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JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 18B

electrophoresis a survey of techniques and applications part B: applications

JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1

Chromatography of Antibiotics, by G.H. Wagman and M.J. Weinstein

Volume 2

Extraction Chromatography, edited by T. Braun and G. Ghersini

Volume 3

Liquid Column Chromatography. A Survey of Modern Techniques and Applications, edited by Z. Deyl, K. Macek and J. Jangk

Volume 4

Detectors in Gas Chromatography, by J.ievEik

Volume 5

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods, by N.A. Parris

Volume 6

Isotachophoresis. Theory, Instrumentation and Applications, by F .M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen

Volume 7

Chemical Derivatization in Liquid Chromatography, by J.F. Lawrence and R.W. Frei

Volume 8

Chromatography of Steroids, by E. Heftmann

Volume 9

HPTLC - High Performance Thin-Layer Chromatography,edited by A. Zlatkis and R.E. Kaiser

Volume 10

Gas Chromatography of Polymers, by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovs kaya

Volume 11

Liquid Chromatography Detectors, by R.P.W. Scott

Volume 12

Affinity Chromatography, by J. Turkovif

Volume 13

Instrumentation for HighPerformance Liquid Chromatography, edited by J.F.K. Huber

Volume 14

Radiochrornatography. The Chromatography and Electrophoresis of Radiolabelled Compounds, by T.R. Roberts

Volume 15

Antibiotics. Isolation, Separation and Purification, edited by M.J. Weinstein and G. H. Wagman

Volume 16

Porous Silica. I t s Properties and Use as Support in Column Liquid Chromatography, by K.K. Unger

Volume 17

76 Years of Chromatography - A Historical Dialogue, edited by L.S. Ettre and A. Zlatkis

Volume 18A Electrophoresis. A Survey of Techniques and Applications. Part A: Techniques, edited by Z. Deyl Volume 188 Electrophoresis. A Survey of Techniques and Applications. Part B: Applications, edited by Z. Deyl Volume 19

Chemical Derivatization in Gas Chromatography, by J. Drozd

Volume 20

Electron Capture. Theory and Practice in Chromatography, edited by A. Zlatkis and C.F. Poole

Volume 21

Environmental Problem Solving using Gas and Liquid Chromatography, by R.L. Grob and M.A. Kaiser

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 18B

electrophoresis a survey of techniques and applications part B: applications

editor Z.Deyl Physiological Institute, Czechoslovak Academy of Sciences, Prague

co-editors A. Cbrambach, E M . Everaerts and 2. Prusik

ELSEVIER SCIENTIFIC PUBLISHING COMPANY Amsterdam - Oxford - New York 1983

ELSEVIER SCIENTIFIC PUBLISHING COMPANY Molenwerf 1 P.O. Box 21 1,1000 AE Amsterdam, The Netherlands Distributors for the United States and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY INC. 52, Vanderbilt Avenue New York, N Y 10017

Library of Congress Cataloging in Publication Data

(Revised )

Main entry under title: Electrophoresis : a survey of techniques and applications. (Journal of chromatography library ; 18 A-B) CONTENTS: pt. A. Techniques.--pt. B. Applications. Go-editors pt. B: A. Ghrambach, F. Everaerts and 2. Prusik. ' I n c lud es bibliographic a 1 references and index. 1. Electrophoresis. I. Deyl, Zdenek. 11. Series. QW9.344345 541.3'7 79-22525 V.

.

ISB!'i 9-41:i-41721-4 pt. A ISBN 0-44.4-42114-9 [pt B )

.

ISBN 044442114-9 (Vol. 188) ISBN 0 4 4 4 4 1 6 1 6 1 (Series) 0 Elsevier Scientific Publishing Company, 1983 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, 1000 A H Amsterdam, The Netherlands

Printed in The Netherlands

V CONTENTS Contributors

..............................................................

XI

Introduction

..............................................................

XIV

.

.............................. ...........................................................

1 Alcohols and phenolic compounds ( Z . Deyl) References 2 . Aldehydes and ketones ( Z Referentes

1 7

. Deyl) ........................................

9 12

...........................................................

.

3 Carbohydrates ( Z . Deyl) ................................................ Introduction Mono- and o l i gosacchari des ........................................... Polysaccharides Sugar phosphates Polyhydric alcohols Amino sugars ......................................................... Acids and lactones of carbohydrates References ...........................................................

13 13 113 15 25 30

......................................................... ...................................................... .....................................................

..................................................

30

..................................

31 32

...................................... ............................................... ................................. ..................................... ...........................................................

35 35 35 36 39

. Deyl) .............................. ...........................................................

41 44

4 . Carboxylic acids (F.M. Everaerts) General considerations Separations via zone electrophoresis Separations via isotachophoresis References 5 . Steroids and s t e r o i d conjugates ( Z References 6

. Amines ( Z . Deyl) ....................................................... References

.

45 53

...........................................................

.

............................ ......................................................

7 Amino acids and t h e i r d e r i v a t i v e s ( Z Deyl) General aspects Amino acids n a t u r a l l y occurring i n proteins .......................... Amino acids a r i s i n g during post-translational reactions in proteins Amino a c i d d e r i v a t i v e s used i n sequence analysis Other amino acids and amino acid conjugates .......................... References

.....................

...........................................................

..

55 55 55 66

73 74 77

VI

.

. Prusik) ............... 81 Selection of the method of electromigration separation ............... 81 Zone electrophoresis ................................................. 84 I s o e l e c t r i c focusing ................................................. 94 97 I s o t ac hop ho res i s .................................................... Detection of pept des i n electromigration systems .................... 101 104 References ...... ....................................................

8 Peptides and s t r u c t u r a l analysis of proteins ( Z

9 . Gel electrophoresis and electrofocusing o f proteins ( e d i t e d by A Chrambach) 9.1. Usefulness of second-generation gel electrophoret 001s i n protein f r a c t i o n a t i o n ( A Chrambach) General aspects Optimal pH and solvent Optimal pore s i z e S t a t i s t i c a l l y defined molecular weight and net charge Objectively defined separation s t r a t e g y Preparative PAGE Gel electrofocusing Summary References ...................................................... 9.2. Membrane proteins. native (L.M. Hjelmeland) References 9.3. Membrane proteins. denatured ( H . Baumann and D Doyle) References 9.4. Protein membrane receptors ( U Lang) References 9.5. Steroid receptors ( S Ben-Or) References ................. 9.6. Cell surface antigens (R.A. Reisfeld and M.A. Pel egrino) References 9.7. Lysosomal glycosidases and sulphatases ( A . L . Fluharty) General aspects Separation and analysis of major enzyme subtypes Enzyme p u r i f i c a t i o n Enzyme microheterogeneity Enzyme subunit composition Biological processing of lysosomal enzymes Conclusion Acknowledgement References

.

........................................

.

.............. 109

.............. 110 .............. 110 ........................ .............. 111 ............................................... 112 ........... 113 ......................... 113 ................................................ 114 ............................................. 114 ......................................................... 115 ............

...............................

115

....................... 117 ...................................................... 119 . ............ 120 ..................................... ................. 123 . ............ ................. 125 .................................... ................. 127 . ................... ................. 129 ....................................

......... ...................................................... ............ ................................................. ................ ............................................. ....................................... ...................................... ...................... ...................................................... ................................................. ......................................................

139 141 146 149 149 149 150 151 152 153 154 154 154

VII

.

. Bonaventura and C . Bonaventura) ... 156

9.8.

Haemocyanins (M B r e n o w i t z . J

9.9.

..................................................... 158 Human haemoglobins (A.B. Schneider and A.N. S c h e c h t e r ) ........... 161 General a s p e c t s ................................................ 161 References

............................................. 161 ............................................. 163 164 References ..................................................... 9.10 . I s o e l e c t r i c f o c u s i n g o f immunoglobulins (M.H. Freedman) .......... 166 Acknowledgements ............................................... 169 169 References ..................................................... 9.11. C o n t r a c t i l e and c y t o s k e l e t a l p r o t e i n s (P . Rubenstein) ............ 172 175 References ..................................................... 9.12. P r o t e i n s o f c o n n e c t i v e t i s s u e ( Z . Deyl and M . Horakova) .......... 177 Col lagens ...................................................... 177 E l a s t i n ........................................................ 182 Connective t i s s u e p r o t e o g l y c a n s ................................ 182 S t r u c t u r a l studies

Functional studies

References 9.13.

.....................................................

M i c r o t u b u l a r p r o t e i n s (K.F. General aspects

S u l l i v a n and L

183

. W i l s o n ) .............. 185

................................................

185

T u b u l i n . t h e h e t e r o d i m e r i c s t r u c t u r a l subun t o f t h e m i c r o t u b u l e 186 Mu1 t i p l e t u b u l i n s : m o l e c u l a r s p e c i a l i z a t i o n f o r d i v e r s e 188 functions?

9.14.

9.15.

................................ .................... S i g n i f i c a n c e o f mu1t i p l e t u b u l i n s ......... .................... M i c r o t u b u l e a s s o c i a t e d p r o e i n s ........... .................... Acknowledgements ......... ..................................... Re f e rences ............... ..................................... P r o t e i n hormones (A.D. Rogol .................................... General aspects .......... ..................................... P r o l a c t i n ...................................................... Growth hormone ................................................. Human c h o r i o n i c g o n a d o t r o p i n ................................... References .....................................................

189 190 191 191 194 194 195 196 198 199

E l e c t r o p h o r e s i s o f plasma p r o t e i n s : a contemporary c l i n i c a l

............................................. 201 .............. 202 Acute phase p r o t e i n response ................................... 207 I m p a i r e d s y n t h e s i s and c a t a b o l i c l o s s o f plasma p r o t e i n s ....... 209 211 Conclusions .................................................... 212 References ..................................................... .

approach (M E n g l i ? ) Monoclonal and p o l y c l o n a l hypergammaglobulinaemia

VIII 9.16.

.

.......................... PAGE and SDS-PAGE ............................................ IEF ..........................................................

A l l e r g e n s (H Baer and M.C. Anderson)

.................................................... I mmunoe1e c t r o p hores is ........................................ References ...................................................

CIE. CRIE

.

213 214 214 215 216 216

10 G l y c o p r o t e i n s and g l y c o p e p t i d e s ( a f f i n i t y e l e c t r o p h o r e s i s ) (T.C.

Bbg-Hansen and J

. Hau) .........................................

.................................................... ...................................... I n t e r p r e t a t i o n o f t h e p a t t e r n s ..................................... D e t e r m i n a t i o n o f a f f i n i t y .......................................... Q u a n t it a t i o n o f m i c r o h e t e r o g e n e i t y forms ........................... O t h e r e l e c t r o p h o r e t i c methods ...................................... Biomedical a p p l i c a t i o n s ............................................ Acknowledgements ................................................... References ......................................................... General aspects

219

Description o f the technology

220

.

11 L i p o p r o t e i n s ( H

. P e e t e r s ) ............................................

General aspects Synthetic data

............................................ l i p o p r o t e i n p a t t e r n ............................................ developments ...................................................

13

.

227 235 241 248 249 250 253 253 253 255

The

262

References

.

.................................................... on l i p o p r o t e i n s .....................................

226

M e t h o d o l o g i c a l problems New

12

219

.........................................................

. G a b r i e l ) .................... ................................................... .........................................................

L i p o p o l y s a c c h a r i d e s (P.F. Coleman and 0

277 277 281

Acknowledgements

284

References

284

E l e c t r o p h o r e t i c e x a m i n a t i o n o f enzymes ( W

. O s t r o w s k i ) ................ 287

.................................................... 287 289 E l e c t r o p h o r e t i c s e p a r a t i o n o f enzymes .............................. P r e p a r a t i v e e l e c t r o p h o r e s i s ........................................ 290 298 A n a l y t i c a l s e p a r a t i o n o f enzymes ................................... Examination o f some physico-chemical p r o p e r t i e s o f enzymes ......... 325 References ......................................................... 333 General aspects

IX

.

14 Nucleotides. nucleosides. nitrogenous c o n s t i t u e n t s of nucleic acids ( S Zadra'iil)

.

........................................................ General aspects .................................................... Nucleosides and nucleotides ........................................ Oligonucleotides ................................................... 01 i gonucleotide sequence analysi s .................................. References ......................................................... .

.......................................... .................................................... Ri bonuclei c acids .................................................. Deoxyribonucleic acids ............................................. Sequence a n a l y s i s of nucleic acids ................................. References .........................................................

15 . Nucleic acids (S . Zadrazll) General aspects V

.................................................. .........................................................

341 341 343 348 353 357 361 361 363 375 383 388

16 . Alkaloids ( Z . Deyl)

395

References

399

................................................... .........................................................

17 . Vitamins ( Z . Deyl)

401

References

408

.............................................. 409 .................................................... 409 ...................................................... 409 P e n i c i l l i n s and cephalosporins ..................................... 411 Aminoglycoside and o t h e r carbohydrate a n t i b i o t i c s .................. 412 Peptide a n t i b i o t i c s ................................................ 414 Miscellaneous ...................................................... 416 417 References .........................................................

18. A n t i b i o t i c s ( V . Betina) General aspects Bioautography

.......................................... .........................................................

19 . Dyes and pigments ( Z . Deyl) References 20

.

419 434

Inorganic compounds (F.M. Everaerts a n d Th.P.E.M. Verheggen) ......... 437 General aspects .................................................... 437 Separations via zone electrophoresis ............................... 437 Separation via ion focusing ........................................ 439 Separations via isotachophoresis ................................... 439

................................... .........................................................

Separations in non-aqueous media

440

References

441

X

..........................

443

............................................................

447

.............................................

450

Contents o f E l e c t r o p h o r e s i s . P a r t A: Techniques S ubjec t in dex

Index o f compounds s e p a r a t e d

XI

CONTRIBUTORS H. Baumann, Department of Molecular Biology, Rosewell Park Memorial I n s t i t u t e , 666 Elm S t r e e t , Buffalo, NY 14263, U.S.A. S. Ben-Or, Department of Physiology, Hadassah Medical School , The Hebrew

University, Jerusalem, I s r a e l V . Betina, Department of Chemistry and Technology o f Environment, Faculty of Chemical Technology, Slovak Technical University, JBnska 1, 880 37 B r a t i s l a v a ,

Czechoslovakia T.C. B6g-Hansen, The Protein Laboratory, University of Copenhagen, 34 Sigurdsgade, DK 2200 Copenhagen N , Denmark

C. Bonaventura, Department o f Biochemistry and Marine Biomedical Center, Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A.

J . Bonaventura, Department o f Biochemistry and Marine Biomedical Center, Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A. M. Brenowi t z , Department of Biochemistry and Marine Biomedical Center, Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A.

A. Chrambach, Endocrinology and Reproduction Research Branch, National I n s t i t u t e f o r Child Health and Human Development, National I n s t i t u t e s of Health, Bethesda, MD 20205, U.S.A. Z. Deyl, I n s t i t u t e o f Physiology, Czechoslovak Academy of Sciences, Videsski 1083, 142 20 Prague 4, Czechoslovakia D. Doyle, Department of Molecular Biology, Rosewell Park Memorial I n s t i t u t e , 666 Elm S t r e e t , Buffalo, N Y 14263, U.S.A.

M. Engli:, Department of Clinical Biochemistry, Thomayer's Hospital, VidefiskZ 800, 142 00 Prague 4 , Czechoslovakia F.M. Everaerts, Department o f Instrumental Analysis, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands

XI1

A.L. F l u h a r t y , Mental R e t a r d a t i o n Center Group a t Lanterman S t a t e H o s p i t a l , UCLA School o f Medicine, P.O.

Box 100-R,

Pomona, CA 91766, U.S.A.

M.H. Freedman, F a c u l t y o f Pharmacy and I n s t i t u t e o f Immunology, U n i v e r s i t y o f Toronto, T o r o n t o M5S 1A1, Canada

L.M. Hjelmeland, Developmental Pharmacology Branch, N a t i o n a l I n s t i t u t e o f Chi 1d H e a l t h and Human Development , N a t i o n a l I n s t i t u t e s o f H e a l t h , Bethesda, MD 20205, U.S.A. M. H o r i k o v z , I n s t i t u t e o f P h y s i o l o g y , Czechoslovak Academy o f Sciences,

VideEski 1083, 142 20 Prague 4;

Czechoslovakia

U. Lang, B i o l o g y o f Growth and Reproduction, Department o f P e d i a t r i c s and Genetics , Uni v e r s i t y o f Geneva , Medical School , Geneva , S w i t z e r l a n d W. Ostrowski,

I n s t i t u t e o f M e d i c i n a l Chemistry, N i k o l a u s Copernicus Academy

o f Medicine, u l . Kopernika 7, 3 1 034 Krakow, Poland

H. Peeters, Department o f L i p i d s and P r o t e i n s , I n s t i t u t e f o r M e d i c i n a l B i o l o g y , Alsembergsesteenweg 196, 1180 B r u s s e l s , Belgium

M.A.

P e l l e g r i n o , Department o f M o l e c u l a r Immunology, S c r i p p s C l i n i c and

Research Foundation, 10666 N. T o r r e y Pines Road, La J o l l a , CA 92037, U.S.A.

Z . P r u s i k , I n s t i t u t e o f Organic Chemistry and B i o c h e m i s t r y , Czechoslovak Academy o f Sciences, Flemmingovo nim. 2, 166 10 Prague 6, Czechoslovakia R.A.

R e i s f e l d , Department o f M o l e c u l a r Immunology, S c r i p p s C l i n i c and Research

Foundation, 10666 N. T o r r e y Pines Road, La J o l l a , CA 92037, U.S.A. A.D.

Rogol, Department o f P e d i a t r i c s , U n i v e r s i t y o f V i r g i n i a , Medical Center,

C h a r l o t t e s v i l l e , VA 22908, U.S.A. P. Rubenstein, Department o f B i o c h e m i s t r y , C o l l e g e o f Medicine, U n i v e r s i t y o f Iowa, Iowa C i t y , I A 52242, U.S.A.

A.N.

Schechter, L a u o r a t o r y o f Chemical B i o l o g y , N a t i o n a l I n s t i t u t e o f

A r t h r i t i s , Metabolism and D i g e s t i v e Diseases, N a t i o n a l I n s t i t u t e s o f H e a l t h , Bethesda, MD 20205, U.S.A.

XI11 A.B.

Schneider, Michael Reese H o s p i t a l and Medical Center, Chicago,

IL 60616, U.S.A. K.F. S u l l i v a n , Department o f B i o l o g i c a l Sciences, U n i v e r s i t y o f C a l i f o r n i a , Santa Barbara, CA 93106, U.S.A. Th.P.E.M.

Verheggen, Department o f I n s t r u m e n t a l Analysis, Eindhoven U n i v e r s i t y

o f Technology, 5600 MB E i ndhoven, The Nether1 ands

L. Wilson, Department o f B i o l o g i c a l Sciences, U n i v e r s i t y o f C a l i f o r n i a , Santa Barbara, CA 93106, U.S.A. S. Z a d r a z i l ,

I n s t i t u t e o f M o l e c u l a r Genetics, Czechoslovak Academy o f Sciences,

Flemmingovo nim. 2, 166 10 Prague 6, Czechoslovakia

XIV INTRODUCTION The p r e s e n t work i s t h e second p a r t o f a t w o - p a r t s e t devoted t o e l e c t r o m i g r a t i o n t e c h n i q u e s and t h e i r a p p l i c a t i o n s . P a r t A d e a l s w i t h t h e p r i n c i p l e s , t h e o r y and i n s t r u m e n t a t i o n o f t h e s e t e c h n i q u e s . P a r t

B i s concerned w i t h t h e

d e t a i l e d a p p l i c a t i o n s o f e l e c t r o m i g r a t i o n methods t o many d i v e r s e c a t e g o r i e s o f compounds. A d e t a i l e d c o n t e n t s o f P a r t A i s l i s t e d on pages 443

-

446.

1

Chapter 1

ALCOHOLS AND PHENOLIC COMPOUNDS Z. DEYL

F o r obvious reasons, a1 cohol s have t o be c o n v e r t e d i n t o s u i t a b 1 e d e r i v a t i v e s p r i o r t o e l e c t r o n h o r e s i s . T h i s c o u l d be done by r e a c t i o n w i t h 2 - f l u o r o - 1 - m e t h y l 1 I n t h e presence o f dioxane t h e c o r r e s p o n d i n g P y r i d i n i u m p - t o 1 uenesul phonate

.

2 - a l k o x y d e r i v a t i v e s a r e formed. The r e a g e n t i s a l l o w e d t o r e a c t w i t h a l c o h o l s i n t h e presence o f t r i d o d e c y l a m i n e . The s e p a r a t i o n o f most a l c o h o l d e r i v a t i v e s can be achieved a t pH 1.7 i n a b u f f e r c o n s i s t i n g o f 0.05 m o l / l o r t h o p h o s p h o r i c a c i d and 0 . 1 m o l / l o r t h o b o r i c a c i d , t o which i s added 10% ( v / v ) o f e t h y l e n e g l y c o l . Detection i s e f f e c t e d w i t h Dragendorff's reagent. E l e c t r o p h o r e t i c separation i s a r o u t i n e h i g h - v o l t a g e run. I n a d d i t i o n t o a l c o h o l s i t i s a l s o p o s s i b l e t o separ a t e e s t e r s b y t h i s t e c h n i q u e ; t h i s r e l a t e s t o t h e i d e n t i f i c a t i o n o f a l k o x y groups o f e s t e r s ( T a b l e 1.1).

TABLE 1.1 CHARACTERIZATION OF ALCOHOLS AS 2-ALKOXY-1-METHYLPYRIDINIUM p-TOLUENESULPHONATES Mobi 1 it y o f t h e d e r i v a t i ve (relative t o 2-fluoro-lm e t h y l p y r i d i n i u m x 100)

A1 cohol

Methanol Ethanol Propan- 1-01 Propan-2-01 Butan-1-01 2-Methyl propan-1-01 Butan-2-01 Pentan-1-01 3-Methyl butan- 1-01 3-Me t h y 1b u t an- 2 o 1 Hexan-1-01 Cyclohexan-1-01 2-Ethylhexan-1-01 Nonan-1-01 Decan-1-01 2-Ethoxyethanol 2-Propen-1-01 Pheny 1met hano 1 2-Phenylethanol

-

90 82 76 75 70 70 70 67 66 67 61 55 51 48 45 70 79 63 60

2

I n addition t o t h e 2-alkoxy-1-methylpyridinium s a l t s , d e r i v a t i z a t i o n of alcohols could be done in two other ways: the xanthate and S-alkylation o f thiourea with alcohols 4 (Table 1.2). Results f o r the separation of sulphur analogues ( t h i o l s ) a r e given i n Table 1.3. TABLE 1.2 RELATIVE RATES OF MIGRATION OF XANTHATE IONS Compounds were detected a f t e r paper electrophoresis i n sodium hydrogen carbonate solution (0.1 m l / l ) a t 21 V/cm and 4OC f o r 1 h . For each parent compound, the molecular weight of the derived xanthate ion i s given. Parent compound Aliphatic alcohols 1. Methyl 2. Ethyl 3. n-Propyl 4. Isopropyl 5. n-Butyl 6. Isobutyl 7. sec.-Butyl 8. n-Amy1 9. Isoamyl 10. n-Hexyl 11. Cyclohexyl 12. n-Heptyl 13. n-Octyl 14. Capryl (octan-2-01) 15. n-Nonyl 16. n-Decyl 17. Benzyl Unsaturated aZcohoZs la. Aiiyi 19. a-Furfuryl Glyco 2 e t h e r s 20. 2-Met hoxye t h ano 1 (methyl Cellosolve) 21. 2- E t hoxye t h an o 1 (ethyl Cellosolve)

Derived ion M m X 100

143 122

ioa

111 100 97 100

aa aa

a4 86 76 63 65 54 49 a7

No1 . w t .

107 121 135 135 149 149 149 163 163 177 175 191 205 205 219 233 183 133 173

111

151

101

165

90

195

a3

209

22. 2-(2-Methoxyethoxy)ethanol (methyl carbi to1 )

23. 2-(2-Ethoxyethoxy)ethanol (ethyl c a r b i t o l )

3

TABLE 1.3 CHARACTERIZATION OF THIOLS AS 2-ALKYL(ARYL)THIO-l-METHYLPYRIOINIUM p-TOLUENESULPHONATES pH 7.6 b u f f e r : 0.5 mol/l b o r i c a c i d , 0.1 mol/l d i e t h a n o l a m i n e and 0.025 mol/l disodium s a l t o f ethylenediaminetetraacetic a c i d . Run

R i n thiol

Derived c a t i o n

(M, x 1

Methyl Ethyl n- P r o p y l Isopropyl n-Butyl I sobutyl tert.-Butyl n-Amy1 Isoamyl n-Hexyl Cycl ohexyl n-Nonyl n-Dodecyl Benzyl Phenyl 2 Pyr idy 1 p-Cresyl 2-Naphthyl A1 l y l Diethyldithiophosphate

2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

-

100 93 87 88 76 76 79 71 70 62 94 37 16 64 71 75 63 45 87 19

Sensitivity l i m i t b

(pmol)

1000 750 500 500 500 500 500 500 500 500 1000 500 500 250 500 1000 750 750 250

500

a

v a l u e s express m o b i l i t i e s r e l a t i v e t o t h e 2-methylthio-1-methylpyridinium i8n; w h i c h moved a p p r o x i m a t e l y 23 cm. b S e n s i t i v i t y l i m i t d a t a are, i n f a c t , t h e combined e f f e c t s of two procedures, f o r m a t i o n of t h e c a t i o n and d e t e c t i o n .

M

E l e c t r o p h o r e t i c s e p a r a t i o n s of p h e n o l i c compounds a r e r a r e . I n o r d e r t o o b t a i n charged groups i n t h e s e compounds, t h e s e p a r a t i o n s a r e c a r r i e d o u t above'pH 7 when p h e n o l i c groups a r e a t l e a s t p a r t l y i o n i z e d . E x c e p t i o n s e x i s t , however. F o r example, i t i s p o s s i b l e t o r u n s e p a r a t i o n s a t pH 5.2 w i t h sodium a c e t a t e o r ammonium molybdate as t h e s e p a r a t i o n b u f f e r . The a d d i t i o n o f i n o r g a n i c o x y a c i d s , e.g.,

b o r i c a c i d , serves t h e same p.urpose.

I n the l a t t e r instance polyphenolic

compounds ( t h o s e w i t h OH groups i n t h e ortho p o s i t i o n ) f o r m n e g a t i v e l y charged complexes. The c a p a b i l i t y f o r complex f o r m a t i o n can a l s o be used f o r t h e determinat i o n o f an unknown s t r u c t u r e t o g e t h e r w i t h s p e c i f i c c o l o u r r e a c t i o n s . Most s e p a r a t i o n s a r e r u n on c o n v e n t i o n a l chromatographic papers a t about 10-20 V/cm ( w i t h c o o l i n g of t h e paper sheet o r s t r i p a t h i g h e r v o l t a g e s ) .

Running t i m e s

4

TABLE 1.4 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME PHENOLS5 a

Compound

Mu

p-Bromophenol o-Chlorophenol m-Chl orophenol p-Chl orophenol o-Cresol m-Cresol p-Cresol 2 ,2 ' - D i hy d r o x y d ip heny 1 2,3-Di hydroxynaphthalene 2,6-Di hydroxynaphthalene 2,7-Di hydroxynaphthalene Hydroquinone 1-Hydroxyanthraquinone 2-Hydroxybenzyl a l c o h o l o-Hydroxybiphenyl Hydroxyhydroquinone p-Iodophenol 1-Naphthol 2-Naphthol o-Ni t r o p h e n o l m-Ni t r o p h e n o l p-Ni t r o p h e n o l p-Ni t r o t h i o p h e n o l Phenol Py r o c a t e c ho 1 Phlorogl u c i n o l Pyrogal l o 1 Resorcinol Salicylic acid Sal i c y l a l d e h y d e p-Thi ocresol 1,2,5-Xylenol 1,3,5-Xylenol 1,4,5-Xylenol

122 144 125 127 120 101 110 107 82 16 1 143 161 44 153 65 204 (190d) 112 100 89 145 125 117 139 126 181 233 (248d) 193 f221d) 189 162 138 160 96 98 94

X

100

Colour

b

Ye1 1ow-orange Yellow Yellow Rose Orange-ye1 1ow Go1 den ye1 1ow Rose Yellow Brown Brown Brown v i o l e t Brownish y e l l o w Red Go1 den ye1 1ow Orange Brown Yellow Carmine r e d Orange Orange-ye1 1owc Ye1 1owc Ye1 1owc Ye1 1owc Lemon ye1 l o w Violet Ye1 l o w brown Brown Ye1 1ow Violet Lemon y e l l o w Yellowe Orange Go1 den y e 1 1 ow Orange

a0.05 m o l / l NaOH; S c h l e i c h e r and S c h i i l l 2043b paper; 20-25 V/cm; 20-30 min; b m o b i l i t y r e l a t i v e t o 1-naphthol. Colour produced w i t h d i a z o t i z e d s u l p h a n i l i c a c i d u n l e s s s t a t e d o t h e r w i s e . :Natual c o l o u r o f compound. 2% Na25.03 added t o r e t a r d o x i d a t i o n . Change i n m o b i l i t y i s due t o change i n ptH o f e l e c t r o l y t e s o l u t i o n on a d d i t i o n o f Na2S03. eColour developed w i t h l e a d a c e t a t e .

5

TABLE 1.5 RELATIVE

ELECTROPHORETIC

MOBILITIES

Compound

Caffeic acid Catechol Chlorogenic a c i d p-Coumari c a c i d 3,4-Di hydroxyphenyl a1 a n i ne Ferulic acid Gallic acid Hyd r oq u inone p-Hydroxybenzaldehyde o-Hydroxybenzoic a c i d a b s o l u t e m i g r a t i o n (cm/h/1200 V) m-Hydroxybenzoic a c i d p-Hydroxybenzoic a c i d Hydroxyhydroqui none 4-Methyl umbel 1iferone Phloroglucinol Protocatechuic a c i d Pyrogal l o 1 Resorcinol Syringaldehyde Umbel 1iferone (7-hydroxycoumarin) Vanillin Flavonoid d e r i v a t i v e s Aescul i n Arbutin Catechi n Genistein I r igeni n Kaempherol Luteolin 4-Methyl a e s c u l e t i n Myri c e t i n Phloridzin Q u e r c e t in

OF SOME PHENOLIC

COMPOUNDS~a

R e l a t i v e m o b i l i t y i n b u f f e r x 100

lb

2c

3d

4e

5f

20 0 33 34 0 21 45 0 0 100 8.8 81 64 0 0 0 50 0 0 0 0 0

79 107 103 44 81 21 103 0 0 100 8.8 79 70 107 0 0 100 989 0 0 0 0

50 1 44 58 1 42 59 1 279 100 8.0 85 77 1259 0 1 67 1 1 21 4 27

104 67 83 98 72 87 120 9 849 100 9.7 105 121 1209 52 127 119 759 .~ 35 71 76 79

105 51 74 86

9 799 100 10.8 95 110 1169 50 105 102 9 44 72 63 76

0 0 0 0 0 0 0 0 0 0 0

0 0 23 0 0 0 0 61 0 0 5

26 0 0 1 4 0 0 11 0 8 0

51 22 65 25 52 12 11 48 18 42 21

24 9 63 16 35 0 0 27 0 14 0

9 77 93

aWhatman No. 3 paper; 22 V/cm; 1.5-2 h; m o b i l i t y r e l a t i v e t o o-hydroxybenzoic baci d. 0.2 m o l / l acetate, pH 5.2. dc8.1 m o l / l molybdate, pH 5.2. 0.2 m o l / l phosphate, pH 7.2. e0.2 m o l / l borate, pH 10.0. f~ m o. l / l lg l y c i n e , p~ 10.0. %ompound decomposed o r " t a i l e d " . Some t a i l i n g due t o o x i d a t i o n .

6

TABLE 1.6 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME FLAVONOID COMPOUNDS7 a

Compound

Re1a t i ve m o b i l i t y x 100

Acacetin Afzelechin Ampeloptin Aromadendri n Butein Butin d- Cat e c h in epi-Catechi n epi-Catechin g a l l a t e Chrysin D a h l i a chalcone D i hydrorobi n e t i n 4 ' , 7 - D i h y d r o x y f l avanone Eriodictyol F is e t i n Fisetinidol Formononeti n Fustin Gal l o c a t e c h i n epi-Gal 1o c a t e c h i n epi-Gallocatechin g a l l a t e Genistein Hesperidin 4 ' - H y d r o x y f l avanone 7-Hydroxyhl avanone I some1a c a c i d i n Kaempferol Khell i n

3d 33 148 99 33 172 100 92 146 11 16 182 a4 154

38 104 25b 185 93 80 138 lb 3d 0 95 164 6 -30b ,C

Re1a t ive m o b i l i t y x 100

Compound Leucofi s e t i n id i n Leucorobi n e t i n i d i n Lutedin-7-91 u c o s i d e Ye1 a c a c i d i n Morin Myri c e t i n Naringin Phloridzin P i nobanksin P i nocembri n Quercetin Q u e r c e t r in Robi n e t i n R o b n e t i n i do1 Robinin Robtein Robtin Rutin Taxi f o l i n Tectochrysin

105 96 3d 171 3b 12 3d 67b 93 a5 25 9ab 24 96 2d 17 149 88b 164 0

3',4',5',7'-Tetrahydroxy84

flavan

3',4',5',7'-Tetrahydroxyf l avan-46-01 Tricin 3 ' ,4' , 7 ' - T r i h y d r o x y f l a v a n 3 ' ,4' ,7 ' - T r i h y d r o x y f l avan-06-01

88 -3ob,c 97 100

a

B o r a t e u u f f e r (12.6 g o f sodium b o r a t e and 3.1 g o f b o r i c a c i d p e r l i t r e ) , pH 8.8; S c h l e i c h e r and 'FchU11 2043b paper ( 4 x 4 1 cm); 0.31 mA/cm f o r 6 h; m o b i l i t y r e l b a t i v e t o d-catechin. Sodium t e t r a b o r a t e (9.54 g / l ) ; Whatman No. 4 paper (15 x 6 1 cm); 10 V/cm; 7 h; mobi 1 it y r e l a t i v e t o DNP-glycine. :Movement towards cathode; t h e compound d i d n o t move i n i t i a l l y . 2% Borax s o l u t i o n ; Toyo No. 50 paper; 1.5 V/cm; m i g r a t i o n i s movement i n m i l l i m e t r e s p e r 4 h. u s u a l l y do n o t exceed 4 h. Sodium h y d r o x i d e (0.05-0.1 m o l / l w i t h t h e a d d i t i o n of 2% Na2S03 f o r e a s i l y o x i d i z a b l e phenols such as hydroxyhydroquinone, p h l o r o g l u c i n o l and p y r o g a l l o l ) , sodium a c e t a t e (0.2 m o l / l , pH 5.2), (8.1

pH 5.2),

( 0 . 1 m o l / l , pH 10.0),

phosphate b u f f e r (0.2 m o l / l , pH 7.2), b o r a t e b u f f e r pH 8.8-10)

suitable electrophoresis

ammonium molybdate

glycinate buffer

and sodium t e t r a b o r a t e b u f f e r a r e

7

TABLE 1 . 7 ELECTROPHORETIC BEHAVIOUR OF SOME PHENOLIC COMPOUNDS’ S e p a r a t i o n on Whatman No. 1 paper i n 0 . 1 m o l / l NaOH a t 450-500 V f o r 90 min. ~

Compound Acaceti n 1-Anthiol Api geni n Astralgin Chrysi n 1,2- D i hyd r o x y a n t hraq u inone 1,4-Di h y d r o x y a n t h r a q u i none 6 , l l - D i hydroxynaphthacenequi none Escul i n 1-Hydroxyanthraquinone 4-Hydroxyxanthone 2-Hydro xy ca r b azo 1e 6-Hydroxychrysene 2-Hydroxydibenzofuran 0-Hydro xyd ip he ny 1 ,p-Hydroxydi phenyl 2 - H y d r o x y f l uorene 3 - H y d r o x y f l uorene 5-Hydroxyisoquinoline 7-Hydroxy-4-methyl coumari n 1- Hyd r o x y py r e ne 5-Hydroxyqui no1 ine 8-Hydroxyqui no1 ine Ma1 v i n o n Morin 1-Naphthol 2-Naphthol Robi n i n Techtochrysi n

M o b i l i t y r e l a t i v e t o 1-naphthol x 100 41 66 56 92 39 tailing tailing tailing 113 59 64 72 20 80 96 90 70 81 93 98 44 93 91 106 75 100 97 82 origin

Spots a r e r e v e a l e d w i t h i r o n ( I I 1 ) reagent, ammonical s i l v e r n i t r a t e s o l u t i o n , b i s d i a z o t i z e d b e n z i d i n e , v a n i 11 i n - t o 1 uene-p-sul phoni c a c i d r e a g e n t , d i a z o t i z e d p - n i t r o a n i l i n e o r d i a z o t i z e d s u l p h a n i l i c a ~ i d ~ -Many ~ . f l a v o n o i d compounds can be d e t e c t e d d i r e c t l y under u l t r a v i o l e t l i g h t ( f o r a r e v i e w , see r e f . 8 ) . Examples o f s e p a r a t i o n s a r e p r e s e n t e d i n Tables 1.4-1.7. REFERENCES

1 E. B a l d and 8. M a z u r k i e v i c z , Chromatographia, 13 (1980) 295. 2 H. Berbalk, Monatsh. Chem., 90 (1959) 24. 3 J.L. Fahn, J . Chromatogr., 37 (1968) 279. 4 B. Mazurkiewicz, Chem. Anal. (Warsaw), 24 (1979) 699.

8 5 6 7 8

H. Berbal k, Monatsh. Chern., 90 (1959) 198. J . Chrornatogr., 2 (1965) 396. D.R. Cooper and D.G. Roux, J . Chrornatogr., 17 (1965) 396. G. Zweig and J.R. Whitaker, Paper Chromatography and EZectrophoresis, Academic Press, New York, 1967, p. 302. 9 E. S a w i c k i , M. Guyer, R. Schumacher, R. E b e r t and C.R. Engel, Microchirn. Acta, (1968) 1025.

J.B. Pridham,

9

Chapter 2 ALDEHYDES AND KETONES Z. DEYL

The absence o f a n e t charge makes u n d e r i v a t i z e d aldehydes and ketones u n s u i t a b l e f o r e l e c t r o p h o r e t i c s e p a r a t i o n s , and t h e y s h o u l d be c o n v e r t e d i n t o s u i t a b l e charged d e r i v a t i v e s . D e r i v a t i v e s can be prepared w i t h hydrogen s u l p h i t e , benzenesulphohydroxamic a c i d , ( 4 - s u l phobenzyl ) x a n t h y d r a z i d e o r ( 8 - c a r b o x y e t h y l ) x a n t h y drazide. The advantage o f c o n v e r t i n g aldehydes i n t o hydroxamic a c i d s ' i s t h a t ketones do n o t i n t e r f e r e i n t h i s s e p a r a t i o n . The d e r i v a t i v e s a r e p r e p a r e d i n w a t e r o r aqueous methanol by h e a t i n g t h e aldehyde s o l u t i o n w i t h s o l i d benzenesulphohydroxamic a c i d . F i r s t , t h e r e a c t i o n m i x t u r e i s made a l k a l i n e w i t h 2 m o l / l NaOH t o o b t a i n t h e sodium s a l t o f benzenesulphohydroxamic a c i d . The e l e c t r o p h o r e t i c separ a t i o n i t s e l f i s c o n v e n t i o n a l zone e l e c t r o p h o r e s i s on paper: 0 . 1 m o l / l NaOH serves as t h e e l e c t r o p h o r e s i s b u f f e r . D e t e c t i o n i s e f f e c t e d by s p r a y i n g t h e d r i e d e l e c tropherograms w i t h i r o n ( I I 1 ) c h l o r i d e s o l u t i o n : hydroxamic a c i d s appear as v i o l e t s p o t s on a w h i t e background. B e n z e n e s u l p h i n i c a c i d , which i s formed as a b y p r o d u c t , can be e a s i l y d i s t i n g u i s h e d because o f i t s h i g h e r m o b i l i t y and y e l l o w c o l o u r on s p r a y i n g w i t h i r o n ( I I 1 ) c h l o r i d e s o l u t i o n . The r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s o f some hydroxamic a c i d d e r i v a t i v e s o f aldehydes a r e g i v e n i n Table 2.1.

TABLE 2 . 1 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME ALDEHYDES .4S HYDROXAFIIC A C I D DERIVATIVES A1 dehyde

Relative m o b i l i t y a x 100

A1 dehyde

Relative mobi 1 it y a x 100

Formaldehyde n-Butanal n-Pentanal Isopentanal n-Hexanal 2-Ethyl butanal n-Hep t a n a l n- Oc t ana 1

100 64 57 59 52 44 47 41

2- Et hy 1hexana 1 Decanal Isododecanal C r o t o n a l dehyde C it r a l Citronella1 Benzal dehyde Cinnamal dehyde

47 30 13 66 46 81 67 44

a M o b i l i t y r e l a t i v e t o formaldehyde; 0 . 1 rnol/l NaOH, S c h l e i c h e r and S c h i j l l 2043b c e l l u l o s e paper, 20 V/cm f o r 30 min. Movement i s towards t h e anode.

10 Another means o f s e p a r a t i n g aldehydes and ketones is t o r u n t h e e l e c t r o p h o r e s i s i n hydrogen s u l p h i t e - a c e t a t e b u f f e r a t DH 4.7 ( r e f . 2 ) . Conventional paper e l e c t r o p h o r e s i s i s used and t h e samples a r e s p o t t e d on t h e s t a r t e i t h e r as aqueous s o l u t i o n s i n t h e s e p a r a t i n g b u f f e r o r d i s s o l v e d i n e t h a n o l . The m o b i l i t i e s a r e usually given r e l a t i v e t o the m o b i l i t y o f v a n i l l i n . The aldehydes t h a t have moved towards t h e anode as h y d r o x y s u l p h o n i c a c i d s can be d e t e c t e d under UV 1 i g h t ( a r o m a t i c aldehydes) o r w i t h d i n i t r o p h e n y l h y d r a z i n e spray r e a g e n t (0.05% 2,4-dinitrophenylhydrazine i n 2 m o l / l HC1). The s p o t s can a l s o be r e v e a l e d w i t h s i l v e r n i t r a t e - s o d i u m e t h o x i d e reagent, prepared by d i l u t i n g s a t u r a t e d s i l v e r n i t r a t e s o l u t i o n 2 0 0 - f o l d w i t h acetone and adding w a t e r drop-wise t o d i s s o l v e t h e p r e c i p i t a t e formed. The e l e c t r o p h e r o g r a m i s d r i e d , d e t e c t e d by d i p p i n g i n t o t h e above s o l u t i o n , d r i e d a g a i n and sprayed

w i t h 0.5 mol/l NaOH ( i n aqueous e t h a n o l ) . Brown s i l v e r o x i d e i s produced. The excess o f s i l v e r o x i d e i s washed away by immersing t h e e l e c t r o p h e r o g r a m i n 6 m o l / l ammonia s o l u t i o n and washing i t w i t h r u n n i n g w a t e r f o r about 1 h b e f o r e d r y i n g . Reducing compounds appear as d a r k s p o t s on a w h i t e background. R e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s o f v a r i o u s 0x0 compounds i n hydrogen s u l p h i t e - a c e t a t e b u f f e r a r e g i v e n i n Table 2.2. TABLE 2.2 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME ALDEHYDES AND KETONES I N HYDROGEN SULPHITE-ACETATE BUFFER Compound

Re1a t i ve m o b i l i t y a x 100

3-A1dehydo-4-hydroxy-5-methoxybenzaldehyde ( f o r m y l vani 11i n ) Benzal dehyde Cinnamal dehyde C itral Cyclohexane-1,3-dione Cycl ohexanone D iacetyl 2,4-Di hydroxybenzal dehyde 3,4- Dih y d r oxy benz a 1d e hy de ( p r o t o c a t e c h u a 1de hy de ) 2,6-Di hydroxy-$-methyl benzal dehyde ( a t r a n o l ) 3,4-Dimethoxybenzaldehyde ( v e r a t r a l d e h y d e ) Furfural n- Hept anal o-Hydroxybenzal dehyde ( s a l ic y l a1 dehyde) m-Hydroxybenzal dehyde 4-Hydroxy-3,5-dimethoxybenzal dehyde ( s y r i n g a l dehyde) 2-Hydroxy-3-methoxybenzaldehyde ( o r t h o v a n i l l i n ) 4-Hydroxy-3-methoxybenzaldehyde ( v a n i 11i n ) w- Hydroxyme t hy 1 f u r f u r a l 3,4,5-Trimethoxybenzal dehyde

153 116 118 136 178 114 69 82 100 74 103 129 98 95 116 95 110 100 107 101

a M o b i l i t y r e l a t i v e t o v a n i l l i n ; 0 . 1 m o l / l hydrogen s u l p h i t e - a c e t a t e b u f f e r , pH 4.7; Whatman No. 3 paper; 13-18.5 V/cm f o r 3-5 h; room temperature.

TABLE 2 . 3 SEPARATION OF ALDEHYDES AND KETONES AS (4-SULPH0BENZYL)XANTHYDRAZONES AND

(0-CARB0XYETHYL)XANTHYDRAZONES

Data r e p r e s e n t m o b i l i t y r e l a t i v e t o acetone o r acetaldehyde x 100. Ketones

A1 dehydes

(4-Sulphobenzyl ) xanthydrazones Acetone Met hy 1 e t hy 1 ketone Met hy 1 n - p r opy 1 ketone Methyl n - b u t y l k e t o n e Methyl n - h e x y l ketone Cycl ohexanone A c e t y l acetone Acetophenone D i - n - b u t y l ketone Methyl n-amyl ketone Benzilideneacetophenone D i e t h y l ketone Benzi 1 ideneacetone Benzyl acetone Benzophenone Bromoacetophenone Reagent

100 93.5 84.5 75.5 62.2 73.6 109.0 51.2 56.5 69.3 0.0 84.2 37.0 43.0 0.0 29.0 165

( = 1 2 . 5 cm)

(56.4)

(64.5) (134.0) (128.0)

(0-Carboxyethyl ) xanthydrazones 100

(=11.0cm)

86.0 73.5 70.0 60.0 66.5 112.0 53.0 55.0 65.0 0.0 76.0 41.0 40.0 ( 7 6 . 0 ) 0.0 32.0 ( 7 3 . 0 ) 182

Acetaldehyde Propionaldehyde n - B u t y l a1 dehyde n-Val era1 dehyde Enantal dehyde Caoryl a1 dehyde Benzal dehyde p-Hethoxybenzal dehyde Cinnamaldehyde Vani 11i n p - D i m e t h y l aminobenzaldehyde D-Aminobenzaldehyde p-Chlorobenzaldehyde p-Ni t r o b e n z a l dehyde P h e n y l a c e t a l dehyde Reagent

(4-Sul phobenzyl ) xanthydrazones

( 8-Carboxyethyl ) xanthydrazones

100 (=11.0 cm) 95.0 89.0 84.5 79.2 76.5 64.2 57.5 51.6 0.0 0.0 44.5 ( 7 3 . 0 )

100

0.0

32.5 69.0 175

(=10.3 cm) 87.5 72.0 70.0 64.0 60.0 42.3 73.0 47.0 0.0 0.0 35.0 ( 6 2 . 0 ) 0.0 0.0 56.0 200

c c

12 F i n a l l y , i t i s p o s s i b l e t o t r e a t aldehydes and ketones w i t h ( 4 - s u l p h o b e n z y l ) x a n t h y d r a z i d e o r ( B - c a r b o x y e t h y l ) x a n t h y d r a z i d e and s e p a r a t e t h e r e s u l t i n g hy3 . F o r (4-sulphobenzy1)xanthy-

drazones by h i g h - v o l t a g e e l e c t r o p h o r e s i s on paper

drazones e l e c t r o p h o r e s i s i s c a r r i e d o u t i n 0.05 mol/l t e t r a b o r a t e , whereas ( B - c a r b o x y e t h y l )xanthydrazones a r e s e p a r a t e d i n 0 . 1 m o l / l NaOH-dimethylformamide ( 9 : 1 ) (Table 2 . 3 ) . The d e r i v a t i v e s a r e c o l o u r e d and no s p e c i f i c d e t e c t i o n r e a c t i o n s a r e needed. The advantage o f t h i s procedure i s t h a t c a r b o h y d r a t e s f a i l t o f o r m t h e c o r r e s p o n d i n g hydrazones w i t h t h e above r e a g e n t s .

TABLE 2.4 ELECTROPHORETIC SEPARATION OF SOME CARBONYL COMPOUNDS REACTED WITH GIRARD

T

REAGENT Compound

M o b i l i t y o f the product (towards c a t h o d e ) a (cm)

Glyoxal P y r u v i c a1 dehyde Phenyl g l y o x a l D iacetyl Benzal dehyde

16 16 10, 14.5b 9, 11, 13, 15b 9

:o-Phenylenediamine ( s t a n d a r d ) t r a v e l l e d 15 cm. A f t e r p r o l o n g e d r e a c t i o n p h e n y l g l y o x a l showed one s p o t w i t h a m o b i l i t y o f 14.51 cm and d i a c e t y l showed two s p o t s w i t h m o b i l i t i e s o f 13 and 15 cm.

Less g e n e r a l l y a p p l i c a b l e i s t h e r e a c t i o n o f a - d i c a r b o n y l compounds w i t h Girard

T r e a g e n t (trimethylaminoacetohydrazide ~ h l o r i d e ) ~w,h i c h r e s u l t s i n t h e

f o r m a t i o n o f s t r o n g l y UV-absorbing a d d i t i o n compounds. For paper e l e c t r o D h o r e s i s , a c e t i c a c i d - f o r m i c a c i d - w a t e r (87:25:588,

v / v ) i s used as a r u n n i n g b u f f e r (pH 1 . 9 ) .

However, e x c e p t f o r g l y o x a l and p l y r u v i c aldehyde, aldehydes g i v e m u l t i p l e s p o t s upon e l e c t r o p h o r e s i s ( T a b l e 2.4).

REFERENCES

1 2 3 4

C.L. Brown and P.L. K i r k , Microchim. Acta, (1957) 72C. G.B. M a r i n i - B e t t t i l o and G.C. C a s i n o v i , J . Chromatogr.. 1 (1958) 411. S. Plaza, Chem. AnaZ. (Warsaw), 17 (1972) 379. R.E.J. M i t c h e l and A.C. Birnboim, AnaZ. Biochm., 6 1 (1977) 2734.

13

Chapter 3 CARBOHYDRATES

Z. DEYL INTRODUCTION Polyhydroxy compounds such as c a r b o h y d r a t e s and t h e i r d e r i v a t i v e s may become charged as t h e r e s u l t o f complex f o r m a t i o n w i t h o t h e r i o n s . The most commonly used a r e b o r a t e complexes”*

b u t o t h e r i n o r g a n i c o x y a c i d s f o r m a n i o n i c complexes

w i t h n e u t r a l p o l y h y d r o x y compounds, e.g.,

s u l p h o n a t e d p h e n y l b o r o n i c a c i d 3 , sodium 5 6 Basic l e a d a c e t a t e and

germanate4, sodium s t a n n a t e l Y 5 and sodium t u n g s t a t e c a t i o n s o f t h e a l k a l i and a l k a l i n e e a r t h m e t a l s

.

6 f o r m c a t i o n i c complexes. I n a d d i -

t i o n , a number o f c a r b o h y d r a t e s t r a v e r s e a n o d i c a l l y i n sodium h y d r o x i d e , presuma b l y because o f t h e i o n i z a t i o n o f t h e i r h y d r o x y l groups ( f o r a r e v i e w , see r e f .

7 ) . A l l o f t h e s e p r o p e r t i e s can be e x p l o i t e d f o r t h e s e p a r a t i o n of complex carboh y d r a t e m i x t u r e s . Amino sugars, sugar phosphates and a c i d i c p o l y s a c c h a r i d e s can be r e a d i l y separated f r o m n e u t r a l p o l y h y d r o x y compounds by e l e c t r o p h o r e t i c separ a t i o n i n non-complexing b u f f e r s ( a c e t a t e , v e r o n a l

, phosphate!.

Early investiga-

t i o n s on c a r b o h y d r a t e e l e c t r o p h o r e s i s r e v e a l e d d i s t i n c t s t r u c t u r e - m o b i l i t y r e l a t i o n s h i p s ( f o r r e v i e w s , see F o s t e r 8 and Weige15).

MOliO- AND OLIGOSACCHARIDES Host e l e c t r o p h o r e t i c s e p a r a t i o n s o f s i m p l e mono- and o l i g o s a c c h a r i d e s a r e c o n v e n t i o n a l e l e c t r o p h o r e s i s i n t h e presence o f t h e above-mentioned complexing agents. There a r e two systems a v a i l a b l e t h a t o f f e r some i n f o r m a t i o n about t h e 9 m o l e c u l a r s i z e o f aldoses. The f i r s t method i s based on t h e c o n v e r s i o n o f t h e a l d o s e t o t h e N-benzylglycosamine d e r i v a t i v e (which can be done d i r e c t l y on t h e sheet o f f i l t e r - p a p e r by t r e a t m e n t w i t h benzylamine). E l e c t r o p h o r e s i s i s t h e n c a r r i e d o u t a t pH 1.8 i n f o r m a t e b u f f e r . I n t h e o t h e r method’’

a l d o s e s a r e con-

v e r t e d i n t o b i s u l p h i t e complexes by r u n n i n g t h e s e p a r a t i o n i n 0.4 m o l / l sodium bisul phite solution. As chromatographic and e l e c t r o p h o r e t i c s e p a r a t i o n s o f mono- and o l i g o s a c c h a r i d e s a r e governed b y c o m p l e t e l y d i f f e r e n t p r i n c i p l e s , t h e y have f r e q u e n t l y been combined i n t h e p a s t . A l t h o u g h D-glucose, D-galactose and D-mannose a r e e a s i l y separated by e l e c t r o p h o r e s i s , t h e i r s e p a r a t i o n b y chromatography i s c o m p l i c a t e d ;

TABLE 3.1 RECOMMENDEDBUFFER SYSTEMS FOR THE SEPARATION OF TIONO- AND OLIGOSACCHARIDES Buffer

Composition

Reference

Borate (pH 9.2) Borate (pH 10.0) Sodium molybdate (pH 5.0)

0.05 m o l / l Na2B4O7.10H 0 0.2 m o l / l borate, pH a i j u s t e d w i t h NaOH 20.8 g/1 o f sodium molybdate, a d j u s t e d t o t h e d e s i r e d pH w i t h conc. s u l p h u r i c a c i d 0.05 m o l / l germanium d i o x i d e a d j u s t e d t o t h e d e s i r e d pH w i t h sodium hydroxide 2% sodium stannate 0.05 m o l / l sulphonated phenylboronic a c i d a d j u s t e d t o pH 6.5 w i t h sodium hydroxide 0.2 m o l / l arsenious a c i d adjusted t o pH 9.6 w i t h sodium hydroxide. During e l e c t r o p h o r e s i s t h e r u n should be p r o t e c t e d from CO 58 g of b a s i c l e a d a c e t a t e a r e shaken i n 1 1 o f water and $he undissolved r e s i d u e i s f i l t e r e d o f f 0.1 m o l / l NaOH; should be p r o t e c t e d from C02 90% f o r m i c a c i d (400 m l ) + 5% sodium hydroxide (600 m l j

6, 75 2 16, 17

Sodium germanate (pH 10.7) Sodium stannate (pH 11.5) Sulphonated phenyl boronic a c i d (pH 6.5) Sodium a r s e n i t e (pH 9.6) Basic l e a d acetate (pH 6.8) Sodium hydroxide Formate (pH 1.8;

4

6 9

15

on t h e o t h e r hand, t h e p a i r s D - x y l o s d - g l u c o s e and D - a r a b i n o s d - g a l a c t o s e have v e r y c l o s e e l e c t r o p h o r e t i c m o b i l i t i e s and t h u s chromatography i s p r e f e r r e d f o r t h e i r separation. I n electrochromatography, e l e c t r o p h o r e t i c separations a r e c a r r i e d o u t f i r s t ; however, t h i s r e q u i r e s t h e a p p l i c a t i o n o f v o l a t i l e b u f f e r s . The d e t e c t i o n procedure depends c o n s i d e r a b l y on t h e b u f f e r system used18. F o r r e commended b u f f e r systems see Table 3.1 and f o r d e t e c t i o n procedures see T a b l e 3.2. For e l e c t r o p h o r e t i c m o b i l i t i e s o f common mono- and o l i g o s a c c h a r i d e s see Tables 3.3 and 3.4. TABLE 3.2 SOME REAGENTS FOR DETECTION OF SUGARS I N V A R I O U S ELECTROLYTES

Electrolyte

Detection reagenta

A n i l i n e phosphate; u r e a - t a r t r a t e ; AgNO -NaOH-pentae r y t h r i t o l ; C r O -KPlnO,-H SO ; p - a n i s i d j n e - H C 1 ; a n i 1 ine-dipheny?amine'ph$spfloric a i cd; urea-phosphoric acid A c e t o n e - A g N 0 ~ - a l c o h o l i c NaOH; p - a n i s i d i n e - H C 1 Sodium molybdate AgNOQ-sodium e t h o x i d e ; ammonical AgN03 Sodium germanate Sodium s t a n n a t e A c e t o n e - A g N 0 ~ - a l c o h o l i c NaOH Sulphonated phenyl b o r o n i c a c i d p-Anisidine-HC1 f o r r e d u c i n g sugars; p e r i o d a t e b e n z i d i n e f o r non-reducing sugars A n i l i n e phosphate; u r e a - t a r t r a t e ; heat-UV l i g h t ; Sodium a r s e n i t e H20 Cr03-H2SO@. A n i ? f n e phosphate; u r e a - t a r t r a t e ; Cr03-Ki.ln04-H2S04; Basic l e a d acetate H20 a c e t i c a c i d ; CrO3-HpSOq A n i f i n e phosphate; u r e a - t a r t r a t e p l u s b o r a t e ; Sodium h y d r o x i d e AgN03 ; Cr03-KHn04-H2S04 Borate

-

P e r i o d a t e - b e n z i d i n e , r e f . 15. Heat-UV l i g h t , r e f . 6. H202-Cr0 H2SO r e f . 6. AgN03-NaOH-pentaerythritol , r e f . 6. p-Anisidine-HC1, r e f . 14. Cr03-KMn&-H2S& , r e f . 6. Aniline-diphenylamine-phosphoric a c i d , r e f . 7. H202-acetic a c i d , r e f . 6 Urea-phosphoric a c i d , r e f . 11. Cr03-H2S04, r e f . 6 . U r e a - t a r t r a t e plus borate, r e f . 6 A c e t o n e - A g N 0 ~ - a l c o h o l i c NaOH, r e f . 12. AgN03, r e f . 12. AgND3-sodium e t h o x i d e , r e f . 12. Ammoniacal AgNO3, r e f . 13.

a A n i l i n e phosphate, r e f . 10. U r e a - t a r t r a t e , r e f . 6.

POLYSACCHARIDES The b u f f e r systems used d i f f e r a c c o r d i n g t o t h e n a t u r e of t h e p o l y s a c c h a r i d e s t o be separated. N e u t r a l p o l y s a c c h a r i d e s a r e s e p a r a t e d i n e i t h e r sodium b o r a t e (0.1 m o l / l , pH 9 . 3 ) o r sodium h y d r o x i d e (2 m o l / l ) . F o r a c i d i c p o l y s a c c h a r i d e s

0.06 m o l / l b a r b i t u r a t e (pH 8 . 5 ) , 0 . 1 m o l / l a c e t a t e (pH 4 . 7 ) o r 0.01 mol/l c i t r a t e (pH 3 . 5 ) a r e recommended7 The above e l e c t r o l y t e systems a r e used w i t h paper as

.

TABLE 3.3 RELATIVE ELECTROPHORETIC MOBILITIES Compound

(X

100) OF THE COFTION HONO- AND OLIGOSACCHARIDES (ACCORDING TO REF. 7)

Electrolyte

Trioses and tetroses 1,3-Dihydroxy-Z-propanone

D-Erythrose DL-G1 yceraldehyde L-Threose Aldopentoses D-Arabinose L-Arabinose

73 79

0

91

2-Deoxy-D-erythro-pentose

96 33

1,2-Dideoxy-D-eqthro-pentose

D-Lyxose D-Ribose D-Xylose Ketopentoses D-erythro-Pentu 1 ose D-threo-Pentulose A ldohexoses D-A11ose D-A1 trose

107 94 103 200e 150

0 0

71 75 101

71(3) 77 100

110 40 0

190 210 140

84 24 19 115 104 81

240

30

7

79

230 470 180

42 100 17

30 100 8

97 100 93

209 194

73 41

75 77

33 10

68 96

22 28 16 53 115 35 32 119

6 10 6 31 42 41 28 110

60 65 80 70 96

90 75 83 97

2-Deoxy-D-arabino-hexose

2-Deoxy-D-lyxo-hexose 2-Deoxy-D-ribo-hexose 3-Deoxy-D-ribo-hexose L-Fucose(6-deoxy-L-galactose) D-Galactose 0-Glucose D-Gu1 ose L- Idose D-iiannose L-Rhamnose(6-deoxy-L-mannose) D-Talose

90 0 60

83EC 93 100 32 102 69 49 87

180 97(3) 29;?) 37 85 ( 4 1 89 93 100 72 52

0

0 O(5) O(5)

580 0 160

31 23 52

O(51 0 0 110

130 100

69 78 63 107

180 100

9osc 60Sc 70

140 130

100 100

100 50

84 88 103

Ketoheccoses D-Fructose 0- P s i cose

L- So r b o se

D- Ta ga t o se Oligosaccharides Cellobiose Genti b i o s e I soma1 t ose Isomal t u l ose Lactose L a m i n a r ib i o s e Leucrose ilaltose Ma1 t u l ose l l e l ib i o s e N ige r o se Raf f i nose Sophorose Sucrose a ,a-Trehal ose Turanose

89 76 97 95

90

22

29 72 69(2) 60(19)

37 30 77

64

95

50 30 105(5j

0 0 0 64(19) <10(17:. 69j2) 0 35(19) 56(19! 0 34 63(19': 15(19) <10(17: 69 0 28 0 33 O(19) lO(19) 19 O(5) 69(19j lOj19;

210

91

200 240

94

30 100 90

62 65 58

70 110

75

40

65

140 130

57

930 850 860

75 188 73 103

22 16 65

89 125 88 82

15

10

68

72 (19) 24

13

73 (19) 61

62(19) 15 76(19) 32

7

28(19! 68 60( 19) 62

91Sc

10

50e

57 10e

11

25(19) 80419; 30

7

15(19) 28 19) 79

f

a E l e c t r o l y t e s and e x p e r i m e n t a l c o n d i t i o n s : 1, 0.05 m o l / l b o r a t e (pH 9.2), Whatman No.4 paper, 20-25 V/cm, 90 min; 2, 0.2 m o l / l b o r a t e (pH lo), Whatman No.3 paper, 15 V/cm; 3, 0.1 m o l / l sodium molybdate (pH 5), Whatman No.3 I.111 paper, 15 V/cm; 4, 0.05 m o l / l sodium germanate (pH 10.7), Whatman Mo.1 paper, 25-30 V/cm, 1.5 h; 5, 2% sodium s t a n n a t e (pH 11.5), Whatman No.3 MI4 paper; 6, 0.05 m o l / l s u l p h o n a t e d p h e n y l b o r o n i c a c i d (pH 6.5), Clhatman No.1 paper, 10 V/cm, 3-6 h; 7, 0.2 m o l / l sodium a r s e n i t e (pH 9.6), Uhatman No.4 paper, 20-25 V/cm, 90 min; 8, 0.2 g-atom l e a d p e r . l i t r e o f b a s i c l e a d a c e t a t e (pH 6.8), Whatman No.4 papers, 20-25 V/cm, 90 min; 9, 0.1 m o l / l sodium h y d r o x i d e , Hhatman No.4 paper, 20-25 V/cm, 90 min. Reference numbers i n parentheses. b N ~= m o b i l i t y r e l a t i v e t o D-glucose; 11s = m o b i l i t y r e l a t i v e t o D - g l u c i t o l ; FIRi = m o b i l i t y r e l a t i v e t o D-ribose. 'E, e l o n g a t e d spots; S, s t r e a k i n g . t h i s r e f e r e n c e many o f t h e compounds were assigned a movement of < l o . I n r e f . 5 t h e s e were a s s i g n e d a v a l u e o f 0. eSee r e f . 20; 0.04 m o l / l sodium germanate IpH 10, Uhatman N0.3 paper (14 x 56 cm), 12 V/cm, 2 h, 13-16OC. w i l l be n o t e d t h a t t h e r e p o r t e d movements o f t u r a n o s e and a , a - t r e h a l o s e i n sodium a r s e n i t e and sodium h y d r o x i d e a r e t h e o p p o s i t e o t t h o s e i n r e f s . 6 and 19.

18 TABLE 3.4 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME BENZYLGLYCOSYLAMINES (REF. 9 ) a Sugar

Relative mobility x 100

Pentoses Hexose Hept o s e Hexose d i s a c c h a r i d e s Hexose t r i s a c c h a r i d e s Hexose t e t r a s a c c h a r i d e s Hexose p e n t a s a c c h a r i d e Hexose hexasaccharide

109-115 100 91 71-78 59-63 49-51 42 33

b

a E l e c t r o p h o r e s i s i n pH 1.8 f o r m a t e b u f f e r (600 m l o f 5% NaOH and 400 m l o f 90% b f o r m i c acid!, Whatman No. 3 paper, 10-15 V/cm f o r 6 h. R a t i o between t h e d i s t a n c e which separates t h e N-benzylglycosylammonium i o n f r o m t h e unchanged sugar and t h e d i s t a n c e which separates t h e N - b e n z y l g l u c o s y l ammonium i o n f r o m unchanged glucose ( x 100). TABLE 3.5 RELATIVE ELECTROPHORETIC f4OBILITIES OF SOME NEUTRAL POLYSACCHARIDES (REF. 21)a ~

Tolysaccharide

R e l a t i v e m o b i l i t y b x 100 ~~

F i l ter-paperc

S i 1kC

Glass paperd

F r e e boundary

100 45 125 28 38 45 45 117 73 153 33 33

100 48 133

~~

Yeast mannan Yeast glycogen Snail galactan Elecampane inu1 i n Amylose Amy1 o p e c t i n llheat s t r a w x y l a n Lucerne mannogalactan I v o r y n u t mannan Appl e p e c t i n Dextran A ( N W = 10,000) Dextran B (NLl = 150,000)

100

49 llle 25 origin 27

-

100 58 133 25 o r igin 42 origin origin

-

33 50

-

36 41 40

-

64

-

a I n 0 . 1 m o l / l sodium t e t r a b o r a t e b u f f e r (pH 9 . 3 ) ; movement i s towards t h e anode. bMobil i t y r e l a t i v e t o y e a s t mannan. i 5 0 V/cm (100 mA) f o r 90 min; Whatman No. 54 f i l t e r - p a p e r was used. 33 V/cm (140 mA) f o r 90 min. eStreaked.

19 TABLE 3.6 ELECTROPHORETIC MOBILITIES OF SOHE A C I D I C POLYSACCHARIDES ON CELLULOSE PAPER (REF. 22) Polysaccharide

Mobilitya jxl0

cm

Polysaccharide

V-lsec-l) Heparin N- (2,4-Dini t r o phenyl ) h e p a r i n Chondroitin sulphate From b o v i n e t r a c h e a From b o v i n e septa Rhizobiwri radicico lwri polysaccharide

M o b i l i t y a (x10

5

2 cm

V-’sec-’

13.8 13.5

Pneumococcus polysaccharide Type 1 Type I 1 Type I11 Alginic acid Dextran Amy1 ose

10.7 11.9 9.9

9.1 3.4 8.3 12.9 0.0 0.0

a I n 0.06 m o l j l b a r b i t u r a t e b u f f e r (pH 8 . 5 ) , Whatman No. 1 paper (6.5 x 28 cm), 4-5 V/cm f o r 6-12 h; movement i s towards t h e anode. s o r b e n t . I n a d d i t i o n , methods u s i n g g l a s s paper o r s i l k t h r e a d s have been d e s c r i b e d ( T a b l e 3 . 5 ) . A c i d i c p o l y s a c c h a r i d e s can be e f f e c t i v e l y separated on c e l l u l o s e paper ( T a b l e 3.6)22 o r on s t a r c h g e l 23

.

A w i d e l y s t u d i e d c a t e g o r y o f p o l y s a c c h a r i d e s a r e glycosaminoglycans, which may be p r e s e n t i n t h e sample as v e r y l a r g e polymers ( f o r a r e v i e w , see r e f . 2 4 ) . U s u a l l y a p r e l i m i n a r y p r o t e o l y t i c d i g e s t i o n o f t h e t i s s u e i s needed i n o r d e r t o l i b e r a t e t h e s e compounds f r o m t h e t i s s u e . The r e a c t i o n m i x t u r e i s t h e n p r e c i p i t a t e d w i t h q u a t e r n a r y ammonium bases, m a i n l y c e t y l p y r i d i n i u m c h l o r i d e , o r w i t h e t h a n o l

,

o r may be s u b j e c t e d t o chromatographic f r a c t i o n a t i o n . The methods f o r t h e i s o l a t i o n o f glycosaminoglycans f r o m t i s s u e s a r e w e l l e s t a b l i s h e d 25-31,

b u t some i m -

provements appeared r e c e n t l y , m a i n l y c o n c e r n i n g t h e c o m p o s i t i o n o f t h e r u n n i n g buffers3‘ y33. A l t h o u g h most s e p a r a t i o n s a r e c a r r i e d o u t on c e l l u l o s e a c e t a t e , f i l t e r - p a p e r , agarose o r p o l y a c r y l a m i d e g e l s can a l s o be used. The s e p a r a t i o n o f h y a l u r o n i c a c i d and c h o n d r o i t i n s u l p h a t e s on f i l t e r - p a p e r i n sodium phosphate b u f f e r (pH 6.71 i s o f h i s t o r i c a l value34. H a r u k i and K i r k 35 were a b l e t o s e p a r a t e c h o n d o r i t i n s u l p h a t e B f r o m a m i x t u r e o f c h o n d r o i t i n s u l phate A and C on paper u s i n g z i n c s u l p h a t e o r z i n c a c e t a t e as e l e c t r o l y t e . C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s (Separax) i n 0.05 m o l / l LiC1-0.1 m o l / l HC1 (pH 2.0) o r 0.1 m o l / l KH2P04-0.1 m o l / l K2HP04 (pH 8 ) can be used f o r t h e separat i o n o f f l uorescence-label l e d glycosaminoglycans36. The compounds a r e s e p a r a t e d a f t e r r e a c t i o n w i t h 5-isothiocyanatofluorescein i n 0.1 m o l / l c a r b o n a t e b u f f e r (pH 8.5, 35OC, 6 h ) t o y i e l d t h e c o r r e s p o n d i n g N - f l u o r e s c e i n t h i o c a r b a m o y l hepar i n s (and o t h e r glycosaminoglycans).

C e l l u l o s e a c e t a t e s e p a r a t i o n s o f glycosamino-

g l y c a n s was f u r t h e r adapted t o t h e m i c r o s c a l e b u f f e r systems shown i n T a b l e 3.7 a r e i n use.

For t h e s e s e p a r a t i o n s t h e

20

TABLE 3.7 BUFFERS RECOHilENDED FOR THE SEPARATION OF GLYCOSAI4IMOGLYCANS

1. f o r m i c a c i d (0.48 m o l / l 1 - p y r i d i n e ( 0 . 1 m o l / l ) , pH j 4 0 (Eat thews38 ) 2. 0 . 1 m o l / l sodium phosphate b u f f e r , pH 6.7 ( R i e n i t s ) 3. sodium b a r b i t a l b u f f e r , pH 8.6; p = 0.1 (Wessler39; 4.. 0.2 m o l / l b u t y l a m i n e (Hsu e t al.4O) 5. 0 . 1 m o l / l HC1 (Wessler41) 6. 0.1 m o l / l barium a c e t a t e ( W e ~ s l e r ~ ~ ; 7. 0.1 m o l / l z i n c a c e t a t e (Prout4.2) 8. 0 . 1 m o l / l c o p p e r ( I 1 ) a c e t a t e z 4 9. 0 . 1 m o l / l lanthanum n i t r a t e (Hata and PIagai43) 10. 0.1-0.3 m o l / l c a l c i u m a c e t a t e (Seno e t a1.44)

I n t h e f i r s t f i v e b u f f e r systems i n Table 3.7 t h e m o b i l i t y o f glycosaminoglycans i s determined by t h e n e t charge o f t h e m o l e c u l e s . I n 0 . 1 m o l / l HC1 g l u c u r o n i c a c i d i s n o t d i s s o c i a t e d and t h e m o b i l i t y is determined by t h e number o f S03H groups i n t h e molecule. Conversely, i n 0.2 m o l / l b u t y l a m i n e t h e c a r b o x y l groups a r e completel y d i s s o c i a t e d and t h e s e p a r a t i o n i s governed b y t h e t o t a l amount o f a n i o n i c

groups i n t h e m o l e c u l e . I n b u f f e r systems c o n t a i n i n g barium, z i n c , copper, lanthanum o r c a l c i u m , t h e glycosaminoglycan molecules a r e complexed w i t h t h e m e t a l i o n s and t h e i r m i g r a t i o n depends on t h e backbone s t r u c t u r e 3 9 y 4 5 . C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s w i t h v a r y i n g i o n i c s t r e n g t h shows t h a t f o r t h e s e p a r a t i o n o f a c i d i c glycosaminoglycans p h y s i o l o g i c a l c o n d i t i o n s seem most s u i t a b l e . A decrease i n i o n i c s t r e n g t h r e s u l t s i n i n c r e a s e d m o b i l i t y b u t l e s s compact s p o t s . With i n c r e a s 46 i n g i o n i c s t r e n g t h t h e r e s u l t s a r e r e v e r s e d ( T a b l e 3.8)

.

It i s perhaps unnecessary t o s t r e s s t h a t none o f t h e above b u f f e r systems i s

capable o f e f f e c t i n g a complete s e p a r a t i o n o f a l l members o f t h i s group. Theref o r e , two-dimensional s e p a r a t i o n s were i n t r ~ d u c e d ~U~s .u a l l y i n t h e f i r s t r u n t h e compounds a r e separated a c c o r d i n g t o charge whereas t h e second s e p a r a t i o n i s based on d i f f e r e n c e s i n t h e backbone s t r u c t u r e 3 9 y 4 3 y 4 5 ( T a b l e 3.9).

The a d d i t i o n o f

c e t y l p y r i d i n i u m c h l o r i d e has no e f f e c t on t h e e l e c t r o p h o r e t i c b e h a v i o u r o f t h e compounds in v o l ved. The most d i f f i c u l t t a s k i n t h e c e l l u l o s e a c e t a t e s e p a r a t i o n o f glycosaminoglycans i s t h e s e p a r a t i o n o f c h o n d r o i t i n s u l p h a t e A and C. These can be r e s o l v e d i n c a l c i u m a c e t a t e s o l u t i o n , p r o v i d e d t h a t h e p a r i n and k e r a t a n s u l p h a t e have p r e v i o u s l y been removed 44 . Sorbents o t h e r than c e l l u l o s e a c e t a t e can be used. Homer4’

used agarose g e l ,

H i l b o r n and A n a s t a ~ s i a d i sp~o l~y a c r y l a m i d e g e l and M c D e v i t t and F l ~ i combined r~~ agarose-polyacrylamide g e l s . Recent improvements i n t h e agarose g e l e l e c t r o p h o r e s i s o f glycosaminoglycans r e l a t e t o t h e c o m p o s i t i o n o f t h e b u f f e r s used32. Various d i a m i n e b u f f e r s ( I y 2 - d i -

TABLE 3.8 MIGRATION SPEEDS OF THE GLYCOSAIIINOGLYCANS STUDIED IN AN ELECTRIC FIELD AT VARIOUS pH VALUES FOR THE BUFFER USED The desired pH range was produced by t i t r a t i n g the b u f f e r with 0.1 mol/l HC1 o r 0.1 mol/l NaOH. The i o n i c strength was adjusted in each instance (except f o r b a r b i t u r i c acid b u f f e r ) t o I = 0.164 with NaC1.

Buffer

0.1 mol/l HC1 0.08 mol/l HC1 0.06 mol/l HC1 0.04 mol/l HC1 0.02 mol/l HC1 C.01 mol/l HC1 0.02 mol/l Histidine.HC1 0.02 mol/l C i t r i c acid 0.02 mol/l HistidineVHCl 0.01 mol/l Urocanic acid 0.02 mol/l C i t r i c acid 0.01 mol/l Urocanic acid 0.02 mol/l C i t r i c acid 0.01 mol/l Urocanic acid 0.01 mol/l Urocanic acid 0.02 mol/l C i t r i c acid 0.02 mol/l C i t r i c acid 0.01 mol/l Urocanic acid 0.01 mol/l Urocanic acid 0.01 mol/l Urocanic acid 0.02 mol/l Imidazole 0.02 mol/l Imidazole 0.02 mol/l Imidazole Barbituric acid 0.02 mol/l Histidine-HC1 0.02 mol/l Histidine-HC1 0.02 mol/l Histidine.HC1 0.02 mol/l Glycine 0.02 mol/l Glycine 0.02 mol/l Histidine 0.02 mol/l Glycine

PH

Nobility (nun)

HA

CS-A

CS-C

CS-B

KS-1

HeS

HeP

Start

Anode

Cathode

19.0 18.0 17.0 13.5 10.0 11.0 12.5 12.0 13.5 14.5 17.0 19.0 18.2 20.5 21.0 20.5 19.5 21.0 19.5 21.0 20.5 21 .o 21.5 20.0 22.0 22.5 22.0 21.5 21.0 20.0 10.0

22.5 2 1 .o 22.0 21.2 19.0 21.5 21.5 22.0 23.0 23.5 26.0 28.5 28.0 28.5 30.0 28.0 26.5 28.7 29.0 29.0 28.5 30.2 31.0 31.5 31.0 31.0 30.5 29.7 30.5 29.0 25.5

22.5 21.0 22.0 21.5 19.0 22.5 22.2 22.7 24.0 23.5 26.0 28.5 28.2 29.5 30.5 28.5 27.0 30.0 29.5 30.5 29.5 31.0 31.7 31.5 31.5 31.5 31.0 30.5 31.0 29.5 26.0

23.2 21.0 23.0 22.7 20.0 22.7 22.2 22.7 23.0 23.5 25.0 27.0 27.0 28.5 30.7 29.0 27.0 30.2 29.7 30.7 29.5 31.0 31.7 31.5 32.0 31.5 31.5 30.7 32.0 30.0 26.0

23.7 22.0 23.0 23.7 21.0 24.0 23.0 23.0 23.0 22.5 24.0 25.2 24.7 24.0 25.2 23.5 22.0 24.0 24.0 25.5 24.2 25.5 25.5 23.5 25.0 25.5 25.0 25.0 26.0 24.0 22.0

21.7 20.0 21.0 20.5 17.0 19.0 19.5 19.5 19.5 20.5 23.0 25.2 25.0 25.0 26.5 25.2 24.0 25.7 25.5 26.5 26.2 27.0 27.5 26.5 27.0 26.5 26.7 26.0 27.0 26.0 22.5

25.7 25.0 26.0 28.0 26.0 29.5 27.0 28.7 26.5 28.0 29.0 31.5 32.0 30.7 32.5 30.5 29.5 30.5 31.0 31.5 31.0 32.0 33.0 32.5 32.0 32.5 32.0 30.5 32.5 31.0 27.0

1.42 1.44 1.50 1.61 1.85 2.12 2.39 2.44 2.66 2.87 3.30 2.45 3.65 3.98 4.37 4.83 5.08 5.48 5.83 6.20 6.63 7.10 7.59 8.50 8.64 9.08 9.60 10.02 10.68 11.27 11.41

1.42 1.44 1.50 1.61 1.85 2.11 2.35 2.44 2.63 2.74 3.25 3.37 3.62 3.79 4.25 4.79 5.04 5.35 5.66 6.07 6.60 7.06 7.57 8.36 8.57 9.07 9.54 9.99 10.57 11.15 11.35

1.42 1.44 1.50 1.61 1.86 2.12 2.46 2.50 2.79 3.04 3.32 3.70 3.72 4.17 4.67 4.90 5.19 5.61 6.02 6.36 6.77 7.12 7.69 8.51 8.69 9.26 9.61 10.13 10.70 11.32 11.53

22

TABLE 3.9 MOBILITIES OF GLYCOSAMINOGLYCANS (mm) ON CELLULOSE ACETATE STRIPS I N DIFFERENT SOLVENTS4' G1ycosami nogl ycan

Hyaluronic a c i d Chondroitin s u l phate B Keratan s u l p h a t e Chondroitin s u l phate A / C Heparin

Sodi um c i t r a t e

Zinc acetate

Formic a c i d - p y r i d i n e

(0.1 m o l / l , pH 3.5)

(0.15 m o l / l , pH 6 . 0 )

(pH 3 . 0 )

39 47

37.5 41

45 52

47 51

43 47.5

56 59

59

47.5

59

aminoethane, l Y 3 - d i a m i n o p r o p a n e, l Y 4 - d i a m i n o b u t a n e, 1,6-diaminohexane,

lY7-diamino-

heptane and l Y 8 - d i a m i n o o c t a n e ) , prepared by a d d i t i o n o f a c e t i c a c i d t o 0.1 m o l / l amine s o l u t i o n s a t pH up t o 8.5 ( f o r ethane-, propane- and butanediamines) o r 10 ( f o r t h e r e m a i n i n g d i a m i n e s ) , were t e s t e d w i t h c o n s i d e r a b l e success f o r t h e separ a t i o n o f h e p a r i n , c h o n d r o i t i n s u l p h a t e A, B and C and h e p a r i t i n s u l p h a t e C and A. The d i a m i n e b u f f e r systems and b a r b i t a l have a l s o been a p p l i e d t o micro-separa-

ti on^^^.

The i d e n t i f i c a t i o n o f most a c i d i c glycosaminoglycans i s p o s s i b l e i n t h r e e

d i f f e r e n t b u f f e r systems. I n b a r b i t a l b u f f e r t h e glycosaminoglycans a r e f r a c t i o n a t e d a c c o r d i n g t o t h e i r n e t charge, whereas i n d i a m i n e b u f f e r s t h e s e p a r a t i o n p r o b a b l y occurs a c c o r d i n g t o t h e degree t o which t h e y a r e bound t o t h e diamine ( T a b l e 3.10).

Two-dimensional s e p a r a t i o n s u s i n g b a r b i t a l b u f f e r ( f i r s t d i r e c t i o n )

and a d i a m i n e b u f f e r (second d i r e c t i o n ) have a l s o g i v e n good r e s u l t s (see a l s o 5 9 ) . Agarose d i s c g e l s e p a r a t i o n s o f glycosaminoglycans f o r p r e p a r a t i v e purposes were d e s c r i b e d by Funderbergh and Chandler5'.

The s e p a r a t i o n was r u n i n 0 . 3 m o l / l

barium a c e t a t e (pH 5.6). B i a n c h i n i e t a1.51 r e p o r t e d a d i s c o n t i n u o u s method f o r t h e f r a c t i o n a t i o n o f h e p a r i n . The 5% agarose g e l i s r u n f i r s t i n 0.04 m o l / l b a r i u m a c e t a t e (10 m i n ) , t h e n t h e s l a b g e l i s t r a n s f e r r e d t o a n o t h e r chamber and e l e c t r o p h o r e s i s i s cont i n u e d i n diaminopropane a c e t a t e b u f f e r f o r 15 min. The m i g r a t i o n i s towards t h e anode and t h e s e p a r a t i o n o f h e p a r i n i n t o t h r e e components t o g e t h e r w i t h chondroi t i n s u l p h a t e A and B i s achieved. For m o l e c u l a r w e i g h t d e t e r m i n a t i o n s o f glycosaminoglycans, 6% p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s can be a p p l i e d . The s e p a r a t i o n i s c a r r i e d o u t i n 0.2 m o l / l phosphate b u f f e r (pH 11.5) and 0.25 m o l / l sodium f ~ r m a t e ~As~ .glycosaminoglycans always r e p r e s e n t s i z e - c h a r g e isomers, t h e r e l a t i v e w i d t h o f t h e bands may be t a k e n as a measure o f m i c r o h e t e r o g e n e i t y . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s can a l s o be r u n a t pH 7.5 (0.05 m o l / l phosphate b u f f e r ) i n a s e r i e s o f 3, 4, 5, 8 and 10% g e l s .

23 TABLE 3.10 RELATIVE ELECTROPHORETIC ilIGRATION OF GLYCOSAIIINOGLYCANS I N THREE EUFFERS Re1a t i v e e l e c t r o p h o r e t i c m i g r a t i o n ( RChqn)

G1ycosaminogl ycana

C h o n d r o i t i n s u l p h a t e A (1) Chondroitin sulphate A Chondroitin sulphate B Chondroitin sulphate B Chondroitin sulphate C (1) Chondroitin sulphate C ( 2 ) H e p a r i t i n s u l p h a t e (1) H e p a r i t i n sulphate B ( 3 ) Heparin (1) Heparin ( 4 ) H y a l u r o n i c a c i d (1) Hyaluronic a c i d 2 Kerato s u l p h a t e 111

b

Barbital

1,3-Diaminopropane

1,lO-Diaminodecane

1.00

1.00 1.00 0.89 0.89 0.97 0.97 0.82 0.82 0.67 0.76 0.83 0.83 0.89

1.00 1.00 0.91 0.91 0.96 0.96 0.81 0.81 0 0

0.87 1.00 0.77 0.77 1.00, 1.15 1.15 0.51 0.52 0.69

-

0

aGlycosaminoglycan p r e p a r a t i o n : ( 1 ) U n i v e r s i t y o f Chicago; ( 2 ) i l i l e s Lab. ; b ( 3 ) prepared as d e s c r i b e d i n r e f . 75; ( 4 ) Upjohn Co. RChSA = m i g r a t i o n o f t h e glycosaminoglycan/migration of c h o n d r o i t i n s u l p h a t e A.

The d i s t a n c e s m i g r a t e d a r e t h e n d i v i d e d by t h e d i s t a n c e m i g r a t e d by t h e l o w e s t m o l e c u l a r w e i g h t p o l y s a c c h a r i d e i n each t y p e i n 3% g e l . The values t h u s o b t a i n e d e l i m i n a t e t h e e f f e c t o f e l e c t r i c f o r c e s a c t i n g on each c a t e g o r y of m o l e c u l e s and are r e l a t e d t o t h e logarithm o f t h e r e l a t i v e molecular weight o f t h e separated 53 compounds

.

So f a r t h e problem of s e p a r a t i n g t h e s i x common glycosaminoglycans appears n o t t o be t o o complicated.

I t must be k e p t i n mind, however, t h a t t h e r e a r e l i k e l y t o

be two o r t h r e e c h e m i c a l l y u n s p e c i f i e d t y p e s of h e p a r i n ”

and a l s o t h a t t h e r e i s

a d i s t i n c t m i c r o h e t e r o g e n e i t y i n i n d i v i d u a l glycosaminoglycan s p e c i e s w i t h r e s p e c t t o t h e degree of s u l p h a t i o n . Hata and Nagai54 and Hata e t a1.55 used twodimensional e l e c t r o p h o r e s i s t o demonstrate t h e presence o f a l o w - s u l phated chond r o i t i n s u l p h a t e i n b l o o d . Oreste and T ~ r r used i ~ c~e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s i n 0.1 m o l / l barium a c e t a t e ;pH 5 . 8 ) t o demonstrate t h e s t r u c t u r a l h e t e r o g e n e i t y o f v a r i o u s commercial h e p a r i n p r e p a r a t i o n s . A w a r n i n g must be g i v e n here, however. E a r l y i s o e l e c t r i c f o c u s i n g e x p e r i m e n t s 5 7 y 5 8 showed t h e f r a c t i o n a t i o n o f h e p a r i n i n t o as many as 2 1 components. T h i s band p a t t e r n appears t o be artificial,

a r i s i n g from a s t r o n g i n t e r a c t i o n between t h e p o l y s a c c h a r i d e and

d i f f e r e n t amphoteric s p e c i e s i n t h e Ampholine m i x t u r e used5’,

m a i n l y a t pH 3.5 t o

5.1. T h i s can be demonstrated by a l t e r i n g t h e Ampholine t o h e p a r i n r a t i o o r by l o a d i n g t h e sample e i t h e r b e f o r e f o c u s i n g o r t o a p r e - f o c u s e d s l a b g e l and by r e -

TABLE 3.11 SEPARATION OF SOME POLYSACCHARIDES REACTED WITH PROCION DYES BEFORE ELECTROPHORESIS Polysaccharide

R. t r i f o l i i TA1 p o l y s a c c h a r i d e C. parapsilosis g l ucan C. aZbicans mannan T . sabourandi galactomannan I E. fZoccoswn g a l actomannan I Larch a r a b i n o g a l a c t a n E. fZoccoswn g a l actomannan II + glucan S. sabourandi g l u c a n T . sabourandi g a l actomannan M. quinckeanim g l y c o p e p t i d e

E l e c t r o p h o r e t i c m o b i l i t y r e l a t i v e t o f r e e dye P r o c i o n Red 285

P r o c i o n B l u e 365

0 0.21

0 0.19

0.43 0.55 0.47 0.63

0.43 0.51 0.52 0.65 0.34 0.51 0.39 0.48 0 0.40 0.55

0.32

0.49 0.30 0.50 0

0.45 0.59

25

running s i n g l e heparin bands. A d e t a i l e d study and a s t r u c t u r a l model explaining t h i s behaviour were published by Righetti e t a l . 60 . The a n a l y s i s of glycosaminoglycans i s widely used in biomedical research and f o r c l i n i c a l purposes. For example, t h e u r i n e of healthy individuals contains chondroitin sulphates and a small amount of heparin ~ u l p h a t e ~ l -I ~t ~i s. beyond the scope of t h i s review t o deal with a l l of these a p p l i c a t i o n s , which include t i s s u e c u l t u r e s t ~ d i e s ~ ~ -serum ~ ' , analysis71y72 and screening f o r hereditary connective t i s s u e diseases 72-74 and neopl a s i as75 76. Detection i s commonly c a r r i e d out with Alcian blue77 by s t a i n i n g t h e g e l s and removing t h e excess o f t h e unbound dye. Quantitation can be e f f e c t e d by the atomic-absorption determination of copper complexes78 a f t e r Alcian blue s t a i n i n g (see a l s o Newton e t a1 .79). During the e l u t i o n of t h e dye the polysaccharides remain within the gel and can be re-stained". The actual procedure of Alcian blue s t a i n ing was l a t e r s l i g h t l y modified. Instead of t h i s dye, s t a i n i n g can a l s o be c a r r i e d out w i t h t o l u i d i n e blue ( i n water a t pH 6 - 7 ) , which i s p a r t i c u l a r l y s u i t a b l e f o r c e l l u l o s e a c e t a t e s t r i p s . The excess of t h e dye i s removed by washing with ethanol. Dudman and Bishop81 used r e a c t i v e dyes, Procion Blue 3GS and Procion Red 2BS, with which the polysaccharides a r e stained before electrophoresis so t h a t they move a s already coloured zones. This method i s generally applicable. The degree of s u b s t i t u t i o n v a r i e s with t h e dye t o polysaccharide r a t i o used i n the reaction and w i t h the s t r u c t u r e of the polysaccharide. Electrophoresis of t h e reaction products can be c a r r i e d o u t on c e l l u l o s e a c e t a t e (0.1 mol/l sodium t e t r a b o r a t e 0.1 mol/l sodium c h l o r i d e , 25-35 V/cm) (Table 3.11). Other polysaccharides a r e a l s o amenable t o e l e c t r o p h o r e t i c s e p a r a t i o n s ; procedures have been described f o r hemicel luloses82, agar components, agarose and a g a r ~ p e c t i n ~pneumococcal ~, polysaccharides84 and carboxymethylated s t a r c h 85 . Microelectrophoretic separations of polysaccharides on c e l l u l o s e a c e t a t e membranes were described by S t r a s s e r and Miserez 86 . Polyacrylamide gel e l e c t r o p h o r e s i s (4.6% acrylamide) can be used f o r the separ a t i o n of amylopectin, dextrans, laminarin, sargssan and ~ o s t e r i n e ~Pectin ~. substances can a l s o be separated on polyacrylamide g e l . After completion of the run t h e gels a r e pre-fixed w i t h A1C13 and CuS04 and stained with HI04-Schiff's reagent. Typical e l e c t r o p h o r e t i c p a t t e r n s can be discerned f o r p e c t i n , deesteri f i e d pectin and polygalacturonic acid 88 . SUGAR PHOSPHATES

The properties of sugar phosphates permit t h e i r separation from most of other n a t u r a l l y occurring phosphate e s t e r s , b u t separations within t h i s group of compounds may be d i f f i c u l t . Methylation of t h e e s t e r s before electrophoresis can be

26

TABLE 3.12 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME SUGAR PHOSPHATES Reference numbers i n parentheses. Compound

E ryt h r o se 4- phosphate Deoxyribose 1-phosphate Ribose 5-phosphate Ribose 1,5-diphosphate C y c l i c r i b o s e diphosphate 5-Phospharylribose 1-pyrophosphate R i b u l o s e 5-phosphate R i bulose 1,5-diphosphate Xylose 1-phosphate X y l u l o s e 5-phosphate F r u c t o s e 1-phosphate F r u c t o s e 6-phosphate F r u c t o s e 1,6-diphosphate Galactose 1-phosphate Galactose 6-phosphate Glucose 1-phosphate Glucose 6-phosphate G1 ucose 1,6-diphosphate G l u c o n i c a c i d 6-phosphate G1ucosamine 6-phosphate Mannose 1-phosphate Mannose 6-phosphate Sedoheptulose 7-phosphate Sedoheptul ose 1,7-di phosphate Sedulose 7-phosphate Octulose l Y 8 - d i p h o s p h a t e I n o s it o 1 d i phosphate triphosphate tetraphosphate hexaphosphate Orthophosphate

R e l a t i v e m o b i l i t y x 100 1 ( 9 0 ) a 2 ( 9 1 ) b 3(89)‘

4(89)d

5(92)e 6(93)f

(“pi

(“pi

(”pi )

70

(“‘G)

165

(“pi

36

81

109

132

65 70 69 104 118 120

(Mpi

75

169 113 169 62 100

161 189

64 64 64

158 158

80

201

64

162 154

27 27

132;152 81

65 27 85 36

81 60 106 60

70 52

120 94

63 63 95

70 70 107

63 64 61 95 91 0

68 69 68

63 59 93

70

64 88

111 138 156 1709 100

100

100

100

a P l o b i l i t y r e l a t i v e t o orthophosphate; Whatman No. 3 paper (15.5 x 38 cm), 1.0 m o l / l bsodium b u t y r a t e b u f f e r (pH 3.2), 10 V/cm f o r 4.5 h, 2OoC. M o b i l i t y r e l a t i v e t o glucose, which moved 9 cm f r o m o r i g i n i n 7 h due t o e l e c t r o endosmosis; Whatman No. 1 paper (45 x 56 cm), 5% p y r i d i n e - 0 . 5 % a c e t i c a c i d (pH 6 ) , 9 V/cm f o r 6-8 h, room t e m p e r a t u r e . ‘ M o b i l i t y o f u n t r e a t e d e s t e r r e l a t i v e t o orthophosphate; Whatman No. 1 paper (50 cm d l o n g ) , 0.1 m o l / l c e t y l t r i m e t h y l a m m o n i u m b o r a t e (pH 9 . 6 ) , 20 V/cm f o r 5 h w i t h c o o l i n g . C o n d i t i o n s as i n c except t h e phosphate e s t e r s a r e m e t h y l a t e d . e P l o b i l i t y r e l a t i v e t o orthophosphate, which moved about 30 cm i n 2 h; washed Whatman No. 3 MFI paper (19 x 57 cm), p y r i d i n e - a c e t i c a c i d - w a t e r b u f f e r (20:64:916) (pH 3.9), f36 V/cm f o r 2 h. c o r r e c t e d f o r e l e c t r o e n d o s m o s i s w i t h glucose. M o b i l i t y r e l a t i v e t o orthophosphate; washed S c h l e i c h e r and S c h d l l 589 Green Ribbon paper, 0.25 m o l / l ammonium a c e t a t e b u f f e r which c o n t a i n e d 0.05 g/1 o f EDTA (pH 3.6), 600 V f o r 2.5 h. gCompound t r a i 1s .

TABLE 3.13 BUFFER SYSTEllS RECOLIFIENDED FOR THE SEPARATION OF SUGAR PHOSPHATES Buffer

Composition

Reference

B u t y r a t e (pH 3.2: P y r i d i n e a c e t a t e (pH 3.9)

1.0 m o l / l sodium n - b u t y r a t e G l a c i a l a c e t i c a c i d - w a t e r - p y r i d i n e (64:916:20); pH a d j u s t e d w i t h p y r i d i ne 5% p y r i d i n e - 0 . 5 4 a c e t i c a c i d Cetyltrimethylam_monium [ f r e e base o b t a i n e d by p a s s i n g t h r o u g h , e.g. Dowex 1-X8 (OH-LI; 1 m o l / l s o l u t i o n o f t h e base i s added t o 10 g boric acid 0.05-0.20 rnol/l b o r a t e ; pH a d j u s t e d w i t h NaOH

90 92

P y r i d i n e a c e t a t e (pH 6.0) Cetyltrimethylammoniurn b o r a t e (pH 9.6; B o r a t e b u f f e r s (pH 9, 9.5,

10)

,

91 89 94

N -3

TABLE 3.14 RELATIVE ELECTROPHORETIC F.IOBILITIES ( x 100) Compound

Acyclic t r i o l s and t e t r i t o l s G1y c e r o l Erithritol L-Threi to1 Pent a e r y t h r it o 1 Acyclic pentitols D-Arabini t o 1 L- Ara b i nit o 1 R i b ito1 Xyl i t o l Acyclic hexitols A1 1 it o 1 0-A1 t r i t o l ( D - t a l i t o l ) Galactitol (dulcitol ) D-Glucitol (D-sorbitol ) L-Idi to1 D-Manni t o 1 Reduced disaccharides Cel l o b i i t o l ( 8 - 1 : 4 ) G e n t i o b i it o 1 (8-1:6j I s o m a l t i t o l ja-1:6) L a c t i t o l (B-1:4) L a m i n a r i b i i t o 1 (8-1:3) Q l a l t i t o l (a-1:4) Me1 ib i it o 1 (8-1:6) N i g e r i t o l (a-1:3) S o r p h o r i t o 1 (8- 1 :2)

OF SOPIE SUGAR ALCOHOLS

Electrolytea

1(6)

2(16)

3(17)

4i4)

5(1)

in,)b

;“G)

(Ids)

(11,)

(tl,)

49 75 75 85

44 75

< l o (101)

40 100

87

90

100;101) 50(101)

23 57 62

6(3) ),$I(

0 10 30

<10;101)

110

72 100

85 79

110 110

90 89 97 83 81 91

98 89

84;ll) 99jll) 100 100

210 190

88 95 99 100

90

100

190

93

40 80 80 40

120

90

(FIRi )

(PIRi )

(PIRi )

24 53 96 21

3 3 11 0

0 3 3 12

14

7

76 155

4 25

10 3

100 130

92 138 145 161 173 130

9 17 32 47 57 23

23 16 8 11 7 12

100 110

60 150 10

40 80 0

9(6)

124

140

0

8(6)

60 30 90

95 180 120 170

7(6)

150 120

aElectrolytes and experimental conditions: 1, 0.05 mol/l borate (pH 9.2:, Whatman No.4 paper, 20-25 V/cm; 90 m i n ; 2 , 0.2 mol/l borate (pH l o ) , Whatman No.3 paper, 15 V/cm; 3, 0.1 mol/l sodium molybdate (pH 5;, Whatman N0.3 Mil paper, 15 V/cm; 4, 0.05 mol/l sodium germanate ;pH 10.7), Whatman No.1 paper, 25-30 V/cm, 1.5 h ; 5 , 2% sodium stannate (pH 11.5), Whatman No.3 PlM paper; 6, 0.05 mol/l sulphonated phenylboronic acid (pH 6.5), Whatman No.1 paper, 10 V/cm, 3-6 h ; 7 , 0.2 mol/l sodium a r s e n i t e (pH 9.6!, Whatman No.4 paper, 20-25 V/cm, 90 min; 8 , 0.2 mol/l lead per l i t r e o f basic lead a c e t a t e ipH 6.8), Whatman No.6 paper, 20-25 V/cm, 90 min; 9 , 0.1 mol/l sodium hydroxide, bWhatman No.4 paper, 20-25 V/cm, 90 min. Reference numbers i n parentheses. MG = mobility r e l a t i v e t o D-glucose; !Is = mobility r e l a t i v e t o D-glucitol; PIPI = mobility r e l a t i v e t o D-mannitol; M . = mobility r e l a t i v e t o D-ribose. ‘DEthrei to1 .

30 recommended i n t h i s r e s p e c t ( T a b l e 3.12) t i o n and pH may a l s o help”.

, and

v a r i a t i o n o f t h e borate concentra-

E l e c t r o p h o r e s i s on paper i n 0.2 m o l / l b o r a t e b u f f e r

(pH 9 . 5 ) p e r m i t s t h e s e p a r a t i o n o f glucose 1-phosphate ( l ) , a combined zone o f glucose 6-phosphateY f r u c t o s e 1-phosphate and f r u c t o s e 6-phosphate ( 2 ) , f r u c t o s e 1,6-diphosphate

( 3 ) and orthophosphate ( 4 ) . F r u c t o s e 1-phosphate i s separated

f r o m g l u c o s e 6-phosphate i n 0.05 m o l / l b o r a t e b u f f e r (pH 9.0) and g l u c o s e 6-phosphate i s separated f r o m f r u c t o s e 6-phosphate a t pH 9 o r 10 i n 0.05 m o l / l b o r a t e b u f f e r . F r u c t o s e 1-phosphate i s separated f r o m f r u c t o s e 6-phosphate i n 0.3 m o l / l b o r a t e b u f f e r a t pH 9.5.

B u f f e r s t h a t can be used f o r t h e s e p a r a t i o n o f t h i s c a t e g o r y o f compounds on paper a r e l i s t e d i n T a b l e 3.13. Ammonium molybdate, a l c o h o l i c s i l v e r n i t r a t e and i r o n ( I1 I ) c h l o r i d e - s u l phosal i c y 1 i c a c i d have been used f o r many y e a r s t o d e t e c t t h e zones o f sugar phosphates ( f o r a r e v i e w , see r e f . 7 ) . A method f o r two-dimensional c h r o m a t o e l e c t r o p h o r e s i s o f sugar phosphates 95 on c e l l u l o s e l a y e r s i n p y r i d i n e - a c e t a t e (pH 4 . 0 ) was r e p o r t e d by Schirmann

.

POLYHYDRIC ALCOHOLS S e p a r a t i o n procedures f o r p o l y h y d r i c a l c o h o l s a r e v e r y s i m i l a r t o t h o s e used f o r mono- and 01 i g o s a c c h a r i d e s . For d e t e c t i o n o f p o l y h y d r i c a l c o h o l s separated i n b o r a t e b u f f e r s , Cr03-KMn04-H2S04 r e a g e n t i s used. With sodium a r s e n i t e as t h e r u n n i n g b u f f e r , d e t e c t i o n i s e f f e c t e d w i t h hydrogen p e r o x i d e f o l l o w e d by Cr03H2S04. Basic l e a d a c e t a t e i s u s u a l l y combined w i t h H202-acetic a c i d . A l k a l i n e s i l v e r n i t r a t e can a l s o be used. The Cr03-KMn04-H2S04 r e a g e n t i s s p e c i f i c a l l y u s e f u l f o r n o n - v i c i n y l a l c o h o l s . The r e l a t i v e m o b i l i t i e s o f t h e most common sugar a l c o h o l s a r e g i v e n i n Table 3.14.

F o r a d d i t i o n a l d a t a see r e f . 7, p. 264.

AMINO SUGARS Amino sugars a r e i m p o r t a n t c o n s t i t u e n t s o f many p o l y s a c c h a r i d e s and g l y c o p r o t e i n s . T h e i r s e p a r a t i o n on paper i s m o s t l y c a r r i e d o u t i n b o r a t e b u f f e r s (0.05 pH 10) o r i n a c e t i c a c i d ( 2 mo1/1)7. A wide range o f v o l t a g e s has been

mol/l,

a p p l i e d w i t h o u t a pronounced e f f e c t on s e p a r a t i o n . N i n h y d r i n , p - a n i s i d i n e o r p-dimethylaminobenzaldehyde can be used f o r d e t e c t i o n . With p-dimethylaminobenzaldehyde t h e N-acetylamino sugars can be assayed d i r e c t l y . For u n a c e t y l a t e d sugars, t h e d r i e d e l e c t r o p h e r o g r a m i s f i r s t sparayed w i t h a c e t i c a n h y d r i d e i n acetone ( 1 : 4 ) , heated a t 95OC f o r about 5 min and t h e n d e t e c t e d . An i m p r e s s i o n o f t h e s e p a r a t i o n p o s s i b i l i t i e s can be g a i n e d f r o m T a b l e 3.15.

