter and also relates it to S. VII.

4-

Section

VI

The promising technique of photochemical labelling is advancing rapidly. The Symposium provided the first platform to discuss the work pertinent to insulin. The chemical preparation of a series of homogeneous photo-insulins with the label in either position A1,B1,B2 or B29, or in two of these positions, was described as well as their chemical, physical and biological characterization. Specific covalent binding to liver cell receptors was demonstrated for some of these derivatives. Analysis of the covalent insulin/receptor complex yields a molecular weight of 300,000 which, upon reduction, drops to 130,000. Initial reports on the covalent binding of photo-insulins to living fat cells show that permanent fixation generates a continuous signal. Even from the relatively sparse data available at present it can be predicted that the photo-insulins will be very important in defining and investigating the central steps of insulin action, and clarifying the process of internalization. Section

VII

In this central part of the Proceedings hormone chemistry is linked to receptor binding and subsequent effects. Structurefunction studies with glucagon substantiate the pronounced differences which can occur between the capacity of the hormone to bind to the receptor and to trigger a biological response. Previously, comparable differences in insulin binding and response have only been observed with hagfish insulin. New examples are covalently crosslinked insulin dimers (see S.III) and some A1-substituted derivatives. The most exciting case is an abnormal human insulin recently identified in a patient with severe endogenous hyperinsulinaemia. The replacement of B24- or 25-phenylalanine by leucine produces an insulin which not only is devoid of biological activity but also inhibits the action of normal insulin. These findings underline the importance of systematic structure/function studies and provide a strong stimulus for an approach started more than a decade ago • Modifica.ti.ons of ths N—tGirminus of th.s B — chain

5 show that this region deserves more detailed study.

This is

in agreement with the results of photo-affinity labelling (S. VI), but in contrast to the irrelevance of B1-phenylalanine to biological activity.

Insulin crosslinked either to itself

or to soluble polymers yielded a complementary group of derivatives particularly suitable for the exploration of receptor topology in various cells, site-site interaction or negative cooperativity, as well as internalization and stimulation of mitochondrial pyruvate dehydrogenase.

Further points in the

discussion about insulin action were whether

H J O J

is a poten-

tial second messenger, and whether cleavage by glutathioneinsulin-transhydrogenase is a necessary step for action.

The

insulin-like activity of some anti-(insulin-receptor)-antibodies indicates it is not.

Experimental evidence for the

occurrence of internalization was presented, but none for its relevance to the mechanism of insulin action. Section

VIII

The interest in degradation is obvious from its pharmacokinetic importance.

Tracer-dependent differences in degra-

dation were reported for an insulin protease isolated from muscle.

The insulin degrading enzyme of erythrocytes has now

been characterized chemically and appears to be a sulfhydryldependent proteinase.

Tritiated insulin was used to follow

degradation in vitro and in vivo.

The degradation of subcut-

aneously administered insulin can efficiently be depressed by inhibitors such as Trasylol® or ophthalmic acid. Section

IX

The availability of numerous species-variants, modified insulins and partial synthetic sequences make insulin an ideal molecule for investigating a number of basic immunological questions.

Special emphasis was laid on the genetic regulation

of the immune response to insulin which was studied in animals and in man. Immune response genes in the major histocompatability locus are responsible for the recognition of carrier determinants on the insulin molecule. Evidence was presented that

6 these genes also determine the response of isolated macrophages.

Immunogenicity and antigenicity were tested with a

large variety of modified insulins.

Allotypic antibody popu-

lations were not observed. Section

X

The papers on insulin biosynthesis range from its quantification by means of chemical and immunological techniques to the elucidation of the amino acid sequence of human preproV

insulin via the primary structure of the corresponding messenger RNA.

A high molecular weight precursor was also

postulated for somatostatin.

Electron microscope, X-ray dif-

fraction and model building studies partially explain the structure of the storage form of the pancreatic hormones.

An

investigation of the secretion and metabolic effect of insulin in hagfish suggests that the regulatory mechanisms of insulin have undergone only minor quantitative variations during the 500 million years of vertebrate evolution. Section

XI

Both with respect to structure-function relationship, and under the aspect of evolution, the insulin-like growth factors and relaxin have steadily gained in interest and were now studied in considerable detail. IGF I was shown to be a true somatomedin.

Fractionation of relaxin preparations

indicated a certain heterogeneity.

This may in part be due to

non-uniform proteolytic conversion of the precursor, the existence of which has now been established.

SECTION I STRUCTURE OF INSULIN

A COMPARISON BETWEEN THE INSULIN MOLECULES IN 2-ZINC AND 4-ZINC INSULIN CRYSTALS

E. J. Dodson, G. G. Dodson, C. D. Reynolds and D. Vallely, Department of Chemistry, University of York, Heslington, York, YOl 5DD, England.

Introduction Recent work on the refinement of the crystal structures of 2-zinc and 4-zinc pig insulins (1,2) using high resolution data (to 1.58 spacing) by Fourier and fast Fourier leastsquares methods allows us to compare in detail the independent molecules present in the different crystals and their interactions in the monomer and in their various aggregation states. Careful comparisons between the four independent molecules in 2- and 4-zinc insulin reveals some parts of the molecule are very well defined. The differences in structure that occur between the two independent molecules in 2-zinc insulin are not the same as those found in 4-zinc insulin crystals nor do they appear to explain the nature of the structural change between the two crystal forms. In both the 2-zinc and 4-zinc insulin rhombohedral crystals the insulin molecule is present as a hexamer assembled from three identical dimers. Each dimer is related by the threefold rotation axis in the crystal. The dimers in both 2-zinc and 4-zinc insulins are formed from crystallographically independent molecules (I(2Zn), II(2Zn) from 2-zinc insulin and I(4Zn), II(4Zn) from 4-zinc insulin respectively) and are related by a local 2-fold axis. In Figure 1 ,the polypeptide

© 1980 Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

10

backbone for each molecule is shown viewed down,the 2-fold axis, (a is 2 zn and b is 4 zn insulin). Molecule^ r.(2zn) and II(2zn) in the 2-zinc pig insulin dimer show distinct differences in conformation at the N terminus of the A chain and in the arrangement of the B5 histidine and B25 phenylalanine side-chains.

Molecules I(4zn) and II(4zn)

of the 4-zinc insulin dimer exhibit much greater differences in conformation.

There is a major rearrangement of the

first eight residues of the B-chain of molecule I(4zn), the B5 histidine is moved (by about

158) close to the BIO

residue of a neighbouring molecule.

In addition the A-chain

of molecule I(4zn) has shifted away from the B-chain in response to the movement of the A7-B7 disulphide bond. cm ^k ÍC~r\

Figure 1

The Zinc Co-ordination. In 2-zinc and 4-zinc insulins each hexamer is co-ordinated to two and four zinc ions respectively as shown in Fig. 2. There are two zinc ions on the three-fold axis in 2-zinc insulin both of which are octahedrally co-ordinated.

The upper ion

is bonded to three symmetry-related BIO histidine residues from molecule I and the lower ion is attached to three BIO histidine residues from molecule II of each insulin dimer. In each case the remaining sites are occupied by water molecules.

11

Figure 2(a). 2 Zn Insulin Hexamer.

Figure 2(b). 4 Zn Insulin Hexamer

/

12 In the so called 4-zinc insulin we have refined at 1.58 resolution,there are disordered zinc sites in molecule I. Two zinc ions appear on the three-fold axis, one at full weight co-ordinated to molecule II BIO and one at half weight co-ordinated to molecule I BIO (itself disordered). The general zinc ion site is co-ordinated to B5 and a disordered BIO,both from molecule I. In all cases the co-ordination is completed by water molecules. The co-ordination geometry is octahedral for the lower zinc ion linked to molecule II, whereas for the upper zinc ion it appears to be tetrahedral at this stage of the refinement. The off axial zinc site has tetrahedral co-ordination geometry. The presence of two co-ordination geometries suggests that the metal ion co-ordination is not an essential feature of the 4zlnc insulin structure and that the transformation between the 2Zn and 4Zn crystalline forms is directed by other interactions.

Common Structural Features The B9-B19 a-helix is common to all of the independent insulin molecules and is a region in which the closest match in structure occurs (Table 1 and Fig. 4). There is also good agreement in the main-chain structure for the residues B23 to B26. The side-chains for the residues involved in dimer formation have similar conformations, apart from B25 of molecules I(2Zn) and I(4Zn) (Figure 3). In fact the conformation of the majority of the B-chain backbone (B9-B28) is similar in all four insulin molecules. In Figure 4 the B-chain of 2-zinc insulin, molecule II is shown overlapped onto the B chains of the other insulin molecules. Although there is no extensive and well-defined secondary structure in the A-chain, and some overall movement relative to the B-chain, there is generally similar folding- of the polypeptide backbone

13 F i g u r e 3.

O v e r l a p of d i m e r f o r m i n g r e s i d u e s f r o m 2 Zn

insulin, molecule 2 Zn I n s u l i n Molecule I

F i g u r e 4. molecule

II on: 4 Zn I n s u l i n M o l e c u l e II

4 Zn I n s u l i n Molecule I

O v e r l a p of B chain b a c k b o n e for 2 Zn

insulin,

II on:

2 Zn I n s u l i n Molecule I

4 Zn I n s u l i n M o l e c u l e II

F i g u r e 5. O v e r l a p of A c h a i n b a c k b o n e m o l e c u l e II on: 2 Zn I n s u l i n Molecule I

4 Zn I n s u l i n M o l e c u l e II

4 Zn I n s u l i n Molecule I

for 2 Zn

insulin,

4 Zn I n s u l i n Molecule I

14 in the r e g i o n A 7 - A 2 1

for all t h e i n s u l i n m o l e c u l e s

(shown

in

F i g u r e 5. ) .

Structural

Differences

The two i n s u l i n m o l e c u l e s

in t h e 2 - z i n c d i m e r are r e l a t e d b y

a local or n o n - c r y s t a l l o g r a p h i c conformational

t w o - f o l d axis.

differences between molecules

The major

I(2Zn)

I I ( 2 Z n ) o c c u r at the N - t e r m i n u s of the A - c h a i n

and

involving

r e s i d u e s A 1 - A 6 a n d in the a r r a n g e m e n t of the B5 h i s t i d i n e B 2 5 p h e n y l a l a n i n e s i d e - c h a i n s as i l l u s t r a t e d in Fig. 6.

and The

a l t e r e d s t r u c t u r e at the A - c h a i n N - t e r m i n u s is due to a r o t a t i o n a b o u t the b o n d b e t w e e n r e s i d u e s A5 and A6 of approximately

30°.

The B5 histidines interact closely

with

the h e l i c a l r e g i o n A 1 - A 8 a n d m a y p l a y an i m p o r t a n t r o l e mediating these conformational

changes

T h e r e s i d u e s i n v o l v e d in d i m e r f o r m a t i o n are c l o s e l y by the local t w o - f o l d a x i s - apart f r o m the B 2 5 of m o l e c u l e

in

(3). related

phenylalanine

I(2Zn) w h i c h l i e s a c r o s s the r o t a t i o n axis.

d i m e r f o r m i n g r e s i d u e s in 4 - z i n c i n s u l i n are a l s o

The

closely

r e l a t e d by the local t w o - f o l d axis a n d s h o w s i m i l a r

structural

f e a t u r e s to t h e c o r r e s p o n d i n g r e s i d u e s in 2 - z i n c i n s u l i n shown in F i g u r e 3. molecule

The phenylalanine

s i d e - c h a i n at B 2 5

I(4Zn) t a k e s up a s i m i l a r c o n f o r m a t i o n

f o u n d in m o l e c u l e

to that

I(2Zn).

H o w e v e r in the 4 - z i n c p i g i n s u l i n d i m e r as a w h o l e the two-fold symmetry

as in

local

is m u c h less c o m p l e t e s i n c e in m o l e c u l e

I(4Zn) there is t h e m a j o r r e a r r a n g e m e n t

of the B - c h a i n

N - t e r m i n u s r e s i d u e s B 1 - B 8 a n d a m o v e m e n t of the A - c h a i n from the B - c h a i n (2).

In 4 - z i n c i n s u l i n , m o l e c u l e

I,

away

the

r e s i d u e s B1 to B 8 h a v e a h e l i c a l c o n f o r m a t i o n a n d f o r m a c o n t i n u a t i o n of t h e B 9 - B 1 9 ct-helix (2). the B - c h a i n in b o t h 2 Z n a n d 4 Z n i n s u l i n

At the C - t e r m i n u s (molecule

II) B 3 0

of

TABLE I

R.M.S. Differences for Malnchain Atom« of Various Inaulln Molecules After Optimlaiag by a LeastSquares Procedure to Holecule II(2Zn).

Molecule IK2ZS)

I(aZn)

1I( 4Za)

I( 4ZD )

A1-A21

0.00

0.80

0.49

0.89

A7-A21

0.00

0.54

0.48

0.66

B1-B30

0.00

0.81

0.65

B1-B28

0.00

0.27

0.67

B9-B30

0.00

0.96

0.25

B9-B28

0.00

0.19

0.25

0.47

B9-B19

O.OO

0.11

0.18

0.29

Figure

3.60*

6

J 2 Zn

J

Insulin

Xi^

cr

^ *x ^ P

Molecule

II

Molecu le

Molecule

II

Molecule

4 Zn Insulin

16

turns back and forms a H-bond to B27 threonine in a $ turn type structure. In contrast the C-terminal residues B29 and B30 in molecule I(2Zn) have an extended conformation, the B29 lysine forms close contacts with A4 glutamic acid and with the carbonyl oxygens of B29 and B30 of molecule II(2Zn). B30 alanine only makes contacts with solvent structure. In 4-zinc insulin molecule I the residues B29 lysine and B30 alanine appear even more disordered than in 2-zinc insulin, molecule I and we do not have a satisfactory description of the contacts at present.

Conclusion The remarkable changes in the insulin molecule I structure and, in contrast, the absence of any significant structural changes in molecule II raises some fascinating questions. We do not for example fully understand what constraints stabilise molecule II nor do we know how these structures relate to the monomer in solution and the biologically active species.

References (1)

Blundell, T. L., Cutfield, J. F. , Cutfield, S. M., Dodson, E. J., Dodson, G. G., Hodgkin, Di C., Mercola, D. A. and Vijayan, M. (1971) Nature, 231, 506-511.

(2)

Bentley, G., Dodson, E. J., Dodson, G. G., Hodgkin, D.C. and Mercola, D. A. (1976) Nature, 261, 166-168.

(3)

Dodson, E. J., Dodson, G. G., Hodgkin, D. C. and Reynolds, C. D. (1979) Can. J. Biochem., 57, 469-479.

CRISTAL S T R U C T U R E , A G G R E G A T I O N A N D B I O L O G I C A L P O T E N C Y I N S U L I N C R O S S - L I N K E D AT A1 A N D B29 BY D I A M I N O S U B E R I C

G.G. D o d s o n a n d S.

Cutfield

D e p a r t m e n t o f C h e m i s t r y , U n i v e r s i t y of Y o r k , E. H o e n j e t a n d A.

OF BEEF ACID

U.K.

Wollmer

F a c h g e b i e t S t r u k t u r u n d F u n k t i o n der P r o t e i n e , A b t e i l u n g P h y s i o l o g i s c h e C h e m i e , RWTH, D-5100 Aachen, Federal Republic of Germany D.

Brandenburg

Deutsches Wollforschungsinstitut D - 5 1 0 0 A a c h e n , F e d e r a l R e p u b l i c of

Germany

Introduction The three-dimensional

s t r u c t u r e of the i n s u l i n

molecule

(1,2) h a s b e e n r e l a t e d to the b i o l o g i c a l p o t e n c y of natural and modified insulins.

This evidence has

different

suggested

the e x i s t e n c e of an a c t i v e s u r f a c e r e g i o n r e s p o n s i b l e h o r m o n e ' s b i n d i n g at the cell w a l l a n d for the e v e n t s that l e a d t o

for the

subsequent

'.nsulin's m e t a b o l i c e f f e c t s (3, 4 ).

There are a number < E difficulties with the inferences on r e d u c e d a c t i v i t y

in the m o d i f i e d h o r m o n e .

is that a c h e m i c a l or

One of

based

these

ructural difference can affect

the

h o r m o n e ' s o v e r a l l s t r u c t u r e a n d p o s s i b l y a l t e r the s u r f a c e some d i s t a n c e from the m o d i f i c a t i o n .

If a r e d u c t i o n

p o t e n c y is to be i d e n t i f i e d w i t h a p a r t i c u l a r it is t h e r e f o r e e s s e n t i a l to e s t a b l i s h that the s t r u c t u r e of the m o l e c u l e

at

of

modification,

(as far as

is o t h e r w i s e

possible)

unaltered.

Spectral measurements, especially circular dichroism, b e e n u s e d to a s s e s s s t r u c t u r a l a l t e r a t i o n s , b u t s u c h

© 1980 Walter de Gruyter & Co., Berlin • N e w York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

have evidence

18

is limited by the difficulty in relating spectral differences to specific structural changes (5). Most of the evidence suggests insulin circulates in the body and produces its biological effects as a monomer.

Any change

in the molecule's structure that reduces its potency however may be disguised in the crystal through the reassertion of the native structure when it aggregates and crystallises.

This

limits the value of crystal structure analysis in determining the relationship between structure and activity.

The

measurement of the aggregation constants provides a sensitive test of the monomer's structural integrity, since a large proportion of the molecule's surface is involved in dimer and hexamer formation. This paper is concerned with the aggregation, crystal structure and biological potency of beef insulin crosslinked at A^ & B^g by diamino-suberic acid (referred to as DAS-A^ - B 2g -insulin). This, and other crosslinked insulins, were prepared when the 8-loS separation of the A-^ a and Bgg e -amino groups was established by X-ray analysis (3).

The extended length of

the suberic acid chain is also about loS and it proves to be the shortest cross link that Can be made without seriously perturbing the molecule's crystal structure.

The cross link

is able to guide the correct folding of the molecule during reoxidation of the reduced disulphide bonds.

Addition of the

a-amino groups to the cross link leaves the cross linked insulin molecule with the same general charge distribution as in the native hormone.

Interestingly, DAS-insulin has a

circular dichroic spectrum even more like the native insulin than suberic acid cross linked insulin (6).

So far crystals

of cross linked insulin have only been obtained with DASinsulin.

The ability to form crystals suggests the cross

link hasnot seriously disturbed the hormone's folding.

19 The binding affinity of DAS insulin to the fat cell is, however, 5% that of native insulin; its biological potency is similarly reduced to 5% (7). The reduction in potency might be explained by alterations at the A chain N terminal glycine, which is a surface invariant residue with some sensitivity to chemical modifications. Alternatively, the steric effects of the diamino suberic acid crossbridge could perturb the B chain C terminal residues B 2 4 - B 2 6 have also been implicated in biological activity. Alteration in the structure of these residues would affect dimer formation (figure 1) (4).

Experimental Aggregation The equilibrium constants of both native and A^ - Bgg crosslinked beef insulin (Zn-free) and in the presence of Zn-ions has been measured by the column elution method. Table 1 lists the values obtained for the dimerization constant (Zn-free) together with some earlier values taken from the literature. The values for native and crosslinked insulin are close, implying that the structure of the dimerforming residues is essentially the same in the two insulins before and after dimerisation. In the presence of zinc-ions (2Zn/Hex), the dimerisation constant of the two insulins is somewhat lower. This is attributed to the very low increase of molecular weight in the highest concentration range between 1 - 5 mg/ml for native Zn-free insulin. To fit the data in the highest concentration range, the tetramerization and hexamerization constant must adopt too low a value, which in turn elevates the dimerisation constant. The variation in the higher aggregation constants for cross-

20 linked insulin can be explained.

In the crosslinked insulin

the linkage of the A chain N terminus presumably limits its flexibility.

Because the A chain N terminal residues

interact with the B chain N terminus at B5 histidine and at the disulphide A7 - B7, there may be some impairment of the B chain N terminal residues ability to assume the proper interactions (which are complex) for hexamer formation (Fig. 1).

The X-ray Analysis The A^ - Bgg crosslinked insulin crystal structure has been determined at 3.28 spacing. The crystals are rhombohedral and the phases determined by two isomorphous heavy atom derivatives. The crystals did not diffract well and the heavy atom substitution tended to reduce diffraction quality even further and this had led to the low figure of merit. (See Table 2 for crystallographic details.) Nonetheless, the electron density is often well defined and it is clear the folding and side chain conformation of the crosslinked beef insulin while not identical is very close to that of native pig insulin.

Results The electron density calculated with the isomorphous phases shows that the folding of the crosslinked molecule is essentially unchanged.

Figure 2 illustrates the electron

density for some of the dimer-forming residues and its match to the refined native 2 zinc insulin coordinates.

Because

the analysis extends only to 3.28 spacing, small differences in conformation, particularly of sidechains, will however not be detected.

Table I 2 Zn/Hexamer

Zn free Insulin

K

K

l,2 5

2.8 x 10

12'

1 5

1.8 x IO

1.7 x 10

5.00 x 10

1.3 x 103

5.5 x 103

2.2 x 105)a -5 ) 1.4 x 10 jb

Native

1.1 x 105)c 2.4 x 105

DAS Aj-Bjg

1.8 x 105

based on the 'pie model' of Axel Wolljiier a

.114 NaClj pH 8 (Pekar, A.H. and Frank, D.H. 1972, Biochemistry, 11, "»013-6.)

b

.1M NaCl; pH 8. (Goldman, J. I Carpenter, F.H. 1974, Biochemistry, 13, 1566-74.)

c

.1H NaCl; pH 2 (Jeffrey, P.D. I Coates, J.H., 1966, Biochemistry, 489-493.)