31

TABLE 3.15 ELECTROPHORETIC MIGRATIONS (RELATIVE MOBILITY x 100) OF SOME A M I N O SUGARS AND DERIVATIVES Compound

Buffera 1(96)b (MG)

N-acetyl-D-allosamine N-Acetyl-D-fucosamine

N-Acetyl-D-galactosamine N-Acetyl -D-gl ucosamine N-Acetyl-D-glucosamine N-Acetyl-D-mannosamine N-Acetyl-D-talosamine N-Acetyl-D-xylosamine

N-Acetyl-D-chondrosamine Gal actosamine G1 ucosami ne Mannosamine blethyl 3-ami no-3-deoxy-a-0-91 ucopyranosi de Methyl 6-ami no-6-deoxy-a-0-91 ucopyranosi de G1 ucosamini c a c i d Muramic a c i d S i a l i c a c i d (not defined f u r t h e r )

42 14 33 23 63 65 60 23 35

2(97)‘ (cm)

3(98)d (cm)

4.5 2.7 8.4

0.5(51) -1.0(51) 3.6(51) -3.1(51) -6.6 ( 5 1 )

-24.2

-10.0 -14.8 8.5(51)

a B u f f e r s : 1, sodium t e t r a b o r a t e (pH 10.0; 23.4 g o f sodium t e t r a b o r a t e and 30 m l o f 1 m o l / l NaOH p e r l i t r e ) ; 2, 0.025 m o l / l potassium t e t r a b o r a t e ; 3, a c e t i c b a c i d ( 2 m o l / l ) . Reference numbers i n parentheses. Movement towards t h e anode r e l a t i v e t o t h a t o f glucose; Whatman No. 3 paper, 1300 V (25 mA), 2.5 h. , Ilovement towards t h e cathode; t , movement towards t h e anode; Whatman No. 3 MM o r No. 3 paper, 6 V/cm, 6 h. d- , Movement towards t h e cathode; Whatman No. 3 paper, 15 V/cm, 250 min, 2 m o l / l acetic acid.

‘-

ACIDS AND LACTONES OF CARBOHYDRATES Acids o f c a r b o h y d r a t e s a r e amenable t o paper e l e c t r o p h o r e t i c s e p a r a t i o n i n b o r a t e (0.2 m o l / l sodium t e t r a b o r a t e , pH l o ) , s u l p h a t e (0.05 m o l / l ,

pH 2 ) , ace-

t a t e (0.05 m l / l , pH 4 ) and phosphate (0.05 m o l / l , pH 7 ) b u f f e r s . The s e p a r a t i o n i t s e l f i s c o n v e n t i o n a l paper e l e c t r o p h o r e s i s 7 . A n i l i n e hydrogen p h t h a l a t e ” can be used f o r r e d u c i n g sugar a c i d s , b u t i n most d e t e c t i o n s t h e a c i d i c c h a r a c t e r o f t h e s e compounds i s made use o f and d e t e c t i o n i s e f f e c t e d by s p r a y i n g t h e e l e c t r o p h e r o g r a m w i t h i n d i c a t o r solutions100.

Bromocresol p u r p l e o r bromocresol

b l u e can be used f o r t h i s purpose. E l e c t r o p h o r e t i c m i g r a t i o n s o f some sugar a c i d s a r e summarized i n T a b l e 3.16.

32 TABLE 3.16 ELECTROPHORETIC MIGRATIONS OF SOME SUGAR ACIDS Reference numbers i n parentheses. Compound

Buffer

Glyoxylic acid Erythronic aicd Arabionic a c i d Xylonic a c i d 2-0- (4-0-Methyl -a-g1 ucuronosyl ) x y l o s e Galactonic a c i d Galacturonic a c i d D-Galacturonic a c i d l-phosphate D i galacturoni c acid d.5; Gluconic a c i d 2-Ketogluconic a c i d 5-Ketogluconic a c i d Glucuronic a c i d D-Glucuronic a c i d 1-phosphate 4-0-Methyl g l ucuronic a c i d Glucaric acid <1.5; a-Saccharinic a c i d a- Isosacchari n i c a c i d

15.4 10.5 0; 8.6 0; 9.1 0; 6.9

24.7 18.1 15.3 15.8 9.9

0;

8.4 7.9

14.5 13.7

1.7 <1.5 3.2 1.5 1.9

8.1; 10.2 0; 7.7 10.7 9.3 9.3

13.2; 14.3 14.9 14.6 14.0

<1.5 1.5; 2.6 <1.5 <1.5

0; 9.2 9.4; 10.8; 12.6 0; 7.7 0; 8.1

13.8 15.6; 24.6 13.7 13.9

<1.5 (1.5 <1.5 ~1.5 4.5

107 163 16.5

117 140

;Sulphate b u f f e r , 0.05 m o l / l (pH 2 ) , Whatman No. 1 paper, 35 V/cm. As a, except 0.05 m o l / l a c e t a t e b u f f e r (pH 4 ) . ‘As a, except 0.05 m o l / l phosphate b u f f e r (pH 7 ) . dSodium b o r a t e b u f f e r , 0.2 m o l / l (pH l o ) , Whatman No. 3 paper, m o b i l i t y r e l a t i v e t o glucose.

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, 22 (1979) 121; C - A - Y 93 (1980) 40730q. 67 D.G. Chukaeva, G.E. Tsimarkina and D.Ya. Dyachlcova, Lab. DeZo, (1977) 619. 68 N. Nakamura, Y. M o r i , Y. T a n i g a k i and J. Kojima, Biochim. Biophys. Acta, 627 (1980) 60. 69 Y. Nanomiya, R . - I . Hata and Y. Nagai, Biochim. Biophys. A c t a , 629 (1980) 349. 70 C.P. D i e t r i c h and H. Montes de Ora, Biochem. Biophys. Res. Comun., 80 (1978) 805. 7 1 K. F u j i t a , Rinsho Kensa, 23 (1979) 943; C.A., 91 (1979) 189031d. 72 S. R u f f i n i , M. Ghebregzabher, G. C a s t e l l u c c i and M. L a t o , Clin. Chim. Acta, 95 (1979) 443. 73 P. Whiteman and H. Henderson, C l i n . Chirn. A c t a , 79 (1977) 99. 74 H.B. Nader, D.M. Cohen and C.P. D i e t r i c h , Biochim. Biophys. Acta, 582 (1979) 33. 75 L.O. Sampaio, C.P. D i e t r i c h and O.G. F i l h o , Biochim. Biophys. A c t a , 498 (1977) 123. 76 H.B. Nader, W. E a r x and L. S p o l t e r , Biochim. Biophys. Acta, 631 (1980) 463. 77 R. Hata and Y . Nagai, Anal. Biochem., 52 (1973) 652. 78 J.F. O ' B r i e n and M.E. Emmerling, Anal. Biochem., 85 (1978) 377. 79 0.3. Newton, J.E. S c o t t and P. Whiteman, Anal. Biochem., 62 (1974) 268. 80 G. Manley and J. Hawksworth, Arch. Dis. Childh., 41 (1966) 91. 8 1 W.F. Dudman and C.T. Bishop, Can. J . Chem., 46 (1968) 3079. 82 M.S. Dudkin and N.G. Shkantova, Zh. P r i k l . Khim. (Leningrad), 43 (1970) 206; C.A . , (1970) 123246d. 83 K. Katsuura, T. Akahane and S. Abe, Nippon Xugaku Zasshi, 89 (1968) 983; C.A. , 70 (1969) 6 5 0 2 2 ~ . 84 T. M i y a z a k i , T. Yadomae and J.K.N. Jones, J . Biochem. ( T o k y o ) , 68 (1970) 755. 85 S. Tomita, K. T e r a j i m a and Y. Yoshida, Kogyo Kagaku Zasshi, 72 (1969) 532; C . A . , 7 1 (1969) 8 1 6 8 0 ~ . 86 R. S t r a s s e r and A. Miserez, EqevYientia, 27 (1971) 239. 87 A.F. Pavlenko and Yu.S. Ovodov, J . Chromatogr., 52 (1970) 165. 88 J.Y. Do, J. Idannou and N.F. Hard, J . Food S c i . , 36 (1971) 1137. 89 R. P i r a s and E. Cabib, Anal. Chern., 35 (1963) 755. 90 H.E. Wade and D.M. Morgan, Biochem. J . , 60 (1955) 264. 91 V.C. Runeckles and G. Krotkov, Arch. Biochem. Biophys., 70 (1957) 442. 92 B.S. Vanderheiden, A n d . Biochem., 8 (1964) 1. 93 R.L. B i e l e s k i and R.E. Young, Anal. Biochem., 6 (1953) 54. 94 S. Schwimmer, A. Bevenue and W.J. Weston, Arch. Biochem. Biophys., 60 (1956) 279. 95 P . Schiirmann, J . Chromatogr., 39 (1969) 507. 96 M.J. Crumpton, Biochem. J . , 72 (1959) 479. 97 S. Ohkuma and T. Shinohara, Nature (London), 202 (1964) 593. 98 H.R. P e r k i n s , Biochem. J., 74 (1960) 182. 99 S.M. P a r t r i d g e , Nature (Londonl, 164 (1949) 443. 100 0. Theander, S u . Kern. T i d s k r . , 70 (1958) 393.

36

Chapter 4 CARBOXYLIC A C I D S

F. M. EVERAERTS GENERAL CONSIDERATIONS C a r b o x y l i c a c i d s g e n e r a l l y have h i g h m o b i l i t i e s compared w i t h t h o s e o f , e.g., p r o t e i n s . I n t e r e s t i n s e p a r a t i n g t h e s e compounds v i a e l e c t r o p h o r e t i c t e c h n i q u e s has i n c r e a s e d because t h e y can e a s i l y be s e p a r a t e d qua1 it a t i v e l y and q u a n t i t a t i v e l y by i s o t a c h o p h o r e s i s . F o r t h i s reason, c a r b o x y l i c a c i d s have f r e q u e n t l y been used as model substances f o r c h e c k i n g b o t h t h e o r e t i c a l aspects and t h e c o r r e c t f u n c t i o n i n g o f t h e equipment. Most e l e c t r o p h o r e t i c work, e x c e p t i s o t a c h o p h o r e s i s , has been p e r f o r m e d i n equipment i n which s t a b i l i z i n g media (such as paper, c e l l u l o s e a c e t a t e and s i l i c a g e l ) can be a p p l i e d . Common i s o t a c h o p h o r e t i c e q u i p m e n t l Y 2 makes use o f narrowb o r e PTFE tubes w i t h i n s i d e d i a m e t e r s o f 0.2-0.6 mm. The s e p a r a t i o n o f c a r b o x y l i c a c i d s q u a l i t a t i v e l y and q u a n t i t a t i v e l y v i a moving boundary e l e c t r o p h o r e s i s " *

o r i o n f o c u s i n g 3 i s l e s s e f f i c i e n t i n comparison t o

isotachophoresis. SEPARATIONS V I A ZONE ELECTROPHORESIS I n zone e l e c t r o p h o r e t i c experiments problems w i t h c o n v e c t i o n and d e t e c t i o n have t o be overcome. There a r e t h r e e ways o f a l l e v i a t i n g t h e problem o f convect i o n . One emphasizes t h e use o f a d d i t i o n a l f o r c e f i e l d s , e.g., G r a v i t a t i o n a l o r e l e c t r o m a g n e t i c 1 I n a second, more p r a c t i c a l approach, s t a b i l i z i n g media such

.

as paper, c e l l u l o s e a c e t a t e o r s i l i c a g e l 4 a r e a p p l i e d . The t h i r d approach, which has proved t o g i v e s t a b l e e l e c t r o p h o r e t i c performance, i s t o use t h e a n t i - c o n v e c t i v e " w a l l e f f e c t " 5 y 6 . S t a b i l i t y i s achieved by d e c r e a s i n g t h e r a t i o o f t h e c r o s s - s e c t i o n o f t h e s e p a r a t i o n compartment t o i t s s u r f a c e a r e a . Because i n general t h e c o n c e n t r a t i o n o f t h e sample compounds is s m a l l compared w i t h t h e c o n c e n t r a t i o n o f t h e i o n i c s p e c i e s i n t h e background e l e c t r o l y t e , u n i v e r s a l d e t e c t o r s a r e seldom used i n s e p a r a t i o n s i n which zone e l e c t r o p h o r e t i c p r i n c i p l e s a r e used. Over t h e y e a r s ( n o t a l l r e f e r e n c e s w i l l b e g i v e n ) , zone e l e c t r o p h o r e s i s has been a p p l i e d t o t h e d e t e r m i n a t i o n o f s t a b i l i t y c o n s t a n t s o f substances w i t h v a r -

36

i o u s complexing agents, e.g.,

S t e y e r t h a l e t a1 .lo determined

c a r b o x y l i c acids7-’.

homogentisic a c i d by h i g h - v o l t a g e e l e c t r o p h o r e s i s .

Homogentisic a c i d was n o t found

i n t h e u r i n e o f c o n t r o l i n d i v i d u a l s , b u t c o u l d be d e t e c t e d i n nanomole amounts ( u s i n g s i l v e r n i t r a t e as r e a g e n t ) i n t h e u r i n e o f a l k a p t o n u r i c p a t i e n t s . M i k k e r s e t a1.6 used an apparatus‘

equipped w i t h a narrow-bore PTFE t u b e ( I . D . ca. 0 . 2 mm)

t o s e p a r a t e a s e r i e s o f c a r b o x y l i c a c i d s u s i n g b o t h UV a b s o r p t i o n and c o n d u c t i m e t r i c 2 d e t e c t i o n . F i g . 4.1 shows t h e s i g n a l s d e r i v e d f r o m a c o n d u c t i m e t r i c d e t e c t o r

.

8

7 I

1

9 FJII b

I

I

12 I

13

14

15

id

I

I

1

I

E”- E’

-

2

3

4

t (min)

5

6

7

8

9

F i g . 4.1. Zone e l e c t r o p h o r e t i c s e p a r a t i o n o f a 16-component sample. 1 = C h l o r i d e ; 2 = s u l p h a t e ; 3 = c h l o r a t e ; 4 = malonate; 5 = chromate; 6 = p y r a z o l e - 3 , 5 - d i c a r b o x y l a t e ; 7 = a d i p a t e ; 8 = a c e t a t e ; 9 = p r o p i o n a t e ; 10 = 6 - c h l o r o p r o p i o n a t e ; 11 = u n i d e n t i f i e d ; 12 = benzoate; 13 = naphthalene-2-monosulphate; 14 = g l utamate; 15 = enanthate; 16 = benzyl-DL-aspartate. Sample l o a d : 17.5 .lo-12 mol o f each c o n s t i t u e n t . ES-EZ = d i f f e r e n c e i n e l e c t r i c a l f i e l d s t r e n g t h between t h e c a r r i e r e l e c t r o l y t e and t h e sample zone. t ( m i n ) = t i m e o f a n a l y s i s . SEPARATIONS V I A ISOTACHOPHORESIS C a r b o x y l i c a c i d s can be s e p a r a t e d e f f e c t i v e l y b y i s o t a c h o p h o r e s i s . Commonly t h e s e p a r a t i o n i s performed w i t h w a t e r as solvent‘, stances methanol has been recommended11’12.

b u t f o r more a l i p h a t i c sub-

Carboxyl i c a c i d s can o f t e n be sepa-

r a t e d i n t h e h i g h l y d i s s o c i a t e d form, b u t i f t h e s e p a r a t i o n f a i l s t h e e f f i c i e n c y can be improved by s e l e c t i n g an a p p r o p r i a t e pH o f t h e l e a d i n g e l e c t r o l y t e ( a n d / o r pH o f t h e sample i n some instances)13y14, t h e common c o u n t e r i o n l 3 3 I 4

o r a (non)-

i o n i c substance f o r m i n g an ( i r ) r e v e r s i b l e complex w i t h one o r a l l o f t h e substances under i n ~ e s t i g a t i o n ’ ~ .E x t e n s i v e i n f o r m a t i o n on t h e o p e r a t i n g c o n d i t i o n s i s g i v e n i n r e f . 2. An e a r l y p r a c t i c a l a p p l i c a t i o n was p u b l i s h e d by E v e r a e r t s and

37

Konz16, who d e s c r i b e d t h e a n a l y s i s of p r o d u c t s formed d u r i n g t h e homogeneous o x i d a t i o n o f sugars. The a n a l y t i c a l r e s u l t s were used f o r t h e o p t i m i z a t i o n o f t h e r e a c t i o n c o n d i t i o n s and t o s t u d y t h e k i n e t i c s . The c o m p o s i t i o n o f c a r b o x y l i c a c i d s i n , e . g . ,

f r u i t s , f r u i t j u i c e s and jams

has been e x t e n s i v e l y s t u d i e d . The analyses can e a s i l y be performed w i t h o u t sample p r e - t r e a t m e n t i n t h e column-coupling system17 o r w i t h a s i m p l e e x t r a c t i o n and/or centrifugation.

With t h e column-coupling system17 i o n i c substances a t l o w concen-

t r a t i o n s i n complex m i x t u r e s (e.g.,

serum, u r i n e , j u i c e ) can be d i r e c t l y determined T h i s column-coupling system w i l l i n c r e a s e

q u a l i t a t i v e l y and

t h e range o f a p p l i c a t i o n s o f i s o t a c h o o h o r e s i s , because h i g h sample l o a d s a r e p e r m i t t e d , t h e a n a l y s i s t i m e i s i n c r e a s e d n e g l i g i b l y by h i g h sample l o a d s , h i g h r a t i o s o f c o n c e n t r a t i o n s between t h e samDle comipounds a r e p e r m i t t e d and, i f necessary, d i f f e r e n t o p e r a t i n g c o n d i t i o n s can be a p p l i e d i n t h e s e p a r a t i o n and p r e s e p a r a t i o n compartments. Using t h e coupled-column system, M i k k e r s e t a1 .I8 det e r m i n e d v a l p r o i c a c i d a t serum l e v e l s and compared t h e d a t a w i t h t h o s e o b t a i n e d w i t h a r o u t i n e gas chromatographic method. A l s o u s i n g t h e coupled-column system, Verheggen e t a1.l'

determined u r i c a c i d a t b o t h u r i n e and serum l e v e l s and com-

pared t h e r e s u l t s w i t h t h o s e o b t a i n e d by a r o u t i n e enzymatic method. E x c e l l e n t agreement was o b t a i n e d , s t a n d a r d d e v i a t i o n s s m a l l e r t h a n 2% b e i n g o b t a i n e d , even i n t h e "day-to-day'' v a r i a t i o n s . The d e t e c t i o n l i m i t i s a few picomoles and t h e

2 o r the "UV spike"

average a n a l y s i s t i m e i s ca. 8 min. U s i n g t h e "mixed-zone'' method method',

even l o w e r l i m i t s can be reached.

Comparison w i t h and c o m b i n a t i o n w i t h high-performance l i q u i d chromatography i s s t i l l under i n v e s t i g a t i o n . B a l d e s t e n e t a1 .20 s e p a r a t e d a s c o r b i c a c i d i n j u i c e and y o g h u r t and a l s o separ a t e d c i t r a t e , i s o c i t r a t e and cyclamate. Yagi e t a1.21 determined t h e l a c t i c a c i d i n l a c t i c a c i d beverages. K a i s e r and Hupf 2 2 y 2 3

described t h e determination o f

amongst a l l c a r b o x y l i c a c i d s i n d i f f e r e n t k i n d s o f foods. The p o s s i b i l i t y o f a n a l y s i n g compounds f r o m enzymatic r e a c t i o n s i n v i v o and i n v i t r o was c o n s i d e r e d n e a r l y 10 y e a r s ago by BeckersZ4 and K ~ p w i l l e m ~Dunn ~ . and KempZ6 o b t a i n e d glucose6-phosphate and f r u c t o s e - 6 - p h o s p h a t e as a mixed zone i n t h e e x t r a c t f r o m p e r f u s e d mouse l i v e r c e l l s . S j o d i n e t a l . 27 y 2 8

analysed s e v e r a l sugar d e r i v a t i v e s and

BoEek and co-workers 2 9 y 3 0 s t u d i e d t h e Krebs c y c l e . Tschope's group analysed o x a l a t e i n u r i n e i n o r d e r t o s t u d y r e n a l s t o n e d i s e a s e and problems r e l a t e d t o o x a l a t e metabolism. T h e i r most r e c e n t work was p u b l i s h e d by Bommer e t a ~ ~ F' u .r t h e r i n f o r m a t i o n about o x a l a t e d e t e r m i n a t i o n i s g i v e n i n r e f s . 32 and 33; t h e o x a l a t e zone has been v e r i f i e d by Tschope and co-workers by means of t h e enzyme o x a l a t e d e c a r b o x y l ase. Sol l e n b e r g and Bal d e ~ t e nanalysed ~ ~ v a r i o u s c a r b o x y l i c a c i d s i n u r i n e a f t e r exposure o f humans t o s t y r e n e , t o l u e n e and xylene. P h e n y l g l y o x y l i c , mandelic, h i p p u r i c and m e t h y l h i p p u r i c a c i d s were r e c o v e r e d by a s i m p l e e t h e r e x t r a c t i o n . The minimal measurable amount ( w i t h t h e

LKB Tachophor i n s t r u m e n t ) was 0.5 nmol and t h e a n a l y s i s t i m e was ca. 20 min. Van d e r Hoeven and co-workers 35y36 r e p o r t e d t h e d e t e r m i n a t i o n o f v a r i o u s o r g a n i c a c i d s produced by t h e microorganisms on t e e t h . The a n a l y s i s o f t h e s e a c i d s i s i m p o r t a n t i n i d e n t i f y i n g anaerobic b a c t e r i a . They q u a n t i f i e d t h e 1ow-molecul a r - w e i g h t c a r b o x y l i c a c i d s i n d e n t a l plaque f r o m g n o t o b i o t i c r a t s i n o c u l a t e d w i t h S t r e D t o coccus mutans and determined t h e a c i d f e r m e n t a t i o n p r o d u c t s f r o m c u l t u r e s o f Streptococcus mutans grown i n v a r i o u s media. The q u a l i t y o f s i l a g e e x t r a c t s has been determined by BoEek e t a1.37, who q u a n t i f i e d b u t y r a t e , a c e t a t e and l a c t a t e w i t h o u t p r e - t r e a t m e n t o f t h e sample. The c o n c l u s i o n o f Tschope and co-workers,

u s i n g t h e enzyme o x a l a t e decarboxylase,

t h a t t h e t o t a l o x a l a t e i n u r i n e must be found i n two s e p a r a t e zones, was conf i r m e d by F r e d e r i k ~ s o n ~who ~ , s t u d i e d t h e f o r m a t i o n o f o x a l a t e - i r o n ( I I 1 ) complexes. He showed t h a t a v e r y l o w amount o f i r o n ( I I 1 ) i o n s ( o r i g i n a t i n g , f o r i n s t a n c e , f r o m t h e needle o f t h e s y r i n g e used f o r i n j e c t i o n o f t h e sample) was s u f f i c i e n t t o be d e t e c t e d as an o x a l a t e - i r o n ( I I 1 ) i o n complex. T h e r e f o r e , Tschb'pe and coworkers recommended t h e use o f a complexing agent (EDTA o r EGTA) i f o x a l a t e needs t o be analysed. M i k k e r s e t a1 .39 and Verheggen e t a1 .40 measured v a r i o u s c a r b o x y l i c a c i d s a t serum l e v e l s . They p o i n t e d o u t t h a t , u s i n g u l t r a f i l t r a t i o n techniques, mistakes can e a s i l y be made owing t o t h e p r o t e i n - b i n d i n g c a p a c i t y o f t h e v a r i o u s a c i ds. F i g . 4.2 shows t h e a f f i n i t y o f u r i c a c i d t o albumin. U s i n g u l t r a f i l t r a t i o n f a l s e r e s u l t s a r e e a s i l y o b t a i n e d . The u r a t e - a l b u m i n complex i s u n s t a b l e under isotachophoretic

condition^^^.

Examples can be g i v e n o f substances ( h e p a r i n ) t h a t

form i r r e v e r s i b l e complexes w i t h p r o t e i n s . P o s s i b l y v i a t h e s e i r r e v e r s i b l e comp l e x e s t h e p r o t e i n s can be m o b i l i z e d i n o r d e r t o improve t h e i s o t a c h o p h o r e t i c s e p a r a t i o n o f these h i g h - m o l e c u l a r - w e i g h t substances.

J ....

F i g . 4.2. I s o t a c h o p h o r e t i c a n a l y s i s o f u r a t e i n serum ( a ) w i t h o u t s a q p l e p r e t r e a t m e n t and ( b ) a f t e r u l t r a f i l t r a t i o n . The f i l t e r used had a m o l e c u l a r w e i g h t c u t - O f f o f 25,000.

39 REFERENCES

1 Z. Deyl ( E d i t o r ) , EZectrophoresis, A Survey of Tehcniques and AppZications, Part A: Techniques, E l s e v i e r , Amsterdam, Oxford, New York, 1979. 2 F.M. E v e r a e r t s , J.L. Beckers and Th.P.E.PI. Verheggen, Isotachophoresis, Theory, Instrumentation and AppZications, E l s e v i e r , Amsterdam, Oxford, New York, 1976. 3 F. Schonhofer and F. Grass, J . Chromatogr., 110 (1975) 265. 4 J.R. Sargent and S.G. George, Methods in Zone FZectrophoresis, BDH Chemicals, Poole, 3 r d ed., 1975. 5 Th. P.E.M. Verheggen, F.E.P. M i k k e r s and F.M. E v e r a e r t s , J . Chromatogr., 132 (1977) 205. 6 F. E.P. N i k k e r s , F. M. E v e r a e r t s and Th.P. E.M. Verheggen, J. Chromatogr., 169 ( 1 9 l 9 ) 11. 7 F. S p r t a .and M. BartuSek, CoZZect. Czech. Chem. Commun., 39 (1374) 563. 8 P.C. Yadava, A.K. Ghose, K.L. Yadava and A.K. Dey, J . Chromatogr., 119 (1976) 563. 9 V. Vajgand and T. S u r A n y i - M i h a j l o v i t , TaZanta, 22 (1975) 803. 10 N.L. S t e y e r t h a l , A.B. Nesse and J.M. Castagnino, Acta Bioquim. CZin. Latinoam., 11 (1977) 139. 11 J.L. Beckers and F.M. E v e r a e r t s , J . Chromatogr., 51 (1970) 339. 12 W. Houtermans, Isotachophoresis in non-aqueous solutions, G r a d u a t i o n r e n o r t , Eindhoven U n i v e r s i t y o f Technology, Eindhoven, 1972. 13 F.E.P. M i k k e r s , F.M. E v e r a e r t s and J.A.P. Peek, J. Chromatogr., 168 (1979) 293. 14 F.E.P. Mikkers, F.M. E v e r a e r t s and J.A.F. Peek, J . Chromatogr., 165 (1979) 317. 15 D. Kaniansky and F.M. E v e r a e r t s , J. Chromatogr., 148 (1978) 441. 16 F.M. E v e r a e r t s and W.J.M. Konz, J . Chromatogr., 65 (1972) 287. 17 F.M. E v e r a e r t s , Th. P.E.N. Verheggen and F.E. P. M i k k e r s , J . Chromatogr., 169 (1979) 21. 18 F. Mikkers, T. Verheggen, F. E v e r a e r t s , J. Hulsman and C. M e i j e r s , J . Chromatogr., 182 (1980) 496. 19 T. Verheggen, F. M i k k e r s , F. E v e r a e r t s , F. Oerlemans and C. de Bruyn, J . Chromatogr., 182 (1980) 317. 20 A. Baldesten,S.-G. H,jalmarsson and G. Neumann, 2 . Anal. Chem., 290 (1978) 148. 21 T. Yagi, Y. S h i o g a i and J. Akiyama, Shimadzu, Hydron, 34, 4 (1977) 229. 22 K. K a i s e r and H. Hupf, Deut. Lebensm.-Rundsch., 75 (1979) 346. 23 K. K a i s e r and H. Hupf, Deut. Lebensm.-Rundsch., 75 (1979) 300. 24 J.L. Beckers, Isotachophoresis: Some FundmentaZ Aspects, Thesis, Eindhoven U n i v e r s i t y o f Technology, J.H. Pasmans, ' s Gravenhage, 1973. 25 A. Kopwillem, Acta Chem. Scand., 27 (1973) 2426. 26 J. Dunn and R. Kemp, Protides BioZ. FZuids, 22 (1975) 727. 27 B. S j i j d i n , A. Kopwillem and J. Karlsson, J . Scand. C Z i n . Lab. Invest., 35 (1975) 699. 28 B. S j o d i n , A. KoDwillem and J. Karlsson, Protides BioZ. Fluids, 22 (1975) 733. 29 P. BoEek, M. Deml and J . JanAk, J . Chromatogr., 106 (1975) '283. 30 P. Botek, K. Lekova, M. Deml and J. Janiik, J . Chromatogr., 117 (1976) 97. 31 3. Bommer, E. R i t z , !J. Tschope, R . Waldherr andiM1. Gebhardt, Kidney I n t . , 16 (1979) 722. 32 K. Smidt, G. B r u c h e l t and V. Hagmaier, J . CZin. Chem. C Z i n . Biochem., 17 (1979) 187. 33 W. Tschope, R. Brenner, A. B a l d e s t e n and E. R i t z , i n A. Adam and C. Schots ( E d i t o r s ) , BiochemicaZ and BioZogicaZ AppZications o f Isotachophoresis, E l s e v i e r , Amsterdam, Oxford, New York, 1980, NPD. . . 117-126. 34. J. S o l l e n b e r g and A: Baldesten, J . Chrornatogr., 132 (1977) 469. 35 J.S. van d e r Hoeven and H.C.M. Franken, i n A. Adam and C. Schots ( E d i t o r s ) , BiochernicaZ and BioZogicai! AppZications o f Isotachophoresis, E l s e v i e r , Amsterdam, Oxford, New York, 1980, pp. 69-79.

40

36 J.S. van d e r Hoeven, H.C.M. Franken, P.J.M. Camp and C.W. D e l l a b a r r e , A p p l . Envirown. Microbiol. , 35 (1978) 17. 37 P. BoEek, S. Pavelka, K. G r i g e l o v B , I!. Deml and J . JanBk, J . Chrornatogr.. 154 (1978) 356. 38 S. F r e d e r i k s s o n , J . Chrornatogr., 188 (1980) 266. 39 F. M i k k e r s , S. R i n g o i r and R. de Smet, J . Chrornatogr., 162 (1979) 341. 40 Th.P.E.M. Verheggen, F.E.P. M i k k e r s and F.Y. E v e r a e r t s , Protides BioZ. F l u i d s , 27 (1979) 723.

41

Chapter 5 STEROIDS AND STEROID CONJUGATES

2 . DEYL The s e p a r a t i o n o f f r e e s t e r o i d s and s u l p h a t e and g l u c u r o n i d e c o n j u g a t e s i s f a i r l y s i m p l e . U r i n e i s t h e m a t e r i a l most commonly analyzed. U r i n e samples a r e a d j u s t e d t o pH 10 w i t h 2.5 m o l / l NaOH, t h e s o l u t i o n i s made 5% w i t h r e s p e c t t o NaCl and t h e s t e r o i d s and t h e i r c o n j u g a t e s a r e o b t a i n e d by r e p e a t e d e x t r a c t i o n w i t h n-butanol'.

Washing o f t h e e x t r a c t s w i t h 0.04 m o l / l NaOH h e l p s t o o b t a i n

good r e s u l t s i n subsequent e l e c t r o p h o r e s i s . The most c o n v e n t i o n a l paper separat i o n i s r u n a t pH 7.4 i n 0 . 1 m o l / l phosDhate b u f f e r . The c o n j u g a t e d 17-ketos t e r o i d s a r e d e t e c t e d w i t h a l k a l i n e m - d i n i t r o b e n z e n e r e a g e n t . The c o l o u r s a r e developed by h e a t i n g t h e chromatogram. Spots o f s t e r o i d s u l p h a t e s move towards t h e anode; s t e r o i d g l u c u r o n i d e s move i n t h e same d i r e c t i o n b u t more s l o w l y . Plasma s t e r o i d s u l p h a t e s and g l u c u r o n i d e s can a l s o be seDarated by p a p e r e l e c t r o p h o r e s i s i n 0.1 m o l / l v e r o n a l b u f f e r (pH 8.6) c o n t a i n i n g 20% o f e t h a n o l 2 . The s u l p h a t e and g l u c u r o n i d e c o n j u g a t e s m i g r a t e towards t h e anode, t h e f o r m e r more r a p i d l y t h a n t h e l a t t e r ; unconjugated s t e r o i d s remain a t t h e s t a r t . The zone o f g l u c u r o n i d e c o n j u g a t e s can be r e v e a l e d by d i p p i n g t h e e l e c t r o p h e r o g r a m i n t o a f r e s h l y prepared s o l u t i o n o f d i p h e n y l t e t r a z o l i u m c h l o r i d e ( 2 % o f t h e r e a g e n t ,

4 volumes o f 10% NaOH i n 60% m e t h a n o l ) . S u l p h a t e c o n j u g a t e s can be r e v e a l e d w i t h Zimmerman r e a g e n t ( f r e s h l y p r e p a r e d a l k a l i n e m - d i n i t r o b e n z e n e i n e t h a n o l ) E s t r i o l c o n j u g a t e s can be s e p a r a t e d i n 0 . 2 m o l / l a c e t a t e (PH 4 . 5 ) , b u f f e r ( 0 . 1 m o l / l ) (pH 6.8) o r i n 0 . 1 m o l / l v e r o n a l b u f f e r (pH 8.9)

3

.

i n phosphate ( T a b l e 5.1)

( r e f . 4). TABLE 5.1 ELECTROPHORETIC MIGRATIONS OF ESTRIOL CONJUGATES (REF. 4 ) Electrolyte

0.2 m o l / l A c e t a t e (pH 4.5) 0.1 m o l / l Phosphate (pH 6.8) 0 . 1 m o l / l Verona1 (pH 8.9)

Sodium e s t r i o l - 3 s u l p h a t e (cm) 6.5 7.7, 6.0a 6.0

aVal ues determined a t d i f f e r e n t t i m e s .

Sodi um e s t r i 01 - 16 ( 1 7 ? )g l u c o s i d u r o n a t e (cm) 3.0 4.5 4.5

42

TABLE 5.2 ELECTROPHORETIC MIGRATIONS OF SOME STEROID ESTERS (REF. 5) Compound

Androsterone s u l p h a t e Cortisone g l y c i n a t e Dehydroepiandrosterone s u l p h a t e D i c o r t i s o l e phosphate

75 -80 85 53

7-Hydroxydehydroepiandrosterone s u l p h a t e 7-Hydroxydehydroepi androsterone d i s u l phate 7-Oxodehydroepiandrosterone s u l p h a t e P r e d n i s o l one hemi s u c c i n a t e P r e d n i s o l o n e phosphate Prednisone g l y c i n a t e Testosterone g l y c i n a t e

46 -56 59 35

65 -27 32

73 -55 73 39

-

60

62 96 61 52 82 -87 -97 -

13 24 -53 -5 1

63 75 -56 -72

46

-

- 32 -

asodium c i t r a t e - H C 1 (pH 3 . 5 ) , p = 0.05; S c h l e i c h e r and S c h i i l l 2043b paper, 10 V/cm, 2 h. b0.05 m o l / l P y r i d i n e - a c e t i c a c i d (pH 3.5); Whatman No. 1 paper, 40 V/cm, 1 h. :Phosphate b u f f e r (pH 7.3), = 0.055; o t h e r c o n d i t i o n s as i n a. Phosphate b u f f e r (pH 7 . 3 ) , p = 0.055; o t h e r c o n d i t i o n s as i n b. eRef. 5; 0.1 m o l / l veronal b u f f e r (pH 9 . 2 ) ; Whatman No. 1 paper, 20 V/cm, 2 h; mobi 1 i t y r e 1 a t i v e t o p - n i t r o p h e n o l = 100. E l e c t r o p h o r e t i c m i g r a t i o n s o f s t e r o i d e s t e r s ( c o n v e n t i o n a l paper e l e c t r o p h o r e s i s ) a r e summarized i n Table 5.2 ( r e f . 5 ) . These compounds a r e d e t e c t e d e i t h e r d i r e c t l y under UV l i g h t o r w i t h Zimmerman reagent. G l y c i n a t e c o n j u g a t e s a r e n i n h y d r i n p o s i t i v e and can be d e t e c t e d a c c o r d i n g l y6

.

S t e r o i d s c o n j u g a t e d w i t h s u l p h u r i c a c i d (dexamethasone-SO3-) t o i s o t a c h o p h o r e t i c s e p a r a t i o n u s i n g 0.02 m o l / l ammediol

can be s u b j e c t e d

, hydrochloric

a c i d and

0.25% methyl c e l l u l o s e as l e a d i n g e l e c t r o l y t e (pH 6 . 5 ) and 0.01 m o l / l phenol and barium h y d r o x i d e (pH 10.0) as t e r m i n a t o r 7 . Unconjugated s t e r o i d s can be separated i n b o r a t e b u f f e r (0.01-0.3 a c i d , pH 8.5-9.4,

mol/l b o r i c

a d j u s t e d w i t h NaOH, which c o u l d be d i l u t e d w i t h

S t e r o i d s a r e a p p l i e d on t o t h e paper sheet i n m e t h a n o l i c s o l u t i o n . S t e r o i d - c o n t a i n i n g zones a r e r e v e a l e d under UV l i g h t . More s p e c i f i c i d e n t i f i c a t i o n o f compounds i s p o s s i b l e by u s i n g one o f t h e many r e a g e n t s r e p o r t e d by Neher’’. o f t h e e l e c t r o p h o r e t i c b e h a v i o u r i s g i v e n i n Table 5.3.

A survey In a d d i t i o n t o borate, i t

i s a l s o p o s s i b l e t o e x p l o i t t h e p r o p e r t i e s o f molybdate o r a r s e n i t e complexes f o r s e p a r a t i o n ; i n t h e s e s e p a r a t i o n s 0.005 m o l / l ammonium molybdate (pH 9.4) o r 0.01 m o l / l sodium a r s e n i t e (pH 9.4) a r e used as r u n n i n g b u f f e r s (50 V/cm, t i m e ) ( T a b l e 5.4).

1 h running

43 TABLE 5.3 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME STEROIDS I N BORATE BUFFER (REF. 9 ) Steroid

R e l a t i v e mobi 1 it y a i n e l e c t r o l v t e ( x 100)

Ib C-16 and (7-17 oxygenated steroids

16a-Hydroxyandrost-4-ene-3,17-dione 16a,l7g-Dihydroxyandrost-4-en-3-one

50

28

d d 91 74 d

16a,17 B- D i hydroxy- 17a-me t h y 1 es t r - 4 - e n - 3-one

16a,l7g-Dihydroxy-17~-methylestr-4-en-3-one 16a,l7~-Dihydroxy-16~-methylestr-4-en-3-one

55

16a-Hydroxypregn-4-ene-3 ,20-dione

17a-Hydroxypregn-4-ene-3,ZO-dione 16a,l7a-Dihydroxypregn-4-ene-3,2O-dione 16a-Acetoxy-17a-hydroxypregn-4-ene-3,20-dione 16a,21-Dihydroxypregn-4-ene-3,2O-dione 17a,21-Di hydroxypregn-4-ene-3 ,20-dione 16a, 1 7 a - T r i hydroxypregn-4-ene-3,ZO-di one 16a, 17a,21-Tri hydroxypregn-4-ene-3 ,20-dione

0 100 0 100

20-Ethylenedioxy-11~,17~,21-trihydroxypregn-4-en-3-one 21-Acetoxy-20-ethyl enedi oxy- 1l g ,16a, 1 7 a - t r i hydroxypregn4-en-3-one 48 9a-F1 uoro- 116,16a, 17a ,21-tetrahydroxypregn-4-ene100 3,20-di one

109 7

b 0 100 59,100 0 86 100

9a-F1uoro-ll~,l6a,l7a,20a,2l-pentahydroxypregn4-en-3-one

79

100

16a,l7a-Dihydroxy-17~-hydroxymethyl-D-homoandrost4-ene-3,17a-dione

7 8e

16a,l7aa-Dihydroxy-l7aa-hydroxymethyl-D-homoandrost4-ene-3,17-dione 9a-F1 uoro- 11@,16a, 1 7 a a - t r i hydroxy- 17a6-hydroxymethyl -

D-homoandrost-4-ene-3,17-dione

66e 74

59e

C-20 hydroxysterols

S l i g h t streak

17a ,20a-Di hydroxypregn-4-en- 3-one

17a,ZOa-Dihydroxypregn-4-en-3-one 11B,20B,21-Trihydroxypregn-4-en-3-one 17a,20gY21-Tri hydroxypregn-4-en-3-one 208,2 1 - D i acetoxy- 17a-hydroxyDregn-4-en-3-one

31

9a-Fluoro-ll~,17a,20~,2l-tetrahydroxy~regn-4-en-3-one 23

21 11 72 0 69

a M o b i l i t y toward t h e anode and r e l a t i v e t o t h a t o f 90.-fluoro-16a-hydroxycortisol ; bWhatman No. 3 MM c e l l u l o s e paper, 500 V ( 1 1 V/cm) f o r 5 h a t room temperature. 0.3 m o l / l b o r i c a c i d a d j u s t e d t o pH 8.5 w i t h 10% NaOH, t h e n d i l u t e d 1:l w i t h methanol. :As i n b, e x c e p t pH a d j u s t e d t o 9.3. S l i g h t movement towards t h e cathode. e M i g r a t i o n r a t e s o b t a i n e d a t pH 8.0.

44

TABLE 5.4 MOBILITY OF SOME STEROIDS (cm/h) AS BORATE, MOLYBDP.TE AND ARSENITE COMPLEXES (REF. 10) Steroid

Mobility in 0.01 mol/l

A1 dosterone Corticosterone Tetrahydrocorticosterone

Desoxycorticosterone Cortisol Tetrahydrocortisol Cortizone Tetrahydrocortizone Pregnane-3a,llf3,17a,2Oa-tetrol Pregnane-3a,118,17ny21-tetrol-2O-one Pregnane-3aY178,20a,2l-tetrol-ll-one Pregnane-3a,17a,208-triol

A5- Pregnen-3a,17a ,ZOa-triol A5- Andros ten-3a-01 - 17-one

Na3B03

0.005 mol/l ammoni um

(DH 9.4)

molybdate (PH 9.4)

4.0 5.2 5.0 3.6 5.3 5.1 5.0 5.1 4.5 5.0 4.0 0.0 0.0 2.1

5.3 5.3 5.3 4.4 5.3 5.7 5.4 5.5 5.2 5.3 5.0 0.0 0.0 2.0

0.01 mol/l

NagAsOg (PH 9.4)

5.0

4.8 5.4 4.2 5.2 5.0 5.3 4.8 5.2 5.7 4.1 0.0 0.0 3.5

REFERENCES

G. Cavina and L. Tentori, CZin. Chirn. Acta, 3 (1958) 160. G.L. Cohen and P.K. Bondy, J . BioZ. Chem., 234 (1959) 31. C.J. Migeon and J.E. Plager, Recent Progr. Horm. Res., 9 (195 ) 235. P. Troen, B. Nilsson, N . lsliqvist and E. Diczfalusy, Acta Endocrinol., 38 (1961) 361. 5 L. StBrka, J. sulcova and K. Silink, CZin. Chim. Acta, 7 (1962) 309. 6 A.L. Levy and D. Chung, Anal. Chern., 25 (1953) 396. 7 Shimadzu application data NO. 1, CapiZZary type isotachophoretic analyzer, p. 11. 8 M. Bulaschenko, E.M. Richardson and F.C. Dohan, Arch. Biochern. Biophys., 87 (1960) 81. 9 R.H. Blank, W.K. Hausmann, C.E. Holmlund and N . Bohonos, J . Chrornatogr., 17 (1965) 528. 10 J. FerenEikovB, Chem. L i s t y , 61 (1967) 1505. 11 R. Neher, J . Chromatogr., 1 (1958) 205. 1 2 3 4

45

Chapter 6 AMINES 2. DEYL

I n most b u f f e r systems amines, owing t o t h e p r o t o n a t i o n o f t h e amino group up t o pH 9, m i g r a t e towards t h e cathode, except i f t h e r e are o t h e r s u b s t i t u e n t s t h a t might a f f e c t t h e behaviour o f these compounds i n an e l e c t r i c a l f i e l d . F u r t h e r , the behaviour o f amines upon e l e c t r o p h o r e s i s depends on t h e s i z e o f t h e molecule, a b s o r p t i v i t y t o t h e sorbent (mostly paper and l o c a t i o n o f t h e amino group along t h e carbon chain. TABLE 6.1 BUFFERS USED FOR THE SEPARATION OF AMINES (REF. 1) No.

Components

PH

1

( a ) HC1, 0.075 m o l / l ( b ) HC1, 0.01 m o l / l i n 50% ethanol ( c ) HC1, 0.1 m o l / l ( d ) HC1-KC1 b u f f e r , 0.004 A c e t i c a c i d (85% aqueous s o l u t i o n ) Citrate buffer: 10.5 g o f c i t r i c a c i d + 2 g o f NaOH a d j u s t e d t o pH 3.8 and d i l u t e d t o 500 m l w i t h water C i t r a t e phosphate b u f f e r s : ( a ) 0.40 m l o f 0.2 m o l / l K2HP04 t 19.60 m l o f 0.1 m o l / l c i t r i c a c i d , d i l u t e d t o 40 m l ( b ) 4.11 m l o f 0.2 m o l / l K2HP04 t 15.99 m l o f 0.1 m o l / l c i t r i c a c i d , d i l u t e d t o 40 m l ( c ) 7.71 m l o f 0.2 m o l / l K2HP04 + 12.29 m l o f 0.1 m o l / l c i t r i c acid, d i l u t e d t o 40 m l ( d ) 10.30 m l o f 0.2 m o l / l K2HP04 t 9.70 m l o f 0.1 mol/l c i t r i c a c i d d i l u t e d t o 40 m l ( e ) 12.63 m l o f 0.2 m o l / l K2HP04 + 7.37 m l o f 0.1 mol/l c i t r i c acid, d i l u t e d t o 40 m l ( f ) 16.47 m l o f 0.2 m o l / l K2HP04 + 3.53 m l o f 0 . 1 m o l / l c i t r i c a c i d , d i l u t e d t o 40 m l ( 9 ) 19.45 m l o f 0.2 m o l / l K2HP04 + 0.55 m l o f 0.1 m o l / l c i t r i c acid, d i l u t e d t o 40 m l Phthalate buffer: 100 m l o f 0.1 m o l / l potassium a c i d p h t h a l a t e + 0.8 m l of 0.1 m o l / l NaOH, d i l u t e d t o 200 m l

-

2 3 4

5

-

1.75 2.7

-

3.8

2.35 3.1 4.2 5.2 5.8 7.2 8.7 4.0

(Continued on p . 4 6 )

46

TABLE 6.1 ( c o n t i n u e d ) No. 6 7

Components

PH

Sulphosalicylate buffer: 0.05 m o l / l s u l p h o s a l i c y l i c a c i d a d j u s t e d t o pH 4.0 w i t h NaOH Pyridine-formate b u f f e r : 19 g o f 88-90% f o r m i c a c i d and 2 g o f p y r i d i n e a r e mixed and d i l u t e d w i t h w a t e r t o 100 m l F o r use 2 m l o f t h i s s t o c k s o l u t i o n a r e mixed w i t h 7.5 m l o f formamide and d i l u t e d w i t h w a t e r t o 25 m l 2-Dimethylaminopropionitrile-acetate b u f f e r : 19.6 g o f 2 - d i m e t h y l a m i n o p r o p i o n i t r i l e and 6 g g l a c i a l a c e t i c a c i d a r e d i l u t e d t o 100 m l ; 3 m l o f t h e s t o c k s o l u t i o n and 7.5 m l o f formamide a r e d i l u t e d w i t h w a t e r t o 25 m l 2-Dimethylaminoethanol-acetate b u f f e r : 18 g o f 2-dimethylaminoethanol and 6.6 g o f g l a c i a l a c e t i c a c i d a r e d i l u t e d w i t h w a t e r t o 100 m l . F o r use 2 m l o f t h e s t o c k s o l u t i o n and 7.5 m l formamide a r e d i l u t e d t o 25 m l w i t h w a t e r Sodium b a r b i t u r a t e b u f f e r s : ( a ) 0.04 m o l / l sodium b a r b i t u r a t e ( b ) 10.3 g o f sodium v e r o n a l and 1.84 g o f d i e t h y l b a r b i t u r i c a c i d are dissolved i n 1 l i t r e o f water Ammonia s o l u t i o n , 0.065 m o l / l

4.0

3.3

.

8

9

10

11

7.2

9.3

8.0 8.6

10

E l e c t r o p h o r e t i c s e p a r a t i o n s on paper have been c a r r i e d o u t o v e r a v e r y wide pH range (see t h e l i s t o f b u f f e r s i n Table 6 . 1 ) . The c h o i c e o f t h e a p p r o p r i a t e b u f f e r depends on t h e m i x t u r e o f amines t o be separated. When t h e sep,aration i s based on m o l e c u l a r s i z e alone, t h e n any pH a t which a l l t h e components o f t h e m i x t u r e a r e p r o t o n a t e d i s s u f f i c i e n t ; when, however, t h e s i z e s o f t h e molecules a r e s i m i l a r , t h e n b e s t s e p a r a t i o n s a r e achieved i n t h e neighbourhood o f t h e p~ o f t h e p a r t i c u l a r amines 1 F o r an e f f i c i e n t s e p a r a t i o n o f amines f r o m amino

.

a c i d s 2 i t i s p o s s i b l e t o r u n c o n v e n t i o n a l paper e l e c t r o p h o r e s i s i n a c e t i c a c i d f o r m i c a c i d - w a t e r (200:60:740);

e t h y l e n e d i a m i n e , h i s t a m i n e , e t h y l a m i n e and

rn-phenylenediamine a r e w e l l s e p a r a t e d b o t h f r o m each o t h e r and f r o m t h e t w e n t y common amino a c i d s . Various r e a g e n t s can be used f o r d e t e c t i o n . Of many t h a t were l i s t e d by B l a u o n l y a few a r e mentioned here: F o l i n ' s reagent, E h r l i c h ' s r e a g e n t , n i n h y d r i n ,

3

p-nitrobenzenediazonium f l u o r o b o r a t e and UV l i g h t ( f o r a r o m a t i c amines). Colours a r i s i n g f r o m t h e r e a c t i o n w i t h p - n i t r o b e n z e n e d i a z o n i u m f l u o r o b o r a t e a r e o f cons i d e r a b l e d i a g n o s t i c v a l u e ( T a b l e 6.2) ( r e f . 4 ) . E l e c t r o p h o r e t i c m i g r a t i o n o f homologous a1 kylamines , some s i m p l e a r o m a t i c amines and a number o f amino compounds w i t h o t h e r s u b s t i t u e n t s a r e summarized i n Tables 6.3-6.5.

,

TABLE 6.2

COLOUR REACTIONS OF SOME AROMATIC AMINES WITH p-NITROBENZENEDIAZONIUM FLUOROBORATE (REF. 4 ) a

Compound

Neutral o r a c i d i c s o l u t i o n

Alkaline solution

p - h i nobenzoi c a c i d o-Aminophenol m-Aminophenol Aniline Anthranilic acid Benz id i ne o-Chloroaniline m-Chl o r o a n i 1 ine p - C h l o r o a n i 1 ine D i e t h y l a n i 1i n e Diethyl-a-naphthylamine D i e t h y 1 -m-t o 1 u id i ne Dimethyl a n i 1 ine Ethylanil ine Ethyl-a-naphthylamine Methylaniline a-Naphthyl ami ne 8-Naphthylami ne p-Nitroaniline N i t r o s o - 6-naphthol Phenol m-Phenyledediamine p-Phenylenediamine P i crami c a c i d Pyrogal l o 1 Sulphanilic acid o-Toluidine m- To 1u id ine p-To1 u i d i ne m- Xy 1id i ne

L i g h t ye1 1ow Ye1 1ow brown Ochre Orange Bright yellow Yellow-green Light yellow Orange Lemon-ye1 1ow Dark r e d Dark v i o l e t Pink-red Brown-red Ye1 1ow-orange Purple Ye1 low-orange Violet Pink Bright yellow L i g ht ye1 1ow L i g h t ye1 1ow Light rust Orange- brown Pale y e l l o w B r i g h t orange Greenish y e l l o w Ye1 1ow-orange Orange-red Green-ye1 1ow P i nk-orange

Pink Black-violet Purple Crimson Red Blue-black V i 01 e t - r e d Dark r e d Violet-red Dark r e d Dark v i o l e t Red Brown-red Ye1 1ow-orange Purple Ye1 1ow-orange Dark v i o l e t Light violet Pale v i o l e t Light yellow Dark r e d Red-violet Grey-brown Light violet V i 01 e t - b l ack Pink-red Brown-vi o l e t Dark v i o l e t Light violet Brown-red v i o l e t

a 0.5% aqueous s o l u t i o n o f p - n i t r o b e n z e n e d i a z o n i u m f l u o b o r a t e . I f one t h e n sprays w i t h 1 m o l / l NaOH t h e r e i s a change i n c o l o u r t h a t can be r e v e r s e d t o t h e o r i g i n a l c o l o u r b y a c i d i f y i n g w i t h HC1.

L a t e r t h e a p p l i c a t i o n o f o r g a n i c s o l v e n t s was i n v e s t i g a t e d f o r t h e s e p a r a t i o n o f h i g h e r a l i p h a t i c amines ( u p t o CI8), i n p u r e l y aqueous b u f f e r s " .

m a i n l y because o f t h e i r p o o r s o l u b i l i t y

A 0.1 m o l / l s o l u t i o n o f NalCl d i l u t e d w i t h a l i p h a t i c

a l c o h o l s ( o r mixed w i t h d i m e t h y l s u l p h o x i d e ) was u s e f u l i n t h e paper e l e c t r o p h o r e s i s o f t h e s e compounds.

48

TABLE 6.3 ELECTROPHORETIC MIGRATION OF HOMOLOGOUS ALKYLAMINES ( C1-C18) A1 k y l ami ne

(REF. 5 ) a

M i q r a t i o n fcm) i n e l e c t r o l y t e b

Methylamine Ethylamine Propylamine B u t y l ami ne Hexyl ami ne O c t y l ami ne Nonylamine Oecyl ami ne Dodecyl ami ne T e t r a d e c y l ami ne Hexadecyl ami ne Octadecyl ami ne

1

2

3

4

-16.0 -14.0

-16.5 -13.1 -10.4 -9.6 -7.0 -1.8

-7.4 -6.0

-5.1

-5.0 -4.2 -3.8

-3.8 -3.2 -2.8

-2.9 -2.8 -2.5

-2.4 -2.2 -2.0 -1.8 -1.7

-

-10.8 -9.0 -8.3 -8.0 -7.0 -5.4 0 0 0

-

-

0 0

-

-4.4

-

:Whatman No. 3 paper, 18.5 V/cm f o r a h, 1 5 . 5 O C . E l e c t r o l y t e s : 1, 7.5 10-3 m o l / l HC1; 2, 0.014 m o l / l potassium c a p r y l a t e ; 3, 0.01 m o l / l HC1 i n 50% e t h a n o l ; 4, 85% a c e t i c a c i d . The m i g r a t i o n i s towards t h e cathode.

.

10

15

20 min

F i g . 6.1. I s o t a c h o p h o r e t i c s e p a r a t i o n o f methylamines ( s t a n d a r d s o l u t i o n ) . Leading e l e c t r o l y t e : 0.01 m o l / l potassium a c e t a t e , a c e t i c a c i d and 0.2% T r i t o n X-100 (pH 4.3). Terminal e l e c t r o l y t e : 0.01 m o l / l h i s t i d i n e and 0.01 m o l / l a c e t i c a c i d (pH 4.3). C a p i l l a r y tube 20 cm; 100 PA, 20oC.

49

Ethanolamines and methylamines can be s e p a r a t e d b y isotachophoresis''.

For

ethanolamines 0.005 m o l / l b a r i u m h y d r o x i d e and g l u t a m i c a c i d (pH 4 . 2 ) s e r v e s as t h e l e a d i n g e l e c t r o l y t e and 0.01 mol'/l h i s t i d i n e and h y d r o c h l o r i c a c i d (pH 4.0) i s used as t h e t e r m i n a t o r . Methylamines a r e s e p a r a t e d w i t h 0.01 m o l / l potassium a c e t a t e , a c e t i c a c i d and 0.2% T r i t o n X-100 (pH 4.3) as t h e l e a d i n g e l e c t r o l y t e and 0.01 m o l / l h i s t i d i n e and a c e t i c a c i d (pH 4.3) as t h e t e r m i n a l e l e c t r o l y t e (Fig. 6.1).

C o n s i d e r a b l e a t t e n t i o n i n i s o t a c h o p h o r e s i s has been p a i d t o t h e

s e p a r a t i o n o f polyamines owing t o t h e i r d i a g n o s t i c s i g n i f i c a n c e i n neoplasms; 0.005-0.01 m o l / l b a r i u m h y d r o x i d e and v a l i n e (pH 9.8-9.9)

a r e used as l e a d i n g

e l e c t r o l y t e s and 0.02 m o l / l t r i e t h y l e n e d i a m i n e i s used as t h e t e r m i n a t o r . A l t e r n a t i v e l y , 0.01 m o l / l barium h y d r o x i d e and g l u t a m i n e (pH 9.25) can be a p p l i e d as t h e l e a d i n g e l e c t r o l y t e ( F i g . 6.2).

0

10 min

F i g . 6.2. I s o t a c h o p h o r e t i c s e p a r a t i o n o f polyamines. Standard m i x t u r e o f p u t r e s c i n e , s p e r m i d i n e and spermine. Leading e l e c t r o l y t e : 0 . 0 1 m o l / l b a r i u m h y d r o x i d e and g l u t a m i n e (pH 9.25). Terminal e l e c t r o l y t e : 0.02 m o l / l t r i e t h y l e n e diamine. C a p i l l a r y t u b e 15 cm; 75 PA.

rn 0

TABLE 6.4 ELECTROPHORETIC MIGRATION OF SOME AMINES (REF. 1) Compgund

Agmatine A1 lylamine y-Aminobutyric acid N-Aminoethylpiperazine 2-Amino-2-hydroxymethyl-1,3-propanediol 3-Amino-1-propanol 1-Amino-2-propanol Amphetamine n-Amylamine Isoamylamine Arcaine Argi ni ne n-Butylamine Isobutylamine Cadaverine Chol ine Creatine Creatini ne Cytosine 1,4-Diaminobutane 1,5-Diaminopentane 1,3-Diaminopropane Diethanolamine Diethylamine Dimethylamine

Migration in electrolyte 1(3)a 2(6)b 3(7)C 4(7)e 1 (cm) (cm) (cm)

5(7)f (cm)

6(7)g

7(8)h

(cm)

(y.el)l(Mrel)’

-16.0 -15.4 -22.5 -15.2 -137 -13.0d -74 -180 - 80 -120

-117 -90

8(8)i

-137 -2

-156 - 80 -119 -117 -89

9(8)j

(Mrel)l

-123

-68

0 -95 - 14 -92 -70

-40

-64

-62 -61

-54

-7.0 -6.8 -lO.Zd -5.1 -26 -7.2

10(9)k (Mrel)’

-124 -8.3 -11.4 -12.4 -5.4 -68 -11.2d -11.2d -17.6 -17.2 -23.5 -15.0 -160 -13.4d

-62

-124 -45

-149

-137

-163

-5 -131

-36 -60 -76

-37 -0.7 -10.2 -10.4

-75 -163

- 82

-6.1

-7.9 -11.7 -81

-61 - 88

3-Dimethylamino-n-propylamine Ephedrine Epinephrine Ethanol ami ne E t h y l ami ne Ethylenediamine D-Galactosamine D-G1 ucosami ne Glycinamide Glycine Glycyl l y s i n e Grami ne Histamine H i s t i d i ne

-3.5 -8.5 -11.4

-73

-2.5

-16.5 -14.5d -8.5

-17.9

-12.7

-20.8

-9.0

-8.5

-3.9

-156

-90

-66 -134

-67 -135

-35 -93

- 197 -59 -61 -125

-159 -58 -58 -112

-73 -10 -6 -3

-89

-55

-5

-70 -79

-3 -6.2

-29 -58 -27

-17.1

-16.2

-16.0

-16.0

N-Hydroxyethylpiperazine Hydroxylamine 3-Hydroxypiperidine 3-Hydroxytryptamine 5-Hydroxytryptamine 3-Hydroxytyramine Kynurenine L y s i ne L y s y l g l y c i ne Methylamine Mescaline Neurine Nicotine Norepinephrine Orni t h i ne 2-Phenylethylamine Piperazine P i peri dine N-Propyl ami ne Putrescine Pyrrolidine Spermidine

-175 -32

-70 -37 -153

-82

-50

-105 -105

-102 - 102

-78 -78

-74

-74

-1.4

-4.3 -3.1

-13.4

-28

-10.2

-92 -25

-19.5d

-12.4

-13.4

-4.7

-88 -80 -180

-49 -187

-55 -8

-11 -40

-100

-14.6d -3.6 -3.5 -5.9 -6.6 -8.1 -9.4

-43

- 30 -41

8.5d

-61 -67

-12.4d -18.9

-90 -100 -175 -18.7

-24.3

-100 -111

-31 -75 - 82

-49 -68 - 80

-12.6

- 140 -178

-140 -147

-131 -97

-6 1

(Continued on p . 52)

2

TABLE 6.4 (continued) Compound

Spermine Taurine Tetraethylenepentamine Triethyl ami ne Trimethylamine Tryptamine Tyramine Urea

Miaration in electrolvte

-49 -10

4;7 -5.2 -30

-11.7d -16.0d -8.9 -11.4 -12.6 -10.2 -12.3 -14.4

-8.8 -6.0

-164

-144

-74

-79 -84

-79 -84

-70 -61

-35 -41

0

a b0.04 mol/l sodium barbiturate buffer (pH 8.0); Whatman No. 1 paper, 20 V/cm, 1 h. Citrate buffer, pH 3.8 (10.5 g of citric acid and 2 g of NaOH adjusted to pH 3.8 and diluted to 500 ml); 50-100 min; mobility relative to NH4+, Kf = 100; 7.5 V/cm. :0.05 mol/l potassium acid phthalate, pH 4.0; Whatman No. 3 paper, 400 V, 2 h. As c. except Whatman No. 1 paper was used; 1.5 h. ;As c, except electrolyte was 0.05 mol/l sulphosalicylic acid (pH 4.0); 2.5 h. As c, except electrolyte was veronal buffer (pH 8.6) (10.3 g of sodium veronal and 1.84 g of diethylbarbituric acid diluted to 1 litre); 2.5 h. ;As c, except buffer was 0.065 mol/l NH3 (pH 10.0); 3 h. Pyridine-formate buffer (pH 3.3) containing 30% (v/v) of formamide; !Jhatman No. 4 paper (3.8-5 x 24 cm), 10 V/cm, 1-2 h; mobility relative to amaranth (FD and C Red No. 2). i AS h, except buffer was 2-dimethylaminopropionitrile-acetate(pH 7.2) containing 30% (v/v) of formamide. ;As h, except buffer was 2-dimethylaminoethanol-acetate (OH 9.3) containing 30% (v/v) of formamide. lCitrate buffer (pH 3.8) (see b ) ; 1980 V, 35 min; mobility relative to methylamine = 100. Relative mobility x 100.

63 TABLE 6.5 RELATIVE ELECTROPHORETIC MOBILITIES OF SOME AROMATIC AMINES (REF. 4 ) a Ami ne

Relative mobi 1it y b x 100

p-Aminobenzoic a c i d An i1ine o-Chloroanil i n e m-Chloroanil i n e p-Chloroaniline D i met h y 1 a n i 1ine

86 -133 - 106 - 106 106 - 123 -101

Dimethyl-a-naphthylamine

-

-

Amine

Methylaniline a-Naphthylamine 6-Naphthyl ami ne 0-Toluidine m-Toluidine m-Xylidine

Re1a t i ve mobi 1 it y b x 100 -166

- 100 - 100 -144 -132 -106

aHC1-KC1 b u f f e r (pH 2.7), p = 0.004; Whatman No. 4 paper (28 cm l o n g ) , 8 V/cm b f o r 2 h. M o b i l i t y r e l a t i v e t o a-naphthylamine = 100. REFERENCES

1 G. Zweig and J.R. Whitaker, Paper Chromatography and EZectrophoresis, Academic Prkss, New York, 1967, p. 50. 2 Y . Kitaoka, J . Chromatogr., 147 (1978) 449. 3 K. Blau, Biochem. J . , 80 (1961) 193. 4 C. Hanot, BUZZ. Soc. Chim. Belg., 66 (1957) 76. 5 J.T. Edward and R. Crawford, Chem. Ind. (London), (1956) 1274. 6 R. Weber, HeZv. Chim. Acta, 34 (1951) 2031. 7 J. Blass, A. S a r r a f f and M.B. N i c o l a s , J . Chromatogr., 3 (1960) 168. 8 W.W. Thornburg, L.N. Werum and H.T. Gordon, J . Chromatogr., 6 (1961) 131. 9 D.L. van Rheenen, Rec. Trav. Chim. Pays-Bas, 82 (1963) 225. 10 A. Wronski, Chem. AnaZ. (Warsaw), 14 (1969) 837. 11 Shimadzu A p p l i c a t i o n d a t a No. 1, CapiZZary isotachophoretic analyzer, p. 30.

This Page Intentionally Left Blank

55

Chapter 7 AMINO A C I D S AND THEIR DERIVATIVES

2. DEYL GENERAL ASPECTS Separation methods f o r amino a c i d s are w e l l e s t a b l i s h e d ; t h e D r e f e r r e d procedures today a r e chromatographic. However, e l e c t r o p h o r e t i c separations s t i l l possess considerable importance as they can be used e f f e c t i v e l y f o r screening purposes. Equally, e l e c t r o p h o r e t i c methods can h e l p i n t h e search f o r t h e r a r e r amino acids t h a t a r i s e d u r i n g p o s t - t r a n s l a t i o n a l r e a c t i o n s i n p r o t e i n s o r t h a t occur as t h e r e s u l t o f d i v e r s e m e t a b o l i c processes and d i s o r d e r s . F i n a l l y , e l e c t r o p h o r e s i s can be used f o r t h e s e p a r a t i o n o f amino a c i d d e r i v a t i v e s t h a t a r e used i n p r o t e i n sequence a n a l y s i s . The approach t o t h e s e p a r a t i o n o f u n d e r i v a t i z e d amino a c i d s semains olmast.the same, a p a r t from small v a r i a t i o n s , whether one i s d e a l i n g w i t h t h e " c l a s s i c a l twenty" o r w i t h r a r e l y o c c u r r i n g amino acids.

AMINO ACIDS NATURALLY OCCURRING I N PROTEINS Three independent separations i n d i f f e r e n t b u f f e r systems a r e necessary i n o r d e r t o o b t a i n a complete s e p a r a t i o n o f t h e n a t u r a l l y o c c u r r i n g amino a c i d s found i n p r o t e i n hydrolysates. This procedure i s sometimes r e f e r r e d t o as u n i d i mensional m u l t i - e l e c t r o p h o r e t i c s e p a r a t i o n 1 The f i r s t e l e c t r o p h o r e s i s i s c a r -

.

r i e d o u t a t pH 5 . 2 i n , e.g.,

p y r i d i n e a c e t a t e (paper, r u n n i n g t i m e about 200 min

a t 75 V/cm). Under these c o n d i t i o n s b a s i c amino a c i d s ( l y s i n e , h i s t i d i n e , a r g i n i n e , h y d r o x y l y s i n e and o r n i t h i n e ) move as c a t i o n s , and a c i d i c amino a c i d s ( c y s t e i c acid, a s p a r t i c and g l u t a m i c a c i d s ) move as anions. N e u t r a l amino a c i d s form an unresolved zone t h a t i s d i s p l a c e d about 1.5-2 cm towards t h e cathode owing t o t h e endo-osmotic flow. The second e l e c t r o p h o r e t i c r u n i s c a r r i e d o u t a t pH 1.85 i n acetate-formate b u f f e r (100 V/cm, 270.min).

I n t h i s r u n most o f t h e n e u t r a l

amino a c i d s a r e separated. I n t h e t h i r d s e p a r a t i o n step, which i s c a r r i e d o u t a t pH 1.81 (cadmium a c e t a t e b u f f e r ) , t h e s e p a r a t i o n o f n e u t r a l amino a c i d s i s completed. The s e p a r a t i o n i t s e l f may be b e s t i l l u s t r a t e d by t h e m o b i l i t y d a t a i n Table

7.1.

Sometimes a s i n g l e h i g h - v o l t a g e (50-70 V/cm) e l e c t r o p h o r e s i s a t

DH

1.9 could

be s u f f i c i e n t f o r s e p a r a t i n g a p a r t i c u l a r m i x t u r e ; however, some i p a i r s o f com-

56

TABLE 7 . 1 RELATIVE ELECTROPHORETIC MOBILITIES OF A M I N O A C I D S AT pH 5.2, Compound

1.85 AND 1.81

Relative m o b i l i t y i n e l e c t r o l y t e

A1 ani ne 8-Alanine a-Aminobutyr,ic a c i d Argi n i ne Asparagine Aspartic acid Cystei ne Cysteic a c i d Cystine G1 utami c a c i d G1 utami ne Glycine H i s t i d i ne Hydroxyprol ine Is01e u c i ne aZZo- I s o l e u c i n e Leuci ne Lysine Hydroxylysine Methionine

la

2b

- 100 - 145

- 100

-90 -131 -71 -6 1 -60

3c

- lOOd -59

121e 130e

-59 -6 7 -69 -114 -131 -54 -77

- 78 -147 -71 -57 -60 -152 -61 -69 -83 -3 -75 - 46 -53 -81

Orni t h i n e Phe ny 1a 1an in e Pro1 ine Seri ne Taurine Threoni ne Tryptophan Tyrosi ne Valine

-66

lOOe -lOfjd

-80 -76 -78 -1lld

- 104d -55 -6 1 -69 -81 -73 -53 -81

a

2.5% (w/v) f o r m i c acid-7.8% (w/v) a c e t i c a c i d (pH 1.85); 100 V/cm; m o b i l i t y b r e l a t i ve t o a1 anine. 2.0% (w/v) formic acid-ilO% (w/v) a c e t i c acid-0.4 mmol/l cadmium a c e t a t e (DH 1.81); 100 V/cm; m o b i l i t y r e l a t i v e t o alanine. C d2.0% (w/v) pyridine-0.95% (w/v) a c e t i c a c i d (pH 5.2); 70 V/cm. M o b i l i t y r e l a t i v e t o arginine. e M o b i l i t y r e l a t i v e t o g l u t a m i c acid.

pounds remain unresolved. Low-voltage e l e c t r o p h o r e s i s can a l s o be a p p l i e d f o r t h e separation o f amino a c i d s s u c c e s s f u l l y . Over 30 years ago a s u i t a b l e procedure was i n t r o d u c e d by Durrum2 The separation i s c a r r i e d o u t on paper i n 0 . 2

.

m o l / l ammonia s o l u t i o n (pH 11.3). A f t e r r u n n i n g a t 10-15 V/cm f o r 90 min, the paper i s removed, a i r d r i e d , and then turned through 90'.

I n t h e second dimension

separation i s performed i n 1 m o l / l a c e t i c a c i d (pH 2.3). Wetting o f t h e paper f o r

57

t h e second dimension r u n i s a c h i e v e d by l e t t i n g t h e b u f f e r p e n e t r a t e by c a p i l l a r y a c t i o n from b o t h s i d e s . T h i s procedure, p r o v i d e d t h a t b o t h streams coalesced i n t h e a r e a where t h e amino a c i d s have been s e p a r a t e d i n t h e f i r s t r u n , r e s u l t s i n sharpening o f t h e amino a c i d zones. Then, e l e c t r o p h o r e s i s i s a l l o w e d t o proceed i n t h e second dimension a t 30 V/cm f o r 30 min. A t pH 11.3 t h e amino a c i d s behave as anions, whereas a t pH 2.3 t h e y behave as c a t i o n s . The s e p a r a t i o n o f b a s i c amino a c i d s can be f u r t h e r improved by u s i n g two subsequent e l e c t r o p h o r e t i c r u n s a t pH 11.7 (0.5 m o l / l ammnnia s o l u t i o n ) and a t pH 6 . 5 ( p y r i d i n e - a c e t i c a c i d - w a t e r , 200:8: 1972). I n c i d e n t l y , two-dimensional paper s e p a r a t i o n s can a l s o be r u n a t c o n s i d e r a b l y 3 . I n p r i n c i p l e , however, always one o f t h e r u n s i s c a r r i e d o u t

higher voltages

a t pH around 3 and t h e o t h e r e i t h e r a t pH 9-10 o r a t pH 5.5-6.5.

Preference i s

g i v e n t o t h o s e methods i n w h i c h s e p a r a t i o n i s c a r r i e d o u t a t pH 5.5-6.5

followed

by s e p a r a t i o n i n t h e second d i r e c t i o n near pH 2.0 ( r e f s . 4, 5 ) . I t was soon r e c o g n i z e d t h a t amino a c i d s can be s h a r p l y s e p a r a t e d on c e l l u l o s e l a y e r s a t l o w v o l t a g e s . Formic a c i d o r f o r m i c a c i d - a c e t i c a c i d (pH 2 . 0 ) i s used as t h e r u n n i n g b u f f e r ( T a b l e 7.2)

(see a l s o r e f . 7 ) . C e l l u l o s e a c e t a t e paper o r 8

t h i n l a y e r s can be used f o r s i m i l a r purposes

.

TABLE 7.2 RELATIVE ELECTROPHORETIC MOBILITIES OF COMMON A M I N O A C I D S ON THIN LAYERS OF CELLULOSE (REF. 6 ) I N 0.7% ( v / v ) FORMIC A C I D ( 3 5 V/cm, Compound Tryptophan Hydroxyproline Aspartic acid T y r o s i ne Pro1 ine Phenyl a1 a n i ne G1 utami ne Asparagine G1 utami c a c i d Methi o n i ne Threonine Serine Leucine Valine A1 a n i ne Glycine A r g i n i ne H i s t i d i ne Lys ine a-Arninobutyric a c i d 6-A1 a n i ne

15 m i n ) Mobility relative t o proline -0.84 -0.86 -0.93 -0.94 -1.00 -1.00 -1.03 -1.05 -1.05 -1.07 -1.13 -1.15 -1.15 -1.20 -1.32 -1.45 -1.97 -2.03 -2.09 -2.16 -2.22

58

I n c h r o m a t o e l e c t r o p h o r e s i s (on paper o r c e l l u l o s e l a y e r s ) i t i s necessary t o a v o i d p o s s i b l e i n t e r f e r e n c e s f r o m t h e e l e c t r o p h o r e t i c b u f f e r s i n t h e subsequent chromatographic s t e p , As t h e s e methods a r e n o t c l a s s i f i e d as p u r e l y e l e c t r o p h o r e t i c , 9 t h e y w i l l be mentioned h e r e o n l y b r i e f l y ( f o r d e t a i l e d i n f o r m a t i o n , see Munier ) . I n these s e p a r a t i o n s f o r m i c a c i d serves as t h e r u n n i n g b u f f e r i n t h e f i r s t dimens i o n . Thus, on c e l l u l o s e t h i n l a y e r s e l e c t r o p h o r e s i s can be c a r r i e d o u t i n 1.04 m o l / l f o r m i c a c i d a t 11 V/cm (7.5 mA) f o r 3 h. I n t h e second d i r e c t i o n v a r i ous a l i p h a t i c a l c o h o l - p y r i d i n e - f o r m i c a c i d m i x t u r e s can be used. The apparent pH

o f t h e m o b i l e phase used f o r t h e chromatographic s e p a r a t i o n has a c o n s i d e r a b l e e f f e c t on t h e f i n a l appearance o f t h e electropherogram. Coup1i n g e l e c t r o D h o r e s i c and chromatography on amorphous c e l l u l o s e t h i n l a y e r s was reviewed r e c e n t l y by Munier and D r a p i e r "

w i t h special reference t o t h e i n f l u e n c e o f i o n i c e q u i l i b r i a

and s o l v e n t components on chromatographic s e p a r a t i o n s w i t h a l c o h o l - a c i d - w a t e r systems. A procedure t h a t i n c r e a s e s t h e r e l i a b i l i t y o f i d e n t i f i c a t i o n o f amino a c i d s i n a two-dimensional map was r e c e n t l y p u b l i s h e d by Munier and Meunier 11

.

When s e v e r a l amino a c i d analogues a r e r u n c o n c o m i t a n t l y w i t h t h e m i x t u r e t o be analysed and t h e i r p o s i t i o n s a r e connected w i t h s t r a i g h t l i n e s , t h e n e t o b t a i n e d serves as a r e f e r e n c e system f o r t h e i d e n t i f i c a t i o n o f common amino a c i d s r u n n i n g i n t h e neigh-bourhood o f t h e i n t e r n a l standards used. Sometimes two e l e c t r o p h o r e t i c s e p a r a t i o n s f o l l o w e d by a chromatographic s t e p (on t h e same sheet o f paper o r t h i n - l a y e r p l a t e ) a r e used i n t h e s o - c a l l e d " t h r e e dimensional s e p a r a t i o n " . The f i r s t r u n i s an e l e c t r o p h o r e t i c s e p a r a t i o n , which can be done on a c e l l u l o s e t h i n - l a y e r p l a t e i n p y r i d i n e - f o r m i c a c i d - w a t e r (90:207: 1908; pH 6.5,

40 V/cm, 4 mA, r u n n i n g t i m e 1.5 h ) . Then, t h e p o i n t o f a p p l i c a -

t i o n of t h e sample i s e l i m i n a t e d and a second r u n i s c a r r i e d o u t i n t h e same d i r e c t i o n w i t h 1.04 m o l / l f o r m i c a c i d (10 V/cm, 6 . 2 mA, 3 h ) . F i n a l l y , a chromat o g r a p h i c s e p a r a t i o n i s a p p l i e d p e r p e n d i c u l a r l y i n n-propanol-ethanol-formic a c i d - w a t e r (40:38:2:20).

The d i f f e r e n c e s o b t a i n e d when t h e f i r s t e l e c t r o p h o r e t i c

s e p a r a t i o n i s o m i t t e d a r e shown i n F i g . 7 . 1 (see a l s o Munier e t a1.12 and Munier 13 and Tommegay ). S i m i l a r s e p a r a t i o n s can be c a r r i e d o u t on paper. Thus, Lewandowski and Wronski14 succeeded i n s e p a r a t i n g 26 amino a c i d s i n a d d i t i o n t o f i v e s i m p l e p e p t i d e s by u s i n g e l e c t r o p h o r e t i c s e p a r a t i o n on paper a t pH 5.2,

then c u t t i n g

t h e paper sheet i n t o two a t t h e s t a r t and s u b j e c t i n g t h e a n o d i c a l l y moving f r a c t i o n t o a n o t h e r e l e c t r o p h o r e s i s and t h e c a t h o d i c a l l y moving m i x t u r e t o chromatography. The second e l e c t r o p h o r e t i c r u n on t h e a n o d i c components was c a r r i e d o u t i n t h e p e r p e n d i c u l a r d i r e c t i o n t o t h e f i r s t one a t pH 1.5. I n c o n t r a s t , t h e f i n a l chromatographic s e p a r a t i o n o f t h e c a t h o d i c m i x t u r e was c a r r i e d o u t i n t h e same d i r e c t i o n as t h e f i r s t e l e c t r o p h o r e t i c r u n , w i t h n - b u t a n o l - a c e t i c w a t e r (4:1:1.5)

as t h e m o b i l e phase.

acid-

59

F i g . 7.1. Comparison o f r e s u l t s o b t a i n e d d u r i n g e l e c t r o c h r o m a t o g r a p h y and t h r e e dimensional e l e c t r o c h r o m a t o g r a p h y . Electrochromatography: 1 s t d i r e c t i o n : 1.04 m o l / l f o r m i c a c i d , 398 V, 6 mA, 3.3 h. 2nd d i r e c t i o n : chromatography i n n - p r o p a n o l e t h a n o l - f o r m i c a c i d - w a t e r (40:38:2:20:) ( A ) . Three-dimensional s e p a r a t i o n : 1 s t d i r e c t i o n : p y r i d i n e - f o r m i c a c i d - w a t e r (90:2.07:1907) (pH 6 . 5 ) , 400 V , 4 mA, 1.15 h. The s t a r t p o i n t i s e l i m i n a t e d . 2nd d i r e c t i o n ( t h e same as t h e f i r s t ) : 1.04 mol/l f o r m i c a c i d , 400 V, 3 h, 6.2 mA. 3 r d d i r e c t i o n ( p e r p e n d i c u l a r t o t h e f i r s t two r u n s ) : n-propanol-ethanol-formic a c i d - w a t e r (40:38:2:20), 8 h; mc = methyl orange; t h e number a f t e r mc r e f e r s t o t h e p o s i t i o n o f t h e dye a f t e r t h e p a r t i c u l a r r u n . Sorbent : c e l l u l ose 1a y e r . The arrangement o f t h e " t h r e e - d i m e n s i o n a l "

s e p a r a t i o n c o u l d be m o d i f i e d i n

such a way t h a t i n t h e f i r s t r u n t h e complex m i x t u r e o f amino a c i d s i s s e p a r a t e d by e l e c t r o p h o r e s i s on a c e l l u l o s e a c e t a t e p l a t e , c u t i n t o two p a r t s , one o f which ( c o n t a i n i n g t h e a c i d i c amino a c i d s ) i s s u b j e c t e d t o a n o t h e r (per-pendicular) e l e c t r o p h o r e t i c separation, w h i l e the o t h e r p a r t o f the p l a t e (containing the n e u t r a l and b a s i c amino a c i d s ) i s s e p a r a t e d c h r o m a t o g r a p h i c a l l y 8 . An example o f t h e s e p a r a t i o n o f a c i d i c and b a s i c amino a c i d s i s shown i n F i g . 7.2. The s e p a r a t i o n i s s t a r t e d w i t h an e l e c t r o D h o r e t i c s e n a r a t i o n i n p y r i d i n e a c e t i c a c i d - w a t e r (180:7.7:1812,

pH 6.5,

10 V/cm, 7 mA, 5 h ) . The p l a t e i s t h e n

c u t i n t o two a s h o r t d i s t a n c e f r o m t h e s t a r t (which i s l o c a t e d i n t h e m i d d l e o f t h e p l a t e ) towards t h e anode. The a n o d i c a l l y moving amino a c i d s (Asp, Glu, CysCH and CyS03H) a r e s e p a r a t e d by a n o t h e r ( p e r p e n d i c u l a r ) e l e c t r o p h o r e s i s i n p y r i d i n e -

aa

0

TABLE 7.3 ELECTROPHORETIC MIGRATIONS

OF

COMFION AMINO ACIDS

Compound

Miaration i n electrolvte

Alanine B-A1 a n i ne a-Aminobutyric a c i d y-Ami nobutyri c a c i d A r g i n i ne Asparagine Asparti c acid S-Carboxymethylcysteine C it r u l l ine Cysteic a c i d Cyst ine 3,4-Di hydroxyphenyl a1 a n i ne Glutamic a c i d G l y c i ne H i s t i dine Hydroxyprol i n e Leucine Lys ine Met h ion ine M e t h i o n i n e sulphone Ornit h i n e Phenyl a l a n i ne 0-Phosphoethanolamine 0-Phosphoserine Pro1 ine S e r i ne

-24.2

-15.0

-21.7

-13.1

-5 -6 1

10 3

-74 -68

-2 -62

0 -45

21

76

81

- 100

-73 -8.0

-14.3

6.8

= -8.5

-15.5 -26.9

-3.8 -16.4

-13.0 -20.9

-8.0 -12.8

-17.6

-10.9

-16.6

-10.1

-15.4 -19.7

-46 -62 25 -6 7

-9.6 -11.9

-69 7 -7 -68

72 0 -8

229 77 12 10

-115 -60

-141 -71 -60 -141 -62 1 60 0

33 74 0

57 95

0 25

-75

Taurine Threonine Tryptophan Tyrosine Val i ne

-6.4 -18.2 -15.5

-5.8 -11.2

-16.2

9.3 -12.8

-21.2

- 78

-48

aRef. 16; 2 mol/l a c e t i c acid-0.06 mol/l formic acid ( 1 : l ) ( ~ H 1 . 9 ) ;llhatman No. 1 paper, 70 V/cm, 200 min. bAs a, except e l e c t r o l y t e 1 mol/l a c e t i c acid ( O H 2.3) and running time 180 min. ‘Ref. 17; pyridine-formic acid buffer (pH 3.3) 119 g (17 ml) of 88-90% formic acid and 9 g (9.2 ml) of pyridine were mixed, cooled and d i l u t e d t o 100 ml. For use, mix 2.0 ml of stock buffer and 7.5 ml of formamide and add water t o 25 ml]; Whatman dNo. 4 paper, 10 V/cm, 1-2 h ; mobility r e l a t i v e t o amaranth. As c , except buffer 2-dimethylaminopropionitrile-acetic acid (pH 7.2) (19.6 g o f 2-dimethylaminopropionitrile and 6 g of g l a c i a l a c e t i c acid d i l u t e d t o 100 ml. For use, mix 3 ml of stock buffer and 7.5 ml of formamide and add water t o 25 ml). t o alanine. “As c , mobility r e l a t i v e

62

Ii

0

I

. %

md.

li_ _ _ _ _ _ _ _ . _ _ _I

I I

I

j.i

F i g . 7.2. S e p a r a t i o n o f a c i d i c and b a s i c amino a c i d s by two s e p a r a t e two-dimens i o n a l r u n s . 1 s t d i r e c t i o n : p y r i d i n e - a c e t i c a c i d - w a t e r (180:7.7:1812) (pH 6.5), 400 V, 7 mA, 5 h. 2nd d i r e c t i o n : ( p a r t I o f t h e p l a t e ) : D y r i d i n e - a c e t i c a c i d w a t e r (30:100:4870) (pH 3 . 9 ) , 450 V, 10 mA, 1 h. 3 r d d i r e c t i o n (2nd d i r e c t i o n , p a r t I1 o f t h e p l a t e ) : chromatography i n n - b u t a n o l - a c e t i c a c i d - w a t e r (70:7:23); mc = orange G as marker; t h e number a f t e r mc r e f e r s t o t h e p o s i t i o n o f t h e marker a f t e r i n d i v i d u a l runs. Sorbent: c e l l u l o s e l a y e r . a c e t i c a c i d - w a t e r (30:100:487u, pH 3.9, ca. 20-25 V/cm, 10 mA). N e u t r a l and b a s i c amino a c i d s a r e separated c h r o m a t o g r a p h i c a l l y i n n-butanol - a c e t i c a c i d - w a t e r (70: 7: 23).

There a r e , o f course, o t h e r v a r i a t i o n s o f t h i s " m u l t i d i m e n s i o n a l " t y p e of s e p a r a t i o n , which may r e s u l t i n r a t h e r complex s e p a r a t i o n system. F o r i n s t a n c e , i n a m i x t u r e o f amino a c i d s , a c i d i c and b a s i c components may be s e p a r a t e d f i r s t a t pH 3.9 f o l l o w e d by chromatoyraphy i n t h e second dimension w i t h n - b u t a n o l a c e t i c a c i d ; n e u t r a l components a r e t h e n s e p a r a t e d by e l e c t r o p h o r e s i s a t pH 2.4 and chromatography i n t h e above s o l v e n t system. B a s i c components a r e s e p a r a t e d by one-dimentional e l e c t r o p h o r e s i s a t pH 6.5,

11.7 and 8.9 s u c c e s s i v e l y (Paysant 15 ).

Some o t h e r s e p a r a t i o n s a r e shown i n Tables 7.3-7.5. Amino a c i d m i x t u r e s were one o f t h e f i r s t t y p e s o f compounds t o be s e p a r a t e d by i s o t a c h o p h o r e s i s ( E v e r a e r t s and Van d e r Put 20 ) . T h e i r amphoteric c h a r a c t e r i s t h e main problem: i n i s o t a c h o p h o r e s i s t h e amino a c i d s may be analysed e i t h e r as anions o r as c a t i o n s . U s u a l l y a n i o n a n a l y s i s i s p r e f e r r e d as t h e r e i s a g r e a t e r d i f f e r e n t i a t i o n o f t h e pK values of t h e c a r b o x y l groups. The s e p a r a t i o n i l l u s t r a t e d i n F i g . 7.3 was c a r r i e d o u t w i t h s - c o l l i d i n e (0.02 m o l / l ) mol/l)

-

-

f o r m i c a c i d (0.01

formaldehyde (17.5%) as t h e l e a d i n g e l e c t r o l y t e a t pH 7.1.

The t e r m i n a l

e l e c t r o l y t e was prepared by m i x i n g t h e b u f f e r - f o r m a l d e h y d e s o l u t i o n w i t h an i o n w i t h a l o w n e t m o b i l i t y such as v a n i l l i n , R e c e n t l y t h e i s o t a c h o p h o r e t i c separat i o n o f amino a c i d s was m o d i f i e d 2 ' i n such a way t h a t 0.01 m o l / l h y d r o c h l o r i c

63

I-

I

I

I

35

40

45

I

50 min

F i g . 7.3. I s o t a c h o p h o r e t i c s e p a r a t i o n of u r i n a r y amino a c i d s . Sample: 5 v l o f u r i n e w i t h o u t p r e - t r e a t m e n t . Leading e l e c t r o l y t e : 0.01 m o l / l h y d r o c h l o r i c a c i d , ammediol and 0.5% p o l y ( v i n y 1 a l c o h o l ) (pH 8 . 7 ) . Terminal e l e c t r o l y t e : 0.01 m o l / l 6 - a l a n i n e and b a r i u m h y d r o x i d e (pH 10.9); c a p i l l a r y t u b e , 20 cm, 100 PA, 2OoC. TABLE 7.4 ELECTROPHORETIC MIGRATION DISTANCES OF SUCPHUR AMINO ACIDSa

M i g r a t ion (cm)

Compound

Migration (cm)

Compound

Cystine C y s t e i ne Methyl c y s t e i ne Methyl c y s t e i n e s u l p h o x i d e M e t h y l c y s t e i n e s u l phone Ethyl cysteine a u t y l c y s t e i ne Cysteic a c i d Cyst e ine s u 1p h in ic a c id Homocystine

-3.3 -3.1 -2.1 -1.8 -1.7 -2.0 -2.1 15.5 14.0 -4. a

Homocysteic a c i d 13.0 T a u r i ne -1.9 Cyst a t h io n i ne -1.2 -4.0 Met h ion ine Methionine sulpnoxide -2.7 M e t h i o n i ne s u l Dhone -2.6 Methionine sulphoximine -3.0 Methylmethi o n i ne s u l phonium chloride -19.0 Djenkolic acid -3.2

18; p h o s p h a t e - c i t r a t e b u t f e r (pH 2.7); ldhatman 3 MM Paper, 20 V/cm, 4 h.