M=M D, = K,.H? »2

K,'. H3

T T, • K P

= K

H

. K

3

.K,,3.H6

U,'6.K6

. H * 2(D, + 0 2 ) + 3 T + 4(T, + Tj) + 5 P • 6 H + 12 D 0 • —

K^ K,' Kg

(M + 4(0, + Dj) • 9 T + 16

®

Fig. 5: Aspects of insulin association (the "pie model").

The association scheme in Fig. 5 was derived from the crystal structure which shows that the protomer has two potential contact areas and that contacts are possible only between corresponding regions. This scheme remains complicated enough to be analyzed from the concentration dependence of the average molecular weight even though it is narrowed down by the fact that one of the two contacts is energetically favoured. This makes association predominantly follow the steps dimers->-tetramers+hexamers, whereas odd intermediates are negligeable. The calculated CD of the single association products is obtained by deliberate restriction of the pairwise interactions to the species under consideration. This, of course, implies the assumption that the isolated intermediates in any detail maintain the conformation they have in the hexamer, which is simplest but without straightforward alternative. With the rotational strength calculated for all insulin species participating in the association equilibrium and with the association constants determined in the molecular weight studies one is in the position to describe how the calculated CD varies with insulin concentration.

34-

This was done not only for normal insulin but also for the tentative cases that quaternary structure formed exclusively with monomers I or monomers II respectively.

Fig. 6: Concentration dependence of subunit population and tyrosyl CD of insulin (b), calculated and observed. The concentration range shown extends to 0.6 mg/ml. Conditions: 0.025 M Tris/HCl buffer pH 7.8 containing 5-10 M EDTA, zinc-free. Calculations based on 1978 coordinates; 1.5 A resolution, R=17.1%. I/I; II/II monomer I respectively II were assumed to be the only conformation existing in solution. I/II designates coexistance of both monomers as in the crystal. The experimental curve is found between the normal and the homo-I case which gives the highest negativity (see Fig. 6). Any of the three cases calculated correlates very well with the observed values except for a constant term. In the presence of zinc, however, correlation fails. The experimental values are generally more negative than those calculated, and in the low concentration range it seems that different effects overlap. One of the interfering processes probably is the binding of zinc prior to promoting hexamerization. Experiments were as yet conducted at constant zinc per insulin molar ratio. More have to be run under varied conditions in order to allow for a more refined analysis.

35 The monomer I/monomer II problem may, again, benefit from B1 des-Phe -insulin. Table IV: Comparison of monomer I and II in insulin and des-PheB1 -insulin with respect to their calculated tyrosyl CD (based on 1978 coordinates; 1.5 A resolution, R=17.1%) [275 nm; M

cm2- 103]

INSULIN

DES-PHE61-INSULIN

HONOMER I

-1 .42

-0.61

MONOMER II

-0.56

-1 .30

AVERAGE

-0.99

-0.96

.If monomer I were the favoured conformation, des-Phe should exhibit much smaller a value than insulin at high dilution. If it were II, the opposite should result (see Table IV). CD measurements at sufficiently low concentrations are being studied.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft and by the Kroc Foundation.

References 1. 2. 3. 4.

Tinoco, I.: J. Advan. Chem. Phys. _4, 113 (T962). Hsu, M.C., Woody, R.W. : J. Amer. Chem. Soc. 9J3 , 351 5 (1 971 ). Strickland, E.H.: Biochemistry 11, 3465 (1972). Strickland, E.H., Mercola, D.A.: Biochemistry 15, 3975 (1976). 5. Wollmer, A., Fleischhauer, J., Straßburger, W., Thiele, H., Brandenburg, D., Dodson, D., Mercola, D.: Biophys. J. 20, 233-243 (1977). 6. Momany, F.A., McGuire, R.F., Burgess, A.W., Scheraga, H.A.: J. Phys. Chem. 79, 2361 (1975).

SECTION II PEPTIDE SYNTHESIS

AN ASSESSMENT OF PROTECTING GROUP STRATEGIES IN SYNTHESIS OF LARGE PEPTIDES

Johannes Meienhofer Chemical Research Department, Hoffmann-La Roche I n c . , Nutley, NJ 07110 USA

Introduction

The proper choice of protecting groups has always been an important c o n s i deration in peptide s y n t h e s i s but i t assumes c r i t i c a l dimensions for l a r g e r molecules such as p r o i n s u l i n , lysozyme, bungarotoxin, or Kazal t r y p s i n i n hibitor.

F a i l u r e to remove a l l protective groups completely at the end of

synthesis have been reported for several larger synthetic peptides and thus the exploration of a l t e r n a t i v e approaches becomes an important concern. One of the presently preferred protecting group s t r a t e g i e s i s based on d i f f e r e n t i a l or graded a c i d o l y s i s , i . e . cleavage of «//"-blocking groups during synthesis by mild acid and cleavage of semipermanent side chain protection by hard acid at the end of s y n t h e s i s .

C h a r a c t e r i s t i c for these

s t r a t e g i e s i s the use of the strongest cleaving agents, f o r example l i q u i d hydrogen f l u o r i d e (1,2) for the treatment of the f i n a l s e n s i t i v e and v a l u able synthetic product.

Experience now shows that l i q u i d HF can be a very

destructive reagent f o r peptides of increased chain length. In t h i s paper the exploration of some a l t e r n a t i v e approaches i s proposed in which the mildest a v a i l a b l e cleaving agents would be u t i l i z e d for the f i n a l deprotection of the synthetic target molecule.

© 1980 Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

40 Discussion and Evaluation of Some Reported Problems In a recent report on the studies toward the synthesis of a lysozyme analogue, Kenner, Ramage, and Galpin (3) attributed the failure to obtain any enzyme activity to incomplete cleavage of the 8 thiol protecting groups from the synthetic 127-peptide. that the acetamidomethyl protecting group.

These authors concluded: "...it is clear

(Acm) function is far from adequate as a cysteine

Using mercuric acetate the recovery of thiol was only

about 75% of the expected figure."

Only 67% cleavage of the Acm groups

from six cysteine residues was obtained by Rocchi et al. (4) with their 52residue peptide of Kazal-type trypsin inhibitor.

Very low bioactivity was

obtained and the synthesis declared as unsuccessful.

Even with the synthe-

tic 14-peptide somatostatin the mercuric acetate cleavage of the Acm thiol protection never yielded more than 60-75% of dihydrosomatostatin in our laboratory (5).

Direct formation of disulfide A7-B7 from two acetamido-

methyl cysteine residues in the last stage of the CIBA-GEIGY insulin synthesis (6) proceeded with a yield of 70% using iodine in 60% acetic acid. While these yields are acceptable for the formation of one disulfide, the situation deteriorates rapidly with larger numbers of residues.

Assuming,

for example, an equal 75% Acm group cleavage for all 8 cysteine residues of synthetic lysozyme, one would expect no more than 10% of completely deprotected material and no more than 9% in the case of the synthetic Kazal trypsin inhibitor.

It is evident that the yields of protecting

aleavage from large peptides

must be as close to quantitative

group as

possible.

Similar considerations should guide the application of very powerful

cleav-

age reagents in peptide synthesis such as liquid HF (7) or sodium in liquid ammonia (8).

Both procedures have been and continue to be of enormous

value in syntheses of small and medium sized peptides..

However, the rou-

tine and indiscriminate use of liquid HF is clearly counterindicated by the rather large list of side reactions (9) and incomplete cleavage, Table 1.

41 Table l.

Protective Group Cleavage by Liquid Hydrogen Fluoride

Ser Thr

N + 0 peptidyl shift

Sakakibara et

peptide bond cleavage

Lenard, Hess (11)

deamidation

Robinson et

Orn formation

Yamashiro et

Tyr

C-alkylation

Erickson, Merrifield (14)

1973

Glu

acylation or

86

81

fcOBU pBZL

^BZL JCHjCO-g

0BZL

¿»ZL

«zl

mzL

KjBU

100

90

95

WCM^CO f

Jel

Figure 1. Stepwise synthesis on 2-oxoethyl polystyrene gel phase of fragment 4-14 of the bovine insulin A-chain from excess Ddz-amino acids/dicyclohexylcarbodiimide in dichloromethane/dimethylformamide.

100

—

OBU

,och 2 CC|

^ZL 96

klBU iC»2C0| •OBU

DDZ-

95 I OVERALL YIELD (MEAN VALUE) IN THE SYNTHESIS OF FRAGH. 15 - 21 W

THE BOVINE INSULIN A CHAIN III POLYHER PHENACYL GEL PHASE.

Figure 2. Synthesis of fragment 15-21 of the bovine insulin Achain as in figure 1. All synthetic operations were performed 3-times aiming at optimized and photometrically determined yield,

Utilizing the mode of action of the centrifugal reactor and the spectroscopic properties of the Ddz-protecting

group,

53 all synthetic steps on polymer phase were followed photometrically (3), as demonstrated in figure 3.

Figure 3. The original photometric recording of all synthetic operations on polymer phase in the synthesis of the insulin Achain from Ddz-amino acids. Upper left to lower right: Deprotection, washings, deprotection, washings, deprotonations»washings, peptide synthesis, washings, both repeated for completion. In the positions A5, A15 and A18 we introduced benzyl glutamate and aspartate, respectively. At the C-terminus aspartic acid a-tert.butyl ester was bound to the 2-oxoethyl link of the polymer support by its

13 — carboxy function. In this way

during the synthesis we avoided nitril formations at amide side functions, which all were incorporated later at the end of the synthesis by ammonolysis of the benzyl and the 2-oxoethyl ester bonds. We obtained the fragment 11(4-14) in 90 % and the fragment IV(15-21) in 93 % overall yield on polymer. To build up insulin A from the fragments, the sterically hindered Nterminal tripeptide (synthesized in solution from mixed anhydrides in 84 % yield) was condensed in a 5-fold excess three times with fragment II on the polymer support. This yielded fragment 111(1-14) in 86 %. After base-hydrolytic detachment from the carrier of fragment III, as well as

54 ammonolytic release of fragment IV, both were purified chromatographically. One equivalent of III and two equivalents of IV were subjected to the final condensation in dimethylformamide by the aid of benzotriazole

dicyclohexylcarbodiimide/1-hydroxy-

(4). After chromatography on Sephadex LH 20/

methanol we obtained the fully protected insulin A-chain in 78 % yield

(1.3 gr.). From this material tert.butyl mercaptane

was cleaved by reduction with tributyl phosphine and the liberated SH-funtions in position A6 and

1 immediately were

oxidized to yield the small disulfide ring in 84 % (5). Subsequently, all tert.butyl groups were cleaved with

trifluoro-

acetic acid and the p-methoxybenzyl protector in position was selectively removed

A20

(ca. 80 %) with HF in pyridine/anisole

(6). After work-up by ether precipitation followed by alternated washings with ether and centrifugations, one equivalent of the remaining A-chain containing a single free SH-group in position

A20 was dissolved in 30 % acetic acid. To this

solution one equivalent of reduced natural B-chain from bovine insulin was added in the same solvent and oxidized with 0.1 N iodine/acetic acid

(7). Because this oxidant also removes

the acetamidomethyl group, this last remaining group in position same reaction bridge

~

protecting

A7 was cleaved so that subsequently in the

mixture the second interchain w a s

disulfide

formed.