Q,

P

TABLE 7.5 ELECTROPHORETIC

MOBILITIES

OF AROMATIC

AMINO

ACIDS~ ~

Compound

Benzene 2-Aminobenzoic a c i d

~~~~

Mobi 1it y i n e l e c t r o l y t e b 1

2

3

4

5

6

7

8

9

10

-8.1

-5.0

-2.6

-0.8

5.6

6.7

6.3

5.4

11.0

10.4

-5.8

-3.9

-1.6

-1.5

3.1

5.1

5.3

4.8

8.9

9.5

0.0

0.0

0.0

2.gC

3.7

5.5

3.2

3.2

7.0

7.9

8.3

3.3

6.2

9.0

8.8

7.5

6.5

6.2

11.2

12.7

0.0

0.0

0.0

0.2

3.8

3.7

3.2

2.7

8.2

8.7

-6.1 0.4 -0.1 -16.3

-5.8 0.2 -0.1 -23.6

2.3 0.1 0.0 .19.9

4.8 0.3 0.5 -8.1

5.0 3.7 3.1 -0.9

4.6 3.9 4.0 -0.3

2.8 3.0 2.9 -0.1

2.9 2.9 2.7 -0.1

5.8 5.6 7.1 -0.3

5.8 6.4 6.8 -0.2

-3.6

-2.9

-2.4

-1.6

2.3

3.4

2.1

1.8

6.3

7.2

-3.6

-6.9

-1.3

3.3

3.5

3.2

3.2

3.1

5.2

5.2

-5.3 1.3 -0.4 -1,3 -2.8

-7.5 12.6 -0.7 -0.9 -4.3

-3.1 14.0 0.7 0.7 -2.1

1.0 12.8 4.4 10.7 0.0

3.4 12.3 5.5 9.2 3.9

3.1 10.6 7.8 7.6 4.0

2.6 7.9 6.2 7.0 4.2

2.1 7.2 5.2 4.4 3.0

5.8 14.5 12.6 13.8 6.1

6.1 13.5 11.8 11.9 5.2

2-Amino-6-hydroxybenzoic acid Naphthalene 1-Amino-2-hydroxynaphthalene4-sulphonic a c i d 2-Amino-8-hydroxynaphthalene3,5-di sul phoni c a c i d 2-Aminonaphthalene-6-sulphonic acid Dipheny 2

4-Aminodiphenyl-3-sulphate 4-Amino-3-sulpho4-Ami no-4'-sul pho4,4'-Di(carboxymethy1amino)3,3'-Dicarboxymethyl e t h e r -4,4 ' -diamino 2 ' ,4-Di aminodiphenyl-3sulphate 4,4'-Diaminodiphenyl-3-sulphate 2,4'-Diamino-3,5'-disul pho4,4'-Diamino-2,2 '-disulpho4,4'-Diamino-3,3'-di s u l pho2,4'-Diamino-5-sulpho-

- r

L n ' t

m

L

"

n

L

W

3

L

ar

$

E c , V L

X

aRef. 19; Whatman No. 1 paper ( 2 . 5 x 55 cm), 250 V , 3 h ; m o b i l i t i e s i n 105cm2V-1~-1. S t a n d a r d m i g r a t i n g compound was b3-nitrophthalic acid. E l e c t r o l y t e s ( e l e c t r o l y t e s 2-10 were f u r t h e r d i l u t e d 1 : l w i t h d i s t i l l e d w a t e r ) : (1) 0 . 1 mol/l HC1 (pH 1 . 7 5 ) ; ( 2 ) 0 . 4 0 m l of 0 . 2 mol/l K2HP04 t 19.6 ml o f 0 . 1 mol/l c i t r i c a c i d (pH 2 . 2 5 ) ; ( 3 ) 4 . 1 1 ml o f 0 . 2 mol/l K2HP04 + 15.99 mol of 0 . 1 rnol/l c i t r i c a c i d ( P H 3 . 1 ) ; ( 4 ) 7.71 ml of 0 . 2 mol/l K HPO4 t 12.29 ml of 0 . 1 mol/l c i t r i c a c i d (pH 4 . 2 ) ; ( 5 ) 10.30 ml o f 0.2 mol/l KzHP04 + 9 . 7 0 ml of 0 . 1 mol/l c i t r i c a c i d f p H 5 . 2 ) ; ( 6 ) 1 2 . 6 3 ml o f 0 . 2 mol/l K7HP04 + 7.37 ml o f 0 . 1 mol/l c i t r i c a c i d (pH 5 . 8 ) ; ( 7 ) 16.47 ml o f 0.2 mol/l K2HP04 + 3.53 ml of 0 . 1 mol/l c i t r i c a c i d ( p k 7 . 2 ) ; ( 8 ) 19.45 ml of 0.2 mol/l K2HP04 t 0 . 5 5 ml of 0 . 1 mol/l c i t r i c a c i d (pH8.7); ( 9 ) 0 . 0 5 mol/l borax (pH 8 . 9 ) ; ( 1 0 ) 6 . 0 m l o f 0 . 1 mol/l borax t 4.0 ml o f 0 . 1 rnol/l NaOH (pH 9 . 7 ) . ‘Elongated spots. N U

v

-4

bco

CUL Y Q

-

u o . .r a m u s

m

65

66

a c i d , 0.02 m o l / l ammediol and 0.5% p o l y ( v i n y 1 a l c o h o l ) (pH 8.7) was used as t h e l e a d i n g e l e c t r o l y t e and 0.01 mol/l 6 - a l a n i n e and b a r i u m h y d r o x i d e (pH 10.9) s e r v e d as t h e t e r m i n a t o r (see a l s o r e f . 73). I s o t a c h o p h o r e t i c a n a l y s i s a l s o a l l o w s t h e r a p i d s e p a r a t i o n o f some s u l p h u r - c o n t a i n i n g amino a c i d s i n human u r i n e . For example, S - ( 1 , 2 - d i c a r b o x y e t h y l ) c y s t e i n e , S-(carboxymethy1)cysteine and S-(2-methyl2 - c a r b o x y e t h y l ) c y s t e i n e ( i s o b u t e i n e ) can be c l e a r l y s e p a r a t e d and q u a n t i t a t e d . The l e a d i n g e l e c t r o l y t e c o n s i s t e d o f 0.01 m o l / l h y d r o c h l o r i c a c i d and 6 - a l a n i n e (pH 3.1) and t h e t e r m i n a l e l e c t r o l y t e was 0.01 m o l / l c a p r o i c a c i d . An a t t e m p t t o s e p a r a t e UV-absorbing amino a c i d s by i s o e l e c t r i c f o c u s i n g was d e s c r i b e d by Catsimpoolas and Campbellz2. F o r a comparison o f t h e c l a s s i c a l n i n h y d r i n method w i t h t h e more advanced f l u o r e s c a m i n e and o - p h t h a l a l d e h y d e d e t e c t i o n methods, see r e f . 23. AMINO A C I D S A R I S I N G DURING POST-TRANSLATIONAL REACTIONS I N PROTEINS Over 140 compounds a r i s i n g f r o m p o s t - t r a n s l a t i o n a l r e a c t i o n s o f amino a c i d s can be t r a c e d i n t h e l i t e r a t u r e ( f o r a review, see Uy and Woldz4 and H o r l k o v a 25 ). The a p p l i c a t i o n o f e l e c t r o p h o r e t i c t e c h n i q u e s t o t h i s c a t e g o r y o f

and Deyl

compounds i s l i m i t e d , however, and t h e f o l l o w i n g examples may s e r v e as a guide. E l e c t r o p h o r e t i c s e p a r a t i o n o f m e t h y l a t e d amino a c i d s was used by Kakimoto and Akazawae6 f o r m o n i t o r i n g column e f f l u e n t s . An A m b e r l i t e IR-120 column was e l u t e d w i t h ammonia s o l u t i o n o f i n c r e a s i n g c o n c e n t r a t i o n and t h e f r a c t i o n s c o l l e c t e d were s u b j e c t e d t o e l e c t r o p h o r e s i s [Toyo-Roshi

No. 51 paper, pH 3.6 b u f f e r con-

s i s t i n g o f p y r i d i n e - a c e t i c a c i d - w a t e r (5:50:945) 100 V/cm,

r u n n i n g t i m e 30 min].

using a potential gradient o f

I t i s obvious t h a t paper e l e c t r o p h o r e s i s cannot

be g e n e r a l l y recommended f o r s e p a r a t i n g t h e s e amino a c i d s because, f o r example, m e t h y l a t e d l y s i n e s merge i n t o a s i n g l e zone t o g e t h e r w i t h l y s i n e . H i s t i d i n e and E - N - t r i m e t h y l l y s i n e f o r 35 m i n a t pH 1.9 and 3000 VZ7.

can be s e p a r a t e d by paper e l e c t r o p h o r e s i s Under t h e s e c o n d i t i o n s t r i m e t h y l l y s i n e moves

s l i g h t l y ahead o f h i s t i d i n e . M e t h y l a t e d l y s i n e s m i g r a t e , as a s i n g l e zone, however, and i n a m i x t u r e t h e y cannot be d i s t i n g u i s h e d f r o m a r g i n i n e . M e t h y l a t e d h i s t i d i n e s , u n l i k e m e t h y l a t e d l y s i n e s , can be s e p a r a t e d f r o m t h e p a r e n t amino a c i d by paper e l e c t r o p h o r e s i s u s i n g Whatman 3MM paper, p y r i d i n e

(5%, v / v ) - a c e t i c a c i d (0.2%, v / v ) b u f f e r (pH 6 . 5 ) and a p o t e n t i a l g r a d i e n t o f 40 V/cm. 3 - M e t h y l h i s t i d i n e moves s l i g h t l y b e h i n d h i s t i d i n e (towards t h e cathode) whereas 1-methyl h i s t i d i n e moves f a s t e r z 8 . Also, t h e s e p a r a t i o n f r o m l y s i n e does n o t cause any problems, t h e d i s t a n c e between l y s i n e and 1 - m e t h y l h i s t i d i n e b e i n g s u f f i c i e n t l y g r e a t t o a l l o w a c l e a r s e p a r a t i o n . The p o t e n t i a l can be i n c r e a s e d t o about 120 V/cm w i t h o u t a f f e c t i n g t h e s e p a r a t i o n r e s u l t s . As r e p o r t e d by Hardy and P e r r y z 7 , l y s i n e and E-N-monomethyllysine f o r m t h e f a s t e s t zone towards t h e

67 cathode, f o l l o w e d by a t r i p l e zone o f 1 - m e t h y l h i s t i d i n e , h i s t i d i n e and 3-methylh i s t i d i n e . 1-Methyl- and 3 - m e t h y l h i s t i d i n e can a l s o be s e p a r a t e d a t 2500 V/cm i n p y r i d i n e - a c e t i c a c i d (pH 6.1) u s i n g Whatman 3MW paper, as r e p o r t e d by Asatoor 24 and Armstrong

.

E-N-

and 6 - N - m e t h y l o r n i t h i n e ,

and o t h e r r a r e l y seen amino a c i d s , can be sepa-

r a t e d by paper e l e c t r o p h o r e ~ i s ~t h~ e; 6-N-methyl d e r i v a t i v e moves f a s t e r t h a t t h e E-N-methyl

compound. I n a m i x t u r e o f t h e "common t w e n t y " amino a c i d s 6-N-methyl-

o r n i t h i n e cannot be d i s t i n g u i s h e d f r o m t h e zone o f l y s i n e . M e t h y l p r o l i n e s , w h i c h o c c u r i n some a n t i b i o t i c s , can be seRarated by e l e c t r o 30 p h o r e s i s on paper u s i n g 4% f o r m a t e b u f f e r (DH 1 . 9 ) ( T a b l e 7.6)

.

TABLE 7.6 ELECTROPHORETIC SEPARATION OF PROLINE AND METHYLPROLINES Paper e l e c t r o p h o r e t i c m o b i l i t i e s a r e expressed r e l a t i v e t o s a r c o s i n e = 1.00. Amino a c i d

Relative mobility

Pro1 ine cis-3-Methylproline trans-3-Methylproline cis-4-methyl pro line trans-4-Methylproline cis-5-Methylproline trans-5-methyl pro i n e

0.78 0.74 0.77

1

0.69

1

0.72

F o r t h e a n a l y s i s o f b i o l o g i c a l samples r e p r e s e n t i n g multi-component p r o t e i n m i x t u r e s , i t i s sometimes necessary t o know t h e p r o p e r t i e s o f u n l i g a n d e d T3 and T4 ( t r i - and t e t r a i o d o t h y r o n i n e ) .

I t t h e r e f o r e seems w o r t h w h i l e m e n t i o n i n g t h a t

i n i s o e l e c t r i c f o c u s i n g (LKB 8101 a n a l y t i c a l column a t 5OC, 4

W

f o r 72 h ) i n an

ampholyte-sucrose g r a d i e n t t h e s e two amino a c i d s o c c u r a t d i f f e r e n t pHs. Whereas T4 focuses a t pH 3.8-4.3

(peak 4 . 3 ) , t h e c o r r e s p o n d i n g v a l u e s f o r Tg a r e 4.6-5.3

(peak 5 . 1 ) (Handwerger e t a1 .31).

Electrophor.esis o f i o d i n a t e d amino a c i d s can 32

be c a r r i e d o u t on Sephadex G-25 t h i n l a y e r s , as r e p o r t e d by R e i t h and Brown

.

These workers used a c o m b i n a t i o n o f t h i n - l a y e r g e l f i l t r a t i o n and e l e c t r o p h o r e s i s t o s e p a r a t e d i n i t r o p h e n y l (DNP) d e r i v a t i v e s o f d i - and m o n o i o d o t y r o s i n e . O f h y d r o x y l a t e d amino a c i d s , t h e commonest, h y d r o x y p r o l i n e , has n o t been con-

s i d e r e d f o r e l e c t r o p h o r e t i c s e p a r a t i o n . On t h e o t h e r hand, 6 - N - h y d r o x y o r n i t h i n e , a c o n s t i t u e n t o f f e r r i o c h r o m e s , albomycin, f u s a r i d i n e s and r h o d o t o r u l i c a c i d , can be separated i n a s i m i l a r manner t o h y d r o x y l y s i n e , e.g., a c i d - w a t e r (7:5:465,

pH 5.0,

with pyridine-acetic

10 V/cm, 3 h ) as t h e r u n n i n g b u f f e r . 6-N-Hydroxy-

o r n i t h i n e g i v e s a r e d zone when d e t e c t e d w i t h t r i p h e n y l t e t r a z o l i u m c h l o r i d e 33

.

68 S - C a r b o x y v e t h y l c y s t e i n e occurs as an i n t e g r a l p a r t o f some p r o t e i n s such as immunoglobulin G, and i t may a r i s e d u r i n g s t r u c t u r a l s t u d i e s by r e a c t i o n o f t h e t e r m i n a l r e s i d u e w i t h iodoacetamide. For i t s e l e c t r o p h o r e t i c s e p a r a t i o n , h i g h v o l t a g e paper e l e c t r o p h o r e s i s can be recommended, i t s m o b i l i t y r e l a t i v e t o a s p a r t i c a c i d b e i n g 1.87. When s t u d y i n g amino a c i d s w i t h a m o d i f i e d s u l p h u r f u n c t i o n one can meet t h e problem o f s e p a r a t i n g c y s t e i c a c i d , S - s u l p h o c y s t e i n e , S - s u l p h o t h i o c y s t e i n e and S - s u l p h o g l u t a t h i o n e .

A method f o r t h e s e p a r a t i o n o f t h e s e compounds

was developed by Ubuka e t a l . 3 4 , u s i n g c o n v e n t i o n a l paper e l e c t r o p h o r e s i s a t 85 V/cm f o r 45 min i n p y r i d i n e - a c e t i c a c i d - w a t e r (0.5:10.0:79.5) 25 a d d i t i o n a l d a t a a r e g i v e n i n Table 7.7 .

(pH 3 . 1 ) . Some

TABLE 7.7 ELECTROPHORETIC M I G R A T I O N DISTANCES AND RF VALUES ON DEAE-PAPER

OF SULPHUR AMINO

ACIDS Amino a c i d 3

Cyst ine Cysteine Met hy 1cys t e ine M e t h y l c y s t e i n e s u l phoxide M e t h y l c y s t e i n e sulphone E t h y l c y s t e i ne Butylcysteine Cysteic a c i d Cysteinesulphinic a c i d Homocystine Homocysteic a c i d T a u r i ne Cyst a t h ione Methionine Me t h io n i ne M e t h i o n i n e sulphone Methionine sulphoximine Methyl methi o n i n e s u l phoni um chloride Djenkolic acid

Electrophoretic migration,

DEAE c e l l u l o s e , PH 4.7

pH 2.7

RF

N i n h y d r in c o l o u r

-3.3 -3.1 -2.1 -1.8 -1.7 -2.0 -2.1 t15.5

0.14 0.11 0.36 0.25 0.26 0.38 0.40 0.05

+14.0

0.10 0.15 0.10

-4.8 +13.0 -1.9 -1.2 -4.0 -2.7 -2.6 -3.0

0.33 0.18 0.43 0.34 0.28

Brown Brown Brown-grey Ye1 1ow-brown Ye1 1ow-brown Brown-grey Brown-grey Grey-bl ue B1 ue B1 ue B1 ue Grey-bl ue B1 ue Purple Purple Purple Purple

-19.0 -3.2

0.80 0.15

Purple Grey

0.29

i 2 0 pg o f each amino a c i d a p p l i e d t o t h e paper. D i s t a n c e m i g r a t e d (cm) towards anode o r cathode i n 4 h. A l a n i n e d e r i v a t i v e s ( a l k y l a m i n o d e r i v a t i v e s ) can a l s o be sub.jected t o e l e c t r o p h o r e t i c s e p a r a t i o n on paper a t v e r y a c i d i c pH ( T a b l e 7 . 8 ) . Under i d e n t i c a l c o n d i t i o n s i t i s p o s s i b l e t o s e p a r a t e d e h y d r o a l a n y l r e s i d u e s and o t h e r compounds a r i s i n g f r o m t h e a d d i t i o n a l r e a c t i o n s o f d e h y d r o a l a n i n e w i t h amino and t h i o l 35

groups ( l y s i n o a l a n i n e , B-aminoalanine and l a n t h i o n i n e )

.

69 TABLE 7.8 CONDITIONS FOR THE SEPARATION OF ALANINE OERIVATIVES (A) a t pH 1.85, 75 V/cm f o r 1.75 h, 18OC, and ( 6 ) a t pH 1.1, 50 V/cm f o r 2.25 h, 18OC. Conditions

A B

Amino a c i d 8-Ethylaminoalanine B-Propylaminoalanine 8-Bu t y l ami noa 1an ine 8-Amy1 ami n o a l a n i ne

R, r e l a t i v e

R, r e l a t i v e

t o glycine

t o alanine

1.13 1.07 0.85 0.82

1.3 1.21 0.962 0.83

N - A c e t y l a t e d amino a c i d s t h a t o c c u r i n p r o t e i n s a t t h e i r N - t e r m i n i a r e access i b l e t o an i n d i r e c t assay by f l a t - b e d t e c h n i q u e s o r can be analysed d i r e c t l y by gas chromatography. The s t r a t e g y i s t o s p l i t t h e a c e t y l a t e d N - t e r m i n a l p e p t i d e w i t h a s u i t a b l e enzyme, e.g.,

pronase, remove a l l f r e e amino a c i d s and p e p t i d e s

w i t h o u t t h e b l o c k e d N-terminus and s u b j e c t t h e N - a c e t y l a t e d D e p t i d e t o h y d r a z i n o l y s i s . Then chromatography i s c a r r i e d o u t i n o r d e r t o s e p a r a t e a c y l h y d r a z i d e s , f r e e h y d r a z i n e and amino a c i d h y d r a z i d e s . F o r t h i s purpose Satake e t a1.36 r e c ommended paper chromatography w i t h p y r i d i n e - a n i l i n e - w a t e r

(9:1:4).

In o r d e r t o

o b t a i n a good r e s o l u t i o n , e l e c t r o p h o r e t i c s e p a r a t i o n i n p y r i d i n e - a c e t i c a c i d w a t e r (pH 6 . 6 ) a t 5 0 0 V/cm has t o be c a r r i e d o u t b e f o r e chromatography. The assignment o f t h e a c y l group t o a p a r t i c u l a r amino a c i d p r e s e n t i n t h e combined s p o t o f amino a c i d h y d r a z i d e s can be a c h i e v e d by p a r t i a l h y d r o l y s i s and b y comparison o f l i b e r a t e d N-termini.

Other N-blocked amino a c i d s such as f o r m y l s e r i n , a c e t y l -

s e r i n e and 0 - p h o s p h o r y l a t e d s e r i n e can be r e a d i l y s e p a r a t e d i n p y r i d i n e formate a t pH 3.5 on paper (10 V/cm f o r 4 h a t 4°C)37.

Under these c o n d i t i o n s p y r o g l u t a m i c

a c i d , f o r m y l g l u t a m i c a c i d and a c e t y l g l u t a m i c a c i d a r e a l s o w e l l s e p a r a t e d . A c y l s e r i n e moves f a s t e r t h a n a c y l g l u t a m i c a c i d and formylamino a c i d s move f a s t e r t h a n a c e t y l a m i n o a c i d s . D e t e c t i o n i s e f f e c t e d by s p r a y i n g t h e e l e c t r o p h e r o g r a m w i t h an i n d i c a t o r (0.02% m e t h y l r e d i n 0.03 m o l / l b o r a t e b u f f e r , pH 7 . 8 ) . The a c i d i c components a r e r e v e a l e d as r e d spots. E l e c t r o p h o r e t i c s e p a r a t i o n o f g a l a c t o s y l h y d r o x y l y s i n e and g l u c o s y l g a l a c t o s y l 25 h y d r o x y l y s i n e i s p o s s i b l e i n p y r i d i n e - a c e t i c a c i d - w a t e r (1:10:89) (pH 3.8)

.

The s e p a r a t i o n i s r u n a t 4000-5000 V p e r paper s h e e t and t a k e s 1-2 h. S e p a r a t i o n o f b o t h m o d i f i e d amino a c i d s and a l s o t h e i r s e p a r a t i o n f r o m h y d r o x y l y s i n e , l y s i n e and o r n i t h i n e o c c u r w i t h o u t o v e r l a p , t h e g l y c o s y l a t e d amino a c i d s moving c o n s i d e r a b l y more s l o w l y t h a n t h e u n m o d i f i e d a c i d s .

TABLE 7.9 RELATIVE ELECTROPHORETIC MOBILITIES OF DINITROPHENYL AMINO ACIDS AND RELATED COMPOUNDS Compound

Relative mobility in electrolyte

DNP-a-a1aninex DNP-8-a1 anine DNP-alanylglycylglycine DNO-alanylleucine DNP-allylglycine DNP-a-aminobutyric acid DNP-y-aminobutyric acid DNP-a-aminoisobutyric acid DNP-E-aminocaproic acid DNP-aminomethylene sulphonic acid a-DNP-arginine . DNP-aspartic acid DNP-asparagine DNP-cysteic acid DNP-2,4-diaminobutyric acid DNP-a,E-diaminopimel i c acid DNP-djenkolic acid DNP-gal actosamine DNP-g1ucosamine DNP-glutarnic acid DNP-g1utamine DNP-glycine DNP-glycylaspartic acid DNP-glycylglutamic acid DNP-glycylhistidine DNP-glycylphenylalanine

100 98

100 92

100 75

100 48

100 18

100 20

97

97

93

91

aa

90

-5 174 a5 188

-2 167 a7 190

-4 156 a3 207

1 125 a2 213

-4 97 83 201

-290 101 69 430

162 86 98

151 a3 90

124 a3 86

103 86

a2

68-87 89 79

109 96 77

48 11 la 41 46 48 4 47 2 34

67 51 44 55 55 56 39 55 30 72

65 5a 43 55 59 59 56 56 50 70

62 63 43 55 61 60 60 58 49 69

25

a9

97

96

0 14 16 0

0 41 39 0

a1 41 38 0

ao

0

0

0

48 15

65 70 68 32 44

68 82 80 33 44

12

5 22

49 70 0 0 66

a2 a1 43 45

DNP-glycylpro1 i ne DNP-glycyltryptophan DNP-glycyltyrosi ne a-DNP-histi dine DNP-6-hydroxylysi ne DNP-hydroxyproli ne DNP-isoleuci ne DNP-leucine DNP-leucyltyrosine a-DNP-lysine E-DNP-lysine DNP-lysylglycine DNP-methioni ne DNP-methionine sul phone DNP-methionine sulphoxide

-7

-40

-57

-70

93

100

101

105

111

141

90

88

86

86

83

101

-46 -5

- 70

-75

-

-

-,1100

-2

-62 -3

-4

-280

83 90 88

89 92 91

84 92 91

83 96 96

45-89 102 104

88 143 141

73 96 109 94

68 98 108 94

61 99 107 93

61 102 100 94

24-69 104 91 98

86 111 69 109

92 26 92 20 50 25 15 24 0

94 24 93 18 21 19 6 27 0

96 27 91 25 0 0 5 23 0

109 31 102 36 0 0 0

15.7

16.3

14.3

-800

DNP-methioninesulphoximine

DNP-phenylalanine DNP-proline DNP-sarcosine DNP-serine DNP-taurine DNP-threonine DNP-tryptophan DNP-valine Di-DNP-cysteine Di-DNP-cystine Di -DNP-histidi ne Di -DNP-1ysi ne Di -DNP-ornithi ne Di-DNP-tyrosine ‘DNP-alanine (cm moved in 2 h)

91 40 92 38 96 45 38 45 2 37.7

93 37 91 27 31-84 36 23 32 0 19.0

24 17 21 0 15 48 42

46 37 40 0 37 53 52

50 42 45 32 36 53 54

50 41 46 48 37 56 56

16

38

37

36

6 45

32 53

32 54

38 54

52 39 43 47

50 52 48 55

52 52 48 57

57 54 48 58

47 34 45 31

62 65 53 41

61 63 56 41

60 65 55 44

0 0

4.2

aRef. 40; pyridine-acetic acid ( D H 6.5-6.6) [Dyridine-glacial acetic acid-water (100:3:800r] filter-paper (30 x 60 cm); 67 V/cm, 120 min; mobility relative t o DNP-alanine x 100. bAs a, except pH 5.9 buffer kyridine-glacial acetic acid-water (20:3:1200)]. ‘As a, except pH 4.8-4.9 buffer kyridine-glacial acetic acid-water (19:16:1500)].

;

Schleicher and Schiill 204 3a 4

(Continued on p. 721

w

TABLE 7.9 (continued) dAs a, except pH 4.2 buffer [pyridine-glacial acetic acid-water (19:47:3000)]. :As a, except pH 3.2-3.3 buffer pyridine-glacial acetic acid-water (6:79:1200)]. As a, except pH 1.8-1.9 buffer formic acid (85%)-glacial acetic acid-water (3:1:40)]. gRef. 41; pH 3.3 buffer El9 g (17 ml) of 88-90% formic acid and 9 g (9.2 ml) of Fyridine mixed, cooled and diluted to 100 ml with water. For use, mix 2.0 ml of stock buffer and 7.5 ml of formamide and add water to 25 ml], Whatman No. 4 paper (3.8-5 x 24 cm), 10 V/cm; mobility relative to amaranth and Apolon used as electro-endosmotic indicator. hAs g, except pH 4.7 buffer (10 g of 2-dimethylaminoethanol and 13.5 g of glacial acetic acid diluted to 100 ml with water. iFor use, mix 2 ml of stock buffer and 7.5 ml of formamide and add water to 25 ml). As g, except pH 7.2 buffer (19.6 g of 2-dimethylaminopropionitrileand 6 g of glacial acetic acid diluted to 100 ml with .water. For use, mix 3 ml of stock buffer and 7.5 ml of formamide and add water to 25 ml). 'As g, except pH 9.3 buffer (18 g of 2-dimethylaminoethanol and 6.6 g of glacial acetic acid diluted to 100 rnl with water. For use, mix 2 ml of stock buffer and 7.5 ml of formamide and add water to 25 ml).

E

73 AMINO A C I D DERIVATIVES USED I N SEQUENCE ANALYSIS S e p a r a t i o n o f DNP d e r i v a t i v e s o f amino a c i d s l o s t most o f i t s p o p u l a r i t y w i t h t h e i n t r o d u c t i o n o f more s e n s i t i v e ( f l u o r e s c e n t ) l a b e l s f o r t h e f r e e N - t e r m i n a l amino a c i d s . Both low- and h i g h - v o l t a g e e l e c t r o p h o r e t i c s e p a r a t i o n s on paper can be used f o r s e p a r a t i o n (10-67 V/cm)38y3g, The r u n n i n g b u f f e r s used and t h e separ a t i o n s o b t a i n e d a r e g i v e n i n Table 7.9. C h r o m a t o e l e c t r o p h o r e s i s o f DNP-amino a c i d s was once commonly used. T h i n l a y e r s o f c e l l u l o s e can be used f o r t h i s purpose w i t h e l e c t r o p h o r e t i c s e p a r a t i o n i n 0.033 m o l / l d i e t h y l a m i n e w h i c h c o n t a i n s 0.02 m o l / l o f sodium c h l o r i d e (25 V/cm). Separation i n the f i r s t run i s e f f e c t e d chromatographically using toluene-ethylene

chlorhydrin-pyridine-0.8 m o l / l ammonia s o l u t i o n (150:90:45:90)

as t h e m o b i l e

phase. When paper i s used i n s t e a d o f c e l l u l o s e l a y e r s , 0.02 m o l / l b o r a t e (pH 8.92) (10 V/cm) o r 0.25 m o l / l t r i e t h y l a m i n e (10 V/cm) can be recommended f o r e l e c t r o p h o r e t i c s e p a r a t i o n i n t h e second r u n . O t h e r b u f f e r s can be used (Munier and Sarrazin4')

f o r the e l e c t r o p h o r e t i c step o f electrochromatography: f o r separations

on paper 0.025 m o l / l d i e t h y l a m i n e o r 0.25 m o l / l ammonia s o l u t i o n can be recommended. A l l o f these s e p a r a t i o n s a r e r u n a t 10 V/cm. E l e c t r o p h o r e t i c s e p a r a t i o n o f p h e n y l t h i o h y d a n t o i n (PTH) amino a c i d d e r i v a t i v e s i s a r a r e t e c h n i q u e f o r i d e n t i f y i n g these compounds. As r e p o r t e d b y Nakajima e t a1.43, e l e c t r o p h o r e s i s can be c a r r i e d o u t on paper u s i n g a h i g h p o t e n t i a l (20003000 V/cm) w i t h a r u n n i n g t i m e o f 2-3 h. The f i n a l appearance o f t h e e l e c t r o pherograms i s s t r o n g l y dependent on t h e pH: w h i l e PTH-Leu,

PTH-Pro and PTH-Lys a r e

w e l l s e p a r a t e d a t pH 1.0, a t pH 2.0 i t i s p o s s i b l e t o i d e n t i f y PTH-Ala, PTH-Gly, PTH-Thr,

i s p o s s i b l e t o o b t a i n a s a t i s f a c t o r y s e p a r a t i o n o f PTH-Asp, PTH-Glu, PTH-Tyr,

PTH-Ser,

PTH-Leu, PTH-Ile and PTH-Met. I d i t h a f u r t h e r i n c r e a s e i n pH i t

PTH-Gly and PTH-Thr. F i n a l l y , a t pH 6.0 zones o f PTH-Ser,

PTH-Trp,

PTH-Asp, PTH-

Glu, PTH-Arg, PTH-His and PTH-Cys can be d i f f e r e n t i a t e d . Another method f o r t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f PTH-amino a c i d s was sugg e s t e d by S ~ h r o e d e r( ~f o~r a p p l i c a t i o n i n f l a t - b e d Edman d e g r a d a t i o n , see Rosmus 45 and Deyl ) . S t a r c h e d Whatman 3MM paper i s used t o g e t h e r w i t h p y r i d i n e - a c e t a t e b u f f e r (pH 6 . 4 ) .

The r u n i s c a r r i e d o u t a t 15-20 V/cm f o r 2 h. D e t e c t i o n i s

e f f e c t e d w i t h i o d i n e - a z i d e r e a g e n t i n a s i m i l a r manner t o t h a t used i n chromat o g r a p hy . Kawauchi e t a1 .46 a t t e m p t e d t o s e p a r a t e f l u o r e s c e i n t h i o c a r b a m y l amino a c i d s by paper e l e c t r o p h o r e s i s a t a p o t e n t i a l g r a d i e n t o f 10 V/cm i n 0 . 0 1 m o l / l sodium phosphate b u f f e r (pH 4 . 0 ) .

However, n o t a l l amino a c i d s commonly o c c u r r i n g i n

p r o t e i n s were i n v e s t i g a t e d and t h e method was n o t e l a b o r a t e d i n enough d e t a i l t o be r e a d i l y a p p l i c a b l e .

74

For t h e s e p a r a t i o n o f dansyl amino a c i d s on paper i t i s p o s s i b l e t o r u n t h e sample i n p y r i d i n e - a c e t a t e b u f f e r (pH 4.4)38 a c c o r d i n g t o a method suggested o r i g i n a l l y by Gray and Hartley3’.

The paper i s t h e n c u t i n t o two p a r t s a t t h e

s t a r t i n g p o i n t , p e r p e n d i c u l a r t o t h e e l e c t r o p h o r e t i c r u n , and each p a r t i s subj e c t e d t o a s e p a r a t e chromatographic r u n . N e g a t i v e and n e u t r a l amino a c i d s a r e separated i n l i g h t p e t r o l e u m - a c e t i c a c i d - w a t e r (100:90:10)

and b a s i c dansyl de-

r i v a t i v e s a r e chromatographed i n d i i s o p r o p y l e t h e r - 2 - b u t a n o n e - a c e t i c (8:2:5:5)

acid-water

(see a l s o r e f . 47).

OTHER AMINO A C I D S AND AMINO A C I D CONJUGATES

D e t o x i f i c a t i o n r e a c t i o n s w i t h i n t h e organism l e a d t o amino a c i d c o n j u g a t e s t h a t a r e found i n u r i n e . Some i d e a o f t h e e l e c t r o p h o r e t i c b e h a v i o u r o f t h e s e compounds i s p r e s e n t e d i n Tables 7.10 and 7.1148.

I n g e n e r a l , m e t a b o l i c processes

i n b o t h t h e animal and p l a n t kingdoms a r e a r i c h source o f a v e r y d i v e r s e range

TABLE 7.10

PAPER ELECTROPHORESIS OF BENZOIC A C I D CONJUGATES AT 60 V/cm

B u f f e r s : pH 1.85, a c e t i c a c i d - f o r m i c a c i d (20 m l / l ) ; pH 6.0, 6.8 3/1 o f KH2P04 and 0.27 g/1 NaOH; pH 8.0, 6.8 g/1 o f KH2P04 and 1.86 g/1 o f NaOH; pH 12.3, 0.02 m o l / l NaOH. M i g r a t i o n r e l a t i v e t o b e n z o i c a c i d (t = towards anode; = towards cathode).

-

Compound

Benzoic a c i d Benzoylglycine B nzoyl g l utami c a c i d N$-Benzoylglutamine N2-Benzoyl a r g i n i n e N2-Benzoyl agmati ne N2-Benzoylci t r u l l i n e N2 - Benz oy 1h is t id in e N5-[en?oylorni t h i n e N2N - D i b e n z o y l o r n i t h i n e

M i g r a t i o n (cm) pH 1.85

pH 6.0

PH 8

?H 12.3

0 0 0 0 -4.4 -5.0 0 -4.1 -3.0 0

0 -0.5 t1.2 -2.0 -6.6 -9.6 -2.0 -6.2 -6.1 -1.8

0 -1.2 t1.4 -2.8 -7.6 ,11.5 -3.2 -4.6 -7.4 -4.0

0 -1.6 t1.4 -3.4 -9.2 .11.4 -4.0 -4.4 -4.4 -4.4

I5 TABLE 7.11

PAPER ELECTROPHORESIS OF p-AMINOBENZOIC ACID CONJUGATES M i g r a t i o n d i s t a n c e s (cm) a r e measured r e l a t i v e t o p-aminobenzoic a c i d . Compound

M i g r a t i o n (cm)

p-Aminobenzoic a c i d p - A m i nobenzoyl g l y c i ne

p-Aminobenzoyl g l utami c a c i d N2-p-Aminobenzoylgl utamine

N2-p-Aminobenzoylarginine N2-p-Aminobenzoylorni t h i n e N5- -Aminobenzoylorni t h i n e

N2Nf-Di-p-aminobenzoylorni t h i n e

N2-p- Aminobenzoyl c i t r u l 1 ine N2-p- Ami no be nzoy 1h i s t id i ne

pH 1.85

pH 6 . 0

pH 12.3

0 0 +0.8 +1.0 -2.2 -2.8 -1.8 -1.2 +1.2 -1.8

0 0 t2.2 -0.4 -5.4

0 0 t2.0 -1.6 -6.6 -3.8 -3.2 -3.2 -2.4 -3.0

-5.0 -4.8 -2.2 -0.8 -4.8

o f compounds b e a r i n g b o t h amino and c a r b o x y l groups. These amino a c i d s a r e r a r e l y analysed a l o n g w i t h t h e common t w e n t y . U s u a l l y t h e y a r e s e p a r a t e d f r o m t h e c l o s e s t p r o t e i n amino a c i d o r f r o m t h e i r p r e c u r s o r s and m e t a b o l i t e s . P y r i d i n e - a c e t i c a c i d b u f f e r s o f d i f f e r e n t pH values a r e p o p u l a r . It i s almost i m p o s s i b l e t o l i s t a l l o f these compounds. Table 7.12 i s t h e r e f o r e i n t e n d e d s i m p l y as an i l l u s t r a t i o n o f t h e v a s t d i v e r s i t y o f these compounds and t h e i r e l e c t r o P h o r e t i c behaviour. TABLE 7.12 CONDITIONS

FOR THE ELECTROPHORETIC

SEPARATION

OF SOME RARE AMINO ACIDS ON PAPER^

Amino a c i d

pH o f r u n n i n g b u f f e r

N- Ace t y 1cys t a t h ion ine N-Acetyl m e t h i o n i ne d, I-erythro-4-Ami no-2,3-di hydroxy-3methyl b u t a n o i c a c i d N-Ami d i nohomoseri ne

49 p y r i d i n e a c e t a t e (pH 4 . 8 ) 50

N-Amidinohomoserine a n h y d r i d e

4-Amino-2-amidinoperhydro-l,2-oxazin3-one 5-hino-4-hydroxyvaleric acid 5-Aminolevulinic a c i d 2-Ami no-5-methyl -6-hydroxy-4-enoi c a c i d

Reference

51

0.05 m o l / l p h t h a l a t e b u f f e r (pH 3.5) 0.05 m o l / l p h t h a l a t e b u f f e r (pH 3.5) 0.05 m o l / l p h t h a l a t e b u f f e r (pH 3 . 5 ) pH 6 . 0 pH 6 . 0 p y r i d i n e acetate (pH 3.4 o r 5.3)

52 52 52 53 53 54

(Continued on p . 761

76

TABLE 7.12 ( c o n t i n u e d ) Amino a c i d

pH o f r u n n i n g b u f f e r

Canavanine

0.05 m o l / l p h t h a l a t e b u f f e r (DH 3.5) formic acid-acetic acidw a t e r (61:97:1842) (PH 2-01 or a c e t i c a c i d-pyri dinew a t e r (1O:l:lOO) (PH 3.5) formic acid-acetic acidw a t e r (61:97:1842) (PH 2.0) or acetic acid-pyridinew a t e r (1O:l:lOO) (PH 3.5) p y r i d i n e acetate (pH 3.5 o r 5.3) 1 m o l / l NH3-l m o l / l a c e t i c acid-water (9:l:lO) p y r i d i n e acetate (pH 3.1) 0.05 m o l / l p h t h a l a t e b u f f e r (pH 3.5)

cis-a- (Carboxycycl o p r o p y l ) g l y c i ne

trans-a-( C a r b o x y c y c l o p r o p y l ) g l y c i n e

trans-a-2-(Carboxymethyl c y c l o p r o p y l ) g l y c i n e Carnosine 3-(S-Cysteinyl ) g l u t a r i c a c i d Desaminocanavanine

Reference

S-(1,2-Dicarboxyethy1)homocysteine Dirnethylargi n i n e

S-(1,2-Dimethyl-2-carboxyethy1)cysteine N-Formylmethionine L-Homomethionine (L-5-methylthionorvaline)

S-(2-Hydroxy-2-carboxyethy1)cysteine y-Hydroxy-y-methylglutamic a c i d Hypus ine N-Ma1 o n y l - 8 - m e t h i o n i n e N-Malonyl - D - s e r i ne ezo-3,4-Methanoproline

D-B-Methioni ne 3-He t hoxy - 5-me t hy 1naphtha 1 ene- 1carboxylic acid N-Me t h y 1 - DL- a1 1o i s o l e u c i ne 5-Methyl-2-amino-4-hexenoic acid

S-(1-Methyl-2-carboxyethy1)cysteine S-Methyl c y s t e i ne y-Methylenegl utamic a c i d y-Wethylenegl utamine y - M e t h y l g l utamic a c i d

52 55

55

54 56 57 52 58

a c e t i c acid-formic acidw a t e r (4:1:45) (pH 2.1) 59 p y r i d i n e a c e t a t e (pH 3.1) 57 p y r i d i n e a c e t a t e (pH 4 . 8 ) 50 60 61 p y r i d i n e a c e t a t e (OH 3.4) 62 p y r i d i n e a c e t a t e (DH 3.4) 63 p y r i d i n e a c e t a t e (pH 6.5) 64 p y r i d i n e a c e t a t e (DH 6 . 5 ) 65 formic acid-acetic acidw a t e r (61:97:1842) (PH 2.0) 55 or acetic acid-pyridinew a t e r (1O:l:lOO) (PH 3.5) p y r i d i n e a c e t a t e (DH 6.5) 64 4% f o r m a t e 1 mol/l acetic acid (PH 2.4) p y r i d i n e a c e t a t e (DH p y r i d i n e a c e t a t e (OH p y r i d i n e a c e t a t e (pH p y r i d i n e a c e t a t e (oH p y r i d i n e a c e t a t e (DH

51 66 3.1 5.3 3.4 3.4 3.4

54 57 67 62 62 62

77 TABLE 7.12 ( c o n t i n u e d ) ~

~~

Amino a c i d

pH o f r u n n i n g b u f f e r

N1- Me t hy 1h i s t id ine

1 m o l / l NH3-l m o l / l a c e t i c acid-water ( 9 : l : l O ) 56, 59 or a c e t i c acid-formic acidw a t e r (4:1:45) (pH 2 . 1 ) 4% f o r m a t e 66 p y r i d i n e a c e t a t e (pH 5 . 3 ) 67 4% f o r m a t e 66 a c e t i c acid-formic acidw a t e r (4:1:45) (DH 2.1) 59 acetic acid-formic acidw a t e r (4:1:45) (DH 2.1) 59 p y r id ine-ace t ic a c i dw a t e r b ( 1:10: 190) (PH 3 . 6 ) 68 s e p a r a t i o n a t pH 7.5 also possible acetic acid-pyridinew a t e r (61:97:1842) (PH 2 . 0 ) 55 or acetic acid-pyridinew a t e r (10:1:100)(pH 3.5) 20% f o r m i c a c i d (pH 1.4) 69 20% f o r m i c a c i d (pH 1.4) 69 p y r i d i n e a c e t a t e (pH 3.6) 70 0.05 m o l / l f o r m a t e b u f f e r (PH 3.7) 71 formic acid-acetic acidw a t e r ( 1 : 3 : 1 6 ) (UH 1.9) 72 p y r i d i n e a c e t a t e (OH 3 . 5 ) 63

N-Methyl-D,L-isoleuci ne Se-Methylselenocysteine N- Flet hy 1- D ,L- Val i ne Monome t h y 1 a r g i n ine

Mon ome t h y 1 1y s ine p-N-Oxalyl -L-a, p - d i ami n o p r o p i o n i c a c i d

P i pecol ic a c i d

Phosphoserine Phosphothreoni ne Putreanine Pyrrolidonecarboxylic acid T h i a l y s i n e IS-(Z-amino-Z-carboxyethyl) cys t e i ne] 4-Thia-Al-pi peridine-6-carbonate

Reference

aSee a l s o t h e s e c t i o n on amino a c i d s a r i s i n g d u r i n g p o s t - t r a n s l a t i o n a l r e a c t i o n s i n proteins. bAlso a p p l i c a b l e t o o x a l y l d e r i v a t i v e s o f a,B-diaminopropionic a c i d , a,B-diaminob u t y r i c a c i d , homoserine, o r n i t h i n e , l y s i n e , g l u t a t h i o n e , a - a m i n o b u t y r i c a c i d , y - a m i n o b u t y r i c a c i d , B-alanine, g l y c i n e , s e r i n e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11

G.T. A t f i e l d and C.J.O.R. M o r r i s , Biochem. J . , 81 (1961) 606. E.L. Durrum, J . CoZZoid S c i . , 6 (1951) 274. D. Gross, Nnture (London), 184 (1959) 1298. V. Richmond and B.S. H a r t l e y , Nature (London), 184 (1959) 1869. G.H. Dixon, D.L. Kauffman and H. Neurath, J . BioZ. Chem., 233 (1958) 1373. N. Nybom, Physiol. PZant., 17 (1964) 434. R.S. E r s s e r and S . Keywawych, Med. Lab. lechnol., 31 (1974) 235. G.H. S c h e r r , Anal. Chem., 34 (1962) 777. R.L. Munier, 2. AnaZ. Chem., 236 (1968) 358. R.L. Munier and A.M. D r a p i e r , Chromatographia, 11 (1978) 74. R.L. Munier and S. Meunier, Chromatographia, 12 (1979) 773.

78

12 R.L. Munier, A. Peigner and C. Tommegay, Ckromatographia, 3 (1970) 205. 13 R.L. Munier and C. Tommegay, BuZZ. Soc. Chim. F r . , 9 (1967) 3179; 9 (1967) 3176. 14 A. Lewandowski and A. Wronski, Chem. Anal. (Warsaw), 14 (1969) 841. 15 P. Paysant, Pharm. BioZ., 5 (1968) 135. 16 B. K i c k h o f e n and 0. Westphal , Z. Naturforsch. B, 7 (1952) 659. 17 L.N. Werum, H.T. Gordon and W. Thornburg, J . Chromatogr., 3 (1960) 125. 18 P.J. Peterson and G.W. B u t l e r , J . Chromatogr., 8 (1962) 70. 19 J. Franc and V. KoviF, J . Ckromatogr., 18 (1965) 100. 20 F.M. E v e r a e r t s and A.J.M. van d e r Put, J . Chromatogr., 52 (1970) 415. 21 Shimadzu, A p p l i c a t i o n d a t a No. 1, CupiZZary type isotackopkoretic analyzer. 22 N. Catsimpoolas and T. Campbell, AnaZ. Biochern., 46 (1972) 674. 23 E. S c h i l t z , K.D. Schnackerz and R.W. Gracy, AnaZ. Biochem., 79 (1977) 33. 24 R. Uy and F. Wold, Science, 198 (1977) 890. 25 M. Hor%kov%and Z. Deyl , J . Chromatogr. (Chromatogr. Rev.), 159 (1975) 227. 26 Y. Kakimoto and S. Akazawa, J . BioZ. Ckem., 245 (1970) 5751. 27 M.F. Hardy and S.V. P e r r y , Nature (London), 223 (1969) 300. 28 A.M. Asatoor and M.D. Armstrong, Biockem. Biopkys. Res. C o m n . , 26 (1967) 168. 29 Y. Matsuoka, A. Kumon, T. Nakajima, Y. Kakimoto and I. Sano, Biochim. Biopkys. Acta, 192 (1969) 136. 30 A.B. Mauger, E. Katz and K.T. Mason, J . G'krornatogr., 85 (1973) 167. 31 B.S. Handwerger, P.J. Davis and F. Glaser, J . Ckromatogr., 106 (1975) 225. 32 W.S. R e i t h and B.L. Brown, Biockem. J . , 100 (1966) 1OP. 33 G. Tomlinson and T. Viswanatha, C a n . J . Biochem., 5 1 (1973) 754. 34 T. Ubuka, M. K i n u t a , R. Akagi and S. K i g u c h i , J . Chromatogr., 188 (1980) 442. 35 R.S. A s q u i t h and P. Carthew, Biochim. Biophys. Acta, 278 (1972) 8. 36 K. Satake, S. Sasakawa and T. Maruyama, J . Biockem., 53 (1963) 516. 37 A. Yoshida, AnaZ. Biochem., 49 (1972) 320. 38 G.L. Moore and R.S. A n t o n o f f , A n d . Biockem., 39 (1971) 260. 39 W.R. Gray and B.S. H a r t l e y , Biockem. J . , 89 (1963) 379. 40 S. F i t t k a u , J . Ckromatogr., 18 (1965) 331. 41 W.W. Thornburg, L.N. Werum and H.T. Gordon, J . Chrornatogr., 6 (1961) 131. 42 R.L. Munier and G. S a r r a z i n , J . Chromatogr., 12 (1963) 542. 43 N. Nakajima, A. Araya, M. Nakajima, N. F u j i w a r a , K.C. Wu and K. Mizuno, Acta Med. (Nagasaki), 12 (1967) 7; C.A., 69 (1968) 65042j. 44 W.A. Schroeder, Methods EnzyrnoZ., 11 (1967) 445. 45 J. Rosmus and Z. Deyl, J . Chromatogr., 70 (1972) 221. 46 H. Kawauchi, K. Tuzimura, H. Maeda and N. I s h i d a , J . Biochem., 66 (1969) 783. 47 A. Massa, R. C o c c h i e r i and L. T e n t o r i , Ann. I s t . Super. Sanita, 5 (1969) 48. 48 T.W. Jordan, R.W. McNaught and J.N. Smith, Biockem. J . , 118 (1970) 1. 49 T.L. P e r r y , S. Hansen, D. Love and C.A. F i n c h , Nature (London), 219 (1968) 178. 50 G. Poez and G. K r e i l , Biochem. Biophys. Res. C o m n . , 39 (1970) 516. 5 1 M. Onda, Y. Konda, S. Omura and T. Hata, Ckem. Pharm. BUZZ., 19 (1971) 2013; I.C., 44 (1972) 186390. 52 W.S. R i c k e r t and T. Viswanatha, Biockim. Biopkys. Acta, 170 (1968) 123. 53 K. Shigasada, T. Ebisuno and H. K a t s u k i , Biochem. Biophys. Res. C o m n . , 39 (1970) 135. 54 G. Dardenne, J. C a s i m i r and J. J a d o t , Phytochemistry, 7 (1968) 1401. 55 L. Fowden, A. Smith, D.S. W i l l i n g t o n and R.C. Sheppard, Phytochemistry, 8 (1969) 437. 56 T. Yambe and Y. T e r a k i , Shma Igakkai Zasski, 27 (1967) 410; C.A., 60 (1968) 36585~. 57 H. Kodama, Biochim. Biophys. Acta, 165 (1968) 432. 58 S. Mizuhara, H. Kodama, H. Haraguchi and M. Hirosawa, P h y s i o l . Ckem. Pkys., 1 (1969) 600; I.C., 37 (1970) 139754. 59 G.S. Baldwin and P.R. Carnegie, Biochem. J . , 123 (1971) 69. 60 Y. Suketa, M. S u g i i and T. Suzuki, Ckem. P h a n . BUZZ., 18 (1970) 249; I . C . , 37 (1970) 141074.

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81

Chapter 8 PEPTIDES AND STRUCTURAL ANALYSIS OF PROTEINS

z.

PRUS~K

SELECTION OF THE METHOD OF ELECTROMIGRATION SEPARATION P e p t i d e s d i f f e r c o n s i d e r a b l y i n t h e q u a l i t a t i v e and q u a n t i t a t i v e o c c u r r e n c e and sequence o f t h e i r c o n s t i t u e n t amino a c i d s , and t h e s e d i f f e r e n c e s a r e expressed by t h e d i f f e r e n t shapes, m o l e c u l a r w e i g h t s and charges o f t h e molecules. The v a l u e o f t h e charge i s a l s o s t r o n g l y dependent on t h e medium i n which t h e p e p t i d e o c c u r s .

A l l o f these e f f e c t s a r e r e f l e c t e d i n d i f f e r e n t e l e c t r o m i g r a t i o n p r o p e r t i e s . Whereas i n amino a c i d s g e n e r a l l y a broad pH range e x i s t s i n which t h e amino a c i d occurs i n t h e f o r m o f a z w i t t e r i o n w i t h an e f f e c t i v e charge c o n v e r g i n g t o zero, i n p e p t i d e s , as i n p r o t e i n s , t h e i s o e l e c t r i c p o i n t ( P I ) a t w h i c h t h e e f f e c t i v e charge becomes z e r o i s w e l l d e f i n e d . The r e s u l t i n g charge o f a p e p t i d e depends on t h e degree o f d i s s o c i a t i o n o f i n d i v i d u a l i o n o g e n i c groups i n t h e amino a c i d r e s i d u e s

o f t h e p e p t i d e chain. Hence t h e e f f e c t i v e charge o f p e p t i d e s may be changed d i s t i n c t l y by a p p r o p r i a t e s e l e c t i o n o f t h e pH o f t h e medium. Owing t o t h e r e l a t i v e s t a b i l i t y o f t h e p e p t i d e bond, t h e pH can be changed i n aqueous s o l u t i o n s o f p e p t i d e s by up t o 9 u n i t s and t h u s can change t h e charge o f t h e s e a m p h o t e r i c substances f r o m a d i s t i n c t l y p o s i t i v e charge a t l o w e r pH t o a d i s t i n c t l y n e g a t i v e charge a t h i g h e r pH. The c o u r s e o f p r o t o n a t i o n u s u a l l y d i f f e r s i n v a r i o u s p e p t i d e s and so s u i t a b l e c o n d i t i o n s f o r t h e s e p a r a t i o n o f p e p t i d e s may be f o u n d by changing t h e pH b o t h on t h e b a s i s o f d i f f e r i n g e l e c t r o p h o r e t i c m o b i l i t y and on t h e b a s i s o f differing isoelectric point. F o r t h e s e p a r a t i o n o f p e p t i d e m i x t u r e s , t h r e e b a s i c e l e c t r o m i g r a t i o n methods may be used, which p e r m i t complete r e s o l u t i o n o f i o n o g e n i c substances i n s o l u t i o n , viz.,

zone e l e c t r o p h o r e s i s (ZE), i s o e l e c t r i c f o c u s i n g ( I F ) and i s o t a c h o p h o r e s i s

( I T P ) , o r a c o m b i n a t i o n o f t h e s e . A l t h o u g h t h e r e a r e no c o m p l e t e l y unambiguous c r i t e r i a f o r t h e c h o i c e o f t h e method o f s e p a r a t i o n o f complex m i x t u r e s o f p e p t i d e s , 1 c e r t a i n r u l e s may be f o r m u l a t e d t h a t d e f i n e t h e a p p l i c a b i l i t y o f i n d i v i d u a l methods a c c o r d i n g t o t h e n a t u r e o f t h e m i x t u r e o f p e p t i d e s b e i n g separated. Among t h e main c r i t e r i a a r e t h e m o l e c u l a r w e i g h t (M.W.),

t h e i s o e l e c t r i c p o i n t ( P I ) and t h e

l i m i t i n g e l e c t r o p h o r e t i c m o b i l i t y . A c r i t e r i o n t h a t i s n o t n e g l i g i b l e , and somet i m e s even predominates, i s t h e s o l u b i l i t y o f t h e p e p t i d e i n r e l a t i o n t o t h e pH v a l u e and t h e i o n i c s t r e n g t h o f t h e medium, and t h e chemical s t a b i l i t y o f t h e

82

p e p t i d e . From t h e p o i n t o f view o f a f f i n i t y t o t h e e l e c t r o p h o r e t i c c a r r i e r , i . e . , t h e s t a b i l i z i n g medium, t h e c h o i c e o f t h e method w i l l a l s o be a f f e c t e d by t h e presence o f hydrophobic amino a c i d r e s i d u e s and o f n o n - p e p t i d i c components (sugars, l i p i d s , p r o s t h e t i c groups, e t c . ) and by t h e tendency o f t h e p e p t i d e components t o aggregate. F i n a l l y , a v e r y i m p o r t a n t c r i t e r i o n f o r t h e s e l e c t i o n o f t h e e l e c t r o m i g r a t i o n method and o f t h e c a r r i e r medium i s t h e method o f d e t e c t i o n .

Crite ri o n of t h e moZecuuZar weight F o r o l i g o p e p t i d e s o f l o w M.W.

(102 -2.10 3 ) t h e methods o f c o o l e d ZE w i t h a

medium- (MVE) and h i g h - v o l t a g e g r a d i e n t (HVE) ( i . e . , f r o m about 3500 up t o usua 3 3 8.10 -12-10 V m - l ) a r e s t i l l most commonly used. Paper and, l e s s f r e q u e n t l y , a c e t a t e c e l l u l o s e f o i l s a r e commonly used as c a r r i e r s . O f t e n f r e e l y spread c a r r i e r s o f t h e c e l l u l o s e type o r loose layers o f microgranulated g e l s ( s i l i c a gel Sephadex, e t c . ) a r e a l s o used. So f a r c a r r i e r s f r o m homogeneous g e l s have been l e s s o f t e n used, o b v i o u s l y owing t o t h e d i f f i c u l t i e s w i t h t h e d e t e c t i o n o f r a p i d l y d i f f u s i n g zones o f small p e p t i d e s . These d i f f i c u l t i e s do n o t a r i s e i n a n a l y t i c a l capillary ITP i n free solution. For p e p t i d e s o f medium m o l e c u l a r w e i g h t (103 -10 4 ) t h e c h o i c e o f t h e s e p a r a t i o n method i s most d i f f i c u l t . F o r l o w e r M.W. p e p t i d e s f i b r o u s ( p a p e r ) o r l o o s e l y spread c a r r i e r s may s t i l l by used, b u t t h e s u i t a b i l i t y o f f i b r o u s c a r r i e r s ( c e l l u l o s e , paper) decreases r a p i d l y w i t h i n c r e a s i n g M.W. Above M.W.

3000 a paper

c a r r i e r i s c o m p l e t e l y u n s u i t a b l e , owing t o t a i l i n g . When e l u t i o n o f p e p t i d e s w i t h w a t e r i s a p p l i e d a l o s s o f a t l e a s t 50% o f p e p t i d e c o n t e n t i n t h e zone should be expected, and a l s o c o n t a m i n a t i o n o f t h e p e p t i d e w i t h m a i n l y g l y c i n e , a s p a r t i c a c i d 2 and v a l i n e . B e t t e r r e s u l t s a r e o b t a i n e d by e l e c t r o e l u t i o n o r by e l u t i o n w i t h concentrated p y r i d i n e buffers 3 V i r t u a l l y n e g l i g i b l e losses a r e achieved w i t h

.

c a r r i e r - f r e e methods o f s e p a r a t i o n . Flow-through c o n t i n u o u s ZE i s v e r y s u i t a b l e f o r p r e p a r a t i v e s e p a r a t i o n s o f p e p t i d e s o f medium m o l e c u l a r w e i g h t , d i s p l a y i n g r e l a t i v e l y h i g h l i m i t i n g e l e c t r o p h o r e t i c m o b i l i t i e s . A n a l y t i c a l l y and m i c r o p r e p a r a t i v e l y c a p i l l a r y I T P i n f r e e s o l u t i o n has a l s o been a p p l i e d s u c c e s f u l l y i n t h i s M.W.

range.

In t h e M.W. range up t o Z.103 t h e methods o f d e t e c t i o n w i t h

a d s o r p t i o n dyes, which a r e used commonly f o r t h e s t a i n i n g o f p o l y p e p t i d e s and p r o t e i n s , s t i l l cannot be a p p l i e d r e l i a b l y . The l o w e r l i m i t ranges between M.W.

b 1 0 3 and 2-103, where t h e method o f e l e c t r o p h o r e s i s i n g e l w i t h a p o l y a c r y l a m i d e m a t r i x (PAGE) may be a p p l i e d 4 , sometimes i n t h e presence o f sodium dodecyl s u l p h a t e (SDS-PAGE) and/or urea. F o r M.W. c l o s e t o t h e 104 l i m i t , b o t h t h e ZE method and I F i n g e l may u s u a l l y be used. P o l y p e p t i d e s w i t h M.W. above 104 can be s e p a r a t e d s u c c e s s f u l l y u s i n g e i t h e r ZE o r I F and ITP. F o r ZE and i t s m o d i f i c a t i o n s d i s continuous e l e c t r o p h o r e s i s , MZE ( m u l t i p h a s i c zone e l e c t r o p h o r e s i s ) , g e l c a r r i e r s ( p o l y a c r y l a m i d e , agarose) i n t h e f o r m o f a b l o c k a r e used',

and a l s o g r a n u l a t e d

83 ones (Sephadex) and c e l l u l o s e a c e t a t e . The a p p l i c a t i o n o f a r e s t r i c t i v e g e l p r e v a i l s i n t h e ZE method and i t s v a r i a n t s , i . e . ,

the e f f e c t o f t h e pore diameter

of p o l y a c r y l a m i d e g e l on t h e e l e c t r o p h o r e t i c m o b i l i t y o f p e p t i d e s as a f u n c t i o n o f t h e e f f e c t i v e r a d i u s o f p e p t i d e m o l e c u l e s i s o p e r a t i v e (Ferguson p l o t 5 y 6 ) . The i n t r i n s i c charge o f t h e p o l y p e p t i d e s can be suppressed 'almost c o m p l e t e l y by t h e presence o f i o n i c mono-polar d e t e r g e n t s (e.g.,

SDS),

t h e molecules o f which

a r e bound t o t h e p o l y p e p t i d e s i n amounts p r o p o r t i o n a l t o t h e number o f p e p t i d e bonds i n t h e molecule. The l o w e r M.W.

l i m i t o f u t i l i z a b i l i t y o f t h e method ex-

p l o i t i n g t h e p o r o s i t y gradient o f a r e s t r i c t i v e polyacrylamide gel7 f o r l i m i t i n g t h e e l e c t r o p h o r e t i c m o b i l i t y o f p o l y p e p t i d e s t o zero, most commonly as SDS-PAGE, 4 i s c l o s e t o 2-10 On t h e o t h e r hand, a g e l w i t h l a r g e pores i s i n d i s p e n s a b l e

.

f o r I F o f h i g h e r M.W.

p e p t i d e s i n g e l s and ITP i n g e l s w h i c h s h o u l d n o t possess

r e s t r i c t i v e p r o p e r t i e s . F o r h i g h e r M.W.,

i t i s combined i n a two-dimensional

arrangement w i t h SDS-PAGE. These two-dimensional combinations, g e n e r a l l y w i t h I F i n t h e f i r s t d i r e c t i o n , a f f o r d t h e maximal r e s o l u t i o n f o r complex m i x t u r e s o f p o l y p e p t i d e s , as was demonstrated by O ' F a r r e l 18. Such two-dimensional arrangements g i v e i n f o r m a t i o n on b o t h pT v a l u e s and a p p a r e n t m o l e c u l a r w e i g h t s (ISO-DALT and BASO-DALT systems). F o r q u a n t i t a t i v e e v a l u a t i o n on t h e m i c r o s c a l e , c a p i l l a r y ITP can be used even f o r h i g h - m o l e c u l a r - w e i g h t p o l y p e p t i d e s . F o r p r e p a r a t i v e purposes b o t h I F i n g r a n u l a t e d g e l b l o c k s and I T P i n macroporous g e l s may be used. Steadys t a t e c o n c e n t r a t i o n s o f p o l y p e p t i d e s a r e v e r y h i g h under t h e c o n d i t i o n s o f I F and e s p e c i a l l y ITP',

1-10 w t . % .

so t h a t focused zones may r e p r e s e n t t h e c o n c e n t r a t i o n o f

Thus, f o r p r e p a r a t i v e purposes I F and ITP a r e most e f f i c i e n t e s p e c i a l l y

a t h i g h e r m o l e c u l a r w e i g h t s , and t h e s o l u b i l i t y o f t h e separated substances becomes t h e l i m i t i n g f a c t o r . F o r p o l y p e p t i d e s w i t h a r e l a t i v e l y h i g h e l e c t r o 10 p h o r e t i c m o b i l i t y f l o w - t h r o u g h c o n t i n u o u s ZE may s t i l l be used p r e p a r a t i v e l y and w i t h o u t l o s s e s . Hence f o r t h e s e p a r a t i o n o f p o l y p e p t i d e s above M.W.

4 the

10

c u r r e n t 1y used methods f o r t h e s e p a r a t i o n o f p r o t e i n s may be a p p l i e d .

Criterion of the e l e c t r i c charge Sing1 e i o n o g e n i c groups o f p e p t i d e s i n c r e a s e t h e i r c o n t r i b u t i o n t o t h e t o t a l charge as t h e degree o f d i s s o c i a t i o n o f t h e group under c o n s i d e r a t i o n i n c r e a s e s . The t o t a l e f f e c t i v e charge o f p e p t i d e s i s t h e sum o f t h e charges o f i n d i v i d u a l i o n o g e n i c groups. F o r a c l e a r i d e a o f t h e c o n t r i b u t i o n o f i n d i v i d u a l groups i n ._ 8.1. The p K - p l r e p e p t i d e s , a survey o f t h e i r pK v a l u e s i s p r e s e n t e d i n Table

l a t i o n s h i p s i n p o l y p e p t i d e s were d i s c u s s e d b y R i g h e t t i ' ' .

The e l e c t r o p h o r e t i c

m o b i l i t y i s p r o p o r t i o n a l t o t h e t o t a l charge o f t h e p e p t i d e . I f t h e l i m i t i n g e l e c t r o p h o r e t i c m o b i l i t i e s of p e p t i d e s a r e s u f f i c i e n t l y d i f f e r e n t and i n c o n t r a s t , t h e p l values o f t h e components o f t h e m i x t u r e a r e v e r y c l o s e , t h e components o f a p e p t i d e m i x t u r e can be separated e i t h e r a t v e r y l o w o r a t v e r y h i g h pH. A t l o w

84

TABLE 8.1 APPROXIMATE RANGE OF pK VALUES OF IONOGENIC PEPTIDE GROUPS12y13 Ionogenic group

PK

Amino a c i d r e s i d u e , f u n c t i o n a l qroup

-SO H a-CJOH 8-COOH -S-CH2-COOH Y-COOH imidazolyl a-NH2 E-NH2 -SH phenolic g u a n i d i no

1.3 2.0-3.5 3.5-4.5 3.5-4.0 4.0-4.5 5.6-6.9 7.5-8.6 9.0-11 9.0- 10.5 9.8-11 >12

cysteic acid COOH-end o f p e p t i d e aspartic acid S-carboxymethyl c y s t e i n e glutamic a c i d h i s t i d i ne NH2-end o f p e p t i d e lysine c y s t e ine tyrosine arginine

pH, f o r example, t h e e f f e c t o f t h e d i f f e r e n c e i n t h e charges o f g l u t a m i n e and g l u t a m i c a c i d r e s i d u e s , o r o f asparagine and a s p a r t i c a c i d r e s i d u e s , i s suppressed. U s u a l l y t h e pH o f t h e medium ( w i t h t h e e x c e p t i o n o f ITP) i s chosen i n t h e range 1.8 ( f o r p e p t i d e s t h a t do n o t c o n t a i n sugar components) t o 10. I n t h e s e l e c t i o n o f pH i t i s i m p o r t a n t t o ensure t h a t t h e chemical s t a b i l i t y o f t h e p e p t i d e components i s n o t a l t e r e d i n t h e course o f t h e e l e c t r o m i g r a t i o n s e p a r a t i o n . The presence o f amides, d i s u l p h i d e bonds and o t h e r l a b i l e groups r e s t r i c t s t h e u t i l i z a b l e pH range t o some e x t e n t . Thus, f o r example, on l o n g e r exposure i n t h e a l k a l i n e r e g i o n and e s p e c i a l l y a t e l e v a t e d temperatures produced by J o u l e ' s h e a t , t r a n s - s u l p h i d a t i o n may t a k e p l a c e , i .e.

t h e I F method i s o f t e n u n s u i t a b l e f o r

l a b i l e p e p t i d e s t h e p I o f which exceeds 10. The I F method, where p e p t i d e s m i g r a t e t o and remain i n a pH r e g i o n i d e n t i c a l w i t h t h e i r , IP

i s analogous t o t h e method o f d e s a l t i n g o f p e p t i d e s a c c o r d i n g t o

K6st and K6st-Reyes14 by t h e ZE method i n t h e p r o x i m i t y o f t h e PI

o f t h e peptide.

F o r o t h e r cases, however, a pH d i f f e r e n t f r o m t h e p l o f t h e p e p t i d e components

i s s e l e c t e d , which r e s u l t s i n a b e t t e r s o l u b i l i t y o f t h e s e p a r a t e d substances. D u r i n g t h e s e p a r a t i o n o f p e p t i d e s a c c o r d i n g t o t h e i r d i f f e r e n t pK v a l u e s , t h o s e pH r e g i o n s which a r e between t h e pK values o f t h e d i f f e r e n t p e p t i d e s a r e used w i t h advantage, f i n e r e s o l u t i o n o f e l e c t r o p h o r e t i c m o b i l i t i e s b e i n g p o s s i b l e .

ZONE ELECTROPHORESIS

Carrier zone electrophoresis of peptides on paper and thin-layer electrophoresis The c h a r a c t e r i z a t i o n and t h e p r e p a r a t i v e s e p a r a t i o n o f l o w - m o l e c u l a r - w e i g h t p e p t i d e s by zone e l e c t r o p h o r e s i s w i t h a medium v o l t a g e g r a d i e n t 1 5 and by c o o l e d

85 TABLE 8.2 VOLATILE BUFFERS FOR ZONE ELECTROPHORESIS OF PEPTIDES15’ 17’19-24

HVE = c o o l e d h i g h - v o l t a g e paper e l e c t r o p h o r e s i s ; MVE = medium-voltage e l e c t r o p h o r e s i s on paper w i t h o u t c o o l i n g ; TLE = t h i n - l a y e r e l e c t r o p h o r e s i s on c e l l u l o s e o r s i l i c a g e l sheets.

PH 1.9 2.1 3.5 3.8 4.4 4.7 5.3 5.6 6.0 6.5

Formic a c i d (ml)

Acetic a c i d (ml)

100 90 40

300 120 160 200 100 1000 10 200 80 10 15 10 20

Pyr i d i ne (m1)

20 10 100 20 200 200 300 50 250 200

Water

Met hod

(ml) 1600 1790 1800 1780 1900 800 2470 960 1720 2700 1500 2250 1780

HVE HVE HVE HVE, TLE HVE TLE HVE TLE HVE MVE TLE TLE HVE

ZE w i t h a h i g h v o l t a g e g r a d i e n t became r o u t i n e i n t h e 1960s f o r t h e s e p a r a t i o n o f complex p e p t i d e m i x t u r e t h a t a r e formed i n t h e c o u r s e o f t h e s t r u c t u r a l a n a l y s i s o f p r o t e i n s by enzymatic o r chemical cleavage. The d e s c r i p t i o n o f t h e methods f o r t h e s e p a r a t i o n o f p e p t i d e s by ZE may be a l s o found i n Chapter 5 i n P a r t A o f t h i s 16 book ( O s t r o w s k i ). l h e need t o decrease t h a amount necessary f o r t h e sequence a n a l y s i s o f m i n o r p r o t e i n s o r f o r t h e c h a r a c t e r i z a t i o n o f t h e minor, b i o l o g i c a l l y a c t i v e p e p t i d e s l e d t o t h e development o f ZE on t h i n l a y e r s (TLE) o f c e l l u l o s e , g r a n u l a t e d g e l s (Sephadex) and s i l i c a g e l , which decreases t h e amounts necessary f o r t h e d e t e c t i o n o f p e p t i d e s by a t l e a s t one o r d e r o f magnitude w i t h t h e same o r even b e t t e r r e s o l v i n g power, on a s m a l l e r f o r m a t and i n a p r o p o r t i o n a l i y s h o r t e r s e p a r a t i o n t i m e . F o r t h e s e p a r a t i o n o f complex m i x t u r e s and f o r t h e i r easy d e t e c t i o n , v o l a t i l e b u f f e r s c o n t a i n i n g p y r i d i n e , f o r m i c a c i d and a c e t i c a c i d i n v a r i o u s p r o p o r t i o n s o r a c e r t a i n amount o f o r g a n i c s o l v e n t s (e.g.,

a c e t o n e ) used t o i n -

crease t h e s o l u b i l i t y of t h e p e p t i d e s were f o u n d t o be t h e most s u i t a b l e . A s e l e c t i o n of t h e s e b u f f e r s i s g i v e n i n T a b l e 8.2. I n a d d i t i o n t o one-dimensional s e p a r a t i o n s t h a t s e r v e f o r comparison o f l a r g e numbers o f samples and f o r p r e p a r a t i v e purposes, HVE and ILE a r e used f o r twoand mu1 t i - d i m e n s i o n a l s e p a r a t i o n s ( s e e Vandereckhove and Van M o n t a g ~and ~ ~Krause 23 ) w i t h a c o n s i d e r a b l y h i g h e r r e s o l v i n g power. Ingram26 i n t r o d u c ed the and Chen

TABLE 8.3 DIAGONAL ELECTROPHORETIC METHODS FOR SELECTIVE IDENTIFICATION OF PEPTIDE FUNCTIONAL GROUPS Specific functional group, amino acid residue

Modification, reagent

Histidine

Methionine Cystine

Cysteine + lysine Cysteine

Lysine C-Termi nal tryptic peptide

PH

Increase in net charge

Ref.

o f buffer

citraconylation, dinitrophenylation, proteolysis, thiolysis, 2-mercaptoetnanol photo-oxidation

3,5 or 6.5

+

29

2.1 or 3.5 or 4.0 or 6.5

+

30

ethoxycarbonylation by diethyl pyrocarbonate methoxycarbonylation by iodoacetamide oxidation by performic acid sul phitolysis, proteolysis, I. electrophoresis, cystamine hydrochloride oxidation by performic acid reduction, substitution by iodoacetic acid S-aminoethylcysteine, S-ethyl trifluoroacetate thiol exchange, oxidation by performic acid substitution by iodoacetic acid a1 kylation by N-ethylmaleimide trifluoroacetylation, enzyme cleavage pH%. 2, E - N H ~regeneration after I. electrophoresis carboxypeptidase B after I. electrophoresis, all peptides with the exception of C-terminal decrease or lose the positive charge

5

31

2.0

+ +

3.6

+

34

5.6 or 6.5

-

35

6.5

36

6.5

+ -

6.5

+

38

6.4

0

39

32 33,66

6.5 6.5;

6.5

2.0

37 37 117

" f i n g e r p r i n t i n g " method, i.e.

,a

combination o f consecutive paper HVE and chro-

matography i n a p e r p e n d i c u l a r d i r e c t i o n f o r t h e d e t e r m i n a t i o n o f d i f f e r e n c e s i n t h e composition o f peptides i n enzymatic h y d r o l y s a t e s o f human haemoglobins. His method i s used i n several m o d i f i c a t i o n s , which d i f f e r i n t h e composition and t h e pH o f t h e e l e c t r o p h o r e t i c b u f f e r s and t h e chromatographic s o l v e n t s and a l s o i n t h e method o f cleavage o f t h e p r o t e i n s . A summary o f two-dimensional mapping o f peptides was g i v e n by James27 and o f TLE two-dimensional mapping by Sargent and Vadlamudi18 and Gracy".

The TLE method f o r t h e p r e p a r a t i o n o f sub-nanomole-scale 28 p e p t i d e maps was described by Bates and Perham A combination o f ZE/ZE i n t h e form o f diagonal e l e c t r o p h o r e s i s was found t o

.

be convenient f o r t h e s e l e c t i v e i s o l a t i o n o f peptides. A f t e r t h e f i r s t s e p a r a t i o n a s e l e c t i v e m o d i f i c a t i o n o f some o f t h e groups i n t h e p e p t i d e s i s c a r r i e d o u t d i r e c t l y on t h e e l e c t r o p h o r e t i c c a r r i e r . The changes i n t h e charge r e s u l t i n g from t h i s m o d i f i c a t i o n cause a d e v i a t i o n o f t h e p e p t i d e s from t h e diagonal a f t e r a f u r t h e r s e p a r a t i o n b y ZE i n a p e r p e n d i c u l a r d i r e c t i o n , i n t h e d i r e c t i o n o f e i t h e r t h e cathode o r t h e anode, depending on t h e c h a r a c t e r o f t h e change i n t h e charge o f t h e m o d i f i e d group o f t h e peptide. A survey o f t h e diagonal ZE/ZE methods i s presented i n Table 8.3.

Determination of the charge of peptides from t h e i r reZative electrophoretic mobility Although t h e q u a l i t a t i v e d e t e r m i n a t i o n o f t h e charge o f peptides by ZE'was used i n t h e e a r l y days o f t h e a p p l i c a t i o n o f t h e paper ZE o f peptides, t h e quant i t a t i v e d e t e r m i n a t i o n o f t h e charges of p e p t i d e s from t h e i r r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s became w i d e l y used when Offord4O found a l i n e a r dependence between t h e e l e c t r o p h o r e t i c m o b i l i t y o f p e p t i d e s and t h e l o g a r i t h m o f t h e i r M.W. a t pH 6.5 on a paper c a r r i e r . From O f f o r d ' s r e l a t i o n s h i p (eqn. 8.1), t h e number o f amide groups i n a p e p t i d e may be determined, e.g., asparagine can be r e s o l v e d from a s p a r t i c a c i d r e s i d u e s and glutamine f r o m g l u t a m i c a c i d r e s i d u e s . This f i n d i n g i s e s p e c i a l l y v a l u a b l e if t h e method o f sequence a n a l y s i s (Edman degradation) does n o t a f f o r d these data, i.e., i f t h e N-terminal group o f t h e p e p t i d e i s blocked. The p r a c t i c a l u t i l i z a t i o n o f eqn. 8 . 1 i s n o t l i m i t e d t o t h e determinat i o n o f amides o f t h e d i c a r b o x y l i c amino a c i d residues, and i t was f u r t h e r extended t o t h e l o c a l i z a t i o n of p r o s t h e t i c groups, t h e d e t e c t i o n o f new amino acids, and t h e c h a r a c t e r i z a t i o n of t h e a c t i v e s i t e i n enzymes. A survey o f t h e uses was given by Offord41. For t h e e l e c t r o p h o r e t i c m o b i l i t y o f a p e p t i d e i n t h e ZE regimen i n a paper c a r r i e r a t pH 6.5 t h e f o l l o w i n g equation a p p l i e s :

m = keM

-213

2 ,o

I

I

I

I

I I I l l

1

I

l

l

1.0 -

--

-

-

-

-

L

E

-

e=3

-

e=2

-

-

-

e =1

0.1

I

I

I

I

I I l l 1

I

I

I

I .

f i g . 8.1. L o g a r i t h m i c p l o t o f t h e m o l e c u l a r w e i g h t o f p e p t i d e s v e r s u s t h e i r r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s a t pH 1.9 a c c o r d i n g t o B a i l e y and Ramshawlg. e = Net charge; M = m o l e c u l a r w e i g h t o f t h e p e p t i d e ; mr = r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y ( r e l a t i v e t o N - d a n s y l a r g i n i n e ) . Paper c e l l u l o s e c a r r i e r ; b u f f e r composit i o n , f o r m i c a c i d - a c e t i c a c i d - w a t e r (9:12:179 o r 1:4:45, v / v ) .

where m i s t h e r e l a t i v e m o b i l i t y o f t h e p e p t i d e r e f e r r e d t o a s t a n d a r d ( u s u a l l y an e l e c t r o p h o r e t i c a l l y m i g r a t i n g amino a c i d ) , e i s t h e n e t charge o f t h e p e p t i d e ,

k i s a c o n s t a n t and M i s t h e m o l e c u l a r w e i g h t o f t h e p e p t i d e . On e x c l u d i n g p e p t i d e s containing h i s t i d i n e residues, o r c y s t e i c a c i d residues, t h e p o i n t s i n t h e logar i t h m i c p l o t l i e on a s e r i e s o f p a r a l l e l s t r a i g h t l i n e s , always c h a r a c t e r i s t i c o f a whole-number m u l t i p l e o f t h e n e t charge o f t h e p e p t i d e s . The e m p i r i c a l l y d e t e r mined s l o p e i s v e r y c l o s e t o t h e t h e o r e t i c a l v a l u e o f -2/3, by B a i l e y and Ramshaw19 ( s l o p e -0.69+0.01).

as was a l s o c o n f i r m e d

As f o r u n m o d i f i e d p e p t i d e s , t h e r e -

l a t i o n s h i p i s a l s o v a l i d f o r d a n s y l a t e d p e p t i d e s , w i t h a s l o p e o f -0.6.

Using

t h i s method Gray42 checked t h e p r o p e r t i e s o f t h e p e p t i d e s formed g r a d u a l l y d u r i n g t h e Edman d e g r a d a t i o n by d a n s y l a t i o n and t h e ZE o f an a l i q u o t a f t e r each degradat i o n cycle. Vandereckhove and Van M o n t a g ~used ~ ~ TLE s u c c e s s f u l l y f o r t h e c h a r a c t e r i z a t i o n o f t h e n e t charge o f t h e p e p t i d e . Eqn. 8 . 1 a l s o a p p l i e s ZE a t pH 1.9, as shown by B a i l e y and Ramshaw”.

I n t h i s case t h e s l o p e i s -0.62,

as i s e v i d e n t f r o m

F i g . 8.1. F o r d a n s y l a t e d p e p t i d e s , t h e n e t charge on a paper c a r r i e r can be determined r e 1 i a b l y w i t h o u t t a i l i n g f o r up t o o c t a p e p t i d e s . F o r u n m o d i f i e d p e p t i d e s t h e method was used i n some i n s t a n c e s even f o r p e p t i d e s w i t h m o l e c u l a r w e i g h t s h i g h e r t h a n 3000.

89

TABLE 8.4 BUFFERS FOR CONTINUOUS FREE-FLOW PREPARATIVE ELECTROPHORESIS OF PEPTIDES Three t i m e s h i g h e r e l e c t r o l y t e c o n c e n t r a t i o n s a r e g e n e r a l l y used f o r e l e c t r o d e buffers. Sample

Carrier electrolyte

PH

Ref.

K i n i n s , neu-oh p o p h y s i a l hormones, b a s i c and n e u t r a l peptides

0.5 m o l / l a c e t i c a c i d

2.6

45, 48, 49

P r o t e i n hydrolysates

25.8 in1 o f p y r i d i n e t 85.8 m l o f a c e t i c a c i d i n 10 1

3.9

50

Thymus e x t r a c t p e p t i d e s

37 m l o f p y r i d i n e 29.6 m l o f a c e t i c a c i d i n 10 1

4.9

51

Angiotensin, bradykinin 0.033 m o l / l p y r i d i n e and t h e i r s y n t h e t i c analogues a d j u s t e d t o pH 5 w i t h acetic acid

5.0

47

Neurohypophysial e x t r a c t , ACTH, MSH, v a s o p r e s s i n

5.6

46, 49

+

0.08 m o l / l p y r i d i n e a d j u s t e d t o pH 5.6 w i t h acetic acid

Flow-through carrier-free zone electrophoresis o f peptides Continuous d e v i a t i o n ZE a c c o r d i n g t o Hannig has been d e s c r i b e d i n Chapter 11 i n P a r t A o f t h i s book44. C a r r i e r - f r e e ZE has t h e advantage o f a l m o s t a l o s s - f r e e s e p a r a t i o n o f p e p t i d e s and i t i s e s p e c i a l l y s u i t a b l e f o r t h e s e p a r a t i o n o f p e p t i d e s o f h i g h e r e l e c t r o p h o r e t i c m o b i l i t y , independent o f t h e M.W.

I t has p r o v e d e s p e c i a l l y

useful f o r t h e i s o l a t i o n o f b i o l o g i c a l l y a c t i v e peptides from t i s s u e e x t r a c t s

45,46

and f o r t h e p u r i f i c a t i o n o f s y n t h e t i c p e p t i d e s f r o m c r u d e r e a c t i o n m i x t u r e s 48 Mashburn and Hoffman ) . A s o l u t i o n o f a m i x t u r e o f (Schroder and p e p t i d e s o f c o n c e n t r a t i o n up t o 5% (w/v) i s i n j e c t e d c o n t i n u o u s l y i n t o a separa-

matt he^^^,

t i o n chamber c o n t a i n i n g a l a y e r o f c a r r i e r e l e c t r o l y t e f l o w i n g p e r p e n d i c u l a r l y t o t h e d i r e c t i o n o f t h e e l e c t r i c f i e l d . I n view o f t h e s h o r t t i m e o f s e p a r a t i o n (usua l l y n o t exceeding 20-30 m i n ) a t a t e m p e r a t u r e c l o s e t o O°C

and t h e l o w s o r p t i o r l

o f p e p t i d e s on a r e l a t i v e l y s m a l l a r e a o f t h e chamber w a l l s , t h e s e p a r a t i o n o f p e p t i d e s by c o n t i n u o u s ZE i s v e r y m i l d even f o r p e p t i d e s u n d e r g o i n g e a s i l y t r a n s s u l p h i d a t i o n r e a c t i o n s . High y i e l d s a l l o w t h e e l u t e d f r a c t i o n s t o be s u b m i t t e d t o f u t h e r c o n t i n u o u s ZE a t a d i f f e r e n t pH, r e s u l t i n g i n a good y i e l d . Using t h i s quasi-two-dimensional method, p u r e p r e p a r a t i o n s o f n a t u r a l b i o l o g i c a l l y a c t i v e p e p t i d e s of t h e neurohypophysial hormone t y p e were prepared4’.

For t h e i s o l a t i o n

and d e t e c t i o n o f p e p t i d e s , v o l a t i l e c a r r i e r e l e c t r o l y t e s were g e n e r a l l y used,

90

among which t h e most commonly used and t h e most s u i t a b l e f o r t h e e v a l u a t i o n b y b i o l o g i c a l t e s t s i s 0.5-1 m o l / l a c e t i c a c i d . Examples o f s e p a r a t i o n s and t h e c o m p o s i t i o n o f t h e c a r r i e r e l e c t r o l y t e s used a r e surveyed i n T a b l e 8.4.

The capac-

i t y o f t h e s e p a r a t i o n i s u s u a l l y about 100 mg/h.

Zone electrophoresis o f peptides i n polyacrylamide g e l P o l y a c r y l a m i d e g e l a f f o r d s a c h e m i c a l l y and m e c h a n i c a l l y s t a b l e medium w i t h a l o w e l e c t r o e n d o s m o t i c f l o w , s u i t a b l e f o r t h e s e p a r a t i o n o f p e p t i d e s and f o r t h e i r c h a r a c t e r i z a t i o n on t h e b a s i s o f t h e i r s i z e and charge (see, f o r example, 4 Haber e t a l . ) . A more d e t a i l e d a n a l y s i s o f ZE i n p o l y a c r y l a m i d e g e l was g i v e n by Hrka15 i n Chapter 6 i n P a r t A o f t h i s book. I n c o n t r a s t t o p o l y a c r y l a m i d e ZE (PAGE) o f p r o t e i n s , t h e PAGE o f p e p t i d e s encounters t h e problems o f t h e f a s t e r spreading o f zones by d i f f u s i o n and t h e - e s c a p e f r o m d e t e c t i o n o f s m a l l p e p t i d e 3 molecules, b u t a t m o l e c u l a r w e i g h t s o f ca. 10 t h e advantages o f p o l y a c r y l a m i d e g e l b e g i n t o p r e v a i l o v e r t h e c l a s s i c a l f i b r o u s and l o o s e l y spread c a r r i e r s . The t a i l i n g i s suppressed and i n p r e p a r a t i v e a p p l i c a t i o n s h i g h e r y i e l d s and/or p u r i t y may be achieved on e l u t i o n o r e l e c t r o e l ~ t i o nf r o~m~ t~h ~i s ~ i n e r t c a r r i e r t h a n f r o m c e l l u l o s e c a r r i e r s t h a t s e t f r e e s t r u c t u r a l l y bound amino a c i d s on e x t r a c 3 tion

.

The e f f e c t i v e d i a m e t e r o f t h e pores i n t h e p o l y a c r y l a m i d e m a t r i x i s so l a r g e 7 t h a t t h e p o r e - l i m i t i n g e l e c t r o p h o r e s i s a c c o r d i n g t o S l a t e r cannot be a p p l i e d t o smaller oligopeptides, b u t i n combination w i t h I F i t o f f e r s t h e b e s t r e s o l u t i o n o f p o l y p e p t i d e s o f M.W. ca. 104 and h i g h e r , a c c o r d i n g t o O ' F a r r e l 1 8 . On t h e o t h e r hand, h i g h e r c o n c e n t r a t i o n s o f p o l y a c r y l a m i d e g e l (15% f o r M.W. up t o 104 t o 20% f o r M.W.

up t o 1.5.10

4

) a f f o r d a medium, a c c o r d i n g t o Weber e t a1.54, i n which

t h e s e p a r a t i o n o f p e p t i d e s i n t h e f o r m o f a n i o n i c complexes w i t h sodium dodecyl s u l p h a t e (SDS-PAGE) t a k e s p l a c e s a t i s f a c t o r i l y even f o r p e p t i d e s w i t h M.W.

o f the

o r d e r o f 103

extended

.

The r e s o l u t i o n can u s u a l l y be improved and t h e range o f M.W.

t o l o w e r values even w i t h o u t t h e presence o f SDS o r o t h e r i o n i c d e t e r g e n t s by a d d i t i o n o f a h i g h e r c o n c e n t r a t i o n o f urea, u s u a l l y a b o u t 8 m o l / l . Poole e t a l . 4 separated p e p t i d e s o f M.W. l e s s t h a n 1.5010 i n a 10% g e l w i t h 8 m o l / l u r e a and

55

0.9 m o l / l a c e t i c a c i d . Even though t h i s method does n o t a c h i e v e a r e s o l u t i o n as

good as SDS-PAGE,

i t p e r m i t s , c o n t r a s t t o SDS-PAGE,

o f i d e n t i c a l s i z e (M.W.),

a r e s o l u t i o n o f polypeptides

b u t o f d i f f e r e n t i n t r i n s i c charge. I n t h e presence o f

a h i g h e r c o n c e n t r a t i o n o f urea, SDS-PAGE ( i . e . ,

SDS-urea-PAGE)

i s especially

s u i t a b l e f o r t h e s t u d y o f p r o t e i n s t r u c t u r e s and f o r comparative s t u d i e s o f p r o t e i n s on t h e b a s i s o f one-dimensional p e p t i d e maps f o r t h e m o n i t o r i n g o f t h e e x t e n t o f t h e p r o t e i n cleavage, f o r example as a f u n c t i o n o f t i m e , pH, t e m p e r a t u r e o r c o n c e n t r a t i o n . O f course, maximal r e s o l u t i o n can be achieved by two-dimensional s e p a r a t i o n , t h e second dimension b e i n g m o s t l y SDS-PAGE. A number o f workers ( s e e

91 t h e s e c t i o n "Peptide mapping i n g e l s " ) used SDS-PAGE o r SDS-urea-PAGE f o r t h e a n a l y s i s o f p e p t i d e mixtures, o b t a i n e d by s e l e c t i v e cleavage o f p r o t e i n s . I f t h e p o l y p e p t i d e c o n t a i n s an S-S bond, e.g.,

c y s t i n e residues, t h e r e p r o -

d u c i b i l i t y o f t h e separation increases i f i t i s e l i m i n a t e d , f o r example, by r e because t h e amount o f SDS i n t h e SDS-peptide com-

d u c t i o n w i t h mercaptoethanol,

p l e x a t t a i n s t h e maximal values. The degree o f s a t u r a t i o n o f t h e p e p t i d e c h a i n by SDS molecules i s e s p e c i a l l y i m p o r t a n t f o r t h e accuracy o f t h e molecular weight d e t e r m i n a t i o n o f t h e peptide.

Determination of the molecular weight of the peptide from electrophoretic mobility A l i n e a r r e l a t i o n s h i p between t h e l o g a r i t h m o f t h e M.W. o f a p e p t i d e and i t s m o b i l i t y under t h e c o n d i t i o n s o f ZE i n a gel i s v a l i d w i t h c e r t a i n l i m i t a t i o n s , f o r peptides w i t h lower M.W. (see Ferguson p l o t i n chapters by Deyl' and H r k a l 5 i n P a r t A of t h i s book E v e n when t h e i n t r i n s i c charge o f t h e p e p t i d e i s almost completely suppressed and t h e p e p t i d e ' s m o b i l i t y i s determined by t h e dominating charge ( u s u a l l y a n e g a t i v e one) o f t h e c r e a t e d p e p t i d e - i o n i c d e t e r g e n t complex]. This f a c t was f i r s t made use o f by ShapiroS6 i n t h e e l e c t r o p h o r e s i s o f SDS com-

3 plexes w i t h polypeptides w i t h a h i g h M.M. For p e p t i d e s w i t h M.W. between 8.5.10 3 57 and 1.8.10 t h i s dependence was confirmed i n SDS-urea-PAGE by Swank and Munkres Urea 8 m o l / l improves t h e l i n e a r i t y o f t h e p l o t o f l o g M.W. peptides w i t h a low M.W.,

and a t t h e same t i m e i t increases t h e r e s o l u t i o n o f t h e

zones ( i n c r e a s i n g v i s c o s i t y ) ,

t h e aggregation o f t h e peptides i s suppressed, t h e i r

s o l u b i l i t y i s improved and t h e t a i l i n g o f t h e zones disapnears owing t o t h e suppressed hydrogen bond i n t e r a c t i o n s . The s o l u b i l i t y o f p o l y p e p t i d e s a l s o may be improved w i t h z w i t t e r i o n i c detergents58y59. A1 though t h e i n t r i n s i c charge and shape of small peptides has a s t r o n g e r e f f e c t on t h e m o b i l i t y o f t h e SDS-peptide complex than i n t h e case o f protein-SDS complexes, t h e e r r o r o f t h e determinat i o n d i d n o t exceed 218% f o r eleven p e p t i d e s w i t h checked M.W.

values. Kato e t

a1.60 and l a t e r Chan e t a1.61 found f o r SDS-urea-PAGE (0.1% SDS, 12.5-15% p o l y acrylamide gel i n 9 m o l / l u r e a ) t h a t d a n s y l a t i o n o f peptides does n o t have a n e g a t i v e e f f e c t on t h e l i n e a r i t y of t h e l o g M.W. versus m o b i l i t y p l o t i n t h e 4 3 4 3 2.2-10 -4.6-10 o r 1.2010 -1.4010 ranges. T h i s r e l a t i o n s h i p i s demonstrated i n Fig. 8.2a and b. Thus, t h e . i n t r o d u c t i o n o f a f l u o r o p h o r e w i t h a good quantum y i e l d (see, f o r example, t h e r e a c t i o n o f methoxydiphenylfuranone with p e p t i d e s

.

according t o Chen-Kiang e t a1 62 o r f l uorescamine and o-phthalaldehyde) increased t h e s e n s i t i v i t y of t h e M.W.

.

versus m o b i l i t y f o r

d e t e r m i n a t i o n considerably. For t h e d e t e r m i n a t i o n o f

t h e dependence o f t h e m o b i l i t y of peptides on M.W.

i n m i c r o c a p i l l a r i e s Chan e t

a1.61 needed n o t more than 25-50 pmole o f dansylated peptide.

92

4.5

3

i

4.0

0 L11 -1

3 .s

3.0

0.1

0.3

0.2

0.4

0.5

0.6

0.7

"r

4s

(b) 4.0

I

3

\ ACTH

3.5 0)

2 0

3.0

2.5

0.1

0.2

0.3

0.4

0.5

mr

Fig. 8.2. SDS- olyacrylamide gel electrophoresis o f dansylated peptides according to Chan et a1.l1: plot o f the logarithm o f the molecular weight o f DNS-peptides versus their relative electrophoretic mobilities. (a) 15% polyacrylamide gel; (b) 12.5% polyacrylamide gel. M = molecular weight; m = relative electrophoretic mobility (relative to DNS-OH, i.e. 1-dimethylaminonaphhalene 5-sulphonic acid); OVA = ovalbumin; SOY = soybean inhibitor; RIB = 3ribonuclease A; L Y S = lysozyme; CYT = cytochrome C; L I M = lima-bean trypsin inhibitor; ACTH = adrenocorticotropic hormone; GLU = glucagon; POL = polylysine; BAC = bacitracin; ANG = angiotensin; GRA = gramicidin S; MET = methionine-enkephalin.

93

Peptide mapping in g e l s

One- and two-dimensional peptide mapping in gel s l a b s , using e i t h e r an enzymatic or a chemical fragmentation technique, becomes an e f f i c i e n t tool in the determinat i o n of the degree o f s t r u c t u a l s i m i l a r i t y of polypeptides. I n c o n t r a s t t o t h e c l a s s i c a l peptide mapping ( f i n g e r p r i n t i n g ) o f smaller peptides on paper ( s e e , f o r 64 example, Meloun e t a l . 6 3 o r Prusik ) o r on loose c a r r i e r s ( s e e , f o r example Gerday e t a l . l 7 or Pardee and B a m b ~ r g ~where ~ , chromatographic separation princ i p l e s a r e a l s o applied, electromigration separation p r i n c i p l e s in b o t h dimensions in t h e gel medium a r e used f o r t h e comparison of l a r g e peptide fragments of prot e i n s . This comparison i s based e i t h e r on the d i f f e r i n g mobility due t o t h e int r i n s i c charge ( s e e , f o r example, Nomura e t a1.24, Nadeau e t a1.66 and Yamamoto e t a1.67) and t h e i r shape and s i z e , o r on t h e SDS-PAGE regimen in t h e medium of a r e s t r i c t i v e g e l , on t h e basis of the comparison of fragments according t o t h e apparent minimum M.W. 24368-72. The l a t t e r method i s applied both in one-dimensional maps ( c f . , Kraus a n d H i l ~ n a nand ~ ~ S ~ i k e and r ~ ~in the two-dimensional a r r a n g e ~ n e n t ~Between ~. a p p l i c a t i o n of t h e f i r s t and the second d i r e c t i o n of t h e separation a cleaving or modifying reaction can be c a r r i e d o u t . Rapid peptide mapping of proteins t h a t were cleaved enzymatically with limited proteolysis d i r e c t l y in t h e polyacrylamide gel a f t e r SDS-PAGE in the f i r s t d i r e c t i o n (using the discontinuous system according t o L a e m ~ n l iwas ~ ~ developed by Cleveland e t a l . 7 7 . The digestion of t h e polypeptides separated in SDS-gel s l i c e s by means of chymotrypsin or Staphylococcus uureus protease takes place d i r e c t l y in t h e stacking gel in t h e second dimension, during t h e stacking phase o f SDSPAGE. Then t h e peptides formed a r e separated in 15% SDS-gel. This method was adjusted f o r peptide mapping of heterogeneous membrane protein samples by Bordier and Crettol-Jarvinen7*. The proteins were separated by SDS-PAGE in a l i n e a r polyacrylamide gel concentration gradient from 5 t o 17% and, a f t e r digestion in t h e stacking phase, t h e fragments were separated in a homogeneous 15% gel using SDS-

PAGE. The method of Cleveland e t a l . mentioned above was f u r t h e r extended by Nikodem and Fresco7' f o r cyanogen bromide cleavage in the g e l . Lam and Kasper8' characterized the sequence homology of polypeptide p r o t e i n s by means of peptide maps obtained b o t h by enzymatic cleavage w i t h t r y p s i n , chymotrypsin and S. uureus protease and by chemical cleavage of polypeptides separated in t h e f i r s t d i r e c t i o n in a c y l i n d r i c a l gel with a 5 4 . 7 5 % concentration of polyacrylamide by SDSPAGE. The enzymatic cleavage takes place during t h e co-stacking of proteases with t h e polypeptide in t h e stacking g e l , using a decreased current in the prolonged i n i t i a l phase of the second d i r e c t i o n of SDS-PAGE. The chemical cleavage took place a l t e r n a t i v e l y with hydroxylamine o r cyanogen bromide d i r e c t l y in t h e cyl i n d r i c a l separation g e l , a f t e r electrophoresis in t h e f i r s t d i r e c t i o n on uncuba-

94 t i o n i n a s o l u t i o n o f the cleaving reagent. A f t e r repeated e q u i l i b r a t i o n , stacking was c a r r i e d o u t and t h e s e p a r a t i o n i n t h e second d i r e c t i o n i n 12% g e l by SDS-PAGE, as w i t h enzymatic h y d r o l y s a t e s . B e t t e r r e p r o d u c i b i l i t y may be expected where t h e chemical cleavage takes p l a c e . T h i s method g i v e s a v a l u a b l e comparison o f t h e fragments o f homologous p o l y p e p t i d e s f r o m t h e p o i n t o f view o f t h e d i s t r i b u t i o n o f m o l e c u l a r w e i g h t s , n a t u r a l l y w i t h e x c e p t i o n o f s m a l l p e p t i d e fragments w h i c h m i g h t escape d e t e c t i o n ( s e e t h e s e c t i o n " D e t e c t i o n o f p e p t i d e s i n e l e c t r o m i g r a t i o n systems"). Obviously, t h e method does n o t r e s o l v e s m a l l e r changes i n t h e amino a c i d sequence. F o r such s m a l l changes i n t h e sequence, I F i n a g e l g i v e s b e t t e r r e s o l u t i o n , a c o m b i n a t i o n of I F w i t h subsequent SDS-PAGE, a1 f o r example, by Goldsmith e t a l .

as c a r r i e d o u t ,

.

ISOELECTRIC FOCUSING A l l p e p t i d e s can be focused u s i n g t h e I F method a c c o r d i n g t o Svensson ( R i l b e )

a2

w i t h o u t r e g a r d t o t h e i r m o l e c u l a r w e i g h t . The e f f e c t o f t h e s i z e o f t h e p e p t i d e m o l e c u l e ( o r o f t h e d i f f u s i o n c o e f f i c i e n t ) on t h e degree o f f o c u s i n g d u r i n g t h e a p p l i c a t i o n o f t h e e l e c t r i c f i e l d i s n o t i m p o r t a n t , as was shown e x p e r i m e n t a l l y by F i n l a y s o n and Chrambach"

and C a t ~ i m p o o l a si ~n ~t h e I F o f d i p e p t i d e s . D u r i n g

t h e I F process, t h e f o c u s i n g o f t h e p e p t i d e zones i n t h e pH g r a d i e n t t o o k p l a c e , t h e l a t t e r b e i n g formed by t h e e l e c t r o l y s i s - e l e c t r o p h o r e s i s o f c a r r i e d s y n t h e t i c ampholytes s i m i l a r l y t o t h e case w i t h p r o t e i n s , o r even more r a p i d l y . The s i t u a t i o n changes when t h e c u r r e n t i s i n t e r r u p t e d , e.g., when t h e d e f o c u s i n g o f t h e zones o f s m a l l p e p t i d e molecules, caused by d i f f u s i o n , t a k e s p l a c e much more r a p i d l y (even i n t h e g e l ) t h a n i t does w i t h p r o t e i n s . T h i s u n f a v o u r a b l e s i t u a t i o n i s f u r t h e r d e t e r i o r a t e d by t h e d i f f i c u l t i e s stemming f r o m t h e comparable s i z e s o f t h e molecules o f t h e c a r r i e r s y n t h e t i c ampholytes, as shown by Goerth and R a d 0 1 a ~ ~These . d i f f i c u l t i e s a r e most s t r i k i n g i n t h e p r e p a r a t i v e I F o f p e p t i d e s , where i t should be t a k e n i n t o c o n s i d e r a t i o n t h a t t h e M.W. o f t h e c a r 2 4 r i e r ampholytes i s about 10 -lo3, o r even up t o 10 C a r r i e r ampholytes h a v i n g a 3 l o w a p p a r e n t M.W. o f up t o 2-10 may be prepared f o r t h e s e p a r a t i o n o f p e p t i d e s 86 w i t h an a p p a r e n t M.W. h i g h e r t h a n 5*103, as was shown by GaspariE and Rosengreen

.

.

The i n t e r f e r e n c e of c a r r i e r ampholytes m a n i f e s t s i t s e l f even i n chemical r e a c t i v i t y , i.e.,

some u n i v e r s a l d e t e c t i o n s ( n i n h y d r i n , Lowry, c h l o r i n a t i o n and s i m i l a r ) can-

n o t b e used. On t h e o t h e r hand, t h e u n d o u b t e d l y e x c e l l e n t r e s o l v i n g power o f t h e I F o f p e p t i d e s i n g e l s s t i m u l a t e d developments t o such an e x t e n t , t h a t R i g h e t t i and C h i l l e m i B 7 f o r m u l a t e d t h e p r i n c i p l e s f o r t h e a p p l i c a t i o n of I F i n p o l y a c r y l a m i d e g e l i n t h e a n a l y s i s o f o l i g o p e p t i d e s and p o l y p e p t i d e s f o r t h e c h a r a c t e r i z a t i o n o f t h e charge homogeneity and f o r c o m p a r a t i v e s t u d i e s o f p r o t e i n fragments. The

95

F i g . 8.3. I s o e l e c t r i c f o c u s i n g o f l o w - m o l e c u l a r - w e i q h t p e p t i d e s i n p o l y a c r y l a m i d e urea g e l a c c o r d i n g t o Faupel and Von ArxgO. H i s t i d i n e - c o n t a i n i n g p e p t i d e s were a p p l i e d on s m a l l paper s t r i p s i n d i c a t e d by b l a c k r e c t a n g l e s ; d e t e c t i o n was c a r r i e d o u t w i t h m o d i f i e d P a u l y ' s r e a g e n t . pH markers: whale myoglobin, h o r s e myog l o b i n , 6 - l a c t o g l o b u l i n A and B. P e p t i d e s : 1 = p-Glu-His-NH2; 2 = p-Glu-His-OCHg; 3 = p-Glu-His-Pro-NH2.HOAc; 4 = H-Asn-Gln-His-OH; 5 = p-Glu-His- Pro-OH.HC1 ; 6 = V a l 5 - a n g i o t e n s i n 11; 7 = human c a l c i t o n i n ; 8 = p o r c i n e ACTH ( 1 - 3 9 ) ; 9 = human insulin.

main p o s s i b i l i t y o f t h e a p p l i c a t i o n o f t h e I F o f p e p t i d e s i s i n t h e comparison o f t h e c o m p o s i t i o n o f m i x t u r e s o f p e p t i d e s formed by d i r e c t e d c l e a v a g e o f p r o t e i n s and i n t h e c o n t r o l o f t h e p r o d u c t s o f s y n t h e s i s o f t h e p e p t i d e s o f medium and h i g h P1.W.

Examples o f t h e s e a p p l i c a t i o n s a r e p r e s e n t e d i n Table 8.5.

F o r p e p t i d e s w i t h l e s s t h a n 15-20 amino a c i d s , u n i v e r s a l d e t e c t i o n b y Coomassie Blue s t a i n i n g i s u n r e l i a b l e , b u t t h e s e l e c t i v e d e t e c t i o n o f some amino a c i d s can be used (as demonstrated by Faupel and Von Arxg0, F i g . 8 . 3 ) , and o f a r g i n i n e and t r y p t o p h a n , a c c o r d i n g t o Gianazza e t a1.94. F o r t h e I F o f p e p t i d e s i n p o l y a c r y l amide g e l R i g h e t t i and C h i l l e ~ n recommended i~~ t h e use o f 6-8 m o l / l u r e a o r some o t h e r d i s s o c i a t i o n agent, so t h a t t h e f o r m a t i o n o f a r t i f i c i a l zones o f t h e i n t e r a c t i o n o f p e p t i d e s w i t h t h e c a r r i e r ampholytes does n o t t a k e p l a c e . Working i n v e r y t h i n l a y e r s o f g e l i s recommended where t h e e l u t i o n o f u r e a and c a r r i e r ampholytes i s s i m u l t a n e o u s l y f a c i l i t a t e d .

P e r s u l p h a t e and t r a c e s o f cyanate f r o m

urea a r e e l i m i n a t e d by means o f a p r e - r u n b e f o r e a p p l i c a t i o n o f t h e p e p t i d e sample. The use o f d e t e r g e n t s t h a t f o r m complexes w i t h c a r r i e r ampholytes, e.g.,

Triton

TABLE 8.5 ISOELECTRIC FOCUSING OF PEPTIDES PAG = polyacrylamide gel; HGH = human growth hormone; GH cyanogen bromide cleavage at methionine residue.

=

growth hormone; ACTH

=

adrenocorticotropic hormone; CNBr

=

Sample

Stabilizing medium

Detection

Ref.

HGH and ovine GH synthetic fragments (8-54 amino acid residues) Tyrosine dipeptides

PAG, 8 mol/l urea, 2% Ampholine of PI3.5-10

Coomassie Blue 6-250 staining

87

sucrose density gradient, carrier ampholytes of p1 3-10 PAG, 8 mol/l urea, 2% Ampholine of PI5-8 PAG, 1.5% Ampholine of PI3-10

UV absorbtion scanning in situ during IF Coomassie Blue 6-250 staining

88

modified Pauly reagent

90

PAG (5%), 6 mol/l urea, 2% Ampholine of PI3-10 PAG (5%), 8 mol/l urea, Ampholine o f PI3.5-10 PAG, u mol/l urea PAG, 8 mol/l urea, 0.4% Ampholine of PI3-6 PAG, ultra-thin layer

Coomassie Blue staining

91

Coomassie Blue R-250 staining

92

Coomassie Blue R-250 staining

93

Coomassie Blue staining specific detections (Pauly, Sakaguchi, etc.)

83 94

PAG

visible light absorption scanning at 600 nm

95

N-Terminal HGH fragment (54 amino acid residues) Human insulin, porcine ACTH 1-31 fragment, human calcitonin, Val5-angiotensin, peptides o f M.W. 265-5808 CNBr fragments o f human i soferritins CNBr fragments of bovine ribonuclease, catalase CNBr fragments of tubulins CNBr fragments of a-chain o f collagen and hemoglobin Oligopeptides containing arginine, histidine, tyrosine or tryphophan residues HGH synthetic fragment (position 125-156), histidine residue dinitrophenylated

89

97

X-100 and N o n i d e t P-40, s h o u l d be avoided. B i b r i n g and Baxandallg2 and R i g h e t t i and C h i l l e ~ n di ~i f ~f e r t o some e x t e n t i n t h e i r o p i n i o n s on t h e o p t i m a l s i t e o f sample a p p l i c a t i o n . I f t h e sample o f p e p t i d e s i s a p p l i e d t o t h e gel on a paper S t r i p , t h e n t h e most c o n v e n i e n t s i t e o f a p p l i c a t i o n i s c l o s e r t o t h e anode, where sorpt i o n on t o t h e paper c a r r i e r i s suppressed and t h e sample i s a l s o b e t t e r p r o t e c t e d f r o m c a r b a m y l a t i o n . A c c o r d i n g t o B i b r i n g and Baxandall",

t h e r i s k o f formation

of a r t i f i c i a l c a r b a m y l a t i o n p r o d u c t s d u r i n g t h e r e a c t i o n o f p e p t i d e s and cyanates formed from u r e a does indeed e x i s t , b u t i t does n o t p r e v e n t t h e i n t e r p r e t a t i o n of t h e r e s u l t s o f t h e

IF

s e p a r a t i o n o f p e p t i d e s . Whereas w i t h p r o t e i n s a n a r r o w e r

pH range o f c a r r i e r ampholytes i s o f t e n used, i t can o n l y seldom be used f o r a m i x t u r e o f p e p t i d e s . The charge o r o p e r t i e s o f p e p t i d e s and t h u s a l s o t h e i r PI values are, i n view o f t h e i r l o w e r M.W.,

more dependent on t h e charge p r o p e r t i e s

of i n d i v i d u a l amino a c i d r e s i d u e s , and can t h u s assume extreme values. As i s e v i d e n t f r o m T a b l e 8.5 f o r t h e I F o f p e p t i d e s , a pH g r a d i e n t f r o m 3 t o 10 i s g e n e r a l l y used. I f a knowledge o f t h e IP

o f i n d i v i d u a l p e p t i d e s i n t h e presence o f

ureag6 i s necessary, t h e pH g r a d i e n t i s determined b y p a r a l l e l focused s t a n d a r d p o l y p e p t i d e s , w i t h known PI, o r a c o r r e c t i o n o f t h e measured v a l u e s o f pH acc o r d i n g t o U i g 7 o r Gelsema e t a1.98 f o r t h e g e l c o n t a i n i n g u r e a may be used. Duri n g t h e judging o f t h e heterogeneity o f t h e mixture without t h e determination o f P theI p e p t i d e m i x t u r e can be l a b e l l e d by f l u o r e s c e n c e .

I n such a case p e p t i d e

can be d e t e c t e d r a p i d l y even i n c y l i n d r i c a l g e l s . The p o l y a c r y l a m i d e g e l i s even b e t t e r u t i l i z a b l e f o r t h e I F o f p e p t i d e s t h a n f o r p r o t e i n s , as i t s c o n c e n t r a t i o n can be s e l e c t e d , w i t h simultaneous p r e s e r v a t i o n o f n o n - r e s t r i c t i v e p r o p e r t i e s , f r o m t h e l o w e s t l i m i t o f i t s mechanical s t r e n g t h upwards, commonly i n t h e 4-6% o f a c r y l a m i d e c o n c e n t r a t i o n range. The c o n t e n t o f t h e c r o s s - l i n k i n g component s h o u l d be k e p t low, o r o t h e r w i s e non-polymerized monomers c o u l d be e x t r a c t e d t o g e t h e r w i t h p e p t i d e s . F o r p r e p a r a t i v e use t h e method o f c o n t i n u o u s I F a l s o comes i n t o c o n s i d e r a t i o n , where t h e d e t e c t i o n i s s i m p l y c a r r i e d o u t a f t e r t h e o u t f l o w o f t h e f r a c t i o n s f r o m t h e s e p a r a t i o n chamber. ISOTACHOPHORESIS The s e p a r a t i o n o f p e p t i d e s u s i n g t h e ITP method began t o be developed o n l y i n t h e f i r s t h a l f o f t h e 1970s. C a p i l l a r y ITP i n f r e e s o l u t i o n was a p p l i e d i n a number o f m i c r o a n a l y t i c a l d e t e r m i n a t i o n s o f t h e q u a l i t a t i v e and e s p e c i a l l y o f q u a n t i t a t i v e c o m p o s i t i o n o f p e p t i d e m i x t u r e s o f n a t u r a l and s y n t h e t i c o r i g i n . F o r a n a l y s i s by c a p i l l a r y ITP amounts from 0.1 t o 1 nmole o f p e p t i d e u s u a l l y s u f f i c e . F o r t h e d e t e c t i o n of p e p t i d e s chemical d e t e c t i o n ( r e a c t i o n ) i s n o t necessary, as b o t h u n i v e r s a l and UV a b s o r p t i o n d e t e c t o r s a r e a v a i l a b l e . The t i m e o f ' a n a l y s i s

does n o t

exceed s e v e r a l t e n s o f minutes even f o r p e p t i d e s w i t h a l o w e l e c t r o p h o r e t i c mo8-

TABLE 8.6 ISOTACHOPHORESIS OF PEPTIDES E l e c t r o l y t e systems. HPMC = hydroxypropylmethylcellulose; NC = m e t h y l - c e l l u l o s e ; PVA = p o l y ( v i n y 1 a l c o h o l ) ; HEPES = N-2-hydroxyethy1piperazine-N1-2-ethanesulphonic a c i d , sodium s a l t ; EACA = 6-aminocaproic a c i d ; PG = p o t e n t i a l g r a d i e n t d e t e c t o r ; T = t h e r m a l d e t e c t o r ; C = e l e c t r i c a l c o n d u c t i v i t y d e t e c t o r ; UV = d e t e c t o r o f UV l i g h t a b s o r p t i o n . Sample

Charge

Leading e l e c t r o l y t e

Terminating e l e c t r o l y t e

DH ~

P e p t i d e s i n uraemic body fluids, polyglycine with b l o c k e d N-terminus (M.W.

-

~~

Detection

Ref.

~~

0.005 m o l / l H C l , 0.02 m o l / l 8 - a l a n i n e 0.4% HPNC

4.0

0.005 m o l / l c a p r o i c a c i d

T, UV

+

0.005 m o l / l Ba(OH)2 a d j u s t e d w i t h Val i n e , 0.25% HPYC

5.1

0.005 m o l / l a - a l a n i n e

uv

100

T r i g l y c y l v a s o p r e s s i n , Lysv a s o p r e s s i n , O-methylo x y t o c i n , des-NH2-D-Alavasopressin, b o v i n e pancreatic trypsin inhibitor

-+

0.01 m o l / l KOH adjusted w i t h acetic acid

5.1

0.02 m o l / l 6 - a l a n i n e

uv

10 1

Porcine s e c r e t i n

+

0.005 m o l / l KOAc adjusted w i t h acetic acid

5.1

0.005 m o l / l a - a l a n i n e

uv

102

Secretin, cholecystokinin

+

0.005 m o l / l KOAc adjusted w i t h acetic acid 0.4% HPMC

5.2

0.01 m o l / l a - a l a n i n e

uv

100

99

1000-2000)

VIP ( v a s o a c t i v e i n t e s t i n a l p e p t i d e ) (28 amino a c i d r e s i d u e s , M.W. 3400)

T r y p t i c fragments f r o m c i t r a c o n y l a t e d human haemopexin

0.01 m o l / l HC1, 0.02 m o l / l h i s t i d i n e

6.0

0.01 mol/l HEPES a d j u s t e d t o pH 8.3 w i t h Ba(OH)2

UV, PG

103

Reduced and o x i d i z e d g l utathiones

-

0.02 m o l / l Ammediol a d j u s t e d w i t h HC1, 0.5% HPMC

6.52

0.005 m o l / l phenol a d j u s t e d t o pH 10 w i t h 8a(OH)2

PG

104

Somatostatin s y n t h e t i c fragment (14 amino a c i d residues )

+

0.005 m o l / l KOH adjusted w i t h c a c o c y l i c acid, 0.5% HPMC

6.9

0.01 mol/l c r e a t i n i n e

uv

105

A n g i o t e n s i n I and 11, reduced and o x i d i z e d g l utathiones

-

0.007 m o l / l HC1 adjusted w i t h T r i s

7.0

0.005 m o l / l phenol a d j u s t e d t o pH 10 w i t h Ba(OHI2

Hexapeptide f r o m c h o l e c y s t o k i n i n and i t s degradation products

+

0.005 m o l / l KOAc a d j u s t e d w i t h cacod y l i c acid, 0.25% HPMC

7.0

0.01 m o l / l 8 - a l a n i n e a d j u s t e d t o pH 3.8 w i t h HC1

uv

100

O x y t o c i n , Arg-vasopressin, b a c i t r a c i n , ACTH s y n t h e t i c fragment (18 amino a c i d residues)

+

0.005 m o l / l KOH adjusted w i t h cacodylic acid

7.0

0.01 m o l / l 6 - a l a n i n e a d j u s t e d t o pH 3.8 w i t h HC1

uv

100

A n g i o t e n s i n I and I1

-

0.02 mol/l Ammediol a d j u s t e d w i t h HC1, 0.5% MC

7.05

0.01 mol/l phenol a d j u s t e d t o pH 10 w i t h Ba(OH)2

PG

104

F i b r i n s y n t h e t i c fragments (10 and 11 amino a c i d residues )

-

0.005 mol/l HC1, 0.006 mol/l T r i s

7.2

0.01 mol/l v a l i n e a d j u s t e d t o pH 9 w i t h Ba(OH)2

T, UV

106

HGH s y n t h e t i c fragment ( p o s i t i o n 125-156)

-

0.01 m o l / l HC1, 0.02 mol/l T r i s 0.2% T r i t o n X-100

7.65

0.05 m o l / l g l y c i n e a d j u s t e d t o pH 9 w i t h Ba(OH)2

T, UV

107

(Continued on p . 1 0 0 )

(c, (0

TABLE 8.6 (continued] Sampl e

Leading e l e c t r o l y t e

pH

Terminating e l e c t r o l y t e

Detection

Ref.

0.0025 mol/l KOAc

8.0

0.05 m o l / l a-alanine a d j u s t e d t o DH 1.82 w i t h HC1

PG

108

Human apo-HDL-polypepti des

0.005 m o l / l HC1, 0.02 mol/l T r i s , 0.2% T r i t o n X-100

8.05

0.04 m o l / l 8-alanine a d j u s t e d t o pH 8.65 w i t h Ba(OH)2

T, U V

109

Diglycine, t r i g l y c i n e , dialanine, t r i a l a n i n e

0.05 mol/l 2-amino-2methyl -1-propanol adjusted w i t h HC1, 0.5% MC

8.97

0.01 mol/l 8-alanine adjusted t o pH 10.9 w i t h Ba(0H)p

PG

110

Sarcosine, d i s a r c o s i n e

0.01 m o l / l HC1 8.97 adjusted w i t h Amediol, 0.2% PVA

0.01 m o l / l 6-alanine a d j u s t e d t o pH 10.9 w i t h Ba(OH)2

PG

104

Diglycine, tetraglycine, g l u t a t h i one

0.04 m o l / l 5-bromo9.0 2,4-dihydroxybenzoate adjusted w i t h L-lysine

a-alanine

c, uv

111

0.01 m o l / l Ba(OH)2 a d j u s t e d w i t h methionine

9.25

0.02 mol/l T r i s , 0.005 m o l / l HC1, pH 8.0

PG

104

Baci t r a c in

0.005 mol/l HC1 adjusted w i t h Ammediol, 0,3% HPMC

9.3

0.005 m o l / l EACA a d j u s t e d t o pH 10 w i t h Ba(OH)2

uv

100

Arg-vasopressin, Lys-vasopressin

0.005 mol/l Ba(OH)2 adjusted w i t h valine, 0.25 HPMC

9.3

0.02 m o l / l T r i s 0.005 m o l / l HC1 pH 8.4

uv

100

Kallidin, bradykinin

0.01 mol/l Ba(OH)2 adjusted w i t h methionine

9.55

0.02 m o l / l T r i s 0.005 m o l / l HC1 DH 8.0

PG

104

Basic peptides, M.W. and 3300

Met-Lys-bradykinin,

Charge 1900

k a l 1i d i n

101 b i l i t y . The p r i n c i p l e o f ITP p e r m i t s t h e s e p a r a t i o n o f e i t h e r p o s i t i v e l y o r nega t i v e l y charged p e p t i d e s . B a s i c p e p t i d e s , w h i c h i n c l u d e a number o f a b i o l o g i c a l l y a c t i v e substances, a r e b e s t s e p a r a t e d i n p r o t o n a t e d f o r m by c a t i o n i c ITP, most commonly i n t h e pH 4-5.2

range. I n t h i s r e g i o n hypophysial hormones, k i n i n s ,

v a s o a c t i v e and s e c r e t o r i c hormones o f t h e d i g e s t i v e t r a c t were s e p a r a t e d w i t h t h e potassium o r sodium as t h e l e a d i n g i o n and a c e t a t e as t h e c o u n t e r i o n . 6 - A l a n i n e was s u i t a b l e as t h e t e r m i n a t i n g i o n . F o r t h e a n a l y s i s o f n e u t r a l p e p t i d e s and p e p t i d e s w i t h p I l o w e r t h a n 7, a n i o n i c ITP i s more common. The c h o i c e o f t h e e l e c t r o l y t e system f o r t h e a n i o n i c ITP o f p e p t i d e s i s a f f e c t e d (above pH 6 ) by t h e n e c e s s i t y t o suppress t h e d i s t u r b i n g e f f e c t o f t h e c a r b o n a t e i o n s i n t h e t e r m i n a t i n g e l e c t r o l y t e w i t h t h e a d d i t i o n o f b a r i u m h y d r o x i d e , so t h a t t h e pH o f t h e t e r m i n a t i n g e l e c t r o l y t e i s u s u a l l y h i g h e r t h a n 8 . The c h l o r i d e i o n i s u s u a l l y s e l e c t e d as a l e a d i n g e l e c t r o l y t e c o n s t i t u e n t . The t e r m i n a t i n g i o n can be s e l e c t e d e i t h e r among t h e s e r i e s o f Good's a m p h o l y t i c components, w i t h a r e l a t i v e l y l o w m o b i l i t y , f o r example HEPES, MOPS and MES, whereas f o r p e p t i d e s w i t h a l o w mob i l i t y even some n e u t r a l amino a c i d s a r e s u i t a b l e as "spacers" o r as t e r m i n a t o r s , e.g.,

a l a n i n e , v a l i n e and 6-aminocaproic a c i d . Examples o f t h e a p p l i c a t i o n o f

c a p i l l a r y ITP t o t h e a n a l y s i s o f p e p t i d e s a r e g i v e n i n T a b l e 8 . 6 . The e l e c t r o l y t e systems a p p l i e d i n t h e c a p i l l a r y ITP o f p e p t i d e s may, o f course, be e q u a l l y w e l l a p p l i e d i n n o n - r e s t r i c t i v e g e l s and on a p r e p a r a t i v e s c a l e (e.g., r e f s . 9, 112, 113). The w i d e r a p p l i c a t i o n o f m i c r o a n a l y t i c a l c a p i l l a r y ITP w i t h i t s s i m p l e q u a n t i t a t i v e i n t e r p r e t a t i o n i s p o s s i b l e e s p e c i a l l y i n analyses connected w i t h t h e s t u d y o f t h e p r o t e i n fragments, i n t h e c o n t r o l o f t h e p u r i f i c a t i o n procedure used f o r p o l y p e p t i d e s and o l i g o p e p t i d e s which a r e n o t e a s i l y de2 e s p e c i a l l y f o r p e p t i d e s w i t h M.W. o f 10 -

t e c t e d by t h e I F method i n g e l s , e.g.,

3

10 which a r e formed b y s e l e c t i v e cleavage o r by s y n t h e s i s . An example o f t h e ITP a n a l y s i s o f n a t u r a l p o l y p e p t i d e s , i n h i b i t o r s o f proteases, i s g i v e n i n F i g . 8.4. DETECTION OF PEPTIDES I N ELECTROMIGRATION SYSTEMS The methods o f d e t e c t i o n o f p e p t i d e s a r e determined t o a c o n s i d e r a b l e e x t e n t by t h e medium i n which e l e c t r o m i g r a t i o n t a k e s p l a c e . The l i m i t o f d e t e c t a b i l i t y o f p e p t i d e s i s a f f e c t e d by a number o f p r o p e r t i e s o f t h e s t a b i l i z i n g medium, e s p e c i a l l y p o r o s i t y , v i s c o s i t y , mechanical s t r e n g t h , o p t i c a l p r o p e r t i e s , chemical a f f i n i t y and r e a c t i v i t y . The r e l a t i v e l y l o w M.W.

o f p e p t i d e s and t h e above-men-

t i o n e d ensuing r a p i d zone s p r e a d i n g a f t e r t h e t e r m i n a t i o n o f t h e s e p a r a t i o n subs t a n t i a l l y l i m i t t h e t i m e d u r i n g w h i c h t h e o r i g i n a l r e s o l u t i o n o f p e p t i d e zones may be m a i n t a i n e d . One o f t h e s o l u t i o n s c o n s i s t s i n i n s i t u d e t e c t i o n d u r i n g t h e s e p a r a t i o n . T h i s method i s used i n procedures w i t h a s t e a d y - s t a t e regimen, esp e c i a l l y i n t h e c a p i l l a r y ITP o f p e p t i d e s by means o f s t a t i o n a r y d e t e c t o r s . I n

102 dU

u, x

UV-ABS

F i g . 8.4. A n a l y t i c a l c a p i l l a r y i s o t a c h o p h o r e s i s o f two b a s i c a c r o s i n i n h i b i t o r s a c c o r d i n g t o KaSiEka and P r u s i k l o 3 . C a t i o n i c I T P s e p a r a t i o n was performed u s i n g t h e apparatus developed by P r u s i k e t a l , 1 1 4 . Right-hand main zone, 4.5 pg o f B S I 11; l e f t - h a n d main zone, 3.5 p g o f BUS1 I I b ; b o t h i s o i n h i b i t o r s o f M.W. 6.2-10 were i s o l a t e d f r o m b u l l seminal plasma by techova e t alaii5. The sample was app l i e d as 2 p1 o f a s o l u t i o n i n water. Leading e l e c t r o l y t e , 0 . 0 1 m o l / l Na+; c o u n t e r i o n , a c e t a t e ; pH 4.8; a d d i t i v e , 0.02% Mowiol; t e r m i n a t i n g e l e c t r o l y t e , 0.01 m o l / l B-alanine, a d j u s t e d t o pH 4.25 w i t h a c e t i c a c i d . C a p i l l a r y l e n g t h , 450 mm; s t a b i l i z e d c u r r e n t , 80 uA (20 pA d u r i n g d e t e c t i o n ) ; t i m e o f a n a l y s i s , 30 min. U = pot e n t i a l g r a d i e n t s i g n a l ; UV-ABS = o p t i c a l absorbance s i g n a l a t 254 nm; dU/dt = d i f f e r e n t i a t e d p o t e n t i a l gradient signal; t = time.

Y

c o n t r a s t , i n I F a scanning d e t e c t o r f o r t h e UV r e g i o n i s i n d i s p e n s a b l e , moving i n t h e d i r e c t i o n o f t h e e l e c t r i c f i e l d , However, when I F i s a p p l i e d t h e presence o f c a r r i e r ampholytes r e s t r i c t s t h e p o s s i b i l i t y o f d e t e c t i o n t o p e p t i d e s t h a t cont a i n UV-absorbing amino a c i d r e s i d u e s o f t y r o s i n e , t r y p t o p h a n o r p h e n y l a l a n i n e . I n most s e p a r a t i o n s o f p e p t i d e s on c a r r i e r s , d e t e c t i o n i s performed o n l y a f t e r t h e t e r m i n a t i o n o f t h e e l e c t r o m i g r a t i o n process. F o r a paper c a r r i e r , which was

TABLE 8.7

DETECTION OF OLIGOPEPTIDES AND POLYPEPTIDES IN ELECTROMIGRATION SYSTEMS If not otherwise defined, sensitivity is defined as the spot detection limit. Detection o f

Reagent

Sensitivity

Carrier

Ref. (selected)

Histidine, tyrosi ne

Pauly reagent, diazosulphanilic acid, a-nitroso-B-naphthol Isatin

5 pmole/mm2

90, 132

1 ng/mm2

Paper PAG Sephadex (TLE) Paper

Phosphorescence at low temperature (1 iquid N2) after short-wave UV light excitation Chlorination, KI + starch, o-tolidine, benzidine, etc. p-Dimethylaminobenzaldehyde, Ehrl ich reagent Silver reagent

Medi um

PAG

125

0.1 ug

Paper

118

Paper

132

Ver high, 10- pmol e/mm2

PAG

122, 123, 126

Ni n hydri n

1-2 mg, 0.12-0.02 mg 5 pmol e/mm2

Paper, cell ul ose, silica gel Sil ica gel, cellulose, paper, PAG Paper, cellulose PAG

22

Pro1 ine oxyproline Tyrosine tryptophan Peptide bond Tryptophan Universal Universal, NH2-group Universal, NH2-group Argi n i ne Universal

Fluorescamine (long-wave

f

UY light excitation)

Sakaguchi reagent Staining by Coomassie Brill iant B1 ue R-250, 6-250

Medi um 1-2 pmol e/mm2

131

4, 43 127, 128, 133 52, 87, 121, 129, 130

CL

0

w

104 used most o f t e n f o r more t h a n a q u a r t e r o f t h e c e n t u r y and which i s s t i l l used i n i n s t a n c e s where s u f f i c i e n t amounts o f p e p t i d e m a t e r i a l a r e a v a i l a b l e , a s e r i e s of u n i v e r s a l o r s p e c i f i c reagents116 can be used. N i n h y d r i n i s t h e most p o p u l a r

reagent, b u t d e t e c t i o n by c h l o r i n a t i o n o f t h e p e p t i d e bond118 i s a l s o common. I n a d d i t i o n t o more s e n s i t i v e methods, such as u n i v e r s a l l y u t i l i z a b l e i s o t o p i c l a b e l l i n g , r e a c t i o n s a r e a l s o used i n which f l u o r o p h o r e s a r e i n t r o d u c e d i n t o t h e mole c u l e . I n cases when quenching does n o t t a k e p l a c e t h e d e t e c t i o n s e n s i t i v i t y can be i n c r e a s e d by two o r t h r e e o r d e r s o f magnitude i n comparison w i t h n i n h y d r i n de120 t e c t i o n . These methods i n c l u d e d e t e c t i o n w i t h f l u o r e ~ c a m i n eo -~p~h t~h a l a l d e h y d e and methoxydiphenylfuranone62. I n a l l o f t h e s e cases, e x c i t a t i o n by UV r a d i a t i o n e l i c i t s f l u o r e s c e n c e i n t h e v i s i b l e p a r t o f t h e spectrum. The above-mentioned d e t e c t i o n methods a r e a l s o u t i l i z a b l e on c e l l u l o s e a c e t a t e f o i l s and on l o o s e l a y e r s i n TLE on c e l l u l o s e , s i l i c a g e l , e t c . L a r g e r p e p t i d e s separated i n p o l y a c r y l a m i d e g e l a r e most c o n v e n i e n t l y d e t e c t e d by s t a i n i n g w i t h Coomassie B r i l l i a n t 3 B l u e (Xylene B r i l l i a n t Cyanine G) i f t h e i r M.W. exceeds 1.5-2.10 Their f i x a t i o n

.

and d e t e c t i o n may b e s t be performed by t h e method o f B l a k e s l e y and Boezi121, where f i x a t i o n t a k e s p l a c e d i r e c t l y i n t h e s t a i n i n g s o l u t i o n i n t h e presence o f s u l p h u r i c a c i d . P e p t i d e s t h e b a s i c groups o f which a r e b l o c k e d o r which do n o t c o n t a i n bas i c groups a r e more d i f f i c u l t t o s t a i n w i t h Coomassie B r i l l i a n t Blue. I n such a case s p e c i f i c d e t e c t i o n may h e l p , as mentioned i n t h e s e c t i o n " I s o e l e c t r i c focusi n g " and i n Table 8.7.

P r o m i s i n g b u t s t i l l l i t t l e used i s t h e " s i l v e r method" o f

d e t e c t i o n a c c o r d i n g t o S w i t z e r e t a1 .123,

t h e s e n s i t i v i t y o f which i s comparable 123

t o t h a t o f a u t o r a d i o g r a p h i c d e t e c t i o n and w h i c h exceeds, a c c o r d i n g t o A l l e n

,

t h e s e n s i t i v i t y o f s t a i n i n g w i t h Coomassie B l u e a b o u t 2 0 0 - f o l d . The s i l v e r method makes use o f t h e f i x a t i o n o f p e p t i d e s w i t h paraldehyde. F i x a t i o n w i t h formaldehyde a l s o proved e f f e c t i v e

i n t h e d e t e c t i o n o f l o w M.W.

B l u e a c c o r d i n g t o Steck e t a1.124,

p e p t i d e s by means o f Coomassie

b u t i t cannot be used f o r d e t e c t i o n a f t e r I F

i n t h e presence o f c a r r i e r ampholytes. An a l t e r n a t i v e f o r t h e d e t e c t i o n o f nond e s t r u c t i v e d e t e c t i o n o f l a r g e r p o l y p e p t i d e s i n p o l y a c r y l a m i d e g e l i s based on t h e phosphorescence o f p o l y p e p t i d e s a t v e r y l o w temperatures, f o l l o w i n g e x c i t a t i o n 125 by UV l i g h t a t l i q u i d n i t r o g e n temperature, a c c o r d i n g t o Mardian and I s e n b e r g

.

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Chapter 9 GEL ELECTROPHORESIS AND ELECTROFOCUSING OF PROTEINS* EDITED BY ANDREAS CHRAMBACH CONTENTS 9.1. 9.2. 9.3. 9.4. 9.5. 9.6. 9.7. 9.8. 9.9. 9.10. 9.11. 9.12. 9.13. 9.14. 9.15. 9.16.

Usefulness o f second-generation g e l e l e c t r o p h o r e t i c t o o l s i n p r o t e i n f r a c t i o n a t i o n (A Chrambach) Membrane p r o t e i n s . n a t i v e (L.M. H j e l m e l a n d ) Membrane p r o t e i n s . denatured (H Baumann and D D o y l e ) P r o t e i n membrane r e c e o t o r s (U Lang) S t e r o i d r e c e p t o r s ( S Ben-Or) C e l l s u r f a c e a n t i g e n s (R.A. R e i s f e l d and M.A. P e l l e g r i n o ) Lysosomal g l y c o s i d a s e s and s u l p h a t a s e s (A.L. F l u h a r t y ) Haemocyanins (M B r e n o w i t z . J Bonaventura and C Bonaventura) Human haemoglobins (A.B. Schneider and A.N. S c h e c h t e r ) I s o e l e c t r i c f o c u s i n g of immunoglobulins (M.H. Freedman) C o n t r a c t i l e and c y t o s k e l e t a l p r o t e i n s ( P Rubenstein) P r o t e i n s o f c o n n e c t i v e t i s s u e ( Z Deyl and M Horakova) M i c r o t u b u l a r p r o t e i n s (K.F. S u l l i v a n and L W i l s o n ) P r o t e i n hormones (A.D. Rogol) E l e c t r o p h o r e s i s o f y p l a s m a p r o t e i n s : a contemporary c l i n i c a l approach (M E n g l i s ) A l l e r g e n s (H Baer and M.C. Anderson)

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. . . . . . . . . . . . . . . . . 110 . . . . . . . . . . . . . . 117 . . . . . . . . . . 120 . . . . . . . . . . . . . . . . . . 125 . . . . . . . . . . . . . . . . . . . . . 129 . . . . . . 141 . . . . . . . . 149 . . . . . . 156 . . . . . . . . 161 . . . . . . . 166 . . . . . . . . . 172 . . . . . . . . . 177 . . . . . . . . . . 185 . . . . . . . . . . . . . . . . . . . . 194 . . . . . . . . . . . . . . . . . . . . . . . . . 201 . . . . . . . . . . . . . . . . 213

*Because o f t h e c o m p l e x i t y o f t h i s c h a p t e r . r e f e r e n c e s a r e a t t a c h e d t o each section

.

110

USEFULNESS OF SECOND-GENERATION GEL ELECTROPHORETIC TOOLS I N PROTEIN FRACTIONATION ANDREAS CHRAMBACH GENERAL ASPECTS Experts i n some r e p r e s e n t a t i v e f i e l d s o f p r o t e i n b i o c h e m i s t r y have summarized i n t h i s c h a p t e r t h e use made o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and e l e c t r o f o c u s i n g i n t h e i r r e s p e c t i v e f i e l d s . T h i s use, on t h e a n a l y t i c a l s c a l e , r e f l e c t s t h e g r e a t advance t h a t p r o t e i n f r a c t i o n a t i o n made when i t became p o s s i b l e , f i r s t on s t a r c h l Y 2 , t h e n on p ~ l y a c r y l a m i d e ~ t' ~o , s e p a r a t e serum n o t i n t o 5 o r 6, b u t i n t o 30 s t a i n a b l e p r o t e i n components. T h i s b r e a k t h r o u g h two decades ago was due,

o f course, t o t h e p o s s i b i l i t y w i t h t h e s e two media t o v a r y t h e e f f e c t i v e p o r e s i z e c o n t i n u o u s l y by t h e c h o i c e o f g e l c o n c e n t r a t i o n . P o l y a c r y l a m i d e soon d i s p l a c e d s t a r c h i n t h a t a p p l i c a t i o n , as i t a l l o w e d f o r a w i d e r range o f g e l c o n c e n t r a t i o n s and was more m e c h a n i c a l l y s t a b l e . The o r i g i n a l t e c h n i q u e o f p r o t e i n s e p a r a t i o n employed s i n g l e g e l c o n c e n t r a t i o n s , and u s u a l l y an e i t h e r s t r o n g l y b a s i c o r s t r o n g l y 3 a c i d i c pH R e s u l t s o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) were i n t e r p r e t e d

.

e i t h e r by c o u n t i n g t h e number o f p r o t e i n zones as a s t a t e o f homogeneity

-

-

a s i n g l e zone b e i n g i n t e r p r e t e d

o r by comparing r e l a t i v e m i g r a t i o n d i s t a n c e s o f t h e

zones, e i t h e r v i s u a l l y o r d e n s i t o m e t r i c a l l y . Much o f t h e p r e s e n t p r a c t i c e o f p r o t e i n f r a c t i o n a t i o n s t i l l f o l l o w s t h i s o r i g i n a l p a t t e r n , a l t h o u g h i n appearance i t f r e q u e n t l y d i f f e r s f r o m t h e " d i s c e l e c t r o p h o r e s i s " o f two decades ago b y t h e a d d i t i o n o f sodium dodecyl s u l p h a t e (SDS) and by t h e use o f g e l s l a b t e c h n i q u e s . I n t h e a p p l i c a t i o n o f PAGE t o w a t e r - i n s o l u b l e p r o t e i n s such as membrane p r o t e i n s , a d d i t i o n o f a d e t e r g e n t i s o f c o u r s e r e q u i r e d , a l t h o u g h one may w i s h t o a p p l y a non-denaturing d e t e r g e n t

5 r a t h e r t h a n SDS f o r t h i s purpose. However, SOS i s a l s o

f r e q u e n t l y used f o r s o l u b l e p r o t e i n s were i t o b l i t e r a t e s n e t charge d i f f e r e n c e s and c o n f o r m a t i o n a l d i f f e r e n c e s , so d i m i n i s h i n g t h e r e s o l v i n g c a p a c i t y o f t h e method. The SDS-PAGE method o f p r o t e i n s e p a r a t i o n g i v e s t h e appearance o f b e i n g used as a p h y s i c a l method, because m i g r a t i o n d i s t a n c e s a r e expressed ( e r r o n e o u s l y ) i n mass u n i t s ( d a l t o n s ) , and because l i n e a r p l o t s , such as log(MW) v e r s u s RF, a r e used t o t r a n s l a t e m i g r a t i o n d i s t a n c e s i n t o MW ( d a l t o n s ) . However, t h e s e t r a n s l a t i o n s a r e f r e q u e n t l y i n a c c u r a t e , as t h e p l o t s a r e s i g m o i d a l and, more i m p o r t a n t , i n a p p l i c a b l e t o g l y c o - o r l i p o p r o t e i n s which, when s a t u r a t e d w i t h SDS, do n o t

111 share t h e charge d e n s i t y o f u n c o n j u g a t e d p r o t e i n s . M o l e c u l a r i d e n t i f i c a t i o n by m i g r a t i o n d i s t a n c e a t a s i n g l e g e l c o n c e n t r a t i o n remains t h e predominant p r a c t i c e . T h i s " f i r s t - g e n e r a t i o n PAGE" remains r e l a t i v e l y low i n e f f i c i e n c y as i t a p p l i e s t o s e p a r a t i o n s w i t h non-optimal g e l c o n c e n t r a t i o n s , non-optimal pHs and non-optimal s o l v e n t s . I t a l s o f a i l s t o e x p l o i t m i g r a t i o n d i s t a n c e s p h y s i c o - c h e m i c a l l y as l e g i t i m a t e measures o f t h e s i z e (geometry, n o t mass) and n e t charge o f a p r o t e i n . I t f a i l s t o assess t h e s t a t i s t i c a l s i g n i f i c a n c e o f PAGE d a t a , o r t o u t i l i z e t h e

o b j e c t i v e guidance towards an o p t i m a l s e p a r a t i o n method ( f r a c t i o n a t i o n p a t h ) which can be deduced f r o m t h o s e d a t a . "Second-generation PAGE" p r o v i d i n g o p t i m i z a t i o n , s t a t i s t i c a l t r e a t m e n t o f d a t a and an o b j e c t i v e b a s i s f o r a f r a c t i o n a t i o n s t r a t e g y , has been developed6-8 b u t has r a r e l y been a p p l i e d t o p r o t e i n s e p a r a t i o n s . A l s o , p r e p a r a t i v e PAGE has h a r d l y been used i n b i o c h e m i c a l s e p a r a t i o n s a1 though many s u i t a b l e t e c h n i q u e s and types of apparatus have e v o l v e d9

.

F a i l u r e of most o f t h e s e p r e p a r a t i v e methods i s due t o t h e absence, t o d a t e , o f p r a c t i c a l means o f o p t i m i z i n g r e s o l u t i o n 8 - 1 0 and t o t h e r e l a t i v e p r o c e d u r a l c o m p l e x i t y o f " e l u t i o n PAGEto7. T h e r e f o r e , s l i c i n g and e x t r a c t i o n o f g e l s i d e n t i c a l w i t h t h e a n a l y t i c a l g e l s e x c e p t f o r an i n c r e a s e i n g e l t h i c k n e s s appears a t p r e s e n t t o be t h e b e s t means o f p r e p a r a t i v e PAGE a v a i l a b l e f o r b i o c h e m i c a l p r a c 11,12

tice

I n t h e f o l l o w i n g s e c t i o n s we s h a l l c o n s i d e r t h e f i v e m a j o r aspects o f "secondg e n e r a t i o n PAGE" which p r o v i d e advantages i n s e p a r a t i o n e f f i c i e n c y o v e r o t h e r methods commonly used a t p r e s e n t and which have developed t h e h i g h degree o f o p e r a t i o n a l s i m p l i c i t y r e q u i r e d o f any p r a c t i c a l method. OPTIMAL pH AND SOLVENT Gel e l e c t r o p h o r e s i s of w a t e r - s o l u b l e p r o t e i n s i s c o n v e n t i o n a l l y c a r r i e d o u t a t an i o n i c s t r e n g t h of a p p r o x i m a t e l y 0 . 0 1 m o l / l and pH v a r y i n g f r o m 2.5 t o 11.0. B u f f e r systems o p e r a t i v e under these c o n d i t i o n s and s i m u l t a n e o u s l y capable o f p r o t e i n concentration p r i o r t o electrophoresis t o provide a concentrated s t a r t i n g zone a r e a v a i l a b l e i n m i c r o f i c h e form13. The r e l e v a n t computer o u t p u t , p r i n t e d

on m i c r o f i c h e , which i s based on complex e l e c t r o p h o r e t i c theory14, p r o v i d e s b u f f e r r e c i p e s and t h e o p e r a t i o n a l p r o p e r t i e s o f t h e b u f f e r systems a t v a r i o u s pH i n an e x p l i c i t and a c c e s s i b l e , s i m p l e

w i t h o u t need f o r c a l c u l a t i o n s

o r computations. Gel e l e c t r o p h o r e s i s a t l o w i o n i c s t r e n g t h ( 0 . 0 1 m o l / l ) i s c o m p a t i b l e w i t h t h e a c t i v i t i e s ( t h e " n a t i v e s t a t e " ) o f many p r o t e i n s . High c o n c e n t r a t i o n s (10-50%) o f g l y c e r o l , e t h y l e n e g l y c o l o r sucrose can be used t o s t a b i l i z e n a t i v e c o n f i g u r a t i o n s . I n o t h e r i n s t a n c e s , however, p r o t e i n s i n t e r a c t w i t h themselves ( a g g r e g a t e ) o r w i t h o t h e r p r o t e i n s o r l i g a n d s v i a e i t h e r h y d r o p h i l i c or hydrophobic bonds

112 under t h o s e c o n d i t i o n s . I f t h e i n t e r a c t i o n s a r e hydrophobic, a d d i t i o n o f d e t e r g e n t s t o t h e b u f f e r s i s i n d i c a t e d . D e t e r g e n t s c o m p a t i b l e w i t h a t l e a s t some o f t h e act i v i t y o f t h e p r o t e i n a r e a v a i l a b l e 5'16-18.

I f the interactions are hydrophilic,

g e l e l e c t r o p h o r e s i s may be conducted a t h i g h i o n i c s t r e n g t h (0.1-0.6

mol/l). This

may be i m p r a c t i c a b l y slow and t h e r e f o r e i n c o m p a t i b l e w i t h a c t i v i t y maintenance, as t h e l i m i t e d e f f i c i e n c y o f d i s s i p a t i o n o f t h e J o u l e h e a t o f t h e g e l e l e c t r o p h o r e s i s apparatus p r e c l u d e s o p e r a t i o n a t i n c r e a s e d c u r r e n t l e v e l s . I f t h e i n t e r a c t i o n s i n v o l v e hydrogen bonds, b u f f e r s w i t h h i g h c o n c e n t r a t i o n s o f u r e a (4-8 m o l / l ) a r e s u i t a b l e d i s s o c i a t i n g media. W a t e r - i n s o l u b l e p r o t e i n s can be separated by PAGE i n d e t e r g e n t - c o n t a i n i n g b u f f e r s 5y16-18.

I f p o s s i b l e w i t h maintenance o f a c t i v i t y , o r a t l e a s t o f charge d i f -

ferences ( n o n - i o n i c and z w i t t e r i o n i c d e t e r g e n t s ) . I f t h e p r o t e i n c a n n o t b e s o l u b i l i z e d o t h e r w i s e , SDS o r t h e c o r r e s p o n d i n g q u a t e r n a r y ammonium a l k y l compounds a r e r e q u i r e d . These r e a c t w i t h a l l p r o t e i n s , y i e l d i n g a u n i f o r m n e t s u r f a c e charge, l o s s o f n a t i v e c o n f o r m a t i o n ( a c t i v i t y ) and s e p a r a t i o n between n o n - c o v a l e n t l y bound p r o t e i n subunits. OPTIMAL PORE S I Z E Two p r o t e i n s d i f f e r i n g i n m o l e c u l a r s i z e a r e o p t i m a l l y s e p a r a t e d a t a g e l 2 x 11, where l.~ d e s i g n a t e s t h e m o b i l i t y ( o r r e l a t i v e m o b i l i t y )

concentration o f l / e

c o r r e s p o n d i n g t o t h e i n t e r s e c t i o n between t h e Ferguson p l o t s u l o g RF)/%T] f o r t h e two p r o t e i n s . Thus, t o f i n d t h e o p t i m a l l y r e s o l v i n g p o r e s i z e f o r t h e separat i o n between any two p r o t e i n s , one needs(a) g e l e l e c t r o p h o r e s i s a t s e v e r a l g e l c o n c e n t r a t i o n s (%T),w i t h r e l a t i v e m o b i l i t y ( R ~ measurement ) i n each, and ( b ) a calculation. ( a ) To make RE' measurements r e p r o d u c i b l e , one needs r e p r o d u c i b l e g e l p o r e s i z e s . These can be o b t a i n e d w i t h m i n i m a l e f f o r t t h r o u g h c o n t r o l o f t h e c o n d i t i o n s o f t h e p o l y m e r i z a t i o n r e a c t i o n f o r m i n g c r o s s - l i n k e d p ~ l y a c r y l a m i d e ~e, s p e c i a l l y t h e temperature o f p o l y m e r i z a t i o n and t h e i n i t i a t o r and i n h i b i t o r (atmospheric oxygen) l e v e l s 7 . E l e c t r o p h o r e s i s must e q u a l l y b e r i g o r o u s l y t e m p e r a t u r e c o n t r o l l e d , w i t h c a r e t o p r e v e n t J o u l e h e a t i n g i n t h e i n t e r i o r o f t h e g e l f r o m exceeding t h e h e a t d i s s i p a t i o n c h a r a c t e r i s t i c s o f t h e a p p a r a t u s . Use o f a moving boundary as a " f r o n t " , as p r o v i d e d by use o f m u l t i p h a s i c b u f f e r systems8, f u r t h e r i n c r e a s e s t h e accuracy o f RF measurement i n view of t h e extreme sharpness o f t h a t zone, independent o f m i g r a t i o n d i s t a n c e . ( b ) To p r e v e n t e r r o r s and save time, c a l c u l a t i o n o f t h e o p t i m a l l y r e s o l v i n g g e l c o n c e n t r a t i o n s h o u l d employ a s e t o f computer programs, t h e "PAGE-PACK" o f Rodbard".

A l l t h a t i s r e q u i r e d i s t o t y p e t h e RF

values i n t o a remote t e r m i n a l , f o l l o w e d b y a few s i m p l e commands p r o v i d e d by t h e i n s t r u c t i o n s 15'19. The r e s u l t i n g s l o p e ( r e t a r d a t i o n c o e f f i c i e n t , K R ) and y i n t e r -

113 c e p t ( Y o ) o f t h e Ferguson p l o t s f o r t h e two p r o t e i n s t o be separated a r e then entered i n analogous fashion. Once t h i s has been done f o r each b u f f e r system (pH), t h e same i s done f o r molecular weight (MW) standards t o e s t a b l i s h t h e r a t i o between KR and molecular r a d i u s ( o r w e i g h t ) . W i t h i n a few minutes t h e d e s i r e d o p t i m a l l y r e s o l v i n g g e l c e n c e n t r a t i o n i s computed on t h e b a s i s o f t h e KR and

xQ

values and molecular r a d i i ( o r w e i g h t s ) . STATISTICALLY DEFINED MOLECULAR WEIGHT AND NET CHARGE Molecular i d e n t i f i c a t i o n by i n s p e c t i o n , and even by RF measurement o f bands on an electropherogram, i s u s u a l l y n o t p o s s i b l e over an extended p e r i o d o f f r a c t i o n a t i o n experiments o r f o r separations f r o m d i f f e r e n t l a b o r a t o r i e s , unless one knows t h e s t a t i s t i c a l d i s t r i b u t i o n o f band p o s i t i o n s or RF values. More important, bands on an electropherogram f a i l t o g i v e an i n s i g h t i n t o t h e n a t u r e o f t h e r e l a t i o n s h i p between those bands. This problem i s a l s o overcome by use o f t h e PAGE-PACK through computation o f t h e j o i n t (i.e.,

c o r r e l a t e d ) confidence l i m i t s

o f KR (molecular s i z e ) and Y, (molecular n e t charge). These e l l i p s o i d a l "confidence envelopes" f o r t h e v a r i o u s zones i n a gel electropherogram a l i g n i n a semi-logar i t h m i c p l o t o f yo versus K R , e i t h e r v e r t i c a l l y a l o n g t h e y-axis,

indicating

molecules o f t h e same s i z e v a r y i n g preponderantly i n n e t charge and thus presumably d e r i v e d from g a i n o r l o s s o f charged groups from t h e same molecule d u r i n g t h e h i s t o r y o f the preparation, o r h o r i z o n t a l l y along the z-axis,

i n d i c a t i n g several ag-

g r e g a t i o n s t a t e s of t h e same molecule, o r t h e y d i s t r i b u t e on t h e p l o t a t random, suggesting r e a l molecular heterogeneity. Such molecular c h a r a c t e r i z a t i o n d e r i v e s d i r e c t l y from t h e experimental Ferguson p l o t s and i s n o t s u b j e c t t o e r r o r s due t o erroneous assumptions. I n a d d i t i o n , t h e PAGE-PACK p r o v i d e s a t r a n s l a t i o n o f KR i n t o conventional s i z e parameters, molecular r a d i u s and MW, and o f Yo i n t o conventional n e t charge parameters, f r e e e l e c t r o p h o r e t i c m o b i l i t y and molecular valence ( n e t p r o t o n s p e r molecule). These t r a n s l a t i o n s , however, depend on e i t h e r a g l o b u l a r o r a randomc o i l model f o r unknown p r o t e i n and MW standards, and on n e c e s s a r i l y o v e r s i m p l i f y i n g assumptions w i t h regard t o h y d r a t i o n , p a r t i a l s p e c i f i c volume, e t c . Again, t h e computation o f KR-Yo e l l i p s e s and t r a n s l a t i o n s i n t o conventional s i z e and n e t charge parameters i s e a s i l y and r a p i d l y c a r r i e d o u t w i t h o u t e r r o r a t t h e remote t e r m i n a l i n t h e l a b o r a t o r y , immediately f o l l o w i n g t h e RF measurements. OBJECTIVELY DEFINED SEPARATION STRATEGY

A t present, t h e choice o f gel e l e c t r o p h o r e s i s versus g e l e l e c t r o f o c u s i n g i s s t i l l f r e q u e n t l y a r b i t r a r y . The Ferguson p l o t s computed by means o f t h e PAGE-

114

PACK allow one to avoid such arbitrariness. If the two Ferguson plots intersect but are at a maximal distance from another at 0 %T, or if they are parallel, electrofocusing or isotachophoresis is objectively indicated as the most effective separation method. In contrast, if the intersecting Ferguson plots are at a maximal distance from another at a finite %T value, or if they intersect at 0 %T, gel electrophoresis is the separation method of choice (see Fig. 19 of ref. 15). PREPARATIVE PAGE The most important function of a separation method such as gel electrophoresis or electrofocusing is preparative, providing macromolecules in sufficiently homogeneous form to allow for their organic- or physico-chemical analysis. However, this has been the most neglected and unsuccessful aspect of these methods. An objective definition of the conditions of preparative elution PAGE by computer methods does not yet existgy10. At present, the safest and simplest mode of protein recovery from PAGE or electrofocusing gels appears to be the electrophoretic extraction and simultaneous concentration of the protein from gel slices, followed by a gel filtration step (to exchange the buffer for a volatile one and to remove non-proteinaceous impurities) and lyophil ization1"12. The preparative gel electrophoretic separation at the optimally resolving pore size (see above) employs the analytical technique15, using the analytical apparatus except for an upper buffer reservoir with grommets for 18 mm I . D . 20 mm O.D. tubes. The load capacity of each of these preparative gels is ten times larger than that of analytical-scale 6 mm diameter gels. With 6-8 such gels per apparatus, milligram amounts of protein derived from a single zone can be prepared in one run1*. Representative overall yields are 70%. The product purity is 90% if the isotachophoretic extraction and concentration step has been carried out on an agarose gel (which produces far less impurities than polyacrylamide). However, as the zones, despite their sharp appearance on staining, have relatively broad distributions on the gel, homogeneous protein can usually be gained only by pooling of the ascending, central and descending parts of the distribution from several experiments, and re-electrophoresis of these fractions, with a necessary decrease in total yield. GEL ELECTROFOCUSING First-generation gel electrofocusing, even more than PAGE, has been a rigid "cookbook" method. The failure to adapt and optimize the pH gradient, together with an uncritical acceptance of any band as being isoelectrically resolved, has led to low resolving efficiencies and large errors. Second-generation electrofocusing overcomes this basic rigidity2'. Adaptation o f the pH gradient to the

115 s e p a r a t i o n problem i s made p o s s i b l e t h r o u g h a f r e e c h o i c e o f c a r r i e r ampholytes, i n c l u d i n g a l l o f t h e a v a i l a b l e amphoteric o r even non-amphoteric b u f f e r s , and t h r o u g h s e l e c t i v e d i s p l a c e m e n t of c a r r i e r ampholytes f r o m t h e g e l by c h o i c e o f a n o l y t e l c a t h o l y t e a t t h e pH v a l u e s c i r c u m s c r i b i n g t h e d e s i r e d pH g r a d i e n t 2 1 . To ensure f u l l r e s o l u t i o n and avoidance o f t r a n s i e n t zones, t h e p r o t e i n p a t t e r n i s f o l l o w e d as a f u n c t i o n o f e l e c t r o f o c u s i n g t i m e u n t i l q u a l i t a t i v e p a t t e r n constanc y o f pH f o r each o f t h e zones has been a t t a i n e d ( F i g . 7 i n r e f . 2 0 ) . To be a b l e t o do t h i s , a c o n s i d e r a b l e degree o f pH g r a d i e n t s t a b i l i t y i s r e q u i r e d . T h i s can be secured t h r o u g h t h e use o f t h e t e r m i n a l a n o d i c / c a t h o d i c c a r r i e r ampholytes o f t h e pH g r a d i e n t as a n o l y t e / c a t h o l y t e 2 ' ,

through admixture o f conducting species

(presumably a t t h e t r a n s i e n t s t a t e ) such as 0.01 m o l / l amino a c i d s 2 3 o r b u f f e r c a r r i e r a m p h o l y t e ~ ~and ~ , t h r o u g h f i r m adherence o f t h e g e l t o t h e a p p a r a t u s w a l l s b y c o a t i n g t h e w a l l s w i t h l i n e a r p o l y a c r y l a m i d e , agarose o r y - m e t h a c r y l oxypropyl t r i m e t h o x y s i l ane and s u p p o r t o f t h e t u b e w i t h n y l o n mesh25726. Preparat i v e g e l e l e c t r o f o c u s i n g can be c a r r i e d o u t by t h e same procedures as p r e p a r a t i v e PAGE (see above), e x c e p t t h a t c a r e must be t a k e n t o s l i c e t h e g e l as soon as t h e i s o e l e c t r i c pH i s b e i n g approached ( e a r l i e r r a t h e r t h a n l a t e r ) , and i f necessary t o r e - s o l u b i l i z e t h e i s o e l e c t r i c p r o t e i n p r e c i p i t a t e by exposure t o an extreme 27 pH v a l u e f o r a s h o r t t i m e p r i o r t o i s o t a c h o p h o r e t i c e x t r a c t i o n

.

SUMMARY Second-generation methods i n g e l e l e c t r o p h o r e s i s and e l e c t r o f o c u s i n g a r e now a v a i l a b l e t h a t promise t o enhance t h e u s e f u l n e s s and r e s o l v i n g power o f c u r r e n t a n a l y t i c a l and p r e p a r a t i v e p r o t e i n s e p a r a t i o n s . REFERENCES

1 2 3 4 5

0. S m i t h i e s , Biochern. J . , 6 1 (1955) 629. 0. S m i t h i e s , Advm. Protein Chem., 14 (1959) 65. L. O r n s t e i n , Ann. Iv. Y . Acad. S c i . , 121 (1964) 321. S. Raymond and M. Nakamichi, AnaZ. Biochern., 7 (1961) 225. L.M. Hjelmeland, D.W. Nebert and A. Chrambach, i n N. Catsimpoolas ( E d i t o r ) , EZectrophoresis '78, E l s e v i e r / N o r t h H o l l a n d , Amsterdam, New York, 1978, p. 29. 6 A. Chrambach and D. Rodbard, Science, 172 (1971) 440. 7 A. Chrambach, T.M. Jovin, P.J. Svendsen and D. Rodbard, i n N. Catsimpoolas ( E d i t o r ) . Methods of protein Serxrution. V o l . 2, Plenum Press, New York, 1976, Ch, 2, p. 27. 8 A. Chrambach, MoZ. CeZZ. Biochern., 29 (1980) 23. 9 A. Chrambach and N.Y. Nauven. i n P.G. R i a h e t t i , C.J. Van Oss and J.W. Vanderhoff ( E d i t o r s ) , EZectrokinetFc-Separation Metiods, E l s e v i e r , Amsterdam, 1978, p. 337. 10 D. Rodbard, A. Chrambach and G.H. Weiss, i n R.C. A l l e n and H.R. Maurer ( E d i t o r s ) , Electrophoresis and IsoeZectric Focusing on PoZyacrylamide GeZ. W. de G r u y t e r , B e r l i n , 1974, p. 62. 11 N.Y. Nguyen and A. Chrambach, J . Biochern. Biophys. Methods, 1 (1979) 171. I

-

116 12 N.Y.

Nguyen, G. Baumann, D.E.

Arbegast, R. G r i n d e l a n d and A. Chrambach, Prep.

Biochem., 11 (1980) 139. 13 T.M. J o v i n , M.L. Dante and A. Chrambach, Multiphasic E u f f e r Systems Cutput, N a t i o n a l T e c h n i c a l I n f o r m a t i o n Service, S p r i n g f i e l d , VA, 1970, P u b l i c Board Numbers 196090, 259309-259312. 14 T.M. J o v i n , Biochemistry, 12 (1973) 871, 879, 890. 15 A. Chrambach and D. Rodbard, i n B.D. Hames and D. Rickwood ( E d i t o r s ) , Gel Electrophoresis: A Practical Approach, I n f o r m a t i o n R e t r i e v a l L t d . , London, 1981, p. 93 ( a v a i l a b l e f r o m t h e a u t h o r on r e q u e s t ) . 16 A.C. Newby and A. Chrambach, Biochem. J., 177 (1978) 623. 17 A.C. Newby, M. Rodbell and A. Chrambach, Arch. Biochem. Biophys., 190 (1978) 109. 18 L.M. Hjelmeland, Proc. Nut. Acad. S c i . U.S.A., 77 (1980) 6368. 19 Biomedical Computing Technology I n f o r m a t i o n Center, Room-1302, V a n d e r b i l t Medical Center, N a s h v i l l e , TN, program i d e n t i f i c a t i o n number MED-34 PAGE-PACK. 20 A. Chrambach, L.M. Hjelmeland, N.Y. Nguyen and B. An d e r Lan, i n B.J. Radola ( E d i t o r ) , Electrophoresis ‘79, W. de G r u y t e r , B e r l i n , New York, 1980, p. 3. 21 N.Y. Nguyen and A. Chrambach, Electrgphoresis, 1 (1980) 14. 22 N.Y. Nauven and A. Chrambach. Anal. Biochem.. 79 (1977) 462. 23 N.Y. N&&en and A. Chrambach; Anal. Biochem.; 82 (1977) 226. 24 N.Y. Nguyen and A. Chrambach, Anal. Bicohem., 82 (1977) 54. 25 N.Y. Nguyen, A.G. McCormick and A. Chrambach, Anal. Biochem., 88 (1978) 186. 26 N.Y. Nguyen and A. Chrambach, i n B.D. Hames and D. Rickwood ( E d i o r s ) , Gel EZectrophoresis: A Practical Approach, I n f o r m a t i o n R e t r i e v a l L t d , London, 1981, p. 145 ( a v a i l a b l e from t h e a u t h o r on r e q u e s t ) . 27 N.Y. Nguyen, R. G r i n d e l a n d and A. Chrambach, Prep. Biochem., 11 1980) 173.

117

Chapter 9 . 2 MEMBRANE PROTEINS, NATIVE L.M. HJELMELAND Two c l a s s e s o f p r o t e i n s e x i s t which demonstrate abnormal s o l u b i l i t y p r o p e r t i e s i n aqueous media: t h o s e which r e q u i r e h i g h i o n i c s t r e n g t h f o r s o l u b i l i t y and t h o s e which a r e hydrophobic and t h u s r e q u i r e d e t e r g e n t s . The membrane p r o t e i n s , t h o s e which r e q u i r e d e t e r g e n t s f o r s o l u b i l i t y , w i l l be s p e c i f i c a l l y c o n s i d e r e d i n t h i s review. A l t h o u g h t h e s o l u b i l i z a t i o n and c h a r a c t e r i z a t i o n o f membrane p r o t e i n s have been e x t e n s i v e l y studied"*,

most r e f e r e n c e s t o p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s

(PAGE) and i s o e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e (IFPA) r e f e r t o t h e use o f d e n a t u r i n g c o n d i t i o n s . Perhaps t h e two most g e n e r a l methods a r e SDS-PAGE and IFPA i n T r i t o n X-100-9 m o l / l urea, and as b o t h methods a r e designed t o work w i t h den a t u r e d p r o t e i n s t h e y w i l l n o t be c o n s i d e r e d f u r t h e r here. More t h a n 90% o f t h e r e m a i n i n g r e f e r e n c e s deal w i t h PAGE and IFPA i n t h e presence o f n o n - i o n i c d e t e r g e n t s such as T r i t o n X-100 and L u b r o l PX3. U n f o r t u n a t e l y , n o t o n l y t h e use o f t h e s e d e t e r g e n t s b u t a l s o t h e i r s e l e c t i o n as u s e f u l d e t e r g e n t s a r e e f f e c t e d on an e x t r e m e l y ad hoc b a s i s . The fundamental p r o p e r t i e s o f d e t e r g e n t s and t h e i r use i n e l e c t r o p h o r e t i c systems has been r e v i e w e d e l s e 4 The use o f n o n - i o n i c d e t e r g e n t s such as T r i t o n and L u b r o l , w h i c h b e l o n g

where

.

t o t h e p o l y o x y e t h y l e n e t y p e , i s u n f o r t u n a t e l y f r a u g h t w i t h problems. The most i m p o r t a n t i s t h e n a t u r e o f t h e p r o t e i n - d e t e r g e n t complex a f t e r s o l u b i l i z a t i o n . N o n - i o n i c d e t e r g e n t s g e n e r a l l y g i v e v e r y l a r g e complexes (500,000 d a l t o n s o r g r e a t e r ) , i n which s e v e r a l s p e c i e s o f p r o t e i n a r e p r e s e n t t o g e t h e r w i t h v a r i a b l e amounts o f l i p i d . These d e t e r g e n t s a r e w e l l known t o be o n l y p a r t i a l l y e f f e c t i v e i n b r e a k i n g p r o t e i n - p r o t e i n i n t e r a c t i o n s 5 , and t h u s l e a v e a r t i f a c t u a l aggregates i n s o l u t i o n a f t e r d e t e r g e n t t r e a t m e n t o f membranes. These aggregates can c o n t a i n m u l t i p l e c o p i e s o f t h e same p r o t e i n , o r c o m p l e t e l y u n r e l a t e d p r o t e i n s which may have no b i o l o g i c a l r e l e v a n c e t o each o t h e r . I n t h e few i n s t a n c e s where aggregates o f p r o t e i n a r e n o t p r e s e n t , t h e s i z e o f complexes o f s i n g l e p r o t e i n s p e c i e s and non-ionic detergents i s u s u a l l y large, l e a d i n g t o special considerations i n elect r o p h o r e t i c experiments such as i n c r e a s e d p o r e s i z e by t h e use o f h i g h l y c r o s s l i n k e d g e l s 3 , and l o w e l e c t r o p h o r e t i c m o b i l i t i e s o f v e r y l a r g e d e t e r g e n t - p r o t e i n complexes. There a r e many i m p l i c a t i o n s o f t h e s e problems f o r b o t h PAGE and IFPA. The f i r s t i s u n c e r t a i n t y c o n c e r n i n g t h e meaning o f t h e " p r " o r t h e PAGE parameters

118 d e s c r i p t i v e o f m o l e c u l a r s i z e and n e t charge ( K R and Y o ) o f t h e s e complexes. Although most workers i n t h e f i e l d o f membrane p r o t e i n s measure t h e s e v a l u e s w i t h no concern f o r t h e above problems, i t i s c l e a r t h a t i n many i n s t a n c e s t h e s e measurements a r e i n f a c t meaningless. I n a d d i t i o n , i t i s now c l e a r t h a t many d i f f e r e n t t y p e s o f d e t e r g e n t s , polymers and i n o r g a n i c s a l t s a r e c a p a b l e o f b i n d i n g t o t h i s t y p e o f d e t e r g e n t , t h u s changing t h e i r s o l u t i o n p r o p e r t i e s . The b i n d i n g o f Ampholines, u r e a nd p o l y a c r y l i c a c i d a r e

The b i n d i n g o f charged

species t o membrane p r o t e i n s , which i n t u r n a l t e r t h e e l e c t r o p h o r e t i c m o b i l i t y o f t h e s e p r o t e i n s , i s i n f a c t t h e b a s i s o f t h e "charge s h i f t " t e c h n i q u e s which have r e c e n t l y been d e ~ c r i b e d ~ 'I ~n . t h e s e t e c h n i q u e s , t h e p o l a r i t y o f t h e d e t e r gent which i s complexed w i t h t h e p r o t e i n ( a n i o n i c o r c a t i o n i c ) determines t h e r e l a t i v e change i n t h e m o b i l i t y o f t h e p r o t e i n w i t h r e f e r e n c e t o t h e o p e r a t i n g pH. I n a d d i t i o n , i t i s o f t e n p o s s i b l e t o a l t e r t h e m o l e c u l a r w e i g h t o f t h e det e r g e n t - p r o t e i n complex by u s i n g m i x t u r e s o f d e t e r g e n t s . I n an e l e g a n t s t u d y o f a d e n y l a t e cyclase",

t h i s p r i n c i p l e was demonstrated by m i x i n g i o n i c d e t e r g e n t s

w i t h L u b r o l PX. Not o n l y was t h e m o b i l i t y o f t h e p r o t e i n i n c r e a s e d as expected, b u t a l s o t h e e f f e c t i v e s i z e o f t h e s o l u b i l i z e d s p e c i e s was h a l v e d . These s t u d i e s a l s o p o i n t o u t t h e u t i l i t y o f t h e b i l e s a l t anions, which, a l though e x t e n s i v e l y used i n s o l u b i l i z a t i o n , a r e n o t r o u t i n e l y used i n PAGE o r IFPA. These d e t e r g e n t s , c h o l a t e , deoxycholate and t h e i r t a u r i n e c o n j u g a t e s , a r e u s u a l l y n o n - d e n a t u r i n g l i k e t h e n o n - i o n i c s , b u t i n a d d i t i o n o f f e r s e v e r a l advantages o v e r t h e p o l y e t h o x y l a t e - t y p e d e t e r g e n t s . The most i m p o r t a n t i s t h a t t h e s e compounds a r e much more e f f e c t i v e a t d i s a g g r e g a t i n g t h e l a r g e complexes t h a n noni o n i c d e t e r g e n t s . T h i s i s undoubtedly due n o t o n l y t o t h e s m a l l m i c e l l a r s i z e 4 o f t h e s e d e t e r g e n t s b u t a l s o t o fundamental d i f f e r e n c e s i n t h e s t r u c t u r e o f t h e hydrophobic segments which a r e r i g i d a l i c y c l i c m o i e t i e s . The obvious d i s a d v a n t a g e o f t h e s e d e t e r g e n t s i s t h a t t h e y a r e i n c a p a b l e o f p r e s e r v i n g t h e n a t i v e n e t charge o f p r o t e i n s , a f e a t u r e r e q u i r e d f o r IFPA.

A general s o l u t i o n t o t h e problem o f n o n - d e n a t u r i n g d e t e r g e n t s which p r e s e r v e t h e n e t charge o f membrane p r o t e i n s and a r e t h u s u s e f u l f o r IFPA was r e c e n t l y proposed1lYl2. These systems employed N-a1 k y l s u l p h o b e t a i n e s , which behave e l e c t r i c a l l y l i k e n o n - i o n i c d e t e r g e n t s b u t have t h e advantage o f b e i n g s u p e r i o r p r o t e i n s o l u b i l i z e r s and d i s a g g r e g a t i n g agents. I n a d d i t i o n , t h e s e compounds do n o t form many o f t h e u n d e s i r a b l e complexes seen w i t h t h e n o n - i o n i c p o l y e t h o x y l a t e s d e s c r i b e d above. U n f o r t u n a t e l y , t h e c l a i m s t h a t t h e s e compounds a r e n o n - d e n a t u r i n g have been s u b s t a n t i a t e d i n o n l y a few o f t h e i r a p p l i c a t i o n s 9,13-15. I n r e t r o s p e c t , t h i s b e h a v i o u r o f N - a l k y l d e t e r g e n t s was p r e d i c t a b l e , as t h e f l e x i b i l i t y o f t h e i r hydrocarbon segments was t h o u g h t t o be r e s p o n s i b l e f o r t h e i r d e n a t u r i n g p r o p e r t i e s According t o t h i s reasoning, r i g i d hydrocarbon t a i l s w i t h s i m i l a r p o l a r groups would p r o v i d e n o n - d e n a t u r i n g d e t e r g e n t s w i t h t h e same e l e c t r i c a l p r o p e r t i e s o f t h e

16

.

119 s u l p h o b e t a i n e p o l a r group. T h i s i n f a c t appears t o be so. The s y n t h e s i s o f a s u l p h o b e t a i n e d e r i v a t i v e o f c h o l i c a c i d was r e c e n t l y r e p o r t e d , and t h e non-den a t u r i n g p r o p e r t i e s o f t h i s d e t e r g e n t have been w e l l documented17y18. T h i s det e r g e n t i s n o t o n l y n o n - d e n a t u r i n g and e l e c t r i c a l l y n e u t r a l , b u t a l s o appears t o be m a x i m a l l y e f f e c t i v e a t d i s a g g r e g a t i n g cytochrome P-450 and p o s s i b l y many o t h e r membrane p r o t e i n s now b e i n g s t u d i e d . The d e t e r g e n t a l l o w s one t o f r a c t i o n a t e n a t i v e cytochrome P-450 by IFPA

19 .

I n summary, i t now appears p o s s i b l e . t o p e r f o r m m e a n i n g f u l PAGE and IFPA f r a c t i o n a t i o n s on membrane p r o t e i n s i n which t h e f u n c t i o n a l e n t i t i e s a r e s i n g l e c o p i e s o f p r o t e i n s i n p h y s i c o - c h e m i c a l l y d e f i n e d d e t e r g e n t media. REFERENCES

1 A. H e l e n i u s and K. Simons, Biochim. Biophys. Acta, 415 (1975) 29. 2 C. T a n f o r d and J.A. Reynolds, Biochirn. Biophys. Acta, 457 (1976) 133. 3 U. Lang, C.R. Kahn and A. Chrambach, Endocfinology, 106 (1980) 40. 4 L.M. Hjelmeland, D.W. N e b e r t and A. Chrambach, i n N. Catsimpoolas ( E d i t o r ) , Electrophoresis ' 7 8 , E l s e v i e r / N o r t h H o l l a n d , Amsterdam, New York, 1978, pp. 29-56. 5 A. Helenius, D.R. McCaslin, E. F r i e s and C . Tanford, Methods Enzymol., 56 (1978) 734. 6 E. Gianazza, C. A s t o r r i and P.G. R i g h e t t i , J . Chromatogr., 171 (1979) 161. 7 F.E. B a i l e y and J.V. Koleske, i n M.J. S c h i c k ( E d i t o r ) , Nonionic Surfactants, Marcel Dekker, New York, 1966, pp. 794-822. 8 A. H e l e n i u s and K. Simons, Proc. Nut. Acad. S c i . U.S., 74 (1977) 529. 9 T.C. Bog-Hansen, I. Lorenc-Kubis and O.J. Bjerrum, i n B.J. Radola ( E d i t o r ) , Electrophoresis '79, W. de G r u y t e r , B e r l i n , New York, 1980, pp. 173-192. 10 A.C. Newby and A. Chrambach, Biochem. J . , 177 (1978) 623. 11 J.C. A l l e n and C. Humphries, i n J.P. A r b u t h n o t t and J.A. Beeley ( E d i t o r s ) , I s o e l e c t r i c Formsing, B u t t e r w o r t h s , London, 1975, pp. 347-354. 12 A. Gonenne and R. E r n s t , A m Z . Biochem., 87 (1978) 28. 13 L.M. Hjelmeland, D.W. N e b e r t and A. Chrambach, Anal. Biochem., 95 (1979) 201. 14 F. M a l p a r t i d a and R . Serrano, FEBS L e t t . , 111 (1980) 69. 15 E.M. B a i l y e s , J.P. L u z i o and A.C. Newby, Transactions Biochemical Society, London, i n p r e s s .

16 C. Tanford, The Hydrophobic E f f e c t , John W i l e y , New York, 1973. 17 L.M. Hjelmeland, Proc. Nat. Acad. S c i . U.S., 77 (1980) 6368. 18 W.F. Simonds, G. Koski, R.A. S t r e a t y , L.M. H j e l m e l a n d and W.A. Klee, Proc. Nut. Acad. Sci. U.S., 77 (1980) 4623. 19 L.M. H j e l m e l a n d e t a l . , i n p r e p a r a t i o n .

120

Chapter 9.3 MEMBRANE PROTEINS, DENATURED

HEINZ BAUMANN and DARRELL DOYLE Because an e x c e l l e n t summary o f t h e t e c h n o l o g y o f t h e s o l u b i l i z a t i o n o f membrane p r o t e i n s and t h e i r s e p a r a t i o n and d e t e c t i o n i n v a r i o u s g e l systems has a l r e a d y been g i v e n by Maddy and D u n n l Y 2 , we l i m i t o u r d i s c u s s i o n h e r e t o t h e problems t h a t a r i s e when one t r i e s t o a n a l y s e t h e complex m i x t u r e o f p r o t e i n s and g l y c o p r o t e i n s t h a t c o n s t i t u t e c e l l u l a r membranes. B e f o r e t h e a n a l y s i s o f these c o n s t i t u t e n t s i s begun, i t i s i m p o r t a n t t o e s t a b l i s h whether membrane p r o t e i n s can be b r o u g h t i n t o s o l u t i o n w i t h o u t m a j o r p r e f e r e n t i a l l o s s o f c e r t a i n m o l e c u l a r s p e c i e s and a l s o whether t h e y remain i n t h e “ s o l u b l e s t a t e ” under t h e c o n d i t i o n s used f o r t h e a c t u a l s e p a r a t i o n , u s u a l l y be e l e c t r o p h o r e s i s i n p o l y a c r y l a m i d e g e l s . Membrane p r o t e i n s , which r e s i d e i n t h e p h o s p h o l i p i d b i l a y e r and, t h e r e f o r e , a r e exposed t o a hydrophobic environment, possess i n t h e i r s t r u c t u r e one o r more 1 i p o p h i l i c

Consequently, many i n t r i n s i c membrane p r o -

t e i n s e x h i b i t a l i m i t e d s o l u b i l i t y i n aqueous media. Techniques used f o r t h e i r s t r u c t u r a l and f u n c t i o n a l c h a r a c t e r i z a t i o n , t h e r e f o r e , have t o be adapted accordingly.

To b r i n g membrane p r o t e i n s i n t o s o l u t i o n , t h e l i p i d b i l a y e r , i n w h i c h t h e p r o t e i n s a r e embedded, has t o be d i s r u p t e d and a t t h e same t i m e t h e l i b e r a t e d p r o t e i n s have t o be prevented f r o m a g g r e g a t i o n . I n most i n s t a n c e s a m p h i p a t h i c compounds ( s u r f a c t a n t s o r d e t e r g e n t s ) which a c t as l i p i d s u b s t i t u t e s have been used t o e f f e c t , a l b e i t t o v a r y i n g e x t e n t s , t h e s o l u b i l i z a t i o n o f membrane comp o n e n t ~ ~Among . t h e more g e n e r a l l y used d e t e r g e n t s (sodium dodecyl s u l p h a t e , deoxycholate, c h o l a t e , T r i t o n X-100, NP-40, L u b r o l

, Brij,

Tween e t c . ) ,

o n l y so-

dium dodecyl s u l p h a t e and d e o x y c h o l a t e c o n s i s t e n t l y show a h i g h e f f i c i e n c y o f i n i t i a l s o l u b i l i z a t i o n o f membranes. The o t h e r d e t e r g e n t s a r e c h a r a c t e r i z e d by a l o w e r o v e r a l l y i e l d i n t h e s o l u b i l i z a t i o n o f t o t a l membrane p r o t e i n s , b u t some have t h e a b i l i t y t o s o l u b i l i z e s p e c i f i c proteins6-‘.

Only l i m i t e d success i n

membrane p r o t e i n e x t r a c t i o n from t h e 1 i p i d b i l a y e r has been achieved u s i n g c h a o t r o p i c agents, a c i d i c o r b a s i c c o n d i t i o n s o r h i g h s a l t c ~ n c e n t r a t i o n ~ ’ ~ . G e n e r a l l y , however, most methods o f s o l u b i l i z a t i o n l e a d t o a l t e r a t i o n s o r even 6,9-11

complete l o s s o f t h e b i o l o g i c a l a c t i v i t i e s o f membrane p r o t e i n s

The most p o w e r f u l method f o r b o t h a n a l y t i c a l and p r e p a r a t i v e s e p a r a t i o n s o f membrane p r o t e i n s o f v a r i o u s c e l l u l a r and membrane o r i g i n has proved t o be e l e c t r o p h o r e s i s i n p o l y a c r y l a m i d e g e l s under d e n a t u r i n g c o n d i t i o n s i n t h e presence

121

of sodium dodecyl ~ u l p h a t e ’ ~ ’ ~The ~ . separation occurs almost exclusively, according to the logarithm of the molecular size of the protein. Because membranes contain complex mixtures of proteins covering a wide range o f molecular weights, optimal resolution of all membrane proteins, such as those labeled in situ at the cell surface with lactoperoxidase and NalZ5I, can be obtained by using gels 20-30 cm in length and consisting of a linear gradient of acrylamide concentration14’15. The gradient can range between 5 and 15% acrylamide, but we found that in most instances in which analyses were carried out with plasma membrane proteins a gradient of 7.5-12% acrylamide yielded the best results16. The limitations of this one-dimensional method of separation were apparent, however, when membrane glycoproteins were analysed. Because many of the intrinsic membrane glycoproteins, which mostly have apparent molecular weights above 50,000, show extensive size heterogeneity (up to +20,000 dal tons), no satisfactory separation for these groups of membrane proteins could be obtained in the one-dimensional system16-18. Membrane glycoproteins differ from sectory glycoproteins in this respect in that secretory glycoproteins seldom show such extensive size heterog e n e i t i e ~ ~ ~To ’ ~resolve ~. adequately the complex nature of glycoproteins in the plasma membrane, it was necessary to effect separations based on both the charge and the size o f these constituents using a two-dimensional electrophoretic system (see below). Electrophoresis of complex mixtures of membrane proteins in polyacrylamide gels in the presence of detergents other than sodium dodecyl sulphate, such as NP-40, Triton X-100 or deoxycholate, are generally characterized by very poor resolutionz1. In most instances in the presence of these other detergents the proteins precipitate upon entering the gel or migrate as large aggregates. For this reason, large-pore (4-7.5%) polyacrylamide gels are usually used. Predipitation of proteins can be minimized by inclusion of 6-9.5 mol/l urea both in the solution used for membrane “solubilization” and in the gel system itself (Baumann, unpubl ished work). Isoelectrofocusing of membrane proteins in polyacrylamide gels i s feasible in the presence of both high concentrations of urea and non-ionic detergents. In the absence o f urea, aggregation and precipitation of the proteins is a serious problem. Even the presence of urea will not always prevent precipitation of some membrane proteins8 . The non-glycosylated membrane proteins exhibit no or only minor charge heterog e n e i t i e ~ ~ ~The ’ ~ ~separation . of glycosylated membrane proteins by isoelectric focusing, however, because of the extensive charge heterogeneities , generates extremely complicated patterns. It is often very difficult to identify individual proteins among the set of glycoproteins resolved by this method. The major factor contributing to the charge heterogeneity of membrane glycoproteins is the varying

122 numbers o f s i a l i c a c i d r e s i d u e s p e r p r o t e i n molecule24. Some a d d i t i o n a l minor d i f f e r e n c e s i n charge can be i n t r o d u c e d by h e t e r o g e n e i t y i n t h e mannose r e s i d u e c o n t e n t o f a p a r t i c u l a r member o f a g l y c o p r o t e i n s p e c i e s . The more mannose r e s i d u e s a r e p r e s e n t on t h e p r o t e i n t h e more t h e i s o e l e c t r i c p o i n t i s s h i f t e d towards t h e b a s i c r e g i o n 2 5 . Because h e t e r o g e n e i t y i n b o t h mannose and s i a l i c a c i d c o n t e n t a r e combined i n membrane g l y c o p r o t e i n s , one g l y c o p r o t e i n s p e c i e s can show a range o f 24,26 i s o e l e c t r i c p o i n t s o f up t o 4 pH u n i t s

.

As p o i n t e d o u t above, one-dimensional s e p a r a t i o n o f membrane p r o t e i n s by e i t h e r e l e c t r o p h o r e s i s i n sodium dodecyl s u l p h a t e o r i s o e l e c t r o f o c u s i n g has i n h e r e n t problems. When b o t h techniques a r e combined i n a two-dimensional system, however, o p t i m a l r e s o l u t i o n f o r most o f t h e membrane p r o t e i n s can be o b t a i n e d . A t t h e same time, e l e c t r o p h o r e t i c s e p a r a t i o n i n two dimensions g i v e s v a l u a b l e b i o c h e m i c a l i n f o r m a t i o n , such as i s o e l e c t r i c p o i n t , apparent m i n i m a l m o l e c u l a r w e i g h t s and e x t e n t s o f s i z e and charge h e t e r o g e n e i t i e s o f t h e r e s o l v e d p r o t e i n s . B a s i c a l l y , t h r e e two-dimensional e l e c t r o p h o r e t i c g e l systems have been d e s c r i b e d and can be employed s u c c e s s f u l l y f o r t h e r e s o l u t i o n o f d i f f e r e n t c l a s s e s o f membrane p r o t e i n s . The w i d e l y used system d e s c r i b e d by 0 ' F a r r e l l and

y i e l d s reasonable

r e s u l t s f o r n o n - g l y c o s y l a t e d membrane p r o t e i n s 22y29y30 and f o r g l y c o p r o t e i n s w i t h low degrees o f s i z e and charge h e t e r o g e n e i t y , such as t h e m a j o r h i s t o c o m p a t i b i l i t y antigens31. However, some membrane p r o t e i n s do n o t e n t e r t h e s e p a r a t i n g g e l i n t h i s system. F o r t h e s e p a r a t i o n o f h i g h l y g l y s o s y l a t e d p r o t e i n s , t h e g e l system d e s c r i b e d by Ames and Nikaido3'

i s preferable,

i n t h a t t h e samples a r e s o l u b i l i z e d

i n i t i a l l y i n sodium dodecyl s u l p h a t e and t h e n a r e a p p l i e d on an i s o e l e c t r o f o c u s i n g s l a b g e l i n t h e presence o f n o n - i o n i c d e t e r g e n t s g e n e r a t i n g a s t e e p pH g r a d i e n t . Using abbroad pH range, t h e g l y c o p r o t e i n s showing e x t e n s i v e charge h e t e r o g e n e i t y a r e more compressed t h a n i n t h e i s o e l e c t r o f o c u s i n g t u b e g e l s d e s c r i b e d by

.

0 ' F a r r e l l e t a1 27y28. I n some i n s t a n c e s , f o r example, w i t h t h e h i g h - m o l e c u l a r - w e i g h t a c i d i c g l y c o p r o t e i n s , sodium dodecyl s u l p h a t e has t o be a v o i d e d because t h e p r o 26 t e i n s do n o t f o c u s a t t h e i r p r o p e r i s o e l e c t r i c p o i n t s

.

H o r s t e t a1 .33 developed a s p e c i a l s o l u b i l i z a t i o n procedure f o r membrane p r o t e i n s which i s s u i t a b l e f o r subsequent i s o e l e c t r ~ f o c u s i n g ~The ~ . membranes were s o l u b i l i z e d by u l t r a s q n i c a t i o n i n 9.5 m o l / l u r e a c o n t a i n i n g 5 mmol/l K2C03 (pH

10.3). Using t h i s a1 k a l i n e u r e a s o l u t i o n , g l y c o p r o t e i n s c o u l d be e x t r a c t e d a l m o s t q u a n t i t a t i v e l y from t h e membrane. Recovery f r o m t h e membrane was l o w e r , however, f o r n o n - g l y c o s y l a t e d p r o t e i n s 3 3 (Baumann, u n p u b l i s h e d w o r k ) . The a1 k a l i n e u r e a e x t r a c t i o n procedure o f f e r s t h e advantage o v e r most o t h e r e x t r a c t i o n procedures t h a t t h e m a n i p u l a t i o n s can b e c a r r i e d o u t a t O°C w i t h o u t r i s k o f r e c r y s t a l l i z a t i o n o r urea. The s e p a r a t i o n of membrane p r o t e i n s i n a s p e c i a l l y m o d i f i e d two-dimensional gel system a l l o w s one a t l e a s t t o some e x t e n t t o e s t i m a t e i n t e r a c t i o n s between membrane components, I n t h i s approach, t h e p r o t e i n s a r e s e p a r a t e d i n t h e f i r s t

123 dimension, w h i l e t h e y a r e s t i l l i n t e g r a t e d i n complexes. To accomplish t h i s , t h e membranes a r e s o l u b l i z e d g e n t l y , a v o i d i n g c h a o t r o p i c a g e n t s o r s t r o n g d i s s o c i a t i n g d e t e r g e n t s . The " s o l u b i l i z e d " p r o t e i n i s t h e n s u b j e c t e d t o e l e c t r o p h o r e s i s i n a l a r g e - p o r e p o l y a c r y l a m i d e g e l under c o n d i t i o n s t h a t p r e s e r v e t h e p r o t e i n complexes. The s e p a r a t i o n i n t h e second dimension i s t h e n c a r r i e d o u t under d i s s o c i a t i n g o r even c o m p l e t e l y d e n a t u r i n g c o n d i t i o n s 2 1 y 3 5 . Such s e p a r a t i o n schemes can be d e v i s e d t o determine, f o r example, i f d i s u l p h i d e bonds o r bonds through d i v a l e n t c a t i o n s a r e i n v o l v e d i n complex f o r m a t i o n . I n t h e f o r m e r i n s t a n c e t h e membrane p r o t e i n s would be d i s s o l v e d on complex f o r m a t i o n . I n t h e f o r m e r i n s t a n c e t h e membrane p r o t e i n s would be d i s s o l v e d i n sodium dodecyl s u l p h a t e , a v o i d i n g b o i l i n g and i n t h e absence o f s u l p h y d r y l r e d u c i n g agents. A f t e r t h e f i r s t s e p a r a t i o n , t h e complexes would be d i s r u p t e d by b o i l i n g i n t h e presence o f r e d u c i n g agents''.

I n t h e l a t t e r i n s t a n c e , t h e s o l u b i l i z a t i o n and t h e f i r s t -

dimensional s e p a r a t i o n a r e c a r r i e d o u t i n t h e presence o f t h e a p p r o p r i a t e d i v a l e n t c a t i o n s (e.g.,

5 mmol/l Mg2+ o r La"),

whereas i n t h e second dimension t h e separa-

t i o n i s c a r r i e d o u t i n t h e presence o f c h e l a t i n g agents (EDTA, EGTA)26. I n a l l t h r e e o f t h e above systems t h e p r o t e i n s i n v o l v e d i n t h e f o r m a t i o n o f aggregates can e a s i l y be r e c o g n i z e d by t h e i r p o s i t i o n o f f t h e d i a g o n a l e . By a p p l y i n g two-dimensional g e l analyses t o membrane p r o t e i n s , i t i s p o s s i b l e

n o t o n l y t o a c q u i r e i n f o r m a t i o n about t h e p r o t e i n and g l y c o p r o t e i n c o m p o s i t i o n o f a p a r t i c u l a r membrane b u t a l s o , i n c o m b i n a t i o n w i t h o t h e r methods and s t r a t e g i e s , t o (1) i d e n t i f y t h o s e p r o t e i n s spanning t h e membrane36y37, ( 2 ) l o c a l i z e p r o t e i n s w i t h i n a c e l l z 4 and ( 3 ) e l u c i d a t e t h e whole pathway o f e p i g e n e t i c m o d i f i c a t i o n s 38,39 t h a t e v e n t u a l l y produce t h e n a t i v e membrane g l y c o p r o t e i n s

.

REFERENCES

1 A.H. Maddy and M.J. Dunn, i n A.H. Maddy ( E d i t o r ) , BiochemicaZ AnaZysis of Membranes, Chapman and H a l l , London, 1976, pp. 177-196. 2 M.J. Dunn and A.H. Maddy, i n A.H. Maddy ( E d i t o r ) , BiochemicaZ AnaZysis of Membranes, Chapman and H a l l , London, 1976, pp. 197-251. 3 M. Tomita and V.T. Marchesi, Proc. Nat. Acad. S c i . U.S., 72 (1975) 2964. 4 T.L. Steck, J.J. K o z i a r z , M.K. Singh, G. Reddy and H. Kohler, Biochemistry, 17 (1978) 1216. 5 T. Gul ik - K r z y w i c k i , Biochim. Biophys. Acta, 415 (1975) 1. 6 M.L.P. C o l l i n s and M.P.J. S a l t o n , Biochim. Biophys. Acta, 553 (1979) 40. 7 R.L. J u l i a n o and G. L i , Biochemistry, 17 (1978) 678. 8 J. Tweto, E. Hou and D. Doyle, Arch. Biochem. Biophys., 193 (1979) 422. 9 Y. H a t e f i and W.G. H a n s t e i n , Proc. Nut. Acad. Sci. U.S., 62 (1969) 1129. 10 J.N. Umbriet and J.L. S t r o m i n g e r , Proc. Nat. Acad. S c i . U.S., 70 (1974) 2997. 11 M.L. Sussman and J.B. Hays, Biochim. Biophys. Acta, 465 (1977) 559. 12 U.K. Laemmli, Nature (London), 227 (1970) 680. 13 G. F a i r b a n k s , T.L. Steck and D.F.H. Wallach, Biochemistry, 10 (1971) 2606. 14 J. Tweto and 0. Doyle, J . B i o l . Chem., 251 (1976) 872. 15 D. Owerbach, D. Doyle and T.B. Shows, Sometic CeZZ Genet., 5 (1979) 281. 16 0. Doyle and H. Baumann, L i f e S c i . , 24 (1979) 951.

124 17 K.D. Noonan, Biochim. Biophys. Acta, 551 (1979) 22. 18 D. Doyle, H. Baumann, B. England, E. Friedman, E. Hou and J . Tweto, J . Biol. Chem.. 253 (1978) 965. 19 L. Anderson'and N.G. Anderson, Proc. flat. Acad. Sci. U.S., 74 (1977) 5421. 20 H. Baumann, T.D. G e l e h r t e r and D. Doyle, J . CeZZ Biol., 85 (1980) 1. 2 1 C. B o r d i e r , W.F. Loomis, J . E l d e r and R. L e r n e r , J . B i o l . Chem., 253 (1978) 5133. 22 D. Doyle, H. Baumann, B. England, E. Friedman, E. Hou and J . Tweto, i n H. Segal and D. Doyle ( E d i t o r s ) , Protein !Turnover and LysosomaZ Function, Academi Press, New York, 1978, pp. 689-717. 23 H. Bussey, D. S a v i l l e , M.R. C h e v a l l i e r and C.H. Rank, Biochim. Biophys. Acta, 553 (1979) 185. 24 H. Baumann and D. Doyle, J . BioZ. Chem., 254 (1979) 2542. 25 H. Baumann and W.A. Held, J . BioZ. Chem., 256 (1981) 10145. 26 H. Baumann and D. Doyle, J . B i o l . Chem., 254 (1979) 3935. 27 P.H. O ' F a r r e l l , J . BioZ. Chem., 250 (1975) 4007. 28 P.Z. O ' F a r r e l l , H. Goodman and P.H. O ' F a r r e l l , CeZZ, 12 (1977) 1133. 29 K.J. Clemetson, A. C a p i t a n i o and E. Luscher, Biochim. Biophys. Acta, 553 (1979) 11. 30 S.D.J. Pena, G. M i l l s and R.C. Hughes, Biochim. Biophys. Acta, 550 (1979) 100. 3 1 P.P. Jones, J . E q . Ned., 146 (1977) 1261. 32 F.L.G. Ames and K. N i k a i d o , Biochemistry, 15 (1976) 616. 33 M.N. H o r s t , S.M. Basha, G.A. Baumbach, E.H. M a n s f i e l d and R.M. Roberts, Anal. Biochem., 102 (1980) 399. 34 M.N. H o r s t , G. Baumbach and R.M. Roberts, FEBS L e t t . , 100 (1979) 385. 35 M. Imada, P. Hsieh and N. Sueoka, Biochim. Biophys. Acta, 507 (1978) 459. 36 R.M. Evans, D.C. Ward and L.M. F i n k , Proc. Nat. Acad. S c i . U.S., 76 (1979) 6235. 37 D.B. Archer, A.W. Rodwell and E.S. Rodwell, Biochim. Biophys. Acta, 513 (1978) 268. 38 M.L. P r i v a l s k y and E.E. Penhoet, Proc. Nab. Acad. S c i . U.S., 73 (1978) 3625. 39 H. Baumann, J . Supramol. S t m c t . , 12 (1979) 151.

125

Chapter 9.4 PROTEIN MEMBRANE RECEPTORS URSULA LANG Over t h e l a s t few y e a r s , a number o f p r o t e i n c e l l w a l l r e c e p t o r s have been s o l u b i l i z e d by d e t e r g e n t s w i t h maintenance o f a c t i v i t y , i n c l u d i n g t h e c e l l w a l l 4 r e c e p t o r s s p e c i f i c f o r human p r o l a c t i n ’ , i n s u l i n z y 3 , n e r v e g r o w t h f a c t o r , glucagon’, t h y r o i d s t i m u l a t i n g hormone 6 and c h o r i o n i c gonadotropin7’*. I n a l l i n s t a n c e s t h e s o l u b i l i z i n g d e t e r g e n t has been o f t h e n o n - i o n i c type, p r o v i d i n g maintenance o f a c t i v i t y a t t h e p r i c e o f r e l a t i v e l y p o o r s o l u b i l i z i n g and d i s a g g r e g a t i n g power o f t h i s t y p e o f d e t e r g e n t 9 ’ l 0 .

I n n e a r l y a l l i n s t a n c e s t h e mo-

l e c u l a r c h a r a c t e r i z a t i o n o f t h e r e c e p t o r was c a r r i e d o u t e i t h e r ( a ) by g e l f i l t r a t i ~ n ~ ’ ’ i’ n~ a n o n - i o n i c d e t e r g e n t , l a c k i n g i n r e s o l v i n g power (number o f t h e o r e t i c a l p l a t e s ) i n t h e usual and c o n v e n i e n t e x p e r i m e n t a l column set-ups, o r ( b ) by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n SDS-containing b u f f e r s , a f t e r f u l l d e n a t u r a t i o n o f t h e r e c e p t o r and cleavage i n t o i t s p r o t e i n s u b u n i t s . Gel e l e c t r o p h o r e s i s i n d e t e r g e n t - c o n t a i n i n g b u f f e r s o f n a t i v e p r o t e i n r e c e p t o r s has been a p p l i e d o n l y t o t h e physical c h a r a c t e r i z a t i o n o f t h e i n s u l i n receptorzy3. I n a l l instances t h e d e t e r g e n t T r i t o n X-100 was employed, which r e s u l t e d i n e s t i m a t e s o f m o l e c u l a r w e i g h t f o r t h e i n s u l i n r e c e p t o r r a n g i n g f r o m 300,000 t o 1,000,000.

There was no

evidence i n any o f t h e s e i n s t a n c e s t h a t t h e p r o t e i n s p e c i e s i n t h e e l e c t r o p h o r e t i c system would n o t i n c l u d e s e v e r a l c o p i e s o f t h e r e c e p t o r molecule, o r o t h e r membrane components, p r o t e i n s and/or l i p i d s . The w e i g h t r a t i o between d e t e r g e n t and p r o t e i n s i n t h e s o l u b i l i z e d complex i s unknown, as i s t h e p h y s i c a l s t a t e (e.g., m i c e l l a r ) o f t h e d e t e r g e n t i n t h e complex. Thus, an a t t e m p t w i l l be made n o t so much t o r e v i e w t h e ( b a r e l y e x i s t i n g ) l i t e r a t u r e on t h e f r a c t i o n a t i o n and p h y s i c a l c h a r a c t e r i z a t i o n o f s o l u b i l i z e d membrane r e c e p t o r s by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE), b u t r a t h e r t o p o i n t t o t h e u n i q u e s u i t a b i l i t y and p o t e n t i a l of PAGE f o r t h i s purpose, and t o a number of p i t f a l l s and problems t h a t we have encountered and t h a t t h e f u t u r e i n v e s t i g a t o r i n t h i s f i e l d s h o u l d be aware o f .

PAGE i s u n i q u e l y s u i t e d f o r t h e c h a r a c t e r i z a t i o n o f membrane r e c e p t o r s i n n a t i v e f o r m because i t a l l o w s one e a s i l y and e f f i c i e n t l y t o m o n i t o r two e s s e n t i a l parameters, t h e degree o f a c t i v i t y maintenance and t h e m o l e c u l a r s i z e . T h i s i s i n c o n t r a s t t o g e l f i l t r a t i o n o r s e d i m e n t a t i o n v e l o c i t y methods, w h i c h i n p r i n c i p l e a l s o l e n d themselves t o m o l e c u l a r s i z e d e t e r m i n a t i o n , b u t do so w i t h much l e s s e f f i c i e n c y ( i .e., numbers o f t h e o r e t i c a l p l a t e s under t h e usual e x p e r i m e n t a l

126 c o n d i t i o n s ) . The procedure t h u s c o n s i s t s o f two s t e p s : f i r s t , PAGE f r a c t i o n a t i o n i n d i f f e r e n t d e t e r g e n t s , g e l s l i c i n g and assay o f g e l s l i c e s a f t e r e l u t i o n t o p r o v i d e an a c t i v i t y p r o f i l e on t h e g e l ; and secondly, a Ferguson p l o t i n t h e o p t i m a l d e t e r g e n t - c o n t a i n i n g g e l b u f f e r . L e a s t e x p e r i m e n t a l work i s necessary i f t h e pH and d e t e r g e n t a r e o p t i m i z e d , u s i n g s t a c k i n g g e l s o f m u l t i p h a s i c zone electrophoresis".

N o n - i o n i c and z w i t t e r i o n i c d e t e r g e n t s a r e p r e f e r a b l y a p p l i e d

t o e l e c t r o p h o r e s i s because t h e y m a i n t a i n t h e charge d i f f e r e n c e s between s p e c i e s d u r i n g s e p a r a t i o n . The c o n c e n t r a t i o n range between 0.01 and 1.0% has been u s u a l l y e x p l o r e d , a l t h o u g h t h e r e l e v a n t v a r i a b l e s h o u l d n o t be t h e a b s o l u t e d e t e r g e n t 9 Another p o s s i b l e proconcentration, b u t r a t h e r t h e protein:detergent r a t i o

.

cedure a t t h i s s t a g e i s t o m i x d i f f e r e n t d e t e r g e n t t y p e s , i n p a r t i c u l a r t h o s e w i t h d i f f e r e n t p o l a r headgroups".

Thus, d i f f e r e n t d e t e r g e n t s , d e t e r g e n t con-

cent.rations and d e t e r g e n t m i x t u r e s a r e v a r i e d i n t h e s t a c k i n g g e l u n t i l a s e t o f c o n d i t i o n s i s o b t a i n e d i n which e s s e n t i a l l y a l l o f t h e r e c e p t o r a c t i v i t y m i g r a t e s w i t h i n t h e s t a c k o r , i f t h e experiment i s c a r r i e d o u t on r e s o l v i n g g e l s , under t h e a c t i v i t y p r o f i l e . As i t appears t h a t r e c e p t o r molecules a r e l a r g e , i . e . , have m o l e c u l a r w e i g h t s l a r g e r t h a n 100,000, i t i s advantageous t o use r e l a t i v e l y n o n - r e s t r i c t i v e g e l media (1%agarose, 15% CDATD c r o s s - l i n k e d g e l s n o t exceeding

5%T), and l o w s t a c k i n g l i m i t s ' ' .

To m a i n t a i n b u f f e r i n g w i t h i n t h e s t a c k , m u l t i -

p h a s i c b u f f e r systems p r o v i d i n g a s h a l l o w pH g r a d i e n t across t h e s t a c k a r e desirable".

Having t h u s found t h e o p t i m a l d e t e r g e n t , PAGE i s s i m p l y c a r r i e d o u t

a t s e v e r a l g e l c o n c e n t r a t i o n s t o o b t a i n t h e Ferguson p l o t ( l o g RF versus IT), t h e s l o p e o f which ( d e s i g n a t e d as t h e r e t a r d a t i o n c o e f f i c i e n t , K R ) i s a measure o f m o l e c u l a r s i z e . T h i s s i z e i n a d e t e r g e n t p r o v i d i n g f u l l r e c e p t o r a c t i v i t y may 5 6 be enormous, e q u i v a l e n t t o m o l e c u l a r w e i g h t s o f 3 - 1 0 t o 1-10 f o u n d f o r t h e i n s u l i n receptor2y3. To e s t a b l i s h whether t h i s s p e c i e s i s t h e s m a l l e s t p o s s i b l e a c t i v e molecule, one can now r e v e r t t o o t h e r d e t e r g e n t s , d e t e r g e n t c o n c e n t r a t i o n s o r d e t e r g e n t m i x t u r e s t h a t may p r e s e r v e o n l y a p a r t o f t h e a c t i v i t y , e.g.,

50%, b u t a r e more

e f f e c t i v e d i s a g g r e g a t i n g agents. An example i s t h e membrane p r o t e i n a d e n y l a t e cyclase, which was s p l i t i n h a l f by a d m i x t u r e o f i o n i c d e t e r g e n t s t o a n o n - i o n i c d e t e r g e n t , w i t h maintenance o f enough a c t i v i t y f o r d e t e c t i o n 1 3 . PAGE t h u s a l l o w s one t o d e t e c t t h e s m a l l e s t p o s s i b l e a c t i v e s p e c i e s , which must be c o n s i d e r e d a x i o m a t i c a l l y t h e most b i o l o g i c a l l y , or a t l e a s t b i o c h e m i c a l l y , r e l e v a n t . There a r e numerous p i t f a l l s i n PAGE c h a r a c t e r i z a t i o n o f r e c e p t o r s p e c i e s . ( a ) I n e v a l u a t i n g t h e degree o f i n a c t i v a t i o n by any s e t o f d e t e r g e n t c o n d i t i o n s , one must be aware o f t h e p o s s i b i l i t y t h a t n o t a f r a c t i o n o f t h e a c t i v i t y i s l o s t

i n t h e e n t i r e p o p u l a t i o n , b u t r a t h e r t h a t o n l y a s p e c i f i c f r a c t i o n o f t h e populat i o n i s inactivated. (b) I n assessing the molecular species being fractionated, t h e p o s s i b i l i t y has t o be k e p t i n mind t h a t m u l t i p l e c o p i e s o f t h e r e c e p t o r may

127 complex w i t h t h e d e t e r g e n t , o r t h e r e c e p t o r t o g e t h e r w i t h o t h e r p r o t e i n s and/or l i p i d s . ( c ) As t h e r e c e p t o r - d e t e r g e n t complex, o r even more a complex i n v o l v i n g these o t h e r s p e c i e s , d i f f e r s f r o m homogeneous d e t e r g e n t systems, no c e r t a i n t y e x i s t s about t h e a p p l i c a b i l i t y o f i n f o r m a t i o n c o n c e r n i n g t h e m i c e l l a r s i z e (agg r e g a t i o n number), i t s geometry and t h e dependence o f t h e CMC on i o n i c s t r e n g t h and t e m p e r a t u r e as may be known f r o m s i m p l e systems".

(d) Although they a r e

q u a n t i t a t i v e l y unknown, t h e e f f e c t s o f . d e t e r g e n t c o n c e n t r a t i o n , i o n i c s t r e n g t h , temperature and pH on t h e p h y s i c a l s t a t e o f t h e s e l e c t e d d e t e r g e n t ( s ) s h o u l d be c o n s i d e r e d g ' l O . ( e ) In t r a n s l a t i n g t h e PAGE parameter d e s c r i p t i v e o f m o l e c u l a r size, K weight

ZR ,

t o c o n v e n t i o n a l parameters such as m o l e c u l a r radius''

o r molecular

by means o f a s t a n d a r d graph, i t i s i m p o s s i b l e t o a s s i g n e i t h e r s p h e r i c a l

geometry t o t h e d e t e r g e n t - r e c e p t o r complex ( t o f i t t h e s t a n d a r d graph o f square r o o t o f KR versus m o l e c u l a r r a d i u s ) , n o r i s i t p o s s i b l e t o a s s i g n a random-coil c o n f i g u r a t i o n t o i t ( w h i c h would a l l o w us t o use t h e s t a n d a r d graph o f KR v e r s u s molecular weight)".

F u r t h e r , even i f i t were p e r m i s s i b l e t o a p p l y e i t h e r o f t h e

two broad c o n f o r m a t i o n a l models t o t h e r e c e p t o r - d e t e r g e n t complex, one would need a c h e m i c a l l y homogeneous s e r i e s o f m o l e c u l a r w e i g h t standards,

i.e.,

proteins

t h a t share t h e degree o f h y d r o p h o b i c i t y and t h e n o n - p r o t e i n m o i e t i e s w i t h t h e membrane r e c e p t o r s . Such s t a n d a r d s o b v i o u s l y do n o t e x i s t . F o r a l l t h e s e reasons, KR s h o u l d be used d i r e c t l y i n e v a l u a t i n g m o l e c u l a r s i z e , whenever p o s s i b l e , r a t h e r t h a n t h e " t r a n s l a t e d " parameters m o l e c u l a r r a d i u s and w e i g h t . These problems i n t h e e v a l u a t i o n o f m o l e c u l a r w e i g h t a r e n o t s p e c i f i c t o PAGE, b u t a p p l y even more so t o g e l f i l t r a t i o n , i n p a r t i c u l a r when i t i s app l i e d t o t h e determination o f molecular radius, c a l c u l a t i o n o f d i f f u s i o n coeff i c i e n t s from t h e radius, c a l c u l a t i o n o f molecular weight using t h i s d i f f u s i o n c o e f f i c i e n t , and f i n a l l y c a l c u l a t i o n o f t h e f r i c t i o n a l c o e f f i c i e n t f r o m t h e s e t h r e e . Such " c h a r a c t e r i z a t i o n " , g o n a d o t r o p i n receptor',

as i t has been a p p l i e d , f o r example, t o t h e

i s f a l l a c i o u s ( s e e pp. 172-174 o f r e f . 14). O t h e r more

obvious p i t f a l l s i n m o l e c u l a r s i z e d e t e r m i n a t i o n s a r e t h e p a u c i t y o f t h e number 14 o f p r o t e i n s t a n d a r d s and n e g l e c t o f c o n f i d e n c e l i m i t s

.

REFERENCES

1 2 3 4

5 6 7 8

R.P.C. Shiu and H.G. F r i e s e n , Biochern. J . , 140 (1974) 301. U. Lang, R.C. Kahn and A. Chrambach, EndocrinoZogy, 106 (1980) 40. L. Kuehn, H. Meyer, M. Rutschmann and P. Thamm, FEBS L e t t . , 113 (1980) 189. N.V. C o s t r i n i , M. Kogan, K. K u k r e j a and R.A. Bradshaw, J . BioZ. Chern., 254 (1979) 11242. N.A. G i o r g i o , C.B. Johnson and M. B l e c h e r , J . BioZ. Chem., 249 (1974) 428. R.L. P r e s t i d g e and M.T.W. Hearn, Anal. Biochern., 97 (1979) 95. M.L. Dufau, E.H. Charreau and K.J. C a t t , J . BioZ. Chern., 248 (1973) 6973. K.K. Sen and K.J. Menon, Biochern. Biophys. Res. C o m n . , 92 (1980) 1250.

128 9 L.M. Hjelmeland, D.W. Nebert and A. Chrambach, i n N. Catsimpoolas ( E d i t o r ) , EZectrophoresis ' 7 3 , E l s e v i e r / N o r t h H o l l a n d , Amsterdam, New York, 1978, pp. 29-56 (copy a v a i l a b l e f r o m t h e a u t h o r on r e q u e s t ) . 10 L.M. Hjelmeland and A. Chrambach, EZectrophoresis, 2 (1981) i n p r e s s (copy a v a i l a b l e f r o m t h e a u t h o r on r e q u e s t ) . 11 A. Chrambach, MoZ. CeZZ Biochem., 29 (1980) 23. 12 A.C. Newby, M. Rodbell and A. Chrambach, Arch. Bioehern. Biophys., 190 (1978) 109. 13 A.C. Newby and A. Chrambach, Biochem. J . , 177 (1978) 623. 14 D. Rodbard, i n N. Catsimpoolas ( E d i t o r ) , Methods of Protein Separation, V o l . 2, Plenum Press, New York, 1976, pp. 145-218.

129

Chapter 9 . 5 STEROID RECEPTORS

SARAH BEN-OR The s t e r o i d hormones a r e r e g u l a t o r y s i g n a l s t h a t modulate t h e e x p r e s s i o n o f t i s s u e - s p e c i f i c gene f u n c t i o n i n e u k a r y o t i c c e l l s ' .

This r e g u l a t o r y r o l e o f t h e

s t e r o i d i s mediated by r e c e p t o r s which e x h i b i t s p e c i f i c i t y and a h i g h b i n d i n g 2 a f f i n i t y f o r t h e p h y s i o l o g i c a l l y a c t i v e s t e r o i d hormone(s)

.

The r e c e p t o r s f o r s t e r o i d hormones a r e s o l u b l e p r o t e i n s t h a t i n s t e r o i d - f r e e c e l l s r e s i d e i n t h e c y t o s o l , t h e s o l u b l e f r a c t i o n o f t h e cytoplasm. Upon e n t r y of t h e l i p o p h y l i c s t e r o i d hormones i n t o t h e c e l l , and t h e i r subsequent b i n d i n g t o t h e r e c e p t o r s , t h e s t e r o i d - r e c e p t o r complexes t r a n s l o c a t e f r o m t h e c y t o s o l t o t h e nucleus and b i n d t o a c c e p t o r s i t e s i n t h e c h r o m a t i n , t h e s i t e o f t h e i r r e g 3 ulatory function

.

The process o f t r a n s f o r m a t i o n o f t h e s t e r o i d r e c e p t o r s f r o m i n a c t i v e c y t o s o l i c p r o t e i n s t o a c t i v e r e g u l a t o r s t h a t induce, i n t h e r e s p o n s i v e c e l l s , a s p e c i f i c d i f f e r e n t i a l i n c r e a s e i n t r a n ~ c r i p t i o n ~i -s ~n o t y e t understood. The e l u c i d a t i o n o f t h e s e processes i s an e s s e n t i a l p r e r e q u i s i t e f o r u n d e r s t a n d i n g t h e mechanism o f hormonal r e g u l a t i o n o f gene a c t i v i t y i n e u k a r y o t i c c e l l s . The s t r u c t u r e and f u n c t i o n o f t h e s t e r o i d r e c e p t o r s have been e x t e n s i v e l y s t u d i e d d u r i n g t h e l a s t 12 y e a r s . T h e i r physico-chemical c h a r a c t e r i z a t i o n by u l t r a c e n t r i f u g a t i o n i n sucrose ( o r g l y c e r o l ) g r a d i e n t s , g e l f i l t r a t i o n , i o n exchange chromatography, g e l e l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g ( I E F ) have r e v e a l e d s t r i k i n g s i m i l a r i t i e s among a l l s t e r o i d r e c e p t o r s s t u d i e d , s u g g e s t i n g t h a t a l l o f t h e s t e r o i d r e c e p t o r s i n t h e v e r t e b r a t e have a common m o l e c u l a r a r c h i t e c t u r e . However, t h e s t r u c t u r e and s u b u n i t c o m p o s i t i o n o f t h e n a t i v e s t e r o i d r e c e p t o r s a r e s t i l l unknown. Many s e r i o u s impediments remain, and t h e s e a r e c o n s i d e r e d be1 ow. The n a t i v e r e c e p t o r s f o r s t e r o i d hormones a r e l a b i l e p r o t e i n s w h i c h a r e v e r y s e n s i t i v e t o t h e i o n i c c o m p o s i t i o n o f t h e b u f f e r s used f o r t h e i r e x t r a c t i o n and f r a c t i o n a t i o n . T h e i r t r a n s f o r m a t i o n t o a c t i v e s t e r o i d - r e c e p t o r complexes, w h i c h i n t h e c e l l i s a temperature-dependent

event3, i s r e a d i l y induced i n t h e i s o l a t e d

c y t o s o l i n t h e c o l d . Exposure o f i n a c t i v e s t e r o i d - r e c e p t o r complex t o a h i g h c o n c e n t r a t i o n o f monovalent c a t i o n s , a n d / o r t o 40 mmol/l T r i s b u f f e r s a t pH 7 . 4 o r a p r o l o n g e d s t a y i n d i l u t e s o l u t i o n s d u r i n g f r a c t i o n a t i o n procedures, causes

130 t h e i r t r a n s f o r m a t i o n t o s t e r o i d - r e c e p t o r complexes w i t h reduced a c i d i t y ' t h a t b i n d t o n u c l e i , c h r o m a t i n and DNA o r t o columns o f p o l y a n i o n s analogous t o DNA8

.

Another s e r i o u s o b s t a c l e i n s t u d y i n g t h e p h y s i c a l p r o p e r t i e s o f t h e s t e r o i d r e c e p t o r s i s t h e i r g r e a t tendency t o f o r m l a r g e i r r e v e r s i b l e aggregates i n hypot o n i c s o l u t i o n s . T h i s tendency t o aggregate i s a l s o m a i n t a i n e d by p u r i f i e d r e ceptor preparations

9.

Sodium dodecyl s u l p h a t e (SDS) i s g e n e r a l l y used t o overcome t h i s o b s t a c l e , b u t under t h e d e n a t u r i n g a c t i o n o f t h i s d e t e r g e n t t h e s t e r o i d r e c e p t o r s l o s e t h e i r b i n d i n g a c t i v i t y . Successful r e s u l t s a v o i d i n g a g g r e g a t i o n w i t h o u t a f f e c t i n g b i n d i n g p r o p e r t i e s o f e s t r o g e n r e c e p t o r (ER) were achieved w i t h l o w doses o f heparin"

o r m i l d d i g e s t i o n w i t h t r y p s i n 1 l Y l 2 . However, b i n d i n g o f l o w doses o f