Biological Methods and Results From a single desalting chromatography of the

semisynthetic

insulin on Sephadex G 50 in 10 % acetic acid we obtained fractions

two

(24.2 mg; 25 % yield) containing the target molecule

electrophoretically

and chromatographically

indistinguishable

from an authentic sample. The biological activity of these two portions was already remarkable were chromatographed

(table 1). The two

fractions

individually on Biogel P6 in 1 % acetic

55 acid. Insulin was pooled and chromatographed once more on the same column. This yielded 18.2 mg (20 %) of bovine insulin, which showed 86 % biological activity in the lipogenesis assay on isolated lipocytes. Finally, we passed our semisynthetic insulin through a carboxymethyl cellulose column and eluted with a gradient of sodium chloride (0 - 0.35 M) in sodium acetate/acetic acid (0.024 M; pH 3.3) buffer (8). Desalting was performed on Biogel P 6 in 1 % acetic acid. The amino acid analysis after performic acid oxidation and total hydrolysis with 6 N HC1, 110° C, 42 h, was in best agreement with the theory: CysSO^H

Asp

Thr

Ser

1 0,86

Calcul. Found

6 5,91

3 3,70

Calcul.

Val 5

He 1

Found

5,12

0,86

Leu 6 6,00

Pro

3

Glu 7

Gly

Ala

1

4

3

2,71 Tyr

6,75 Phe

0,91 His

4 ,03 Lys

3,23 Arg

4

3

2

1

1

3,73

3,38

1 ,89 1,11

1 ,06

The Zn-complex showed the characteristic crystal forms of bovine insulin, as presented in figure 4.

Figure 4. Zn-complex of bovine insulin prepared from a synthetic A- and a natural B-chain by selective disulfide bridging. For the biological assays fat cells were isolated enzymatically from epidydimal fat tissue of Wistar rats by collagenase (9). The biological activities of the individual preparations of semisynthetic insulin on different stages of the chromatographic enrichment have been measured by , (table 1) :

56 a) the incorporation of 2-3H-glucose in the total lipids of the fat cells (lipogenesis)(10) and by b) the inhibition of the catecholamine-induced glycerol release in fat cells (antilipolysis). c) The competitive receptor affinity of the samples was determined in fat cells according to Gammeltoft and Gliemann (11). The protein content of the insulin solutions was measured according to Lowry (12). Chromatographically purified pork insulin (NOVO, MC quality) and 10-times recrystallized bovine insulin (HOECHST) have been utilized for standards.

Table 1. Biological activities at different stages of chromatographic purification of semisynthetic bovine insulin obtained by selective disulfide bridging of the A-chain synthesized in polymer phase with natural B-chain.

Stage of Purification

Yield [%]a)

Lipo-

Antilipo• b) lysis • b) genesis

Sephadex G 50 desalting; c)

25

52+9;

Biogel P6, 3-times; e)

20

86 + 3; 8 d )

Cellulose CM 52f) Biogel P6, 2-times; e)

18 105+7;

5 d)

5 d)

Receptor •b) affinity 65+11; 4 d )

_ 98

_ 2d)

99±7; 3 d )

a) based on chain combination; b) [%] ,in fat cells ofWLstar rat c) in 30% acetic acid; d) measurements; e) in 1% acetic acid f) sodium chloride gradient 0 - 0.35 M in 0.024 M sodium acetate/acetic acid buffer, pH 3.3

The biological activity both in the lipogenesis and in the antilipolysis assay as well as in the competitive receptor affinity test of the semisynthetic insulin after chromatographic purification was 100%. This pure insulin was obtained in 18% yield in the selective formation of the disulfide bridges.

57 Perspectives This positive result on an intermediate state of the total synthesis has encouraged us to proceed with the synthesis of the B-chain by fragment condensation on polymer phase, which is now in progress (13). The synthesis of the B-chain is projected from 6 fragments linked to the polystyrene gel phase by 2-oxoethyl functions. As in the strategy for the synthesis of the A-chain all fragments of the B-chain are protected on sidefunctions to the maximal extent by lipophilic moieties. To mask

the imidazole ring of histidine

we are utilizing the 2,4-dinitrophenyl group. In the positions B7 and B19 we are introducing the same sulfur protecting groups as in A7 (Acm) and in A20 (p-methoxybenzyl) allowing the SH-groups to be selectively liberated also in the B-chain for the directed bridging with the particular thiol functions of the A-chain.

References 1. Birr, Chr., Pipkorn, R.: Peptides 1978, Gdansk. Warszawa University Press, 1979; in press. 2. Birr, Chr., Pipkorn, R. : Angew. Chem. 91_, 571-573 (1979); Angew. Chem. Int. Ed. Engl. 1_8, 536-538 (1 979). 3. Birr, Chr.: Aspects of the Merrifield Peptide Synthesis, Springer, Berlin(1978). 4. König, W., Geiger, R.: Chem. Ber. _UD3, 788-798 (1970). 5. Rietschoten, J., van Granier, C., Rochat, H., Lissitzky,S. Miranda, F.: Europ. J. Biochem. 56^ 35-40 (1975). 6. Matsuura, S., Niu, C., Cohen, J.: Chem. Sve., Chem.Comm. 451-452 (1976). 7. Sieber, P.,Kamber, B., Hartmann, A., Jöhl, A., Riniker,B., Rittel, W.: Helv. Chim. Acta 60, 27-37 (1977). 8. Katsoyannis, P., Trakatellis, A.C., Johnson, St., Zalut,C., Schwartz, G.: Biochemistry 6, 2642-2655 (1967).

58 9. Rodbell, M.: J. Biol. Chem. 239, 375-380 (1964). 10. Moody, A.J., Stan, M.A., Stan, M., Gliemann, J.; Horm. Metab. Res. 6, 12-16 (1974). 11. Gammeltoft, S., Gliemann, J.: Biochim. Biophys. Acta 320, 16-32 (1973). 12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Biol. Chem. JjH, 265-275 (1951). 13. Voss, Chr.: Doctoral Thesis, Heidelberg; in preparation.

[A14-PHENYLALANINE-INSULIN] :

A SYNTHETIC ANALOGUE

W. Danho, A. Sasaki, E. Büllesbach, H.-G. Gattner Deutsches Wollforschungsinstitut D-5100 Aachen, Federal Republic of Germany A. Wollmer Fachgebiet Struktur und Funktion der Proteine, Abteilung Physiologische Chemie, Rheinisch-Westfälische Technische Hochschule, D-5100 Aachen, FRG.

Introduction Quantum chemical calculations based on the atomic coordinates of rhombohedral 2 Zn-insulin suggested that the largest contribution to the tyrosyl circular dichroism originates (1) from the interaction of A14-tyrosine and B1-phenylalanine. However, the abolition of that interaction by elimination of Bt-phenylalanine has no influence on the CD observed in B1 solution. Des-Phe -insulin is indistinguishable from native insulin with respect to all criteria available (2).

The most

plausible explanation for the unchanged CD signal and the poor interaction between B1 and A14 seems to be the thermal mobility of B1-phenylalanine or A14-tyrosine, or both. It is highly desirable to study the interaction not only by altering B1 position, but by altering A14 position as well. The most conservative replacement of A14-tyrosine is a phenylA1 4 alanine residue. [Phe ]-insulin allows one to specifically study the role of the phenolic OH-function of A14-tyrosine in insulin.

Furthermore this replacement yields a cluster of

four phenylalanine residues at the dimer/dimer interface of hexameric insulin, which might exhibit interesting optical properties.

© 1980 Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

60

In this communication we wish to describe the synthesis of the protected [ 14-phenylalanine]-A-rchain, its deprotection with trifluoroacetic acid/thiophenol, conversion to the tetraS-sulphonate form, and its combination with native di-S-sulA1 4 phonate B-chain to yield porcine [Phe ]-insulin.

Results The strategy of fragment condensation and the use of acid labile protecting groups was selected for the synthesis.

Thus

Y-carboxyl functions were protected as tert-butyl esters, the OH-functions of tyrosine, threonine and serine as tert-butyl ethers.

For the temporary protection of a-amino groups, the

benzyloxycarbonyl- and 2-(4-biphenyl)-isopropyloxycarbonyl groups were used. trityl-groups.

Cysteine side-chains were protected with

The plan for the synthesis is outlined in

Figure 1.

13 - 16

1

-

12 | 113

17 - 21

-

21

I 1 Figure 1.

-

21

Plan for the synthesis of [Phe A14 ]-A-chain

Synthesis of fragment 13-16.

The removal of the Z-group from

Z-Gln-Leu-OMe (3) was accomplished with HBr/CH 3 C00H in the usual manner.

The resulting HBr"H—Gin—Leu—OMe was coupled

with Z-Phe-OH by the mixed anhydride method (4) to give the crystalline tripeptide Z-Phe-Gln-Leu-OMe (I) in 85% yield. This was deprotected with HBr/CH^COOH and coupled with BpocLeu-OH by the mixed anhydride method.

After purification by

61

P 3 CQ O

I CJ

I

I 3 AI J I G R-4 0 1

ai

43 A. I N

03 U \ ^ PQ 03

!

I

3 AI

0 1 C 01
I --I m ^ 43 0 B 4-1 O 1 \ ai IN C .H -l-t OI U < 03 TH J I

i C

A I (J O

ft m

• S

3 I—T H AI O I W < W A S U I-

CG 0 1 M

ai

J

•FL ¿ E-I X TA AI O ^ O « O 43 MFT FK, AI O UI M

ai

S

O C G AI ---I 43 PILLI O CN

DB 0 1 TÍ OL < OB I FT ® RN "I OI 0 _ >I »OL U I O

C\J L/L ID O • • IN

3

I C

3

P-) ONO ° Y CQ 03 OJ I TTI OB S 2

' .-I

m fi 1

•H (0

^

I G

^ - > 1 'H E-I U I —

M 01 P

o

I -U I

È Q

(d -P M-l "H C

I M

P 3

I O 43 1

P 3

ra

G

o

0 G

ai

43 FT I 3

. ai to

I

ai

I

u o ft

oi

J I ^

>I

•

u

M

ai

I

ai I •P SJ 3 . . ai M M I P 3 .

CD O EN

>1 — U I

ai

m p

E-I

N

ai

N. EH

W I ^ 43

u

I

•

oi

>I

m

u

O CQ —

u

m

I

P W

G I-L 0 1 3 "R-L U

01 >I U I

oí I

G IH 0 1

tí

T—I 0 1 .H

I

.-I >n)

>id I

ai >I Î-I U I U O M

O O CQ

I >I ÍH 0 1 03

PN

62 CCD in the toluene system (K = 0.2), a yield of 93% of the tetrapeptide derivative Bpoc-Leu-Phe-Gln-Leu-OMe obtained.

(II) was

Quantitative saponification in dioxane/water (9/1)

solution yielded Bpoc-Leu-Phe-Gln-Leu-OH

(III) .

Fig. 2

illustrates the synthetic scheme. Synthesis of fragment 13-21. t

The deprotection of Bpoc-function 1

from Bpoc-Glu (0Bu )-Asn-Tyr (Bu ") -Cys (Trt) -Asn-OBu1" (5) was carried out with HCl/90% trifluorethanol (6). t

fc

HCl-H-Glu(OBu )-Asn-Tyr(Bu )-Cys(Trt)-Asn-OBu

11

The resulting was neutralized

with N-methyl-morpholine and coupled to Bpoc-Leu-Phe-Gln-LeuOH by the mixed anhydride method to yield the protected peptide Bpoc-Leu-Phe-Gln-Leu-Glu (0Bufc) -Asn-Tyr (Bu1-) -Cys (Trt) -AsnOBu1" (IV) in 6 4% yield.

Purification was achieved on Sephadex

LH-20 in DMF. Synthesis of the protected [Phe A 1 4 ]-A-chain. 1

The Bpoc-group

fc

of Bpoc-Leu-Phe-Gln-Leu-Glu(OBu )-Asn-Tyr(Bu )-Cys(Trt)-AsnOBu1" (IV) was removed with HCl/90% trifluorethanol as preThe free base H-Leu-Phe-Gln-Leu-Glu (OBu1") -

viously described.

Asn-Tyr (Bu1")-Cys (Trt)-Asn-OBu1" was obtained after neutralization with triethylamine and gel filtration on Sephadex LH 20 in DMF in a yield of 87%.

This peptide was coupled with Boc-

fc

Gly-Ile-Val-Glu(0Bu )-Gln-Cys(Trt)-Cys(Trt)-Thr(Bufc)-Ser(Bufc)Ile-Cys(Trt)-Ser(But)-0H

(7) by the DCC/HOBT method (8) using

N-methyl pyrrolidone as a solvent.

Since the sequence 1-21

represents the final step of the synthetic procedure, no purification was performed on the crude coupling mixture. Deprotection, conversion to the [A14-phenylalanine]-Ssulphonated A-chain and purification.

The deprotection of

the crude synthetic product was achieved in'a single step with trifluoroacetic acid using thiophenol as trityl-cation scavenger (9).

The conversion to the tetra-S-sulphonate

form was accomplished by reaction with sodium sulphite and

63

•

V [ml]

Figure 3. DEAE-Cellulose chromatography of partially purified [PheA14]-S-sulphonated A-chain (Column 2.5 x 25 cm), 400 ml., 0.05 M NaOAc/7 M urea/acetic acid, pH 5.6. Linear salt gradient of 0.3 M NaCl. freshly-prepared sodium tetrathionate. Purification was achieved by gel filtration on Sephadex G-2 5 in 0.05M NH 4 HC0 3 and then ion-exchange chromatography on DEAE-cellulose at pH 5.6 (Fig. 3). The purity of the [Phe A14 1 - chain was checked by: 1.

Amino acid analysis of the purified material after acid

hydrolysis gave a composition in good agreement with the theoretically expected values: Asp 2.01 (2), Thr 1.03 (1), Glu 4.00 (4), Gly 1.04 (1), Cys 1.31 (2), Val 0.26 (1), lie 1.18 (2), Leu 2.00 (2), Tyr 0.97 (1), Phe 0.91 (1). 2.

Paper electrophoresis at pH 2.2 and 4.8.

A single band

was detected (Pauly reaction) which migrated to the same position as the natural S-sulphonated A chain.

The net yield

for the final coupling, deprotection, conversion to S-sulphonate

and purification

was 19.0%.

A1 4 Synthesis and purification of porcine [Phe ]-insulin. A1 4 Tetra-S-sulphonated [Phe ]-A-chain and native di-S-sulphonated B-chain were reduced with mercaptoethanol at pH 10.6 and oxidized at this pH for 48 h at 4°C to yield the crude A1 4 [Phe ]-porcine insulin (10).

64 e

254

Figure 4 Gel filtration of the crude recombination mixture on Sephadex G-50 (3 x 90 cm) in 1 M acetic acid. 10 ml/fraction, insulin containing fraction 66-72.

Figure 5 DEAE-cellulose chromatography of fraction 66-72 from gel filtration (column 1.5 x 16 cm), 150 ml 0.05 M tris/HCl with 7 M urea pH 8.1, linear salt gradient of 0.2 M NaCl.

65 T h i s w a s p u r i f i e d b y gel f i l t r a t i o n o n S e p h a d e x G - 5 0 i n IM acetic acid

(Fig. 4), a n d i o n - e x c h a n g e

chromatography on

c e l l u l o s e a t p H 8.1 u s i n g a l i n e a r N a C l g r a d i e n t A1 4 The purity of 1-

[Phe

(Fig.

3.06

A c i d h y d r o l y s i s gave a c o m p o s i t i o n values:

1.92

(2), S e r 2.74

(3), G l u 6.76

(7),

Gly 4.00 64), A l a 2 . 0 3

(2), V a l 3.40

(4), H e

1.44

(2),

L e u 5.86

(6), T y r 2 . 2 3

(3), Phe 4.20

(4), L y s 0 . 9 7

(1),

His

(2), A r g

(1).

1.53

(3), T h r

5):

] was checked by:

Amino acid analysis.

in g o o d a g r e e m e n t w i t h the t h e o r e t i c a l l y e x p e c t e d Asp

1.01

-2.Cellulose-acetate electrophoresis band was detected p o s i t i o n as

DEAE-

a t p H 2.2 a n d 8.6:A

single

(Pauly r e a c t i o n ) w h i c h m i g r a t e d to the

same

insulin. A1 4

B i o l o g i c a l a c t i v i t y of [Phe

] -insulin.

The

biological

a c t i v i t y of the i n s u l i n a n a l o g u e as d e t e r m i n e d i n v i t r o

with

r a t e p i d i d y m a l a d i p o c y t e s b y the m e t h o d of M o o d y e t a l .

(11)

w a s 96 ± 6%.

Discussion I n c o n c l u s i o n , t h i s c o m m u n i c a t i o n d e s c r i b e s the s y n t h e s i s of a n e w s y n t h e t i c a n a l o g u e , p o r c i n e

successful [A-14-phenyl-

a l a n i n e ] - i n s u l i n b y t h e s o l u t i o n m e t h o d s of p e p t i d e

synthesis,

u s i n g the s a m e s t r a t e g y a n d t a c t i c s as in the s y n t h e s i s A19 [Phe ] - i n s u l i n (7). T h e b i o l o g i c a l a c t i v i t y of 96 ± 6% as m e a s u r e d b y

lipogenesis

i n r a t e p i d i d y m a l a d i p o c y t e s i n d i c a t e s t h a t this r e s i d u e n o t c r i t i c a l for the b i o l o g i c a l a c t i v i t y of the h o r m o n e . f i n d i n g is s u p p o r t e d by the f a c t t h a t the i o d i n a t i o n o f residue

(12) o r s u b s t i t u t i o n by a l a n i n e

(13) d o e s n o t

any l o s s o f the b i o l o g i c a l a c t i v i t y of the h o r m o n e . n e a r u l t r a v i o l e t c i r c u l a r d i c h r o i s m s p e c t r a o f the

of

is This this

cause Far

and

standard

66 insulin and of [Phe

A1 4 ]-insulin have been measured, and will

be published in a future communication.

Acknowledgements The authors would like to thank C. Diaconescu for the determination of the biological activities.

References 1.

Wollmer, A., Fleischhauer,*J., Strassburger, W. , Thiele, H., Brandenburg, D., Dodson, G., and Mercola, D.: Biophys. J. 20, 233-243 (1977).

2.

Brandenburg, D.: Hoppe Seyler's Z. Physiol. Chem., 350, 741-750 (1969).

3.

Katsoyannis, P.G.,Suzuki, K., and Tometsko, J. Am. Chem. Soc. , 8J5, 1 1 39-1 141 (1963).

4.

Anderson, G.W., Zimmerman, J.E., and Callahan, F.M.: J. Am. Chem. Soc., 89_, 5012-5017 (1967).

5.

Berndt, H.: Hoppe Seyler's Z. Phsiol. Chem., 360, 747-760 (1979).

6.

Riniker, B., Kamber, B., and Sieber, P.: Helv. chim. Acta, 58, 1086-1094 (1975).

7.

Danho, W. , Sasaki, A., Büllesbach, E., Föhles, J., and Gattner, H.-G.: Proc. of 2nd FRG-USSR Symposium on Chemistry of Peptides & Proteins,Grainau/Eibsee (1978).

8. 9.

König, W., and Geiger, R.: Chem. Ber. 103, 788-798 (1970). Büllesbach, E.: Dissertation TH Aachen (1978).

A.:

10. Rüegg, U., and Gattner, H.-G.: Hoppe Seyler's Z. Physiol. Chem. 356, 1527-1533.(1975). 11. Moody, A.J., Stan, M.A., and Gliemann, J.: Horm. Metab. Res. 6, 12-16 (1974). 12. Gliemann, J., Sonne, 0., Linde, S., and Hansen, B.: Biochem. Biophys. Res. Comm., in print (1979). 13. Weber, U., Schneider, F., Köhler, P., and Weitzel, G.: Hoppe Seyler's Z. Physiol. Chem. 34J3, 947-949 (1967).

[B25-TYROSINE]B-CHAIN S-SULFONATE OF PORCINE INSULIN

R. Knorr, W. Danho, E.E. Büllesbach, H.-G. Gattner and H. Zahn Deutsches Wollforschungsinstitut D-5100 Aachen, Federal Republic of Germany

The C-terminal region of the insulin-B-chain is important for dimer formation and hormone-receptor interaction (Figure 1). The aromatic side-chains of B25-phenylalanine and B26-tyrosine seem to protect the hydrogen bonds between the two monomers against the influence of solvents.

It is our intention to

substitute the B25-phenylalanine by other amino acids having smaller side-chains (e.g^ Ala) so as to diminish steric hindrance or having more hydrophilic groups (e.g. Tyr, Ser) so as to diminish hydrophobic protection in this region.

Figure 1: The arrangement of residues B21-B30 in the insulin dimer, viewed in the direction of the local axis. The COOH-terminal chains run antiparallel to each other, forming an antiparallel pleated sheet structure with four hydrogen bonds. From Blundell et al (1).

© 1980 "Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

68 This work describes the synthesis of the [B25-Tyr]B-chain of porcine insulin by solution condensation of fragments using acid labile protecting groups.