h e p a r i n t o g l u c o c o r t i c o i d r e c e p t o r s (GR) o f t h e n e u r a l r e t i n a , which p r e v e n t s a g g r e g a t i o n o f GR i n t h e c y t o s o l , caused an i n c r e a s e i n t h e s e d i m e n t a t i o n c o e f f i c i e n t o f t h e n u c l e a r GR o f t h i s t i s s u e 1 3 . M i l d " t r y p s i n i z a t i o n " t r a n s f o r m s t h e ER t o a l e s s a c i d i c form14 and a l s o causes i t s f r a g m e n t a t i o n upon exposure t o 15

buffers o f high i o n i c strength

.

The l a b i l i t y o f t h e s t e r o i d r e c e p t o r s t o e x p e r i m e n t a l c o n d i t i o n s , t h e m u l t i p l e forms o f s t e r o i d - r e c e p t o r complexes formed ( o r r e s o l v e d ) d u r i n g f r a c t i o n a t i o n and t h e d i s c r e p a n c y i n t h e b e h a v i o u r o f s t e r o i d r e c e p t o r s between g e l f i l t r a t i o n i t d i f f i c u l t , i f n o t imposand sucrose d e n s i t y g r a d i e n t ~ e d i m e n t a t i o n " ~make ~

s i b l e , t o e s t a b l i s h t h e Mr and s u b u n i t c o m p o s i t i o n o f t h e r e c e p t o r i n i t s n a t i v e form. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) i s a l m o s t a r o u t i n e method i n re#search on s t e r o i d r e c e p t o r s . However, s t e r o i d r e c e p t o r s aggregate a t t h e l o w i o n i c s t r e n g t h o f PAGE and by p l u g g i n g t h e s u r f a c e o f t h e r e s o l v i n g g e l c o n s t i t u t e a s e r i o u s o b s t a c l e t o t h e use o f PAGE as an a n a l y t i c a l technique, PAGE was t h e r e (non-restricf o r e 1 i m i t e d t o " n o n - r e s t r i c t i v e " , 5 6 % t o t a l g e l c o n c e n t r a t i o n s 17318 6 t i v e o n l y t o molecules o f M, l e s s t h a n 0.5.10 ) o r t o a p p l i c a t i o n on denatured r e c e p t o r p r e p a r a t i o n s and f r a c t i o n a t i o n under d e n a t u r i n g c o n d i t i o n s i n e i t h e r SDS-PAGE o r PAGE i n a c i d i c urea.

One o f t h e most common a p p l i c a t i o n s o f PAGE has been t o m o n i t o r t h e p u r i f i c a t i o n o f s t e r o i d r e c e p t o r s , a f t e r t h e i r f r a c t i o n a t i o n by v a r i o u s chromatographic techniques. The p u r i f i e d e s t r o g e n r e c e p t o r (ER) o f c a l f u t e r u s was a n a l y s e d by PAGE and proved t o be a s i n g l e homogeneous p r o t e i n , w i t h an apparent Mr o f 70,00010, 19 t e r m i n e d by SDS-PAGE a c c o r d i n g t o Weber and Osborn

de-

.

S i m i l a r M, e s t i m a t e s (60,000-70,000)

were o b t a i n e d by g e l f i l t r a t i o n a n a l y s i s

o f t h e a c t i v e receptor species i s o l a t e d from various steroid-responsive tissues

20,21 .

The progesterone r e c e p t o r (PR) was f r a c t i o n a t e d i n t o two d i s t i n c t r e c e p t o r s u b u n i t s with d i f f e r e n t intranuclear functions: subunit 6, "the specifier", contains the

131 c h r o m a t i n b i n d i n g s i t e , and s u b u n i t A, t h e " r e g u l a t o r " o f RNA c h a i n i n i t i a t i o n s i t e s , c o n t a i n s t h e DNA b i n d i n g s i t e .

I t was proposed t h a t s u b u n i t B d i r e c t s s u b u n i t A t o t h e p r o p e r s i t e s i n t h e genome22

.

The p u r i f i e d s u b u n i t B o f PR was e x t e n s i v e l y analysed by PAGE under v a r i o u s experimental c o n d i t i o r ~ s ~under ~; n o n - d e n a t u r i n g c o n d i t i o n s i n 6.5% g e l s a c c o r d i n g t o T i n d a l l e t a1.l8,

under d e n a t u r i n g c o n d i t i o n s i n SDS-PAGE i n a d i s c o n t i n u o u s

b u f f e r system a c c o r d i n g t o L a e ~ n m l and i ~ ~ i n a c i d i c u r e a PAGE, by a m o d i f i c a t i o n

o f t h e method d e s c r i b e d by Maureri15. P u r i f i e d s u b u n i t B was t h e r e b y proved t o be a s i n g l e homogeneous p r o t e i n w i t h an a p p a r e n t M~ of 117,000 determined by SDSPAGE. The homogeneity o f p u r i f i e d s u b u n i t A o f PR was a l s o assessed by a c i d i c urea PAGE and SDS-PAGE26. S u b u n i t A was a l s o r e s o l v e d as a s i n g l e p r o t e i n w i t h an app a r e n t M, o f 79,000 ( i n SDS-PAGE). The two s u b u n i t model o f PR may, however, t u r n o u t t o be inadequate because, as r e c e n t l y r e p o r t e d 2 7 y 2 8 , DNA b i n d i n g a c t i v i t y was a l s o d e t e c t e d i n s u b u n i t B . The p o s s i b i l i t y t h a t s u b u n i t A may d e r i v e f r o m 27 s u b u n i t B by p r o t e o l y t i c cleavage o f B i s b e i n g i n v e s t i g a t e d . The u n i q u e c o n t r i b u t i o n o f e l e c t r o p h o r e t i c methods i n r e s e a r c h on s t e r o i d r e c e p t o r s was t h e s e p a r a t i o n , on t h e b a s i s o f t h e i r h e t e r o g e n e i t y i n n e t charge, between t h e a c t i v e and i n a c t i v e s t e r o i d - r e c e p t o r complexes. IEF o f s t e r o i d r e c e p t o r s r e v e a l e d t h a t a c t i v a t i o n o f s t e r o i d r e c e p t o r (SR) by h e a t t r a n s f o r m s t h e r e c e p t o r s i n t o a l e s s a c i d i c form. As shown i n T a b l e 9.1,

t h e more b a s i c a p p a r e n t

i s o e l e c t r i c p o i n t s ( p r ) o f t h e a c t i v a t e d r e c e p t o r s seem t o b e a g e n e r a l p r o p e r t y o f s t e r o i d r e c e p t o r s ( e x c e p t p o s s i b l y f o r t h a t o f t h e l i v e r GR35, 36 e v e r open t o doubt ).

which i s how-

I t i s i n t e r e s t i n g t h a t t h e GR e x t r a c t e d f r o m t h e s o l u b l e f r a c t i o n o f l i v e r

n u c l e i ( a f t e r f r e e z i n g and t h a w i n g ) focuses a t pH 6.7 ( s i m i l a r t o t h e p l o f t h e 20 a c t i v a t e d f o r m i n t h e c y t o s o l ) and a t pH 7.5 . IEF was a p p l i e d by Wrange e t a1 . I 2 as an e f f i c i e n t method f o r t h e d e t e c t i o n and q u a n t i t a t i o n o f e s t r o g e n r e c e p t o r s . The a r e a o f t h e r a d i o a c t i v e peak a t t h e c h a r a c t e r i s t i c p l o f t h e r e c e p t o r s p e c i e s was used as a measure o f ER c o n t e n t i n t h e c y t o s o l . A c c o r d i n g t o Wrange e t a1

., a n a l y s i s

and q u a n t i t a t i o n o f ER f r o m

v a r i o u s t i s s u e s by IEF i s most s p e c i f i c and s e n s i t i v e and t h e r e f o r e m i n i m i z e s f a l s e l y h i g h e s t i m a t e s o f r e c e p t o r due t o c o n t a m i n a t i n g n o n - r e c e p t o r e s t r o g e n binding proteins with different P L iI m iS t e d t r y. psin digestion before electrof o c u s i n g r e s u l t e d i n t h e f o r m a t i o n o f s h a r p e r peaks w i t h h i g h e r s p e c i f i c r a d i o a c t i v i t y and improved t h e IEF o f e s t r o g e n r e c e p t o r s i n cancerous b r e a s t t i s s u e , 15 l i v e r and u t e r u s c y t o s o l s The use o f PAGE as a method f o r t h e c h a r a c t e r i z a t i o n o f i n t a c t n a t i v e s t e r o i d

.

r e c e p t o r s i s l i m i t e d p r i m a r i l y because t h e SR complexes aggregate under t h e e l e c t r o p h o r e t i c c o n d i t i o n s and do n o t p e n e t r a t e i n t o t h e r e s o l v i n g g e l , i n v i e w o f

132 TABLE 9.1 APPARENT ISOELECTRIC POINTS OF STEROID RECEPTORS

RIA)^

Steroid

Source

IP

Androgen

Rat epididymis Rat v e n t r a l p r o s t a t e Rat v e n t r a l p r o s t a t e Rat v e n t r a l p r o s t a t e Mouse kidney

5.8 5.8 5.8 5.8 4.8~

Calf uterus Calf uterus Rat uterus Rat u t e r u s Rat uterus Rat 1 i v e r

5.8 6.2 5.84. 5.8

Human mammary carcinoma

5.5

Mouse f i b r o b l a s t

5.9 (74%)

Estrogen

G1 UCOc o r t i c o id

Rat Rat Rat Rat

1i v e r 1i v e r 1i v e r kidney

Rat 1 i v e r

Progesterone Chick o v i d u c t

P I (RAP

18

o

7.1. (6.6,6. l ) h 4.3'

6.5 6.5

29 30 31

6.4 6.6,6.8 6.5-6.7 6.5 6.3-6.7 6.3-6.7e 6.4-6.8f

32 14 33d 34 12 12 15

6.4 (21%) 6.9 (5%) 6.1 (7.1)h 6.7 6.7,7.5j

219

4.9 5.9k 4.7-5.71 5.6-5.9"' 5.7k

38 39

4.8

a Inactive receptor. A c t i v e r e c e p t o r (binds t o n u c l e i , chromatin o r DNA). Both b e f o r e and a f t e r ammonium sulphate p r e c i p i t a t i o n . After proteolysis. Female. Male. Trypsinized. Derived from c y t o s o l and nucleus. More a c i d i c than t r a n s c o r t i n . Nuclear n a t i v e . Mero-receptor. 61 36 Subunit B. Subunit A.

8.

8'.

35 36 20 37

38

4.0n 4.50

5

Ref.

133 t h e r e s t r i c t e d m o b i l i t y o f l a r g e molecules i n t h e s m a l l p o r e s o f c o n v e n t i o n a l p o l y a c r y l a m i d e g e l s . The use o f c o n v e n t i o n a l r e l a t i v e l y open-pore 5% g e l s proved t o be i n s t r u m e n t a l i n t h e i d e n t i f i c a t i o n and q u a n t i t a t i o n o f n a t i v e s t e r o i d r e c e p t o r s i n unknown samples, b u t p r o v i d e s l i m i t e d i n f o r m a t i o n as t o t h e i r m o l e c u l a r a r c h i t e c t u r e . The f i r s t a t t e m p t s t o c h a r a c t e r i z e t h e n a t i v e s t e r o i d r e c e p t o r s i n t h e c y t o s o l by q u a n t i t a t i v e PAGE were made by Sherman e t a1.40 and developed f u r t h e r by M i l l e r e t a1.41 and Sherman e t a 1 . I 6 . The l a t t e r s t u d i e s w i t h p r o g e s t e r o n e r e c e p t o r s were f a c i l i t a t e d by t h e use o f h i g h l y c r o s s - 1 i n k e d p o l y a c r y l a m i d e g e l s (15% o f N,N'-methylenebisacrylamide was used as t h e c r o s s - l i n k e r ) 42 shown t o be l e s s r e s t r i c t i v e t h a n c o n v e n t i o n a l 2-5% c r o s s - l i n k e d g e l s 3 Two [ H] progesterone-bound macromol e c u l a r complexes were d e t e c t e d a f t e r t h e

.

electrophoresis o f

L3 H]progesterone-label

l e d oviduct cytosol

.

Complex I , " c y t o s o l

slow" complex, i s r e s o l v e d as a h e t e r o d i s p e r s e peak o f Mr i n t h e range 609,000899,000 (computed a c c o r d i n g t o Rodbard and Chrambach

43

) ; complex I , which pos-

s i b l y r e p r e s e n t s v a r i o u s s i z e s o f s t e r o i d - r e c e p t o r aggregates, decreases a f t e r t r e a t i n g t h e c y t o s o l w i t h 0.5 m o l / l KC1. Complex 11, " c y t o s o l f a s t " complex, i s r e s o l v e d as a sharp peak w i t h a mean Mr o f 156,000 (range 117,000-201,000);

com-

p l e x I 1 i s a p p a r e n t l y n o t a f f e c t e d by exposure t o h i g h s a l t c o n c e n t r a t i o n s . complex I d i s s o c i a t e s a t h i g h s a l t c o n c e n t r a t i o n s i n t o two s m a l l e r PR

fH]PR

complexes, w h i c h were r e s o l v e d by f i l t r a t i o n t h r o u g h agarose A 0.5 m columns, e q u i l i b r a t e d w i t h 0.4 m o l / l KC1. One i s i d e n t i c a l w i t h complex I1 ( " c y t o s o l f a s t " ) and a n o t h e r , d e s i g n a t e d complex 111, i s r e s o l v e d by PAGE as an M, 115,000 species

.

A c c o r d i n g t o Sherman e t a 1 . l 6 , subunits

forms I1 and I11 correspond t o p r o g e s t e r o n e

B and A, r e s p e c t i v e l y , d e s c r i b e d by Schrader and O ' M a l l e ~ ~The ~ . higher

Mr v a l u e s o b t a i n e d by PAGE (158,000 and 115,000 compared w i t h 117,000 o f s u b u n i t B

and 79,000 o f s u b u n i t A ) were suggested t o b e o v e r e s t i m a t e s due i n p a r t t o n e g l e c t 16

o f t h e h i g h l y asymmetric r e c e p t o r shape

.

I n t h e presence o f CaC12, b o t h forms a r e c o n v e r t e d i n t o f o r m IV, a m i n o r cons t i t u e n t , and f o r m V , t h e "mero-receptor",

a small g l o b u l a r polypeptide t h a t

c o n t a i n s t h e s t e r o i d - b i n d i n g si te37-39. The " m e r o - r e c e p t o r " l a c k s t h e b i n d i n g s i t e t o c h r o m a t i n and i t s p h y s i o l o g i c a l s i g n i f i c a n c e i s unknown. Q u a n t i t a t i v e PAGE as developed by Chrambach e t a1 .45 has been used r e c e n t l y f o r t h e c h a r a c t e r i z a t i o n of t h e n a t i v e g l u c o c o r t i c o i d r e c e p t o r s (GR) i n t h e c y t o s o l o f t h e n e u r a l r e t i n a of t h e c h i c k embryo46. Attempts were made t o o p t i m i z e t h e c o n d i t i o n s of PAGE so as n o t t o l o s e any GR s p e c i e s t h a t a r e p r e f e r e n t i a l l y s e n s i t i v e t o t h e e x p e r i m e n t a l procedures. T h i s was achieved by ( a ) s a t u r a t i n g t h e GR w i t h (fH]TA)

3

H]triamcinolone

acetonide

i n t h e i n t a c t t i s s u e , t h u s p r o t e c t i n g t h e l a b i l e r e c e p t o r s by a s t a b l e

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

134 b u f f e r system c o m p a t i b l e w i t h s t a c k i n g o f a t l e a s t 90% o f t h e s p e c i f i c a l l y bound fqTA

complexes,

( c ) p e r f o r m i n g PAGE i n h i g h l y c r o s s - l i n k e d (15%) r e s o l v i n g

g e l s , u s i n g N,N'-diallyltartardiamide (DATD) as t h e c r o s s - l i n k e r , and ( d ) O p t i 3 HITA-bound m i z i n g t h e c o n d i t i o n s o f PAGE t o ensure t h e q u a n t i t a t i v e r e c o v e r y o f

L

complexes. PAGE was performed a t l o w i o n i c s t r e n g t h , a t l o w power and under s t r i c t temperature c o n t r o l a t -2OC w i t h 10% g l y c e r o l i n a l l phases o f t h e e l e c t r o p h o r e t i c system17. Under t h e s e a p p a r e n t l y o p t i m i z e d PAGE c o n d i t i o n s i t has been p o s s i b l e 3 k f P 175,000 ( d e s i g n a t e d L H I T A complex 1 1 ) , t o

t o c h a r a c t e r i z e a GR complex o f

d e f i n e t h r e e m o l e c u l a r species t h a t comprise t h e GR and t o deduce t h e p o s s i b l e r e l a t i o n s h i p s between them. The e l e c t r o p h o r e t i c a l l y i s o l a t e d TA complex I 1 d i s s o c i a t e s (under t h e cond i t i o n s o f a PAGE r e - r u n ) i n t o two components: "component 8 " o f My 108,000 and "compbnent A",

a f a s t m i g r a t i n g r e c e p t o r s u b u n i t w h i c h c o u l d n o t be c h a r a c t e r i z e d

under t h e c o n d i t i o n s o f PAGE used ("component A" w i l l be r e f e r r e d t o as s u b u n i t

RX). As t h e g e l c o n c e n t r a t i o n i s i n c r e a s e d ( f r o m 6 t o 8 % T ) , t h e TA complex I1 aggregates t o f o r m TA complex I , a l a r g e r e c e p t o r aggregate (kfP>500,000)

that i s

unable t o e n t e r i n t o r e s o l v i n g g e l s o f 6% o r h i g h e r g e l c o n c e n t r a t i o n . The r a t i o o f t h e number of fH]TA-binding o f fH]TA-binding

s i t e s i n f r a c t i o n B t o t h e number

s i t e s i n RX i s c o n s t a n t and c l o s e t o 2, i r r e s p e c t i v e o f t h e i r

r e a s s o c i a t i o n t o f o r m complex I , as a f u n c t i o n o f g e l c o n c e n t r a t i o n . T h i s f i n d i n g

3

i n d i c a t e s t h a t s u b u n i t RX and s u b u n i t B a r e d e f i n e d components o f LH]TA comDlex 11. 3 When LH]TA complex I which f a i l e d t o e n t e r i n t o 8%T, 15%CDATDg e l s was a n a l 3 ysed (4-5.5%T)i t gave r i s e t o an k f r 61,000 LH]TA r e c e p t o r u n i t ( d e s i g n a t e d as "component C " i n r e f . 46). I t has been suggested t h a t t h e Mr 60,000 s u b u n i t i n v a r i o u s s t e r o i d r e c e p t o r systems i s formed by t h e p r o t e o l y t i c c l e a v a g e o f t h e 14,48 l a r g e r i n a c t i v e r e c e p t o r complex

.

The f i n d i n g t h a t t h e kfr 60,000 species i s formed o n l y d u r i n g t h e r e - e l e c t r o p h o r e s i s o f i s o l a t e d L3q T A complex I and n o t upon r e - e l e c t r o p h o r e s i s o f complex

I1 suggests t h a t TA complex I i s e i t h e r contaminated w i t h a p r o t e a s e o r t h a t t h e r e c e p t o r i t s e l f c o n t a i n s i t s own p r o t e o l y t i c u n i t ; however, under t h e c o n d i t i o n s o f PAGE, i t s a c t i v i t y c o u l d be expressed o n l y under t h e c o n d i t i o n s t h a t p r e v a i l i n a h i g h l y c o n c e n t r a t e d p r o t e i n zone (temperature-dependent e f f e c t ) and/or i n a s s o c i a t i o n w i t h another TA-receptor complex i n a f u n c t i o n a l aggregate. The l a t t e r a l t e r n a t i v e i s supported by t h e f o l l o w i n g f i n d i n g s : ( a ) t h e p r o t e o l y t i c cleavage o f l i v e r GR can a l s o o c c u r i n t h e nucleus38; the

(b) subunit A o f

PR i s r e l e a s e d from t h e 6s PR complex t h a t i s bound t o c h r o m a t i n ; i f s u b u n i t A

i s a p r o t e o l y t i c p r o d u c t o f t h e l a r g e r s u b u n i t B y a p o s s i b i l i t y t h a t was r e c e n t l y suggested27, i t would a l s o suggest t h a t t h e p r o t e o l y t i c a c t i v i t y may r e s i d e i n t h e 6s PR complex; ( c ) p r o t e a s e s u b s t r a t e s and p r o t e a s e i n h i b i t o r s i n t e r a c t d i -

135 r e c t l y w i t h t h e DOC r e c e p t o r i n a dog k i d n e y MDCK c e l l l i n e . These i n t e r a c t i o n s 3 49 i n h i b i t binding o f [ HIDOC t o t h e receptor

.

The p o s s i b i l i t y t h a t a c o n s t i t u e n t o f t h e p r o g e s t e r o n e r e c e p t o r may a c t as a Ca2+-dependent p r o t e a s e was suggested by Sherman e t a l .39 t o e x p l a i n t h e c o i n c i dence t h a t t h e p r o g e s t e r o n e r e c e p t o r and Ca2+-binding p r o t e i n s i n t h e c y t o s o l o f t h e o v i d u c t focused a t t h e same i s o e l e c t r i c p o i n t . I n comparing t h e r e s u l t s o b t a i n e d by PAGE o f t h e n a t i v e GR w i t h t h o s e o b t a i n e d by PAGE o f t h e n a t i v e PR and w i t h t h e m o l e c u l a r c h a r w t e r i s t i c s o f t h e s e and some o t h e r s t e r o i d r e c e p t o r s o b t a i n e d by o t h e r s e p a r a t i o n t e c h n i q u e s , i t becomes obvious t h a t s t e r o i d r e c e p t o r s o f t h e v a r i o u s t a r g e t t i s s u e s i n t h e v e r t e b r a t e have a b a s i c s i m i l a r m o l e c u l a r s t r u c t u r e . Table 9.2 p r e s e n t s some examples. F o r comparison, t h e m u l t i p l e forms o f t h e s t e r o i d - r e c e p t o r complexes have been numbered f r o m

I t o V I i n order o f t h e i r

molecular s i z e . Form I i n c l u d e s a wide range o f l a r g e s t e r o i d - r e c e p t o r complexes, p o s s i b l y r e p r e s e n t i n g aggregated s t a t e s o f SR. These aggregates a r e formed, a t l e a s t i n p a r t , d u r i n g t h e p r e p a r a t i o n and f r a c t i o n a t i o n o f t h e c y t o s o l i n h y p o t o n i c b u f f e r s . However, GR complexes o f M, 355,000 were a l s o r e v e a l e d by g e l f i l t r a t i o n 21 experiments i n t h e presence o f 0.15 m o l / l KC1

.

Form I 1 i s r e s o l v e d by q u a n t i t a t i v e PAGE i n h i g h l y c r o s s - l i n k e d g e l s as a s t e r o i d complex o f average M~ 175,000 and by s e d i m e n t a t i o n experiments i n l o w - s a l t sucrose g r a d i e n t s as a 6s complex o f i d e n t i c a l MP2'.

T h i s f o r m o f s t e r o i d - r e c e p t o r com-

p l e x p r o b a b l y r e p r e s e n t s t h e i n a c t i v e s t a t e o f SR complexes i n t h e c y t o s o l . Form I 1 i s t r a n s f o r m e d t o f o r m I 1 1 by two d i f f e r e n t e x p e r i m e n t a l procedures: by i n c r e a s i n g t h e i o n i c s t r e n g t h ( t h e c o n c e n t r a t i o n o f i s o t o n i c monovalent c a t i o n i s s u f f i c i e n t f o r t h i s t r a n s f o r m a t i o n 1 4 y 2 1 ) , o r by r e m a i n i n g i n d i l u t e s o l u t i o n during prolonged f r a c t i o n a t i o n

procedure^^^.

The f o r m e r causes t h e f o r m a t i o n o f

t h e 4s r e c e p t o r complex, r e v e a l e d by s e d i m e n t a t i o n experiments i n 0.15 m o l / l KC1 sucrose ( o r g l y c e r o l ) g r a d i e n t s . The l a t t e r causes t h e f o r m a t i o n o f f o r m I 1 1 d u r i n g t h e PAGE r e - r u n o f i s o l a t e d TA complex 11. The i d e n t i c a l M, e s t i m a t e s o f f o r m 111 (109,000,

108,000) o b t a i n e d by t h e two d i f f e r e n t r o u t e s , c a l c u l a t e d by

two d i f f e r e n t methods, i s s t r i k i n g . As p r e s e n t e d i n T a b l e 9.2,

p r o g e s t e r o n e " s u b u n i t B" may be analogous t o f o r m

111. It appears t h a t form 111 i s t h e f i r s t s t a g e i n t h e s t e r o i d - r e c e p t o r a c t i v a -

t i o n process, c h a r a c t e r i z e d by t h e f o l l o w i n g :

(1) I t i s t r i g g e r e d b y t h e b i n d i n g o f t h e i n d u c i n g s t e r o i d ; ( 2 ) i t i s a c c e l e r a t e d by an i n c r e a s e i n i o n i c s t r e n g t h ; ( 3 ) i t develops w i t h t i m e i n d i l u t e d s t e r o i d - r e c e p t o r p r e p a r a t i o n s ; ( 4 ) i t i s n o t dependent on t e m p e r a t u r e .

TABLE 9.2

Mp ESTIMATES OF MULTIPLE FORMS OF STEROID RECEPTORS Form

I

I1

M,, x 10-3 Estrogen

G1 u c o c o r t i c o i d

U t e r u s (9,13)

F i b r o b l a s t s (20)

Neural r e t i n a ( 4 5 )

O v i d u c t (15,40)

620a 355e

>500h

609-900h

430a

620

230

8*6s

I11

118

IV

61 7oc

4.5s

1

172

6s l b 4s

Progesterone

1

-h

175h

ii -

108 + Xh (2:l)

O v i d u c t (21,26)

1

158h (117-200) 115h

-c

$el f i l t r a t i o n . 0.15 mol/l KC1. %s- PAGE. dprotease ~ a 2 + F0.15 r n o l / l K C i , g e l f i l t r a t i o n . Nuclear receptor. hgIsopycnic s e d i m e n t a t i o n . PAGE.

i PAGE r e - r u n . i 0 . 4 m o l / l KC1. l C a l c u l a t e d f r o m Rs,s2ow. N a t i v e dimer. 'Subunit B. 'Subunit A. 'Native. PAmmonium s u l f a t e p r e c i p i t a t e ; ion-exchange chromatography.

137 The physico-chemical changes induced i n t h e SR complex a t t h i s stage a r e : ( 1 ) decrease i n M, ( f r o m 175,000 t o 108,000);

( 2 ) decrease i n n e g a t i v e n e t charge; and consequently, ( 3 ) increase i n t h e i s o e l e c t r i c p o i n t (Table 9.1). These molecular changes f a c i l i t a t e ( a ) t h e b i n d i n g o f form I 1 1 t o chromatin and ( b ) t h e p r o t e o l y t i c cleavage o f form I11 t o form I V . The second stage i n s t e r o i d r e c e p t o r a c t i v a t i o n may i n v o l v e t h e p r o t e o l y t i c cleavage o f form 111, which i s temperature dependent. Form I 1 1 i s cleaved i n t o a p p a r e n t l y n o n - i d e n t i c a l u n i t s . One, which c o n t a i n s t h e s t e r o i d - b i n d i n g s i t e , i s form I V i n Table 9.2. T h i s My 60,000 u n i t , which presumably i s t h e s m a l l e s t n a t i v e f u n c t i o n a l SR complex i n t h e c y t o s o l , i s a l s o t h e main species o f SR complexes t h a t a r e e x t r a c t e d from steroid-bound n u c l e i o f v a r i o u s responsive t i s s u e s w i t h h i g h - s a l t b u f f e r s . However, a GR complex o f t h i s dimension has been e x t r a c t e d w i t h a l o w - s a l t b u f f e r from r a t l i v e r n u c l e i a f t e r 20 freezing-thawing

.

The second u n i t which i s released by p r o t e o l y t i c cleavage o f form I 1 1 was n o t detected i n these

experiment^^^

as i t i s n o t l a b e l l e d by t h e r a d i o a c t i v e s t e r o i d

marker. It i s tempting t o suggest t h a t t h i s u n i t c o n t a i n s t h e c a t a l y t i c s i t e which

cleaves SR form 111. I n o r d e r t o express i t s p r o t e o l y t i c f u n c t i o n i t may r e q u i r e t h e p r o x i m i t y o f another i d e n t i c a l SR complex as i t s s u b s t r a t e , thus f o r m i n g a f u n c t i o n a l aggregate, SR form I. TA complex R X i n Table 9.2 c o u l d n o t be c h a r a c t e r i z e d under t h e s p e c i f i c cond i t i o n s of PAGE used because i t s m o b i l i t y exceeded t h e boundary displacement r a t e o f t h e " f r o n t " marked by haemoglobin. This would i n d i c a t e , however, t h a t TA-X i s a newly r e s o l v e d a c i d i c macromolecule which i s released under PAGE c o n d i t i o n s from GR form 11. TA-X may r e p r e s e n t a small GR u n i t analogous t o form I V o r form V i n Table 9.2,

o r , as seems more l i k e l y , a GR u n i t o f M, about 67,000,

w i t h a nega-

t i v e charge h i g h e r than t h e n e t n e g a t i v e charge o f haemoglobin. 3 Results from sedimentation experiments o f LHJTA-GR o f t h e n e u r a l r e t i n a i n g l y c e r o l g r a d i e n t s r e v e a l e d t h a t about 1/3 o f t h e s p e c i f i c TA b i n d i n g s i t e s are i r r e v e r s i b l y l o s t i n t h e process o f heat a c t i v a t i o n o f TA-GRs i n t h e presence o f 150 mmol/l KC151. Reduced c a p a c i t y f o r r e b i n d i n g t h e s t e r o i d was a l s o r e p o r t e d f o r t h e a c t i v a t e d GR o f r a t l i v e r c y t o s 0 1 ~and ~ GR o f c u l t u r e d L929

fibroblast^^^.

A pref-

e r e n t i a l l o s s o f t h e s t e r o i d - b i n d i n g s i t e from s u b u n i t RX by s a l t w i l l e x p l a i n t h e f a i l u r e t o d e t e c t a s i m i l a r R s u b u n i t i n t h e s t u d i e s o f o t h e r s t e r o i d R systems. The k i n e t i c s o f g l u c o c o r t i c o i d b i n d i n g t o GR i n t h e neural r e t i n a 5 3 and t h e ext e n s i v e s t u d i e s on t h e k i n e t i c s of g l u c o c o r t i c o i d b i n d i n g t o GR o f mouse f i b r o b l a s t c y t o s o l , r e p o r t e d by P r a t t e t a1.54, a r e compatible w i t h t h i s idea.

138 = Number of Steroid Binding Sines

R = Native Receptor in Cytosol = GR Complex = Labelled Steroid Hormoi

RIA = Inactive Receptor Unit RA = Activated Receptor Unit RR = The Active Regulator C = Catalytic Unit RX = R, = Negative Control Element (7

f i g . 9.1. Sequence o f m o l e c u l a r t r a n s f o r m a t i o n s o f GR o f t h e n e u r a l r e t i n a r e vealed by PAGE under n o n - d e n a t u r i n g c o n d i t i o n s . These f i n d i n g s suggests t h a t t h e t r a n s f o r m a t i o n o f t h e i n a c t i v e GR f o r m I 1 Mp (175,000) i n t o t h e a c t i v a t e d , l e s s a c i d i c GR f o r m I11 M, (108,000) i s due t o t h e removal o f an a c i d i c r e c e p t o r s u b u n i t f r o m t h e i n a c t i v e GR complex. RX may r e p r e s e n t t h e i n h i b i t o r y c o n t r o l u n i t t h a t m a i n t a i n s t h e s t e r o i d r e c e p t o r comp l e x i n t h e i n a c t i v e s t a t e , and hence i n t h e c y t o s o l . The s u g g e s t i o n t h a t an i n h i b i t o r o f a c t i v a t i o n m i g h t e x i s t i n r a t l i v e r c y t o s o l was f i r s t made by M i l g r o m e t a l . 5 5 on t h e b a s i s o f enhanced a c t i v a t i o n a f t e r p a r t i a l p u r i f i c a t i o n o f t h e r e c e p t o r . The i n v o l v e m e n t o f a s m a l l a c i d i c c o n s t i t u e n t o f t h e c y t o s o l i n t h e process o f l i v e r GR a c t i v a t i o n was shown by g e l f i l t r a t i o n experiments50y56, However, i t was a l s o shown t h a t t h e i n h i b i t o r y f a c t o r i n HTC c e l l s i s a s s o c i a t e d w i t h t h e macromolecular f r a c t i o n 57

.

The p o s s i b i l i t y t h a t an a c i d i c c o n s t i t u e n t i s an i n t e g r a l p a r t o f t h e s t e r o i d r e c e p t o r complex r e l e a s e d a f t e r b i n d i n g t h e s t e r o i d l i g a n d by i s o t o n i c s a l t o r by d i l u t i o n may s e t t l e t h i s d i s c r e p a n c y and account f o r t h e e f f e c t o f s a l t and d i l u t i o n on s t e r o i d r e c e p t o r a c t i v a t i o n . F i g . 9.1 summarizes t h e sequence o f events i n t h e t r a n s f o r m a t i o n o f GR o f t h e n e u r a l r e t i n a as r e v e a l e d b y PAGE under s t r i c t l y c o n t r o l l e d e x p e r i m e n t a l c o n d i t i o n s . The e s t i m a t e s o f M, f o r t h e v a r i o u s GR species, a l t h o u g h n o t v a l i d a t e d s t a t i s t i c a l l y , a r e i n s t r i k i n g agreement w i t h much o f what i s known i n t h i s f i e l d o f r e s e a r c h ( T a b l e 9.2). The p h y s i c a l c h a r a c t e r i z a t i o n o f C3q T A component RX seems t o be c r u c i a l f o r o u r u n d e r s t a n d i n g o f t h e mechanism o f s t e r o i d hormone action.

139 REFERENCES

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141

Chapter 9.6 CELL SURFACE ANTIGENS

R.A.

REISFELD and M.A. PELLEGRINO Among t h e v a r i o u s b i o c h e m i c a l t e c h n i q u e s developed d u r i n g t h e l a s t 20 y e a r s ,

p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) has made t h e most d e c i s i v e impact on o u r u n d e r s t a n d i n g o f t h e s t r u c t u r e and f u n c t i o n o f c e l l s u r f a c e macromolecules. I n f a c t , i t was r e a l l y t h e a d v e n t o f PAGE, i s o e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e g e l s (IFPA), PAGE i n t h e presence o f sodium dodecyl s u l p h a t e (SDS-PAGE) and IFPA and SDS-PAGE combined i n t o two-dimensional g e l e l e c t r o p h o r e s i s (2D-PAGE) t h a t made i t p o s s i b l e f o r t h e f i r s t t i m e t o i s o l a t e and c h a r a c t e r i z e t h o r o u g h l y h i g h l y complex and h y d r o p h o b i c c e l l s u r f a c e macromolecules. A f u r t h e r a p p l i c a t i o n o f SDS-PAGE, t h e a n a l y s i s o f i m m u n o p r e c i p i t a t e s o f c e l l s u r f a c e macromolecules obt a i n e d w i t h s p e c i f i c a n t i s e r a (IP-SDS-PAGE),

p r o v i d e d t h e whole PAGE t e c h n o l o g y

w i t h a h i g h l y s i g n i f i c a n t a d d i t i o n a l dimension t h a t was even f u r t h e r extended b y t h e more r e c e n t development and use o f monoclonal a n t i b o d i e s .

PAGE and i t s v a r i o u s a p p l i c a t i o n s have produced a n e x p l o s i o n o f l i t e r a t u r e d e a l i n g w i t h c e l l s u r f a c e macromolecules. Because o f space l i m i t a t i o n s , no a t t e m p t

w i l l be made h e r e t o p r e s e n t a comprehensive r e v i e w o f t h i s e x t e n s i v e amount o f work o r t o even m e n t i o n t h e many c e l l s u r f a c e macromolecules analysed w i t h t h e a i d o f v a r i o u s PAGE t e c h n i q u e s . I n s t e a d , t h e emphasis o f t h i s b r i e f o v e r v i e w i s s i m p l y t o h i g h l i g h t t h e u n i q u e e x p e r i m e n t a l approaches made p o s s i b l e , e s p e c i a l l y by IP-SDS-PAGE,

t h a t l e d t o t h e e l u c i d a t i o n o f t h e m o l e c u l a r and immunogenetic

p r o f i l e s o f human h i s t o c o m p a t i b i l i t y a n t i g e n s , a s e t o f g e n e t i c a l l y determined, h ig h l y pol ymor ph ic int r ins ic membrane g l y c o p r o t e i ns

.

Two s e t s o f human h i s t o c o m p a t i b i l i t y a n t i g e n s w i l l be discussed, namely HLA and I a - l i k e a n t i g e n s . Again, because o f space l i m i t a t i o n s , t h e l a r g e body o f d a t a o b t a i n e d w i t h h i s t o c o m p a t i b i l i t y a n t i g e n s o f o t h e r animal s p e c i e s (mouse, r a t , guinea p i g ) w i l l n o t be mentioned as t h e y a r e o f t e n s i m i l a r i n c o n c e p t t o d a t a o b t a i n e d w i t h human h i s t o c o m p a t i b i l i t y a n t i g e n s and p u b l i s h e d w i t h i n t h e same period. I n i t i a l attemps made w i t h PAGE t o p u r i f y and c h a r a c t e r i z e t h e p o l y m o r p h i c and hydrophobic h i s t o c o m p a t i b i l i t y a n t i g e n s had two p r e r e q u i s i t e s f o r success: (1) s o l u b i l i t y o f t h e m a t e r i a l s t o be a n a l y s e d and ( 2 ) t h e a v a i l a b i l i t y o f t h e f u n c t i o n a l i n v i t r o and i n v i v o assays t o i d e n t i f y t h e h i s t o c o m p a t i b i l i t y a n t i g e n s w i t h i n a h i g h l y complex a r r a y o f c o n t a m i n a n t s . T h i s approach was s u c c e s s f u l i n

142 t h e hands o f s e v e r a l i n v e s t i g a t o r s who f o l l o w e d t h e p r o g r e s s o f b i o c h e m i c a l p u r i f i c a t i o n and a t t e m p t e d t o d e f i n e t h e m o l e c u l a r p r o f i l e o f human h i s t o c o m p a t i b i l i t y a n t i g e n ~ l - ~A. t y p i c a l f e a t u r e o f t h i s t y p e o f e x p e r i m e n t a l approach was t o d e t e r mine t h e r e l a t i v e m o b i l i t i e s o f h i s t o c o m p a t i b i l i t y a n t i g e n s b y i d e n t i f y i n g them w i t h i n v i t r o assays f o l l o w i n g t h e i r e l u t i o n f r o m a number o f small a n a l y t i c a l g e l s o r f r o m a few s e m i - p r e p a r a t i v e ge1s5s6. I n a few i n s t a n c e s p r e p a r a t i v e PAGE was a p p l i e d s u c c e s s f u l l y t o t h e i s o l a t i o n o f h i g h l y p u r i f i e d h i s t o c o m p a t i b i l i t y 5-7 a n t i g e n s , b u t r a r e l y w i t h good r e p r o d u c i b i l i t y o r h i g h y i e l d s

.

The a p p l i c a t i o n o f SDS-PAGE added a c o m p l e t e l y new dimension t o t h e m o l e c u l a r c h a r a c t e r i z a t i o n o f hydrophobic c e l l s u r f a c e markers such as h i s t o c o m p a t i b i l i t y a n t i g e n s , e s p e c i a l l y as i t p r a c t i c a l l y c o i n c i d e d w i t h t h e i n c r e a s e d and s u c c e s s f u l use o f d e t e r g e n t s t o s o l u b i l i z e t h e s e hydrophobic c e l l s u r f a c e macromolecules. I n f a c t , SDS-PAGE,

even w i t h o u t t h e use o f i m m u n o p r e c i p i t a t i o n by s p e c i f i c a n t i b o d y ,

made i t p o s s i b l e t o determine t h e HLA a n t i g e n s which i n i t i a l l y were s o l u b i l i z e d m a i n l y b y e i t h e r h y p e r t o n i c s a l t ( 3 m l / l KC1) o r papain and e x i s t e d as a bimolecul a r complex c o n s i s t i n g o f two n o n - c o v a l e n t l y 1 i n k e d p o l y p e p t i d e c h a i n s w i t h molecul a r w e i g h t s o f 34,000 and 12,0008-12.

The s m a l l e r o f t h e two s u b u n i t s t u r n e d o u t t o

be B 2 - m i c r o g l o b u l i n ( B 2 - p ) , a f u l l y sequenced p o l y p e p t i d e t h a t s i n c e i t s d i s c o v e r y i n 196813 was l i t e r a l l y i n search o f a b i o l o g i c a l f u n c t i o n . D e t e r g e n t s o l u b i l i z a t i o n o f HLA a n t i g e n s from lymphoid c e l l s w i t h NP-40 o r sodium d e o x y c h o l a t e r e vealed by e i t h e r SDS- o r sodium deoxycholate-PAGE t h a t t h e s e c e l l s u r f a c e macromolecules c o n s i s t o f a b i m o l e c u l a r complex c o n t a i n i n g a 45,000 d a l t o n heavy c h a i n and a 12,000 d a l t o n l i g h t chain12y14y15 w i t h o n l y t h e heavy c h a i n b e i n g ' g l y c o s y l ated. Again, a few y e a r s l a t e r , SDS-PAGE made i t p o s s i b l e t o e l u c i d a t e f o r t h e f i r s t t i m e t h e b i m o l e c u l a r complex o f t h e I a - l i k e a n t i g e n s . These c e l l s u r f a c e markers, when i s o l a t e d by d e t e r g e n t e x t r a c t i o n o f lymphoid c e l l s , were shown t o c o n s i s t o f two n o n - c o v a l e n t l y l i n k e d p o l y p e p t i d e c h a i n s , an a - s u b u n i t o f M, 34,000 and a 8 - s u b u n i t o f M, 29,00016y17. 30,000 and 23,000,

These a n t i g e n s s o l u b i l i z e d b y p a p a i n had M, 15,16 r e s p e c t i v e l y , when analysed by SDS-PAGE ,

SDS-PAGE a l s o was used s u c c e s s f u l l y t o d e f i n e t h e monomeric and d i m e r i c forms o f d e t e r g e n t - s o l u b i l i z e d , h i g h l y p u r i f i e d HLA a n t i g e n s . Thus, d i m e r i c forms con-

t a i n e d S-S-linked heavy c h a i n s a t t r i b u t a b l e t o a n e a s i l y r e d u c i b l e SH group i n t h e COOH-terminal h y d r o p h i l i c r e g i o n i n s i d e t h e s t r o n g i n t r a c e l l u l a r r e d u c i n g environment o f t h e c e l l cytoplasm. D i m e r i z a t i o n was found due t o e i t h e r SH o x i d a t i o n o r S-S i n t e r c h a n g e c a t a l y s e d by f r e e SH groups. These modes o f dimer format i o n were c o n f i r m e d , as i s o l a t i o n i n t h e absence o f e i t h e r r e d u c i n g o r a l k y l a t i n g agents r e v e a l e d n e i t h e r monomeric n o r d i m e r i c forms c o n t a i n i n g f r e e SH groups. Dimer f o r m a t i o n o c c u r r e d o n l y d u r i n g p u r i f i c a t i o n , as i t was p r e v e n t e d by pret r e a t m e n t o f c e l l membrane c o n t a i n i n g HLA a n t i g e n s w i t h iodoacetamide. SDS-PAGE analyses o f h i g h l y p u r i f i e d d e t e r g e n t - s o l u b i l i z e d a n t i g e n s showed t h a t t h e y con-

143 t a i n a h y d r o p h i l i c r e g i o n a t t h e COOH t e r m i n u s and a p e n u l t i m a t e hydrophobic region18y19. S p e c i f i c a l l y , t h e h i g h l y p u r i f i e d p44,12 complex when t r e a t e d w i t h papain i s c o n v e r t e d i n t o p39,12,

t h e n i n t o ~ 3 4 ~ 1 2which , retain antigenic activi-

t y . Amino t e r m i n a l sequences o f p34 and p44 were i d e n t i c a l and amino a c i d composi-

t i o n s o f p44, p39 and p34 i n d i c a t e d t h a t t h e COOH-terminal p e p t i d e removed by t h e f i r s t papain cleavage i s h y d r o p h i l i c and c o n t a i n s c y s t e i n e , whereas t h e p e n u l t i mate COOH-terminal removed by t h e second p a p a i n cleavage i s hydrophobic and p r o b a b l y anchors HLA a n t i g e n s t o t h e membrane. These experiments u t i l i z i n g SDS-PAGE were t h e f i r s t t o d e f i n e t h e o r i e n t a t i o n and i n t e g r a t i o n o f HLA a n t i g e n s i n t h e 18,19 lymphocyte membrane and suggested t h a t HLA a n t i g e n s span t h e plasma membrane

.

I s o e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e g e l s (IFPA) a p p l i e d as such has f o u n d o n l y l i m i t e d use f o r t h e c h a r a c t e r i z a t i o n o f h i s t o c o m p a t i b i l i t y a n t i g e n s . I n one a p p l i c a t i o n IFPA was used t o show t h a t much o f t h e apparent e l e c t r o p h o r e t i c h e t e r o g e n e i t y o f h i g h l y p u r i f i e d p a p a i n - s o l u b i l i z e d HLA a n t i g e n s was a t t r i b u t a b l e t o v a r i a b l e numbers o f s i a l i c a c i d r e s i d u e s on each m o l e c u l e . The l a c k o f e l e c t r o p h o r e t i c h e t e r o g e n e i t y found by IFPA, f o l l o w i n g neuraminidase t r e a t m e n t , was i m p o r t a n t as i t i m p l i e d a l a c k o f any c o n s i d e r a b l e amino a c i d sequence m i c r o h e t e r o g e n e i t y and t h u s i n d i c a t e d t h e f e a s i b i l i t y o f amino a c i d sequence s t u d i e s o f t h e s e r o l o g i c a l 1y h i g h l y p o l y m o r p h i c human h i stocompati b i 1 it y a n t i gens15 "O.

A semi -

p r e p a r a t i v e a p p l i c a t i o n o f IFPA was t o s e p a r a t e p a r t i a l l y p u r i f i e d I a - l i k e a n t i gens i n t o t h e i r a- and 8 - s u b u n i t s i n t h e presence o f 9 m o l / l urea".

As w i l l be

p o i n t e d o u t below, IFPA i s now most f r e q u e n t l y used f o r t h e m o l e c u l a r c h a r a c t e r i z a t i o n o f complex and p o l y m o r p h i c c e l l s u r f a c e markers such as h i s t o c o m p a t i b i l i t y a n t i g e n s o n l y when t h e m a t e r i a l t o be a n a l y s e d has a l r e a d y been p a r t i a l l y o r even h i g h l y p u r i f i e d by b i o c h e m i c a l means. I n f a c t , t h e i s o l a t i o n and m o l e c u l a r c h a r a c t e r i z a t i o n o f sapecific m o l e c u l a r s t r u c t u r e s f r o m c r u d e lymphoid c e l l e x t r a c t s has become p o s s i b l e o n l y by u s i n g a c o m b i n a t i o n o f t e c h n i q u e s , i . e . ,

indirect

i m m u n o p r e c i p i t a t i o n o f c e l l s u r f a c e a n t i g e n s by h i g h l y s p e c i f i c a n t i b o d i e s and subsequent analyses o f a n t i g e n - a n t i b o d y complexes b y SDS-PAGE. The c o m b i n a t i o n o f t h e two t e c h n i q u e s , SDS-PAGE and i n d i r e c t i m m u n o p r e c i p i t a t i o n ( I P ) o f antigens w i t h s p e c i f i c antibody, i.e.,

IP-SDS-PAGE,

made by f a r t h e

g r e a t e s t impact on t h e e n t i r e f i e l d o f c e l l s u r f a c e a n t i g e n s . The m a j o r advantage o f IP-SDS-PAGE i s t h e g r e a t s p e c i f i c i t y o f t h e a n t i g e n - a n t i b o d y i n t e r a c t i o n , which makes i t p o s s i b l e t o i d e n t i f y and p u r i f y s i n g l e m o l e c u l a r s p e c i e s w i t h o u t r e s o r t i n g t o biochemical p u r i f i c a t i o n even when such molecules a r e p r e s e n t i n o n l y s m a l l amounts w i t h i n an a r r a y o f many c o n t a m i n a n t s . The a p p l i c a b i l i t y and g r e a t u s e f u l ness o f IP-SDS-PAGE were f u r t h e r enhanced b y t h e f r e q u e n t u s e o f procedures r e l y i n g h e a v i l y on n o n - i o n i c (NP-40) o r i o n i c d e t e r g e n t s (SDS o r sodium d e o x y c h o l a t e ) f o r t h e s o l u b i l i z a t i o n o f hydrophobic c e l l s u r f a c e a n t i g e n s . F u r t h e r developments t h a t made IP-SDS-PAGE t h e method o f c h o i c e were t h e widespread u s e o f c u l t u r e d l y m p h o i d

144 c e l l l i n e s t h a t g r e a t l y f a c i l i t a t e d t h e use o f i n t r i n s i c o r s u r f a c e r a d i o l a b e l l i n g o f c e l l s u r f a c e a n t i g e n s and t h e a v a i l a b i l i t y o f p r o t e i n A f r o m StaphgZococcus

aureus which c o u l d be used i n s t e a d o f a second a n t i b o d y f o r i n d i r e c t immunoprecipi t a t i o n , t h u s a v o i d i n g u n d u l y h i g h p r o t e i n l o a d s on g e l s . IP-SDS-PAGE s t r o n g l y supported t h e i n i t i a l c o n c l u s i o n s drawn f r o m SDS-PAGE a n a l y s e s o f p u r i f i e d HLA a n t i g e n s r e g a r d i n g t h e i r m o l e c u l a r s t r u c t u r e , i.e.,

t h e i r non-covalent association

w i t h B2-p. Thus, many i n v e s t i g a t o r s were a b l e t o c o n f i r m t h i s f a c t , u t t l i z i n g I P o f r a d i o l a b e l l e d lymphoid c e l l e x t r a c t s w i t h s p e c i f i c a n t i b o d y t o HLA and/or B2-p. Papain 22-24 o r h y p e r t o n i c s a l t ( 3 m o l / l KC1)25 e x t r a c t s o b t a i n e d f r o m c u l t u r e d human lymphoid c e l l s were now d e f i n i t e l y shown t o c o n s i s t o f two n o n - c o v a l e n t l y l i n k e d p o l y p e p t i d e c h a i n s o f 34,000 and 12,000 d a l t o n s , whereas t h e same a n t i g e n s when s o l u b i l i z e d by d e t e r g e n t s c o n s i s t e d o f p o l y p e p t i d e c h a i n s o f 45,000 and 12,000 d a l t o n s , r e s p e c t i v e l y19y26-28. IP-SDS-PAGE combined w i t h SDS-PAGE a1 so made

i t p o s s i b l e t o c h a r a c t e r i z e HLA a n t i g e n s f o u n d i n t h e c i r c u l a t i o n . Thus, HLA-A9 a n t i g e n s were f o u n d t o b e a H L A - l i p i d complex shed i n t o plasma as a complex o f a s i n g l e HLA heavy c h a i n and a s i n g l e B2-p c h a i n a s s o c i a t e d w i t h a boundary l i p i d .

IP w i t h a n t i - p 2 - p a n t i s e r u m f o l l o w e d by SDS-PAGE showed a heavy c h a i n o f 45,000 d a l t o n s a s s o c i a t e d w i t h p2-p29y30. HLA-A9 a n t i g e n s i n u r i n e were found t o e x i s t i n s o l u b l e f o r m as a heavy c h a i n of 38,000 a l i g h t chain, i.e.,

daltons non-covalently associated w i t h

B2-u o f 12,000 d a l t o n s . T h i s m o l e c u l a r complex r e a c t e d f u l l y

w i t h anti-HLA o r a n t i - B 2 - p a n t i s e r a , i n d i c a t i n g t h a t t h e hydrophobic C-terminal p o r t i o n o f t h e e n t i r e 45,000 d a l t o n HLA heavy c h a i n i s n o t r e q u i r e d f o r immunol o g i c a l f u n ~ t i o n a l i t y ~ ~T h- i ~s ~f i.n d i n g i s s i m i l a r t o t h a t observed w i t h t h e HLA c h a i n o f 34,000 d a l t o n s s o l u b i l i z e d by papain t r e a t m e n t o f l y m p h o i d c e l l s . Another a p p l i c a t i o n o f IP-SDS-PAGE was t o i s o l a t e i n t r i n s i c a l l y l a b e l l e d HLA-A9 a n t i g e n s f r o m c u l t u r e d lymphoid c e l l s e i t h e r w i t h a n t i - B 2 - p a n t i s e r u m 3 3 o r w i t h anti-HLA-A9 xenoantiserum28 i n s u f f i c i e n t l y p u r e f o r m t o f a c i l i t a t e p a r t i a l aminot e r m i n a l amino a c i d sequence a n a l y s i s of t h e HLA heavy c h a i n . Indeed, such sequence d a t a compared v e r y w e l l w i t h t h o s e o b t a i n e d f r o m HLA a n t i g e n s p u r i f i e d s t r i c t l y by biochemical means16, t h u s i n d i c a t i n g t h e e f f i c a c y o f t h e i s o l a t i o n o f HLA a n t i gen by immunochemical means u t i l i z i n g s p e c i f i c x e n o a n t i b o d i e s . The e x t e n s i v e use o f IP-SDS-PAGE d e f i n i t e l y e s t a b l i s h e d t h e m o l e c u l a r p r o f i l e of d e t e r g e n t - s o l u b i l i z e d I a - l i k e a n t i g e n s w h i c h were shown t o e x i s t as a nonc o v a l a n t l y 1 i n k e d moJecular complex w i t h p o l y p e p t i d e c h a i n s o f 34,000 ( a - c h a i n ) and 29,000 d a l t o n s ( B - c h a i n ) 2 2 y 2 3 y 3 4 - 3 6 . As w i t h HLA a n t i g e n s , IP-SDS-PAGE made

i t p o s s i b l e t o i s o l a t e a- and 8-chains o f I a - l i k e a n t i g e n s f r o m d e t e r g e n t e x t r a c t s o f i n t r i n s i c a l l y r a d i o l a b e l l e d c u l t u r e d l y m p h o i d c e l l s and t o d e t e r m i n e t h e i r p a r t i a l N - t e r m i n a l amino a c i d sequence34. I n f a c t , c o n c u r r e n t amino a c i d sequence analyses o f t h e analogue o f human I a - l i k e a n t i g e n s , i . e . , o f m u r i n e I a a n t i g e n s encoded a t t h e I - E sub-region,

t h e a- and 8 - s u b u n i t s

showed f o r t h e f i r s t t i m e

145

considerable N-terminal sequence homologies between t h e r e s p e c t i v e a- and 8-subu n i t s . An a d d i t i o n a l important f i n d i n g was o b t a i n e d by u t i l i z i n g IP-SDS-PAGE t o g e t h e r w i t h t r y p t i c p e p t i d e mapping o f a- and 8-chains o f I a - l i k e a n t i g e n s . Such s t u d i e s i n d i c a t e d t h a t t h e s t r u c t u r a l polymorphism o f these c e l l s u r f a c e antigens w i t h d i f f e r e n t phenotypes i s r e s t r i c t e d t o t h e 8-chain, suggesting t h a t t h i s s u b u n i t bears t h e a l l o t y p i c

determinant^^^-^'.

IP-SDS-PAGE

together w i t h

t r y p t i c p e p t i d e mapping by high-performance l i q u i d chromatography o f i n t r i n s i c a l l y 3 r a d i o l a b e l l e d ( [ H]glucosamine) a- and @-chains a l s o e s t a b l i s h e d t h e a c t u a l number o f carbohydrate chains per s u b u n i t as one f o r t h e a-chain and two f o r t h e 8-chain (see r e f . 41). Another a p p l i c a t i o n o f IP-SDS-PAGE i s i t s widespread use as a "molecular c o r o l 1a r y " t o s e r o l o g i c a l and immunogenetic analyses o f c e l l s u r f a c e macromolecules , i.e.,

t o o b t a i n a molecular p r o f i l e o f a g i v e n s e t o f c e l l s u r f a c e a n t i g e n s reac-

t i v e w i t h d i f f e r e n t a n t i s e r a on t h e same c e l l type o r on d i f f e r e n t c e l l types. To mention o n l y two examples, t h i s i n c l u d e s t h e analyses o f I a - l i k e a n t i g e n s on t r a n s 43 formed c e l l s such as human melanoma c e l l s 4 2 and on T-lymphocytes

.

Another h i g h l y u s e f u l a p p l i c a t i o n o f IP-SDS-PAGE has been t o i d e n t i f y t h e heavyand l i g h t - c h a i n precursor molecules o f HLA a n t i g e n s o b t a i n e d by c e l l - f r e e t r a n s ~ . amino t e r m i n a l s i g n a l sequences o f about 20-24 amino l a t i o n o f t h e i r ~ R N A s ~Here

a c i d residues were i d e n t i f i e d as b o t h p o l y p e p t i d e chains were about 2000 d a l t o n s l o n g e r than t h e i r r e s p e c t i v e non-glycosylated c o u n t e r p a r t s o b t a i n e d i n v i v o . IP-SDS-PAGE e s t a b l i s h e d t h a t t h e HLA heavy- and 1 i g h t - c h a i n precursors synthesized i n v i t r o a r e n o t associated as they a r e f o l l o w i n g p e n e t r a t i o n o f b o t h chains through t h e membrane f o r expression on t h e c e l l surface. T h i s i s t h e case i r r e s p e c t i v e o f whether t h e p o l y p e p t i d e chains a r e non-glycosylated o r g l y c o s y l a t e d i n v i t r o , i.e.,

t h e l a t t e r by t h e a d d i t i o n o f dog p a n c r e a t i c microsomes t o t h e t r a n s -

l a t i o n system44. Studies a l o n g s i m i l a r l i n e s w i t h I a - l i k e a n t i g e n s u t i l i z i n g an i n v i t r o t r a n s l a t i o n system and IP-SDS-PAGE w i t h mono- and p o l y c l o n a l a n t i b o d i e s i n d i c a t e d t h a t , i n c o n t r a s t t o t h e HLA heavy c h a i n and B2-p, t h e p r e c u r s o r s o f

a- and 8-subunits o f l a - l i k e a n t i g e n s a r e indeed associated i n v i t r o and t h a t i n non-glycosylated form t h e i r molecular weights a r e 29,000 and 27,000 d a l t o n s , r e s p e c t i v e l y , compared w i t h 34,000 and 29,000 d a l t o n s f o r t h e a u t h e n t i c , g l y c o s y l a t e d 45

molecules found on t h e c e l l s u r f a c e

Another a p p l i c a t i o n o f IP-SDS-PAGE,

.

i n e l e g a n t pulse-chase experiments, l e d t o

.

an understanding o f t h e assembly and m a t u r a t i o n o f HLA a n t i g e n s i n v ~ v o Thus ~ ~ . completed heavy chains c a r r y 01 igosaccharides w i t h a h i g h mannose c o n t e n t and a r e l a r g e l y unassociated w i t h p2-p immediately a f t e r synthesis. I n t h e f o l l o w i n g 15 min, c h a i n a s s o c i a t i o n occurs and t h e conformation o f t h e heavy c h a i n i s converted from one a n t i g e n i c form t o another, and 30 min a f t e r s y n t h e s i s t h e h i g h mannose o l i g o saccharides a r e converted i n t o t h e complex o l i g o s a c c h a r i d e s c h a r a c t e r i s t i c of

146 mature HLA a n t i g e n s . F i n a l l y , 60-80 min a f t e r s y n t h e s i s , t h e m a t u r e a n t i g e n s c a n be d e t e c t e d on t h e c e l l s u r f a c e .

A f u r t h e r h i g h l y u s e f u l a p p l i c a t i o n o f IP-SDS-PAGE a l r e a d y i n use i n o u r l a b o r a t o r y i s i t s u t i l i z a t i o n f o r gene c l o n i n g s t u d i e s t o screen i n v i t r o t r a n s l a t i o n p r o d u c t s coded f o r by s p e c i f i c cDNA. The s c r e e n i n g o f l a r g e numbers o f p l a s m i d s c o n t a i n i n g cDNA i n s e r t s c a n b e c a r r i e d o u t e f f e c t i v e l y b y i s o l a t i n g plasmids f r o m p o o l s o f c l o n e s which a r e t h e n bound t o n i t r o c e l l u l o s e paper and h y b r i d i z e d t o mRNA. The i n v i t r o t r a n s l a t i o n p r o d u c t s programmed by t h e s e p c i f i c a l l y h y b r i d i z e d mRNA a r e t h e n i d e n t i f i e d by IP-SDS-PAGE w i t h b o t h p o l y c l o n a l and monoclonal a n t i bodies. Two-dimensional PAGE, i . e . ,

combining IP-SDS-PAGE i n t h e f i r s t dimension and

IF-PAGE i n t h e second dimension, has been used t o demonstrate s t r u c t u r a l p o l y morphism among 8-chains and r e v e a l e d charge m i c r o h e t e r o g e n e i t y among b o t h a- and 8-chains o f I a - 1 i k e a n t i g e n s k i t h d i f f e r e n t phenotypes. The p a t t e r n o f a - c h a i n s d i d n o t v a r y whereas t h e 8-chains o f p h e n o t y p i c a l l y d i f f e r e n t I a - l i k e a n t i g e n s d i f f e r e d i n apparent m o l e c u l a r s i z e and charge d i s t r i b u t i o n . S t r u c t u r a l p o l y morphism o f t h e 8-chain b u t n o t t h e a - c h a i n was m a i n t a i n e d a f t e r t r e a t m e n t w i t h e i t h e r neuraminidase o r t u n i c a m y c i n and a1 though m i c r o h e t e r o g e n e i t y was reduced f o r b o t h c h a i n s t h e y s t i l l focused as two m a j o r components, s u g g e s t i n g e i t h e r t h a t p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s other than glycosyl a t i o n c o n t r i b u t e t o t h e i r m i c r o h e t e r o g e n e i t y o r t h a t t h e r e a r e two n o n - a l l e l i c I a - 1 ike mol e c u l es. The twodimensional g e l s t u d i e s showed s i m i l a r p a t t e r n s f o r 8-chains o f I a - l i k e a n t i g e n s w i t h s e r o l o g i c a l l y c r o s s - r e a c t i v e s p e c i f i c i t i e s and suggested t h a t t h e i r s t r u c t u r a l 47 polymorphism i s t h e b a s i s f o r t h e i r s e r o l o g i c a l l y d e t e c t e d polymorphism

.

I n c o n c l u s i o n , i t i s a p p a r e n t f r o m t h i s b r i e f o v e r v i e w , summarizing o n l y some o f t h e r e s e a r c h h i g h l i g h t s o b t a i n e d w i t h j u s t one s p e c i e s o f c e l l s u r f a c e macromolecules, i . e .

, human

h i s t o c o m p a t i b i l i t y a n t i g e n s , t h a t p o l y a c r y l a m i d e gel e l e c -

t r o p h o r e s i s and i n p a r t i c u l a r IP-SDS-PAGE has been t h e key t e c h n i q u e which made i t possible t o achieve t h e r a p i d progress i n t h i s f i e l d o f research d u r i n g t h e

1a s t decade. REFERENCES R e i s f e l d and B.D. Kahan, Advan. I m n o Z . , 12 (1970) 117. R e i s f e l d , M.A. Pel1egr.ino and B.D. Kahan, Science, 172 (1971) 1134. Mann, i n B.D. Kahan and R.A. R e i s f e l d ( E d i t o r s ) , TranspZantation Antigen, ed., Academic Press, New York, 1972, p. 287. R e i s f e l d and B.D. Kahan, i n R.R. P o r t e r ( E d i t o r ) , Defense and Recognition, MTP I n t . Review of Science, Biochern. Services I , Vol 10, B u t t e r w o r t h , U n i v e r s i t y Park Press, London, 1973, p. 257. 5 R.A. R e i s f e l d and M.A. P e l l e g r i n o , i n B.D. Kahan and R.A. R e i s f e l d ( E d i t o r s ) , TranspZantation Antigen, Academic Press, New York, 1972, pp. 259-271. 6 M.A. P e l l e g r i n o , S. Ferrone, P.G. N a t a l i , A. P e l l e g r i n o and R.A. R e i s f e l d , J. Inmunol., 108 (1972) 573. 1 R.A. 2 R.A. 3 D.L. 1st 4 R.A.

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147 7 R.A.

R e i s f e l d and B.D. Kahan, i n F.P. Inman ( E d i t o r ) , Contemporary Topics i n Immunochemistry, V o l . I, Plenum Press, New York, 1972, pp. 51-143. 8 P. C r e s s w e l l , M.J. Turner and J.L. Strominger, Proc. Nut. Acad. S c i . U.S. , 70 (1973) 1603. 9 P. Parham, C . T e r h o r s t , H. Herrmann, R.E. Humphreys, 11.0. W a t e r f i e l d and J.L. Strominger, Proc. Nut. Acad. S c i . U.S., 72 (1975) 1594. 10 R.A. R e i s f e l d , E.D. Sevier, M.A. P e l l e g r i n o , S . F e r r o n e and M.D. P o u l i k , I m u n o g e n e t i c s , 2 (1975) 183. 11 T.A. S p r i n g e r , J.L. S t r o m i n g e r and D.L. Mann, Proc. Nut. Acad. S c i . U.S., 7 1 (1974) 1539. 12 P. C r e s s w e l l , T. S p r i n g e r , J.L. S t r o m i n g e r , M.J. T u r n e r , H.J. Grey and R.T. Kubo, Proc. Nut. Acad. S c i . U.S., 7 1 (1974) 2123. 13 J. Berggard and A . C . Bearn, J . B i o l . Chem.. 243 (1968) 4095. 14 N. T a n i g a k i and 0. Pressman, i n G. f b l l e r ( E d i t o r ) , BpVicrogZobuZin and HL-A Antigens, llunksgaard, Copenhagen, 1974, pp. 15-34. 15 J.L. Strominger, P. C r e s s w e l l , H. Grey, R.E. Humphreys, D.L. Mann, J. McCune, P. Parham, R . Robb, A.R. Sanderson, T.A. S p r i n q e r , C . T e r h o r s t and M.J. T u r n e r , i n G. M o l l e r ( E d i t o r ) , B2-k%crogZobuZin and HL-A Antigens, Hunksgaard, Copenhagen , 1974, pp. 126-143. 16 T.A. S p r i n g e r , J.F. Kaufman, C . T e r h o r s t and J.L. S t r o m i n g e r , Nature (London), 268 (1977) 213. 17 T.A. S p r i n g e r , J.F. Kaufman, L.A. Siddoway, D.L. Mann and J.L. S t r o m i n g e r , J . B i o l . Chem., 252 (1977) 6201. 18 T.A. S p r i n g e r and J . L . S t r o m i n g e r , Proc. Nut. Acad. Sci. U . S . , 73 (1976) 2481. 19 T.A. S p r i n g e r , R.J. Robb, C. T e r h o r s t and J . L . S t r o m i n g e r , J . BioZ. Chem., 252 (1977) 4694. 20 P. Parham, R.E. Humphreys, M.J. T u r n e r and J . L . S t r o m i n g e r , Proc. Nut. Acud. S c i . U.S., 7 1 (1974) 3998. 21 L. Klareskog, L . Rask, J. Fohlman and P.A. Peterson, Nature (London), 275 (1978) 762. 22 L. Rask, L. Ostberg, B. Lindblom, Y . F e r m s t e d t amd P.A. Peterson, i n G. M o l l e r ( E d i t o r ) , B2-MicrogZobulin and HL-A Antigens, Hunksgaard, Copenhagen, 1974, pp. 85- 105. 23 H.M. Grey, R.T. Kubo, S.M. Colon, H.D. P o u l i k , P. C r e s s w e l l , T. S p r i n g e r , M. T u r n e r and J.L. Strominger, J . E q . Med., 136 (1973) 1608. 24 P.A. Peterson, L.Rask and J.B. Lincvlom, Proc. at. Acad. S c i . U.S., 7 1 (1974) 35. 25 M.D. P o u l i k , S. Ferrone, H.A. P e l l e g r i n o , D.E. S e v i e r , S.K. Oh and R.A. R e i s f e l d , i n G. M o l l e r ( E d i t o r ) , B2-b!icrogZobuZin and HL-A Antigens, Munksgaard, Copenhagen, 1974, pp. 106-126. 26 J.L. Strominger, D.L. Mann, P. Parham, R. Robb, T . S p r i n g e r and C. T e r h o r s t , Cold Spring Harbor Symp. &ant. B i o l . , 4 1 (1976) 323. 27 J. Bridgen, D. Snary, M.J. Crumpton, C . B a r n s t a b l e , P. G o o d f e l l o w and W.F. Bodmer, Nature (London), 261 (1976) 200. 28 J.P. A l l i s o n , S. F e r r o n e , L.E. Walker, M.A. P e l l e g r i n o , J. S i l v e r and R.A. R e i s f e l d , TranspZantation, 26 (1978) 451. 29 J.P. A l l i s o n , M.A. P e l l e g r i n o , S. Ferrone, G.N. C a l l a h a n and R.A. R e i s f e l d , J . ImunoZ. , 118 (1977) 1004. 30 R.A. R e i s f e l d , J.P. A l l i s o n , S. Ferrone, M.A. P e l l e g r i n o and M.D. P o u l i k , Tranplant. Proc. , 8 (1976) 173. 31 R.A. R e i s f e l d , M.A. P e l l e g r i n o and S . Ferrone, J . I m n o Z . , 118 (1977) 264. 32 C. V i n c e n t , J.P. R e v i l l a r d and H. B e t u e l , Transplantation, 22 (1977) 500. 33 B. B a l l o u , D.J. McKean, E.R. Freedlander and 0. S m i t h i e s , Proc. Nut. h a d . Sci. U.S., 73 (1976) 4487. 34 T.A. S p r i n g e r , J.F. Kaufman, L.A. Siddoway, M. G i p h a r t , D.L. Mann, C. T e r h o r s t and J.L. S t r o m i n g e r , Cold Spring Harbor Symp. w a n t . B i o Z . , 4 1 (1976) 387. 35 D. Snary, C . B a r n s t a b l e , W.F. Bodmer, P. G o o d f e l l o w and M.J. Crompton, cold Spring Harbor Symp. &ant. BioZ. , 4 1 (1976) 379. 36 S. Ferrone, J.P. A l l i s o h n and M.A. P e l l e g r i n o , i n F. Inman and R.A. R e i s f e l d ( E d i t o r s ) , Contemporary Topics i n M G ~ e C U k PImunoZogy , v o l 7 , P1enum Press, New York, 1978, pp. 239-281.

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148 37 R . Tosi, N. Tanigaki, D. Centis, G.B. Ferrara and D. Pressman, J . E z p . Med., 148 19781 1592. 38 L.E.’Walkbr, J.P. Allison, J . Silver, M.A. Pellegrino, R.A. Reisfeld and S. Ferrone, i n M . R . Quastel (Editor), CeZZ BioZogy and ImmnoZogy of Leukocyte Function. Academic Press, New York, 1979, pp. 271-275. 39 J . Silver and S . Ferrone, Nature (Londonl,279 (1979) 436. 40 J . Silver and S. Ferrone, TranspZant. Proc., 11 (1979) 1743. 4 1 L . E . Walker, S. Ferrone, M.A. Pellegrino and R.A. Reisfeld, in R . A . Reisfeld and S . Ferrone (Editors) , Current Trends in HistocompatibiZity, P1 enum Press, New York, 1981, pp. 511-529. 42 B.S. Wilson, F. Indiveri, M.A. Pellegrino and S . Ferrone, J . E z p . Med., 149 (1979) 658. 43 B.S. Wilson, F. Indiveri, M.A. Pellegrino and S . Ferrone, TranspZant. Proc., 11 (1979) 712. 44 H . L : Ploegh, L . E . Cannon and J.L. Strominger, Proc. f l a t . Aead. S c i . U . S . , 76 (19791 2273. 45 i. Ri;e and R . A . Reisfeld, in preparation. 46 M.S. Krangel, H.T. Orr and J.L. Strominger, CeZZ, 18 (1979) 979. 47 L A . Shackelford and J.L. Strominger, J . E q . Med., 151 (1980) 144.

149

Chapter 9.7 LYSOSOMAL GLYCOSIDASES AND SULPHATASES*

ARVAN L. FLUHARTY GENERAL ASPECTS Concern w i t h lysosomal g l y c o s i d a s e s and s u l phatases from human sources has been prompted by t h e i r d e f i c i e n c i e s i n a number o f human g e n e t i c d i s o r d e r s . E x t e n s i v e use has been made o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) and i s o e l e c t r i c f o c u s i n g (IFPA) i n i n v e s t i g a t i n g t h e s e enzymes and t h e i r c o r r e s p o n d i n g d e f i c i e n c y c o n d i t i o n s . A p p l i c a t i o n s can be c o n v e n i e n t l y d i v i d e d i n t o f i v e m a j o r c a t e g o r i e s : a n a l y s i s o f enzyme subtypes; enzyme p u r i f i c a t i o n ; m i c r o h e t e r o g e n e i t y w i t h i n a subtype; s u b u n i t c o m p o s i t i o n ; and b i o l o g i c a l p r o c e s s i n g o f enzyme s t r u c t u r e s . SEPARATION AN0 ANALYSIS OF MAJOR ENZYME SUBTYPES Several lysosomal enzyme a c t i v i t i e s , as d e f i n e d b y c o n v e n i e n t chromogenic o r f l u o r o g e n i c s u b s t r a t e s , can be s e p a r a t e d i n t o two o r more d i s t i n c t subspecies. I n g e n e r a l , t h e s e have d i s t i c n t p h y s i o l o g i c a l s u b s t r a t e s p e c i f i c i t i e s , a r e t h e produ c t s o f d i f f e r e n t genes and a r e a s s o c i a t e d w i t h d i f f e r e n t d e f i c i e n c y syndromes. For example, B-N-acetyl hexosaminidase i s r e a d i l y r e s o l v e d i n t o two m a j o r subtypes ( d e s i g n a t e d A and B) by e l e c t r o p h o r e t i c o r ion-exchange procedures. R e c o g n i t i o n o f most m a j o r subtypes o c c u r r e d b e f o r e PAGE was w i d e l y employed and t h e t e c h n i q u e has n o t been used e x t e n s i v e l y i n t h e i r a n a l y s i s . F r i e d l a n d e t a 1 . l i n t r o d u c e d a PAGE system f o r R-N-acetylhexosaminidase a n a l y s i s w h i c h was e f f e c t i v e f o r d i a g n o s i n g 2

Tay-Sachs d i s e a s e and d i f f e r e n t i a t i n g Tay-Sachs c a r r i e r s . Hayase and K r i t c h e v s k y u t i l i z e d IFPA (pH 3-10) t o i n v e s t i g a t e serum 8-N-acetylhexosaminidases w i t h p a r t i c u l a r concern f o r i n t e r m e d i a t e forms which i n c r e a s e d u r i n g pregnancy. However,

t h e use o f d i f f e r e n t i a l thermal i n a c t i v a t i o n by t h e m a j o r Tay-Sachs s c r e e n i n g l a b o r a t o r i e s has p r e c l u d e d any b r o a d s c a l e a d o p t i o n o f t h e p o l y a c r y l a m i d e g e l t e c h niques. P o l y a c r y l a m i d e g e l systems have g a i n e d o n l y l i m i t e d use f o r e v a l u a t i n g a r y l sulphatases i n crude e x t r a c t s . One d i f f i c u l t y was t h a t human a r y l s u l p h a t a s e B *These enzymes a r e c o n s i d e r e d here as an example o f lysosomal p r o t e i n s . F o r d e t a i l e d d i s c u s s i o n on enzyme s e p a r a t i o n s , see Chapter 12.

160 f a i l e d t o e n t e r t h e s e p a r a t i n g g e l o f t h e commonly employed O r n s t e i n - D a v i s a1 k a l i n e b u f f e r system3y4. T h i s was n o t a problem w i t h some o t h e r s p e c i e s where a r y l s u l p h H eI l w; i g e t a1 .5 observed b o t h A and B forms i n s t a n d a r d g e l s a t a s e B has a l o w e r P 6 w i t h r a b b i t k i d n e y c o r t e x enzymes. Dubois and Baumann i n t r o d u c e d a pH 3 b u f f e r system f o r m o n i t o r i n g a r y l s u l p h a t a s e s A and B i n human l e u k o c y t e s on t h e same g e l and H a l t i a e t a1.7 u t i l i z e d i t t o e v a l u a t e f a m i l i e s o f j u v e n i l e metachromatic l e u k o d y s t r o p h y p a t i e n t s . Shapira e t a1.8 used a pH 4 b u f f e r system t o observe a r y l s u l p h atases A and B on a s i n g l e g e l . Other examples o f p o l y a c r y l amide g e l s e p a r a t i o n s o f m a j o r lysosomal enzyme forms a r e sparse. N i c o l e t a l . '

s e p a r a t e d B-glucosidase f r o m human s p l e e n b y PAGE a t pH

4.3 i n t o two forms which d i f f e r e d i n t h e i r a b i l i t y t o be t a k e n u p t o c u l t u r e d f i b r o b l a s t s . Romeo e t a1.l'

used IFPA t o s e p a r a t e A and B forms o f a - g a l a c t o s i d a s e .

A r e l a t e d a p p l i c a t i o n o f i n c r e a s i n g i n t e r e s t i s t h e use o f p o l y a c r y l a m i d e g e l 11 techniques f o r d i f f e r e n t i a t i o n o f enzyme f r o m d i f f e r e n t s p e c i e s . Thorpe e t a l . u t i l i z e d PAGE t o s e p a r a t e b o v i n e and m u r i n e 6-glucuronidases i n a s t u d y o f t h e 12 u p t a k e o f liposome-entrapped enzyme b y c u t l u r e d mouse c e l l s . Mohondas e t a l . d i f f e r e n t i a t e d mouse and human a r y l s u l p h a t a s e C i n h y b r i d c e l l s w i t h a mixed agarose-PAGE system i n t h e course o f mapping t h e enzyme gene on t h e human X chromosome. ENZYME PURIFICATION One o f t h e most common a p p l i c a t i o n s o f PAGE has been f o r m o n i t o r i n g enzyme p u r i f i c a t i o n . I n d i v i d u a l examples a r e f r e q u e n t and o n l y two examples i l l u s t r a t i n g a problem o r u n i q u e a p p l i c a t i o n a r e c i t e d . Most workers r e l y on a s i n g l e PAGE system f o r m o n i t o r i n g p u r i t y which, on occasion, has l e d t o i n c o r r e c t c o n c l u s i o n s . U r i n a r y a r y l s u l phatase A p r e p a r a t i o n s s i m i l a r t o t h a t o f Breslow ans Sloan13 appear homogeneous i n t h e O r n s t e i n - D a v i s a l k a l i n e b u f f e r system. On e x a m i n a t i o n w i t h a n e u t r a l b u f f e r , s e v e r a l p r o t e i n bands can be r e s o l v e d w i t h enzyme a c t i v i t y assoc i a t e d w i t h o n l y a m i n o r c o n ~ t i t u e n t ' ~ When . Pentchev e t a1 .15 were u n a b l e t o g e t 8-glucocerebrosidase i n t o t h e i r s e p a r a t i n g g e l s , t h e y r e l i e d on d e n a t u r i n g g e l c o n d i t i o n s t o m o n i t o r t h e i r p u r i f i c a t i o n . O b v i o u s l y , t h i s r e q u i r e s some assurance as t o which s u b u n i t ( s ) d e r i v e f r o m t h e enzyme, b u t t h i s can o f t e n be i n f e r r e d f r o m other information

.

Examples o f t h e d i r e c t use o f p o l y a c r y l a m i d e g e l t e c h n i q u e s f o r t h e p u r i f i c a t i o n o f lysosomal hydrolases a r e f a i r l y l i m i t e d . Breslow and Sloan13, Stevens e t a l . 14 and James and A u s t i n " u t i l i z e d PAGE p r o t o c o l s i n p u r i f i c a t i o n o f a r y l s u l p h a t a s e A from human l i v e r and u r i n e . Cantz e t a1.17 r e s o l v e d Hunter c o r r e c t i o n f a c t o r i n t o two major bands b y p r e p a r a t i v e PAGE, and t h e s e m a t e r i a l s were l a t e r shown t o have i d u r o n a t e s u l p h a t a s e a c t i v i t y 1 8 . Other examples a r e Von F i g u r a l ' w i t h a - N - a c e t y l hexosaminidase, Basner e t a1 .20 w i t h N-acetylglucosamine 6 - s u l p h a t a s e and Gloss1

151

e t a1 .*l w i t h N - a c e t y l g a l a c t o s a m i n e 6 - s u l p h a t a s e . Stevens e t a1

."

used IFPA (pH

4-6) t o r e c o v e r small amounts o f charge isomers from u r i n a r y a r y l s u l p h a t a s e A. C o n s i d e r i n g i t s p o t e n t i a l f o r h i g h r e s o l u t i o n , t h e i n f r e q u e n t use o f prepa.rat i v e PAGE r e q u i r e s comment. A m a j o r problem seems t o have been i n d e v i s i n g methods f o r t h e l a r g e - s c a l e r e c o v e r y o f p r o t e i n s f r o m a c r y l a m i d e g e l s . Commercial e q u i p ment depends o n sweeping away p r o t e i n s emerging f r o m t h e b o t t o m o f t h e g e l i n a f l o w i n g b u f f e r stream, which r e s u l t s i n e x t e n s i v e d i l u t i o n and l o s s o f enzyme. C o n s i d e r a b l e d e g r a d a t i o n o f t h e f r a c t i o n a t i o n achieved on t h e g e l o c c u r s d u r i n g t h e r e c o v e r y process. T a i l i n g and a r t i f a c t u a l peaking have a l s o been encountered. An a l t e r n a t i v e approach i s t o r u n r e l a t i v e l y l a r g e t u b e o r s l a b g e l s and t o a t t e m p t t o r e c o v e r enzyme f r o m a p p r o p r i a t e s l i c e s . Y i e l d s f r o m such methods a r e t y p i c a l l y low b u t have been r e p o r t e d t o be as h i g h as 50% i n some i n s t a n c e s . E l e c t r o p h o r e s i s

of p r o t e i n o u t o f t h e g e l m a t r i x i n t o a semipermeable t r a p has been a p p l i e d and o f f e r s c o n s i d e r a b l e promisez3. I n a l l i n s t a n c e s t h e g e l l o a d i n g c a p a c i t y was r e l a t i v e l y l o w and p r e p a r a t i v e PAGE was n o t employed u n t i l t h e f i n a l stages o f fractionation.

ENZYME MICROHETEROGENEITY One c h a r a c t e r i s t i c commonly found f o r lysosomal enzymes has been e x t e n s i v e m i c r o h e t e r o g e n e i t y . Most o f t e n t h i s has t a k e n t h e f o r m o f a f a m i l y o f charge i s o mers r e c o g n i z e d by IFPA. A l l forms a r e d e f i c i e n t i n a p a r t i c u l a r d e f i c i e n c y syndrome, i m p l y i n g t h a t a l l a r e d e r i v e d f r o m t h e same gene p r o d u c t . Several f a c t o r s c o n t r i b u t e t o such m i c r o h e t e r o g e n e i t y , i n c l u d i n g neuraminic a c i d s u b s t i t u e n t s , p r o t e i n p h o s p h o r y l a t i o n , g e n e t i c polymorphism and d i f f e r e n t i a l p o s t - s y n t h e t i c p r o c e s s i n g . F o r example, Stevens e t a1 . 2 2 r e s o l v e d p u r e u r i n a r y a r y l s u l p h a t a s e A i n t o a three-banded p a t t e r n on IFPA (pH 4 - 6 ) .

Enzyme f r o m o t h e r t i s s u e s had more

complex p a t t e r n s , b u t these reduce t o t h e three-banded p a t t e r n o f u r i n e on t r e a t ment w i t h b a c t e r i a l neuraminidase. T u r n e r e t a1 .24 showed a s i m i l a r s i t u a t i o n w i t h a-L-fucosidase.

I n a d d i t i o n , t h e y r e p o r t e d charge polymorphism w i t h t h e e n t i r e

p a t t e r n s h i f t e d by one band. M u l t i p l e components i n human p l a t e l e t 8 - g l u c u r o n i d a s e were found by Kaplan e t a1.z5 u s i n g IFPA (pH 6-8). A l k a l i n e phosphatase t r e a t m e n t r e s u l t e d i n a s h i f t i n p a t t e r n c o n c o m i t a n t w i t h a l o s s o f c a p a c i t y f o r enzyme uptake by c u l t u r e d f i b r o b l a s t s . G l a s e r e t a1 .z6 showed t h a t t h e IFPA o f B-gluc u r o n i d a s e changed t o a more a1 k a l i n e p a t t e r n on i n c o r p o r a t i o n i n t o f i b r o b l a s t s . Charge and s i z e h e t e r o g e n e i t y o f u r i n e a-N-acetylhexosaminidase was r e p o r t e d by Von F i g u r a l ' .

PAGE i n t h e presence o f sodium dodecyl s u l p h a t e (SDS-PAGE) was used

t o show v a r i a t i o n i n s u b u n i t c o m p o s i t i o n w i t h change i n enzyme i s o e l e c t r i c p o i n t . L u s i s e t a1 .27 showed e x t e n s i v e h e t e r o g e n e i t y o f mouse 8 - g a l a c t o s i d a s e by PAGE. The same group28 r e s o l ved u r i n e and 1ysosomal 8-91 u c u r o n i d a s e s f r o m mouse i n t o

152

approximately 15 equally spaced bands with PIS between pH 5.5 and 6.0 by IFPA in t h e presence of urea. A mutant a t t h e 8-glucuronidase locus showed a s h i f t i n t h e e n t i r e pattern with heterozygotes producing a more complex intermediate p a t t e r n . IFPA was a l s o u t i l i z e d t o f r a c t i o n a t e cyanogen bromide fragments of the a l l e l i c gene products and i d e n t i f y a s i n g l e peptide modification. ENZYME SUBUNIT COMPOSITION

The most frequent use of PAGE with lysosomal hydrolases has been t o i n v e s t i g a t e enzyme subunit composition. No general p a t t e r n in subunit s t r u c t u r e f o r t h i s c l a s s of enzymes has emerged. 6-Glucuronidase from human placenta was considered t o have a basic tetrameric s t r u c t u r e on t h e basis of 77,000 dalton subunits on SDS-PAGE 29 . Minor 60,000 and 18,000 dalton components were ascribed to some nicking of t h e basic subunit. Ceramidetrihexosidase resolved i n t o 22,000 dalton subunits, implicating a tetrameric structure3'. Norden e t a1 .31 could not demonstrate any subunits smaller than t h e native human l i v e r 8-galactosidase. Human kidney a-L-iduronidase with a molecular weight o f around 60,000 daltons provided 31,000 dalton subunits on reductive SDS-PAGE3'. Human 1 i v e r a-L-fucosidase was reported by A1 hadeff e t a1 .33 t o contain a s i n g l e 50,000 dalton subunit. Kress e t a1 ,34 l a t e r reported a small amount o f a l a r g e r component which was t h e dominant subunit in enzyme from I-cell disease p a t i e n t s . a-L-Fucosidase from r a t epididymus generated 60,000 and 35 50,000 dalton bands on SDS-PAGE . Two d i f f e r e n t p a t t e r n s of s u b u n i t organization have been observed f o r a r y l sulphatase A , depending on the source. Human u r i n e enzyme, purified by Stevens e t a 1 . l 4 , gave r i s e to a s i n g l e subunit of approximately 55,000 daltons suggesting a native enzyme of two equivalent subunits. Similar r e s u l t s had been obtained by Roy and J e r f ~f o ~r ox ~ l i v e r enzyme, except t h a t a small amount of 27,000 dalton material was a l s o reported. Lee and Van Etten37 and Yang and S r i ~ a s t a v areported ~~ a s i n g l e subunit in arylsulphatase A preparations from r a b b i t t i s s u e s . In c o n t r a s t , Draper e t a1.39 and James and Austin16 obtained two subunits w i t h a molecular weight d i f f e r e n c e of about 10,000 daltons from arylsulphatase A of human l i v e r . While t h e actual subunit molecular weights reported by the two groups d i f f e r , they a r e c o n s i s t e n t with a two d i s s i m i l a r s u b u n i t arrangement in t h e native enzyme. The two subunits were not produced i n equivalent amounts and peptide mapping by James and Austin16 suggested t h a t one was derived from the other by t h e l o s s of two peptides. In both s t u d i e s , a small amount of material of rbughly 25,000 daltons was reported. Carlson e t a l . 4 0 observed a s i m i l a r two-subunit p a t t e r n with bovine l i v e r ascorbate-2-sulphatase which i s probably i d e n t i c a l with a r y l s u l p h a t a s e A. The two d i s t i n c t patterns of arylsulphatase A subunit composition might be ascribed t o v a r i a t i o n in t h e SDS-PAGE protocols employed i n d i f f e r e n t l a b o r a t o r i e s , b u t i t

153

i s more l i k e l y t o be due t o d i f f e r e n t p a t t e r n s of post-synthetic modification in various sources. I n c o n t r a s t , arylsulphatase B , with a native molecular w e i g h t of about 50,000 daltons, appears t o c o n s i s t of a s i n g l e subunit. Gold e t a1.41 demonstrated a 50,000 dalton band on SDS-PAGE which did not correspond t o any of t h e bands present i n a p a r t i a l l y purified arylsulphatase A preparation. This does not support t h e concept of arylsulphatase B being one of t h e subunits of arylsulphatase A. A common subunit does occur in p-N-acetylhexosaminidases A and B y on the basis of SDS-PAGE and immunochemical studies42. Both enzymes have molecular weights above 100,000 daltons and gave bands i n the 50,000 dalton region on SDS-PAGE in t h e absence of reducing a g e n t s , In the presence of reductants 25,000 dalton subunits were found, p a r i c u l a r l y from B-N-acetylhexosaminidase A. Both Beutler ~ ~ an (a2)(B2) s t r u c t u r e f o r t h e A enzyme e t a1.42 and Geiger and A r n ~ nsuggest and a (B2)(p2) s t r u c t u r e f o r t h e B enzyme. The p2 dimer seems much more d i f f i c u l t ~~ t o resolve i n t o i t s components and the data o f Hasilick and N e ~ f e l dsuggests t h a t i t may in f a c t c o n s t i t u t e a s i n g l e l a r g e r subunit. Considerable v a r i a t i o n in the subunit molecular weights have been reported by d i f f e r e n t l a b o r a t o r i e s , i l l u s t r a t i n g the problems encountered w i t h SDS-PAGE of glycoproteins. I n f a c t , Wiktorowicz e t a1 .45 favour a basic hexameric s t r u c t u r e of ( c x B ) ~ and (B8)3 f o r t h e A and B enzymes, respectively, on t h e b a s i s of 18,000 and 40,000 dalton components. BIOLOGICAL PROCESSING OF LYSOSOMAL ENZYMES By combining radioisotopic l a b e l l i n g , antibody p r e c i p i t a t i o n and SDS-PAGE resolution of enzyme subunits, even small amounts of enzyme i n crude mixtures can be investigated. Information on the s y n t h e s i s , post-synthetic modifications and turnover of lysosomal enzymes i s beginning t o emerge. Mouse macrophage B-galactosidase was shown t o undergo conversion from an i n i t i a l 82,000 dalton component t o one of 63,000 daltons within 24 h46. SDS-PAGE of cyanogen bromide fragments from t h e two components showed c l o s e homology between t h e two m a t e r i a l s , implicating a precursor-product r e l a t i o n s h i p . Stevens e t a1 .47 used s p e c i f i c antibody p r e c i p i t a t i o n and SDS-PAGE t o obtain the arylsulphatase A subunit from human f i b r o b l a s t s grown in t h e presence of a radioactive amino acid. The h a l f - l i f e of t h e enzyme i n normal c e l l s was found t o be g r e a t e r than 3 weeks. Hasil ick and Neufel d44 u t i l i z e d a s i m i l a r approach t o i n v e s t i g a t e B-N-acetylhexosaminidases, a-gl ucosidase and cathepsin D of human f i b r o b l a s t s . Cell and c u l t u r e medium enzymes from normal and I-cell d i s e a s e p a t i e n t s were compared. In a l l instances l a r g e r protein u n i t s were present a t e a r l y times with conversion t o

154

s t a n d a r d s u b u n i t p a t t e r n s w i t h i n a few hours i n normal c e l l s . The I - c e l l enzymes, l o c a t e d p r i m a r i l y i n t h e c u l t u r e medium, r e t a i n e d t h e l a r g e r s u b u n i t s i z e s . Using t h e same b a s i c techniques w i t h l a b e l l e d phosphate and mannose, t h e presence o f mannose phosphate on t h e normal l a r g e fragments b u t n o t on t h o s e f r o m I - c e l l d i s e a s e m a t e r i a l was shown48

.

CONCLUSION The h i g h r e s o l v i n g power o f PAGE and IFPA has been e x t e n s i v e l y e x p l o i t e d i n s t u d i e s o f t h e lysosomal g l y c o s i d a s e s and s u l p h a t a s e s . Most uses have been a n a l y t ical, typical

a p p l i c a t i o n s b e i n g r e s o l u t i o n o f m a j o r enzyme subtypes, r e c o g n i t i o n

o f m i c r o h e t e r o g e n e i t y and i n v e s t i g a t i o n o f enzyme s u b u n i t c o m p o s i t i o n . A small number o f p r e p a r a t i v e uses have been r e p o r t e d , b u t d i f f i c u l t i e s i n t h e r e c o v e r y o f p r o t e i n s f r o m t h e gel s u p p o r t s remains a m a j o r drawback. SDS-PAGE has f r e q u e n t l y been used t o i n v e s t i g a t e s u b u n i t arrangements, and i n c r e a s i n g l y d e t a i l e d comparison o f a g i v e n enzyme f r o m d i f f e r e n t t i s s u e s and/or s u b c e l l u l a r environments can be expected. Two r e c e n t i n n o v a t i o n s , t h e use o f SDS-PAGE o f IFPA t o compare fragments a f t e r s p e c i f i c cleavage p r o t o c o l s , and t h e combined a n t i b o d y p r e c i p i t a t i o n SDS-PAGE t e c h n i q u e f o r i s o t o p i c a l l y l a b e l l e d enzymes, p r o v i d e s e n s i t i v e methods f o r i n v e s t i g a t i n g t h e " n a t u r a l h i s t o r y " o f enzymes i n l i v i n g systems. A l l o f t h i s has s i g n i f i c a n t l y enhanced o u r u n d e r s t a n d i n g o f lysosomal g l y c o s i d a s e s and s u l phates and cont r i b u t e d t o d e t e c t i o n , p r e v e n t i o n and t h e r a p y i n r e l a t e d d e f i c i e n c y syndromes. ACKNOWLEDGEMENT T h i s work was supported i n p a r t by N I H Grants No. AM-25564,

NS-11665, HD-08855

and HD-04612. REFERENCES 1 J. F r i e d l a n d , L. Schneck, A. S a i f e r , M. F o u r f a r and B.W. Volk, CZin. Chirn. Acta, 28 (1970) 397, 2 K. Hayase and D. K r i t c h e v s k y , C Z i n . Chirn. A c t u , 46 (1973) 455. 3 L. O r n s t e i n , Ann. N.Y. Acad. S c i . , 121 (1964) 321. 4 B.J. Davis, Ann. N.Y. Acad. S c i . , 121 (1964) 404. 5 J.-J. Helwig, A.A. Farooqui; C. B o l l a c k and P. Mandel, Biochern. J., 165 (1977) 127. 6 G. Dubois and N. Baumann, Biochern. Biophys. Res. Cornmun., 50 (1973) 1129. 7 T. H a l t i a , A. I c e n and J. Palo, CZin. Chirn. A c t a , 95 (1979) 255. 8 E. Shapiro, R.R. DeGregorio, R. M a t a l o n and H.L. Nadler, Biochm. Biophys. Res. C o m n . , 6 2 (1975) 448. 9 D.M. N i c o l , 0. L a u g n o f f and P. P r i t z l , Biochern. Biophys. Res. Commun., 59 (1974) 941. 10 G . Romeo, G. DiMatteo, M. D'Urso, S.-C. L i and Y.-T. L i , Biochim. Biophys. Acta, 391 (1975) 349.

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11 S.R. Thorpe, M.B. F i d d e r and R.J. Desnick, Pediat. R e s . , 9 (1975) 918. 12 T. Mohondas, L.J. S h a p i r o , R.S. Sparkes and M.C. Sparkes, Proc. Nat. Acad. S c i . U.S. , 76 (1979) 5779. 13 J.L. Breslow and H.R. Sloan, Biochem. Biophys. Res. Commun., 46 ( 1 9 7 2 ) 919. 14 R.L. Stevens, A.L. F l u h a r t y , M . H . Skokut and H. K i h a r a , J . BioZ. Chem., 250 (1975) 2495. 15 P.G. Pentchev, R.O. Brady, S.R. H i b b e r t , A.E. Gal and D. Shapiro, J . BioZ. Chem., 248 (1973) 5256. 16 G.T. James and J.H. A u s t i n , CZin. Chim. A c t a , 98 (1979) 103. 17 M. Cantz, A. Chrambach, G. Bach and E.F. N e u f e l d , J . BioZ. Chem., 247 (1972) 5456. 18 G. Bach, F. Eisenberg, M. Cantz and E.F. N e u f e l d , Proc. Nat. Acad. S c i . U.S. , 70 (1973) 2134. 19 K. von F i g u r a , Eur. J . Biochem., 8 0 (1977) 525. 20 R. Basner, H. Kresse and K . von F i g u r a , J . BioZ. Chem., 181 ( 1 9 7 9 ) 37. 2 1 J . G l o s s l , W. Truppe and H. Kresse, Biochem. J . , 1 8 1 (1979) 37. 22 R.L. Stevens, A.L. F l u h a r t y , A.R. K i l l g r o v e and H. Kihara, Biochim. Biophys. A c t a , 445 (1976) 661. 23 N.Y. Ngugen, J. DiFonzo and A. Chrambach, A m Z . Biochem., 106 (1980) 78. 24 B.M. Turner, U.S. Turner, N.G. B e r a t i s and K. H i r s c h h o r n , Amer. J . Hum. Genet., 27 (1975) 651. 25 A. Kaplan, D.T. Achord and W.S. Sly, Proc. Nat. Acad. S c i . U.S., 74 ( 1 9 7 7 ) 651. 26 J.H. Glaser, K.J. Roozen, F.E. B r a t and W.S. Sly, Arch. Biochem. Biophys., 166 (1975) 536. 27 A.J. L u s i s , G.A.M. Breen and K. Paigen, J . BioZ. Chem., 252 (1977) 4613. 28 A.J. L u s i s and K. Paigen, J . BioZ. Chem., 253 ( 1 9 7 8 ) 7336. 29 F.E. B r a t , C.E. B e l l and W.S. Sly, Biochemistry, 17 (1978) 385. 30 C.A. Mapes, C.H. S u e l t e r and C.C. Sweeley, J . BioZ. Chem., 248 ( 1 9 7 3 ) 2471. 3 1 A.G.W. Norden, L.L. Tennant and J.S. O ' B r i e n , J . BioZ. Chem., 249 (1974) 7969. 32 L.H. Rome, J . G a r v i n and E.F. Neufeld, Arch. Biochem. Biophys., 189 ( 1 9 7 8 ) 344. 33 J.A. A l h a d e f f , A.L. M i l l e r , H. Wenaas, T. Vedvick and J.S. O ' B r i e n , J . BioZ. Chem. , 250 (1975) 7106. 34 B.C. Kress, H.H. Freeze, J.K. Herd, J.A. A l h a d e f f and A.L. M i l l e r , J . BioZ. Chem., 255 (1980) 955. 35 R.B. C a r l s e n and J.G. P i e r c e , J . BioZ. Chem., 247 (1972) 23. 36 A.B. Roy and A. J e r f y , Biochim. Biophys. A c t a , 207 (1970) 156. 37 G.D. Lee and R.L. van E t t e n , Arch. Biochem. Biophys., 166 ( 1 9 7 5 ) 280. 38 C,-H. Yang and P.N. S r i v a s t a v a , Biochem. J . , 159 (1976) 133. 39 R.K. Draper, G.M. Fiskum and J. Edmond, Arch. Biochem. Biophys., 177 (1976) 525. 40 R.W. Carlson, M. Downing and B.M. T o l b e r t , Biochemistry, 16 ( 1 9 7 7 ) 5221. 4 1 E.W. Gold, D. G u s s l e r and E.R. Schwartz, Connect. Tissue Res., 4 (1976) 237. 42 E. B e u t l e r , A. Yoshida, W. Kuhl and J.E.S. Lee, Biochem. J . , 159 (1976) 541. 43 B. G e i g e r and R. Arnon, Biochemistry, 15 (1976) 3484. 44 A. H a s i l i c k and E.F. N e u f e l d , J . BioZ. Chem., 255 (1980) 4937. 45 J.E. Wiktorowicz, Y.C. Awasthi, A. Kuorsky and S.K. S r i v a s t a v a , Biochem. J . , 165 (1977) 49. 46 M.D. Skudlarek and R.T. Swank, J . B i d . Chem., 254 (1979) 9939. 47 R.L. Stevens, A.L. F l u h a r t y and H. K i h a r a , u n p u b l i s h e d r e s u l t s . 48 A. H a s i l i c k and E.F. Neufeld, J . BioZ. Chem., 255 (1980) 4946.

166

Chapter 9.8 HAEMOCYAN I N S MICHAEL BRENOWITZ, JOSEPH BONAVENTURA and CELIA BONAVENTURA Haemocyanin, a c o p p e r - c o n t a i n i n g i n v e r t e b r a t e r e s p i r a t o r y p r o t e i n , has l o n g been s t u d i e d as a s e l f - a s s o c i a t i n g system because o f i t s h i g h m o l e c u l a r w e i g h t and mu1 t i s u b u n i t s t r u c t u r e l Y 2 . Many y e a r s a f t e r Svedberg's s t u d i e s , h e t e r o g e n e i t y i n t h e c o n s t i t u e n t s u b u n i t s o f t h e s e h i g h - m o l e c u l a r - w e i g h t p r o t e i n s became e v i d e n t t h r o u g h e l e c t r o p h o r e t i c and immunological s t u d i e s . Comprehensive r e v i e w s which d e t a i l s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s i n t h e haemocyanins and t h e o t h e r i n v e r t e b r a t e r e s p i r a t o r y proteins, erythrocruorin, chlorocruorin, haemerythrin and haemoglobin, have been pub1 i s h e d e l ~ e w h e r e ~ - ~ . The e x p l o r a t i o n o f s u b u n i t h e t e r o g e n e i t y i n haemocyanins has l e d t o t h e p u r i f i c a t i o n o f t h e c o n s t i t u e n t p o l y p e p t i d e s and a s t u d y o f how t h e s e s t r u c t u r a l l y d i s t i n c t forms c o n t r i b u t e t o t h e s t r u c t u r e and f u n c t i o n o f t h e assembled aggreg a t e . T h i s has proved t o be i n f o r m a t i v e i n advancing o u r u n d e r s t a n d i n g o f assembly and s u b u n i t i n t e r a c t i o n s i n m u l t i - s u b u n i t p r o t e i n s . T h i s r e v i e w i n c l u d e s a desc r i p t i o n o f t h e u s e f u l n e s s o f e l e c t r o p h o r e t i c techniques i n t h e s t u d y o f s u b u n i t d i v e r s i t y and assembly processes i n haemocyanins. The haemocyanins i s o l a t e d f r o m v a r i o u s s p e c i e s o f t h e p h y l a Arthropoda and Mol l u s c a commonly u t i l i z e a b i n u c l e a r copper s i t e t o b i n d oxygen c o o p e r a t i v e l y , a l t h o u g h t h e c o n s t i t u e n t p o l y p e p t i d e s and aggregated ensembles o f m o l l u s c and a r t h r o p o d haemocyanins d i f f e r

Determination o f t h e molecular weight

o f t h e c o n s t i t u e n t p o l y p e p t i d e c h a i n o f t h e haemocyanin o f t h e m o l l u s c Busycon

canaZicuZatm by sodium dodecyl s u l p h a t e (SDS) e l e c t r o p h o r e s i s shows t h a t SDS e l e c t r o p h o r e s i s c o n s i s t e n t l y underestimates t h e m o l e c u l a r w e i g h t when compared w i t h a n a l y s i s by s e d i m e n t a t i o n e q u i l i b r i u m i n d e n a t u r i n g s o l v e n t s . M o l e c u l a r w e i g h t s of 240,000 and 300,000,

r e s p e c t i v e l y , have been reported8".

The r e s u l t s

suggest t h a t t h i s l o n g p o l y p e p t i d e may become o r i e n t e d w h i l e p a s s i n g t h r o u g h t h e a c r y l a m i d e m a t r i x , t h e r e b y i n c r e a s i n g i n r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y . The observed e q u i v a l e n t SDS b i n d i n g o f Busycon haemocyanin and s t a n d a r d p r o t e i n s , such as b o v i n e serum albumin, d i f f e r s from t h e l o w SDS b i n d i n g t o haemocyanins r e p o r t e d b y S a l v a t o and Z a t t a "

f o r o t h e r s p e c i e s . D i f f e r i n g apparent m o l e c u l a r w e i g h t s

from e q u i l i b r i u m and t r a n s p o r t t e c h n i q u e s f o r o t h e r m o l l u s c a n haemocyanins have a l s o been

E l e c t r o n microscopy o f t h e n a t i v e p o l y p e p t i d e s , produced

by d i s s o c i a t i o n a t a l k a l i n e pH, r e v e a l s a t e r t i a r y s t r u c t u r e f o r t h e m o l l u s c a n

157 haemocyanin c h a i n s which appears as "beads on a s t r i n g " 1 3 . The c u r r e n t l y a c c e p t e d model envisages t h e c h a i n s as b e i n g composed o f domains, each o f which has a s i n g l e oxygen b i n d i n g s i t e , connected by a s h o r t l i n k e r r e g i o n . The S D S - e l e c t r o p h o r e t i c analysis o f t h e products o f c o n t r o l l e d p r o t e o l y t i c d i g e s t i o n (which apparently c l i p s a t t h e 1 i n k e r r e g i o n s ) o f n a t i v e p o l y p e p t i d e s o f m o l l u s c a n haemocyanins shows m u l t i p l e s o f a 50,000 d a l t o n b a n d l 4 - I 6 . E l e c t r o p h o r e s i s o f t h e domains i n t h e absence o f SDS and a p a r t i a l p u r i f i c a t i o n o f i n d i v i d u a l domains r e v e a l a n apparent charge h e t e r o g e n e i t y o f t y p e s o f domains, m i r r o r e d by d i f f e r e n c e s i n t h e i r oxygen-binding

Uncertainty b c t h i n t h e molecular weights o f t h e

p o l y p e p t i d e and t h e domains l e a v e s u n c l e a r t h e e x a c t number o f domains p r e s e n t p e r chain. H e t e r o g e n e i t y m a n i f e s t s i t s e l f n o t o n l y w i t h i n t h e domains b u t a l s o f o r t h e i n t a c t p o l y p e p t i d e , as shown w i t h h r e x fuZvescens haemocyanin17. The e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e c h a i n s , i d e n t i c a l on SDS g e l s , were p u r i f i e d chromatog r a p h i c a l l y . N a t i v e a1 k a l i n e e l e c t r o p h o r e s i s r e v e a l e d t h a t each chromatographic peak behaves as an e l e c t r o p h o r e t i c a l l y p u r e s p e c i e s . R e p e t i t i o n o f experiments w i t h haemocyanin o f a s i n g l e Murex demonstrates t h a t t h e observed h e t e r o g e n e i t y i s w i t h i n t h e s u b u n i t s o f a s i n g l e i n d i v i d u a l , R e a s s o c i a t i o n experiments have shown t h a t b o t h s u b u n i t s a r e r e q u i r e d f o r t h e reassembly o f t h e 100s m o l e c u l e . These reassembled 100s m o l e c u l e s show v e r y s i m i l a r f u n c t i o n a l p r o p e r t i e s compared w i t h t h e n a t i v e , u n d i s s o c i a t e d , 100s m o l e c u l e . A r t h r o p o d haemocyanins a r e composed o f 75,000 d a l t o n p o l y p e p t i d e c h a i n s . Sedim e n t a t i o n e q u i l i b r i a and g e l f i l t r a t i o n i n d e n a t u r i n g s o l v e n t s and SDS e l e c t r o p h o r e s i s a l l g i v e s i m i l a r m o l e c u l a r weights18-21.