The y-carboxyl functions were

protected by tert-butyl esters; the hydroxy functions as tertbutyl ethers ; for the temporary protection of the a-amino functions the benzyloxycarbonyl- or the 2-(4-biphenylyl)isopropyloxycarbonyl groups were used; the e-amino function of lysine was protected by the tert-butyloxycarbonyl group; the cysteine side chains were protected by trityl groups.

The

plan for the synthesis is outlined in Figure 2.

1. Synthesis of Fragment (17-22) The removal of the Z-group from Z-Glu(OBut)-Arg(HBr)-OH (2) was accomplished by catalytic hydrogénation.

The resulting

H-Glu(OBu^)-Arg(HBr)-OH was then coupled by the mixed anhydride method (3) with Bpoc-Leu-Val-Cys(Trt)-Gly-OH

(4). After

purification by CCD in toluene system (K = 1 ) , a yield of 70% of the hexapeptide Bpoc-Leu-Val-Cys(Trt)-Gly-Glu(0Bufc)-Arg (HBr)-OH was obtained. 17

1

1 Figure 2.

+

17

221 + |23

I

30

30

30 The plan for the synthesis of [B25-Tyrosine]B-chain

69 2.

Synthesis of Fragment (23-30)

The removal of the Z-group from Z-Thr(Bufc)-Pro-Lys(Boc)-Ala0Bu t (5) was carried out by hydrogenolysis in methanol.

The

resulting free base was then coupled with Z-Gly-Phe-Tyr-Tyr-OH by the DCC/HOBt (6) method.

After purification by CCD in

carbon tetrachloride system (K = 0.4) a yield of 60% of the octapeptide Z-Gly-Phe-Tyr-Tyr-Thr(Bufc)-Pro-Lys(Boc)-Ala-0But was obtained. 3.

Synthesis of Sequence (17-30)

This fragment was synthesised by condensing the fragments (1722) and (23-30).

The deprotection of the Z-function from

Z-Gly-Phe-Tyr-Tyr-Thr (Bu^") -Pro-Lys (Boc) -Ala-OBu** was carried out by hydrogenolysis in methanol.

The resulting free base

was then coupled with Bpoc-Leu-Val-Cys(Trt)-Gly-Glu(0But)Arg(HBr)-OH by the DCC/HOBt method.

After purification by

gel filtration on Sephadex LH-20 in DMF, a yield of 45% of the tetradecapeptide

Bpoc-Leu-Val-Cys(Trt)-Gly-Glu(0Bufc)-Arg(HBr)-

Gly-Phe-Tyr-Tyr-Thr (Bu^~) -Pro-Lys (Boc) -Ala-OBu*" was obtained. Figure 3 shows the scheme of the synthesis. Z-Gly-Phe-Tyr-Tyr-OH

Bufc Boc 1 > t H-Thrr-Pro-Lys-Ala-OBu l. DCC/HOBt

i

2. 4

Trt Bpoc- Leu-Val-Cya-Gly I -OH Yield: 70«

OBu • H- Glu-Arq I HBr-OH I IM.A.) I Mixed Miydrlde 1 tol ymm ir cbu Br rr It I H• IK"i1' ) Bpoc- Leu-Val-Cya-Gly-Glu-Arg-OH • Ylaldt Trt

I

I

OBu

1

-

HBr

45%

CCD,CCl4-system(K=0.4l

1 1 Bu' Boc Z-Gly-Phe-Tyr-Tyr-Thr-Pro-Lys-AU-OBu t 1 H 2 / Pd ut B c sI ?I t H-Gly-Phe-Tyr-Tyr-Thr-Pro-Lyo-Ala-CBu

I 1. DCCI / HOBt 2. LH 20 In DMF Bul Boc

I I t Bpoc- Leu-Val-Cya-Gly-Glu-Arg-Gly-Phe-Tyr-Tyr-Thr-Pro-Ly«-Ala-OBu 17 18 19 20 21 22 23 24 25 2C 27 2B 29 10

Amino acid analyala: Ala 1.00 (1)i Leu 1.01 11)1 Val 1.01 (111 Cya n.d. (Ill Gly 1.89 (2); Glu 1.03 (1); Arg 1.00 (1); Phe 1.05 (1)i Tyr 2.02 (2I| Thr 0.93 (1)> Lya 1 .OO (II.- Pro n.d. (II.

Figure 3

70 4.

Synthesis of the protected

[B25-tyrosine]B-chain

The deprotection of Bpoc-Leu-Val-Cys(Trt)-Gly-Glu(0Bufc)-Arg (HBr)-Gly-Phe-Tyr-Tyr-Thr(Bu^)-Pro-Lys(Boc)-Ala-OBu^" with 80% acetic acid/pyridine hydrobromide was carried out by the method of Schwertner et al. (7) .

The free base was then

coupled to Boc-Phe-Val-Asn-Gln-His-Leu-Cys(Trt)-Gly-Ser(Bu^")His-Leu-Val-Glu(OBut)-Ala-Leu-Tyr(But)-0H method.

(8) by the DCC/HOBt

Since the sequence (1-30) represents the final step

of the synthetic procedure, no purification was performed on the crude coupling mixture (Figure 4). 5.

Deprotection, conversion to the [B25-tyrosine]B-chain S-sulfonate and purification.

The deprotection of the synthetic product was achieved in a single step with trifluoroacetic acid using thiophenol as trityl cation scavenger (9).

The conversion to the di-S-sul-

fonate was accomplished with sodium sulfite and freshly prepared sodium tetrathionate.

Purification was achieved by gel-

filtration on Sephadex G~25 in 0.05 M NH^HCO^ and ion-exchange chromatography on SP-Sephadex at pH 3.5 using a linear NaClTI rt t Bu* 0Bufc BIu* Boc-Phe-Val-Aan-Gln-Hls-Leu-Cy a-Gly-Ser-Hli-Leu-Val-Glu-AlI a-Leu-T yr-OH Trt OBu* HBr But Boc Is-GlIy-Gl? H-Leu-Val-Cy u-Arg-Gly-Phe-Tyr-Tyr-TI hr-Pro-LyIs-Ala-OBut

•

| DCCI/HOBt I

BOC-1-3Q-OBUC I 1. crjCOOH/Thiophenol 2 » H«a803/N«28406

3. Saphadax G-2S in 0.05 M NHJHCOJ

4. 8P-S«phad«x at pH 3.5 "f

@

Amino acid analysis; Asp 1.00(1); Thr 0.99(1); Ser 0.95(1)f Glu 3.12(3); Gly 2.90(3); Ala 2.03(2); Val 2.96(3) Cy, .. (2); Leu 3.62(4); Tyr 3.00(3); Pho 2.04(2); Lya 0.98(1); His 2.01(2); Arg 1.05(1) Figure 4

71 gradient.

The purity of the [B25-tyrosine]B-chain S-sulfonate

was checked by: 1.

Amino acid analysis of the purified material after acid

hydrolysis.

This gave a composition in good agreement with

the theoretically expected values (Fig. 4 ). 2.

Paper electrophoresis at pH 2.2 and 4.8.

A single band

was detected (Pauly reaction) which migrated to the same position as the natural B-chain S-sulfonate.

The overall

yield, starting from the initial fragments after coupling, deprotection, conversion to the S-sulfonate and purification, was 21%. The results of three experiments indicate that the combination of [B25-tyrosine]B-chain with native A-chain does not occur, neither does the modified B-chain form polymers as observed in other recombination experiments.

The reasons for this have

not yet been investigated.

References 1. 2.

Blundell, T., Dodson, G., Hodgkin, D. and Mercola, D.s Adv.Protein Chem. 26, 279-402 (1972). Naithani, V.K. and Föhles, J.: Hoppe Seyler's Z. Physiol. Chem. 359, 1173-1181 (1979).

3.

Anderson, G.W., Zimmermann, J.E. and Callahan, F.M.: J. Amer. Chem. Soc. 89, 5012-5017 (1967).

4.

Müller, J.: Dissertation RWTH-Aachen (1976).

5.

Walkenhorst, W.: Dissertation RWTH-Aachen (1974).

6.

König, W. and Geiger, R.: Chem. Ber. 103, 788-798 (1970).

7.

Klostermeyer, H. and Schwertner, E.: Z. Naturforsch. 28b, 334-338 (1973). Danho, W. and Föhles, J.: Hoppe Seyler's Z. Physiol. Chem. (1979) sub. for publication.

8. 9.

Büllesbach, E.E.: Dissertation RWTH-Aachen (1978).

THE SYNTHESIS OF

[B25-4-NITROPHENYLALANINE]-OCTAPEPTIDE

B(23-30) OF PORCINE INSULIN

M. Casaretto, W. Danho, H.-G. Gattner and H. Zahn Deutsches Wollforschungsinstitut D-5100 Aachen, Federal Republic of Germany

The C-terminal region of insulin B-chain is important for the aggregation of insulin monomer and for the hormone-receptor interaction (1).

Chemical modification of specific amino acid

residues in this region should give more information about the role these amino acids play regarding the structure-activity, receptor-binding and aggregation behaviour of the insulin molecule.

The synthesis of an analogue

modified at the C-terminal

region of the B-chain can be achieved by an enzyme-assisted coupling (2) of des-octapeptide-insulin with a synthetic modified octapeptide (B23-30).

Figure 1. The residues of the proposed receptor-binding region of insulin (1).

© 1980 Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

74 B1 P. T h a m m u s e d insulin,

[p-azido-Phe

for studies

]-insulin

of the m o l e c u l a r

A z i d o c o m p o u n d s are l i g h t - s e n s i t i v e difficult

to h a n d l e .

worked with. chemically insulin

Aromatic

(3), a

and t h e r e f o r e

insulin.

preparatively

n i t r o c o m p o u n d s are m o r e

a c t i v e p r o b e as d e m o n s t r a t e d by E s c h e r

to be a u s e f u l

1. the a g g r e g a t i o n state and in

(4).

An

in p o s i t i o n

B25

tool for the studies

of i n s u l i n m o l e c u l e s

of:

in the

crystalline

solution

2. the p r e p a r a t i o n of c o v a l e n t l y

crosslinked

insulin

dimers

3. the c o v a l e n t b i n d i n g of the h o r m o n e to the r e c e p t o r cell

easily

T h e s e c o m p o u n d s can also be u s e d as a p h o t o -

analogue with p-nitrophenylalanine

appeared

photo-labelled

i n t e r a c t i o n s of

in

the

membrane

This c o m m u n i c a t i o n d e s c r i b e s octapeptide

(B23-30),

(Boc)-Ala-OBu

.

the s y n t h e s i s of a

photo-sensitive

H-Gly-Phe-Phe(N0_)-Tyr-Thr(Bufc)-Pro-Lys

It is p l a n n e d to c o u p l e this

with des-octapeptide-insulin

to y i e l d the

octapeptide

photo-sensitive

analogue.

S y n t h e s i s of the [ B 2 5 - n i t r o p h e n y l a l a n i n e ] - o c t a p e p t i d e t

1

P h e - P h e (NC>2) - T y r - T h r (Bu ) - P r o - L y s (Boc) -Ala-OBu ^

Trt-Gly-

(B23-30) .

T h e s t r a t e g y of f r a g m e n t c o n d e n s a t i o n

and the use of

labile p r o t e c t i n g

for the s y n t h e s i s .

groups were selected

benzyloxycarbonyl,

the t e r t . - b u t y l o x y c a r b o n y l

m e t h y l g r o u p s w e r e used a-amino groups.

for the t e m p o r a r y p r o t e c t i o n

The h y d r o x y l

as t e r t . - b u t y l e s t e r

and the

e-amino

of

the masked

function of l y s i n e w a s residue.

chosen were Trt-Gly-Phe-Phe(N02)-Tyr-OH (5)

The

triphenyl-

f u n c t i o n of t h r e o n i n e w a s

t e c t e d by the t e r t . - b u t y l o x y c a r b o n y l P r o - L y s (Boc) -Ala-OBu**

and t h e

acid

The

(B23-26)

pro-

fragments

and

Z-ThrtBu 1 )-

(B27-30) .

S y n t h e s i s of the

fragment T r t - G l y - P h e - P h e ( N 0 2 ) - T y r - O H

The crystalline

dipeptide

(B23-26).

Trt-Gly-Phe-OMe was prepared

by

75 coupling Trt-Gly-OH with HCl.Phe-OMe using the mixed anhydride method (6) in 71% yield.

The saponification was carried out

in dioxane/water (1/1) with 1 N NaOH solution in a yield of 70%. The crystalline di-peptide BOC-Phe(N02)-Tyr-OMe was synthesized by coupling BOC-Phe(N02)-OH with HC1.Tyr-OMe using the mixed anhydride method and purified by recrystallisation from ethyl acetate/n-hexane in a final yield of 85%. The Boc-group was then removed from Boc-Phe(N02)-Tyr-OMe by treatment with trifluoroacetic acid in the usual manner. The resulting CF^ COOH'H-Phe(N02)-Tyr-OMe was then coupled with Trt-Gly-Phe-OH by the mixed anhydride method to yield 84% of the tetrapeptide derivative Trt-Gly-Phe-Phe(N02)-Tyr-OMe.

The purification was

achieved by counter current distribution in the system composed of toluene/chloroform/methanol/water

(5/5/8/2) (K = 0.2).

Saponification in dioxane/water with 1 N NaOH gave Trt-GlyPhe-Phe (N02)-Tyr-0H.

Figure 2 shows the scheme of the syn-

thesis .

Trt-Gly-OH + H-Phe-OMe ether trituration I M.A. (71%) 1 Trt-Gly-Phe-OMe

1

ethyl acetate/ I M.A. (85%) n-hexane ^ Boc-Phe(N02)-Tyr-OMe

1N NaOH in

I 1-CF COOH

{ 2-N-liethylmorphine Dioxan/Water (70%) 2)-Tyr-OMe Trt-Gly-Phe-OH + H-Phe(N0 CCD in Toluene I M.A. (84%)

k = 0,21

J

t Z-Thr(Bu )-Pro-Lys(Boc)-Ala-OBu I H ? /Pd ^

Trt-Gly-Phe-Phe(N02)-Tyr-OMe 82% I 1N NaOH in 4 (Dioxan/Water) Trt-Gly-Phe-Phe(NOj)-Tyr-OH LH 20 in Methanol

J

DCCI/HOBT (77%)

Trt-Gly-Phe-Phe(N02)-Tyr-Thr(But)-Pro-Lys(Boc)-Ala-0But |

80 % acetic acid. Pyridine hydrobromide (2N)

HBr. H-Gly-Phe-Phe(N02)-Tyr-Thr(But)-Pro-Lys(Boc)-Ala-OBu1

Figure 2.

The scheme of the synthesis of [B25-4-Nitrophenylalanine] octapeptide B (22-30) of porcine insulin.

76 Synthesis of the fragment Trt-Gly-Phe-Phe(N0_)-Tyr-Thr(Bu )t Pro-Lys(Boc)-Ala-OBu . The removal of the Z-group from Z-Thr(Bufc)-Pro-Lys(Boc)-AlaOBu

was carried out by hydrogenolysis in methanol.

The resulting H-Thr(But)-Pro-Lys(Boc)-Ala-OBu

was then

coupled with Trt-Gly-Phe-Phe(N02)-Tyr-OH by the DCC/HOBt method (7) to yield the octapeptide derivative 11

t

Phe(N02)-Tyr-Thr(Bu )-Pro-Lys(Boc)-Ala-0Bu .

Trt-Gly-PhePurification was

achieved by gel filtration on Sephadex LH-20 in methanol. The final yield was 77%. Two attempts at coupling the photosensitive octapeptide B(23-30) to des-octapeptide-insulin following the method of Inouye et al. (2) gave, after purification, very little product. Further work is under way.

References 1. Blundell, T., Dodson, G., Hodgkin, D. and Mercola, D.: Adv. Protein Chem. 26, 279-402 (1972). 2. Inouye, K., Watanabe, K., Morihara, K., Tochino, Y.,Kanaya, T., Emura, J. and Sakakibara, S.: J. Am. Chem. Soc. 101, 751-752 (1979) . 3. Thamm, P.: Diploma Thesis RWTH-Aachen (1977). 4. Escher, E. and Schwyzer, R.: Helv. Chim. Acta 60, 339(1977). 5. Walkenhorst, W.: Dissertation RWTH-Aachen (1974). 6. Anderson, G.W., Zimmermann, J.E. and Callahan, F.M.: J. Am. Chem. Soc. 89, 5012-5017 (1967). 7. König, W. and Geiger, R.: Chem. Ber. 103, 788-798 (1970).

A NOVEL ROUTE FOR THE PREPARATION OF MONO-IODOTYROSINE AND MONO-IODCTYROSINE CONTAINING PEPTIDES BY ELECTROCHEMICAL REDUCTION OF DI-IODO DERIVATIVES

H.-G. Gattner and W. Danho Deutsches Wollforschungsinstitut, D-5100 Aachen, Federal Republic of Germany

Introduction The chemical synthesis of mono-iodotyrosine is difficult and gives low yields(1).

Commercial mono-iodotyrosine often con-

tains di-iodotyrosine in variable amounts.

On the contrary,

3.5-di-iodotyrosine is easily prepared by direct iodination of tyrosine.

In this paper we describe a method for the prepara-

tion of mono-iodotyrosine from di-iodotyrosine and methods of purification.

Methods and results Polarographic studies have shown that di-iodotyrosine has a reduction wave with e-]/2= ~°-95V in 0.1N HCl measured against the saturated Ag/AgCl-electrode (2).

Mono-iodotyrosine shows

no Polarographie activity at this potential.

The wave of di-

iodotyrosine should correspond to the reduction to mono-iodotyrosine at the dropping mercury electrode. Preparative scale reductions were performed in an H-cell, the potential between working and reference electrode was controlled by an electronic potentiostat (3).

Figure 1 shows the

scheme of the reduction apparatus.

© 1980 Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

78

Figure 1. Scheme for the reduction at controlled potential a) solution of the compound to be reduced b) stirred mercury electrode (working electrode) c) auxiliary electrode (Pt-sheet) d) reference electrode (Ag/AgCl) e) diaphragma (2% agar, 3M KC1) - 2

The cathode compartment (a) contained a 10 iodotyrosine in 0.1N HC1.

M solution of di-

The potential measured between the

magnetically stirred mercury electrode (b) and the reference electrode (d) was -1.0 V.

During reduction a slow stream of

purified nitrogen was bubbled through the solution (a) to remove oxygen.

The course of the reduction could be followed by

polarography or TLC.

The reduction of di-iodotyrosine to mono-

iodotyrosine was complete after 2 hours.

Di-iodotyrosine con-

taining peptides were dissolved in dioxane/0.1N HC1 = 30/70 (v/v).

The extent of reduction was determined by TLC or

polarography.

Fig. 2 shows the CCD pattern of a mixture of

two tetrapeptides, Fig. 3 the analytical separation by HPLC.

In conclusion, the electrolytic reduction at controlled

79 CMA J

Cb O

o

c

O O

O

14

18

o

J

Trt - G l y - P h e - P h e - T y r - O H

o

j Trt-Gly-Phe-Phe-Tyr-OH

0

2

Figure 2.

4

6

8

10

12

16

O

20

22

24

26

28

Mono

Di

CCD pattern of a mixture of Trt-Gly-Phe-Phe-Tyr (I)OH (K= 0.5) and Trt-Gly-Phe-Phe-Tyr (I)2"0H (K=0.25) in a system of toluene/chloroform/methanol/water (5:5:8:2); 80 transfers D

Figure 3. HPLC pattern of a mixture of Trt-Gly-Phe-Phe-Tyr(I) • OH and Trt-Gly-Phe-Phe-Tyr(I)„-OH Elution system: methanol/water (90:10) Flow rate: 1 ml/min Column: NUCLEOSIL R C., g Length: 250tnm , 0: 4.6 mm Detection: UV (2 80 nm)

80

potential of di-iodotyrosine and derivatives gives the raonoiodotyrosine compounds in good yields.

The method works with-

out reducing reagents which would necessitate subsequent separation.

The potential of -1.0 V against the Ag/AgCl-

electrode must be constant because further reduction to tyrosine occurs at higher potential.

The products chosen were:

H-Tyr(I)2~0H H-Gly-Phe-Phe-Tyr(I)2-0H

(B23-26)

H-Gly-Phe-Phe-Tyr(I)2(But)-Thr-(But)-Pro-Lys(Boc)-Ala(OBut) (B23-30) The reduction of the di-iodotyrosine peptides was complete after 4 hours, purification, if necessary, could be achieved either by CCD or HPLC.

References 1.

Pitt-Rivers, R.V.rChem. Ind. 2J (1956).

2.

Simpson, G.K. and Traill, D.: Biochem. J. 40, 116 (1946).

3.

Lingane, J.J.: "Electroanalytical Chemistry", Second Edition, Interscience Publishers, Inc. New York (1958).

ALTERNATIVE APPROACH FOR HUMAN PROINSULIN SYNTHESIS

N. Yanaihara, M. Sakagami and C. Yanaihara Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka 422, Japan

Introduction Evidence

has

been accumulated that s y n t h e s i s of the l i n e a r polypeptide

comprising 86 amino acid r e s i d u e s , based on the proposed primary s t r u c t u r e of human p r o i n s u l i n (1, 2), followed by i t s a i r oxidation leads to the formation of the molecule which i s c h a r a c t e r i s t i c of p r o i n s u l i n having both i n s u l i n and C-peptide immunoreactivities (3, 4).

The human proin-

s u l i n C-peptide radioimmunoassay which had been developed in our laboratory (4, 5) was e f f i c i e n t l y used f o r immunological evaluation of synthetic human p r o i n s u l i n preparation and i t s intermediates.

As we have already

a

reported (3, 4 ) , 10 N - Z - or Boc-protected peptide azides were used as a c y l a t i n g agent in the stepwise fragment condensation f o r the s y n t h e s i s of human p r o i n s u l i n .