S a l v a t o and Z a t t a l '

have r e -

p o r t e d v e r y l o w SDS b i n d i n g by a r t h r o p o d haemocyanins, y e t t h e agreement among a l l o f t h e t e c h n i q u e s g i v e s s u p p o r t t o t h e 75,000 d a l t o n c h a i n s i z e . D i s c o n t i n u o u s SDS g e l t e c h n i q u e s have been a b l e t o r e s o l v e s u b t l e m o l e c u l a r w e i g h t h e t e r o g e n e i t y among t h e p o l y p e p t i d e s o f many a r t h r o p o d haernocyanins22. A1 k a l i n e e l e c t r o p h o r e s i s o f t h e n a t i v e p o l y p e p t i d e c h a i n s o f a number o f C h e l i c e r a t e haemocyanins r e v e a l s unique p a t t e r n s f o r each species22. The s t u d y o f M a r k l e t a1 .22 i s p a r t i c u l a r l y s i g n i f i c a n t i n t h a t t h e y demonstrated complete d i s s o c i a t i o n t o s u b u n i t s and assayed a l l o f t h e i r samples under i d e n t i c a l c o n d i t i o n s . The f r e q u e n t l a c k o f such c o n t r o l s makes t h e comparison o f d i f f e r e n t s p e c i e s f r o m t h e a v a i l a b l e l i t e r a t u r e a l m o s t i m p o s s i b l e . On t h e b a s i s o f t h e i r r e s u l t s M a r k l e t a1.22 have proposed t h e hypot h e s i s t h a t t h e r e i s a d i r e c t r e l a t i o n s h i p between s u b u n i t c o m p l e x i t y and t h e maximum a t t a i n a b l e a g g r e g a t i o n s t a t e . Recent a p p l i c a t i o n o f c r o s s e d immunoelectrophoresis t o a number o f s p e c i e s i m p l i e s t h a t c a u t i o n must be used i n e v a l u a t i n g h e t e r o g e n e i t y by p o l y a c r y l a m i d e e l e c t r o p h o r e s i s . Study o f two horseshoe crabs, a s c o r p i o n and a s p i d e r d e m n s t r a t e s t h a t t h e r e i s n o t always i d e n t i c a l correspondence between e l e c t r o p h o r e t i -

158 c a l l y d i s t i n g u i s h a b l e s u b u n i t s and i m m u n o l o g i c a lly d i s t i n c t

subunit^^^-^^.

As t h e

crossed immunoelectrophoresis i s s e n s i t i v e t o b o t h n e t charge and immunological determinates, t h e l a t t e r o f which a r e i n d i c a t o r s o f d i f f e r e n c e s i n t h e p r o t e i n s u r f a c e and p o s s i b l e p r i m a r y s t r u c t u r e , i t i s perhaps a more r e l i a b l e i n d i c a t o r o f h e t e r o g e n e i t y which m a n i f e s t s i t s e l f e i t h e r f u n c t i o n a l l y o r i n assembly. Curr e n t work i n o u r l a b o r a t o r y shows t h a t t h i s i s t h e case f o r Limulus haemocyanin29

.

One o f t h e most i m p o r t a n t c h a r a c t e r i s t i c s t h a t have made haemocyanins v e r y u s e f u l t o o l s i n s t u d y i n g p r o t e i n assembly i s t h e ease o f p u r i f y i n g t h e e l e c t r o p h o r e t i c a l l y i d e n t i f i e d c o n s t i t u e n t s u b u n i t s f r om a number o f species 23,28,30- 35 Reassembly experiments show t h a t d i f f e r e n t s u b u n i t t y p e s p l a y d i f f e r e n t s t r u c t u r a l r o l e s i n t h e assembly o f t h e aggregate 34s36-38. The d i f f e r e n t s u b u n i t t ypes i n seve ra l i n s t a n c e s a r e a l s o known t o d i f f e r f u n c t i o n a l l y 32 s39-41. Oxygen- b i n d i n g c h a r a c t e r i s t i c s v a r y i n b o t h a b s o l u t e oxygen a f f i n i t y and i n t h e e x t e n t t o which h e t e r o t r o p i c mo dul a t i o n o f oxygen b i n d i n g i s p o s s i b l e . The p h y s i o l o g i c a l s i g n i f icanc e o f t h e f u n c t i o n a l h e t e r o g e n e i t y between s t r u c t u r a l l y d i s t i n c t s u b u n i t s i s s t i l l u n c e r t a i n . The s t r u c t u r a l r o l e s o f t h e heterogeneous s u b u n i t s i n t h e assembly process a r e more c l e a r l y d e f i n e d . Wi t h LimuZus haemocyanin a l l b u t one o f t h e subu n it s has a d e f i n e d r o l e i n assembling t h e 48-subunit aggregate. Experiments w i t h t h r e e C h e l i c e r a t e s , LimuZus, Androetonus and EurypeZma, show t h a t t h e haemocyan in s u b u n i t s o f one species can s u b s t i t u t e f o r homologous s u b u n i t s f r o m " a n o t h e r species3*, These homologous s u b u n i t s show immunoel e c t r o p h o r e t i c c r o s s - r e a c t i v i t y . It i s appare nt t h a t s t r u c t u r a l c h a r a c t e r i s t i c s o f t h e s u b u n i t s have been conserved. I n summary, e l e c t r o p h o r e t i c t e c h n i q u e s have played a s i g n i f i c a n t r o l e i n det e c t i n g , and s t u dy i n g , s u b u n i t h e t e r o g e n e i t y . Charge h e t e r o g e n e i t y i s n o t r e a d i l y d e t e c t a b l e b y o t h e r t e c h n i q u e s . Thus i t has p l a yed a major r o l e i n t h e subsequent p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h e c o n s t i t u e n t s u b u n i t s o f m o l l u s c and a r t h r o p o d haemocyanins. The s y n e r g i s t i c c o m b i n a t ion o f immunology and e l e c t r o p h o r e s i s seen i n immunoelectrophoresis i s v e r y u s e f u l i n i d e n t i f y i n g homology i n s u b u n i t m i x t u r e s and i n r a p i d l y d e t e r m i n i n g t h e uniqueness o f an e l e c t r o p h o r e t i c a l l y i d e n t i f i e d bond. These t e c h n i q u e s w i l l c o n t i n u e t o be i m p o r t a n t i n c u r r e n t

s t u d i e s which a r e f o c u s i n g on t h e s p e c i f i c i n t e r a c t i o n s and mechanisms o f assembly o f th es e high-molec u l a r - w e i g h t p r o t e i n s . REFERENCES

1 T. Svedberg and F. Heyroth, J . h e r . Chem. Soe., 5 1 (1929) 539. 2 T. Svedberg and A . Hedenius, BioZ. B U Z Z . , 66 (1934) 191. 3 K.E. Van Holde and E.F.J. Van Bruggen, i n S.N. T imashef f and G.D. Fasman ( E d i t o r s ) , BioZogicaZ MueromoZecuZar S e r i e s , Vol. 5 , Marcel Dekker, New York, 1971, pp. 1-53. 4 E. A n t o n i n i and C. Chiancone, Ann. Rev. Biophys. Bioeng., 6 (1977) 239.

159 5 J . Bonaventura, C. Bonaventura and B. S u l l i v a n , i n J.V. Bannister (Editor), Structure and Function of Hemocyanins, S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New York, 1977, pp. 206-216. 6 J . Bonaventura and C . Bonaventura, h e r . Zoo. , 20 (1980) 7. 7 N.C. Eickman, R.S. Himmelwright and E . I . Solomon, Proc. f l a t . Acad. S c i . u.S., 76 (1979) 2094. 8 S. Q u i t t e r , L. Watts and R. Roxby, AnaZ. Biochem., 89 (1978) 187. 9 S. Q u i t t e r , L. J a t t s , C. Crosby and R. Roxby, J . BioZ. Chem., 253 (1978) 525. 10 B. S a l v a t o and P. Z a t t a , Comp. Biochern. Phy s i o Z. , 608 (1978) 107. 11 M. Brouwer and H.A. K u i p e r , Eur. J . Biochem., 35 (1973) 428. 12 B. S a l v a t o , A. G h i r e t t i - M a g a l d i and F. - G h i r e t t i , Biochemistry, 18 (1979) 2731. 13 J.F.L. Van Breeman, G.J. Schuurhuis and E.F.J. Van Bruggen, i n J.V. B a n n i s t e r ( E d i t o r ) , Structure and Function of Hemocganins, S p r i nger-Verl ag , B e r l i n , H e i d e l b e r g , New York, 1977, pp. 122-126. 14 J.V. B a n n i s t e r , A. Galdes and W.H. B a n n i s t e r , Comp. Biochem. P h y s i o l . , 518 (1975) 1. 15 E.J. Wood, i n J.V. B a n n i s t e r ( E d i t o r ) , Structure and Function of Hemocyanins, S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New York, 1977, pp. 77-84. 16 C. G i e l e n s , G. Preaux and R. L o n t i e , i n J.V. B a n n i s t e r ( E d i t o r ) , S t r u c t u r e and Function of Hernocyanins, S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New York, 1977, pp. 85-94. 17 M. Brouwer, M. Ryan, J. Bonaventura and C. Bonaventura, Biochemistry, 17 (1978) 2810. 18 D.E. Carpenter and K.E. Van Holde, Biochemistry, 12 (1973) 2231. 19 L.M. Hamlin and W.W. F i s h , Biochim. Biophys. Acta, 491 (1977) 46. 20 M. Johnson and D.A. Yphantis, Biochemistry, 17 (1978) 1448. 21 R. Roxby, K . I . M i l l e r , D.P. B l a i r and K.E. Van Holde, Biochemistry, 13 (1974) 1662. 22 J. M a r k l , A. M a r k l , W. Schartau and B. L i n z e n , J . Comp. PhysioZ., 130B (1979) 283. 23 J . Lamy, J . Lamy, J.F. Penser and J . W e i l l , C.R. Acad. S c i . P a r i s , 2820 (1976) 1995, 24 J . Lamy, J . Lamy, J. W e i l l , J. M a r k l , H.J. Schneider and B. L i n z e n , HoppeS e y l e r ' s Z. PhysioZ. Chem., 360 (1979) 889. 25 3. Lamy, J. Lamy and J . W e i l l , Arch. Biochem. Biophys., 193 (1979) 140. 26 J . Lamy, J . Lamy, J. W e i l l , J . Bonaventura, C . Bonaventura and M. B r e n o w i t z , Arch. Biochem. Biophys., 196 (1979) 324. 27 M. H o v l a e r t s , G. Preaux, R. W i t t e r s and R. L o n t i e , Arch. Int. PhysioZ. Biochem., 87 (1579) 417. 28 H.J. Schneider, J . M a r k l , W. Schartau and B. L i n z e n , Hoppe-SeyZer's 2. PhysioZ. Chem.. 358 (1977) 1133. 29 M. Brenowit;, C.'Bonaventura, J, Bonaventura and E. Gianazza, Arch. Biochem. Biophys. , 210 ( 1381) 748. 33 A.C. Murray and P.D. J e f f r e y , Biochemistry, 13 (1974) 3667. 31 P.D. J e f f r e y , D.C. Shaw and G.B. Treacy, Biochemistry, 15 (1976) 5527. 32 B. S u l l i v a n , J . Bonaventura and C. Bonaventura, Proc. Nat. h a d . S c i . U . S . , 7 1 (1974) 2558. 33 B. S u l l i v a n , J. Bonaventura, C . Bonaventura and G. G o d e t t e , J . BioZ. C h c m . , 251 (1976) 7644. 34 J. Lamy, 2 . Lamy, M.C. B a g l i n and J. W e i l l , i n J.V. B a n n i s t e r ( E d i t o r ) , Structure and Function of Hemocyanins, S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New York, 1977, pp. 37-49. 35 J. Markl, W. S t r y c h , W. Schartau, H.J. Schneider, P . Schobert and B. Linzen, Hoppe-Seyler's Z. Physiol. Chem. , 360 (1979) 639, 36 P.D. J e f f r e y , D.C. Shaw and G.B. Treacy, Biochemistry, 17 (1978) 3078. 37 i1.M.C. B i j l h o l t , E.F.J. van Bruggen and J. Bonaventura, E u r . J . Biocizern., 95 (1979) 399. 38 E.F.J. van Bruggen, 1.1.C. B i j l h o l t , J.W. S c h u t t e r , T. U i c h e r t j e s , J . Bonaventura, C. Bonaventura, J . Lamy, J. Lamy, M. L e c l e r c , H.J. Schneider, J. Hark1 and B. L i n z e n , FEES L e t t , , 116 (1380) 207.

160

39 H. Decker, J . Markl, R. Loewe and 6 . Linzen, Hoppe-SeyZer's

2 . PhysioZ. Chern., 360 (1979) 639. 40 C. Bonaventura, B. Sullivan, J . Bonaventura and S . Bourne, Biochemistry, 13 (1974) 4784. 4 1 J. Lamy, J . Larny, C. Bonaventura and J . Bonaventura, Biochemistry, 19 (1980) 3033.

161

Chapter 9.9 HUMAN HAEMOGLOBINS ARTHUR B. SCHNEIDER and ALAN

N. SCHECHTER

GENERAL ASPECTS I s o e l e c t r i c f o c u s i n g and e l e c t r o p h o r e s i s have provided convenient and extremely s e n s i t i v e methods f o r s t u d y i n g t h e s t r u c t u r e s o f normal , m o d i f i e d and mutant haemog l o b i n s . These methods have a l s o been u s e f u l i n f u n c t i o n a l s t u d i e s o f haemoglobin, b u t l e s s u s e f u l than column chromatography f o r p r e p a r a t i v e purposes. STRUCTURAL STUDIES

Noma 1 haemoglobins Normal human haemoglobins i n c l u d e a d u l t (HbA and HbA2), f a e t a l (HbF w i t h Gy A o r y c h a i n s ) , and embryonic (Gower 1, Gower 2 and P o r t l a n d ) haemoglobins. Elect r o p h o r e t i c and i s o e l e c t r i c f o c u s i n g methods have been used t o i d e n t i f y these haemoglobins and t h e i r component g l o b i n chains. E l e c t r o p h o r e s i s i n a l k a l i n e b u f f e r s and i s o e l e c t r i c f o c u s i n g w i t h pH 6-8 ampholytes separate haemoglobins A, F and A2 ( r e f s . 1 and 2 ) . I n s t a r c h gel e l e c t r o p h o r e s i s a t pH 8.6 Hb-Gower 1 and HbA2 have t h e same m o b i l i t y . Hb-Portland moves s l i g h t l y more towards t h e cathode 3 than HbA,andHb-Gower 2 remains near t h e anode

.

Two methods have been used t o produce haemoglobin subunits. Non-denatured subunits r e t a i n i n g haeme a r e produced by r e a c t i o n w i t h p-chloromercuribenzoate 4Y5

.

This method has t h e advantages o f p e r m i t t i n g b e n z i d i n e d e t e c t i o n 6 and o f a1 l o w i n g recovery o f n a t i v e s u b u n i t s . Component g l o b i n chains a r e produced by d e n a t u r a t i o n A i n urea When t h e chains have t h e same charge, such as t h e y and Gy chains,

s e p a r a t i o n can be achieved by adding n e u t r a l detergents such as T r i t o n

X-100 o r NP-40 t o t h e sample, presumably because o f d i f f e r e n t i a l d e t e r g e n t b i n d 8-11 ing . For p r e p a r a t i v e purposes, ion-exhange chromatography i s u s u a l l y used. It should be noted, however, t h a t Houghten and Chrambach12 have shown t h a t isotachophoresis can be used t o separate r e l a t i v e l y l a r g e amounts o f haemoglobin from albumin. The same group has a l s o shown t h a t p r e p a r a t i v e i s o e l e c t r i c f o c u s i n g can e f f e c t i v e l y 13 separate HbA and HbS w i t h a 70% recovery o f HbA

.

162

Mutant haemogZobins Charge d i f f e r e n c e s , which f i r s t a l l o w e d P a u l i n g e t a1 .I4 t o demonstrate t h e m o l e c u l a r change i n s i c k l e c e l l disease, remain t h e p r i m a r y p r o p e r t y used t o s t u d y mutant haemoglobins. E l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e s t r i p s has been t h e p r i n c i p a l method o f s c r e e n i n g l a r g e numbers o f samples 2 y 7 '15. E l e c t r o p h o r e s i s i n agar g e l s o r on c e l l u l o s e a c e t a t e s t r i p s , a t a l k a l i n e o r a c i d i c pH, w i t h o r w i t h o u t u r e a ( t o observe t h e haemoglobin o r i t s c h a i n s ) , can be used f o r f u r t h e r c h a r a c t e r iz a t i on 2 y 7 y 1 6 , Another u s e f u l method f o r i d e n t i f y i n g t h e m u t a n t c h a i n i s t h e p r e p a r a t i o n o f mixed t e t r a m e r s w i t h o t h e r s p e c i e s such as c a n i n e haemoglobin 17 , by comparing t h e p r o d u c t s o f t h e f o l l o w i n g r e a c t i o n s : HbA:

C C 9 8 2 + "282

a-mutant:

aiB2

L L

t a2y2

-c

C + a2P2

L + a2B2

t a;p2

a282

L

R e c e n t l y , i s o e l e c t r i c f o c u s i n g i n t h i n p o l y a c r y l a m i d e l a y e r s has been i n t r o duced as an e f f e c t i v e s c r e e n i n g procedure18y19. Hb Malmo and s e v e r a l o t h e r m u t a n t haemoglobins a r e e l e c t r o p h o r e t i c a l l y " s i l e n t " b u t a r e d e t e c t e d b y i s o e l e c t r i c 20,21

focusing

.

An e l e c t r o p h o r e t i c method f o r d e m o n s t r a t i n g mutants w i t h abnormal oxygen a f f i n i t y was d e s c r i b e d by Rosa e t a1.22. The m u t a n t haemoglobin i s p l a c e d under anaerobic c o n d i t i o n s and r e a c t e d w i t h iodoacetamide. C y s t e i n e Bg3 r e a c t s o n l y i n t h e oxygenated s t a t e , so a mutant w i t h a h i g h oxygen a f f i n i t y w i l l r e a c t and w i l l n o t change i t s e l e c t r o p h o r e t i c m o b i l i t y a f t e r a subsequent r e a c t i o n w i t h cystamine i n t h e presence o f oxygen.

Post t m n s c r i p t i o n a l modificatiom Post t r a n s c r i p t i o n a l m o d i f i c a t i o n s o f haemoglobin i n c l u d e g l y c o s y l a t i o n , a c e t y l a t i o n , a d d i t i o n o f p h o s p h o r y l a t e d g l y c o l y t i c i n t e r m e d i a t e s and methaemoglobin formation. There i s c u r r e n t l y much i n t e r e s t i n HbAIC7

a g l y c o s y l a t e d haemoglobin which

r e s u l t s f r o m t h e non-enzymatic r e a c t i o n o f t h e amino t e r m i n a l v a l i n e o f t h e 6 - c h a i n w i t h g l t ~ c o s e ~ ~Measurements ' ~ ~ . o f HbAIC have become widespread because o f t h e good c o r r e l a t i o n between t h e m e t a b o l i c c o n t r o l o f d i a b e t e s and t h e l e v e l s o f HbAC I ( r e f s . 25-27). HbAIC

can be measured by e l e c t r o p h o r e s i s , c o l o r i m e t r i c r e a c t i o n s

f o r p r o t e i n-bound carbohydrates, ion-exchange chromatography and i s o e l e c t r i c f o c u s i n g ( s e e r e f . 28 f o r c i t a t i o n s ) . Although i s o e l e c t r i c f o c u s i n g i n a g r a d i e n t between pH 6 and 8 r e s u l t s i n good r e s o l u t i o n 1y28-307 t h e small d i f f e r e n c e i n SIP and t h e l a r g e excess o f HbA make q u a n t i t a t i o n d i f f i c u l t . B e c c a r i a e t a1 .28 showed t h a t t h e s e p a r a t i o n c o u l d be improved by i n t r o d u c i n g h i s t i d y l g l y c i n e i n t o g e l s t o s e r v e as a " s e p a r a t o r " , as i t has a p r which r e s u l t s i n f l a t t e n i n g o f t h e pH g r a d i ent i n t h e area o f i n t e r e s t .

163 I n a d d i t i o n t o i t s non-enzymatic r e a c t i o n w i t h glucose, HbA can r e a c t w i t h a number o f p h o s p h o r y l a t e d g l y c o l y t i c i n t e r m e d i a t e s (such as g l u c o s e 6-phosphate and f r u c t o s e 6-phosphate) t o f o r m p r o d u c t s which f o c u s i n t h e p o s i t i n o f HbAlb ( r e f . 3 1 ) . A c e t y l a t e d HbA a l s o focuses i n t h e p o s i t i o n o f HbAlb

and i s e l e v a t e d

i n p e o p l e who use l a r g e amounts o f a s p i r i n 3 ’ .

When a s p i r i n i s i n c u b a t e d w i t h HbA, 32,33 HbS o r r e d b l o o d c e l l s , a c e t y l a t i o n o c c u r s a t t h r e e o r more l y s i n e r e s i d u e s , Bunn and D r y ~ d a l eshowed ~~ t h a t i s o e l e c t r i c f o c u s i n g c o u l d be used t o i d e n t i f y i.

f u l l y o x i d i z e d methaemoglobin and two p a r t i a l l y o x i d i z e d species, a2B2 and a2B2. F e r r i h a e m o g l o b i n s can be i d e n t i f i e d b y t h e a d d i t i o n o f c y a n i d e because cyanomethaemoglobin and oxyhaemoglobin have t h e same P whereas I S methaemoglobin and oxyhaemoglobin d i f f e r by about 0.2 pH

I s o e l e c t r i c f o c u s i n g has been used

t o i d e n t i f y methaemoglobinaemia, t o s t u d y t h e f o r m a t i o n o f methaemoglobin i n

erythrocyte^^^ reductase

36

.

and t o s t u d y t h e r e d u c t i o n o f methaemoglobin by NADH-cytochrome b5

FUNCTIONAL STUDIES Several f u n c t i o n a l p r o p e r t i e s o f haemoglobin have been s t u d i e d e f f e c t i v e l y by i s o e l e c t r i c f o c u s i n g . These p r o p e r t i e s i n c l u d e t h e t e t r a m e r - d i m e r e q u i l i b r i u m , b i n d i n g o f l i g a n d s t o haeme and b i n d i n g o f o r g a n i c phosphates. The f o r m a t i o n o f mixed t e t r a m e r s , i . e . ,

a2R2 t ahB;

*

2 m ’ B B ’ , has been

t h e most u s e f u l s t r a t e g y i n s t u d y i n g t h e s u b u n i t i n t e r a c t i o n s o f haemoglobin. Mixed t e t r a m e r s can b e demonstrated most r e a d i l y b y m i x i n g l i g a n d e d haemoglobins and t h e n s t a b i l i z i n g t h e mixed t e t r a m e r s by r e d u c i n g t h e t e m p e r a t u r e and c r e a t i n g a n a e r o b i c c o n d i t i o n s . R e c e n t l y , a method t h a t can be used a t t e m p e r a t u r e s as l o w as -3OoC, u s i n g acrylamide-methylacrylate copolymers and d i m e t h y l s u l phoxidewater m i x t u r e s , has been d e s c r i b e d 3 7 . Park38 s t u d i e d t h e r a t e o f d i s s o c i a t i o n o f HbA t e t r a m e r s i n t o d i m e r s by o b s e r v i n g mixed t e t r a m e r f r o m a t i o n w i t h HbC o r c a n i n e haemoglobin. The d i s s o c i a t i o n r a t e f o r deoxyhaemoglobin was s l o w and c o u l d be observed o v e r s e v e r a l hours, whereas t h e d i s s o c i a t i o n r a t e o f l i g a n d e d haemog l o b i n was t o o f a s t t o be observed. T h i s i n d i c a t e d t h a t t h e deoxyhaemoglobin even a t v e r y l o w cont e t r a m e r was h i g h l y f a v o u r e d o v e r t h e dimer (K = lo-’’) eq c e n t r a t i o n s . Park a l s o s t u d i e d t h e p l a n e o f d i s s o c i a t i o n o f HbA t e t r a m e r s i n v a r i o u s c o n d i t i o n s u s i n g mixed t e t r a m e r f o r m a t i o n w i t h c a n i n e h a e m o c ~ l o b i n ~The ~. p l a n e was t h e same f o r l i g a n d e d and deoxyhaemoglobin a t pH 7 and 10.6. Liganded haemoglobin can be separated f r o m deoxyhaemoglobin by i s o e l e c t r i c f o c u s i n g because o f t h e Bohr e f f e c t . By f o c u s i n g p a r t i a l l y l i g a n d e d HbA and t h e n a l l o w i n g ATP t o m i g r a t e t h r o u g h t h e bands. Park c o n f i r m e d t h a t ATP was bound o n l y t o t h e deoxy f o r m w i t h a m o l a r r a t i o o f 1 : l (ATP:haemoglobin t e t r a m e r ) 38

.

I n o r d e r t o d e t e r m i n e t h e pH r a n g e o f t h e t i t r a t a b l e amino a c i d s which a r e

164 changed i n mutant haemoglobins o r which a r e i n v o l v e d i n t h e b i n d i n g o f o r g a n i c phosphate l i g a n d s , R i g h e t t i and co-workers 39’40 combined i s o e l e c t r i c f o c u s i n g w i t h e l e c t r o p h o r e s i s . A p o l y a c r y l a m i d e p l a t e i s f o c u s e d i n one d i r e c t i o n t o c r e a t e a pH g r a d i e n t and t h e sample i s i n t r o d u c e d i n t o a trough, which a l l o w s e l e c t r o p h o r e s i s t o be performed a t r i g h t angles t o t h e f o c u s i n g . I f t h e sample i s a m i x t u r e o f HbA and a mutant, t h e n one l i n e appears e x c e p t i n t h e pH range where t h e normal and mutant amino a c i d s have d i f f e r e n t charges (e.g., l i n e s above pH 5.5 f o r t h e G l u

-+

one l i n e below and two

Val s u b s t i t u t i o n i n HbS). The same t e c h n i q u e has

been used f o r haemoglobin-organic phsophate i n t e r a c t i o n s t o d e t e r m i n e t h e pH range o f s t a b i l i t y , t h e r e l a t i v e r a t e o f d i s s o c i a t i o n and t h e pKs o f t h e groups i n v o l v e d i n t h e complex. E l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g have been used t o measure t h e b i o s y n t h e s i s o f haemoglobin and i t s component g l o b i n c h a i n s , A l t h o u g h i t i s n o t p o s s i b l e t o summarize t h e s e s t u d i e s here, i t should be n o t e d t h a t t h e y have been i m p o r t a n t i n e x t e n d i n g o u r u n d e r s t a n d i n g o f e u k a r y o t i c gene e x p r e s s i o n and c e l l d i f f e r e n t i a t i o n41

.

REFERENCES

1 J.W. Drysdale, P. R i g h e t t i and H.F. Bunn, Biochim. Biophys. A c t a , 229 (1971) 42. 2 Y. Beuzard, J.C. C o u r v a l i n , M. Cohen-Solal, M.C. Garel, J. Rose, A. Gibaud and C. B r i z a r d , FEBS L,ett. , 27 (1972) 76. 3 H.A. Pataryas and G. Stamatoyannopoulos, BZood, 39 (1972) 688. 4 E. Bucci and C. F r o n t i c e l l i , J . BioZ. Chem., 240 (1965) PC551. 5 M.A. Rosemeyer and E.R. Huehns, J . MoZ. BioZ., 25 (1967) 253. 6 R.H. B r o y l e s , B.M. Pack, S. Berger and A.R. Dorn, Anal. Biochem., 94 (1979) 211. 7 R.G. Schneider, CZin. Chem., 20 (1974) 1111. 8 B.P. A l t e r , S.C. Goff, D.D. Efremov, M.E. G r a v e l y and T.H.J. Huisman, Brit. J . Haematol., 44 (1980) 527. 9 B.P. A l t e r , BZood, 54 (1979) 1158. 10 P.G. R i g h e t t i , E. Gianazza, A.M. G i a n n i , P. Comi, B. G i g l i o n i , S. O t t o l e n g h i , C. Secchi and L. R o s s i - B e r n a r d i , J . Biochem. Biophys. Methods, 1 (1979) 45. 11 P. Comi, B. G i g l i o n i , S. O t t o l e n g h i , A.M. G i a n n i , G. Ricco, U. Mazza, G. S a g l i o , 12 13 14 15 16 17 18 19 20

C. Casaschella, P.G. Pich, E. Gianazza and P.G. R i g h e t t i , Biochem. Biophys. Res. Comun., 87 (1979) 1. R. Houghten and A . Chrambach, A n d . Biochem., 77 (1977) 303. A.G. McCormick, H. W a l c h s l i c h t and A. Chrambach, AnaZ. Biochem., 85 (1978) 209. L. Pauling, H.A. I t a n o , S.J. S i n g e r and I . C . Wells, Science, 110 (1949) 543. R.G. Schneider, B. Hightower, T.S. Hosty, H. Ryder, G. Tomlin, R. A t k i s , B. B r i m h a l l and R.T. Jones, BZood, 48 (1976) 629. A.R. Robinson, M. Robson, A.P. H a r r i s o n and W.W. Zuelzer, J . Lab. C Z i n . Med., 50 (1957) 745. H.F. Bunn, Ann. N.Y. Acad. S c i . , 209 (1973) 345. P. Basset, Y . Beuzard, M.C. Garel and 3. Rosa, BZood, 51 (1978) 971. A. G i u l i a n i . M. M a t i n u c c i . M.P. CaDDabianCa. D. M a f f i and L. T e n t o r i , CZin. C h h . Acta,-40 (1978) 19.. S.H. Boyer, S. Charache, U.F. F a i r b a n k s , J.E. Maldonado, A. Noyes and E.E. Gayle, J . CZin. I n v e s t . , 51 (1972) 253.

..

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J. Chen-Marotel , F. B a r a c o n n i e r , Y. B l o u q i t , J. M a r t i n - C a b u r i , J. Kammerer and J. Rosa, Hemoglobin, 3 (1979) 253. 22 J. Rosa, Y . Beuzard, J. T h i l l e t , M.C. Gavel and J. Caburic, i n R.11. Schmidt

21

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

( E d i t o r ) , Abnormal Haemoglobins and Thalassaemia: Diagnostic Aspects, Academic Press, New York, 1975, pp. 79-116. H.F. Bunn, D.N. Haney, K.H. Gabbay and P.M. G a l l o p , Biochem. Biophys. Res. Commun. , 67 (1975) 103. R.J. Koenig, S.H. B l o b s t e i n and A . Carami, J . B i o l . Chem., 252 (1977) 2992. R.J. Koenig, C.M. Peterson, R.L. Jones, C. Saudek, M. Lehrman and A. Cerami, N . Engl. J . Med., 295 (1976) 417. K.H. Gabbay, K. Hasty, J.L. Breslow, R.C. E l l i s o n , H.F. Bunn and P.M. G a l l o p , J . Clin. EndocrinoZ. Metab., 44 (1977) 859. B. Gonen, A.H. R u b i n s t e i n , H. Rochman, S.P. Tanega and D.L. H o r o w i t z , Lancet, ii (1977) 734. L. B e c c a r i a , G. Chiumello, E. Gianazza, B. L u p p i s and P.G. R i g h e t t i , h e r . J . Hematol. , 4 (1978) 367. K.M. S p i c e r , R.C. A l l e n and M.G. Buse, Diabetes, 27 (1978) 384. R. Schoos, S. Barbette-Schoos and C. Lambotte, Clin. Chim. Acta, 86 (1978) 61. V.J. Stevens, H. Vlassava, A. A b a t i and A . Cerami , J . B i o l . Chem., 252 (1977) 2998. K.R. B r i d g e s , G.J. Schmidt, M. Jensen, A. Cerami and H.F. Bunn, J . CZin. I n v e s t . , 56 (1975) 201. M. Shamsuddin, R.G. Mason, J.M. R i t c h e y , G.R. Honig and I . M . K l o t z , Proc. Nut. Acad. S c i . U.S. , 7 1 (1974) 4693. H.F. Bunn and J.W. Drysdale, Biochim. Biophys. A c t a , 229 ( 1 9 7 1 ) 51.A. Tomoda, M. T a k e s h i t a and Y . Yaneyama, FEBS L e t t . , 88 (1978) 247. A. Tomoda, M. Imoto, M. H i r a n o and Y . Yoneyama, Biochem. J . , 1 8 1 (1979) 505. M. P e r r e l l a , A. Heyda, A. Mosca and L. R o s s i - B e r n a r d i , Anal. Biochem., 8 8 (1978) 212. C.M. Park, Ann. N.Y. Acad. S c i . , 209 (1973) 237. P.G. R i g h e t t i , R. Krishnamoorthy, E. Gianazza and D. L a b i e , J . Chromatogr., 166 (1978) 455. R. Krishnamoorthy, A.B. B o s i s i o and P.G. R i g h e t t i , FEBS L e t t . , 94 (1978) 319. G. Stamatoyannopoulos and A.W. N i e n h u i s ( E d i t o r s ) , CelZular and MoZecuZar Regulation o f Hemoglobin Switching, Grune and S t r a t t o n , New York, 1979.

166

Chapter 9.10 ISOELECTRIC FOCUSING OF IMMUNOGLOBULINS MURRAY H. FREEDMAN Immunochemists have been impeded i n t h e i r s t u d y o f immunoglobulins by t h e h e t e r o g e n e i t y o f a n t i b o d i e s . Homogeneous a n t i b o d i e s a r e needed f o r t h e d e t e r m i n a t i o n o f t h e amino a c i d sequence and t h r e e - d i m e n s i o n a l s t r u c t u r e and t h e p r o d u c t i o n o f a n t i - i d i o t y p i c a n t i b o d i e s i n o r d e r t o understand s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s i n t h e immune system. I s o e l e c t r i c f o c u s i n g ( I E F ) i n l i q u i d media, p o l y a c r y l a m i d e g e l s o r ‘Sephadex has been s u c c e s s f u l l y u t i l i z e d b o t h a n a l y t i c a l l y and preparat i v e l y t o f r a c t i o n a t e , p u r i f y and e v a l u a t e t h e s t r u c t u r e and/or g e n e t i c s o f many d i f f e r e n t immunoglobulins. D u r i n g t h e p a s t decade, s e v e r a l r e v i e w s have d e s c r i b e d 1-6 t h e methodology and a p p l i c a t i o n o f l i q u i d and g e l IEF o f immunoglobulins

.

IEF i n l i q u i d media’

i s one o f t h e most s u c c e s s f u l methods f o r o b t a i n i n g l a r g e

amounts o f s t r u c t u r a l l y and f u n c t i o n a l l y homogeneous a n t i b o d i e s f r o m h e t e r o g e neous p o p u l a t i o n s . For example, s p e c i f i c a l l y p u r i f i e d r a b b i t a n t i h a p t e n a n t i bodies, o f p o l y c l o n a l o r i g i n , were separated i n t o a number o f monoclonal f r a c t i o n s by p r e p a r a t i v e l i q u i d IEF8”.

P a r t i a l sequencing (20-40 r e s i d u e s ) o f t h e

amino-terminal v a r i a b l e r e g i o n s o f t h e e l e c t r o f o c u s e d l i g h t c h a i n s was performed i n an a t t e m p t t o d i s t i n g u i s h between germ l i n e , somatic m u t a t i o n and i n s e r t i o n a l models o f a n t i b o d y diversity’’.

The 1 i g h t c h a i n f r o m each e l e c t r o f o c u s e d a n t i b o d y

f r a c t i o n d i d n o t always g i v e a u n i q u e v a r i a b l e r e g i o n sequence, i m p l y i n g t h a t t h e number o f d i f f e r e n t a n t i b o d i e s i s n o t n e c e s s a r i l y equal t o t h e number o f bands i n t h e i s o e l e c t r i c spectrum. T h i s a n t i b o d y h e t e r o g e n e i t y can r e s u l t f r o m d i s t i n c t 9,11,12 s t r u c t u r a l genes and/or p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s I n o r d e r t o o b t a i n s u f f i c i e n t amounts o f i n d i v i d u a l e l e c t r o f o c u s e d a n t i b o d i e s f o r sequencing and g e n e t i c analyses , r e s t r i c t e d o r p a r t i a l l y r e s t r i c t e d r a b b i t a n t i b o d i e s were o b t a i n e d f r o m a l l o t y p e - d e f i n e d p e d i g r e e d r a b b i t s hyperimmunized w i t h e i t h e r s t r e p t o c o c c a l o r pneumococcal

vaccine^'^-'^.

I n some o f t h e s e s t u d i e s ,

t h e e l e c t r o f o c u s e d f r a c t i o n s were used t o i s o l a t e a n t i b o d i e s r e s t r i c t e d i n a l l o t y p e and i d i ~ t y p e l ’ ” ~ . The amounts o b t a i n e d were s u f f i c i e n t f o r f u r t h e r chemical and immunological s t u d i e s . Recently, u s i n g l i q u i d IEF, a l l o t y p e - r e l a t e d charge d i f f e r e n c e s were o b t a i n e d between immunoglobulins and l i g h t c h a i n s from r a b b i t s d i f f e r i n g a t t h e Ab l o c u s 19 The d i f f e r e n c e between t h e average A p l o f immunoglobulins o f a l l o t y p e Ab4 and Ab9 i s due t o charge d i f f e r e n c e s between t h e i r r e s p e c t i v e l i g h t c h a i n s and n o t

.

167 due t o t h e heavy c h a i n s . L i q u i d IEF has a l s o been used s u c c e s s f u l l y as p a r t o f t h e p u r i f i c a t i o n p r o c e d u r e f o r a v a r i e t y o f normal 20-22 and p a t h o l o g i c a l immunoglobulin^^^-*^ as w e l l as f o r s p e c i f i c a n t i b o d i e s 8- 15,17,26

A s i m p l e microcolumn method has been used f o r t h e a n a l y s i s o f immunoglobulins by f o c u s i n g i n l i q u i d media27. T h i s method has been o f g r e a t v a l u e i n t h e a n a l y s i s o f a n t i b o d y produced by s p l e n i c fragments c u l t u r e d i n v i t r o b u t i s l e s s s u i t a b l e 28 f o r comparing a n t i b o d i e s produced b y d i f f e r e n t fragments

.

A n a l y t i c a l IEF i s most c o n v e n i e n t l y performed i n p o l y a c r y l a m i d e g e l . T h i s t e c h n i q u e has become a f r e q u e n t l y used method f o r s t u d y i n g t h e h e t e r o g e n e i t y o f a n t i body responses and t o screen g e n e t i c a l l y d e f i n e d p o p u l a t i o n s o f l a b o r a t o r y a n i m a l s f o r t h e p a t t e r n o f s p e c i f i c a n t i b o d i e s 5 y 2 9 - 3 4 . IEF has a l s o been used t o screen human s e r a f o r t h e o c c u r r e n c e o f n a t u r a l l y a c q u i r e d a n t i b o d i e s t o s t r e p t o c o c c a l group p o l y s a c c h a r i d e a n t i g e n s 3 5 , t o m o n i t o r changes which may o c c u r i n a u t o a n t i bodies d u r i n g t h e autoimmune response36y37, t o screen t h e immunoglobul i n s o f c e r e b r o s p i n a l f l u i d and serum i n m u l t i p l e s c l e r o s i s p a t i e n t s 3 8 , t o m o n i t o r t h e immunog l o b u l i n s o f p a n c r e a t i c f l u i d i n p a t i e n t s w i t h p a n c r e a t i c cancer3’

and t o s t u d y

i m n u n o g l o b u l i n changes a f f e c t e d by a v a r i e t y o f diseases and p h y s i o l o g i c a l s t r e s s e s I t i s one o f t h e most r e l i a b l e methods f o r d e t e c t i n g p a r a p r o t e i n a e m i a s and f o r r e veal i n g whether monoclonal o r p o l y c l onal p a r a p r o t e i n s a r e i n v o l v e d . A n a l y t i c a l p o l y a c r y l a m i d e g e l IEF can be performed i n t w o ways - i n n a r r o w tubes41 o r i n a t h i n l a y e r on a g l a s s p l a t e 4 2 . The t h i n - l a y e r system has s e v e r a l advantages o v e r t h e t u b e system: m u l t i p l e samples can be compared o n t h e same g e l , t h e pH g r a d i e n t can be c o n v e n i e n t l y measured and a v a r i e t y o f o v e r l a y t e c h n i q u e s can b e used. The s p e c i f i c d e t e c t i o n o f a n t i b o d i e s i n t h i n - l a y e r g e l IEF has been extended c o n s i d e r a b l y b y o v e r l a y i n g t h e g e l w i t h a n t i g e n 4 . D e t e c t i o n o f t h e antibody-bound a n t i g e n o r s u b s t r a t e has been based on (1) t h e use o f r a d i o a c t i v e a n t i g e n and a u t o r a d i o g r a p h y 29-35y43-45,

( 2 ) t h e u s e o f t h e enzyme a c t i v i t y o f t h e a n t i g e n

46

and ( 3 ) t h e use of a n t i g e n bound t o e r y t h r o c y t e s and subsequent complement-depend e n t l y s i s ( h a e m ~ l y s i s ) ~F ~o r. d e t e c t i n g s p e c i f i c a n t i b o d i e s , t h e method u s i n g r a d i o a c t i v e a n t i g e n and a u t o r a d i o g r a p h y has such a h i g h r e s o l v i n g power and sens i t i v i t y t h a t s e v e r a l micrograms o f c l o n a l l y d e f i n e d a n t i b o d i e s c a n be d e t e c t e d i n immune s e r a o r i n c u l t u r e s u p e r n a t a n t s c o n t a i n i n g t h e b y p r o d u c t s o f s p e c i f i c lymphocyte c l o n e s s e c r e t e d b y s e v e r a l hundred c e l l s 5 y 4 8 . The r e s o l v i n g power a t t a i n a b l e on t h i n - l a y e r g e l IEF when two bands a r e compared s i d e - b y - s i d e i s a t 4 C o n s i d e r i n g t h a t most a n t i b o d i e s f o c u s i n a 2 pH u n i t range, l e a s t 0.005 pH u n i t

.

t h i s a l l o w s 400 t h e o r e t i c a l l y f o c u s i n g p a t t e r n s f o r a s i n g l e a n t i b o d y band. The method o f enzyme a c t i v i t y of t h e a n t i g e n has t h e g r e a t e s t p o t e n t i a l s e n s i t i v i t y f o r d e t e c t i o n o f a n t i b o d y 4 . U l t i m a t e l y , a s i n g l e m o l e c u l e o f anti-enzyme a n t i b o d y c o u l d be d e t e c t e d . The use of a n t i - i m m u n o g l o b u l i n s e r a f o r development enhances

40

,

168 t h e s e n s i t i v i t y g r e a t l y . Also, e x t r a i n f o r m a t i o n i s o b t a i n e d by u s i n g c l a s s o r subclass s p e c i f i c a n t i s e r a .

A disadvantage o f t h e p o l y a c r y l a m i d e g e l IEF method i s t h a t l a r g e molecules (e.g., ISM) do n o t e n t e r t h e g e l . T h i s d i f f i c u l t y has been overcome by t h e use 6,49 o f p o l y m e r i z a b l e t h i n l a y e r s c o n t a i n i n g Sephadex as an a n t i c o n v e c t i v e medium

.

IEF i s now a f a s t and r e l i a b l e method f o r t h e g e n e t i c a n a l y s i s o f a n t i b o d y responses a t t h e c l o n o t y p e l e v e l . The method may be regarded as b e i n g s u p e r i o r t o i d i o t y p i c a n a l y s i s when s t u d y i n g t h e e x p r e s s i o n o f a number o f c l o n o t y p e s i n t h e course o f an immune response5. A n a l y t i c a l IEF has a r e s o l v i n g power s i m i l a r t o a n t i - i d i o t y p i c a n t i s e r a . I n f a c t , i n w e l l documented cases o f i d i o t y p i c i d e n 50,51

t i t y o f c l o n o t y p e s t h e IEF p a t t e r n s were o v e r l a p p i n g

.

When an i s o e l e c t r i c spectrum o f an a n t i b o d y i s used t o c h a r a c t e r i z e t h e phenot y p e o f an animal i t i s c a l l e d s p e c t r o t y p e 2 7 - 2 9 y

52-56. One can t h e n use t h e

s p e c t r o t y p e , i n a s i m i l a r manner t o t h e use o f i d i o t y p e , as a g e n e t i c marker I n t h e mouse, two o f t h e V

4

.

genes which have been mapped can be f o l l o w e d by c h a r -

ac t e r is t i c s p e c t r o t y p e s 50yg7-59. A g i v e n s p e c t r o t y p e r e q u i r e s t h e e x p r e s s i o n o f p a r t i c u l a r VL,

CL, VH and CH genes. As t h e i n h e r i t a n c e o f a s p e c t r o t y p e maps w i t h

t h e H c h a i n genes, mouse s t r a i n s a l l o w i n g e x p r e s s i o n o f t h e s p e c t r o t y p e must possess t h e necessary L c h a i n genes i n t h e i r r e p e r t o i r e . IEF has been used t o e s t i m a t e s p e c i f i c a n t i b o d y r e p e r t o i r e s 5 2 y 6 0 . R e p e r t o i r e s i z e i s e s t i m a t e d by measuring t h e f r e q u e n c y w i t h w h i c h i d e n t i c a l a n t i b o d y molecules a r e e l i c i t e d i n independent events. A n t i - i d i o t y p i c a n t i b o d i e s a r e powerful t o o l s i n s t u d i e s on a n t i b o d y d i v e r s i t y . These a n t i b o d i e s can themselves be c h a r a c t e r i z e d by a n a l y t i c a l g e l IEF. I d i o t y p i c d e t e r m i n a n t s can be c o r r e l a t e d w i t h h y p e r v a r i a b l e r e g i o n s o f 1 i g h t and heavy c h a i n s . The m u l t i - d e t e r m i n a n t view o f i d i o t y p y p r e d i c t s h e t e r o g e n e i t y o f a n t i - i d i o t y p i c a n t i b o d i e s . T h i s was c o n f i r m e d b y g e l IEF6’,

and t h i s t y p e o f

a n a l y s i s s h o u l d be v a l u a b l e i n c l a r i f y i n g d i f f e r e n c e s i n r e a c t i v i t y o f d i f f e r e n t s e r a and i n m o n i t o r i n g a b s o r p t i o n o f sera t o make them s p e c i f i c f o r i n d i v i d u a l d e t e r m i n a n t s . Examination o f 19s and 7s a n t i - a (1+3) d e x t r a n (Dex) a n t i b o d i e s by IEF r e v e a l e d s u b s t a n t i a l v a r i a t i o n o f i d i o t y p i c and s p e c t r o t y p i c e x p r e s s i o n between i n d i v i d u a l s o f t h e same genotype62. The d a t a demonstrate t h a t s u b s t a n t i a l s t r u c t u r a l v a r i a t i o n e x i s t s among a n t i b o d i e s c o m p r i s i n g a response o f r e s t r i c t e d h e t e r o g e n e i t y . Gel IEF s t u d i e s have shown t h a t murine immunoglobulins w i t h d i f f e r e n t s p e c i f i c i t i e s have s i m i l a r i d i o t y p e s u s i n g t h e VH marker63. L i q u i d and g e l IEF were used t o demonstrate t h a t i n d i v i d u a l r a b b i t s a r e a b l e t o s y n t h e s i z e a n t i bodies d i r e c t a g a i n s t t h e i r own i d i o t y p i c d e t e r m i n a n t s ( a u t o a n t i - i d i o t y p i c a n t i bodies)64. These a n t i b o d i e s modulate t h e i d i o t y p i c e x p r e s s i o n when t h e animal i s s u b s e q u e n t l y r e s t i m u l a t e d by t h e o r i g i n a l a n t i b o d y . Based on t h e g e n e t i c s o f t h e i s o e l e c t r i c f o c u s i n g p a t t e r n s o f m u r i n e a n t i - i n s u l i n a n t i b o d i e s , t h i s system

169 p r o v i d e d a u s e f u l model f o r t h e i d e n t i f i c a t i o n o f v a r i a b l e r e g i o n genes t h a t encode 65 antigen-binding s i t e s s p e c i f i c f o r p r o t e i n molecules

.

S t u d i e s u s i n g a n a l y t i c a l g e l IEF have a l s o demonstrated g e n e t i c polymorphism o f murine l i g h t

chain^^^-^'.

The s p e c t r a l d i f f e r e n c e s were shown t o be i n h e r i t e d

i n a s i m p l e codominant f a s h i o n , b u t t h e g e n e t i c b a s i s o f t h e polymorphism i s n o t y e t known. I t was suggested t h a t t h e g e n e t i c d i f f e r e n c e s among IEF s p e c t r a p o s s i b l y i n v o l v e d t h e presence o f a d d i t i o n a l o r a l t e r e d v a r i a b l e r e g i o n sequences i n t h e l i g h t c h a i n s o f c e r t a i n s t r a i n s o f mice. Two-dimensional e l e c t r o p h o r e s i s , employing gel IEF i n t h e f i r s t dimension and sodium dodecyl s u l p h a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e second7',

sepa-

r a t e s p r o t e i n s a c c o r d i n g t o two d i f f e r e n t parameters. S e n s i t i v i t y , r e p r o d u c i b i l i t y and h i g h r e s o l u t i o n make t h i s t e c h n i q u e a b e t t e r method t h a n g e l IEF a l o n e . T h i s t e c h n i q u e p r o v i d e s t h e p o t e n t i a l f o r s t u d y i n g gene p r o d u c t s a t t h e m o l e c u l a r l e v e l . I n summary, t h e s i m p l i c i t y o f t h e equipment, t h e ease o f o p e r a t i o n and t h e h i g h r e s o l v i n g power and s e n s i t i v i t y make p r e p a r a t i v e IEF and a n a l y t i c a l p o l y a c r y l a m i d e g e l IEF p r e f e r a b l e t o many o t h e r t e c h n i q u e s . The u s e o f IEF t o s e p a r a t e and a n a l y s e complex m i x t u r e s o f immunoglobulins has enhanced o u r u n d e r s t a n d i n g o f a n t i b o d y g e n e t i c s and s h o u l d be v a l u a b l e i n u n r a v e l l i n g t h e o r i g i n o f a n t i b o d y d iversi ty

.

ACKNOWLEDGEMENTS Supported b y a g r a n t f r o m t h e Medical Research C o u n c i l o f Canada. The a u t h o r i s i n d e b t e d t o B.J. Trew-Marshall and C.G.

Cupples f o r many h e l p f u l d i s c u s s i o n s

during the preparation o f t h i s review. REFERENCES

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170 14 M.H. Freedman, H. Yeger, J. M i l a n d r e and M. S l a u g h t e r , I m n o c h e m i s t r y , 12 (19751 553. 15 M. Frkedman, T. Hofmann, S. Rhese, H. Yeger, M. S l a u g h t e r and J. M i l a n d r e , Imunochemistry , 12 (1975) 263. 16 0. James and M. Freedman, I m n o c h e m i s t r y , 14 (1977) 15. 17 L.S. Rodkey, T.K. Choi and A. N i s o n o f f , J . ImunoZ., 104 (1970) 63. 18 R.G. Mage, J.H. Pincus, C. Alexander and M.H. Freedman, Eur. J . ImunoZ., 4 (1974) 560. 19 R. Notenboom, S. D u b i s k i , 6. Cinader and 6. Underdown, MoZ. ImunoZ., 16 (1979) 77. 20 E. Valmet,sci. TooZs, 15 (1969) 8. 21 A. Howard and G. V i r e l l a , Protides BioZ. FZuids, Proc. CoZZoq., 17 (1970) 449. 22 G. V i r e l l a and A. Howard, Experientia, 26 (1970) 901. 23 M. E u l i t z , Protides BioZ. FZuids, Proc. CoZZoq., 17 (1970) 481. 24 P.M. M o r i t z , A.A. C o r b e t t and J.R. Hobbs, Protides BioZ. FZuids, Proc. CoZZoq., 17 (1970) 499. 25 R.A. M u r g i t a and S.I. Vas, J . I m n o Z . , 104 (1970) 514. 26 R.A. M u r g i t a and S . I . Vas, ImunoZogy, 22 (1972) 319. 27 J.L. Press and N.R. Klinman, Immunochemistry, 10 (1973) 621. 28 N.R. Klinman and J.L. Press, J . Exp. Med., 141 (1975) 1133. 29 A.R. Williamson, Eur. J . I m n o Z . , 1 (1971) 390. 30 H.W. K r e t c h and A.R. W i l l i a m s o n , Eur. J . ImurwZ., 3 (1973) 141. 31 M. Cramer and D.G. Braun, J . Exp. Med., 139 (1974) 1513. 32 M. Freedman, J. M i l a n d r e and H. Yeger, J . ImmunoZ. Methods, 6 (1974) 165. 33 H. Yeger and M. Freedman, A n a Z . Biochern., 64 (1975) 450. 34 M. Wabl and L. Du Pasquier, Nature ILondonl, 264 (1976) 642. 35 W.F. Riesen, F. S k v a r i l and D.G. Baun, Scand. J . I m n o Z . , 5 (1976) 383. 36 G.N. Abraham, I m n o Z o g y , 35 (1978) 429. 37 G.N. Abraham, P. Brown, S.L. Johnston, L . N e l l i s , S. Marks and E.H. Welch, ImmunoZogy , 35 (1978) 447. 38 J.E. Olsson and K. N i l s s o n , NeuroZogy, 29 (1979) 1383. 39 R.L. Goodale, R.M. Condie, T.D. D r e s s e l , T.N. T a y l o r and K. G a j l - P e c z a l s k a , Ann. Surg., 189 (1979) 340. 40 R.C. A l l e n , J . Chrornatogr., 146 (1978) 1. 41 C.W. Wrigley, J . Chromatogr., 36 (1968) 362. 42 Z.L. Awdeh, A.R. W i l l i a m s o n and B.A. Askonas, Nature (London), 219 (1968) 66. 43 K. Keck, A.L. Grossberg and 0. Pressman, E u r . J . I m n o Z . , 3 (1973) 99. 44 K. Keck, A.L. Grossberg and 0. Pressman, Immunochemistry, 10 (1973) 331. 45 M. Cramer, M. Schwarz, E. Mozes and M. Sela, Eur. J . I m n o Z . , 6 (1976) 618. 46 G. Kb’hler and F. Melchers, Eur. J . ImunoZ., 2 (1972) 453. 47 J.M. P h i l l i p s and D.W. Dresser, Eur. J . ImmunoZ., 3 (1973) 524. 48 D.G. Baun, J. Ouintans, A.L. L u z z a t i , I . L e f k o v i t s and S.E. Read, J . Exp. Med. , 1 4 3 - ( 1976) 360. 49 A. Z i e g l e r and G. Kohler, FEBS L e t t . , 64 (1976) 48. 50 K. Eichmann, Eur. J . I m u n o t . , 2 (1972) 301. 51 D.G. Braun and A.S. Kelus, J . Exp. Med., 138 (1973) 1248. 52 J.R.L. P i n k and B.A. Askonas, Eur. J . ImunoZ., 4 (1974) 426. 53 P.C. Montgomery, R.L. Kahn and C.A. Skandera, J . Immuml., 115 (1975 904. 54 G. Kohler, Eur. J . ImunoZ. , 6 (1976) 340. 55 N.H. S i g a l and N.R. Klinman, Advan. ImunoZ., 26 (1978) 255. 56 C. Boyer, C. S c h i f f , M. M i l i l i and M. Fougereau, MoZ. ImunoZ., 16 ( 979) 1073. 57 K. Eichmann and C. Berek, &r. J . I m n o Z . , 3 (1973) 599. 58 T. I m a n i s h i and 0. Ma’kela, Eur. J . ImunoZ., 2’(1972) 323. 59 A.J. McMichael, J.M. P h i l l i p s , A.R. W i l l i a m s o n , T. I m a n i s h i and 0. Makela, I m n o g e n e t i c s , 2 (1975) 161. 60 A.R. Williamson, Ann. Reu. Biochem. , 45 (1976) 467. 6 1 R.M. P e r l m u t t e r , D.E. B r i l e s and J.M. Davie, J . Immunot., 118 (1977) 2161. 62 0. Hansburg, R.M. P e r l m u t t e r , D.E. B r i l e s and J.M. Davie, E m . J. I m o Z . , 8 (1978) 352.

171 63 E. Enghofer, C.P.J. Glaudemans and M.J. Bosma, MoZ. I m u n o l . , 16 (1979) 1103. 64 C. Wuilmart, M. W i k l e r and J . U r b a i n , MoZ. Immunol., 16 (1979) 1085. 65 J.A. Kapp, D.S. S t r a y e r , P.F. Robbins and R.M. P e r l m u t t e r , J . ImmunoZ., 123 (1979) 109. 66 J.L. C l a f l i n , Eur. J . ImunoZ., 6 (1976) 666. 67 D. Gibson, J . Exp. Med., 144 (1976) 298. 68 D.M. Gibson, B.A. T a y l o r and M. Cherry, J . I m u n o l . , 121 (1978) 1585. 69 D.M. Gibson and S.J. MacLean, J . Exp. Med., 149 (1979) 1477. 70 P.H. O ' F a r r e l l , J . BioZ. Chen., 250 (1975) 4007.

172

Chapter 9.11 CONTRACTILE AND CYTOSKELETAL PROTEINS PETER RUBENSTEIN T h i s r e v i e w d e s c r i b e s a p p l i c a t i o n s o f p o l y a c r y l a m i d e gel e l e c t r o f o c u s i n g and e l e c t r o p h o r e s i s i n c y t o s k e l e t a l and c o n t r a c t i l e p r o t e i n research, and s e l e c t e d examples o f d i f f e r e n t uses o f t h e s e t e c h n i q u e s a r e discussed. C o n t r a c t i l e and c y t o s k e l e t a l p r o t e i n s a r e p r e s e n t a t h i g h - l e v e l s w i t h i n t h e c e l l , and o f t e n t h e y a r e n o t a s s o c i a t e d w i t h an e a s i l y assayable enzymatic a c t i v i t y . F o r t h e s e reasons, p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n v a r i o u s d e n a t u r i n g b u f f e r systems has been w i d e l y employed t o d e t e c t , c h a r a c t e r i z e and q u a n t i t a t e t h e s e p r o t e i n s . By u s i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e presence o f sodium dodecyl sul phate (SDS-PAGE), one can d e t e c t s i m u l t a n e o u s l y a c t i n ,

a - a c t i n i n , myosin heavy and

l i g h t chains, tropomyosin and t r o p o n i n i n s k e l e t a l and smooth muscle m y o f i b r i l p r e p a r a t i ~ n s l - ~I.n s k e l e t a l muscle p r e p a r a t i o n s one can i d e n t i f y t h e C - p r o t e i n and t h e m a j o r M - l i n e p r o t e i n s w i t h t h i s t e c h n i q u e w h i l e i n smooth muscle c y t o s k e l e t a l p r e p a r a t i o n s , desmin appears as a m a j o r component. SDS-PAGE a l l o w s one 4

t o d i f f e r e n t i a t e between t h e myosin l i g h t c h a i n s o f f a s t and s l o w t w i t c h muscle

.

F u r t h e r , by use o f t h i s t e c h n i q u e , s k e l e t a l muscle myosin i s seen t o c o n t a i n t h r e e d i f f e r e n t m o l e c u l a r w e i g h t l i g h t c h a i n s , whereas smooth muscle and non5 muscle myosin d i s p l a y o n l y two d i f f e r e n t l i g h t c h a i n s

.

The same t e c h n i q u e has been used i n s t u d y i n g non-muscle c y t o s k e l e t o n s . The 6 a r e d e f i n e d by t h e i r m o b i l i t i e s

components o f t h e r e d b l o o d c e l l c y t o s k e l e t o n i n SDS-gels, e.g.,

bands 1 and 2 ( s p e c t r i n ) , band 2.1 ( a n k y r i n ) , band 3, band 4.1

and band 5 ( a c t i n ) . Condeelis and T a y l o r '

i d e n t i f i e d t h e components o f t h e D.

d i s c o i d e m c y t o p l a s m i c g e l s t r u c t u r e w i t h t h e a i d o f p o l y a c r y l a m i d e g e l s , and Brown e t a1.* used SDS-PAGE t o show t h a t t h e T r i t o n - i n s o l u b l e c y t o s k e l e t o n o f c h i c k embryo f i b r o b l a s t s c o n s i s t s l a r g e l y o f f i b r o n e c t i o n , a c t i n and i n t e r m e d i a t e f i 1ament p r o t e i n ( v i m e n t i n )

.

I n c e r t a i n i n s t a n c e s , t h e a d d i t i o n o f h i g h u r e a c o n c e n t r a t i o n s t o SDS-gel systems has been shown t o r e s u l t i n enhanced r e s o l v i n g power. W i t h t h i s t e c h n i q u e , s k e l e t a l muscle tropomyosin can be c l e a r l y s e p a r a t e d i n t o two forms w i t h apparent d i f f e r e n c e s i n m o l e c u l a r weights', w e i g h t c l a s s e s 'OY1'

t u b u l i n can be separated i n t o two m o l e c u l a r

and a v i a n muscle a c t i n and non-muscle a c t i n become r e s o l v a b l e

(see r e f . 1 2 ) . O ' F a r r e l l 13, i n 1975, d e s c r i b e d a h i g h - r e s o l u t i o n , two-dimensional g e l system

173 which u t i l i z e s i s o e l e c t r i c f o c u s i n g i n 9 m o l / l urea i n t h e f i r s t dimension and

SDS-PAGE i n t h e second. This system works o p t i m a l l y w i t h p r o t e i n s o f molecular weight l e s s than 100,000 d a l t o n s and i s o e l e c t r i c p o i n t s lower than 6.5,

although

a second system has been described f o r more b a s i c proteins14. With t h e e x c e p t i o n o f a-actinin,

myosin heavy c h a i n and high-molecular-weight a c t i n - b i n d i n g p r o t e i n s

such as s p e c t r i n and f i l a m i n , most c o n t r a c t i l e and c y t o s k e l e t a l p r o t e i n s can be analysed by t h i s h i g h - r e s o l u t i o n technique. Such analyses demonstrate t h a t these p r o t e i n s a r e a c t u a l l y complex f a m i l i e s o f v a r i a n t polypeptides. I n a v i a n and mammalian t i s s u e s , i t was demonstrated t h a t t h e r e were t h r e e i s o e l e c t r i c v a r i a n t s o f a c t i n , termed a , 6, and y a c t i n s i n o r d e r o f i n c r e a s i n g b a s i c i t y 1 5 - 1 7 . I s o e l e c t r i c f o c u s i n g a n a l y s i s o f t h e a c t i n s from lower eukaryotes such as Acanthmoeba18 and D i c t y o ~ t e Z i w n ' ~showed t h a t t h e s e a c t i n s had i s o e l e c t r i c p o i n t s r e s p e c t i v e l y more b a s i c and more a c i d i c than those o f t h e b i r d s and mammals. Subsequent sequencing s t u d i e s by Vandekerckhove and Weber2'

demonstrated

t h a t t h e t h r e e i s o e l e c t r i c f o c u s i n g classes o f a v i a n and mammalian a c t i n s were made up o f s i x d i f f e r e n t gene products: a non-muscle 8 - a c t i n , a non-muscle y - a c t i n , a smooth muscle y-, a s k e l e t a l muscle a-, a smooth muscle a- and a c a r d i a c muscle a-actin.

Hence, d e s p i t e t h e h i g h r e s o l v i n g power o f t h e O ' F a r r e l l gel system,

t h i s technique can s t i l l y i e l d o n l y a minimum e s t i m a t e o f t h e c o m p l e x i t y o f a p a r t i c u l a r p r o t e i n f a m i l y , as c o n s e r v a t i v e o r completely compensating amino a c i d s u b s t i t u t i o n s cannot be detected. Two-dimensional g e l s have been used by a number o f i n v e s t i g a t o r s t o show t h a t 21-24

a- and 8 - t u b u l i n s f r o m b r a i n a r e each composed o f m u l t i p l e species Experiments by Gozes and L i t t a u e r ' l

.

demonstrated t h a t 1i v e r and spleen t u b u l i n

were composed p r i m a r i l y o f a s i n g l e a and s i n g l e

8 subunit instead o f the large

number of v a r i a n t s observed i n mature b r a i n . They f u r t h e r demonstrated t h a t t h e heterogeneity seen i n b r a i n t u b u l i n increased as a f u n c t i o n o f b r a i n m a t u r a t i o n and probably r e s u l t e d from p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n o f t h e p r o t e i n s . Desmin surrounds t h e Z-disc i n s k e l e t a l muscle and i s t h e major component o f 10 nm f i l a m e n t s i n smooth muscle. Two-dimensional gel a n a l y s i s o f c h i c k c a r d i a c , s k e l e t a l and smooth muscle showed t h a t each c o n t a i n s two i s o e l e c t r i c f o c u s i n g desmin v a r i a n t s o f t h e same molecular weight i n t h e same p r o p o r t i o n i n each t i s s u e 2 5 y 2 6 . S i m i l a r experiments i n mammalian muscle r e v e a l e d o n l y one form o f desmin. Brown e t a1.8 have a l s o shown t h a t t h e c h i c k embryo f i b r o b l a s t 10 nm f i l a m e n t s p r o t e i n i s d i f f e r e n t from t h e a v i a n muscle desmins i n d i c a t i n g t h a t from t i s s u e t o t i s s u e i n t h e same organism, m o r p h o l o g i c a l l y i d e n t i c a l i n t e r m e d i a t e f i l a m e n t s a r e probably composed o f t h e products o f d i f f e r e n t s t r u c t u r a l genes. Tropomyosin, as analysed by two-dimensional

g e l s , has been shown t o be another 25 c o n t r a c t i l e p r o t e i n coded f o r by a l a r g e f a m i l y o f d i f f e r e n t s t r u c t u r a l genes

.

Chick g i z z a r d tropomyosin has two components w i t h s l i g h t l y d i f f e r e n t i s o e l e c t r i c

174

p o i n t s and d i f f e r e n t apparent m o l e c u l a r w e i g h t s (34,000 and 36,000 d a l t o n s ) , and c h i c k c a r d i a c tropomyosin can be s e p a r a t e d i n t o two 34,000 d a l t o n components w i t h d i f f e r e n t isoel e c t r i c points. I s o e l e c t r i c f o c u s i n g a l o n e and i n c o n j u n c t i o n w i t h SDS-PAGE has been employed w i t h g r e a t success i n s t u d y i n g t h e developmental c o n t r o l o f c o n t r a c t i l e p r o t e i n gene e x p r e s s i o n . Whalen e t al.27,

u s i n g embryonic r a t muscle and c u l t u r e d r a t

s k e l e t a l muscle c e l l s , demonstrated t h e presence o f an embryonic f o r m o f myosin l i g h t c h a i n LC1 w h i c h i s r e p l a c e d b y t h e a d u l t f o r m f o l l o w i n g m y o b l a s t f u s i o n . I n a v i a n and mammalian p r e f u s i o n myoblasts, non-muscle 6 and y i s o a c t i n s a r e t h e prevalent

form^.'^-^^.

However, c o n c o m i t a n t w i t h m y o b l a s t f u s i o n , t h e muscle a - a c t i n

gene i s t u r n e d on u n t i l , i n a d u l t s k e l e t a l muscle, a - a c t i n i s t h e o n l y d e t e c t a b l e a c t i n observed. I n s i m i l a r experiments w i t h embryonic c h i c k e n g i z z a r d , S a b o r i o e t a t . 2 8 showed t h a t p r i o r t o t h e 8-day embryo stage, o n l y B - a c t i n was made. S t a r t i n g a t 8 days; however, t h e smooth muscle y - a c t i n gene i s t u r n e d on, r e s u l t i n g i n t h i s 29

f o r m o f a c t i n making up a b o u t 75% o f t h e a c t i n seen i n mature g i z z a r d . G a r r e l s

l o o k e d i n t o t o a t t h e changes o f p h e n o t y p i c e x p r e s s i o n t h a t o c c u r d u r i n g myogenes i s . Using two-dimensional g e l s o f i s o t o p i c a l l y l a b e l l e d c u l t u r e d muscle c e l l s coupled w i t h computerized d e n s i t o m e t r i c scanning o f t h e autoradiograms, he s t u d i e d t h e t u r n i n g on and o f f o f 300 p o l y p e p t i d e s , t h e i d e n t i t i e s o f many o f which a r e known. T h i s s t u d y demonstrates t h e a b i l i t y o f t h e two-dimensional g e l system t o be u t i l i z e d f o r a n a l y s i n g changes i n p h e n o t y p i c e x p r e s s i o n o f many gene p r o d u c t s s i m u l t a n e o u s l y i n response t o changing environment o r i n comparing mutant w i t h normal c e l l s . The t e c h n i q u e s h o u l d t h e r e f o r e p r o v e v a l u a b l e i n l o o k i n g a t t h e g e n e t i c b a s i s o f neuromuscular d i s e a s e and a t t h e response o f c o n t r a c t i l e t i s s u e s t o v a r i o u s d r u g regimens. A number o f c o n t r a c t i l e and c y t o s k e l e t a l p r o t e i n s such as a c t i n , tropomyosin

and t r o p o n i n C a r e permanently m o d i f i e d p o s t - t r a n s l a t i o n a l l y w h i l e o t h e r s such as s p e c t r i n and t h e 20,000 d a l t o n smooth muscle myosin 1 i g h t c h a i n a r e t r a n s i e n t l y m o d i f i e d . I f t h e m o d i f i c a t i o n r e s u l t s i n a change i n t h e n e t charge o f t h e p r o t e i n , t h e m o d i f i c a t i o n s h o u l d be d e t e c t a b l e by i s o e l e c t r i c f o c u s i n g p r o v i d e d t h a t t h e p r o t e i n i n q u e s t i o n e n t e r s t h e g e l . With two-dimensional g e l s t h e r e l a t i v e degree o f m o d i f i c a t i o n o f a number o f p r o t e i n s can, i n t h e o r y , be s i m u l t a n e o u s l y analysed. I n a l l t i s s u e s s t u d i e d t o date, a c t i n has been shown t o have a b l o c k e d N - t e r minus. Where s t u d i e d i n d e t a i l group".

, the

b l o c k i n g group has proved t o be an a c e t y l

When compared w i t h t h e u s u a l l y s t a b l e a c t i n n o r m a l l y observed, r a p i d l y

m e t a b o l i z e d a c t i n species have been observed i n mammal ian3O, i n s e c t 3 1 and s l i m e mould c e l l s 3 * .

I n D. d i s c o i d e m u s i n g two-dimensional g e l s , i t was shown t h a t t h e

s t a b l e f o r m o f a c t i n was a c e t y l a t e d whereas t h e r a p i d l y t u r n i n g - o v e r f o r m was not3'.

Subsequent

experiment^^^

have r e v e a l e d t h a t t h e f u l l y t r a n s l a t e d b u t non-

a c e t y l a t e d a c t i n can be a c e t y l a t e d by an a c e t y l CoA-dependent enzyme system. By

175 s i m i l a r techniques, O'Connor e t a1 .34 demonstrated t h a t i n c h i c k t i s s u e s , f i b r o b l a s t i n t e r m e d i a t e f i l a m e n t p r o t e i n , desmin and a- and 6-tropomyosin a r e p a r t i a l l y phospharylated b o t h i n v i t r o and i n vivo. Brenner and K ~ r have n ~ shown ~ the specific and r e v e r s i b l e enzymatic p h o s p h o r y l a t i o n o f band 2 s p e c t r i n u s i n g autoradiography o f SDS g e l s o f 3 2 P - l a b e l l e d m a t e r i a l . The same one-dimensional SDS-gel system has shown t h a t t h e 20,000 d a l t o n smooth muscle l i g h t c h a i n i s t h e s i t e o f phosphoryla36,37

t i o n by t h e Ca2+-calmodulin a c t i v a t e d myosin l i g h t c h a i n kinase

.

I n summary, SDS-polyacrylamide gel e l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g have proved extremely v a l u a b l e i n assaying f o r t h e presence o f f a m i l i e s o f cont r a c t i l e and c y t o s k e l e t a l p r o t e i n s , i n determining t h e c o m p l e x i t y o f these m u l t i component f a m i l i e s , i n s t u d y i n g t h e developmental r e g u l a t i o n o f t h e expression o f these p r o t e i n s and i n assessing t h e i r degree o f p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n . These techniques w i l l i n e v i t a b l y be u s e f u l i n t h e f u t u r e i n s t u d y i n g t h e enzymes c a r r y i n g o u t t h e m o d i f i c a t i o n o f these p r o t e i n s and i n d i s c o v e r i n g t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e e x t e n s i v e m i c r o h e t e r o g e n e i t y observed i n these c o n t r a c t i l e and c y t o s k e l eta1 elements. REFERENCES

1 J.D. E t t i n g e r , R. Zak and D.A. Fischman, J . CeZZ BioZ., 68 (1976) 123. 2 Y. Camaron, S. Neuman and D. Yaffe, CeZZ, 14 (1978) 393. ' 3 A. Sobieszek and R.D. Bremel, Eur. J . Biochem., 55 (1975) 49. S. Sarkar, C. Mis and F. S t r e t e r , Biochem. Biophys. Res. 4 R. Roy, - . K. Mabuchi, Comun., 89 (1979)-181. 5 M. Clarke and J. Spudich, Ann. Rev. Biochem., 46 (1977) 797. 6 S. LUX, Nature (London), 281 (1979) 426. 7 J . Condeelis and D.L. T a y l o r , J . CeZZ BioZ., 74 (1977) 901. 8 S. Brown, W. Levinson and J . Spudich, J. SupmmoZ. S t r u c t . , 5 (1976) 119. 9 P. Cummins and S.V. Perry, Biochem. J . , 133 (1973) 765. 10 J . Bryan and L Wilson, ,?roc. Nat. Acad. S c i . U . S . , 68 (1971) 7 6 2 . 11 R. Luduena and D.O. Woodward, Proc. Nat. Acad. S c i . U . S . , 70 1973) 3594. 12 R.V. S t o r t i and A. Rich, Proc. Nut. Acad. S c i . U . S . , 73 (1976 2346. 13 P. O ' F a r r e l l , J . B i o Z . Chem., 250 (1975) 4007. 14 P.Z. O ' F a r r e l l , H.M. Goodman and P. O ' F a r r e l l , CeZZ, 12 (1977 1133. 15 R. Whalen, G. Butler-Browne and F. Gros, Proc. Nut. Acad. S c i U.S., 73 (1976) 2018. 16 J . G a r r e l s and W. Gibson, CeZZ, 9 (1976) 793. 17 P. Rubenstein and J . Spudich, &roc.' Nat: Acad. S c i . U.S., 74 (1977) 120. 18 D. Gordon, J . Boyer and E. Korn, J . BioZ. Chem., 252 (1977) 8300. 19 0. Uyemura, S. Brown and J . Spudich, J . BioZ. Chem., 253 (1978) 9088. 20 J. Vandekerckhove and K. Weber, J . MoZ. BioZ., 128 (1978) 783. 21 I . Gozes and U. L i t t a u e r , Nature (London), 276 (1978) 411. 22 L. N e l l e s and J . Bamburg, J . Neurochem., 32 (1979) 477. 23 C. Marotta, J . H a r r i s and J . G i l b e r t , J . Neurochem., 30 (1978) 1431. 24 I. Gozes, D. Saya and U. Lihauer, Brain R e s . , 171 (1979) 171. 25 J . I z a n t and E. Lazarides, Proc. Nut. h a d . S c i . U.S., 74 (1974) 1450. 26 E. Lazarides and D. Balzar, J r . , CeZZ, 14 (1978) 429. 27 R . Whalen, G. Butler-Browne and F. Gros, J . MoZ. BioZ., 126 (1978) 415. 28 J. Sabotio, M. Segura, M. Flores, R. Garcia and E. Palmer, J . BioZ. Chem., 254 (1979) 1119. 29 J . G a r r e l s , DeueZop. BioZ., 73 (1979) 134.

176 30 S. H o r o v i t c h , R. S t o r t i n g , A. R i c h and M.L. Pardue, J . CeZZ BioZ., 82 (1979) 86. 31 E. Berger and G . Cox, J . CeZZ B i o Z . , 8 1 (1979) 680. 32 P. Rubenstein and J . Deuchler, J . BioZ. Chem., 254 (1979) 11142. 33 p. Rubenstein, P. Smith, J . Deuchler and K. Redman, J . BioZ. Chem., 256 (1981) 8149, 34 C.M. O'Connor, D. B a l z e r , Jr. and E. L a z a r i d e s , Proc. fluat. Acud. S c i . u.S., 76 (1979) 819. 35 S. Brenner and E. Korn, J . BioZ. Chern., 254 (1979) 8620. 36 A. Sobieszek, E m . J , Biochem., 73 (1977) 477. 37 S. Chacko, M. C o n t i and R . A d e l s t e i n , Proc. Nat. Acad. S c i . U.S., 74 (1977) 129.

177

Chapter 9.12 PROTEINS OF CONNECTIVE TISSUE ZDENEK DEYL and MILADA HORAKOVA

COLLAGENS Collagens a r e a f a m i l y o f c l o s e l y r e l a t e d p r o t e i n s , and c u r r e n t l y f i v e d i f f e r e n t types a r e recognized. O f these, o n l y c o l l a g e n t y p e I i s p a r t i a l l y s o l u b l e ; a l l o f t h e o t h e r s can be brought i n t o s o l u t i o n a f t e r l i m i t e d p r o t e o l y t i c cleavage. During t h i s step, small c r o s s - l i n k e d , n o n - h e l i c a l t e r m i n a l r e g i o n s o f t h e molecule a r e s p l i t , thus r e n d e r i n g t h e c o n s t i t u e n t a - p o l y p e p t i d e chains s o l u b l e . With t y p e

I V collagen, however, l i m i t e d p r o t e o l y t i c cleavage r e s u l t s i n a heterogeneous m i x t u r e o f fragments t h a t r e s u l t s from t h e presence o f n o n - h e l i c a l r e g i o n s susc e p t i b l e t o d i g e s t i o n w i t h i n t h e c o n s t i t u t i n g a-chains. Moreover, d u r i n g biosynt h e s i s , the s o - c a l l e d procollagens a r e synthesized t h a t bear on b o t h ends o f t h e molecule (and on b o t h ends o f t h e c o n s t i t u t i n g a-chains) propeptides t h a t a r e l i b e r a t e d i n v i v o stepwise by procollagen peptidase by which d i g e s t i o n c o l l a g e n molecules a r e formed. As can be seen, t h e samples o f collagenous p r o t e i n s can be complex; some i d e a about t h e n a t u r e and r e l a t i o n s h i p s o f t h e p o l y p e p t i d e chains t o be separated can be gained from Table 9.3. I t has been demonstrated by Furthmayr and Timpl’ t h a t c o l l a g e n a-chains (and

a l s o t h e i r fragments) e x h i b i t a much lower e l e c t r o p h o r e t i c m o b i l i t y on SDS-polyacrylamide g e l s than g l o b u l a r p r o t e i n s o f s i m i l a r molecular weight.

I t can be

excluded t h a t a s m a l l e r n e g a t i v e charge o f collagenous p r o t e i n s compared w i t h o t h e r p r o t e i n c a t e g o r i e s i s t h e reason f o r t h e a t y p i c a l behaviour d u r i n g SDSpolyacrylamide gel e l e c t r o p h o r e s i s .

I f i t were t r u e , then e.g.

methylated c o l l a g e n s

should e x h i b i t much lower m o b i l i t i e s than t h e u n t r e a t e d p r o t e i n . Hayashi and Nagai2, i n a study of t h e e l e c t r o p h o r e t i c behaviour o f a-chains,

found t h a t a l -

though t h e f r e e e l e c t r o p h o r e t i c m o b i l i t y o f these p o l y p e p t i d e s i s s m a l l e r than t h a t o f o t h e r p r o t e i n s , t h e r e t a r d a t i o n c o e f f i c i e n t i s n o t d i f f e r e n t . Therefore, t h e o v e r a l l SDS-collagen complex may be comparable i n s i z e t o SDS complexes of common p r o t e i n s o f s i m i l a r r e l a t i v e molecular mass. There i s a l s o no d i f f e r e n c e i n t h e amount o f bound SDS ( a l s o , t h e e l e c t r o p h o r e t i c separations o b t a i n e d i n t h e presence and i n t h e absence o f SDS a r e v e r y s i m i l a r ) . The h i g h p r o p o r t i o n of p r o l i n e residues tends t o expand t h e random c o i l s owing t o t h e f r e e r o t a t i o n r e s t r i c t i o n around a-carbon and t h e r e f o r e one c o u l d expect a

TABLE 9 . 3

POLYPEPTIDE CHAINS EXPECTED TO OCCUR SIDE BY SIDE IN COLLAGEN PREPARATIONS Coll agen type

Constituent polypeptide chains

Molecular e n t i t i e s occurring side by side

type type type type

1 I1 I11 IV

a1(1); q ( I ) 01(II) CLl(II1) q ( I V ) ; a2(IV)

, Bayhigher y a y higher polymers b polymersb a 1l ((II1) ), ;c1 I )ya;

type

v

d, aB, aC (sometimes referred t o a s A,B,C)

$;

a (111), [a (III)]3'; higher polymersb 56 K, (68 K\, 70 K, (80-95 100 K, (120 K, 130 K), 140 K; 70 S aA, aB, aC

B: and y represent dimers and trimers of a-polypeptide chains. Higher polymers a r e present i n collagen preparations even a f t e r limited proteolysis. C dBefore S-S cleavage (corresponds to y-fraction); a f t e r reduction a l ( I I I ) occurs. Fractionsin parentheses occur rarely or i n some source tissues only.

i),

179 l a r g e r s i z e o f SDS-collagen random c o i l complexes compared w i t h o t h e r p r o t e i n s of equal r e l a t i v e m o l e c u l a r mass; however, o b v i o u s l y t h e h i g h p r o p o r t i o n o f small amino a c i d r e s i d u e s ( g l y c i n e ) compensates f o r t h e expanding e f f e c t o f p r 0 1 i n e ~ ’ ~ . The r a d i u s o f g y r a t i o n ( d e r i v e d from t h e known p r i m a r y s t r u c t u r e ) i s , i n f a c t , s l i g h t l y s m a l l e r t h a n e x p e c t e d i n o t h e r comparable p r o t e i n s . T h e r e f o r e , i t cannot be concluded t h a t t h e r e t a r d e d m o b i l i t y o f c o l l a g e n prot e i n s i s t h e r e s u l t o f a l t e r e d c o n f o r m a t i o n o f t h e SDS-collagen complex ( i n t h e denatured s t a t e )

, as

o r i g i n a l l y suggested by Furthmayr and Timpl’.

The e x p e r i m e n t a l

worker i s f a c e d h e r e w i t h a p u z z l e f o r which no r a t i o n a l e x p l a n a t i o n i s a v a i l a b l e a t t h e moment. Hayashi and Nagai 5 proposed t h a t t h e anomalous b e h a v i o u r o f c o l lagen-SDS complexes c a n be e x p l a i n e d i n terms o f t h e l o w charge d e n s i t y o f t h i s complex, presumably due t o weaker SDS-collagen i n t e r a c t i o n s , e s p e c i a l l y i f t h e collagen-SDS complex does n o t have a r o d - l i k e shape (as accepted f o r o t h e r p r o teins)

6

.

A l r e a d y i n e a r l y s t u d i e s w i t h SDS-polyacrylamide g e l e l e c t r o p h o r e s i s o f c o l l a g e n a - c h a i n s i t was demonstrated t h a t i t is p o s s i b l e t o s e p a r a t e a l ( I )

and

a 2 ( I I ) c h a i n s i n s p i t e o f t h e i r i d e n t i c a l r e l a t i v e m o l e c u l a r masses. The h i g h e r m o b i l i t y o f a2(I) c h a i n s was a t t r i b u t e d t o i t s l e s s r i g i d s t r u c t u r e as r e v e a l e d by t h e l o w e r d e n a t u r a t i o n temperature. T h i s i s , however, o f r a t h e r l i m i t e d v a l u e

i n view o f t h e above experiments t h a t a r e v a l i d b o t h f o r a l ( I )

and a 2 ( I ) p o l y -

p e p t i d e chains. I n s p i t e o f t h e above problems c o n c e r n i n g t h e e x p l a n a t i o n o f t h e r e t a r d e d m o b i l i t y o f c o l l a g e n a-chains,

t h e d i a g n o s t i c v a l u e o f SDS-polyacrylamide g e l

e l e c t r o p h o r e s i s i n c o l l a g e n c h e m i s t r y i s h i g h and w i d e l y used. I n p r a c t i c e , n o t a l l c o l l a g e n p o l y p e p t i d e c h a i n s shown i n T a b l e 9.3 o c c u r i n a s i n g l e sample s i d e by s i d e , b u t a c c o r d i n g t o t h e n a t u r e o f t h e t i s s u e

investigated only a limited

number o f combinations comes i n t o c o n s i d e r a t i o n .

Separation of colZagen type I and III I n general t h e r e a r e t h r e e means o f s e p a r a t i n g and q u a n t i t a t i n g t h e s e two c o l l a g e n t y p e s by e l e c t r o p h o r e s i s :

(1) A complete e l e c t r o p h o r e t i c s e p a r a t i o n o f al(I),

a 2 ( I ) and a l ( I I I )

poly-

p e p t i d e c h a i n s can be achieved a t r e l a t i v e l y h i g h u r e a c o n c e n t r a t i o n s on SDSp o l y a c r y l a m i d e g e l s . T h i s i s based on t h e o b s e r v a t i o n t h a t t h e presence o f u r e a decreases t h e m o b i l i t y o f a l ( I I I ) a-chains.

and cannot be d i s t i n g u i s h e d . cil(II)

chains t o a g r e a t e r e x t e n t than w i t h o t h e r

I n t h e absence o f urea, al(I)

and a l ( I I I )

m i g r a t e a t i d e n t i c a l speeds

On t h e o t h e r hand, analogous a t t e m p t s t o s e p a r a t e

and a l ( I ) were n o t s u c c e s s f u l .

( 2 ) The second approach i s based on delayed r e d u c t i o n o f d i s u l p h i d e bonds i n t y p e I11 c o l l a g e n , By i n t e r r u p t i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s e i t h e r i n

180 s l a b s o r r o d s a b o u t 1 h a f t e r t h e c o l l a g e n sample has been a p p l i e d w i t h c o n c o m i t a n t r e d u c t i o n of c o l l a g e n t y p e I11 d i s u l p h i d e bonds i n s i t u a complete s e p a r a t i o n o f a l l t h r e e collagen a-chain species involved i s possible. A t t h e time o f reduction,

[a,(I I I ) I 3 c h a i n s ( d i s u l p h i d e bonded) have s c a r c e l y e n t e r e d t h e g e l , whereas al(I)

and a 2 ( I ) have t r a v e l l e d a c e r t a i n d i s t a n c e . When t h e c u r r e n t i s s w i t c h e d

on again, t h e depolymerized a l ( I I I )

c h a i n p e n e t r a t e s t h e g e l and s t a r t s t o t r a v e l

w i t h some d e l a y , t h u s a l l o w i n g a complete d i f f e r e n t i a t i o n f r o m cil(I) 8 I n s t e a d o f 6% s l a b gels7, r o d s can be used

.

and ~ ~ ( 1 ) .

( 3 ) F o r t h e s e p a r a t i o n o f c o l l a g e n t y p e I and I 1 1 i n small s k i n samples ( t i s s u e b i o p s i e s ) , SDS-polyacrylamide e l e c t r o p h o r e s i s p a t t e r n o f t h e CNBr p e p t i d e s was used by Weber e t a1.’

(12% g e l s ) . Most o f t h e bands a p p e a r i n g i n t h e CNBr p e p t i d e

m i x t u r e a r e complex and c o n s i s t o f p e p t i d e s d e r i v e d f r o m d i f f e r e n t c o l l a g e n chains. On t h e o t h e r hand, t h e r e a r e a few bands that c o n s i s t p u r e l y o f t y p e I o r t y p e 111, c o l l a g e n p e p t i d e s t h a t can be used f o r q u a n t i t a t i o n . F u r t h e r improvement i n t h i s c a t e g o r y o f s e p a r a t i o n s can be achieved by a r a t i o n a l s e l e c t i o n o f t h e g e l m a t r i x . Whereas on c o n v e n t i o n a l g e l s t h e s e p a r a t i o n o f i n d i v i d u a l fragments i s i n c o m p l e t e w i t h any p a r e n t c o l l a g e n a-chain,

t h e r e s o l u t i o n on t h e c o n t i n u o u s concave p o l y -

a c r y l a m i d e g r a d i e n t i n t r o d u c e d by Cole and Bean’’

i s much b e t t e r and a l l o w s an

almost complete s e p a r a t i o n o f a l ( I ) , a2(I) and al( 111) p o l y p e p t i d e c h a i n s and a l s o t h e main CNBr fragments.

Separation of basement membrane colZagens P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i s a l s o w i d e l y used f o r t h e c h a r a c t e r i z a t i o n o f p r o d u c t s r e s u l t i n g f r o m 1 i m i t e d p r o t e o l y s i s o f basement membrane c o l l a g e n ( t y p e I V ) . F o r i n s t a n c e , t h e 70 K (70,000 r e l a t i v e m o l e c u l a r mass f r a g m e n t ) and o t h e r s can e a s i l y be d i s t i n g u i s h e d f r o m c o n v e n t i o n a l a - c h a i n s [oll(I) 11 4.5% g e l s a r e used f o r t h i s purpose ,

A, B and C c o l l a g e n s ( t y p e

V)

and a ( I ) ] . M o s t l y

i s o l a t e d f r o m c h i c k embryos, human c h o r i o n i c o r

a l l a n t o i c membrane can be separated by d i s c o n t i n u o u s s l a b g e l e l e c t r o p h o r e s i s . The method used i s i n p r i n c i p l e t h a t o f Laemmli” Mark13. Type

V c o n s t i t u t i n g a-chains (A,

and o f Von d e r Mark and Von d e r

6, C) r e v e a l a h i g h e r e l e c t r o p h o r e t i c

m o b i l i t y than al(I). P e p t i d e p r o f i l e s o b t a i n e d a f t e r p r o t e o l y t i c cleavage can a l s o be used f o r t h e i d e n t i f i c a t i o n o f basement membrane c o l l a g e n , as shown by Kao and Foreman14. I n s p i t e o f t h e complex p a t t e r n s o b t a i n e d , i d e n t i f i c a t i o n o f cil(I), cxB c h a i n s i s p o s s i b l e a f t e r , e.g.,

a 2 ( I ) , aA and

c h y m o t r y p t i c cleavage.

Electrophoretic behaviour of c>llagen precursors Because o f d i s t i n c t d i f f e r e n c e s i n r e l a t i v e m o l e c u l a r mass between i n d i v i d u a l types o f c o l l a g e n p r e c u r s o r s , c o l l a g e n a - c h a i n s and t h e i r polymers, p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i s w i d e l y a p p l i e d f o r t h e s e s e p a r a t i o n s . Thus SDS s l a b g e l

181

electrophoresis i s recommended f o r e l u c i d a t i n g t h e stepwise cleavage of N- and C-terminal extensions during type I procollagen conversion t o type I collagen. As f a r a s collagen type I1 precursors a r e concerned, polyacrylamide gel e l e c trophoresis according t o King and Laemmli15 i n s l a b g e l s r e s u l t s i n a s i n g l e band of pro[a(I1)I3 chain, The mobility of t h i s zone i s not d i s t i n g u i s h i b l e from p r o [ a ( I ) I 3 which could be i s o l a t e d from embryonic tendon c e l l s . When the protein i s reduced w i t h 2-mercaptoethanol p r i o r t o a p p l i c a t i o n t o the g e l , i t migrates a t about t h e same speed a s p r o [ a ( I ) ] chains of type I collagen precurso-r. The e l e c t r o p h o r e t i c separation of component chains of type I11 procollagen and type I procollagen can be c a r r i e d out on 5% SDS-polyacrylamide gels. The method of Furthmayr and Timpl' i s used f o r t h i s purpose ( s e e a l s o r e f . 16). The method of Laemmli12 has a l s o been exploited recently in t h e separation of type IV p r o ~ o l l a g e n ' ~ ;t h e d i f f e r e n c e of collagen type IV and procollagen type IV i s small, however, i f any. Detection procedures s p e c i f i c t o collagen proteins

Differences i n s t a i n i n g the protein bands with Coomassie B r i l l i a n t B l u e allow an easy means of distinguishing collagenous and non-collagenous p r o t e i n s , a task t h a t i s frequently solved in analysing complex protein mixtures such as t i s s u e c u l t u r e media. Coomassie Blue R 250-col lagen complexes i n polyacrylamide gel s t a i n pink, whereas o t h e r protein bands s t a i n blue. The absorbance maxima a r e 550-560 nm f o r non-collagenous proteins and 520-535 nm f o r collagenous proteins. The c r i t i c a l f a c t o r in t h e development of collagen-specific s t a i n i s t h e pH o f the d e s t a i n i n c solution. The development of t h e pink colour requires t h a t t h e destaining solut i o n be maintained a t pH 2-4; on the o t h e r hand, t h e ionic composition of t h e destaining solution i s unimportant. I f t h e g e l s destained i n a c i d i c media a r e transferred t o pH 10 s o l u t i o n , t h e pink collagen bands a r e preserved f o r a t l e a s t 8 h t h e r e a f t e r whereas t h e blue bands of t h e other proteins disappear during t h e same period. However, drying t h e g e l s on a s h e e t of paper r e s u l t s i n a colour change of t h e pink collagen bands t o blue 18 The nature of t h e above e f f e c t i s not known. I t i s speculated t h a t the typical collagenous glycine t r i p e p t i d e repeating s t r u c t u r e s bind the dye in a manner t h a t r e s u l t s i n absorbance maxima a t 530 nm; procollagen extension peptides t h a t a r e devoid of t h i s sequence bind the dye i n a manner s i m i l a r t o o t h e r proteins, which r e s u l t s in a d i s t i n c t shoulder in the absorbance curve of the Coomassie Blue complex around 550-560 nm with a peak a t 520-535 nm 18 I n addition t o conventional s t a i n i n g techniques, i t i s a l s o possible t o use fluorescence label1 ing; t h i s could be e f f e c t e d by reaction of collagen a-chains and t h e i r polymers with 2-methoxy-2,4-diphenyl-3(2H)-furanone. The migration of fluorescence-labelled a-chains i s s i m i l a r to t h a t of t h e unlabelled species ex-

.

.

182 c e p t t h a t t h e l a b e l l e d al(I) and a 3 ( I ) move c l o s e r t o g e t h e r . Q u a n t i t a t i o n i s poss i b l e w i t h 10-5-10-8 g o f t h e l a b e l l e d p r o t e i n . The main advantage o f t h i s t e c h n i q u e i s a w i d e r range o f l i n e a r i t y and a v e n t i o n a l s t a i n i n g 19

.

g r e a t e r s e n s i t i v i t y compared w i t h con-

ELASTIN

I n comparison w i t h c o l l a g e n , i n e l a s t i n c h e m i s t r y e l e c t r o p h o r e t i c s e p a r a t i o n s have been v e r y 1 i t t l e e x p l o i t e d . Conventional SDS-polyacrylamide e l e c t r o p h o r e s i s o f f e r s a s i n g l e band o f t r o p o e l a s t i n ( t h e n o n - c r o s s - l i n k e d b u i l d i n g u n i t o f e l a s t i n 2 O Y 2 l ) . On t h e o t h e r hand, t h e r e i s no method a v a i l a b l e t h a t would d e p o l y m e r i z e c r o s s - l i n k e d e l a s t i n , t h u s making i t a c c e s s i b l e t o e l e c t r o p h o r e t i c separations. The p r o t e o l y t i c system n o r m a l l y a s s o c i a t e d w i t h t r o p o e l a s t i n p r e p a r a t i o n s g i v e s r i s e t o bands a d d i t i o n a l t o t h e p a r e n t 72,000 r e l a t i v e m o l e c u l a r mass t r o p o e l a s t i n band. As shown by Mecham e t a1 .22, 57,000,

45,600, 36,000, 24,700 and 13,000-14,000

d a l t o n fragments can be r e v e a l e d . Gel e l e c t r o p h o r e s i s i n 7.5% u r e a - p o l y a c r y l a m i d e g e l a l s o a l l o w s t h e s e p a r a t i o n o f t h e e l a s t i n monomer and c r o s s - l i n k e d dimer t h a t can be found i n c o p p e r - d e f i c i e n t aortae23. T h i s m a t e r i a l behaves s i m i l a r l y t o t r o p o e l a s t i n p r e c u r s o r s o f r e l a t i v e m o l e c u l a r mass 135,000 and 100,00024y25. As e l a s t i n o c c u r s i n t i s s u e s s i d e by s i d e w i t h c o l l a g e n and as, e.g.,

aortic

media c e l l s a r e capable o f s y n t h e s i z i n g b o t h c a t e g o r i e s o f p r o t e i n s , t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f c o l l a g e n a-chains and t r o p o e l a s t i n i s i m p o r t a n t . On p o l y a c r y l a m i d e g e l r o d s (7.5%) t r o p o e l a s t i n moves f a s t e r t h a n c o l l a g e n a - c h a i n s owing t o d i f f e r e n c e s i n t h e r e l a t i v e m o l e c u l a r mass and t o t h e r e t a r d e d e l e c t r o p h o r e t i c 26

mobil it y o f SDS-col 1agen complexes

.

I f u r e a - p o l y a c r y l a m i d e g e l i s used f o r ~ e p a r a t i o n ~t h~e, separated band o f e l a s t i n c o u l d be coacervated i n t h e g e l by i n c u b a t i n g t h e r o d o r s l a b i n sodium c h l o r i d e - p h o s p h a t e b u f f e r a t 37OC. The e l a s t i n band t h e n appears as a w h i t e opaque zone, which disappears when t h e temperature o f t h e g e l i s lowered i n an i c e - b a t h . The opaque band can be r e v e a l e d by d a r k f i e l d i l l u m i n a t i o n a t 37°C28. F o r s l a b g e l e l e c t r o p h o r e s i s d a n s y l - l a b e l l e d t r o p o e l a s t i n can a l s o b e used. T h i s method was a p p l i e d t o f o l l o w t h e t r o p o e l a s t i n p u r i f i c a t i o n procedures by Sandberg e t a l . 29

.

CONNECTIVE TISSUE PROTEOGLYCANS L o w - p o r o s i t y p o l y a c r y l a m i d e ( p o l y a c r y l amide-agarose about 2% i n p o l y a c r y l a m i d e ) has been w i d e l y a p p l i e d i n t h e p a s t t o d i f f e r e n t i a t e c o n n e c t i v e t i s s u e p r o t e o glycans and t h e i r s ~ b u n i t s ~ ~I n- p~ r~i n,c i p l e we have d e a l t h e r e w i t h t h e i n t a c t

183 proteoglycan, p r o t e o g l y c a n s u b u n i t and 1 i n k p r o t e i n ( f o r nomenclature and model s t r u c t u r e , see r e f . 35). Polyacrylamide gel e l e c t r o p h o r e s i s i n t h e presence o f sodium dodecyl s u l p h a t e

o f t h e proteoglycan complex a l l o w s a d i s t i n c t r e s o l u t i o n o f two d i f f e r e n t l i n k p r o t e i n s 3 6 and r e v e a l s t h e presence o f some minor p r o t e i n components i n t h e proteoglycan moiety37. Most r e s u l t s i n t h i s area, however, a r e l a c k i n g a c l e a r mol e c u l a r i n t e r p r e t a t i o n o f t h e bands seen i n t h e electropherogram. The proteoglycan subunit o b t a i n e d by papain d i g e s t i o n r e v e a l s one t o t h r e e zones on agarose-polyacrylamide gel, depending on t h e age o f t h e i n d i v i d u a l whose t i s s u e s were used f o r preparation. Zones having m o b i l i t i e s o f 0.59,

0.64 and 0.69 r e l a t i v e t o c h o n d r o i t i n

sulphate were seen i n p r e p a r a t i o n s o f aged animals38. I n c o n t r a s t , f r a g m e n t a t i o n lby pepsin y i e l d s much l a r g e r fragments. I n o l d e r specimens, bands moving a t 0.68, 0.74,

0.84 and 0.88 r e l a t i v e t o c h o n d r o i t i n s u l p h a t e can be revealed. The age

changes i n t h e p r o f i l e o f pepsin ( o r papain) l i b e r a t e d fragments a r e a s c r i b e d t o t h e arrangement o f glycosaminoglycan chains o r even t h e s t r u c t u r e o f t h e core 38 protein

.

REFERENCES

1 H. Furthmayr and R. Timpl , A m Z . Biochem. , 4 1 (1971) 510. 2 T. Hayashi and Y. Nagai, J . Biochem., 86 (1971) 453. 3 W.L. M a t t i c e and Mandelkern, Biochemistry, 10 (1971) 1934. 4 W.L. M a t t i c e , J.M. R i s e r and D.S. C l a r k , Biochemistry, 15 (1976) 4264. 5 T. Hayashi and Y. Nagai , J . Biochem. , 87 (1980) 803. 6 J.A. Reynold and C. Tanford, J . BioZ. Chem., 245 (1970) 5161. 7 B.S. Sykes, FEBS L e t t . , 61 (1976) 180. 8 C.A. S h u t t l e w o r t h , J.L. Ward and P.N. Hirschmann, Biochim. Biophys. A c t a , 535 (1978) 348. 9 L. Weber, W.N. Meigel and J . Rauterberg, Arch. Dermatol. R e s . , 258 (1977) 251. 10 W.G. Cole and D.A. Bean, Anal. Biochem., 92 (1979) 133. 11 R.W. G l a n v i l l e , A. Rauter and P.P. F i e t z e k , Eur. J . Biochem., 95 (1979) 383. 12 U.K. Laemml i, Nature (London), 227 (1970) 680. 13 H. von der Mark and K. von der Mark, FEBS L e t t . , 99 (1979) 101. Kao and C.A. Foreman, Eur. J . Biochem., 106 (1980) 41. 14 W.W.Y. 15 J. King and U.K. Laemmli, J . MoZ. B i o Z . , 62 (1971) 465. 16 A. Lenaers and C.M. Lapiere, Biochim. Biophys, A c t a , 400 (1975) 121. 17 K. A l i t a l o , Eur. J . Biochem., (1980) i n press. 18 P.J. McCormick, S. Chandrasekhar and A.J.T. M i l l i s , A m Z . Biochem., 97 (1979) 359. 19 R.L. Goldberg and G.C. F u l l e r , Anal. Biochem., 90 (1978) 69. 20 P. Christener, G. Weinbaum, B. Sloan and J . Rosenbloom, A m Z . Biochem., 88 (1978) 682. 21 L. RyhXnen, P.N. Graves, G.M. Bressan and D.J. Prockop, Arch. Biochem. Biophys., 185 (1978) 344. 22 R.P. Mecham, J.A. F o s t e r and C. Franzblau, Biochim. Biophys. A c t a , 446 (1976) 245. 23 P.A. Abraham and W.H. Carnes, J . BioZ. Chem., 253 (1978) 7993. 24 R.B. Rucker, R.P. Murray and C. Franzblau, Biochem. Biophys. Res. Cormn., 75 (1977) 358. 25 J.A. Foster, R.P. Mecham and C. Franzblau, Biochem. Biophys. Res. Comun., 72 (1976) 1399.

184 26 P.A. Abraham, D.W. Smith and W.H. Carnes, Biochem. Biophys. Res. Comun., 58 (1974) 597. 27 R.A. Reisfeld, U.J. Lewis and D.E. Williams, Nature LLondon), 195 (1962) 281. 28 P.A. Abraham and W.H. Carnes, Anal. Biochem., 9 1 (1978) 115. 29 L.B. Sandberg, E. Bruenger and E.G. Cleary, AnaZ. Biochem., 64 (1975) 249. 30 J.C. Anderson, Biochirn. Biophys. A c t a , 379 (1975) 444. 31 J . Hranisavljevic, D.L. Simpson and E.A. Davidson, Biochemistry, 11 (1972) 2983. 32 T.R. Oegema, J r . , V.C. Hascall and R. Eisenstein, J . B i o l . Chem., 254 (1979) 1312. 33 J.P. Pearson and R.M. Mason, B i o c h h . Biophys. A c t a , 498 (1977) 176. 34 M.B.E. Sweet, E.J.M.A. Thonar and A.R. Immelman, Arch. Biochem. Biophys., 189 (1978) 28. 35 H. Kaiser, Arch. Biochem. Biophys., 168 (1975) 622. 36 B.V. Treadwell, L. Shader, C.A. Towle, D.P. Mankin and H.J. Mankin, Biochem. Biophys. R e s . Cmmun., 94 (1980) 159. 37 L.L. F a l t z , A.H. Reddi, G.K. Hascall , D. Martin, J.C. Pita and V.C. Hascall , J . BioZ. Chem., 254 (1978) 1375. 38 P.J. Roughley and R.J. White, J . Biol. Chem., 255 (1980) 217.

185

Chapter 9.13 MICROTUBULAR PROTEINS

KEVIN F. SULLIVAN and LESLIE WILSON GENERAL ASPECTS Microtubul es, u b i q u i t o u s and m u l t i f u n c t i o n a l supramolecular o r g a n e l l e s found i n eukaryotes, have been t h e s u b j e c t o f i n t e n s i v e s t u d y s i n c e t h e i r d e s c r i p t i o n i n t h e e a r l y 1 9 6 0 ~ " ~ . A number o f methodological approaches have g r e a t l y f a c i l i t a t e d t h e study o f t h e molecular b i o l o g y o f these s t r u c t u r e s : e l e c t r o n microscopy, which has y i e l d e d s t r u c t u r a l and o r g a n i z a t i o n a l i n f o r m a t i o n ; t h e use o f drugs, p r o v i d i n g f u n c t i o n a l and mechanistic i n s i g h t ; i n v i t r o p o l y m e r i z a t i o n techniques, which have been i n s t r u m e n t a l i n t h e e l u c i d a t i o n o f t h e mechanism o f assembly and d i ~ a s s e m b l y ~ -and ~ ; e l e c t r o p h o r e t i c techniques, which have been used t o study t h e molecular compositions o f microtubules.

I t i s t h e l a s t area,

t h e uses and i n f o r m a t i o n gained from t h e v a r i o u s techniques o f polyacrylamide gel e l e c t r o p h o r e s i s (PAGE) and i s o e l e c t r i c f o c u s i n g ( I F ) , which i s t h e s u b j e c t o f t h i s review. The b a s i c s t r u c t u r a l s u b u n i t o f t h e m i c r o t u b u l e i s t u b u l i n , a d i m e r i c g l o b u l a r p r o t e i n o f molecular w e i g h t 110,000-120,000

d a l t o n s . T u b u l i n , as discussed below,

i s a heterogeneous p r o t e i n and t h i s h e t e r o g e n e i t y i s m a n i f e s t a t two l e v e l s . F i r s t , t h e dimer i s composed o f two s i l i l a r y e t e v o l u t i o n a r i l y d i v e r g e n t p o l y p e p t i d e chains, a and

6 ( r e f s , 6 and 7 ) . Secondly, i t appears t h a t each c h a i n may e x i s t

i n a number o f forms w i t h i n an organism, d i s t i n g u i s h e d genetically8-''

o r by post-

t r a n s l a t i o n a l m ~ d i f i c a t i o n ~ ~g -i v~i n~g, r i s e t o a number o f "isozymic" t u b u l i n s . I n a d d i t i o n t o t u b u l i n , a number o f o t h e r p r o t e i n s have been found a s s o c i a t e d w i t h m i c r o t u b u l e s from a v a r i e t y o f sources. These microtubule-assgciated

proteins

probably serve t o c o n t r o l o r mediate m i c r o t u b u l e assembly and f u n c t i o n 2 . F o r example, i n c i l i a and f l a g e l l a , m i c r o t u b u l e s and approximately 130 a s s o c i a t e d p r o t e i n s co-assemble t o form a h i g h l y ordered dynamic molecular engine16. F u r t h e r , several p r o t e i n s c o p u r i f y w i t h m i c r o t u b u l e s from b r a i n and o t h e r t i s s u e sources, 2 These p r o t e i n s a r e thought t o through many c y c l e s of assembly and disassembly

.

be i n s t r u m e n t a l i n t h e c o n t r o l and f u n c t i o n o f cytoplasmic m i c r o t u b u l e systems. E l e c t r o p h o r e t i c techniques have been profoundly important i n e l u c i d a t i n g t h e chemical c h a r a c t e r i s t i c s o f m i c r o t u b u l e systems.