In the present study, the amino acid sequence of

human p r o i n s u l i n was divided into 16 fragments, and Boc group was s o l e l y selected for the protection of the a-amino group.

Thus, 16 Boc-protected

peptides were newly prepared and used in the stepwise fragment condensation for construction of the entire sequence of human p r o i n s u l i n . Naithani et a l . (6) have reported the synthesis of human p r o i n s u l i n by d i f f e r e n t approach using two big fragments i n the f i n a l

coupling.

Synthesis of Hexaoctacontapeptide Related to Human P r o i n s u l i n A l t e r n a t i v e s y n t h e s i s of human p r o i n s u l i n was attempted using Boc-protected peptide fragments comprising l e s s than 9 amino acid residues.

As

shown in Figure 1, 15 Boc-protected peptide hydrazides corresponding to human p r o i n s u l i n (HP) fragments (1-8, 9-16, 17-23, 24-30, 31-34, 35-38,

© 1980 Walter de Gruyter & Co., Berlin • New York Insulin, Chemistry, Stucture and Function of Insulin and Related Hormones

82

Figure 1.

(Positions

1- 8) : Boc-•Phe-Val-Asn-Gln-His-Leu-Cys(Bzl)-Gly-NHNHj

(1)

(Positions

t 9-16) : Boc- Ser-His-Leu-Val-Glu(OBu )—Ala-Leu-Tyr—NHNH^

(2)

1 (Positions 17-23) : Boc-•Leu-Val-Cys(Bzl)-Gly-Glu(OBu )-Arg-Gly-NHNHj

(3)

(Positions 24- 30) : Boc-•Phe-Phe-Tyr-Thr-Pro-Lys(Tos)-Thr-NHNH2

(4)

1 (Positions 31- 34) : Boc- Arg-Arg-Glu(OBu )-Ala-NHNHj

(5)

(Positions 35-•39) : Boc-•Glu(OBu1)-Asp(OBu1)-Leu-Gln-NHNHj

(6)

(Positions 39-•41) : Boc-•Val-Gly-Gln-NHNHj

(7)

(Positions 42-•45) : Boc-•Val-Glu-Leu-Gly-NHNHj

(8)

(Positions 46-•51) : Boc--Gly-Gly-Pro-Gly-Ala—Gly-NHNHj

(9)

(Positions 52-•60) : Boc--Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-NHNHj

(10)

{Positions 61- 66) i Boc--Ser-Leu-Gln-Lys(Tos)-Arg-Gly-NHNH^

(11)

(Positions 67-•70) : Boc-•Ile-Val-Glu(OBu1)-Gln-NHNHj

(12)

(Positions 71-•74) : Boc- Cys(Bzl)-Cys(Bzl)-Thr-Ser-NHNH2

(13)

(Positions 75- 77) : Boc--Ile-Cys(Bzl)-Scr-NHNH2

(14)

(Positions 78- 81) : Boc-•Leu-Tyr-Gln-Leu-NHNHj

(15)

(Positions 82- 86) : Boc-•Glu(OBu')-Asn-Tyr-Cys(Bzl)-Asn-OH

(16)

Protected peptide fragments used for the synthesis of human proinsulin

39-41, 42-45, 46-51, 52-60, 61-66, 67-70, 71-74, 75-77 and 78-81) were prepared, of which those having the amino acid sequences corresponding to HP(1-8), HP(9-16) and HP(71-74) are the same fragments as used in the previous synthesis of human proinsulin (3, 4).

The other 12 N a -Boc-

protected peptide hydrazides were newly synthesized mainly in stepwise manner by the N-hydroxysuccinimido ester method or the mixed anhydride method.

The e-amino group of Lys residue was blocked with tosyl

(Tos)

group and the thiol function of Cys residue with benzyl (Bzl) group. The Boc group and the tert-butyl

(Bu t ) protection for the side chain of Asp or

Glu residue were removed with anhydrous TFA in the presence of ethane dithiol

(7).

As described previously, sodium in liquid ammonia treatment

was planned for removal of the Tos and Bzl protecting groups from the protected peptide having the entire amino acid sequence of human proinsulin.

Purity of the protected peptide fragments was assessed by tic in

different solvent systems, elementary analysis and amino acid analysis of their acid hydrolysates. Using the 15 protected peptide hydrazides, peptide chain was elongated according to the azide fragment condensation in solution starting from the

83 HP( 71 -86): HP{67-86): HP(61 -36): HP(52-86): HPI46-86): HP(42-86): HP(39-86): HP05-86): HPOl-86): HP07-86): HP( 9-86): HP( 1-8«): Figure 2.

^

Outline of the method for construction of protected hexaoctacontapeptide having the proposed amino acid sequence of human proinsulin

C-terminal pentapeptide (positions 82-86) in a manner similar to that employed for the previous synthesis (Figure 2).

This kind of strategy

using peptide fragments with minimum protection has been employed for the synthesis of ribonuclease T-| (8).

Such protected tri- or tetrapeptide

hydrazides as used in the present study could readily be purified.In addition, although these small peptides were used for acylation in large excesses, unreacted acylating agents could be removed after the coupling reaction without difficulty.

For purification of the Boc-depro-

tected peptide intermediates, Sephadex LH-20 or LH-60 chromatography was exclusively employed using dimethylformamide or a mixture of dimethylformamide and dimethylsulfoxide (5:1) as solvent, since the

intermedi-

ates prepared in the present synthesis were sparingly soluble in all the aqueous solvents.

Figure 3 shows elution profiles of the Boc-deprotected

peptides having the sequences corresponding to HP(24-86), HP(17-86) and HP(9-86) from Sephadex LH-60 column with a mixture of dimethylformamide and dimethylsulfoxide (5:1).

Purity of the Boc-deprotected peptide

intermediates was examined by ami no-terminal determination by the dansyl method (9) and amino acid analysis of their acid hydrolysates (Tables 1 and 2).

Content of the SH group in the fully deblocked HP(9-86) was

determined spectrophotometrically using N-ethylmaleimide according to the method of Leslie et al. (10).

The observed SH content in the peptide was

4.39 against theoretical content 5.

The Boc-deblocked hexaoctaconta-

84-

*>

* Column 2.5* 05cm OMF D M S O - 5 1 Malarial 3SSmg 4 175 ms

| 1 1

i

1 H

ll II

" B4m

®

11

Vt

« 1 V 10

Figure 3.

20

30

. A .

40

SO

cm < CoiiMnn OMF tXXISO— 5 11 WMaMfMl n m

1 | 1

Column 2 * 95 cm DM*:DM&O* S 1 Mat«.ill i t m n «

33t-«W Ml

1

a

\

*

m 0

20

30 .

IV (G 1 2 _ 1 5 ) V

(G

8-11)

[a ] j"0

Calcd., %

analyses of

the

Found, %

H 7.26

N 12.37

C 61.83

H 7 .38

N 12.39

230

-38 .5

+

C 62.18

160-162

-26 .5 +

55.17

6.51

10.73

54.76

7 .28

10.28

106-107

- 1 .0 +

53.75

6.27

13.76

53.32

6 .14

13.79

177-178

-15 .5 +

59.18

5.92

6.64

59.49

5 .87

6.36

112-114

- 9 .0 +

62.52

5.55

5.61

62.68

5 .53

5.70

VI (G 5 _

?)

140-142

+11 .5 +

66.73

7.00

6.49

67.31

6 .97

6.64

VIKG

4)

152-154

-12 .0 +

54.99

6.71

11.66

55.15

6 .65

11.38

+

2_

C = 1,

CH3OH

The p r o t e c t e d n o n a c o s a p e p t i d e - r e s i n was o b t a i n e d a f t e r ment w i t h HF/10% a n i s o l e under s t r i c t l y (2)

i n almost q u a n t i t a t i v e

T a b l e 2. Amino Acid

anhydrous

yield.

Amino A c i d C o m p o s i t i o n

+ •

Resin--bound Octadecapeptide Derivative

R e s i n - •bound Nonacosapeptide Derivative

Theory

Theory

Asp Thr Ser Glu Gly Ala Val Met Leu Tyr Phe Lys His Arg * Trp not

Found

3 1 1 2

3.14 1 .09 0 . 85 2.21

1 1 1 2 1 1 1

1 .07 1 .03 0.78 1 .78 0.86 1 .06 0.97

2

1 . 80

determined

treat-

conditions

4 3 4 3 1 1 1 1 2 2 2 1 1 2

Found 4.39 2.81 3.48 3.67 0.94 1 .33 1 .27 1 .07 2 .00 1 .59 2.10 0.97 1 .01 2.13

Pure Synthetic Product Found 3.85 2.88 3.69 3.06 1 .00 1 .00 1 .00 1 .09' 2 .00 2.10 1 .90 1 .00 1 .05 1 .85

94 The cleavage product from the resin

(50 mg

for each batch)

was then purified through a Sephadex G-25 column using dilute acetic acid as eluant.

An unexpectedly large amount of sub-

stance appeared in Peak A which might possibly be the polymerized product formed during the HF-treatment, or an aggregated form of the synthetic product, the properties of which have not yet been ascertained.

Peak B was comprised mainly of

the desired product, synthetic glucagon, as indicated by PAGE, and amounted to 22.5 mg

(45% recovery).

A minute amount of

impurities with smaller molecular weights appeared in Peak C as shown in Figure 1.

Figure

1.

Ordinate: — — — — —

Gel-filtration of synthetic crude product on Sephadex G-25. UV (280 nm) absorption;Abscissa: Tube number chromatographic behaviour of synthetic crude product chromatographic behaviour of natural glucagon

K> 30 30 Figure 2. DEAE-cellulose chromatography of synthetic glucagon Ordinate: UV(280 nm) absorption; Abscissa: Tube number

95

1. Nat. Glucagon II.

I

Part. Purified Qlu-ca^on

111- Syn. Glucagon jy. Part. Purified G-lltcagon

Figure 3.

Polyacrylaraide gel electrophoretogram of purified synthetic glucagon.

The partially purified sample (10 mg for each batch) was further fractionated through a DEAE-cellulose column with a salt gradient (0.0 M to 0.1 M NaCl) in 6 M urea-Tris buffer (pH 8.5), or preferably with gradient elution from 0.03 M to 0.6 M NH^HCO^. The main peak II (Fig. 2), which appeared at the same position as authentic glucagon, was found to be homogeneous and indistinguishable from the latter by PAGE (Fig. 3). The fractions required were pooled and lyophilized several times to give about 2.5 mg of white fluffy product (25% recovery). The overall yield calculated from the beginning was about 10.5%. The above-purified sample was dissolved by warming in a solvent mixture of the following composition: 0.1 M NH.HCO, containing O

Q • e

a.

2

0 ' •

Q

«ì

o

•

' •

«

O

O

••

i®

»

'o

a

0

0 o

•HHSHHhbBI Figure 4.

•Ob

a