186 TUBULIN, THE HETERODIMERIC STRUCTURAL SUBUNIT OF THE MICROTUBULE Tubul in17 was f i r s t i s o l a t e d from s t a b l e f l a g e l l a r o u t e r doublet microtubules (see ref, 18-20) o r as the colchine-binding p r o t e i n from v e r t e b r a t e brainz1. I n many o f these studies, t h e r e c e n t l y developed technique o f discontinuous PAGE upon the a l k a l i n e system o f Davisz2 was used t o analyse the composition o f the p u r i f i e d p r o t e i n preparations. I n 8 mol/l urea, reduced and carboxymethylated microtubule p r o t e i n migrated as a s i n g l e major p r o t e i n band. I n conjunction w i t h hydrodynamic data obtained i n t h e presence and absence o f denaturing agents, i t was generally concluded t h a t t u b u l i n consisted o f two s i m i l a r o r i d e n t i c a l 55,000dalton polypeptide chains j o i n e d non-covalently as a dimer. The e l e c t r o p h o r e t i c band ascribed t o tubul i n was occasionally seen t o s p l i t i n t o two c l o s e l y spaced bands upon PAGE i n 8 mol/l urea, suggesting t h e p o s s i b i l i t y t h a t the p r o t e i n was heterogeneous18y21. However, owing t o t h e capricious nature o f the separation, a number o f e a r l y i n v e s t i g a t o r s were l e d t o suggest t h a t such heterogeneity may have been a r t i f a c t u a l . Bryan and Wilson23, using a modified Davis system, demonstrated t h a t reduced and carboxymethylated c h i c k b r a i n t u b u l i n c o n s i s t e n t l y resolved i n t o two d i s t i n c t bands t h a t were present i n a 1:l r a t i o based on dye binding. The two proteins, termed a and B i n order o f increasing m o b i l i t y , were shown t o d i f f e r i n amino a c i d composition and various attempts t o a l t e r t h e 1:l d i s t r i b u t i o n were unsuccessful. Analysis by sodium dodecyl sulphate i n which a and B are unresolved,

(SDS)-PAGE on t h e system o f Shapiro e t al.24,

gave an estimated molecular weight o f 55,000

?

2000 dalton, and a n a l y s i s on g e l s

o f increasing acrylamide concentrationz5 confirmed t h a t t h e p r o t e i n s were separating on the basis o f charge and n o t s i g n i f i c a n t l y on t h e basis o f molecular weight. The molecular sizes o f the two polypeptide chains a r e now known t o be 54,000

?

1000

daltonZ6. Bryan and Wilson proposed t h a t the a c t i v e colchicine-binding p r o t e i n and MT subunit was the aB heterodimer o f tubulinZ3. These r e s u l t s were r a p i d l y and independently confirmed i n several l a b o r a t ~ r i e s ~ ~F e- ~ i t ~e .t a1 .27, using an SDSurea-PAGE system based upon t h e method o f Davis, demonstrated t h a t t u b u l i n s derived

f r o m A- and 8-subfibres o f o u t e r doublet microtubules and from a v a r i e t y o f verteb r a t e neural t i s s u e s resolved i n t o two e q u a l l y intense bands w i t h apparent molecu l a r weights o f 53,000 and 56,000 dalton. Olmsted e t a1.28,

comparing microtubule

proteins o f ChlamydoPnonasflagellar outer doublets and neuroblastoma c e l l s by SDS-

PAGE, a l s o demonstrated t h a t both polypeptide chains were present i n equal amounts and f u r t h e r showed e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e d i f f e r e n c e s i n the tubul i n s

f r o m the two species, While several p l a u s i b l e models f o r t h e composition o f t h e dimer and f o r i t s organization i n microtubules were suggested, t h e heterodimer model o f Bryan and Wilson was favoured27y29,30.

Luduena e t a1 ,7 u t i l i z e d a chemical c r o s s - l i n k i n g

187 s t r a t e g y t o probe t h e s t r u c t u r e o f t h e dimer i n s o l u t i o n . The SDS-PAGE method o f Yang and C r i d d l e 3 1 , which a f f o r d e d a v e r y w i d e s e p a r a t i o n o f reduced and carboxym e t h y l a t e d a- and B - t u b u l i n s , was found t o be capable o f r e s o l v i n g c o v a l e n t l y l i n k e d a-a,

a-P and 13-t? dimers on 5% g e l s . Under c o n d i t i o n s which m i n i m i z e d t h e

s e l f - a s s o c i a t i o n o f dimers, t h e a-B s p e c i e s was t h e predominant p r o d u c t o f c r o s s 1 i n k i n g . O p t i c a l d i f f r a c t i o n s t u d i e s on i n t a c t m i c r o t u b u l e s and z i n c - t u b u l i n sheets 3 2 y 3 3 a l s o s u p p o r t a h e t e r o d i m e r i c s u b u n i t s t r u c t u r e . The s e p a r a t i o n o f a- and 6 - t u b u l i n s

on SDS-PAGE i s an anomalous phenomenon,

t h a t i s , t h e y a r e n o t s e p a r a t e d e n t i r e l y on t h e b a s i s o f m o l e c u l a r w e i g h t , Bryan

34

analysed t h i s s e p a r a t i o n u s i n g t h e t e c h n i q u e o f F e r g ~ s o nand ~ ~found t h a t separat i o n was based on charge and an anomalous i n t e r a c t i o n o f a t u b u l i n w i t h SDS. Furthermore, u s i n g SDS-PAGE systems based on t h o s e o f Weber and O ~ b o r nand ~ ~ Everhart3O, Bryan determined t h a t w h i l e t h e a p p a r e n t m o l e c u l a r w e i g h t o f 8 , e s t i mated a t 52,000,

was c o n s t a n t o v e r a wide r a n g e o f c o n d i t i o n s , t h e a p p a r e n t molec-

u l a r w e i g h t o f a was a f u n c t i o n o f b o t h pH and i o n i c s t r e n g t h . S e p a r a t i o n was b e s t a t low i o n i c s t r e n g t h and a t h i g h pH, whereas m o l e c u l a r w e i g h t d e t e r m i n a t i o n was most a c c u r a t e a t h i g h i o n i c s t r e n g t h and n e u t r a l pH. Several i n v e s t i g a t o r s have d i s c u s s e d t h e i m p o r t a n c e o f t h e s u l p h y d r y l s t a t u s o f tubulin i n electrophoretic analysis

y7

l8 y1gy34.

Ei

demonstrated t h a t

t h e s e p a r a t i o n o f a- and B - t u b u l i n o c c u r r e d i n t h e absence o f SDS, and was depend e n t upon t h e charge s t a t u s o f s u l p h y d r y l o r m o d i f i e d s u l p h y d r y l groups. Unmodified o r S-carboxymethylated t u b u l i n d i d n o t r e s o l v e , b u t S-carboxyamidomethylation d i d r e s o l v e t h e a- and @-chains, t h e q u a l i t y o f r e s o l u t i o n i n c r e a s i n g w i t h i n c r e a s i n g pH. I n t h e presence o f SDS s e p a r a t i o n may be a c h i e v e d w i t h o r w i t h o u t p r i o r s u l p h y d r y l m o d i f i c a t i o n6 y 3 6 y 3 7 ,

b u t r e s o l u t i o n i s h i g h l y dependent on t h e s p e c i f i c

c o n d i t i o n s o f e l e c t r o p h o r e s i s . We have found t h e system o f Sheir-Ness e t a l . 3 8 t o y i e l d o p t i m a l s e p a r a t i o n a f t e r r e d u c t i o n o n l y , and t h e system o f Yang and C r i d d l e

31

t o be e x c e l l e n t f o r t h e s e p a r a t i o n o f reduced and c a r b o x y m e t h y l a t e d s u b u n i t s . The anomalous b e h a v i o u r o f t u b u l i n on SDS-PAGE n e c e s s i t a t e s c a u t i o n i n t h e i n t e r p r e t a t i o n o f electropherograms. F o r example, w i t h t h e w i d e l y used system o f Laemml i some l a b o r a t o r i e s have achieved t h e s e p a r a t i o n o f a- and B - t u b u l i n s whereas o t h e r s have not3'y4'.

Such d i s p a r i t i e s a r e p r o b a b l y due t o s u b t l e v a r i a t i o n s i n t e c h n i q u e

and r e a g e n t q u a l i t y . The importance o f r e a g e n t s o r r e a g e n t q u a l i t y i s s t r i k i n g l y i l l u s t r a t e d by a r e p o r t 4 ' d e m o n s t r a t i n g t h a t two d i f f e r e n t batches o f SDS o b t a i n e d f r o m t h e same s u p p l i e r r e s u l t e d i n t h e s e p a r a t i o n o f s u b u n i t s , b u t w i t h one b a t c h a was t r a i l i n g whereas w i t h t h e second b a t c h t h e m o b i l i t i e s were r e v e r s e d , w i t h

trailing.

B

188 MULTIPLE TUBUL I N S : MOLECULAR SPECIALIZATION FOR DIVERSE FUNCTIONS? I n a d d i t i o n t o t h e fundamental h e t e r o d i m e r i c n a t u r e o f t u b u l i n , a n i n c r e a s i n g number o f i n v e s t i g a t o r s , o v e r t h e p a s t decade, have d e s c r i b e d t h e o c c u r r e n c e o f m u l t i p l e forms o f t h i s p r o t e i n . The use o f I F , SDS-PAGE and two-dimensional

elec-

t r o p h o r e t i c techniques has c o n t r i b u t e d s u b s t a n t i a l l y t o t h e s e d i s c o v e r i e s . R e s u l t s o b t a i n e d w i t h a v a r i e t y o f e x p e r i m e n t a l approaches aimed a t c o r r e l a t i n g b i o l o g i c a l phenomena w i t h t u b u l i n c h e m i s t r y have g i v e n r i s e t o t h e emerging view t h a t t h e t u b u l i n s r e p r e s e n t a c l a s s o f v e r y c l o s e l y r e l a t e d p r o t e i n s . F u r t h e r , t h e chemical d i f f e r e n c e s among t h e t u b u l i n s may be t h e b a s i s f o r t h e a b i l i t y o f m i c r o t u b u l e s t o 42,43 engage i n a v a r i e t y o f c e l l u l a r r o l e s , Stephens44 f i r s t demonstrated e l e c t r o p h o r e t i c and chemical d i f f e r e n c e s i n t h e t u b u l i n s s e l e c t i v e l y s o l u b i l i z e d f r o m A- and B-Eubfibres o f StronggZocentrotus

drobachiensus sperm t a i l o u t e r d o u b l e t m i c r o t u b u l e s . Analogous r e s u l t s were r e p o r t e d f o r Chlamydornonas o u t e r d o u b l e t t u b u l i n ~ However, ~ ~ . w i t h the discoveries t h a t t u b u l i n s f r o m a l l sources c o n s i s t o f two m o l e c u l a r species, i t was i n i t i a l l y assumed t h a t t h e s e i n v e s t i g a t o r s had been d e s c r i b i n g t h e e l e c t r o p h o r e t i c a l l y separable a- and 8-tubul i n s . Stephens has s i n c e demonstrated by p e p t i d e mapping and amino a c i d a n a l y s i s t h a t t u b u l i n s from A- and B - s u b f i b r e s ,

as w e l l as f r o m

o t h e r s t r u c t u r a l l y and f u n c t i o n a l l y d i f f e r e n t sources w i t h i n t h e same organism, a r e indeed c h e m i c a l l y d i s t i n c t

43

.

The a p p l i c a t i o n o f I F and two-dimensional techniques, which a f f o r d v e r y h i g h r e s o l u t i o n s e p a r a t i o n o f p r o t e i n s , has r e v e a l e d t h a t t u b u l i n s i s o l a t e d f r o m a number of sources e x h i b i t some degree o f h e t e r o g e n e i t y . F e i t e t a1.27 r e p o r t e d t h a t c h i c k b r a i n t u b u l i n r e s o l v e d i n t o f o u r bands on I F , and s i m i l a r l y Witman e t a1 .46 d e s c r i b e d f i v e bands upon I F o f t u b u l i n i s o l a t e d f r o m ChZamydomonas f l a g e l l a . 13,47-51 These r e s u l t s have been r e f i n e d and expanded by a number o f i n v e s t i g a t o r s The e x t e n t o f h e t e r o g e n e i t y observed u s i n g I F i s dependent on t h e t i s s u e source 52

.

Thus, as few as t h r e e bands, as f o u n d i n t h e sea u r c h i n egg v i n b l a s t i n e t u b u l i n c r y s t a l s 5 3 , o r as many as f o u r t e e n , as i n r a t b r a i n t ~ b u l i n ~have ~ , been described. By two-dimensional

e l e c t r o p h o r e s i s ( I F i n t h e f i r s t and SDS-PAGE i n t h e

second dimension) t h e a - c h a i n s have an average p I o f 5.3-5.5, 8-chains t h e average i s 5.1-5.2.

whereas f o r t h e

Although I F i s an e x t r e m e l y s e n s i t i v e t e c h n i q u e ,

t h e r e s o l u t i o n o f such c l o s e l y spaced bands i s v e r y dependent on t h e r u n n i n g cond i t i o n s and t h e s l o p e of t h e pH g r a d i e n t i n t h e pH range 5.0-5.5.

I n view o f t h e

apparent c o m p l e x i t y o f many t u b u l i n pools, much c a r e f u l work w i l l be r e q u i r e d i n o r d e r t o r e s o l v e t h e t r u e e x t e n t arid chemical s i g n i f i c a n c e o f t h e h e t e r o g e n e i t y o f tubulin. I n a d d i t i o n t o I F , SDS-PAGE t e c n n i q u e s have been u t i l i z e d t o probe t h e i n f r a s t r u c t u r e of t u b u l i n p o o l s . B i b r i n g e t a1 .54, u s i n g SDS-urea-PAGE,

demonstrated

189 t h e e x i s t e n c e o f two a-chains i n c i l i a r y and m i t o t i c , b u t n o t f l a g e l l a r , t u b u l i n s f r o m S. pupmatus. S t u d y i n g t h e p h o s p h o r y l a t i o n o f t u b u l i n s i n ChZqdomonas flagella,

P i p e r n o and Luck''

developed a c o n t i n u o u s SDS-PAGE system which a l l o w e d

t h e r e s o l u t i o n o f a t l e a s t f o u r a - t u b u l i n s , two o f which a r e phosphorylated, and two B - t u b u l i n s . S h e i r - N e i s s e t a1 .38, u t i l i z i n g t h e p e r p e n d i c u l a r g r a d i e n t s t r a t e g y o f Gross e t a1.55,

optimized t h e conditions f o r the separation o f t u b u l i n s from

AspergiZZus niduZans. T h e i r system, e s s e n t i a l l y a m o d i f i c a t i o n o f t h e Laemml i formulation b u t d i f f e r i n g i n t h e concentration o f bisarylamide, i s capable o f r e s o l v i n g m u l t i p l e t u b u l i n s . StephensS6 has r e c e n t l y d e s c r i b e d t h e use of two PAGE systems 5 7 y 5 8 w h i c h r e s o l v e m u l t i p l e a- and 8 - t u b u l i n s f r o m 5'. drobachiensus, and he has d i s c u s s e d t h e u t i l i t y o f a two-dimensional t e c h n i q u e f o r t h e h i g h - r e s o l u t i o n s e p a r a t i o n o f t u b u l i n components, i n which these two PAGE systems a r e used sequentially. SIGNIFICANCE OF MULTIPLE TUBULINS The s i g n i f i c a n c e o f m u l t i p l e t u b u l i n s i s s t i l l n o t w e l l understood, b u t e v i d e n c e o b t a i n e d t h r o u g h a v a r i e t y o f e x p e r i m e n t a l approaches aimed a t c o r r e l a t i n g t h e presence o f s p e c i f i c t u b u l i n subspecies w i t h v a r i o u s b i o l o g i c a l s t r u c t u r e s o r processes s u p p o r t s t h e i d e a t h a t m u l t i p l e t u b u l i n s a r e i m p o r t a n t i n e s t a b l i s h i n g t h e v a r i e t y o f s t r u c t u r a l l y , f u n c t i o n a l l y and b e h a v i o u r a l l y d i s t i n c t m i c r o t u b u l e s which c o e x i s t w i t h i n a s i n g l e organism. F o r example, w i t h i n f l a g e l l a r o u t e r d o u b l e t s , s e v e r a l r e p o r t s i n d i c a t e d t h a t

A- and B - s u b f i b r e s 43-46 and p o s s i b l y t h r e e p a r t i t i o n p r ~ t o - f i l a m e n t sa~r ~e composed o f c h e m i c a l l y d i s t i n c t t u b u l i n components. The m i n o r t u b u l i n s p e c i e s d e s c r i b e d by P i p e r n o and Luck''

were p r e s e n t i n amounts comparable t o t h o s e o f n o n - t u b u l i n

axonemal p r o t e i n s and, i n view o f t h e p r e c i s e g e o m e t r i c arrangement o f f l a g e l l a r p r o t e i n s , these a u t h o r s suggested t h a t s p e c i f i c t u b u l i n s may be s p a t i a l l y i n t e g r a t e d i n t o m i c r o t u b u l e s i n a comparably p r e c i s e manner. I t s h o u l d b e n o t e d t h a t F u l t o n and Simpson have proposed a m u l t i t u b u l i n h y p o t h e s i s based on t h e a n t i g e n i c uniqueness 42 o f f l a g e l l a r t u b u l i n i n Naegleria

.

I n v e r t e b r a t e systems, t u b u l i n subspecies have been e l e c t r o p h o r e t i c a l l y a n a l y s e d i n d e v e l o p i n g r a t 52y59 and chick6'

b r a i n . I n each o f t h e s e s t u d i e s , t h e t u b u l i n

pool was found t o v a r y s y s t e m a t i c a l l y d u r i n g development. Changes i n t h e c o l c h i c i n e b i n d i n g c h a r a c t e r i s t i c s 6 1 and assembly p r o p e r t i e s 6 2 o f t u b u l i n d u r i n g development have a l s o been d e s c r i b e d though t h e r e l a t i o n s h i p s between t h e s e p r o p e r t i e s and t u b u l i n subspecies c o m p o s i t i o n have y e t t o be e s t a b l i s h e d . G e n e t i c approaches, coupled w i t h e l e c t r o p h o r e t i c a n a l y s i s , may be of g r e a t u t i l i t y i n i n v e s t i g a t i n g t h i s problem. Thus Kemphues e t a1.8 demonstrated t h a t a mutat i o n i n D r o s o p h i l a c a u s i n g male s t e r i l i t y i s due t o a l t e r a t i o n of a t e s t e s - s p e c i f i c

190 8 - t u b u l i n . S h e i r - N e i s s e t a1 .35 used two-dimensional

e l e c t r o p h o r e s i s (IF f o l l o w e d

by PAGE) t o i d e n t i f y 8 - t u b u l i n as t h e s i t e o f a m u t a t i o n c o n f e r r i n g r e s i s t a n c e t o benzimidazole, an i n h i b i t o r o f m i c r o t u b u l e assembly, i n A . nidutans. The o b s e r v a t i o n t h a t o t h e r organisms, such as c h i c k e n s and sea u r c h i n s , c o n t a i n m u l t i p l e genes f o r tubul

suggests t h a t t h e combined g e n e t i c / e l e c t r o p h o r e t i c approach may be o f

fundamental importance t o f u t u r e i n v e s t i g a t i o n s o f t u b u l i n f u n c t i o n . M I CROTU BULE ASSOCIATED PROTEINS

I n s e v e r a l systems m i c r o t u b u l e s have been found i n a s s o c i a t i o n w i t h a v a r i e t y o f proteins. Operationally i t i s convenient t o d i s t i n g u i s h associated proteins which c o p u r i f y w i t h m i c r o t u b u l e s t h r o u g h c y c l e s o f i n v i t r o assembly and disassemb l y f r o m t h o s e which a r e observed u l t r a s t r u c t u r a l l y i n a s s o c i a t i o n w i t h w e l l d e f i n e d s t r u c t u r e s such as c i l i a and f l a g e l l a . However, w i t h t h e e x c e p t i o n o f t h e flagellar

dyne in^^^,

t h e r e a r e few d a t a a v a i l a b l e r e g a r d i n g t h e t r u e c e l l u l a r

f u n c t i o n s o f t h e a s s o c i a t e d p r o t e i n components, a1 though i t i s t h o u g h t t h a t t h e y may be i n v o l v e d i n t h e r e g u l a t i o n o f m i c r o t u b u l e assembly and disassembly, i n t h e g e n e r a t i o n o f f o r c e , i n t h e maintenance o f c e l l shape and i n t h e a s s o c i a t i o n s between m i c r o t u b u l es and o t h e r c e l l u l a r components. The e l e c t r o p h o r e t i c a n a l y s i s o f b r a i n m i c r o t u b u l e p r e p a r a t i o n s p u r i f i e d by c y c l e s o f assembly and disassembly has r e v e a l e d s e v e r a l m i c r o t u b u l e a s s o c i a t e d p r o t e i n s (MAPS) which c o p u r i f y w i t h t u b u l i n i n c o n s t a n t s t o i c h i o m e t r i c r a t i o s , s u g g e s t i n g some s p e c i f i c i t y o f i r ~ t e r a c t i o n ~ ’ ~Two ~ - ~high-molecul ~. ar-weight (HMWj MAPs, r e f e r r e d t o v a r i o u s l y as HMW 1 and 2, MAP 1 and 2 o r HMW MAPs, o f MW 325,000 d a l t o n s and 260,000-280,000*, 65,000 d a l t o n s termed

T

and a group o f p o l y p e p t i d e s o f MW 55,000 t o

( r e f s . 67 and 68), comprise t h e b u l k o f t h e s o - c a l l e d MAP

f r a c t i o n . Both HMW MAPs and

T

have been shown t o s t i m u l a t e m i c r o t u b u l e assembly

i n v i t r o and t h e s e r e s u l t s have generated broad d i s c u s s i o n as t o t h e i r p o s s i b l e s i g n i f i c a n c e i n t h e c o n t r o l o f m i c r o t u b u l e assembly i n vivo67-77.

I t must be

stressed, however, t h a t a l t h o u g h MAPs have been demonstrated t o be a s s o c i a t e d w i t h m i c r o t u b u l e s i n v ~ v o ~ ~ -t h~ e’ r ,e i s no d i r e c t evidence b e a r i n g on t h e i r f u n c t i o n a l s i g n i f i c a n c e . Several e l e c t r o p h o r e t i c s t u d i e s o f b o t h n e u r a l and nonn e u r a l t i s s u e s i n d i c a t e t h a t t h e p r e c i s e c o m p o s i t i o n and q u a n t i t a t i v e c h a r a c t e r i s t i c s o f MAPs a r e dependent on s o u r c e and p u r i f i c a t i o n procedures82-86.

I n sum-

mary, a l t h o u g h i t seems c l e a r t h a t t h e backbone o f t h e m i c r o t u b u l e must i n t e r a c t w i t h o t h e r c e l l u l a r components i n o r d e r t o f u n c t i o n , t h e b i o l o g i c a l s i g n i f i c a n c e o f t h e e l e c t r o p h o r e t i c a l l y d e f i n e d MAPs has y e t t o be e s t a b l i s h e d .

“HMW-1 and HMW-2, t h e nomenclature o f B o r i s y e t a ~ r e~f e r ,t o he 271,000 and 286,000 d a l t o n species, r e s p e c t i v e l y , whereas Sloboda e t a l . 6k r e f e r t o these two p r o t e i n s as MAP2 and t o a t h i r d species o f 345,000 d a l t o n s as MAP1.

191 The s t u d y o f p r o t e i n s a s s o c i a t e d w i t h w e l l d e f i n e d s t r u c t u r e s such as c i l i a and f l a g e l l a o f f e r s t h e advantage o f c o r r e l a t i n g e l e c t r o p h o r e t i c a n a l y s i s w i t h u l t r a s t r u c t u r a l i n f o r m a t i o n g a i n e d by e l e c t r o n microscopy. However, a1 though some p r o t e i n s , most n o t a b l y

dyne in^^^,

have been we1 1 c h a r a c t e r i z e d b i o c h e m i c a l l y , t h e

s t u d y o f axonemal p r o t e i n s has been h i n d e r e d by t h e i r g r e a t c o m p l e x i t y . A t l e a s t 30 m a j o r components have been r e v e a l e d by t h e one-dimensional SDS-PAGE o f whole axonemal

preparation^^^.

Indeed, L i n c k 8 8 has shown t h a t one-dimensional PAGE

r e s o l v e s 30-35 p r o t e i n s a s s o c i a t e d w i t h h i g h l y p u r i f i e d o u t e r d o u b l e t s , once t h o u g h t t o c o n t a i n a l m o s t p u r e t u b u l i n . T h i s c o m p o s i t i o n a l c o m p l e x i t y has been r e v e a l e d a t h i g h e r r e s o l u t i o n by two-dimensional e l e c t r o p h o r e s i s , w h i c h s e p a r a t e s a t l e a s t 130 p o l y p e p t i d e s 1 6 . Piperno, Luck and o t h e r s have used two-dimensional IF-PAGE t o a n a l y s e f l a g e l l a r mutants i n ~hlamydomonas. The e l e g a n t c o m b i n a t i o n o f b i o c h e m i c a l , g e n e t i c and u l t r a s t r u c t u r a l approaches promises t o be p o w e r f u l i n t h e a n a l y s i s o f t h e f u n c t i o n a l p r o p e r t i e s o f axonemal

p r o t e i n s . F o r example,

Luck e t a1.89 have i d e n t i f i e d t w e l v e p o l y p e p t i d e s a s s o c i a t e d w i t h t h e r a d i a l spoke s t r u c t u r e o f t h e axonemal, s i x o f w h i c h a r e a p p a r e n t l y a s s o c i a t e d w i t h t h e spokeheads. I n summary, PAGE t e c h n i q u e s have p l a y e d a m a j o r r o l e i n t h e i d e n t i f i c a t i o n and biochemical c h a r a c t e r i z a t i o n o f t h e t u b u l i n s and a s s o c i a t e d p r o t e i n s . As t h e d i s c o v e r y of m i c r o t u b u l e s and t h e i n t r o d u c t i o n o f PAGE as a valua.ble a n a l y t i c a l method o c c u r r e d a t about t h e same t i m e , t h e h i s t o r i c a l development o f o u r u n d e r standing of t u b u l i n offers

an i n t e r e s t i n g p e r s p e c t i v e on t h e development o f e l e c -

t r o p h o r e s i s i t s e l f . Many i m p o r t a n t chemical and f u n c t i o n a l q u e s t i o n s r e m a i n open, such as t h e s i g n i f i c a n c e o f m u l t i p l e t u b u l i n s and t h e mechanisms o f f l a g e l l a r m o t i l i t y . I t i s e v i d e n t t h a t PAGE t e c h n i q u e s w i l l remain as p o t e n t t o o l s i n t h e e l u c i d a t i o n o f these questions. ACKNOWLEDGEMENTS Supported by USPHS G r a n t NSD9335 and American Cancer S o c i e t y G r a n t CD-3E. K.F.S.

i s s u p p o r t e d by an NSF P r e d o c t o r a l F e l l o w s h i p . The a u t h o r s thank H.W.

D e t r i c h , 111, f o r many h e l p f u l d i s c u s s i o n s d u r i n g t h e p r e p a r a t i o n o f t h i s r e v i e w . REFERENCES

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R.E. Stephens, J . CeZZ BioZ., 83 (1979) 351a. D.M. N e v i l l e , J . BioZ. Chem., 246 (1971) 6328. A. Zweidler, Methods CeZZ BioZ., 17 (1978) 223. J.L. Dahl and V.J. Weibel, Biochem. Biophys. Res. Comun., 86 (1979) 822. K.F. S u l l i v a n , K.W. F a r r e l l and L. Wilson, J . CeZZ BioZ., 83 (1979) 351a. J.R. Bamburg, E.M. Shooter and L. Wilson, Biochemistry, 12 (1973) 1476. J. Francon, A. F e l l o u s , A.M. Lennon and J. Nunez, Nature (London), 266 (1977) 188. 63 I.R. Gibbons, E. Fraonk, B.H. Gibbons and K. Ogawa, i n R. Goldman, T. P o l l a r d and J.L. Rosenbaum ( E d i t o r s ) , CeZZ Motility, C, Cold S p r i n g Harbor Laboratory, Cold Spring Harbor, 1976, pp. 915-932. 64 R.G. Burns and T.D. P o l l a r d , FEBS L e t t . , 40 (1974) 274. 65 W.L. D e n t l e r , S. G r a n e t t and J.L. Rosenbaum, J . CeZZ BioZ., 65 (1975) 237. 66 R.D. Sloboda, W.L. Dentler, R.A. Bloodfood, B.R. Telzer, S. G r a n e t t and J.L. Rosenbaum, i n R. Goldman, T. P o l l a r d and J.L. Rosenbaum ( E d i t o r s ) , CeZZ MotiZity, C, Cold S p r i n g Harbor Laboratory, Cold Spring Harbor, 1976, pp. 1171-1212. 67 M.D. Weingarten, A.H. Lockwood, S.Y. Hwo and M.W. Kirschner, h o e . Nut. Acud. S c i . U.S. , 72 (1975) 1858. 68 S.M. Penningroth, D.W. Cleveland and M.W. Kirschner, i n R. Goldman, T. P o l l a r d and J.L. Rosenbaum ( E d i t o r s ) , CeZZ MotiZity, C, Cold S p r i n g Harbor Laboratory, Cold S p r i n g Harbor, 1976, pp. 1233-1257. 69 G.G. Borisy, J.B. Olmsted, J.M. Marcum and C. A l l e n , Ann. N.Y. Acad. S c i . , 253 (1975) 107. 70 R.D. Sloboda, W.L. D e n t l e r and J.L. Rosenbaum, Biochemistry, 15 (1976) 4496. 7 1 K.A. Johnson and G.G. Borisy, J . MoZ. BioZ., 117 (1977) 1. 72 D.B. Murphy, R.B. V a l l e e and G.G. Borisy, Biochemistry, 16 (1977) 2598. 73 D.B. Murphy, K.A. Johnson and G.G. Borisy, J . MoZ. BioZ., 117 (1977) 33. 74 D.W. Cleveland, S.Y. Hwo and M.W. Kirschner, J . MoZ. BioZ., 116 (1977) 207. 75 D.W. Cleveland, S.Y. Hwo and M.W. Kirschner, J . MoZ. BioZ., 116 (1977) 227. 76 W. Herzof and K. Weber, E u r . J . Biochem., 92 (1978) 1. 77 J.C. Lee, N. Tweedy and S.N. Timasheff, Biochemistry, 17 (1978) 2783. 78 J.B. K i r k p a t r i c k , L. Hyams, V. Thomas and P.N. Howley, J . CeZZ Biol., 47 (1970) 384. 79 P. S h e r l i n e and K. Shiavone, Science, 198 (1977) 1038. 80 J.A. Connoly, V.I. Kalnins, D.W. Cleveland and M.W. Kirschner, J . CeZZ BioZ., 76 (1978) 781. 8 1 A.H. Lockwood, CeZZ, 13 (1978) 613. 82 B.W. Nagle, K.H. Doenges and J. Bryan, CeZZ, 12 (1977) 573. 83 J.A. Weatherbee, R.B. L u f t i g and R.R. Weighing, J . CeZZ BioZ., 78 (1978) 47. 84 B.C. Berk and P.M. H i n k l e , J . BioZ. Chem., 255 (1980) 3186. 85 J.C. B u l i n s k i and G.G. Borisy, J . CeZZ BioZ., 83 (1979) 377a. 86 H. F e i t , J.W. F u s s l e r and J.W. Shay, J . CeZZ BioZ., 83 (1979) 346a. 87 G.B. Witman, R. Fay and J. Plummer, i n R. Goldman, T. P o l l a r d and J.L. Rosenbaum ( E d i t o r s ) , CeZZ MotiZity, C, Cold S p r i n g Harbor Laboratory, Col d Spring Harbor, 1976, pp. 969-986. 88 R.W. Linck, J . CeZZ S c i . , 20 (1976) 405. 89 D. Luck, G. Piperno, Z. Ramanis and B. Huang, Proc. Nut. Acud. S c i . U.S., 74 (1977) 3456.

194

Chapter 9.14 PROTEIN HORMONES ALAN D. ROGOL GENERAL ASPECTS P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) and i s o e l e c t r i c f o c u s i n g i n p o l y a c r y l amide g e l s ( I E F ) a r e p o w e r f u l t o o l s i n d e t e r m i n i n g t h e homogeneity ( p u r i t y ) o f pept i d e s , p r o t e i n s and g l y c o p r o t e i n s .

It ,should be n o t e d t h a t t h i s c o n c e p t o f p u r i t y

i s an o p e r a t i o n a l d e f i n i t i o n because, as we s h a l l see below, i t i s t h e power o f t h e s e t e c h n i q u e s t h a t makes t h e d e f i n i t i o n o f a s i n g l e p r o t e i n s p e c i e s a r e l a t i v e one. Employing PAGE and IEF, many i n v e s t i g a t o r s have demonstrated a number o f p e p t i d e components, some o r a l l o f which may be synthesized, s e c r e t e d o r processed p r o d u c t s o f s p e c i f i c t i s s u e s , The s e p a r a t i o n procedures a r e b u t a f i r s t , a l b e i t e s s e n t i a l , s t e p f o r t h e u l t i m a t e goal o f t h e d e t e r m i n a t i o n o f s p e c i f i c b i o l o g i c a l a c t i v i t y o f each component and may shed some l i g h t on t h e b i l o l o g i c a l r e l e v a n c e o f t h e i n d i v i d u a l components. A l t h o u g h t h e m a j o r t h r u s t i s t o s t u d y t h o s e b i o l o g i c a l l y a c t i v e components, one can d e t e r m i n e many p h y s i c a l p r o p e r t i e s o f each p e p t i d e species; f o r example, t h e s t a t e o f a g g r e g a t i o n (Ferguson p l o t on PAGE) and charge isomerism (Ferguson p l o t as above, o r IEF employing l o n g e r g e l s and more narrow pH g r a d i e n t s ) , These i n d i v i d u a l procedures a r e c r u c i a l f o r t h e d e t e r m i n a t i o n o f t h e r e l a t i o n s h i p o f s t r u c t u r e t o f u n c t i o n , f o r t h e d e t e r m i n a t i o n o f t h e s p e c i f i c b i o l o g i c a l act i v i t y and f o r t h e comparison o f a l t e r a t i o n s induced i n v i t r o t o t h e n a t u r a l l y occ u r i n g products. I n t h e f i e l d o f s e c r e t e d p r o t e i n s t h i s l a s t a s p e c t i s o f g r e a t c u r r e n t i n t e r e s t , as many o f t h e s e molecules undergo s p e c i f i c p r o t e o l y t i c c l e a v a g e f o r a c t i v a t i o n and f o r d i s p o s a l (metabolism).

F o r t h e purposes o f t h i s d i s c u s s i o n ,

t h i s s e c t i o n w i l l be l i m i t e d t o t h r e e human hormones hormone (GH) and c h o r i o n i c gonadotropin (CG)

-

- prolactin

( P r l ) , growth

as t h e r e have been a number o f r e l -

e v a n t s t u d i e s f o r each, t h e r e is r e l a t i v e agreement among s e v e r a l l a b o r a t o r i e s and t h e r e a r e i n v i v o b i o a s s a y d a t a f o r each.

195 PROLACTIN Human p r o l a c t i n has been u n e q u i v o c a l l y s e p a r a t e d f r o m g r o w t h hormone w i t h i n t h e l a s t decade. PAGE has been p i v o t a l i n t h e s e p a r a t i o n o f t h e s m a l l amount o f t h i s p e p t i d e hormone f r o m t h e much more abundant growth hormone. Lewis e t a l . ’ vantage o f t h e r e l a t i v e l y n e u t r a l i s o e l e c t r i c p o i n t o f a p p r o x i m a t e l y 6.5

t o o k ad(refs. 2

and 3 ) t o remove t h e more abundant s o l u b l e p r o t e i n s f r o m p i t u i t a r y e x t r a c t s and t o c o n c e n t r a t e a f r a c t i o n r i c h i n P r l . A f t e r s e p a r a t i o n by DEAE-cellulose chromato4 graphy , t h e y i d e n t i f i e d a component o f l o w e r m o b i l i t y t h a n GH on PAGE a t pH 9.5. I t showed minimal growth-promoting a c t i v i t y i n t h e t i b i a 1 l i n e assay, b u t p o t e n t 4 p r o l a c t i n a c t i v i t y ( 2 2 IU/mg) i n t h e pigeon c r o p sac assay I t s molecular weight

.

as determined by t h e method o f Ferguson was i n d i s t i n g u i s h a b l e f r o m t h a t o f growth hormone. A s i m i l a r m o l e c u l e was n o t e d i n t h e plasma o f a male p a t i e n t w i t h g a l a c t o r r h e a and a chromophobe adenoma as w e l l as i n plasma f r o m a l a c t a t i n g woman Chrambach e t a l ?

5

.

i d e n t i f i e d P r l i n t h e plasma o f t h e s e p a t i e n t s and showed t h a t

t h e component had b i o l o g i c a l a c t i v i t y i n t h e i n v i t r o mouse mammary g l a n d o r g a n c u l t u r e system. L i n e a r Ferguson p l o t s a t pH 7.8 and 10.2 i n d i c a t e d t h a t t h e molec u l e was homogeneous and t h e j o i n t 95% c o n f i d e n c e envelopes d e r i v e d f r o m t h e s e Ferguson p l o t s i n d i c a t e d t h a t P r l was d i s t i n c t f r o m t h e m a j o r GH component

5

. Ben-

David e t a1.2 c h a r a c t e r i z e d t h e P r l components i n plasma, a m n i o t i c f l u i d and p i t u i t a r y e x t r a c t s by PAGE and IEF. They n o t e d t h a t t h e p r o l a c t i n components f r o m plasma o f a post-partum l a c t a t i n g woman, a p a t i e n t w i t h a chromophobe adenoma arid g a l a c t o r r h e a , a m n i o t i c f l u i d and a p u r i f i e d p i t u i t a r y p r e p a r a t i o n were i n d i s t i n g u i s h a b l e on t h e b a s i s o f m o l e c u l a r s i z e , n e t charge and apparent i s o e l e c t r i c p o i n t . L i n e a r Ferguson p l o t s were determined a t pH 7.8 and 10.2.

T h i s component

was e a s i l y d i s t i n g u i s h e d f r o m hGH and was a c t i v e i n t h e pigeon c r o p sac assay. A n a l y s i s o f t h e s e c r e t o r y p r o d u c t o f a human p i t u i t a r y tumour removed f r o m an acromegalic p a t i e n t and grown i n c u l t u r e r e v e a l e d an analogous P r l component when analysed b y PAGE; however, when s u b m i t t e d t o radioimmunoassay u s i n g m u l t i p l e a n t i s e r a , t h i s component r e a c t e d w i t h one o f t h e s e a n t i s e r a i n a manner t h a t d i f f e r e d f r o m t h e P r l standard6.

These d a t a emphasize t h e n e c e s s i t y o f v a l i d a t i n g

t h e components d e r i v e d by p h y s i c a l methods by immunological and, p r e f e r a b l y , by b i o l o g i c a l assays. Several groups o f i n v e s t i g a t o r s have n o t e d t h a t P r l c o n s i s t s o f two o r more isohormones whether i s o l a t e d f r o m p i t u i t a r y 4 y 7 o r a m n i o t i c f l u i d

source^^'^.

U t i l i z i n g t h e t e c h n i q u e o f f l a t t e n e d pH g r a d i e n t s i n e l e c t r o f o c u s i n g l O , Ben-David and Chrambach”

were n o t o n l y a b l e t o d i s t i n g u i s h two charge i s o m e r i c forms o f

P r l (P 5.62, 5.96) I b u t were a l s o a b l e t o e l e c t r o f o c u s these d i s t i n c t p r o t e i n species p r e p a r a t i v e l y t o p r o v i d e a method f o r t h e i s o l a t i o n o f homogeneous human p r o l a c t i n isohormones. The a b i l i t y t o i s o l a t e t h e s e isohormones can p r o v i d e t h e

196 m a t e r i a l necessary t o a s s i g n s p e c i f i c b i o l o g i c a l a c t i v i t i e s u n e q u i v o c a l l y t o c h e m i c a l l y and p h y s i c a l l y d e f i n e d p r o t e i n species. These d a t a d e s c r i b e d above f o r p r o l a c t i n e x e m p l i f y w e l l t h e p r e c i s i o n o f PAGE and IEF. I n a s i n g l e decade a "new" hormone was i d e n t i f i e d as an e l e c t r o p h o r e t i c component o f a p i t u i t a r y homogenate. E l e c t r o p h o r e t i c t e c h n i q u e s showed t h a t t h e m a j o r components o f P r l i n plasma, a m n i o t i c f l u i d and p i t u i t a r y homogenates were n o t d i s t i n g u i s h a b l e . Enough m a t e r i a l c o u l d be prepared by t h e s e methods t o v e r i f y b i o l o g i c a l a c t i v i t y . F i n a l l y , d i s t i n c t isohormones c o u l d be d e t e c t e d and prepared so t h a t s p e c i f i c b i o l o g i c a l a c t i v i t i e s c o u l d be determined and v a l i d s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s may be e x p l o r e d . GROWTH HORMONE Mu1 t i p l e e l e c t r o p h o r e t i c components o f animal g r o w t h hormones had been n o t e d i n t h e e a r l y 1960s12-14,

b u t i t was n o t u n t i l

t h e e l e g a n t PACE and IEF s t u d i e s by

Lewis e t a1 .15 and Chrambach e t a1.16 t h a t m u l t i p l e n a t u r a l l y o c c u r r i n g components o f human growth hormone were d e s c r i b e d and compared w i t h t h o s e components d e r i v e d by i n v i t r o p r o t e i n a s e t r e a t m e n t . A s e r i e s o f s t u d i e s by Lewis e t a l . has been sumnarized"

and i n d i c a t e s t h a t GH c o n s i s t s o f a f a m i l y o f p r o t e i n s p e c i e s t h a t

r e p r e s e n t " i n t a c t " GH, s e v e r a l desamido forms

,a

number o f " c l i p p e d " and "cleaved"

forms and a l s o d i s u l p h i d e and n o n - c o v a l e n t l y l i n k e d o l i g o m e r i c forms. I t a t t e s t s t o the specificity,

p r e c i s i o n and power o f PAGE and IEF t h a t t h e s e forms c o u l d

be i d e n t i f i e d , prepared ( o n l y i n p a r t by e l e c t r o p h o r e t i c t e c h n i q u e s ) , c h e m i c a l l y d e f i n e d ( p r i m a r y sequence) , s p e c i f i c b i o l o g i c a l a c t i v i t y measured and " s p e c i e s " s p e c i f i c a n t i s e r a produced. Many o f t h e o r i g i n a l d a t a have been summarized r e c e n t 17 1Y A t t h e same time, Chrambach e t a1.16 showed t h a t s t a n d a r d p r e p a r a t i o n s o f human growth hormone w i t h w i d e l y v a r y i n g p r o l a c t i n b i o a c t i v i t i e s c o n t a i n e d a t l e a s t f o u r components t h a t were c l e a r l y s e p a r a b l e by PAGE and IEF. Each was homogeneous and y i e l d e d l i n e a r p l o t s upon a n a l y s i s by t h e method o f Ferguson. The p r o l a c t i n b i o a c t i v i t y was r e l a t e d t o t h e r e l a t i v e abundance o f t h e more a c i d i c species. I t became apparent t h a t s p e c i f i c b i o l o g i c a l a c t i v i t i e s c o u l d be determined

o n l y i f enough o f each isohormone c o u l d be prepared. U t i l i z i n g t h e newly r e - d i s covered t e c h n i q u e s o f s t e a d y - s t a t e s t a c k i n g 1 8 and i s o t a c h ~ p h o r e s i s ~ ~Chrambach -~~, and co-workers began t o c a t a l o g u e and p r e p a r e l a r g e amounts o f t h e i n d i v i d u a l components t o b e g i n t o s o r t o u t s t r u c t u r e - f u n c t i o n ( b i o l o g i c a l a c t i v i t y ) r e l a t i o n s h i p s ( s e e below). I n seeking t o undlerstand t h e o r i g i n o f t h e m u l t i p l e components, b o t h groups o f i n v e s t i g a t o r s t u r n e d t o t h e a n a l y s i s o f enzymic d i g e s t i o n , whether n a t u r a l l y o c c u r r i n g l 4 ' I 6 o r produced i n v i t r o 2 2 - 2 5 . Employing a s p e c i f i c p l a s m i n p r e p a r a t i o n ,

197 Yadley and Chrambach2’ were a b l e t o show t h a t t h e most abundant GH isohormone was p r o g r e s s i v e l y t r a n s f o r m e d t o t h e more a c i d i c isohormones t h a t showed i n c r e a s i n g p r o l a c t i n s p e c i f i c b i o a c t i v i t y . I n a f u r t h e r study23, these i n v e s t i g a t o r s showed an i n c r e a s e i n t h e s p e c i f i c growth hormone b i o l o g i c a l a c t i v i t y and i n t e r p r e t e d t h e i r f i n d i n g s as p r o t e o l y t i c c l e a v a g e a c t i v a t i n g t h e molecule. T h e i r d a t a i n d i c a t e d t h a t s i m p l e d e a m i d a t i o n ( a l s o y i e l d i n g more a c i d i c components) c o u l d n o t e x p l a i n t h e changes i n GH o r P r l b i o l o g i c a l a c t i v i t y . Lewis e t a1.24 employed s u b t i l i s i n t o m o d i f y GH. They n o t e d t h e p r o d u c t i o n o f t h r e e m o d i f i e d forms, a l l o f which were two-chain m o d i f i c a t i o n s s e p a r a b l e on PAGE a t pH 10. When t r e a t e d w i t h 2-mercaptoethanol

each d i s s o c i a t e d i n t o two components,

i n d i c a t i n g t h a t these two c h a i n forms were j o i n e d by a d i s u l p h i d e bond. S p e c i f i c b i o l o g i c a l a c t i v i t i e s (hyperglycaemic and h y p e r i n s u l inaemic a c t i v i t i e s i n t h e dog versus t i b i a l 1 i n e a c t i v i t y ) a p p a r e n t l y segregated w i t h i n d i v i d u a l s u b t i l i s i n components w i t h a 2 - 4 - f o l d (compared w i t h t h e s t a r t i n g m a t e r i a l ) t i b i a l l i n e (equated w i t h growth-promoting) a c t i v i t y f o r one component. F u r t h e r a n a l y s i s of e n d - t e r m i n a l r e s i d u e s , p e p t i d e mapping and sequence d a t a u n e q u i v o c a l l y a s s i g n e d t h e s t r u c t u r a l b a s i s f o r t h e c l e a v e d forms. Each d i f f e r e d by a few amino a c i d s i n a s i n g l e p o r t i o n o f t h e m o l e c u l e i n d i c a t i n g a g a i n t h e power and s p e c i f i c i t y o f t h e e l e c t r o p h o r e t i c t e c h n i q u e s used i n t h e a n a l y s i s o f t h e cleavage p r o d u c t s .

A second s e r i e s o f m o d i f i c a t i o n s o f GH have been made by Chrambach and coworkers w i t h p l a ~ m i n ~ ~They ’ ~ ~n o.t e d a p r o g r e s s i v e t r a n s f o r m a t i o n o f t h e s t a r t i n g m a t e r i a l t o more a c i d i c ( b u t n o t deamidated) forms t h a t were homogeneous on PP,GE, corresponded t o t h e more a c i d i c forms found i n much l o w e r y i e l d i n p i t u i t a r y e x t r a c t s , and showed a p a r a l l e l i n c r e a s e i n p r o l a c t i n and growth hormone b i o 23 logical activities

.

Taken i n t h e i r e n t i r e t y , t h e s t u d i e s o f t h e s e two groups o f i n v e s t i g a t o r s have d e f i n e d t h e c o m p l e x i t y o f n a t u r a l l y o c c u r r i n g forms o f GH, r i g o r o u s l y determined t h e p h y s i c a l p r o p e r t i e s o f t h e c l o s e l y r e l a t e d s p e c i e s i s o l a t e d by PAGE and IEF and i n many i n s t a n c e s determined t h e s p e c i f i c b i o l o g i c a l a c t i v i t i e s . T h i s l a s t p o i n t i s c r u c i a l f o r an u n d e r s t a n d i n g o f t h e s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s o f t h e f a m i l y o f p r o t e i n s c o l l e c t i v e l y c a l l e d GH, as t h e hyperglycaemic, hyperi n s u l i n a e m i c , p r o l a c t i n and g r o w t h - p r o m o t i n g a c t i v i t e s a p p a r e n t l y r e s i d e i n s l i g h t l y d i f f e r e n t m o l e c u l a r species

-

a l l o f which may be s e p a r a b l e b y t h e use

o f s o p h i s t i c a t e d e l e c t r o p h o r e t i c techniques.

I n addition, the application o f

i s o t a c h o p h o r e s i s may a l l o w t h e p r e p a r a t i o n o f l a r g e amounts o f t h e s e isohormones. I t would i n d e e d b e an e l e g a n t s o l u t i o n t o t h e problem o f t h e a v a i l a b i l i t y o f

human growth hormone f o r G H - d e f i c i e n t c h i l d r e n i f t h e t o t a l s u b t i l i s i n d i g e s t t h a t may show some augmentation o f growth-promoting a c t i v i t y ( n i t r o g e n and potassium r e t e n t i o n i n s h o r t - t e r m e x p e r i m e n t s ) i n c h i l d r e n i 1 6 c o u l d be s e p a r a t e d i n t o i t s t h r e e components,

t h e most a c i d i c o f which shows t h e g r e a t e s t growth-promoting 24 and t h e l e a s t hyperglycaemic and h y p e r i n s u l i n a e m i c a c t i v i t y i n v i v o i n dogs

.

198

I n a comparable study, Baumann and N i ~ s l e yhave ~ ~ shown t h a t t h e more a c i d i c forms o f GH o b t a i n e d from p l a s m i n d i g e s t i o n show i n c r e a s e d somatomedin g e n e r a t i o n i n p a r a l l e l w i t h t h e i r augmented growth-promoting a c t i v i t y compared w i t h t h e s t a r t i n g m a t e r i a l . Again, t h e use o f p r e p a r a t i v e t e c h n i q u e s t o s e p a r a t e t h e s e charge-isomeric p r o t e i n s c o u l d y i e l d c l i n i c a l l y i m p o r t a n t b i o l o g i c a l pharmaceutic a l s. HUMAN C H O R I O N I C GONADOTROPIN

Although i t i s n o t s e c r e t e d b y t h e a n t e r i o r p i t u i t a r y , c h o r i o n i c g o n a d o t r o p i n (CG) has analogous chemical p r o p e r t i e s when compared w i t h l u t e i n i z i n g hormone (LH

Y

f o l l i c l e - s t i m u l a t i n g hormone (FSH) and t h y r o t r o p i n - s t i m u l a t i n g hormone (TSH). A l l a r e g l y c o p r o t e i n s composed o f a common a - s u b u n i t and a h o r m o n e - s p e c i f i c 6 - s u b u n i t CG most c l o s e l y resembles LH i n i t s b i o l o g i c a l a c t i v i t y . Chemicslly, t h e B-subunit

has a c a r b o x y l - t e r m i n a l e x t e n s i o n compared w i t h LH. I t i s t o t h i s c a r b o x y l t a i l that specific antisera

-

t h o s e which d i s t i n g u i s h LH f r o m CG

-

can be d e r i v e d . As

CG i s made i n l a r g e amounts d u r i n g pregnancy ( e s p e c i a l l y d u r i n g t h e f i r s t t r i m e s t e r )

and i s e x c r e t e d i n t o t h e u r i n e , r e l a t i v e l y l a r g e amounts (compared w i t h LH) can be prepared. Most o f t h e s t u d i e s c i t e d below have used u r i n a r y m a t e r i a l t o b e g i n t h e i s o l a t i o n procedure. Radioimmunoassays f o r CG, i t s a- and 6 - s u b u n i t s and t h e CG 6-carboxyl - t e r m i n a l p e p t i d e , a r a d i o r e c e p t o r assay f o r hormone b i n d i n g and

s p e c i f i c i n v i v o and i n v i t r o bioassays a r e a v a i l a b l e so t h a t v a l i d s t r u c t u r e f u n c t i o n r e l a t i o n s h i p s f o r t h e separated g l y c o p e p t i d e s can be d e r i v e d . CG i s o l a t e d f r o m human pregnancy u r i n e has been s u b m i t t e d t o PAGE and IEF i n

a c i d i c a m p h ~ l y t e s ~ ~Each - ~ ~component . i d e n t i f i e d by IEF was s u b m i t t e d t o b i o assay and s i a l i c a c i d a n a l y s i s . I n i t i a l l y s i x bands were noted28y29, t h e b i o l o g i c a l a c t i v i t y o f which was m a i n l y r e l a t e d t o t h e s i a l i c a c i d c o n t e n t . F u r t h e r a n a l y s i s o f t h e amino a c i d and c a r b o h y d r a t e c o m p o s i t i o n o f CG32333 r e v e a l e d heterogeneity o f t h e carbohydrate s t r u c t u r e ( l o s s o f s i a l i c a c i d residues). I n a re-assessment o f t h e h e t e r o g e n e i t y problem, Nwokoro e t a1.34 employed a f l a t t e n e d pH g r a d i e n t 3 5 and an i n c r e a s e d g e l l e n g t h t o d e t e r m i n e t h e number o f components i n h i g h l y p u r i f i e d (11,600 IU/mg) u r i n a r y CG. They n o t e d 14 s t a i n a b l e (Coomassie B l u e ) p r o t e i n zones, 11 o f which had r e l a t i v e l y s t a b l e s t a i n i n g c h a r a c t e r i s t i c s . Each component was r e c o v e r e d f r o m t h e f o c u s i n g g e l and s u b m i t t e d t o radioimmunoassay u s i n g a n t i s e r a a g a i n s t CG, CGa and CGB c a r b o x y l - t e r m i n a l pept i d e o r t o a r a d i o l i g a n d r e c e p t o r assay. A l l assays r e v e a l e d t h e same components; t h e r e f o r e , a l l were complete CG and none was a CG s u b u n i t . S i x o f t h e s e components were c o n s i d e r e d m a j o r ( r e c o v e r y 1.9-12.58 o f t h e s t a r t i n g m a t e r i a l ) and had s i m i l a r i n v i t r o b i o l o g i c a l a c t i v i t y . When s t u d i e d i n v i v o (mouse u t e r i n e w e i g h t assay) t h e y showed b i o l o g i c a l a c t i v i t y r o u g h l y i n p a r a l l e l w i t h t h e s i a l i c a c i d

199 c o n t e n t . Amino a c i d a n a l y s e s were n o t s i g n i f i c a n t l y d i f f e r e n t f o r any component, 33 as had been p r e v i o u s l y d e s c r i b e d

.

The d a t a d e s c r i b e d above p o i n t o u t t h e g r e a t d i s c r i m i n a t i n g power o f i s o e l e c t r i c f o c u s i n g i n s e p a r a t i n g charge-isomers.

U s i n g t h e newly d e s c r i b e d t e c h n i q u e o f

f l a t t e n e d pH g r a d i e n t s f o r a c r y l a m i d e g e l s , Nwokoro e t a1.34 were n o t o n l y a b l e t o s e p a r a t e a v a s t number o f d i s t i n c t CG components, b u t were a l s o a b l e t o s t u d y them b y immunological, r e c e p t o r and bioassays. T h e i r t e c h n i q u e , however, would n o t a l l o w t h e r e c o v e r y o f t h e l a r g e amounts o f ' C G isohormones necessary f o r s t r u c 35 t u r e - f u n c t i o n r e l a t i o n s h i p s i n v i t r o and i n v i v o . To t h a t end, Kapadia e t a l . have employed s t e a d y - s t a t e s t a c k i n g i n p o l y a c r y l a m i d e g e l t o o b t a i n m i l l i g r a m amounts o f CG i n a s i n g l e i s o l a t i o n step. Such t e c h n i q u e s , when a p p l i e d t o b i o l o g i c a l l y r e l e v a n t components, w i l l p r o v i d e t h e m a t e r i a l s necessary t o work o u t t h e complex s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s o f c l o s e l y r e l a t e d members o f a f a m i l y o f peptides. REFERENCES

1 U.J. Lewis, R.N.P. Singh, Y.S. Sinha and W.P. Vander Laan, J . CZin. EndocrinoZ. Metab., 33 (1971) 153. 2 PI. Ben-David, D. Rodbard, R.W. Bates, W.E. B r i d s o n and A. Chrambach, J . CZin. EndocrinoZ. Metah., 36 (1973) 951. 3 A.D. Rogol, M. Ben-David, R. Sheats, D. Rodbard and A. Chrambach, Endoer. Res. Commun., 2 (1975) 379. 4 U.J. Lewis, R.N.P. Sinqh and B.K. Seavey, Biochem. Biophys. .~ _ . Res. Commun. , 44 (1971) 1169. 5 A. Chrambach, W.E. B r i d s o n and R.W. T u r k i n g t o n , Biochem. Biophys. Res. Commun.. 43 (1971) 1296. 6 J.S. Skyler,'A.D.'Rogol , W. Lovenberg and R.A. Knazek, EndocrinoZogy, 100 (1977) 283. 7 P. Hwang, H. Guyda and H.G. F r i e s e n , J . BioZ. Chem., 247 (1972) 1955. B M. Ben-David and A. Chrambach, Endocr. Res. Comun., 1 (1974) 193. 9 A.D. Rogol and A. Chrambach, EndocrinoZogy, 97 (1975) 406. 10 N.Y. Nguyen and A. Chrambach, AnaZ. Biochem., 79 (1977) 462. 11 M. Ben-David and A. Chrambach, J . EndocrinoZ., 84 (1980) 125. 12 K.A. Ferguson and A.L.A. Wallace, Nature (London), 190 (1961) 632. 13 R.J. B a r r e t t , H. F r i e s e n and E.B. Astwood, J . BioZ. Chern., 237 (1962) 432. 14 U.J. Lewis, J . BioZ. Chern., 237 (1962) 3141. Singh, S.M. P e t e r s o n and W.P. Vander Laan, Exceurpta Med. 15 U.J. Lewis, R.N.P. I n t . Congr. S e r . , 381 (1976) 64. 16 A. Chrambach, R.A. Yadley, M. Ben-David and D. Rodbard, EndocrinoZogy, 93 (1973) 848. 17 U.J. Lewis, R.N.P. Singh, G.F. T u t w i l e r , M.B. S i g e l , E.F. Vander Laan and W.P. Vander Laan, Rec. Progr. Horn. Res., 36 (1980) 477. 18 A. Chrambach and J.S. S k y l e r , Protides BioZ. FZuids, Proc. CoZZoq., 22 (19773 701. 19 J.S. S k y l e r , G. Baumann and A. Chrambach, Acta EndocrinoZ. (Copenhagen), 85 (Suppl. 211) (1977) 5. 20 G . Baumann and A. Chrambach, Proc. Nat. Acad. Sci. U.S. , 73 (1976) 732. 21 N.Y. Nguyen, G. Baumann, D. Arbegost and A. Chrambach, i n p r e p a r a t i o n . 22 R.A. Yadley and A. Chrambach, EndocrinoZogy, 93 (1973) 858. 23 R.A. Yadley, D. Rodbard and A. Chrambach, EndocrinoZogy, 93 (1973) 866.

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24 U.J. Lewis, R.N.P. Singh, W.P. Vander Laan and G.F. T u t w i l e r , EndocrinoZogy, 101 (1977) 1587. 25 R.N.P. Singh, B.K. Seavy, V.P. Rice, T.T. L i n d s e y and U.J. Lewis, Endocrinol o g y , 94 (1974) 883. 26 D.L. Bunner, U.J. Lewis and W.P. Vander Laan, J . C Z i n . EndocrinoZ. Metab., 48 ( i 9 7 5 ) 293. 27 G . Baumann and S.P. N i s s l e y , J . CZin. EndocrinoZ. Metab., 48 (1979) 246. 28 R. Brossmer, W.E. iilerz and U. H i l g e n f e l t , FEBS Letz;., 18 (1979) 246. 29 D. G r a e s s l i n , H.C. Weise and P.J. Czygan, FEBS L e t t . , 20 (1972) 87. 30 H. van H e l l and A.H.W.M. S c h u r i e r s , i n W.R. B u t t , A.C. Crooke, and M. Ryle ( E d i t o r s ) , Gonadotropins and marim DeveZopment, E.S. L i v i n g s t o n e , Edinburgh, London, 1970, pp. 70-76. 3 1 R.D. M a f f e z z o l i , G.N. Kaplan and A. Chrambach, J . CZin. EndocrinoZ. Metab., 34 (1972) 361. 32 W.E. Flerz, U. H i l g e n f e l t , M. Db’rner and R. Brossmer, Boppe-Seyler‘s 2. Physiol. Chem., 355 (1974) 1035. 33 W.E. Merz, U. H i l g e n f e l t , R. Brossmer and G. Rehberger, Hoppe-SeyZer’s Z. PhysioZ. Chem., 355 (1974) 1046. 34 N. Nwokoro, H.-C. Chen and A. Chrambach, Endocrinology, 108 (1981) 291. 35 C. Kapadia, J.L. V a i t u k a i t i s and A. Chrambach, Prep. Biochem., 11 (1981) 1.

201

Chapter 9.15 ELECTROPHORESIS OF PLASMA PROTEINS: A CONTEMPORARY CLINICAL APPROACH

M.

ENGLIS Over t h e p a s t decades, e l e c t r o p h o r e s i s has developed i n t o one o f t h e most i m -

p o r t a n t methods f o r t h e i n v e s t i g a t i o n o f b i o l o g i c a l m a t e r i a l s and i s probably t h e most e f f i c i e n t procedure f o r t h e a n a l y s i s o f p r o t e i n s . The e a r l y r e c o g n i t i o n o f t h e apparent d i a g n o s t i c u t i l i t y o f some t y p i c a l e l e c t r o p h o r e t i c p a t t e r n s l e d t o t h e widespread i n t r o d u c t i o n o f e l e c t r o p h o r e t i c techniques i n t o c l i n i c a l p r a c t i c e .

A l i t e r a t u r e e x p l o s i o n has f o l l o w e d t h e e x t e n s i v e work on plasma p r o t e i n e l e c t r o phoresis. The o b j e c t o f t h i s review i s t o compare contemporary e l e c t r o p h o r e t i c techniques w i t h o t h e r methods o f plasma p r o t e i n a n a l y s i s i n c l i n i c a l p r a c t i c e . The e l e c t r o p h o r e t i c s e p a r a t i o n o f plasma p r o t e i n s according t o t h e i r charge i n t o f i v e o r s i x d i s t i n c t zones gives, i n f a c t , a very poor r e f l e c t i o n o f t h e i r complex p a t t e r n . Under standard c o n d i t i o n s , o n l y about t e n plasma p r o t e i n s cont r i b u t e t o t h e t y p i c a l zone p a t t e r n . Some o f them a r e o f l i m i t e d pathophysiolog i c a l o r d i a g n o s t i c value (a2-macroglobulin)

, and

many p r o t e i n s o f c l i n i c a l

i n t e r e s t (orosomucoid, IgM, IgE) do n o t i n f l u e n c e t h e p a t t e r n c o n s t a n t l y . There i s no c o n v i n c i n g demonstration t h a t t h e s t a i n i n g i n t e n s i t y o f p r o t e i n s dominating t h e i n d i v i d u a l zones i s t h e same, o r t h a t i t i s l i n e a r w i t h p r o t e i n concentrat i o n . Normal o r "reference" values f o r e l e c t r o p h o r e t i c zones a r e q u e s t i o n a b l e owing t o t h e l a r g e i n t e r - i n d i v i d u a l v a r i a t i o n s o f some p r o t e i n s i n h e a l t y subj e c t s and t h e i r independent changes i n disease. Scanning diagrams and c a l c u l a t i o n s may be u s e f u l i n t h e f o l l o w - u p o f some diseases o r i n m o n i t o r i n g t h e i r response t o therapy, b u t t h e r e appears t o be no c l i n i c a l advantage f r o m t h e use o f such c a t e g o r i e s i n general. The i n t r o d u c t i o n o f c e l l u l o s e a c e t a t e p r o v i d e d a s i m p l i f i e d and improved technique o f e l e c t r o p h o r e s i s w i t h t h e subsequent development o f m i c r o e l e c t r o p h o r e t i c systems, a r e d u c t i o n i n c o s t s and improvement i n a n a l y t i c a l accuracy and p r e c i sion. C e l l u l o s e a c e t a t e m i c r o e l e c t r o p h o r e s i s has been o f t e n i n c l u d e d i n t h e s e t o f most comnon l a b o r a t o r y examinations as a d o u b t f u l k i n d o f i n t r o d u c t o r y screening o f plasma p r o t e i n a b n o r m a l i t i e s , d e s p i t e t h e f a c t t h a t t h e approximately

2 cm l o n g separations e s s e n t i a l l y o f f e r o n l y t h e same medical i n f o r m a t i o n as t h e outdated paper technique. These m i c r o s c a l e separations may be s u i t a b l e i n s i t u a t i o n s where a simple and s t e r e o t y p e d zone p a t t e r n g i v e s exhaustive i n f o r m a t i o n on p a r t i c u l a r p r o t e i n s under study (isoenzymes)

. However,

greater electrophoretic

202

r e s o l u t i o n i s r e q u i r e d i f more p a t h o p h y s i o l o g i c a l and d i a g n o s t i c c o n c l u s i o n s a r e t o be drawn. Some m o d i f i c a t i o n s o f c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s and e l e c t r o p h o r e s i s on agarose g e l , which g i v e s u b s t a n t i a l l y b e t t e r , l o n g e r and s h a r p e r r e s o l u t i o n p a t t e r n s , meet t h e s e r e q u i r e m e n t s l S 2 . F o r c l i n i c a l r o u t i n e t h e s e p a r a t i o n c h a r a c t e r i s t i c s o f agarose g e l seem t o be s u p e r i o r t o t h o s e o f c e l l u l o s e a c e t a t e . 8-Lipop r o t e i n , which moves as a narrow band on agarose g e l s , p a r t i c u l a r l y i f c a l c i u m i o n s a r e added t o t h e b u f f e r , does n o t appear i n t h e l e s s d i s t i n c t and homogenous B-area on c e l l u l o s e a c e t a t e . A t r u e m u l t i b a n d p a t t e r n can be l o s t and a f a l s e band-1 i k e p a t t e r n produced on c e l l u l o s e a c e t a t e supports. Some sample a p p l i c a t i o n t e c h n i q u e s o f t e n induce i r r e v e r s i b l e changes i n t h e homogeneous s t r u c t u r e o f t h e h y d r a t e d c e l l u l o s e a c e t a t e , w i t h t h e subsequent appearance o f an a r t i f i c i a l band i n t h e a p p l i c a t i o n p o s i t i o n . The h i g h w a t e r c o n t e n t o f agarose g e l ensures u n i f o r mity o f t h e e l e c t r i c a l f i e l d d u r i n g s e p a r a t i o n and t h e p a r a l l e l r u n o f 10-25

samples i n l a r g e agarose g e l s f a c i l i t a t e s t h e c o m p a r a t i v e e v a l u a t i o n o f i n d i v i d u a l bands i n d i f f e r e n t samples. Depending on t h e degree o f e l e c t r o p h o r e t i c r e s o l u t i o n , more qua1 i t a t i v e i n f o r m a t i o n about i n d i v i d u a l components i s o b t a i n e d and p a t h o p h y s i o l o g i c a l r e l a t i o n s h i p s o f some plasma p r o t e i n s can be e s t a b l i s h e d . T h i s i n t u r n p e r m i t s a more appropriate s e l e c t i o n o f p r o t e i n s o f actual c l i n i c a l i n t e r e s t f o r q u a n t i t a t i v e a n a l y s i s . The q u a n t i t a t i o n o f a few s e l e c t e d p r o t e i n s i n terms o f t h e i r q u a l i t a t i v e changes p r o v i d e s t h e c l i n i c i a n w i t h more medical i n f o r m a t i o n t h a n t h e o u t d a t e d scanning diagrams o r a l a r g e b u t n o n - s e l e c t i v e range o f plasma p r o t e i n q u a n t i f i c a t i o n s 3-8

.

E l e c t r o p h o r e t i c techniques w i t h even g r e a t e r r e s o l v i n g c a p a b i l i t i e s have been developed. These i n c l u d e e l e c t r o f o c u s i n g , p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n homogeneous o r g r a d i e n t g e l s and i s o t a c h o p h o r e s i s , Most o f t h e s e procedures a r e s t i l l w i t h i n t h e p r o v i n c e o f c l i n i c a l resear.ch, a l t h o u g h t h e i r h i g h s e n s i t i v i t y and r e s o l v i n g power have s t i m u l a t e d i n c r e a s i n g i n t e r e s t i n some d e f i n e d areas o f c l i n i c a l practice. Plasma p r o t e i n f r a c t i o n a t i o n by e l e c t r o p h o r e t i c methods i s one o f t h e most i n f o r m a t i v e l a b o r a t o r y t e s t s and many abnormal p a t t e r n s can be r e l a t e d t o known c l i n i c a l c o n d i t i o n s . T h i s r e v i e w w i l l r e s t r i c t i t s coverage t o t h r e e fundamental d i s c r i m i n a t i n g p a t t e r n s o f agarose g e l e l e c t r o p h o r e s i s , which w i l l be d i s c u s s e d i n d e t a i l under s e p a r a t e headings. MONOCLONAL AND POLYCLONAL HYPERGAMMAGLOBULINAEMIA The t e r m monoclonal suggests t h a t t h e p r o d u c t i o n o f excess o f immunoglobulin conponents i s t h e p r o d u c t o f a s i n g l e c l o n e o f c e l l s and t h a t t h e s y n t h e s i z e d

203

p r o t e i n s a r e o f i d e n t i c a l charge, s i z e and shape. The c e l l u l a r k i n e t i c s o f t h e p r o l i f e r a t i n g c l o n e i s c h a r a c t e r i s t i c e i t h e r o f an autonomous, ma1 i g n a n t growth t y p i c a l o f m u l t i p l e myeloma o r Waldenstr5n's n a c r o g l o b u l i n a e n i a , o r o f a b e n i g n t y p e o f c e l l p r o l i f e r a t i o n , s t a b i l i z e d on a s u b l e t h a l l e v e l o f t u m u r c e l l s .

A cornmn f e a t u r e o f b o t h groups o f immunocytomas i s t h e presence o f monoclonal immunoglobulins ( p a r a p r o t e i n s ) i n serum. The p a r a p r o t e i n c o n c e n t r a t i o n i s c o n s t a n t 9 i n benign f o r m , b u t g r a d u a l l y i n c r e a s e s i n t h e m a l i g n a n t ones

.

There i s a d i f f e r e n t and a t l e a s t e q u a l l y l a r g e g r o u p o f diseases, m a n i f e s t i n g themselves by p r o d u c t i o n o f homogeneous immunoglobulins o f mono- o r o l i g o c l o n a l o r i g i n . T h i s t y p e o f c l o n a l p r o d u c t i o n appears t o b e a s i g n o f t e m p o r a r i l y i n creased, b u t non-tumorous s y n t h e s i s i n one o r a few c l o n e s w i t h accompanying accentuation o r depression o f synthesis i n t h e m a j o r i t y o f remaining clones. L i t t l e i s known about t h e e t i o l o g y and pathogenesis o f t h e s e d i s o r d e r s ( t r a n s i e n t paraproteinaemia, monoclonal immunoglobulins i n some i n f e c t i o n s , m a l i g n a n c i e s , 10 immunodeficiencies , a f t e r p r o l o n g e d s t i m u l a t i o n s , e t c . ) , E l e c t r o p h o r e s i s i s by f a r t h e most c o n v e n i e n t l a b o r a t o r y method f o r t h e diagnos i s and m o n i t o r i n g o f p a r a p r o t e i n a e m i a . I d e n t i f i c a t i o n o f a p a r a p r o t e i n i n serum i s h i g h l y dependent on t h e degree o f e l e c t r o p h o r e t i c r e s o l u t i o n ; a l s o o f g r e a t importance i s t h e c h o i c e o f t h e most s u i t a b l e a n a l y t i c a l approach and t h e o v e r a l l e x p e r i e n c e and p r o f i c i e n c y o f t h e i n t e r p r e t e r . I n a n o d i c and c a t h o d i c a r e a s o f t h e y-zone f a i n t bands can be d i s t i n g u i s h e d i f t h e p a r a p r o t e i n c o n c e n t r a t i o n 7 8 exceeds 0.5-1.0 g/1, c o r r e s p o n d i n g t o a tumour c e l l mass o f about 10 -10 ( r e f .

11). The c o n c e n t r a t i o n o f serum p a r a p r o t e i n i n c r e a s e s w i t h p r o g r e s s i o n o f t h e tumour growth. Such a g r a d u a l p r o g r e s s i o n , e.g.,

o f myeloma, i s seldom and o n l y

i n c i d e n t a l l y encountered, Most p a t i e n t s a l r e a d y show c l i n i c a l evidence o f generc.1i z a t i o n w i t h d i s t i n c t p a r a p r o t e i n l e v e l s and a c l e a r e l e c t r o p h o r e t i c band'.

A

demarcated and s h a r p l y edged p a r a p r o t e i n band i s t y p i c a l o f most IgG p a r a p r o t e i n s . P a r a p r o t e i n s o f t h e I g A c l a s s do n o t u s u a l l y f o r m n a r r o w e l e c t r o p h o r e t i c bands. T h e i r m o l e c u l a r s i z e , h e t e r o g e n e i t y and easy p o l y m e r i z a t i o n o r c o m p l e x a t i o n w i t h o t h e r plasma p r o t e i n s a c c o u n t f o r broad, extended o r doubled bands.

IgM parapro-

t e i n bands a r e narrower and doubled, i f IgM polymers a r e formed, w i t h a b l u r r e d m a r g i n p r o x i m a l t o t h e s i t e o f sample a p p l i c a t i o n . l l o s t cases o f I g D myeloma show o n l y f a i n t homogeneous p a r a p r o t b i n bands, Gradual broadening o f t h e band on r e peated a n a l y s e s o f t h e same serum sample may s i g n a l I g D p a r a p r o t e i n a e m i a . The c h a r a c t e r i s t i c f e a t u r e s o f monoclonal bands may a r o u s e s u s p i c i o n o f t h e c o r r e 2

sponding immunoglobulin c l a s s

.

S i m i l a r p r e l i m i n a r y c o n c l u s i o n s c a n be drawn f r o m t h e a n a l y s i s o f a p a t i e n t ' s u r i n e . Unconcentrated and c o n c e n t r a t e d u r i n e s h o u l d be e l e c t r o p h o r e s e d a l o n g s i d e a sample o f t h e p a t i e n t ' s serum, There i s u s u a l l y n e g l i g i b l e r e n a l e x c r e t i o n o f complete IgG, I g A o r even IgM p a r a p r o t e i n s . Even a f t e r c o n c e n t r a t i o n o f u r i n e

204

t h e c o r r e s p o n d i n g bands o f serum p a r a p r o t e i n s a r e - i f p r e s e n t a t a l l - v e r y f a i n t and t h e i r m o b i l i t i e s i n serum and u r i n e a r e i d e n t i c a l . The o p p o s i t e i s t r u e f o r f r e e l i g h t c h a i n s o f monoclonal o r i g i n (Bence Jones p r o t e i n s ) . renal function,

I f t h e r e i s normal

t h e Bence Jones p r o t e i n i s a l m o s t c o m p l e t e l y e x c r e t e d i n t o u r i n e .

One, two o r even more a d j a c e n t bands may appear on agarose g e l e l e c t r o p h o r e s i s , r e f l e c t i n g t h e monomer o r dimer forms of Bence Jones p r o t e i n . The c o r r e s p o n d i n g Bence Jones p r o t e i n band i n serum i s o f t e n v e r y f a i n t o r a b s e n t and may be e a s i l y missed i f t h e e l e c t r o p h o r e t i c r u n i s t o o s h o r t . The u r i n e p r o t e i n p a t t e r n i n Bence Jones p r o t e i n u r i a o f t e n shows a d d i t i o n a l bands o f f$-microglobulin a2-microglobulins

and

c h a r a c t e r i s t i c o f t u b u l a r r e a b s o r p t i o n overload.

The serum l e v e l o f a p a r a p r o t e i n r e s u l t s f r o m t h e i n p u t f r o m p r o d u c i n g c e l l s and i t s subsequent d i s t r i b u t i o n and r a t e of'removal ; consequently, t h e amount o f p a r a p r o t e i n p r e s e n t c o r r e l a t e s w i t h t h e a c t u a l tumour c e l l mass. The most i m p o r t a n t a p p l i c a t i o n o f q u a n t i t a t i o n o f serum p a r a p r o t e i n l e v e l i s t h e management o f myeloma t h e r a p y w i t h r e s p e c t t o t h e i n d i v i d u a l growth k i n e t i c s o f t h e p a t i e n t ' s tumour. Accurate assessment o f t h e p a r a p r o t e i n l e v e l i s a l s o o f m a j o r i m p o r t a n c e i n t h e 9,11,12 d i f f e r e n t i a l d i a g n o s i s o f b e n i g n and m a l i g n a n t immunocytomas Q u a n t i t a t i o n e l e c t r o p h o r e s i s (i.e.,

scanning o r e l u t i o n v a l u e s ) s h o u l d be used

f o r t h e d e t e r m i n a t i o n o f p a r a p r o t e i n c o n ~ e n t r a t i o n l ~I. f t h e p a r a p r o t e i n band coi n c i d e s w i t h one o f t h e main p r o t e i n areas, a v i s u a l s e m i - q u a n t i t a t i v e e s t i m a t i o n

w i l l u s u a l l y p r o v i d e more a c c u r a t e values o f p a r a p r o t e i n l e v e l t h a n o t h e r methods. Q u a n t i t a t i v e e l e c t r o p h o r e s i s i s o f l i t t l e v a l u e f o r l i g h t - c h a i n myeloma m o n i t o r i n g . I f normal r e n a l f u n c t i o n i s preserved, a c o n s t a n t f r a c t i o n o f t h e r e n a l Bence Jones p r o t e i n l o a d s a t u r a t e s t h e t u b u l a r r e a b s o r p t i o n c a p a c i t y and t h e d a i l y u r i n a r y o u t p u t o f Bence Jones p r o t e i n can be used as a s u i t a b l e g u i d e t o t h e management o f l i g h t - c h a i n myeloma, P r e l i m i n a r y s u s p i c i o n o f t h e p a r a p r o t e i n c l a s s can be i n f e r r e d f r o m immunochemical q u a n t i t a t i o n o f immunoglobulins. Low v a l u e s o f p a r a p r o t e i n non-commited immunoglobulin c l a s s e s a r e more o r l e s s i n d i c a t i v e o f t h e p r o g r e s s o f t h e tumour growth. The v a l u e s o b t a i n e d f o r t h e p a r a p r o t e i n c l a s s p r e s e n t u s u a l l y g r o s s l y d i s a g r e e w i t h t h e i n t e n s i t y o f t h e c o r r e s p o n d i n g p a r a p r o t e i n band. Immunochemical g e l techniques ( r a d i a l immunodiffusion, electroimmunoassay) o f t e n g i v e gross o v e r e s t i m a t i o n s o f t h e a c t u a l c o n c e n t r a t i o n , which sometimes exceed t h e t o t a l serum p r o t e i n value. A n t i g e n d e l e t i o n s i n p a r a p r o t e i n m o l e c u l a r s t r u c t u r e , which a f f e c t t h e s e i n c o r r e c t r e s u l t s o f g e l t e c h n i q u e s , seem t o be o f l e s s importance i n immunonephelometric techniques. However, even i n some n e p h e l o m e t r i c systems an unsuspected p a r a p r o t e i n may pass i n t o a n t i g e n excess and produce i n c o r r e c t 13,lri l o w values

.

Immunoelectrophoresis,

c r o s s e d immunoelectrophoresis o r i m m u n o f i x a t i o n r e l i a b l y

i d e n t i f y t h e c l a s s and t y p e o f p a r a p r o t e i n . I m m u n o f i x a t i o n s h o u l d be g i v e n p r e -

206

ference t o l i g h t - c h a i n t y p i n g . T h i s h o l d s above a l l f o r t h e i d e n t i f i c a t i o n o f Bence Jones p r o t e i n i n c o n c e n t r a t e d u r i n e samples, where t h e presence o f p o l y c l o n a l l i g h t chains renders immunoelectrophoretic d i f f e r e n t i a t i o n d i f f i c u l t . Antisera o f h i g h q u i l i t y and r a p i d r e a c t i v i t y a r e i n d i s p e n s a b l e f o r r e l i a b l e i m m u n o f i x a t i o n .

110 o t h e r t e c h n i q u e t h a n h i g h - r e s o l u t i o n e l e c t r o p h o r e s i s i s b e t t e r c a p a b l e o f i d e n t i f y i n g p a r a p r o t e i n a e m i a . With m i c r o e l e t r o p h o r e t i c t e c h n i q u e s most o f t h e f a i n t monoclonal bands t h a t a r e t y p i c a l o f benign paraproteinaemia, remain u n d e t e c t e d and a r e o f t e n i n c o r r e c t l y i n c o r p o r a t e d i n t o t h e p o l y c l o n a l immunoglobulin background. Even though l o n g - t e r m o b s e r v a t i o n i s t h e d e c i s i v e c r i t e r i o n f o r t h e d i a g n o s i s o f benigh paraproteinaemia, t h e e x a m i n a t i o n o f e l d e r l y people w i t h a l o w serum v a l u e of a p a r a p r o t e i n , normal values o f p a r a p r o t e i n non-committed imnunoglobul i n c l a s s e s and no Bence Jones p r o t e i n i n c o n c e n t r a t e d u r i n e can be c o n f i r m e d o n l y t o c o n t r o l determinations o f t h e paraprotein concentration. There i s a gradual t r a n s i t i o n o f c l e a r p a r a p r o t e i n bands t o m u l t i c l o n a l p a t t e r n s , a c c e n t u a t i n g t h e p o l y c l o n a l hypergammaglobulinaemja, o r t o i s o l a t e d o l i g o c l o n a l bands i n immunoglobul i n d e f i c i e n c i e s w i t h i n t h e group o f non-tumorous monoclonal immunoglobulins10,

Again a h i g h - r e s o l u t i o n e l e c t r o p h o r e t i c t e c h n i q u e i s o f p r i m a r y

importance. It s h o u l d be s t r e s s e d t h a t i m m u n o f i x a t i o n o r c r o s s e d immunoelectrop h o r e s i s s h o u l d be used f o r c l a s s and t y p e i d e n t i f i c a t i o n o f f a i n t monoclonal bands, as immunoelectrophoresis cannot be employed i n t h e s e s i t u a t i o n s . Combined immunochemical and h i g h - r e s o l u t i o n e l e c t r o p h o r e t i c techniques, e.g., i s o t a c h o p h o r e s i s , a r e t h e most p r o m i s i n g methods i n t h i s f i e l d

e l e c t r o f o c u s i n g and 15

.

The e x c e l l e n t c a p a b i l i t y o f agarose gel e l e c t r o p h o r e s i s t o demonstrate f a i n t r e s t r i c t i o n s o f p o l y c l o n a l e l e c t r o p h o r e t i c h e t e r o g e n e i t y i s perhaps i t s most d i s t i n g u i s h e d f e a t u r e and cannot be matched by any o t h e r method c u r r e n t l y used i n r o u t i n e p r o t e i n analysis.

I t should be born i n mind t h a t a m u l t i b a n d p a t t e r n i n

t h e y-zone i s n o t n e c e s s a r i l y o f a t r u e m u l t i c l o n a l o r i g i n . An unequivocal

oligo-

c l o n a l p a t t e r n o f t e n develops d u r i n g t h e e a r l y immune response. S i n g l e o r s e v e r a l f a i n t bands, each r e p r e s e n t i n g a p p r o x i m a t e l y 0.5 g/1 o r l e s s o f monoclonal a n t i body, appear on a normal p o l y c l o n a l background and d i s a p p e a r d u r i n g t h e subsequent p o l y c l o n a l i n c r e a s e . I n o t h e r c o n d i t i o n s , p a r t i c u l a r l y i n m a l i g n a n t tumours and i n hyperimmune responses , s i m i l a r bands may be found on t h e p o l y c l o n a l background. P o l y m e r i z a t i o n o f p o l y c l o n a l IgG, complexing o f p o l y c l o n a l IgG w i t h serum p r o t e i n s , c o r i p l e x i n g between IgG and o t h e r immunoglobulin c l a s s e s o r between IgG subclasses and enzymatic breakdown o f immunoglobul i n s can produce a mu1 t i band appearance o f t h e zone4 C i r c u l a t i n g immunocomplexes resemble mono- o r b i c l o n a l bands superim-

.

posed on t h e d i f f u s e p o l y c l o n a l background. Most c i r c u l a t i n g immunocomplexes appear on agarose g e l e l e c t r o p h o r e s i s as r e c t a n g u l a r broad bands w i t h w e l l d e f i n e d edges. Both t h e a c t u a l appearance and t h e e l e c t r o p h o r e t i c m o b i l i t y o f immunocomplexes a r e i n f l u e n c e d by t h e a n t i g e n - a n t i b o d y r a t i o 1 6 . I f c a l c i u m i o n s a r e added t o t h e

206

b u f f e r , a f a i n t b a n d , c o r r e s p o n d i n g t o a h i g h l e v e l o f C - r e a c t i v e p r o t e i n , can be found i n t h e y-zone d u r i n g t h e i n i t i a l stages o f i n f l a m m a t o r y t i s s u e d e s t r u c t i o n . The c o l o u r i n t e n s i t y and d i s t r i b u t i o n o f t h e y-zone i n normal s e r a depends on IgG subclass c o n c e n t r a t i o n s . Normal v a l u e s o f I g A and 1g:l immunoglobulins do n o t cont r i b u t e s i g n i f i c a n t l y t o t h e e l e c t r o p h o r e t i c appearance o f t h e y-zone. The e n t i r e zone i s heterogeneous, and n o t even f a i n t bands a r e accepted as a normal f i n d i n g . A n a l y s i s o f s t o r e d s e r a may o c c a s i o n a l l y produce a s i n g l e , h a r d l y v i s i b l e band i n t h e f i b r i n o g e n p o s i t i o n , r e p r e s e n t i n g an u n s t a b l e d e g r a d a t i o n p r o d u c t o f t h e t h i r d complement f a c t o r (C 3)7,8. P o l y c l o n a l hypergammaglobulinaemia i s a common f i n d i n g i n c l i n i c a l p r a c t i c e , easy t o i d e n t i f y and i n v a r i a b l y a r e s u l t o f i n c r e a s e d immunoglobulin s y n t h e s i s . The p o l y c l o n a l i n c r e a s e o f immunoglobulins i s r a r e l y p a r a l l e l o r even p r o p o r t i o n a l i n a l l immunoglobulin c l a s s e s , b u t t h e r e i s u s u a l l y a n unbalanced s y n t h e s i s o f i n d i v i d u a l immunoglobulin c l a s s e s and subclasses. I t seems f a i r t o assume t h a t e l e v a t e d l e v e l s o f IgM and e s p e c i a l l y o f I g A may a f f e c t t h e appearance o f t h e anodic y-zone.

However, t h e i n c r e a s e i n IgG i s b y f a r t h e most d e t e r m i n i n g f a c t o r

i n t h e r e s u l t i n g c o l o u r i n t e n s i t y o f t h e y-zone.

Compared w i t h o t h e r more homo-

geneous zones, v i s u a l i n s p e c t i o n o f t h e y-zone i s l e s s s u i t a b l e f o r t h e p r e d i c t i o n o f immunoglobulin c o n c e n t r a t i o n s . Q u a n t i t a t i v e d e t e r m i n a t i o n s o f IgG, I g A and I n M immunoglobulins a r e mandatory i f immunoglobulin a b n o r m a l i t i e s a r e suspected. The p o l y c l o n a l i n c r e a s e o f a s i n g l e immunoglobulin c l a s s i s d e t e r m i n e d by t h e p r o p e r t i e s o f t h e p r o v o c a t i n g a n t i g e n , t h e n a t u r a l development o f t h e immune response and t h e body s i t e i n v o l v e d . I f e l e v a t i o n s i n a l l c l a s s e s a r e p r e s e n t , a broad immune response o f l i t t l e d i a g n o s t i c v a l u e can be assumed. An i s o l a t e d i n c r e a s e o f a s i n g l e immunoglobulin c l a s s w i t h a l o w o r delayed response i n t h e o t h e r c l a s s e s i s o f a much h i g h e r d i a g n o s t i c value. I f combined w i t h c l i n i c a l and o t h e r l a b o r a t o r y f i n d i n g s t h i s can be v e r y h e l p f u l i n t h e d i f f e r e n t i a l d i a g n o s i s o f some diseases. D i s t i n c t i v e immunoglobulin p a t t e r n s o f a g e n e r a l l y accepted d i a g n o s t i c v a l u e a r e u s u a l l y a s s o c i a t e d w i t h p r i m a r y b i l i a r y c i r r h o s i s ( i s o l a t e d IgM i n c r e a s e ) and w i t h m i c r o n o d u l a r l i v e r c i r r h o s i s ( l a r g e i n c r e a s e o f I g A compared w i t h IgG and ISM). The immunoglobulin response t o aberr a n t immunity o c c u r s m a i n l y i n t h e IgG c l a s s , r e s u l t i n g i n massive e l e v a t i o n o f serum IgG i n a c t i v e s y s t e m i c l u p u s erythematosus, chroinic a c t i v e h e p a t i t i s o r ma1 ignancy w i t h bone marrow i n v o l ~ e m e n t ~ ’ ~ ~ ~ . IgD immunoglobulin, which i s p r o b a b l y a membrane r e c e p t o r r a t h e r t h a n a genuine serum immunoglobulin, can be excluded f r o m t h e q u a n t i t a t i v e a n a l y s i s i f a p o l y c l o n a l p a t t e r n i s t o be analysed. IgE immunoglobulin has a v e r y s h o r t t r a n s i t t i m e f r o m t h e s y n t h e s i z i n g plasma c e l l t h r o u g h serum t o t h e s u r f a c e o f t h e s e c r e t o r y mast c e l l o r b a s o p h i l e . A s i n g l e v a l u e f o r serum IgE i s t h e r e f o r e o f v e r y l i m i t e d d i a g n o s t i c use and does n o t

p r e c l u d e e l e v a t e d v a l u e s i n subsequent samples. IgE q u a n t i t a t i o n s h o u l d be r e p e a t e d l y performed w i t h due r e s p e c t t o t h e t i m e o f exposure t o t h e a l l e r g e n . The p o l y c l o n a l p a t t e r n which may o c c u r i n p a t i e n t s w i t h a t o p i c d i s e a s e s can by no 17 means be e f f e c t e d by t h e i r e l e v a t e d IgE serum l e v e l s

.

ACUTE PHASE PROTEIN RESPONSE Acute s t r e s s c o n d i t i o n s such as trauma, a c u t e i n f e c t i o n , i n f l a m m a t i o n and p r o g r e s s i v e malignancy w i t h t i s s u e n e c r o s i s e f f e c t d i f f e r e n t changes i n a number o f plasma p r o t e i n s . A c o n c o m i t a n t i n c r e a s e i n leakage o f p r o t e i n s t o t h e i n t e r s t i t i a l space, e x t e r n a l l o s s and i n c r e a s e i n p r o t e i n c a t a b o l i c r a t e s may c o n t r i b u t e t o some o f them, b u t by f a r t h e most d i s t i n c t and c l i n i c a l l y i m p o r t a n t i s t h e r a p i d r i s e i n t h e p r o d u c t i o n and plasma l e v e l s o f a g r o u p o f p r o t e i n s . These "acute-phase r e a c t a n t s " i n c l u d e mai n l y a l - a n t i t r y p s i n , orosonucoid, h a p t o g l o b i n, f i b r i n o g e n , C 3 and C - r e a c t i v e p r o t e i n . Many o t h e r plasma p r o t e i n s can be ranked among a c u t e phase r e a c t a n t s , e.g.,

al-antichymotrypsin,

c a e r u l o p l a s m i n , C 4, C 2,

pregnancy-associated p r o t e i n s and a n t i h a e m o p h i l i c g l o b u l i n . The t e r m acute-phase r e z c t a n t s i s o f t e n extended t o i n c l u d e p r o t e i n s , whose plasma c o n c e n t r a t i o n s decrease under a c u t e s t r e s s c o n d i t i o n s : prealbumin, albumin and t r a n s f e r r i n , i.e.,

negative reactants,

The acute-phase plasma p r o t e i n response produces u s u a l l y d i s t i n c t changes i n t h e e l e c t r o p h o r e t i c p a t t e r n o f plasma. These c o n s i s t i n i n c r e a s e s o f al,

a2,

B2

and f i b r i n o g e n zones. The c o n c o m i t a n t decrease o f albumin and t h e B1 zone i s u s u a l l y l e s s pronounced and l e s s s i g n i f i c a n t . The changes i n t h e c o n c e n t r a t i o n s o f i n d i v i d u a l acute-phase r e a c t a n t s p a r a l l e l t h e i n t e n s i t y and spread o f t h e i n d u c i n g s t i m u l i and r e f l e c t i n a p r e d i c t a b l e way t h e i n v o l v e m e n t o f d i f f e r e n t organs and p a t h o p h y s i o l o g i c a l processes. Orosomucoid i s one o f t h e t r u e s t i n d i c a t o r s o f a c u t e t i s s u e i n j u r y . The i n crease and decrease i n orosomucoid plasma c o n c e n t r a t i o n i s independent o f t h e t y p e o f c e l l i n j u r y , t h e presence o f haemolysis, f i b r i n o l y s i s o r complement a c t i v a t i o n . Because o f t h e l o w d y e - b i n d i n g a b i l i t y o f orosomucoid, even h i g h l e v e l s cannot be r e l i a b l y d e t e c t e d i n t h e e l e c t r o p h o r e t i c p a t t e r n and q u a n t i t a t i v e determination i s t h e r e f o r e essential. The s y n t h e s i s o f h a p t o g l o b i n p a r a l l e l s t h a t o f orosomucoid.

I n acute-phase

r e a c t i o n s w h i c h a r e u n c o m p l i c a t e d by haemolysis t h e r i s e o f b o t h p r o t e i n s i s v e r y s i m i l a r , s t a r t i n g w i t h i n a day o r two, r e a c h i n g a peak on days 4-6 and g r a d u a l l y d e c r e a s i n g d u r i n g days 7-14.

I f t h e r e i s a c o n c u r r e n t r e d c e l l break-

down, t h e expected r i s e i n h a p t o g l o b i n i s e i t h e r l o w e r o r a b s e n t o r h a p t o g l o b i n may a l t o g e t h e r d i s a p p e a r f r o m plasma. D e t e c t i o n o f unsuspected haemolysis i s one o f t h e most u s e f u l f e a t u r e s o f h a p t o g l o b i n q u a n t i t a t i o n . The c o l o u r i n t e n s i t y

208

and d i s t r i b u t i o n o f t h e a2 zone c a n n o t be r e l i a b l y used f o r t h e p r e d i c t i o n o f h a p t o g l o b i n l e v e l owing t o t h e v a r y i n g c o n t r i b u t i o n o f a2-macroglobin t o t h e appearance o f t h e zone. Tne r e s o l v i n g c a p a c i t y o f agarose gel e l e c t r o p h o r e s i s f a c i l i t a t e s t h e s c r e e n i n g o f f i b r i n o g e n l e v e l s . As f i b r i n o g e n i s a r e l i a b l e acute-phase r e a c t a n t , pronounced f i b r i n o l y s i s s h o u l d be suspected i n t h e absence o f a f i b r i n o g e n i n c r e a s e i n a c u t e phase response. U n f o r t u n a t e l y , serum a n a l y s i s i s o f t e n p r e f e r r e d i n many l a b o r a t o r i e s and m i c r o e l e c t r o p h o r e t i c t e c h n i q u e s p r e c l u d e a s u f f i c i e n t l y p r e c i s e separ a t i o n o f t h e f i b r i n o g e n band i f plasma i s analysed. al-Antitrypsin al-band.

i s t h e o n l y plasma p r o t e i n t h a t c o n t r i b u t e s s i g n i f i c a n t l y t o t h e

Even m i c r o e l e c t r o p h o r e t i c t e c h n i q u e s can be used f o r t h e s c r e e n i n g o f

disease-related

c1

1- a n t i t r y p s i n d e f i c i e n c i e s and s l o w o r f a s t a l - a n t i t r y p s i n

vari-

a n t s can be r e c o g n i z e d on agarose g e l e l e c t r o p h o r e s i s . The percentage i n c r e a s e i n a l - a n t i t r y p s i n

i s u s u a l l y s u b s t a n t i a l l y lower than

t h a t o f orosomucoid, h a p t o g l o b i n o r f i b r i n o g e n , and depends much more on t h e t y p e o f s t i m u l u s t h a n t h e o t h e r acute-phase p r o t e i n s . The i n c r e a s e i n a l - a n t i t r y p s i n

i s s i g n i f i c a n t l y h i g h e r i n a c u t e o r subacute l i v e r diseases o r i n o e s t r o g e n - i n duced t i s s u e response, I n c o n t r a s t , a d i s t i n c t l y l o w e r a - a n t i t r y p s i n response 1 characterizes connective t i s s u e inflammation w i t h involvement o f blood vessels. The expected a l - a n t i t r y p s i n

i n c r e a s e may be s i g n i f i c a n t l y d i m i n i s h e d i n

g e n e t i c a l l y dependent a - a n t i t r y p s i n d e f i c i e n c i e s . I n d e f i c i e n c i e s o f t h e i n t e r 1 mediate t y p e , as seen i n most common FIZ o r 11s h e t e r o z y g o t e s , a l - a n t i t r y p s i n i n creases t o t h e normal range d u r i n g t h e acute-phase response. The acute-phase r i s e o f a - a n t i t r y p s i n i n severe, d i s e a s e - r e l a t e d d e f i c i e n c i e s (ZZ, SZ, P i n u l l )

1

i s v e r y s l i g h t o r a b s e n t and t h e r e s u l t i n g plasma c o n c e n t r a t i o n i s r a r e l y w i t h i n t h e i n t e r m e d i a t e range. The d e t e c t i o n o f severe d e f i c i e n c i e s i s t h e i n f o r m a t i o n t h e t t h e c l i n i c i a n a c t u a l l y needs and can be o b t a i n e d even i n t h e presence o f acute-phase response by e x a m i n a t i o n o f t h e e l e c t r o p h o r e t i c p a t t e r n . The absence o f an o b v i o u s y - b a n d i n d i c a t e s t h e need f o r f u r t h e r a n a l y s i s t o demonstrate t h e type o f al-antitrypsin

deficiency.

N e i t h e r q u a n t i t a t i o n n o r agarose g e l e l e c t r o p h o r e s i s i s c a p a b l e o f p r e d i c t i n g the al-antitrypsin

phenotype, because a phenotype may be expressed i n a range o f

plasma c o n c e n t r a t i o n s and s i m i l a r c o n c e n t r a t i o n s can b e a s s o c i a t e d w i t h d i f f e r e n t phenotypes. E l e c t r o f o c u s i n g i s t h e most p r e c i s e method f o r c1 - a n t i t r y p s i n phenol 18 typing C 3 i s a r a t h e r s l o w acute-phase r e a c t a n t and i t s plasma l e v e l c u l m i n a t e s

.

w i t h i n t h e second week o f t h e o n s e t o f acute-phase response. C l h a r a c t e r i s t i c a l ly, C 3 i s consumed i n complement a c t i v a t i o n t h r o u g h b i n d i n g as a p a r t o f t h e a c t i v a -

t i o n u n i t t o a n t i b o d y . Q u a n t i t a t i o n o,f C 3 can be used t o d e t e c t and m o n i t o r diseases, due t o f o r m a t i o n and d e p G s i t i o n o f such immune complexes w i t h i n t h e

209

vascular system. Low values o f C3 i m p l y a c t i v e stages o f autoimmune diseases, e.g.

, systemic

lupus erythematosus and immune complex-mediated forms o f n e p h r i t i s .

The i n t e n s i t y o f t h e B2-band should be c a r e f c r l l y compared w i t h t h e assessed values o f C 3, as low-molecular-mass

fragments o f C 3, produced d u r i n g t h e complement

a c t i v a t i o n , may cause a f a l s e o v e r e s t i m a t i o n o f C 3 values i n j u s t those s i t u a t i o n s i n which low values a r e expected. The concomitant increase i n s y n t h e s i s o f C 3 tends t o normalize i t s consumption and even normal values o f C 3 may thus suggest a C 3 a c t i v a t i o n if o t h e r s i g n s o f an acute plasma p r o t e i n response a r e apparent

19

.

Fresh EDTA plasma should be always used f o r c o r r e c t C 3 a n a l y s i s t o overcome i n v i t r o complement a c t i v a t i o n . The C - r e a c t i v e p r o t e i n i s t h e immediate acute-phase r e a c t a n t i n a l l inflammatory s t a t e s w i t h a v e r y steep increase i n plasma l e v e l s d u r i n g t h e f i r s t few hours o f response.

I f v e r y h i g h l e v e l s o f C-reactive p r o t e i n a r e present, a f a i n t band i n

t h e y-zone may appear, b u t i t s appearance i s n o t constant.

To conclude, q u a n t i t a t i v e a n a l y s i s o f a s e l e c t e d s e t of fundamental acute-phase r e a c t a n t s i s most i n f o r m a t i v e i f an acute-phase response i s suspected. Nevertheless, t h e r e s u l t i n g values f o r acute-phase p r o t e i n s may be i n c o m p l e t e l y o r even i n c o r r e c t l y i n t e r p r e t e d i f t h e corresponding e l e c t r o p h o r e t i c p a t t e r n i s ignored.

IMPAIRED SYNTHESIS AND CATABOLIC LOSS OF PLASMA PROTEINS Primary o r secondary disturbances o f plasma p r o t e i n synthesis, increased cat a b o l i c r a t e and l o s s o f plasma p r o t e i n s a r e d i s o r d e r s o f d i f f e r e n t pathogenetic o r i g i n . Some o f them may be associated w i t h a very c h a r a c t e r i s t i c e l e c t r o p h o r e t i c p a t t e r n , which i s o f t e n t h e reason why t h e c l i n i c i a n asks f o r p r o t e i n e l e c t r o phoresis i f such d i s o r d e r s a r e suspected.

A v a r i e t y o f diseases w i t h p r i m a r y d e f i c i e n c y of immunoglobulins have been described2'.

I n some o f these t h e d e f i c i e n c y i s c o n f i n e d t o a s i n g l e immunoglobulin

class, w h i l e i n o t h e r s a l l t h e t h r e e main classes a r e a f f e c t e d . Secondary d e f i c i e n cies, which a r e much commoner i n c l i n i c a l r o u t i n e than t h e p r i m a r y d e f i c i e n c i e s , are u s u a l l y c h a r a c t e r i z e d by low serum l e v e l s o f a l l immunoglobulin classes. The e l e c t r o p h o r e t i c p a t t e r n shows s i g n i f i c a n t changes o r even a complete l a c k o f t h e y-zone o n l y i t t h e IgG l e v e l i s a l t e r e d . Even gross d e f i c i e n c i e s o f IgA and IgM immunoglobulins remain undetected on e l e c t r o p h o r e s i s , Hypogammaglobulinaemia u s u a l l y i n d i c a t e s a s e r i o u s disease i f i n t e s t i n a l o r r e n a l l o s s e s o r a 8 An o l i g o c l o n a l p a t t e r n i s c a t a b o l i c e f f e c t o f g l u c o s t e r o i d s can be excluded

.

sometimes seen i n severe primary immunodeficiencies and m i g h t be t h e r e s u l t of combined response o f a few r e s i d u a l clones, which a r e present even i n severe 21 hypogammaglobul inaemias

.

I n o t h e r types o f primary immunodeficiencies, t h e c e l l o r organ e f f e c t o r s of t h e immune response a r e d e f i c i e n t o r i n j u r e d . I n some o f these, i m u n o g l o b u l i n

S.

210

d e f i c i e n c i e s o f various types r e s u l t , but i n others a f u n c t i o n a l f a i l u r e o f a n t i gen r e c o g n i t i o n o r processing should be suspected i f normal o r even high l e v e l s of immunoglobulins are present. These disorders manifest themselves by f a i l u r e t o respond t o antigenic s t i m u l a t i o n o r respond i n an unbalanced o r incomplete 20,21 manner Increased s u s c e p t i b i l i t y t o r e c u r r e n t i n f e c t i o n s , which dominates t h e c l i n i c a l p i c t u r e o f most immunodeficiencies, can be found i n p a t i e n t s w i t h normal o r high l e v e l s o f immunoglobulins and apparently w e l l developed and f u n c t i o n i n g c e l l immunity. It i s becoming c l e a r t h a t genetic d e f i c i e n c i e s o f complement components may g i v e r i s e t o such immunodeficiency syndromes. The glomerular c a p i l l a r y w a l l acts as a unique graded f i l t e r , whose successive l a y e r s prevent even smaller molecules from e n t e r i n g the Bowman's space. The h i g h l y anionic s t r u c t u r a l components' o f the glomerul a r capi 11a r y w a l l provide an e l ectros t a t i c functional b a r r i e r t o a l l penetrating p r o t e i n anions. If t h i s molecular sieve e f f e c t i s a l t e r e d , a marked l o s s of p l a s m p r o t e i n s i n t o u r i n e r e s u l t s . Provided t h a t damaged glomerbl.ar c a p i l l a r i e s r e t a i n most o f t h e i r d i s c r i m i n a t o r y a b i l i t y f o r graded f i l t r a t i o n o f d i f f e r e n t sized p r o t e i n molecules, proteins o f lower r e l a t i v e molecular mass are l o s t p r e f e r e n t i a l l y i n t o u r i n e . Simultaneously, l a r g e plasma proteins are r e t a i n e d and a r e l a t i v e increase o f plasma a2-macroglobulin, 8-lipoproteins,

IgW and f i b r i n o g e n develops, This type o f s e l e c t i v e

p r o t e i n l o s s i s t y p i c a l o f nephrotic syndrome. Low albumin and intense a2, B and f i b r i n o g e n zones dominate the e l e c t r o p h o r e t i c p a t t e r n o f advanced nephrotic p r o t e i n losses. I n s p i t e o f the u r i n a r y l o s s o f al-antitrypsin,

i t s plasma l e v e l i s o f t e n

normal o r even elevated. The extent o f immunoglobulin losses varies according t o t h e type o f basic pathology and the plasma l e v e l s o f immunoglobulins. Being the smallest sized imnunoglobulin class, IgG i s r e a d i l y l o s t through i n j u r e d glomerulus, and i t s normally slow production shows no compensatory increase; thus

low con-

centrations o f IgG may be seen i n the nephrotic syndrome. Because o f i t s l a r g e r s i z e and more r a p i d replacement rate, the renal l o s s o f I g A i s lower and i t s plasma concentrations tend t o remain i n t h e normal range. I t i s reasonable t o suspect t h a t t h e disease underlying the nephrotic syndrome i s associated w i t h polyclonal hyperglobulinaemia i f normal plasma concentration o f IgG i s found3y22. C a p i l l a r y l e s i o n s i n o t h e r body areas and more pronounced glomerular l e s i o n s do n o t e x h i b i t the s e l e c t i v e s i e v i n g e f f e c t and a hypoproteinaemia due t o nons e l e c t i v e p r o t e i n l o s s develops, I n these cases, each component o f plasma proteins i s l o s t a t almost t h e same r a t e and the p r o t e i n p a t t e r n does n o t change s i g n i f i cantly. Decreased plasma l e v e l s o f albumin, t r a n s f e r r i n and IgG best r e f l e c t t h e 23 extent o f the non-selective p r o t e i n l o s s

.

211 E l e c t r o p h o r e s i s o f u r i n a r y p r o t e i n s may be h e l p f u l i n d i s t i n g u i s h i n g t h e p a t t e r n and degree o f s e l e c t i v i t y o f renal p r o t e i n l o s s . P a t t e r n s o f glomerular, t u b u l a r and d i f f e r e n t types o f o v e r f l o w p r o t e i n u r i a can be d i s t i n g u i s h e d on agarose gel e l e c t r o p h o r e s i s . High s e l e c t i v i t y and gross n o n - s e l e c t i v e u r i n a r y p a t t e r n s may be r e l i a b l y i d e n t i f i e d on agarose gel e l e c t r o p h o r e s i s , b u t f o r the q u a n t i t a t i v e eval24 u a t i o n o f glomerular s e l e c t i v i t y o t h e r methods a r e recommended

.

I t i s n o t p o s s i b l e t o d i s t i n g u i s h r e l i a b l y t h e plasma p r o t e i n p a t t e r n s o f non-

s e l e c t i v e p r o t e i n l o s s from t h e m a l n u t r i t i o n plasma p a t t e r n s . Decreases i n t h e c o n c e n t r a t i o n s o f plasma albumin and t r a n s f e r r i n a r e g e n e r a l l y regarded as a sens i t i v e index o f n u t r i t i o n a l p r o t e i n d e f i c i e n c y . L i v e r s y n t h e s i s o f albumin i s h i g h l y dependent on t h e supply o f amino a c i d s and a1 bumin p r o d u c t i o n decreases s i g n i f i c a n t l y w i t h i n a few days o f p r o t e i n d e p l e t i o n . N o t w i t h s t a n d i n g t h i s , t h e plasma c o n c e n t r a t i o n o f albumin i s an i n s e n s i t i v e and l a t e i n d i c a t o r o f t h e a c t u a l n u t r i t i o n a l s t a t u s because t h e r a t e o f albumin c a t a b o l i s m a l s o f a l l s when amino a c i d s a r e l a c k i n g and simultaneously a1 bumin i s drawn from t h e e x t r a v a s c u l a r space. Therefore, t h e amount o f plasma albumin i s p r o t e c t e d and does n o t i n d i c a t e t h e f u l l e x t e n t o f t h e n u t r i t i o n a l d e f i c i t , A profound decrease i n plasma albumin 22 u s u a l l y c o i n c i d e s w i t h t h e development o f c l i n i c a l s i g n s o f m a l n u t r i t i o n

.

T r a n s f e r r i n s y n t h e s i s depends even more c l o s e l y on t h e supply o f amino a c i d s than t h a t o f albumin. T r a n s f e r r i n can t h e r e f o r e be a more s e n s i t i v e i n d i c a t o r o f developing p r o t e i n d e f i c i e n c y . L i v e r s y n t h e s i s and t h e r e s u l t i n g plasma t r a n s f e r r i n c o n c e n t r a t i o n are, however, i r o n dependent., and thus t h e plasma l e v e l o f t r a n s f e r r i n i s s u b j e c t e d t o o p p o s i t e s t i m u l i i f b o t h p r o t e i n m a l i n u t r i t i o n and i r o n d e f i c i e n c y occur. This i n v a l i d a t e s , a t l e a s t i n p a r t , t h e use o f t r a n s f e r r i n 25 measurements as a r e l i a b l e index o f t h e a c t u a l n u t r i t i o n a l s t a t u s

.

Inflammatory o r tumorous g a s t r o i n s t e s t i n a l l e s i o n s account f o r most secondary m a l n u t r i t i o n syndromes commonly encountered i n c l i n i c a l p r a c t i c e . The m a l n u t r i t i o n a l e l e c t r o p h o r e t i c p a t t e r n , i f present, may be s u b s t a n t i a l l y m o d i f i e d by t h e i n f l a m matory response o f acute-phase r e a c t a n t s and t h e concomitant i n t e s t i n a l loss o f plasma p r o t e i n s . E v a l u a t i o n o f t h e p a t i e n t ' s medical h i s t o r y , c l i n i c a l s i g n s and o t h e r l a b o r a t o r y f i n d i n g s a r e o f t e n more u s e f u l f o r a c o r r e c t e v a l u a t i o n o f t h e n u t r i t i o n a l s t a t u s than plasma p r o t e i n analyses. CONCLUSIONS The fundamental q u a l i t a t i v e a n a l y s i s , p r e d i c t i v e s e m i q u a n t i t a t i v e screening of some p l a s m p r o t e i n s and case-oriented p r o f i l i n g o f p r o t e i n q u a n t i t a t i o n assays a r e t h e most v a l u a b l e c o n t r i b u t i o n s o f contemporary e l e c t r o p h o r e t i c techniques t o c l i n i c a l p r a c t i c e .

A h i g h - r e s o l u t i o n e l e c t r o p h o r e t i c p r o t e i n a n a l y s i s cannot be used as an o r d i n a r y l a b o r a t o r y t e s t . To make t h e optimal use o f t h e c a p a b i l i t i e s o f e l e c t r o p h o r e s i s ,

212 t h e c l i n i c i a n s h o u l d s u p p l y o v e r a l l and p r o b l e m - o r i e n t e d d a t a i n o r d e r t o o b t a i n p r e c i s e and v a l i d i n f o r m a t i o n f r o m t h e b i o c h e m i s t . REFEXENCES

1 B.G. Johansson, Scand. J . CZin. Lab. I n v e s t . , 29, Suppl. 124 (1972) 7. 2 J. Kohn , i n J. Smith ( E d i t o r ) , Chromatographic and EZectrophoretic Techniques, Heinemann, London, 1976, p. 120. 3 Ch.A. A l p e r , N . EngZ. J . Med., 288 (1974) 287. 4 C.-B. L a u r e l l , Scand. J . CZin. Lab. Invest., 29, Suppl. 124 (1972) 71. 5 T. Sun, Y.Y. L i e n and S. Gross, Ann. CZin. Lab. S c i . , 8 (1978) 219. 6 C.-B. L a u r e l l , CZin. Chem., 19 (1973) 99. 7 J.F. Heremans and P.L. Masson, CZin. Chem., 19 (1973) 294. 8 J.O. Jeppsson, C.-B. L a u r e l l and B. Franzen, CZin. Chem., 25 (1979) 629. 9 J.R. Hobbs, Advan. CZin. Chem., 14 (1971)-219. 10 J. Radl , Protides BioZ. Fluids, Proc. CoZZoq., 23 (1976) 405. 11 S.E. Salomon, Sernid. HematoZ., 10 (1973) 135. 12 J.R. Hobbs, Arch. Int. Med., 135 (1975) 125. 13 J.C. D a n i e l s , T.M. V y v i a l , W.C. L e v i n and S.E. Ritzmann, CZin. Chem., 2 1 (1975) 243. 14 J. Kohn, i n A.M. Ward and J.T. Whicher ( E d i t o r s ) , I m o c h e m i s t r y i n CZinicaZ Laboratory Medicine, MTP Press, L a n c a s t e r , 1979, p. 115. 15 P. Delmote, J . NeuroZ., 215 (1977) 27. 16 R.H. K e l l y , H.A. S c h o l l , V.S. Harvey and A.G. Devenyi, CZin. Chem., 26 (1980) 396. 17 S.G.O. Johansson, H.H. Benich and T. Berg, Progress i n CZinicaZ Irmnunology, Grune and S t r a t t o n , New York, 1972. 18 Ph. Arnaud, C. Chapuis-Cell i e r and R. Creyssel , Protides BioZ. FZuids, Proc. CoZZoq., 22 (1975) 515. 19 J.T. Whicher, i n A.14. Ward and J.T. Whicher ( E d i t o r s ) , Immnochemistq i n CZinicaZ Laboratory Medicine, MTP Press, L a n c a s t e r , 1978, p. 150. 20 M. Seligrnann, H.H. Fudenberg and R.A. Good, Amer. J . Med., 45 (1968) 817. 21 J. Radl and P.v.d.Berg, Protides BioZ. FZuids, Proc. CoZZoq., 20 (1973) 263. 22 T. P e t e r s , Jr., i n S.S. Brown, F.L. I d i t c h e l l and D.S. Young ( E d i t o r s ) , ChemicaZ Diagnosis o f Diseases, E l s e v i e r / N o r t h H o l l a n d Biomedical Press, Amsterdam, 1979, p. 333. 23 T. Kawai, CZinicaZ Aspects of t h e PZasma Proteins, Igaku Shoin, Tokyo, 1973, p. 204. 24 W.H. Boesken, Contrib. NephroZ., 1 (1978) 143. 25 Y. Ingenbleek, H.-G. Van den S c h r i e k , Ph. De Nayer and F!. de V i s s c h e r , CZin. Chim. A c t a , 63 (1975) 61.

213

Chupter 9.16

ALLERGENS

HAROLD BAER and MARTHA C . ANDERSON Allergenic e x t r a c t s a r e used t o diagnose and t r e a t a l l e r g i e s in man, and c o n s i s t of water- o r buffer-soluble proteins derived froni p l a n t s , animals, i n s e c t s , fungi and foods. Since these proteins induce a l l e r g i c response within minutes of exposure, they a r e usually found on t h e surface or a r e r e a d i l y e x t r a c t a b l e or a r e secreted from t h e source material. Common a l l e r g e n s include pollen g r a i n s , dusts from aniral skin surfaces, animal bedding m a t e r i a l s , animal s a l i v a a n d u r i n e , and venoms of s t i n g i n g i n s e c t s such as bees. Because they represent soluble components, e x t r a c t s a r e very complex mixtures, b u t the a l l e r g e n i c a l l y a c t i v e components have been ioent i f i e d as proteins and glycoproteins of r e l a t i v e l y low molecular weight, i . e . generally between 5,000 and 40,000 dal tons1'*. The i s o l a t i o n of pure components has been c a r r i e d o u t f o r some e x t r a c t s , b u t the complexity a n d the v a r i e t y of these products ( a t l e a s t a thousand d i f f e r e n t e x t r a c t s a r e a v a i l a b l e commercially) make p u r i f i c a t i o n impractical as a routine method. Therefore, o t h e r procedures have been developed t o study the crude e x t r a c t s . Various gel electrophoresis techniques have furnished information about t h e number of components in an e x t r a c t , i d e n t i f i e d those important in a l l e r g y and provided physico-chemical data such as molecular weight, pr values and enzymatic characteri s t i c s . When attempts a r e made to i s o l a t e d i f f e r e n t components, these procedures aid in t h e i d e n t i f i c a t i o n of t h e m a t e r i a l , t h e determination of t h e homogeneity of the i s o l a t e and the q u a n t i t a t i o n of t h e purified m a t e r i a l . The procedures t h a t have been found t o be useful a r e polyacrylamide gel electrophoresis ( P A G E ) , a n d PAGE w i t h the addition of SDS (SDS-PAGE), i s o e l e c t r i c focusing ( I E F ) , crossed immunoelectrophoresis ( C I E ) and crossed radioimmunoelectrophoresis ( C R I E ) , and t o a 1 imited e x t e n t , agar o r agarose gel immunoelectrophoresis. Since the e x t r a c t s and t h e i r components a r e examined f o r t h e i r a l l e r g e n i c potency, the various gel procedures a r e usually coupled with one o r more methods of assaying a l l e r g e n i c a c t i v i t y . These t e s t s will n o t be described here, b u t may include skin t e s t i n g in a l l e r g i c humans, t h e radioallergosorbent t e s t (RAST), and ' 5 the r e l e a s e of histamine from t h e peripheral leucocytes of a l l e r g i c individualsJ-

.

214 PAGE AND SDS-PAGE

These procedures have been found t o be helpful i n f o l l o w i n g t h e e f f l u e n t from chromatographic columns used i n t h e f r a c t i o n a t i o n and i s o l a t i o n o f a g l y c o p r o t e i n i n 6 A l t e r n a r i a e x t r a c t and i n s t u d i e s o f dog allergens7. PAGE a n a l y s i s o f honey bee venom was supplemented w i t h enzyme substrate s t a i n s t o reveal several esterases 8 and phosphatases Coupled w i t h p r o t e i n and carbohydrate stains, PAGE was used t o

.

demonstrate the presence o f more t h a t 20 compbnents i n an e x t r a c t o f poplar t r e e pollen, and some o f t h e bands were shown t o s t a i n w i t h both stains, thereby ident i f y i n g them as g l y c o p r ~ t e i n s ~F. r a c t i o n a t i o n o f an e x t r a c t o f house dust mite,

Dermatophagoides pteronyssinus, by column chromatography, was followed by an analysis o f the e f f l u e n t by PAGE, and r e s u l t e d i n t h e d e t e c t i o n o f both p r o t e i n and carbohydrate components, w i t h t h e proteins being responsible f o r t h e a l l e r g e n i c activity".

Using PAGE, albumin was characterized as one o f t h e allergens i n mouse

extracts''.

I n vespid i n s e c t studies, there has been some controversy over t h e

s i m i l a r i t y o f secreted i n s e c t venoms and venom obtained by crushing and e x t r a c t i n g t h e venom sacs. SDS-PAGE was used t o demonstrate t h a t t h e e x t r a c t made from venom sacs contains small q u a n t i t i e s o f high-molecular-weight proteins apparently derived from t h e crushed sac, b u t i n other respects, t h e two products were s i m i l a r i n composition12.

A new allergen, Ra6, was discovered i n s h o r t ragweed p o l l e n e x t r a c t

and was found t o be d i f f e r e n t from a l l other known allergens o f ragweed13. A l l e r g e n Ra3 o f s h o r t ragweed p o l l e n e x t r a c t was shown t o occur as two a n t i g e n i c a l l y i n d i s t i n g u i s h a b l e v a r i a n t s by SDS-PAGE14.

The same procedure was used t o detect t h e two

subunits o f s h o r t ragweed antigen E obtained by degradation, and both subunits were found t o be a l l e r g e n i c a l l y a c t i v e

15

.

IEF

Preparative IEF has been used f o l l o w i n g chromatographic procedures f o r the f u r t h e r p u r i f i c a t i o n o f components from the house dust m i t e D. pteronyssinus 16 and from Kentucky bluegrass pollen17 where a very-low-molecular-weight

protein

w i t h a l l e r g e n i c a c t i v i t y was recovered. I t was also used f o l l o w i n g column f r a c t i o n a t i o n and p u r i f i c a t i o n o f peanut allergens'*

and A1 t e r n a r i a e x t r a c t

.

6 Focusing

o f ryegrass p o l l e n e x t r a c t y i e l d e d f o u r e l e c t r o p h o r e t i c a l l y d i s t i n c t allergens whose molecular weight was estimated by SDS-PAGE".

IEF i s an e x c e l l e n t procedure

f o r p r o v i d i n g a p r o f i l e o f an a l l e r g e n i c e x t r a c t , thus enabling i t t o be compared w i t h o t h e r e x t r a c t s obtained from t h e same source; t h e i n d i v i d u a l IEF bands were eluted and t e s t e d f o r a l l e r g e n i c a c t i v i t y by RASTZoaz1.

This technique i s also

useful f o r i d e n t i f y i n g a p a r t i c u l a r a l l e r g e n i n an e x t r a c t and has been used t o evaluate b i r c h t r e e p o l l e n extract" from f i v e d i f f e r e n t s t r a i n s o f mice

and t o compare the a1 1ergens o f mouse u r i n e 23

,

215 CIE, C R I E The methods and a p p l i c a t i o n s have been described i n

They proved

t o be v e r y v a l u a b l e i n d e t e r m i n i n g - t h e mirimurn number o f p r o t e i n s i n an e x t r a c t and d e f i n i n g those t h a t a r e a l l e r g e n s . C I E r e q u i r e s a p r e c i p i t a t i n g antiserum prepared by immunizing r a b b i t s o r sheep w i t h whole e x t r a c t . T h i s procedure assumes t h a t a l l p r o t e i n s , i n c l u d i n g a l l e r g e n s , w i l l induce p r e c i p i t a t i n g a n t i b o d i e s . Since most e x t r a c t s c o n t a i n between 20 and 50 p r o t e i n s i n w i d e l y v a r y i n g concentrat i o n s ( f r o m 0.1% t o 10% o f t h e t o t a l p r o t e i n ) i t cannot be proven t h a t a l l a l l e r gens o r a n t i g e n s have induced p r e c i p i t a t i n g a n t i b o d i e s . CRIE i s dependent o n t h e C I E p a t t e r n and r e q u i r e s sera from a pool o f a l l e r g i c i n d i v i d u a l s , which i s used t o o v e r l a y t h e C I E plates.

Radiolabelled r a b b i t o r

goat

anti-human IgE i s used as a second a n t i b o d y t o d e t e c t t h e a l l e r g e n i c i t y o f t h e CIE bands. I t i s e s s e n t i a l t o use a pool o f a l l e r g i c i n d i v i d u a l s ,

s i n c e i t i s now known

t h a t such i n d i v i d u a l s may have IgE t o one o r many e x t r a c t components, and t h e second antibody reagent, t h e anti-human IgE, must be s p e c i f i c f o r t h e heavy c h a i n o f human IgE, and f r e s h l y r a d i o l a b e l l e d w i t h 1251, t o p r o v i d e a s a t i s f a c t o r y r a d i o autogram. Mold a l l e r g e n s have been examined i n some d e t a i l by C I E . A l t e r n a r i a c u l t u r e 26 f i l t r a t e s were shown t o have many p r o t e i n s , b u t o n l y f o u r were a l l e r g e n s by C R I E and Cladosporium c u l t u r e f i l t r a t e s were found t o c o n t a i n 57 p r o t e i n a n t i g e n s and o n l y f o u r allergens27y28. A s p e r g i l l u s e x t r a c t s were examined by a v a r i a t i o n o f LIE, fused r o c k e t e l e c t r o p h o r e s i s , t o t r a c e t h e s e p a r a t i o n o f components from t h e mixture2',

and RAST was used t o t e s t these f i l t r a t e s f o r a l l e r g e n i c a c t i v i t y 3 ' .

The

appearance o f a n t i g e n s d u r i n g t h e course o f growth o f A s p e r g i l l u s was f o l l o w e d LIE, showing t h a t 22 a n t i g e n s c o u l d be detected, and t h e a n t i g e n s secreted i n t o t h e medium by t h e organism c o u l d be separated from those antigens r e s u l t i n g from l y s i s o f m y c e l i a31

.

Studies o f p o l l e n e x t r a c t s by C I E have been l i m i t e d , b u t t h i s technique has been used t o analyse r a p i d l y r e l e a s e d b a s i c a l l e r g e n s 3 2 . One o f these p r o t e i n s was i d e n t i f i e d as a new a l l e r g e n , and a l t h o u g h i t appeared t o be homogeneous by SDSPAGE, C I E a n a l y s i s i n d i c a t e d t h a t t h i s p r o t e i n had two components13.

The e f f e c t

o f b o i l i n g b i r c h p o l l e n e x t r a c t was examined by CIE and C R I E , and t h e b o i l e d ext r a c t was found t o c o n t a i n i m p o r t a n t a l l e r g e n s 2 2 . Other e x t r a c t s analysed by C I E i n c l u d e house dust33 where i t was p o s s i b l e t o i d e n t i f y and q u a n t i t a t e m i t e a l l e r 34 gen i n t h i s complex m i x t u r e o f antigens

.

Animal a l l e r g e n s from numerous sources have been examined by C I E and C R I E . A l l e r g e n s from horse35 and

have been studied, and, u s i n g concanavalin A i n

an i n t e r m e d i a t e gel, o r m i x i n g t h i s l e c t i n w i t h e x t r a c t p r i o r t o e l e c t r o p h o r e s i s i n t h e f i r s t dimension, i t was p o s s i b l e t o separate t h e carbohydrate-containing components from t h e p r o t e i n s , thus adding another i d e n t i f y i n g f e a t u r e t o t h e anal-

216 ~ s i s The ~ ~ a. l l e r g e n s o f r a t and mouse have been s t u d i e d and shown t o be d e r i v e d from u r i n e r a t h e r than p e l t 3 8 and e x t r a c t s from s i x breeds o f dog i n d i c a t e d t h a t t h e r e were no b r e e d - s p e c i f i c allergens3’.

Examination o f e x t r a c t s from c a t s l e d

t o t h e conclusion t h a t a l l e r g e n s e x t r a c t e d from c a t p e l t were probably o f s a l i v a r y o r i g i n 40

.

Immunoelectrophoresis i s n o t a w i d e l y used technique s i n c e i t has l i m i t e d sens i t i v i t y , b u t i t has been used t o examine t h e a n t i g e n s produced by A s p e r g i l l u s under d i f f e r e n t growth conditions3’. venoms”

Immunologic c r o s s - r e a c t i v i t y among vespid

and s t r a i n - r e l a t e d d i f f e r e n c e s i n mouse u r i n e 2 3 were a l s o s t u d i e d u s i n g

t h i s procedure. The i n f o r m a t i o n provided by t h e v a r i o u s g e l e l e c t r o p h o r e s i s techniques described here have r e s u l t e d i n t h e expansion o f t h e s t u d y o f a l l e r g e n i c e x t r a c t s t o i n c l u d e more biochemical and physico-chemical aspects o f these complex mixtures. As a d d i t i o n a l s t u d i e s a r e r e p o r t e d and t h e c o r r e l a t i o n between t h i s i n f o r m a t i o n and a l l e r g e n i c a c t i v i t y i s f u r t h e r developed, gel e l e c t r o p h o r e s i s techniques w i l l become i n c r e a s i n g l y important i n t h e study o f a l l l e r g e n s . REFERENCES 1 D.G. Marsh, i n M. Sela ( E d i t o r ) , The Antigens, Vol. 3, Academic Press, New York, 1975. 2 H. Baer, i n E. Middleton, C.E. Reed and E.F. E l l i s ( E d i t o r s ) , AZZergy: PrincipZes and Practice, C.V. Mosby CO., S t . Louis, MO, 1978, pp. 217-230. 3 P.S. Norman, i n E. Middleton, C.E. Reed and E.F. E l l i s , AZZergy: P r k i p Z e s and Practice, C.V. Mosby Co., S t . Louis, 1978, pp. 256-264. 4 P.S. Norman, L.M. L i c h t e n s t e i n and K. I s h i z a k a , J . AZergy C Z i n . I m n o Z . , 52 (1973) 210. 5 L. Wide, H. Bennich and S.G.O. Johansson, Lancet, ii (1967) 1105. 6 J.W. Yunginger, R.T. Jones, M.E. Nesheim and M. G e l l e r , J . AZZergy CZin. I m n o Z . , 6G (1980) 138. 7 L. Yman, R. Brandt and G. Ponterius, I n t . Arch. AZZergg AppZ. I m n o Z . , 44 (1973) 358. 8 A. Benton, J . Apic. Res. , 6 (1967) 91. 9 R.B. Knox and A.E. Ashford, CeZZ. Sci., 44 (1980) 1. 10 G.A. Stewart and K.J. Turner, Aust. J . Ezp. BioZ. Med. Sci., 58 (1980) 259. 11 R.P. Siraganian and A.L. Sandberg, J . Allergy C Z i n . I m m t . , 63 (1979) 435. 12 U. M u e l l e r , W. E l l i o t , f?. Reisman, J. Ishay, S. Walsh, R. Steger, J. Wypych and C. Arbesman, J . AZZergy CZin. I m n o Z . , 67 (1981) 290,. 13 R. Hussain, P.S. Norman and D.G. Marsh, J . AZZergy C Z i n . ImnoZ., 67 (1981) 217. 14 L. Goodfriend, M. Roebber, U. Lundquist and A.M. Choudhoury, J . AZZergy Clin. ImunoZ., 67 (1981) 299. 15 B.R. P a u l l , G.J. G l e i c h and M.Z. Atassi, Imnochernistry, 15 (1978) 199. 16 F1.D. Chapman and T.A.E. P l a t t s - M i l l s , J . ImmunoZ., 125 (1980) 587.

217 17 A.K.M. Ekramoddoullah, S . Chakrabarty, F.T. K i s i l and A.H. Sehon, Int. A r c h . AZZergy AppZ. ImmunoZ. , 63 (1980) 220. 18 14.1. Sachs, R.T. Jones and J.W. Yunginger, J . AZZergy C Z i n . IrnmnoZ., 67 (1981) 27. 19 I.J. Smart and R.B. Knox, Int. A r c h . AZZergy AppZ. ImunoZ., 62 (1980) 179. 20 W.D. B r i g h t o n , Dev. BioZ. S t a n d . , 29 (1975) 362. 21 J.M. Varga and M. Ceska, J . AZZergy CZin. IrnmnoZ., 49 (1972) 274. 22 M. Viander, J. F r a k i , B.M. Djupsund and S . Laine, A Z Z e r g y , 34 (1979) 289. 23 M.J. Schumacher, MoZ. ImunoZ., 17 (1980) 1087. 24 N.H. Axelsen, J . K r o l l and B. Weeke, Scand. J . ImunoZ., Vol. 2 (1973) Suppl. 1. 25 H. Lowestein, B o g . A Z Z e r g y , 25 (1978) 1. 26 S. Gravesen, A Z Z e r g y , 34 (1979) 135. 27 H. Lowestein, L. A u k r u s t and S. Gravesen, Int. A r c h . A l l e r g y AppZ. ImunoZ., 55 (1977) 1. 28 L. A u k r u s t and S.M. Borch, Int. A r c h . AZZergy AppZ. I m n o Z . , 60 (1979) 68. 29 S.J. K i m and S.D. Chaparas, h e r . R e v . R e s p . Dis., 118 (1978) 547. 30 S.J. Kim, S.D. Chaparas, T.M. Brown and I4.C. Anderson, Amer. Rev. R e s p . Dis., 118 (1973) 553. 31 H.F. Kauffman and K. deVries, Int. Arch. AZZergy AppZ. ImunoZ., 62 (1980) 252. 32 D.G. Marsh, L. B e l i n , C.A. Bruce, L.M. L i c h t e n s t e i n and R. Hussain, J . A Z Z e q y CZin. IrnmunoZ., 67 (1981) 206. 33 S.D. Carlsen, B. Weeke and H. Lowenstein, A Z Z e r g y , 34 (1979) 155. 34 P. L i n d , J . Korsgaard and H. Lowenstein, A Z Z e r g y , 34 (1379) 319. 35 H. Lowenstain, B. Markussen and B. Weeke, I n t . Arch. AZZergy AppZ. I m n o Z . , 5 1 (1976) 38. 36 P. P r a h l and J. Roed-Petersen, C o n t a c t D e m a t i t i s , 5 (1979) 33. 37 T.C. Bdg-Hansen, P. P r a h l and H. Lowenstein, J . ImmunoZ. Methods, 22 (1978) 293. 38 J.L. Longbottom, i n H. Dehling, I. Glazer, E. Mathov and C. Arbesman ( E d i t o r s ) , A d v a n c e s i n AZZergoZogy and A p p Z i e d I m n o Z o g y , Pergamon Press , New York, 1979, pp. 483-490. 39 J. Blands, H. Lowenstein and B. Weeke, A c t a A Z Z e r g o Z o g i c a , 32 (1977) 147. 40 M.C. Anderson and H. Baer, J . I m u n o Z o g y , 127 (1981) 972.

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219

Chapter 10 GLYCOPROTEI N S AND GLYCOPEPTIDES (AFFINITY ELECTROPHORESIS) T.C.

BBG-HANSEN AND J. HAU

GENERAL ASPECTS A f f i n i t y e l e c t r o p h o r e s i s i s based on t h e r e a c t i o n between i n t e r a c t i n g components d u r i n g e l e c t r o p h o r e s i s . An e a r l y s y s t e m a t i c s t u d y was conducted by Nakamura 1

.

L a t e r t h e t e r m a f f i n i t y e l e c t r o p h o r e s i s was i n t r o d u c e d by B6g-Hansen2 and H o r e j s i and Kocourek’.

Well known examples o f t h e p r i n c i p l e o f l e t t i n g i n t e r a c t i n g com-

ponents r e a c t d u r i n g e l e c t r o p h o r e s i s a r e c o u n t e r immunoelectrophoresis, r o c k e t immunoelectrophoresis and c r o s s e d immunoelectroohoresis, c o l l e c t i v e l y r e f e r r e d t o as electroimmunoassays o r g e l e l e c t r o i m m u n o p r e c i p i t a t i o n methods (see, f o r 4 i n s t a n c e , Axelsen ) . A f f i n i t y e l e c t r o p h o r e s i s o f o t h e r i n t e r a c t i n g systems has been reviewed b r i e f l y by H o r e j s i e t a l . 5

.

0u r comb ina t ion o f qua n t it a t iv e immunoe 1e c t r oDho re s is and a f f i n ity e 1e c t r0p h o r e s i s w i t h l e c t i n s was developed f o r t h e i d e n t i f i c a t i o n , q u a n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f g l y c o p r o t e i n s and t h e a n a l y t i c a l o r e d i c t i o n o f p r e p a r a t i v e s e p a r a t i o n s . I n t h i s c h a p t e r we s h a l l deal w i t h a n a l y t i c a l a f f i n i t y e l e c t r o p h o r e s i s w i t h l e c t i n s o f g l y c o p r o t e i n s . I n p r i n c i p l e t h e r e a r e t h r e e approaches which can be used f o r s t u d y i n g i n t e r a c t i n g comnonents by a n a l y t i c a l e l e c t r o p h o r e s i s : t h e i n t e r a c t i o n can t a k e p l a c e e i t h e r b e f o r e , d u r i n g o r a f t e r e l e c t r o p h o r e s i s . We have s t u d i e d t h e r e s u l t s o f i n t e r a c t i o n s d u r i n g e l e c t r o p h o r e s i s , because more i n f o r m a t i o n can be o b t a i n e d i n t h i s way. Moreover, t h e c h a r a c t e r i z a t i o n o f e l e c t r o p h o r e t i c a l l y s e p a r a t e d g l y c o p r o t e i n s and g l y c o n e p t i d e s by b i n d i n g o f l e c t i n s , such as f l u o r e s c e i n i s o t h i o c y a n a t e - l a b e l l e d l e c t i n , enzyme-labelled l e c t i n o r a n t i b o d y - l a b e l l e d l e c t i n , has been performed elsewhere (see, f o r i n s t a n c e , B i t t i g e r and S c h n e b l i 6 . A l s o r e a c t i o n s w i t h l e c t i n s a f t e r e l e c t r o D h o r e s i s , i n a system analogous t o t h e c l a s s i c a l i n n n u n o e l e c t r o p h o r e t i c a n a l y s i s a c c o r d i n g t o Grabar have been s t u d i e d b y o t h e r s (Murakawa and Nakamura7, Osunkoya and k l i l l i a m s

9 Spengler and Weber

.

a,

We p r e f e r t o use electroimmunoprecipitation as t h e a n a l y t i c a l r e f e r e n c e method. The main reason i s t h a t p r o t e i n s r e m a i n i n t h e i r n a t i v e s t a t e d u r i n g an e l e c t r o i m m u n o p r e c i p i t a t i o n e x p e r i m e n t and r e t a i n t h e i r b i o l o g i c a l a c t i v i t y . T h e r e f o r e , p r o t e i n - l i g a n d i n t e r a c t i o n s a r e n o t i m p a i r e d . G e n e r a l l y , complex f o r m a t i o n between p r o t e i n s and l i g a n d s t e n d t o change t h e i r e l e c t r o p h o r e t i c and a n t i g e n i c b e h a v i o u r .

220

Consequently, i n t e r a c t i n g p r o t e i n s may be i d e n t i f i e d and s t u d i e d by t h e changes induced by i n c l u s i o n o f l i g a n d s i n crossed immunoelectrophoresis. The changes a r e e v i d e n t w i t h macromolecular l i g a n d s , e x e m p l i f i e d h e r e by l e c t i n s and t h e i r a f f i n i t y t o the carbohydrate parts o f glycoproteins. Another reason why we p r e f e r crossed immunoelectrophoresis as t h e r e f e r e n c e e l e c t r o p h o r e t i c system i s t h a t we want t o t a k e advantage o f t h e e x t r a o r d i n a r y f e a t u r e s o f t h i s method f o r t h e a n a l y s i s and c h a r a c t e r i z a t i o n o f multicomponent p r o t e i n m i x t u r e s . The use o f a n t i b o d i e s o f f e r s t h e p o s s i b i l i t y o f t h e s p e c i f i c i d e n t i f i c a t i o n o f p r o t e i n s , b u t i t l i m i t s t h e a n a l y t i c a l uses o f l e c t i n s because of t h e g l y c o p r o t e i n n a t u r e o f t h e a n t i b o d i e s and t h e n e c e s s i t y f o r n o n - d e n a t u r i n g c o n d i t i o n s , The general usage and p r e p a r a t i o n o f a n t i b o d i e s f o r electroimmuno11 p r e c i p i t a t i o n has been d e s c r i b e d e l sewhere (Svendsen'', B$g-Hansen e t a1 ). The t e c h n i c a l d e t a i 1s o f t h e compound methods o f l e c t i n a f f i n i t y immunoelec2,12-20 t r o p h o r e s i s have been d e s c r i b e d p r e v i o u s l y

.

Many p r o t e i n s have been c h a r a c t e r i z e d and p u r i f i e d by a f f i n i t y chromatograohy

21

~ ( T u r k o v a ) and a l s o s p e c i f i c a l l y by i n t e r a c t i o n w i t h l e c t i n s as reviewed by 3ulaneyZ2. I t i s t h e o b j e c t o f t h i s c h a p t e r t o d e s c r i b e g e n e r a l a n a l y t i c a l t e c h nology t o f a c i l i t a t e : ( 1 ) i d e n t i f i c a t i o n o f l i g a n d - b i n d i n g p r o t e i n s ; ( 2 ) charact e r i z a t i o n o f the reactive s i t e ( s ) ; (3) q u a n t i f i c a t i o n o f ligand-binding proteins; and ( 4 ) p r e d i c t i o n o f p r e p a r a t i v e a f f i n i t y s e p a r a t i o n s . 3ESCRIPTION OF THE TECHNOLOGY

Method A: Reactions with Zectin before electrophoresis Reactions a f t e r i n c u b a t i o n w i t h l e c t i n a r e shown s c h e m a t i c a l l y i n F i g . l O . l a , b and c, which show t h e p a t t e r n s i n crossed immunoelectrophoresis. The c o n t r o l D a t t e r n i s shown i n F i g . 1 0 . l a . F i g . 1 0 . l b shows t h e p a t t e r n a f t e r r e a c t i o n w i t h i m m o b i l i z e d l e c t i n . The i n c u b a t i o n p r i o r t o e l e c t r o p h o r e s i s was oerformed by m i x i n g i m m o b i l i z e d l e c t i n w i t h t h e p r o t e i n sample. Only t h e s u p e r n a t a n t f l u i d was analysed by crossed immunoelectrophoresis. P r o t e i n 1 i s known n o t t o b i n d t o t h e l e c t i n , and i t s p r e c i p i t a t e i s unchanged and may b e used as an i n t e r n a l r e f e r e n c e . The t y p i c a l r e a c t i o n i s disappearance o f p r e c i p i t a t e s f r o m t h e p a t t e r n . E i t h e r t h e disappearance may be t o t a l , as seen f o r p r o t e i n 2, or p a r t i a l , as seen f o r p r o t e i n 3 ( o n l y a f r a c t i o n o f p r o t e i n 3 i s bound). T h i s p a t t e r n o c c u r s f r e q u e n t l y i n t h i s t y p e o f experiment. T h e o r e t i c a l l y o t h e r p a t t e r n s c o u l d occur, i n c l u d i n g s p l i t t i n g o f t h e p r e c i p i t a t e i n t o s e v e r a l l i n e s w i t h t h e same f i r s t - d i m e n s i o n m o b i l i t y (Fig. lO.la, p r o t e i n 4 mobilities (protein 5

-t

-+

4a t 4b) o r w i t h d i f f e r e n t f i r s t - d i m e n s i o n

5 t x ) accompanied by a change i n p r e c i p i t a t e morphology

( p r o t e i n 4) o r p r o f i l e ( p r o t e i n 5 ) .

221

7 7 1

Id

1

lI g

1

1

II

F i g . 10.1. L e c t i n i n t e r a c t i o n immunoelectroDhoresis. Schematic diagrams o f p r o t e i n p r e c i p i t a t e p a t t e r n s i n crossed immunoelectrophoresis w i t h and w i t h o u t l e c t i n . ( a ) The p r o t e i n p r e c i p i t a t e r e f e r e n c e p a t t e r n w i t h o u t l e c t i n ; c o n t r o l experiment. ( b ) The p a t t e r n a f t e r i n v i t r o i n c u b a t i o n w i t h i m m o b i l i z e d l e c t i n . ( c ) The p a t t e r n a f t e r i n v i t r o i n c u b a t i o n w i t h f r e e l e c t i n . ( d ) The r e f e r e n c e p a t t e r n w i t h o u t l e c t i n i n t h e i n t e r m e d i a t e g e l ( i . m . ) ; c o n t r o l exDeriment. ( e ) The o a t t e r n w i t h i m m o b i l i z e d l e c t i n ( l e c t i n - S e p h a r o s e , L-S) i n t h e i n t e r m e d i a t e g e l . ( f ) The o a t t e r n w i t h f r e e l e c t i n (L) i n t h e i n t e r m e d i a t e g e l . ( 9 ) The r e f e r e n c e p a t t e r n w i t h o u t l e c t i n i n t h e f i r s t - d i m e n s i o n g e l ; c o n t r o l experiment. ( h ) The p a t t e r n w i t h i m m o b i l i z e d l e c t i n ( l e c t i n - S e p h a r o s e , L-S) i n t h e f i r s t - d i m e n s i o n g e l . ( i ) The p a t t e r n w i t h f r e e l e c t i n (L) i n t h e f i r s t - d i m e n s i o n g e l . See t e x t f o r d e t a i l s . A b b r e v i a t i o n s : a = a f f i n i t y p r e c i p i t a t e ; i . m . = i n t e r m e d i a t e g e l ; L = l e c t i n ; L-S = l e c t i n - S e o h a r o s e ; 1+ = f i r s t - d i m e n s i o n e l e c t r o n h o r e s i s ; 2t = second-dimension e l e c t r o p h o r e s i s . F i g . 1 0 . 1 ~shows a s i m i l a r p a t t e r n a f t e r t h e r e a c t i o n w i t h f r e e l e c t i n . Some p r o t e i n s ( a r r o w s ) appear as l e c t i n complexes i n t h e s u p e r n a t a n t f l u i d and d u r i n g e l e c t r o p h o r e s i s , r e s u l t i n g i n a t y p i c a l p a t t e r n o f p a r t i a l i d e n t i t y (B#g-'Hansen

N

TABLE 10.1

LIST OF CHANGES IN THE IMMUNOPRECIPITATION PATTERN IN CROSSED IMMUNOELECTROPHORESIS WITH LECTINS Reactions seen f o r various glycoproteins w i t h conA. W i t h immobilized conA-Sepharose reactions 6 and 9 have not been seen. (Modified from T.C. BBg-Hansen e t a1 .20). No.

Reaction w i t h l e c t i n i n f i r s t dimension

Reaction w i t h l e c t i n in i n t e r mediate gel

Interpretation

1. 2.

No reaction. Disappearance of precipitate.

No reaction. Disappearance of precipitate.

3.

5.

S h i f t o f position in electrophoret i c pattern. Appearance of a multi-peak precip i t a t e ("camel" precipitate"). Decrease i n precipitate s i z e .

No molecules have a f f i n i t y f o r the l e c t i n . All molecules have a f f i n i t y f o r the ligand. The b i n d i n g t o immobilized l e c t i n may be observed w i t h glycoprotein enzymes. An aff i n i t y precipitate may appear, indicating that some or a l l molecules contain two o r more binding s i t e s f o r the ligand. Binding to ligand.

Decrease i n precipitate s i z e .

6.

Increase i n precipitate size.

Increase i n precipitate s i z e .

7.

Change i n precipitate profile. Change i n precipitate morphology. A . S p l i t t i n g of precipitate. B. Change t o diffuse precipitate. Reactions of "partial i d e n t i t.y".

Change i n precipitate profile. Change i n precipitate morphology. A. S p l i t t i n g of precipitate. B. Change t o diffuse precipitate. Reactions of "partial identity".

4.

8.

9.

Reveals various molecular forms w i t h d i f ferent a f f i n i t y t o the ligand. Only part of the molecules have binding s i t e s o r the binding i s weak. Binding o f ligand leads to s t e r i c hindrance of anti body-bi ndi ng . As 4. As 6 , o r interrupted precipitation.

As 4 and 6 , o r coupling of different prot e i n s through one molecule of ligand (the ligand mediates the "partial identity").

223 e t a1 .20) mediated by l e c t i n c r o s s - l i n k i n g o f d i f f e r e n t p r o t e i n species (compare below). With small amounts o f l e c t i n , r e l a t i v e t o g l y c o p r o t e i n , t h e p a t t e r n i s a f f e c t e d by t h e l e c t i n concentration, b u t above t h e " s a t u r a t i o n p o i n t " t h e p a t t e r n i s

l i t t l e changed by f u r t h e r a d d i t i o n s o f more l e c t i n . We i n t e r p r e t t h e l o s s o f a p r e c i p i t a t e as b i n d i n g o f t h e p r o t e i n t o t h e l e c t i n . The r e a c t i o n s described here as t h e y appear i n crossed immunoelectrophoresis have analogous c o u n t e r p a r t s i n o t h e r e l e c t r o p h o r e t i c systems.

Method B: Lectin in an intermediate gel The p r i n c i p l e o f i n c o r p o r a t i n g l e c t i n i n t o an i n t e r m e d i a t e gel i n crossed immunoelectrophoresis was i n t r o d u c e d by Bg5g-Hansen2 under t h e term "crossed

immunoaffinoelectrophoresis". Since i t s i n t r o d u c t i o n , t h e method has been used f o r t h e i d e n t i f i c a t i o n o f many g l y c o p r o t e i n s and f o r the p r e d i c t i o n o f s e p a r a t i o n experiments (see, e.g.,

r e f s . 2, 14, 17, 20, 23-32).

A l i m i t e d number o f r e a c t i o n s occur when immobilized l e c t i n i s i n c l u d e d i n an

i n t e r m e d i a t e g e l i n crossed immunoelectrophoresis. C h a r a c t e r i s t i c r e a c t i o n s a r e disappearance o r d i m i n u t i o n o f p r e c i p i t a t e s (see F i g . 1 0 . l d and e ) . T h e o r e t i c a l l y i t i s reasonable t o expect t h a t macromolecular complexes c o n t a i n i n g g l y c o p r o t e i n s

may g i v e more complex p a t t e r n s a f t e r r e a c t i o n w i t h l e c t i n . S p l i t t i n g o f p r e c i p i t a t e s , as shown i n F i g . 1 0 . l e ( p r o t e i n 4

-+

4a t 4b o f t h e same f i r s t - d i m e n s i o n

m o b i l i t y ) , may occur a f t e r r e a c t i o n i n t h e i n t e r m e d i a t e g e l . The change i n prec i p i t a t e p r o f i l e seen f o r p r o t e i n 5 i n F i g . 1 0 . l e i s c h a r a c t e r i s t i c f o r complex g l y c o p r o t e i n s and i s seen, f o r exmaple, w i t h human serum complement C3. Any o f these r e a c t i o n s may be accompanied by changes i n p r e c i p i t a t e morphology. Changes i n t h e p r e c i p i t a t e p a t t e r n s , i n c l u d i n g observations w i t h b o t h f r e e and immobilized l e c t i n s , a r e shown i n Table 10.1. F i g . 1 0 . l f shows t h e r e a c t i o n s w i t h f r e e l e c t i n i n t h e i n t e r m e d i a t e g e l . I n a d d i t i o n t o t h e p r e c i p i t a t e changes mentioned w i t h immobilized l e c t i n , o t h e r n o t a b l e f e a t u r e s appear w i t h f r e e l e c t i n s : f o r m a t i o n o f a f f i n i t y p r e c i p i t a t e s and c o p r e c i p i t a t i o n o f i n d i v i d u a l g l y c o p r o t e i n s (discussed below).

Method C. Lectin in the first-dimension g e l The p r i n c i p l e o f i n c o r p o r a t i n g l e c t i n i n t o t h e f i r s t - d i m e n s i o n g e l was i n t r o duced by B#g-Hansen e t a1.33 and i s analogous t o t h e use o f s p e c i f i c a n t i b o d i e s i n t h e f i r s t - d i m e n s i o n gel

-

a h i g h l y s p e c i f i c and s e n s i t i v e method f o r t h e

d e t e c t i o n and i d e n t i f i c a t i o n of i n t e r a c t i n g antigens and a n t i b o d i e s ( P l a t t e t a1 .34) Since i t s i n t r o d u c t i o n , t h e method w i t h l e c t i n s i n t h e f i r s t - d i m e n s i o n gel has been used f o r t h e i d e n t i f i c a t i o n , c h a r a c t e r i z a t i o n and q u a n t i f i c a t i o n o f g l y c o p r o t e i n s and p r e d i c t i o n o f s e p a r a t i o n experiments (see, e.g., r e f s . 16, 20, 23, 25, 27, 28, 35-61).

224

Fig. 10.2. Lectin i n t e r a c t i o n immunoelectrophoresis o f human serum protein. In each experiment 2 vl of human serum were analysed w i t h multivalent antibodies against human serum proteins (OAK0 Immunoglobulins, code lOOSF, 13 v1 / c d ) o r s o e c i f i c antibodies ( 1 . 3 pl/cm2). ( a ) The control experiment: the reference pat-

226

F i g . 10.3. L e c t i n i n t e r a c t i o n immunoelectrophoresis o f a 1 - a n t i t r y p s i n i n 2 u l o f human serum. The e f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n o f f r e e conA i n t h e f i r s t dimension g e l . ( a ) 0.15 10-5 m o l / l conA. ( b ) 0.9 * 10-5 m o l / l conA. The p o s i t i o n o f a l - a n t i t r y p s i n w i t h o u t conA i s i n d i c a t e d . The a n t i b o d y c o n c e n t r a t i o n was 1.3 p l / c m (DAKO Immunoglobulins, s p e c i f i c a n t i b o d y a g a i n s t a 1 - a n t i t r y p s i n ) . Otherw i s e as f o r F i g . 10.2.

-

F i g . 1 0 . l h and i shows t y p i c a l r e a c t i o n s when p r o t e i n s a r e e l e c t r o p h o r e s e d t h r o u g h a g e l c o n t a i n i n g i m m o b i l i z e d and f r e e l e c t i n , r e s p e c t i v e l y . The p a t t e r n i s h i g h l y dependent on t h e l e c t i n c o n c e n t r a t i o n . P r o t e i n 1 does n o t b i n d t o t h e l e c t i n and i s used as an i n t e r n a l r e f e r e n c e of unchanged o o s i t i o n ( T a b l e 10.1). The r e a c t i o n s a r e disappearance o f p r e c i p i t a t e s , f o r m a t i o n o f an a f f i n i t y p r e c i p i t a t e and changed p o s i t i o n and changed p r o f i l e s : p r o t e i n 2 i s l o s t f r o m t h e

-

10-5 m o l / l f r e e conA i n t h e f i r s t t e r n w i t h o u t l e c t i n . ( b ) The p a t t e r n w i t h 0.9 dimension g e l ( c ) As ( b ) b u t w i t h s p e c i f i c a n t i b o d i e s a g a i n s t G C - g l o b u l i n t o show t h e unchanged p a t t e r n o f G C - g l o b u l i n . ( d ) As ( b ) b u t w i t h s p e c i f i c a n t i b o d i e s a g a i n s t orosomucoid ( a 1 - a c i d g l y c o p r o t e i n ) t o show t h e t r i p l e peak o f orosomucoid w i t h conA. ( e ) As ( b ) b u t w i t h s p e c i f i c a n t i b o d i e s a g a i n s t h a p t o g l o b i n t o show t h e 10-5 double peak o f h a p t o g l o b i n w i t h conA. ( f ) The p a t t e r n o f orosomucoid w i t h 0 . 9 m o l / l conA-Sepharose i n t h e f i r s t dimension [compare w i t h (d)] t o show t h e d o u b l e peak o f orosomucoid w i t h conA-Sepharose. The b a r r e p r e s e n t s 1 cm. A b b r e v i a t i o n s : A = albumin; AT = a l - a n t i t r y p s i n ; GC = GC-globulin; HDL = h i g h - d e n s i t y l i p o p r o t e i n ; Hp = h a p t o g l o b i n ; I g = immunoglobulins; PA = prealbumin; 0 = orosomucoid ( a l - a c i d g l y c o p r o t e i n ) ; T = t r a n s f e r r i n . C o n d i t i o n s o f e l e c t r o p h o r e s i s : 1%agarose i n T r i s v e r o n a l (pH 8.6); f i r s t - d i m e n s i o n e l e c t r o p h o r e s i s 10 V/cm f o r 1 h; second-dimension e l e c t r o p h o r e s i s , 2 V/cm f o r 18 h.

226

p a t t e r n . P r o t e i n 3 i s s h i f t e d c a t h o d i c a l l y t o appear as a double-peak p r e c i p i t a t e , each peak w i t h a c h a r a c t e r i s t i c r e t a r d a t i o n t h a t i s dependent on t h e l e c t i n conc e n t r a t i o n . G l y c o p r o t e i n s t h a t appear r e p r o d u c i b l y as m u l t i - p e a k p r e c i p i t a t e s i n t h i s manner a r e f o r i n s t a n c e human serum a - 1 - a n t i t r y p s i n ,

orosomucoid ( a - 1 - a c i d

g l y c o p r o t e i n , AGP), a - f o e t o p r o t e i n (AFP) , h a p t o g l o b i n and h i g h - d e n s i t y l i p o p r o t e i n (HDL) ( s e e F i g s . 10.2 and 10.3). G e n e r a l l y l e c t i n s w i t h a l o w m o b i l i t y a t pH 8.6 (such as conA f r o m Pharmacia and P h a r m i n d u s t r i e ) i n d u c e a c a t h o d i c s h i f t o f t h e

b i n d i n g g l y c o p r o t e i ns , INTERPRETATION OF THE PATTERNS

VisuaZization of proteins bound t o i m o b i t i z e d t e e t i n s I m m o b i l i z e d l e c t i n s a r e p r o t e i n s themselves and s t a i n w i t h Coomassie B r i l l i a n t B l u e and o t h e r p r o t e i n s t a i n s , so t h a t bound p r o t e i n w i l l n o t b e d e t e c t e d u n l e s s a s p e c i f i c method i s a v a i l a b l e ; c f . , F i g . 10.2f i n w h i c h t h e human serum g l y c o -

F i g . 10.4. V i s u a l i z a t i o n o f b i n d i n g between g l y c o p r o t e i n s and l e c t i n i n l e c t i n i n t e r a c t i o n immunoelectrophoresis. ( a ) Crossed immunoelectrophoresis w i t h conASepharose i n t h e i n t e r m e d i a t e g e l . Two m i c r o l i t r e s o f human serum were analysed. A f t e r e l e c t r o p h o r e s i s t h e p l a t e was s t a i n e d f o r e s t e r a s e a c t i v i t i e s (Brogren and BBg-Hansen86). The arrows i n d i c a t e enzymes bound t o conA ? a r t i c l e s . ArE = HDLa s s o c i a t e d a r y l e s t e r a s e ; ChE = c h o l i n e s t e r a s e . ( b ) Crossed immunoelectrophoresis w i t h f r e e , r a d i o a c t i v e l y l a b e l l e d conA i n t h e i n t e r m e d i a t e g e l ( a u t o r a d i o g r a p h y ) ( c f . , B j e r r u m e t a l . 2 4 ) . Two m i c r o l i t r e s o f human serum were analysed. The m a j o r b i n d i n g p r o t e i n s a r e ( f r o m t h e r i g h t ) : a 2 - m a c r o g l o b u l i n Y h a p t o g l o b i n , LDL, complement C3 and IgM. Otherwise as f o r F i g . 10.2.

227

p r o t e i n s a r e p a r t i a l l y bound t o conA-Sepharose p a r t i c l e s . However, by a u t o r a d i o graphy o f r a d i o a c t i v e l y l a b e l l e d g l y c o p r o t e i n s o r b y h i s t o c h e m i c a l s t a i n i n g o f g l y c o p r o t e i n enzymes, t h e b i n d i n g t o l e c t i n p a r t i c l e s can b e v i s u a l i z e d . Thus, F i g . 10.4 shows I - l a b e l l e d human serum p r o t e i n s i n p r e c i p i t a t e s w i t h conA ( F i g . 10.4a) and human serum c h o l i n e s t e r a s e and h i g h - d e n s i t y l i p o p r o t e i n - a s s o c i a t e d a r y l e s t e r a s e ( a r r o w s ) bound t o conA p a r t i c l e s ( F i g . 10.4b). Appearance of affinity precipitate Most remarkable a f t e r e l e c t r o p h o r e s i s w i t h f r e e l e c t i n i s t h e appearance o f l e c t i n - p r e c i p i t a t e d g l y c o p r o t e i n i n one o r more d i s c r e t e " a f f i n i t y p r e c i p i t a t e s " . T h i s i s seen w i t h human serum p r o t e i n s when t h e y a r e e l e c t r o p h o r e s e d t h r o u g h a f i r s t - d i m e n s i o n g e l o r t h r o u g h an i n t e r m e d i a t e g e l c o n t a i n i n g f r e e conA ( F i g s . 1 0 . l f and i, 10.2, and 10.3). A p r e r e q u i s i t e f o r t h e f o r m a t i o n o f an a f f i n i t y p r e c i p i t a t e i s t h a t b o t h l e c t i n and g l y c o p r o t e i n have a t l e a s t two b i n d i n g s i t e s p e r m o l e c u l e . T h e r e f o r e , we may c h a r a c t e r i z e most o f t h e g l y c o p r o t e i n s i n t h e a f f i n i t y p r e c i p i t a t e as h a v i n g a t l e a s t two b i n d i n g s i t e s . On t h e o t h e r hand, i t cannot be excluded t h a t some g l y c o p r o t e i n s w i t h o n l y one b i n d i n g s i t e w i l l b i n d t o an a l r e a d y e x i s t i n g a f f i n i t y D r e c i p i t a t e . However, i t was p o s s i b l e t o d i s t i n g u i s h between t h e r e a c t i o n s o f two human g l y c o o r o t e i n enzymes [urine a c i d Dhosphatase ( p r o s t a t e ) and serum c h o l inesterase]

i n t h e intermediate g e l technique 14 ) . Both enzymes bound

w i t h conA-Sepharose and f r e e conA (Bdg-Hansen and Brogren

t o conA-Sepharose b u t o n l y c h o l i n e s t e r a s e gave an a f f i n i t y p r e c i p i t a t e w i t h f r e e conA, i n d i c a t i n g two o r more b i n d i n g s i t e s p e r m o l e c u l e f o r c h o l i n e s t e r a s e ( g l y c o p r o t e i n Type 2 ) and o n l y one b i n d i n g s i t e f o r u r i n e a c i d phosphatase ( g l y c o p r o t e i n Type l ) . Reactions of "partiaZ identity" The p r e c i p i t a t i o n p a t t e r n o f p a r t i a l l y i d e n t i c a l p r o t e i n s i n q u a n t i t a t i v e immunoelectrophoresis has been d e s c r i b e d i n d e t a i l by Bock and Axelsen62. When m i x t u r e s o f g l y c o p r o t e i n s a r e a n a l y s e d w i t h f r e e l e c t i n ( c f . , F i g . l O . l c , f and i ) , t h e common c a r b o h y d r a t e m o i e t i e s w i l l m e d i a t e r e a c t i o n s o f p a r t i a l i d e n t i t y and t h e l e c t i n w i l l c r o s s - r e a c t w i t h a l l g l y c o p r o t e i n s h a v i n g b i n d i n g c a p a c i t y . DETERMINATION OF AFFINITY The degree o f r e t a r d a t i o n d u r i n g t h e f i r s t - d i m e n s i o n e l e c t r o p h o r e s i s i n a g e l w i t h l e c t i n i s an e x p r e s s i o n o f t h e a f f i n i t y between t h e g l y c o p r o t e i n and t h e l e c t i n . The c o n d i t i o n s i n t h e e l e c t r o p h o r e s i s g e l a r e analogous t o c o n d i t i o n s i n an a f f i n i t y chromatographic column. H i g h e r a f f i n i t y means s t r o n g e r b i n d i n g and h i g h e r degree o f r e t a r d a t i o n .

228

The retardation coefficient

A s i m p l e way t o express t h e r e l a t i v e a f f i n i t y o f p r o t e i n s t o t h e same l i g a n d i s t o calculate the retardation coefficients, R =

ZdZr -

1, where Zr and Zo a r e

t h e m i g r a t i o n d i s t a n c e s i n t h e f i r s t - d i m e n s i o n e l e c t r o o h o r e s i s w i t h and w i t h o u t l i g a n d , r e s p e c t i v e l y (Bdg-Hansen e t a1 .20). The r e t a r d a t i o n c o e f f i c i e n t can be c a l c u l a t e d f r o m a s i n g l e experiment w i t h l i g a n d and p r o v i d e d t h a t an excess o f l i g a n d i s p r e s e n t , t h e r e t a r d a t i o n c o e f f i c i e n t i s independent o f l i g a n d conc e n t r a t i o n , d i s r e g a r d i n g t h e i n f l u e n c e o f d i f f e r e n c e s i n m o l e c u l a r w e i g h t , charge d e n s i t y , e t c . By d e t e r m i n a t i o n o f t h e r e t a r d a t i o n c o e f f i c i e n t w i t h conA o f human serum g l y c o p r o t e i n s i t was p o s s i b l e t o l i s t t h e s e p r o t e i n s a c c o r d i n g t o t h e i r a f f i n i t y t o conA (Bdg-Hansen e t a1.20). The advantage o f t h i s method i s t h a t o n l y one experiment needs t o be performed w i t h l e c t i n i n c o r p o r a t e d i n t h e g e l . The concentration-dependent retardation The c o n c e n t r a t i o n - d e p e n d e n t r e t a r d a t i o n i s examined u s i n g an i n c r e a s i n g conc e n t r a t i o n o f l e c t i n i n t h e f i r s t - d i m e n s i o n g e l . T h i s i s shown f o r human serum a - 1 - a n t i t r y p s i n i n F i g . 10.3. With f r e e conA i n t h e f i r s t - d i m e n s i o n g e l t h e

F i g . 10.5. Crossed affinoimmunoelectrophoresis o f mouse AFP ( a - f o e t o o r o t e i n ) . The a n t i g e n was a 3-day j u v e n i l e homogenate. The f i r s t - d i m e n s i o n g e l c o n t a i n e d 300 10-5 m o l / l ) . An i n t e r m e d i a t e g e l ( b l a n k ) was i n s e r t e d between pg/cm2 o f conA (1.8 t h e f i r s t - d i m e n s i o n ge1,and t h e a n t i b o d y - c o n t a i n i n g g e l ( g o a t a n t i s e r u m a g a i n s t murine AFP). Note t h e d i f f e r e n c e i n number of oeaks t o F i g . 10.7 (conA f r o m Pharmacia)

-

229

a - 1 - a n t i t r y p s i n shows a c h a r a c t e r i s t i c m u l t i - p e a k p r e c i p i t a t e and a l l components e x h i b i t b i n d i n a t o conA. F o r some c o n A - b i n d i n g g l y c o p r o t e i n s such as orosomucoid ( a - 1 - a c i d g l y c o p r o t e i n , AGP) o n l y some f r a c t i o n s o f t h e p r o t e i n b i n d ( F i g . 10.2d and f ) , whereas one f r a c t i o n does n o t b i n d ( a r r o w , F i g . 10.2d and f ) (Bplg-Hansen e t a1.33, Bplg-Hansen and Takeo16, W e l l s and co-workers60y61), as v e r i f i e d by a f 51 ). F i g . 10.5. shows t h e p a t t e r n s o f m u r i n e a - f o e t o p r o t e i n (rn-AFP) w i t h conA i n

f i n i t y chromatography ( N i l s s o n and B$g-Hansen

t h e f i r s t - d i m e n s i o n g e l s . Four m i c r o h e t e r o g e n e i t y forms can be d i s t i n g u i s h e d by t h e i r r e a c t i o n w i t h conA i n f o e t a l and j u v e n i l e t i s s u e . These were d e s i g n a t e d 0, 1, 2 and 3 a c c o r d i n g t o i n c r e a s i n g conA a f f i n i t y , f o r m 0 b e i n g t h e conA non-

reactive. The e x i s t e n c e o f a m o l e c u l a r f o r m o f f o e t a l m-AFP which c o u l d be c o m p l e t e l y p r e c i p i t a t e d b y f r e e conA was e x c l u d e d by comparison o f r o c k e t s i n affinoimmunoe l e c t r o p h o r e s i s w i t h r o c k e t s i n i m m u n o e l e c t r o p h o r e s i s w i t h o u t conA. The r o c k e t h e i g h t s were i d e n t i c a l i n t h e two t y p e s o f e l e c t r o o h o r e s i s , p r o v i n g t h a t a comp l e t e l y p r e c i p i t a b l e f o r m o f m-AFP was n o t p r e s e n t (Hau and co-workers 46,47 ) . The general T a k e o - N a h r a p l o t The t h e o r e t i c a l background of a f f i n i t y e l e c t r o p h o r e s i s was worked o u t by Takeo and N a k a m ~ r ai ~n ~t h e i r o r i g i n a l experiments w i t h enzymes and s u b s t r a t e s . U s i n g t h e i r t h e o r y , i t i s p o s s i b l e t o c a l c u l a t e t h e a f f i n i t y between a r e t a r d e d p r o t e i n and a l e c t i n as t h e d i s s o c i a t i o n c o n s t a n t f r o m a Takeo-Nakarnura p l o t , which i s a s i m p l e r e l a t i o n s h i p between t h e r e l a t i v e a i g r a t i o n v e l o c i t y and t h e l e c t i n concentration:

1

*mi

= L (1+41) Rmo K

(10.1

where K i s t h e d i s s o c i a t i o n c o n s t a n t o f t h e l e c t i n - g l y c o p r o t e i n complex, c i s t h e c o n c e n t r a t i o n o f l e c t i n expressed as t h e c o n c e n t r a t i o n o f b i n d i n g s i t e s , t h e " n o r m a l i t y " , Rmo i s t h e m o b i l i t y o f t h e g l y c o p r o t e i n w i t h o u t l e c t i n , Rmi i s t h e m o b i l i t y o f t h e g l y c o p r o t e i n i n t h e presence o f l e c t i n and Rmc i s t h e m o b i l i t y o f t h e l e c t i n - g l y c o p r o t e i n complex i n r e l a t i o n t o an i n t e r n a l s t a n d a r d such as bromophenol blue-marked a l b u m i n o r p r e a l b u m i n . U s i n g t h i s method, t h e d i s s o c i a t i o n c o n s t a n t f o r conA complexes o f s e v e r a l g l y c o D r o t e i n s f r o m hunfan serum have been e s t i m a t e d , c f . , T a b l e 10.2. The Takeo-Nakamura p l o t i s c o n f i n e d i m p l i c i t l y t o t h e s i t u a t i o n where t h e complex between t h e i n t e r a c t i n g components i s e l e c t r o D h o r e t i c a l l y immobile, a c o n d i t i o n which i s n o t a p r i o r i met w i t h f r e e macromolecular l i g a n d s such as l e c t i n s , b u t i s w i t h i m m o b i l i z e d l e c t i n s . However, i n many i n s t a n c e s i t would

230 TABLE 10.2 DISSOCIATION CONSTANTS OF COMPLEXES BETWEEN SERUM PROTEINS AND conA

Human orosomucoid component 2 component 3 Human a n t i t r y p s i n component 1 component 2 Human a l - l i p o p r o t e i n associated arylesterase Human a2-HS-glycoprotei n Human h a p t o g l o b i n Human t r a n s f e r r i n Human a - f o e t o p r o t e i n (AFP) component 1 Murine a - f o e t o p r o t e i n (AFP) component 1 component 2 Murine c a r b o x y l e s t e r a s e component 1 component 2 component 3

D i s s o c i a t i o n constant, mean ( m o l / l ) a

nb

1.5 1.1

10-5 10-5

16 12

3.1 1.3

10-5 10-5

7 15

1.1 4.0 3.2 3.9

10-5 10-5 10-5 10-5

2 11 4

5.0

10-6

C

4.5 3.3

10-6 10-6

d d

1.5 1.3 0.9

10-5 10-5 10-5

4

Ref, Bdg-Hansen and Take016

7 T h i s work

Bdg-Hansen e t al.38

4 4

: D i s s o c i a t i o n c o n s t a n t s c a l c u l a t e d a f t e r crossed i m u n o e l e c t r o p h o r e s i s w i t h conA. Number o f s e r a analysed. :Pool o f more t h a n 10 sera. Pool o f more t h a n 20 sera. be d e s i r a b l e t o work w i t h f r e e l e c t i n s because i t would save t h e e f f o r t s i n v o l v e d i n p r e p a r i n g t h e i m m o b i l i z e d l e c t i n s and e l i m i n a t e t h e d i f f i c u l t y o f e s t i m a t i n g t h e e x a c t amount o f bound l e c t i n . Complexes between g l y c o u r o t e i n s and f r e e l e c t i n s would be expected t o have an e i g e n m o b i l i t y . N o n - l i n e a r Takeo-Nakamura p l o t s f o r 63 k i d n e y phosphorylase i n complex w i t h g l y c o p r o t e i n (Takeo and Nakamura ) and human serum g l y c o p r o t e i n s i n complex w i t h conA have been d e s c r i b e d (664-Hansen 16 ). Therefore, t h e general equation f o r e l e c t r o p h o r e t i c determination

and Takeo

o f d i s s o c i a t i o n c o n s t a n t s was d e r i v e d :

(10.2)

where Rmc i s t h e m o b i l i t y o f t h e c o n A - g l y c o p r o t e i n complex, i n r e l a t i o n t o t h e i n t e r n a l s t a n d a r ( o t h e r symbols as i n eqn. 10.1). T h i s e q u a t i o n r e p r e s e n t s a s t r a i g h t l i n e when ( l e c t i n c o n c e n t r a t i o n ) - ' i s p l o t t e d a g a i n s t (Rmo Rh) -1

-

.

231

- 3 - 2 - 1

0

1

2

3

4

5

F i g . 10.6. P l o t f o r d e t e r m i n a t i o n o f d i s s o c i a t i o n c o n s t a n t s f o r conA complexes M u r i n e AFP, m o l e c u l a r f o r m 1; w i t h human and m u r i n e AFP ( a - f o e t o p r o t e i n ) . 04, 0-0, m u r i n e AFP, m o l e c u l a r f o r m 2;0-n8, human AFP, m o l e c u l a r f o r m \1. The i n t e r c e p t w i t h t h e a b s c i s s a i s -K-1 b e e t e x t (ean. 10.1) f o r explanation].

t h e c-l a x i s i s -K-l.

- ~~1 -1

a x i s i s (Rmo - Rmc) The s l o p e o f t h e l i n e i s K/(RmO

The i n t e r c e p t on t h e (Rmo

-1 and t h e i n t e r c e p t on

- R,~).

The e q u a t i o n g i v e s t h e d i s s o c i a t i o n c o n s t a n t K as w e l l as t h e m o b i l i t y o f t h e c o n A - g l y c o p r o t e i n complex, Rmc ( c f . ,

F i g . 10.6). The values o f K and Rmc were

c a l c u l a t e d by t h e l e a s t - s q u a r e s method. The e q u a t i o n and i t s d e r i v a t i o n were subsequently v e r i f i e d and p u b l i s h e d b y H o r e j s i u s i n g o t h e r symbols ( H o r e j s i

64,65

I n t h e s i m p l e case where t h e m o b i l i t y o f t h e c o n A - g l y c o p r o t e i n complex i s z e r o (Rmc = 0 ) , eqn. 10.2 becomes eqn. 10.1). The c a l c u l a t i o n o f t h e d i s s o c i a t i o n c o n s t a n t deoends on t h e accuracy o f t h e d e t e r m i n a t i o n o f t h e l e c t i n c o n c e n t r a t i o n . T h i s a p p l i e s e s p e c i a l l y t o cases w i t h i m m o b i l i z e d l e c t i n , where t h e d e t e r m i n a t i o n o f bound l e c t i n p e r u n i t m a t r i x i s d i f f i c u l t . A c o m p l i c a t i n g f a c t o r i s t h e change i n b i n d i n g p r o p e r t i e s a f t e r i m m o b i l i z a t i o n . I n o u r p r e s e n t e x p e r i m e n t a l work t h e g r e a t e s t d i f f i c u l t y f o r s t a n d a r d i z a t i o n o f t h e s e methods f o r d i a g n o s t i c purposes seems t o b e t h e - i r r e p r o d u c i b i l i t y o f t h e l e c t i n preparation. T a b l e 10.2 l i s t s t h e d i s s o c i a t i o n c o n s t a n t s o f complexes o f conA and g l y c o o r o t e i n s i n human serum. Sets of d a t a were o b t a i n e d f r o m t h e same s e t s o f experiments. I n f a c t , f r o m one s e t o f experiments a s e t o f d a t a c o u l d be o b t a i n e d f o r each g l y c o p r o t e i n r e a c t i n g w i t h conA. When i n d i v i d u a l g l y c o p r o t e i n s f r o m i n d i v i d u a l human s e r a were compared, we found a c o n s i d e r a b l e and u n s y s t e m a t i c v a r i a t i o n i n t h e measured d i s s o c i a t i o n c o n s t a n t s f o r a l l b u t a few g l y c o p r o t e i n s . Among these, orosomucoid was surp r i s i n g l y c o n s t a n t , b o t h w i t h r e s p e c t t o i t s appearance as t h r e e components w i t h

).

232

F i g . 10.7. L e c t i n i n t e r a c t i o n immunoelectrophoresis o f human AFP ( l e f t ) and rnurine AFP ( r i g h t ) i n crossed affinoimmunoelectronhoresis w i t h d i f f e r e n t amounts o f conA added t o t h e f i r s t - d i m e n s i o n g e l . The amounts o f conA added were ( f r o m t h e t o p ) 0, 3.03 and 24.20 10-6 m o l / l . Note t h e number o f components d i f f e r e n t f r o m F i g . 10.5 (conA f r o m I B F ) .

-

233

a c o n s t a n t r a t i o ( W e l l s e t a1.60) and w i t h r e s p e c t t o d i s s o c i a t i o n c o n s t a n t s . When serum f r o m i n d i v i d u a l s l o w i n a - 1 - a n t i t r y p s i n was t e s t e d i n t h i s way, we observed t h e same p a t t e r n o f a m a j o r component and a m i n o r component w i t h t h e normal d i s s o c i a t i o n c o n s t a n t t o conA even though t h e a n t i t r y p s i n c o n c e n t r a t i o n 16 was as low as 1%o f t h e normal c o n c e n t r a t i o n (Bdg-Hansen and Takeo ) . U s i n g eqn. 10.2 we compared t h e d i s s o c i a t i o n c o n s t a n t s o f t h e two m o l e c u l a r forms of human AFP ( 0 and 1) and t h e t h r e e m o l e c u l a r forms o f m u r i n e AFP (0, 1 and 2). The r e a c t i o n o f t h e two m o l e c u l a r forms o f human AFP and t h e t h r e e mol e c u l a r forms o f m u r i n e AFP w i t h v a r i o u s amounts o f conA i s i l l u s t r a t e d i n F i g .

10.7. M i g r a t i o n d i s t a n c e s were determined as f i r s t - d i m e n s i o n m i g r a t i o n d i s t a n c e s f r o m t h e o r i g i n t o t h e p r o j e c t i o n o f t h e peak o f t h e p r e c i D i t a t e on t h e b a s e l i n e .

A l l o f t h e a n t i g e n samples were t e s t e d w i t h t h e same conA p r e p a r a t i o n u s i n g m o n o s p e c i f i c a n t i s e r u m t o t h e r e s p e c t i v e a - f o e t o p r o t e i n and p o l y v a l e n t a n t i s e r u n : which a l s o r e a c t e d w i t h albumin. The d i s s o c i a t i o n c o n s t a n t s were d e r i v e d f r o m t h e p l o t shown i n F i g . 10.6. The d i s s o c i a t i o n c o n s t a n t o f t h e conA-human AFP f o r m 1 complex was 3.9 The d i s s o c i a t i o n c o n s t a n t o f t h e conA-murine AFP f o r m 1 c o m l e x was 1.1

-

mol/l.

m o l / l and t h e d i s s o c i a t i o n c o n s t a n t o f conA-murine AFP f o r m 2 complex was 5.1

.

m o l / l . The c o r r e l a t i o n c o e f f i c i e n t s o f t h e s t r a i g h t l i n e s i n t h e p l o t were i n t h e range 0.97-1.00.

The electrophoretic conditions The e l e c t r o p h o r e t i c parameters were v a r i e d w i t h t h e c o n c l u s i o n t h a t t h e b e s t c o n d i t i o n s a r e t h o s e which a r e o p t i m a l f o r electroimmunoprecipitation.

I n a way

t h i s i s a l i m i t a t i o n t o t h e g e n e r a l use o f t h e method, as t h e v a r i a t i o n o f t h e d i s s o c i a t i o n c o n s t a n t as a f u n c t i o n o f pH and t e m p e r a t u r e can be s t u d i e d o n l y w i t h i n narrow l i m i t s . R o u t i n e l y , immunoelectroprecipitation i s performed i n t h e pH range 8.5-8.9 and i n t h e t e m p e r a t u r e range 16-18OC; e x c e D t i o n a l l y , t h e pH can be chosen as l o w as 5.5 and as h i g h as 9.2, and t h e t e m o e r a t u r e may be f r o m 10 t o 3OoC. Only s m a l l changes i n t h e d i s s o c i a t i o n c o n s t a n t s were observed i n t h e narrow i n t e r v a l s . I t was o u t s i d e t h e scope o f t h i s work t o t e s t extreme c o n d i t i o n s o r l e c t i n s w i t h o t h e r m o b i l i t y p r o p e r t i e s . I m m o b i l i z e d l e c t i n (Sepharosebound conA) was found t o i n f l u e n c e t h e e l e c t r o p h o r e t i c p r o p e r t i e s o f t h e f i r s t dimension g e l . I n a d d i t i o n , c o n t r o l o f t h e b i n d i n g s i t e c o n c e n t r a t i o n was d i f f i c u l t w i t h i m m o b i l i z e d l e c t i n . T h e r e f o r e , f r e e l e c t i n was used i n t h e s e e x p e r i ments as more c o n v e n i e n t and s a t i s f a c t o r y . P r o v i d e d t h a t t h e complex has a l o w e r m o b i l i t y t h a n t h e f r e e o r o t e i n , a h i g h e r a f f i n i t y means a h i g h e r degree o f r e t a r d a t i o n . Q u a l i t a t i v e l y , no d i f f e r e n c e s were found between a n a l y t i c a l a f f i n i t y e l e c t r o p h o r e s i s and a f f i n i t y chromatography (BCg-Hansen2 and K e r c k a e r t a t a l . 4 8 ) . We d i d n o t compare o u r p r e s e n t r e -

234

.2-

I.

1

2

3

PI se

F i g . 10.8. D e v i a t i o n o f t h e r e l a t i v e m o b i l i t y o f a p u r e p r o t e i n w i t h i n c r e a s i n g amounts o f c o n t a m i n a t i n g g l y c o p r o t e i n s : Ro-Rm o f p u r e m u r i n e serum c a r b o x y l i c e s t e r a s e component 1 (CxE-1, l e s s t h a n 1 ng p e r t e s t ) as a f u n c t i o n o f added 10-4 m o l / l ) . serum (0-150 u g p e r t e s t ) a t a f i x e d conA c o n c e n t r a t i o n ( 6

-

sul t s experimentally w i t h determinations o f d i s s o c i a t i o n constants by e i t h e r i n h i b i t i o n p r e c i p i t a t i o n , f r o n t a l a n a l y s i s , e l u t i o n a n a l y s i s o r any o t h e r method. The i n f l u e n c e o f o t h e r g l y c o p r o t e i n s was t e s t e d by comparison o f t h e o a t t e r n o f orosomucoid observed w i t h serum w i t h t h a t observed w i t h D u r i f i e d orosomucoid. The same three-peak p r e c i p i t a t e was observed i n b o t h i n s t a n c e s , which i n d i c a t e d t h a t t h e i n f l u e n c e o f competing g l y c o p r o t e i n s on t h e l e c t i n b i n d i n g i s n e g l i g i b under o u r s t a n d a r d c o n d i t i o n s w i t h a g l y c o p r o t e i n amount o f a b o u t 50 ng p e r ana 37 ) . However, we wondered whether o u r c o n d i t i o n s were i d e a l and

y s i s (Bdg-Hansen

c o u l d be compared w i t h c o n d i t i o n s w i t h e x t r e m e l y s m a l l amounts o f g l y c o p r o t e i n s T h e r e f o r e , we t e s t e d o u r systems w i t h a m i n o r c o n s t i t u e n t o f mouse serum, t h e a - c a r b o x y l e s t e r a s e , which s t a i n s w i t h a h i s t o c h e m i c a l s t a i n o n l y and i s presen i n an amount o f l e s s t h a n 1 ng i n t h e t e s t . W i t h t h i s o u r i f i e d enzyme ( F i g . 10.8) we c o u l d show o n l y small d e v i a t i o n s ( < l o % ) o f r e l a t i v e m o b i l i t i e s and d i s s o c i a t i o n c o n s t a n t s w i t h g l y c o p r o t e i n amounts f r o m l e s s t h a n 1 u g t o more t h a n 150 ug p e r t e s t . T h e r e f o r e , we concluded t h a t t h e i n f l u e n c e f r o m o t h e r g l y c o p r o t e i n s i s n e g l i g i b l e p r o v i d e d t h a t an excess o f l i g a n d (conA) i s p r e s e n t (Bdg-Hansen e t a l .

38,66

).

I t s h o u l d be n o t e d t h a t t h e c o n c e n t r a t i o n o f l i o a n d i s c a l c u l a t e d as t h e monomer

c o n c e n t r a t i o n and i t was n o t p o s s i b l e t o e s t i m a t e t h e e f f e c t o f t h e t e t r a m e r i c n a t u r e o f conA.

Evaluation of the dissociation Constants The method d e s c r i b e d h e r e f o r t h e d e t e r m i n a t i o n o f d i s s o c i a t i o n c o n s t a n t s i s a two-dimensional method t h a t a l l o w s t h e a n a l y s i s o f more complex systems because o f t h e s p e c i f i c i d e n t i f i c a t i o n o f components b y t h e i r immunological c h a r a c t e r i s t i c s . However, compared w i t h f r o n t a l o r e l u t i o n a n a l y s i s a f f i n i t y chromatography, i t i s a drawback t h a t t h e e l e c t r o p h o r e t i c method r e q u i r e s a number o f analyses t o be p e r -

235

formed a t w e l l determined l i g a n d c o n c e n t r a t i o n s . On t h e o t h e r hand, the e l e c trophoreses a r e s i m p l e t o perform and r e q u i r e o n l y a minimum o f m a t e r i a l f o r t h e d e t e r m i n a t i o n and t h e l i g a n d need n o t be immobilized. The values measured f o r d i s s o c i a t i o n constants o f g l y c o p r o t e i n s w i t h conA by o u r method a r e o f t h e o r d e r o f

m o l / l (Table 10.2). T h i s agrees w e l l w i t h

o t h e r measurements o f human serum g l y c o p r o t e i n s . The d i s s o c i a t i o n constants found i n a p r e c i p i t a t i o n i n h i b i t i o n t e s t were 0 . 4

-

mol/l f o r p u r i f i e d transferrin,

m o l / l f o r t r a n s f e r r i n glycopeDtide and

0.7 Leon

67

m o l / l f o r IgM (Young and

). The e a r l i e r s t u d i e s w i t h one-dimensional e l e c t r o p h o r e s i s o f enzymes and

immobile s u b s t r a t e s i n t h e medium y i e l d e d d i s s o c i a t i o n constants i n t h e f o l l o w i n g ranges: 6.1

*

-

27

and N a k a m ~ r a ~and ~ ) 2.5 p l e x (Takeo e t a1.68).

m o l / l f o r phosphorylase-glycogen complexes (Takeo

lo-‘ -

2.6

g/ml f o r amylase-soluble s t a r c h com-

S i m i l a r l y , B l u e Dextran has been used i n t h e medium and mol/l f o r im16

d i s s o c i a t i o n constants were obtained i n t h e range 0.4

-

-

-

m o b i l i z e d dye-complexes w i t h bovine h e a r t l a c t a t e dehydrogenase isoenzymes, 1.3

3

-

m o l / l f o r soybean l a c t a t e dehydrogenase isoenzymes, 2.2-7.9

f o r rape seed a l c o h o l dehydrogenase, 2.2 f o r pyruvate kinase and 8.6

m o l / l and 1.8

isoenzymes (Ticha e t a l . 6 9 ) . Analogously,

mol/l

m o l / l f o r a l d o l a s e , 2.6

mol/l

m o l / l f o r c r e a t i n e kinase

immobilized saccharides have been i n -

corporated i n t h e medium i n one-dimensional e l e c t r o p h o r e s i s t o g i v e d i s s o c i a t i o n constants f o r l e c t i n s i n t h e range 0.2-20 71 r e j s i e t al. ).

. loF4

m o l / l (Hauser e t a1.70 and Ho-

The development o f an easy method f o r d e t e r m i n a t i o n o f t h e a f f i n i t y o f a n t i bodies has h i g h p r i o r i t y . Thus Caron e t a l . 7 2 s t u d i e d D o s s i b i l i t i e s f o r assessing a n t i - a l b u m i n a f f i n i t y i n agarose a f f i n i t y e l e c t r o p h o r e s i s by d e t e r m i n a t i o n of t h e “ p a r t i t i o n i n g c o e f f i c i e n t “ , Kpy between immunosorbent and s o l u b l e a n t i g e n o r antibody. However, a more d i r e c t apDroach f o r a n t i b o d i e s was used by Takeo and Kabat73, i n analogy w i t h t h e o r i g i n a l work on enzymes: A s s o c i a t i o n constants were measured f o r mouse myeloma p r o t e i n s w i t h a n t i d e x t r a n o r a n t i - i s o m a l t o s e o l i g o saccharide a c t i v i t y by e l e c t r o D h o r e s i s i n polyacrylamide g e l s c o n t a i n i n g d e x t r a n 2 4 4 (K, = 3 10 - 6 10 ml/g) o r isomaltose oligosaccharides (K, = 8.3 10 m o l / l ) .

-

.

I t was concluded t h a t a f f i n i t y e l e c t r o o h o r e s i s i s u s e f u l f o r o b t a i n i n g b i n d i n g 2 6 constants r a n g i n g from 10 t o 10 l / m o l .

JUANTITATION OF MICROHETEROGENEITY FORMS The h e t e r o g e n e i t y o f g l y c o p r o t e i n s i n t h e i r r e a c t i o n w i t h conA, as observed w i t h b o t h these methods and o t h e r methods, may now be a s c r i b e d t o t h e e x i s t e n c e o f oopulations o f m i c r o h e t e r o g e n e i t y forms, each p o p u l a t i o n w i t h i t s c h a r a c t e r i s t i c carbohydrate s t r u c t u r e , p o s s i b l y r e f l e c t i n g d i f f e r e n t m e t a b o l i c stages r e l a t e d t o

236

237

238

Fig. 10.9. Q u a n t i f i c a t i o n of microheterogeneous forms o f individual glycoproteins. Crossed immunoelectrophoresis of concentrated human urine ( A ) , with insolubilized conA ( B ) and with f r e e conA i n t h e intermediate gel ( C ) . The r e a l t i v e amounts of glycoprotein with one, with twb o r more and without conA-binding s i t e s were determined by planimetry o f the immunoprecipitates ( s e e t e x t and Table 1 0 . 3 ) .

239 t h e f u n c t i o n o f t h e p r o t e i n . Therefore, i t seems i m p o r t a n t t o develop methods t o assess t h e q u a n t i t a t i v e changes t h a t take p l a c e i n v i v o f o r these microheterogenei t y forms.

&antitation b y t h e intermediate g el technique, Method B P r e c i p i t a t i o n w i t h f r e e l e c t i n versus b i n d i n g t o immobilized l e c t i n forms a b a s i s f o r a c l a s s i f i c a t i o n o f the m i c r o h e t e r o g e n e i t y forms o f a g l y c o p r o t e i n i n t o t h r e e classes: Type 0 (molecules w i t h o u t b i n d i n g s i t e s ) , Type 1 (molecules w i t h one b i n d i n g s i t e ) and Type 2 (molecules w i t h two o r more b i n d i n g s i t e s ) . F i g . 10.9 shows t h e experimental b a s i s f o r q u a n t i f i c a t i o n o f each o f t h e t h r e e types o f microheterogenei t y form mentioned above o f a number o f microheterogeneous g l y c o p r o t e i n s i n human u r i n e . Three d i f f e r e n t experiments were performed w i t h intermediate gels: A without l e c t i n i n the intermediate gel,

B with free lectin

i n t h e . i n t e r m e d i a t e g e l and C w i t h immobilized l e c t i n i n t h e i n t e r m e d i a t e g e l . By c a r e f u l p l a n i m e t r y o f t h e enclosed area o f each p r e c i p i t a t e , a r e l a t i v e e s t i m a t e

was obtained f o r t h e t o t a l amount o f each g l y c o p r o t e i n (A), t h e amount o f glycop r o t e i n o f Type 2 (B) and t h e amount o f g l y c o ? r o t e i n o f Type l p l u s Type 2 ( C ) . 'Table 10.3 shows t h e r e l a t i v e d i s t r i b u t i o n between t h e v a r i o u s m i c r o h e t e r o g e n e i t y classes o f 18 u r i n a r y g l y c o p r o t e i n s . TABLE 10.3 HETEROGENEITY OF URINARY GLYCOPROTEINS R e l a t i v e amount o f p r o t e i n i n each m i c r o h e t e r o g e n e i t y c l a s s f o r 13 human u r i n e p r o t e i n s . Note t h a t o n l y one p r o t e i n appears t o be homogeneous ( a d i c phosphatase); albumin appears t o be g l y c o s y l a t e d ( 1 0 % ) . Most u r i n a r y p r o t e i n s a r e p r e s e n t i n a t l e a s t t h r e e d i f f e r e n t g l y c o s y l a t i o n forms. P r o t e i n component

R e l a t i v e amount o f each h e t e r o g e n e i t y c l a s s (%) Without binding s i t e

u1 U2 = albumin u3 u4 u5 U6 u7 U8 u9 u 10 u11 u12 U13 = a c i d phosphatase

75 90 70 15 35

ao

35 75 50 30 50 55 0

With one binding s i t e

With two o r more binding s i t e s

5 5 0 45 20 10 40 0 15 15 25 10

20 30 40 45 10 25 25 35 55 25 35

100

0

5

240

Quantitation by the f i r s t dimension gel technique, Method C By t h e f i r s t - d i m e n s i o n t e c h n i q u e we d i s t i n g u i s h between m i c r o h e t e r o g e n e i t y forms d i f f e r i n g i n t h e i r a f f i n i t y t o t h e l e c t i n . the r a t i o o f f o u r d i s t i n c t m i c r o h e t e r o g e n e i t y components o f m-AFP i s s h i f t e d i n pregnancy and t h e percentage by which each o f t h e f o u r m o l e c u l a r forms o f m-AFP c o n t r i b u t e t o t h e t o t a l m-AFP l e v e l i n f o e t a l and j u v e n i l e homogenate i s shown i n F i g . 10.10 (Hau and co-workers 46,47 ) . I n 1'2-day-old f o e t u s e s t h e two forms w i t h weakest a f f i n i t y t o conA ( f o r m s 0 and 1) dominate, whereas i n homogenate o f j u v e n i ' l e s t h e two forms w i t h s t r o n g e s t a f f i n i t y t o conA ( f o r m s 2 and 3 ) dominate. A t t h e t i m e o f b i r t h a l l f o u r forms o f m-AFP a r e p r e s e n t and t h e y c o n t r i b u t e a p p r o x i m a t e l y 25% each t o t h e t o t a l AFP l e v e l . T h i s i s p a r a l l e l t o t h e t h r e e forms o f orosomucoid which a r e p r e s e n t n o r m a l l y i n a w e l l defined r a t i o , b u t s h i f t e d d u r i n g pregnancy and d u r i n g t r e a t m e n t w i t h hormone-like

50

12 13 14 15 16 17 18 19 0 1 2 3 4 5

7

10

0%

----.

F i g . 10.10. Q u a n t i f i c a t i o n o f microheterogeneous forms o f i n d i v i d u a l g l ! x o p r o t e i n s . ? l o t o f t h e r e l a t i v e amounts o f f o u r m o l e c u l a r forms o f murine AFP f r o m 12-day mof o e t u s e s t o 10-day j u v e n i l e s . 0---o M o l e c u l a r f o r m 0 ( n o n - b i n d i n g ) ; l ] --l e c u l a r form 1 ( s l i g h t binding); o - - - ~ molecular form 2 (intermediate binding); m o l e c u l a r f o r m 3 ( s t r o n g e s t b i n d i n g ) . Each p o i n t r e p r e s e n t s t h e mean v a l u e f o r 10 samples. I n s i d e t h e p o i n t s i s +1 s.e.m. ( c f . , F i g . 10.5). drugs. However, t h e i r b i n d i n g p r o p e r t i e s t o conA a r e unchanged ( W e l l s and co60,61

workers

).

As mentioned above, t h e number o f m i c r o h e t e r o g e n e i t y forms seems t o depend on t h e l e c t i n p r e p a r a t i o n . Here f o u r forms o f m-AFP were seen w i t h conA f r o m Pharmacia i n c o n t r a s t t o o n l y t h r e e forms w i t h conA f r o m IBF (compare F i g s . 10.7 and 10.10). The reason f o r t h i s i n c o n s i s t e n c y i s n o t c l e a r , b u t may l i e i n d i f f e r e n c e s i n t h e b i n d i n g s o e c i f i c i t i e s o f v a r i o u s commercial l e c t i n s orepared f r o m d i f f e r e n t

241 l i n e s o r c u l t i v a r s o f t h e same p l a n t , as such d i f f e r e n c e s a r e known t o o c c u r 13 (B&-Hansen ) . T h i s i n d i c a t e s t h a t c a r e s h o u l d be t a k e n t o s o e c i f y t h e s o u r c e o f l e c t i n when r e p o r t i n g q u a n t i t a t i v e s t u d i e s . OTHER ELECTROPHORETIC METHODS I n t h e i n t r o d u c t i o n we mentioned t h e h i s t o c h e m i s t r y - l i k e use o f l a b e l l e d l e c t i n s f o r t h e i d e n t i f i c a t i o n o f g l y c o p r o t e i n bands a f t e r e l e c t r o p h o r e s i s . T h i s appears t o be a v e r y s e n s i t i v e means f o r t h e i d e n t i f i c a t i o n o f g l y c o p r o t e i n bands a f t e r a c r y l a m i d e g e l e l e c t r o p h o r e s i s and i s a good supplement t o t h e mentioned t e c h n o l o g y o r f o r t h e p r e l i m i n a r y s c r e e n i n g f o r g l y c o p r o t e i n s . The methodology and r e f e r e n c e s were g i v e n by B i t t i g e r and S c h n e b l i

6

.

Se Zection e Zectrophoresis F o r c e r t a i n purposes i t i s n o t necessary o r d e s i r a b l e t o o e r f o r m t h e e l e c t r o p h o r e s i s as c r o s s e d i m m u n o e l e c t r o p h o r e s i s . S e l e c t i o n e l e c t r o p h o r e s i s i s a s i m p l e r method: a c o m b i n a t i o n o f one-dimensional e l e c t r o p h o r e s i s i n agarose w i t h subsequent d i f f u s i o n o f l e c t i n f r o m a t r o u g h , analogous t o t h e c l a s s i c a l immuno74 e l e c t r o p h o r e t i c a n a l y s i s a c c o r d i n g t o Grabar, c f . , F i g . 10.11 (Harboe e t a l . , Spengler and Weber9’75). The method has n o t been used e x t e n s i v e l y as t h e p a r e n t method w i t h a n t i b o d i e s . However, t h e g r e a t o o t e n t i a l o f g l y c o p r o t e i n a n a l y s i s w i t h l e c t i n s was shown b y Osunkoya and W i l l i a m s 8 i n t h e i r work w i t h human serum g l y c o p r o t e i n s f r o m p a t i e n t s w i t h v a r i o u s d i s e a s e s . F i g . 10.11 shows t h e p r e c i p i t a t i o n p a t t e r n s o f p u r i f i e d human 19s IgM and 7s kappa c h a i n s w i t h a n t i b o d i e s and four different lectins.

One-dimensionaZ a f f i n i t y eZectrophoresis Another example t h a t a r e f e r e n c e p a t t e r n o f c r o s s e d i m m u n o e l e c t r o p h o r e s i s i s n o t necessary i s p r o v i d e d by one-dimensional

a f f i n i t y e l e c t r o p h o r e s i s . Thus, f o r

the i d e n t i f i c a t i o n o f binding t o l e c t i n o r f o r the i d e n t i f i c a t i o n o f precipitat i o n w i t h l e c t i n , e l e c t r o p h o r e s i s may be p e r f o r m e d i n t o a g e l w i t h e i t h e r i m m o b i l i z e d l e c t i n o r f r e e l e c t i n , r e s p e c t i v e l y . T h i s was shown e a r l i e r f o r enzymes f r o m b a r l e y (Bdg-Hansen e t a 1 . l 7 ) . The method was a l s o used f o r t h e q u a n t i t a t i o n o f denatured g l y c o p r o t e i n s . We f o u n d t h a t d e n a t u r a t i o n d i d n o t i n t e r f e r e w i t h t h e s t r u c t u r e o f t h e b i n d i n g s i t e , and g l y c o p r o t e i n s denatured t o v a r i o u s e x t e n t s gave a f f i n i t y p r e c i p i t a t e s o f t h e same h e i g h t (Bfig-Hansen a t a1.l’).

F i g . 10.12a

shows g l y c o p r o t e i n enzymes f r o m a p l a n t e x t r a c t r e a c t i n g w i t h conA i n t h e g e l . F i g . 10.12b shows t h e r e v e r s e d t y p e one-dimensional a f f i n i t y e l e c t r o p h o r e s i s . The g e l c o n t a i n e d g l y c o p r o t e i n s (human serum) and e x t r a c t s o f v a r i o u s p l a n t sources were e l e c t r o p h o r e s e d i p t r t h e g l y c o p r o t e i n - c o n t a i n i n g g e l w i t h f o r m a t i o n

242

Fig. 10.11. Selection electrophoresis with lectins, an analogous method to the

243

Fic. 10.12. One-dimensional a f f i n i t y electrophoresis f o r q u a n t i f i c a t i o n and screening. ( a ) Glycoproteins electroDhoresea i n t o a conA-containing g e l : a plant enzyme e x t r a c t ( s t a i n e d f o r acid phosohatasej a n d p u r i f i e d human serum choline s t e r a s e ( s t a i n e d f o r e s t e r a s e ) . ( b ) Screening f o r l e c t i n a c t i v i t y : plant e x t r a c t s electrophoresed i n t o a gel containing human serum glycooroteins (from l e f t : ext r a c t of barley malted seeds, two d i f f e r e n t e x t r a c t s from UZex europaeus, t h r e e d i f f e r e n t e x t r a c t s of ooke weed, an e x t r a c t from soybeans and p u r i f i e d r i c e seed lectin). of a f f i n i t y p r e c i p i t a t e s as t h e r e s u l t . This tyoe of exoeriment seems t o be a f a s t screening method f o r g l y c o p r o t e i n - o r e c i p i t a t i n g l e c t i n s as well as f o r t h e i r 15 quanti t a t i o n (Bdg-Hansen and liord ) . Compound ge Z method: po Zyacry lamide and agarose

Fig. 10.12 shows how SDS-polyacrylamide gel electrophoresis of denatured glycoproteins can be combined with l e c t i n a f f i n i t y e l e c t r o p h o r e s i s . The protein sample was conA-binding glycoproteins from normal human serum purified by a f f i n i t y chromatography on conA-Sepharose. The samples were t r e a t e d with SDS before applicat i o n t o t h e SDS-polyacrylamide gel s l a b s . After separation i n t h e SDS g e l , lanes were c u t and soaked f o r 15 min in the agarose gel buffer o r stored frozen. The

c l a s s i c a l immunoelectrophoretic analysis according t o Grabar. The p r e c i p i t a t i o n pattern of purified human 19s IgM and 7 s kappa chains with antibodies (anti-IgM) and l e c t i n s (PHA, lens c u l i n a r i s l e c t i n , conA, a n d r i c i n ) i s shown. Reproduced with permission from G . A . Spengler, Bern.

h3

P P

TABLE 10.4 VARIOUS MODIFICATIONS OF AFFINITY ELECTROPHORESIS WTH INTERACTING CONPONENTS AND SOME APPLICATIONS

M o d i f i e d from Bdg-Hansen e t a1 .20. Conventional immunoelectrophoretic methods n o t included. No.

Modification

I n t e r a c t i n g cornponent which i s e l ectroDhoresed

Interacting component i n t h e medium

Purpose

Ref.

1.

Selection electrophoresis analogous = a f f i n i t y electrophoresis analogous t o immunoelectrophoret i c a n a l y s i s according t o Grabar

Glycoproteins

Lectinsa

Identification o f 1e c t in- b i n d i ng glycoproteins

Spengler and Weber75 Harboe e t a1 .74

2.

Crossing diagramsb

Enzyme

Substrate i n h i b i tor

Identification of i n t e r a c t i n g components

Nakamural

3.

One-dimensional a f f i n i t y electrophoresisb

4.

One-di mens ional a f f in it y electrophoresis

Glycoproteins

Lectin

Lectin

Carbohydrate

Identification of lectin

H o r e j s i and Kocourek3

Enzyme

Substrates

Determination o f d i s s o c i a t i o n constants

Takeo and N a k a ~ n u r a ~ ~

A n t i bodies

A n t i gens

Glycoprotein enzymes

Immobilized lectin

Takeo and Kabat73 Identification o f g l y c o p r o t e i n enzymes

Bdg-Hansen e t a1 .I7

5.

Rocket a f f i n i t y electrophoresis

Lectins

Glycoproteins

Polysaccharides

Lectins

G l y c o p r o t e i ns

Lectins

6.

Fused r o c k e t a f f in i ty electrophoresis

Glycoproteins

Lectins

7.

Crossed a f f i n i t y electrophoresis

Lectin

Glycoproteins

8. Crossed immunoelectroPhOreSiS w i t h j i g a n d i n an i n t e r m e d i a t e Ele 1

Screening f o r 1e c t ins Quantification o f polysaccharides Quantification o f g l y c o p r o t e i ns

Bdg-Hansen and Nordl5 Owen and Sal ton84 Bdg-Hansen e t al.20

Analysis o f Bdg-Hansen e t al.20 (1) f r a c t i o n a t i o n ( 2 ) p r o g r e s s i v e changes ( 3 ) treatments Identification o f

Bdg-Hansen and Nordl5

1e c t i ns Polysaccharides

Lectins

Glycoproteins

Lectins

G l y c o p r o t e i ns

Lectins

Lectins

Glycoproteins

Immobilized a f f i n 1t i n s

Biologically active proteins

Identification o f i n t e r a c t i n g components I d e n t i f i c a t i o n and quantification o f l e c t i n-bi n d i n g g l y c o p r o t e i ns

Owen and Salton84 Owen e t a1.85

(1) I d e n t i f i c a t i o n o f interacting components ( 2 ) P a r t i a l characterization of number o f b i n d i n g sites (3) Prediction o f s e p a r a t i o n experiments

Bdg-Hansen*

Bdg-Hansen e t al.20

Bdg-Hansen and Brogrenl4

Bdg-Hansen2 Ramlau and Bock31

(Continued on p. 246)

TABLE 10.4

(continued)

No.

Modification

I n t e r a c t i n g cornponent which i s electrophoresed

Interacting component i n t h e medium

Purpose

9.

Crossed immunoelectrophoresis w i t h ligand i n the f i r s t dimension gel

Glycoprotei ns

Lectins

(1) I d e n t i f i c a BBg-Hansen e t a1 33 tion of intera c t i n g components

L e c t i n s and Glycoproteins

Ref.

( 2 ) Determination b i n d i ng speci f ic t y ( 3 ) Analysis o f BBg-Hansen e t a1 33 m i croheterogenei t y ( 4 ) Determination o f BBg-Hansen and Takeo16 dissociation constants N i l s s o n and Bdg-HansenSl (5) Prediction o f s e p a r a t i o n exper iment s

i L e c t i n a p p l i e d i n a trough b e f o r e d i f f u s i o n . Agarose gel was used as s u p p o r t i n g medium except i n 2' (paper) and 3 (polyacrylamide g e l ) .

247

F i g . 10.13. Combined S D S - p o l y a c r y l a m i d e g e l e l e c t r o o h o r e s i s and a f f i n i t y e l e c t r o p h o r e s i s o f 1 mg o f human serum g l y c o p r o t e i n . The l o w e r D a r t o f t h e p l a t e i s a T r i t o n X - 1 0 0 - c o n t a i n i n g a g a r o s e g e l ( 5 % ) . The w h i t e a r e a w i t h i n t h i s g e l shows where t h e SDS g e l was o v e r l a y e r e d w i t h t h e s e n a r a t e d p r o t e i n s , anode t o t h e r i g h t . 10-6 m o l / i ) . F o r t u r r h e r d e t a i l s , The u p p e r g e l was a g a r o s e c o n t a i n i n g conA (1.6 see t e x t and B d g - H a n ~ e n ~ ~ .

-

s e c o n d - d i m e n s i o n e l e c t r o p h o r e s i s was p e r f o r m e d i n a g a r o s e w i t h n o n - i o n i c d e t e r G e n t ( s e e F i g . 10.13). As t h e p r e c i p i t a t e f o r m a t i o n i s d e p e n d e n t on t h e amount o f 7' g l y c o p r o t e i n and t h e amount o f l e c t i n a p o l i e d (Bfig-Hansen " ) t h i s method may a l s o b e u s e d q u a n t i t a t i v e l y f o r t h e d e t e r m i n a t i o n o f t h e amount o f S l y c o p r o t e i n app l i e d o r t h e amount o f l e c t i n - b i n d i n g g l y c o p r o t e i n i n each band. i t i s r e m a r k a b l e t h a t t h e SDS i n t h e a c r y l a m i d e g e l does n o t d i s t u r b t h e b i n d -

i n g t o t h e l e c t i n . T h i s i s p r e s u m a b l y due t o t h e n e u t r a l i z i n g e f f e c t o f T r i t o n b y i n c l u s i o n o f SDS i n t o m i c e l l e s , s o t h a t t h e l e c t i n i s n o t e x p o s e d t o f r e e SDS. 3n t h e o t h e r hand, l e c t i n s a r e known t o b e s t a b l e and r e a c t i v e i n t h e p r e s e n c e

o f d e t e r g e n t ( s e e , f o r i n s t a n c e , B j e r r u m e t a 1 . 7 7 ) . E a r l i e r we showed t h a t den a t u r a t i o n o f t h e g l y c o p r o t e i n has l i t t l e e f f e c t on t h e b i n d i n g t o l e c t i n ( c o n A ) , w h e t h e r d e n a t u r a t i o n was p e r f o r m e d w i t h d e t e r g e n t , h e a t o r a c i d t r e a t m e n t , and t h a t t h e p r e c i p i t a t i o n i n a f f i n i t y p r e c i p i t a t e s was q u a n t i t a t i v e i r r e s p e c t i v e o f t h e d e g r e e o f d e n a t u r a t i o n (Bdg-Hansen e t a l .

19

).

Preparative a j - f i n i t y electrophoresis One o f t h e m a i n d i f f i c u l t i e s i n o r e p a r a t i v e a f f i n i t y c h r o m a t o g r a p h y i s t h a t t h e a c t i v e component s t i c k s t o o w e l l t o t h e i m m o b i l i z e d l i g a n d . I n o r d e r t o o v e r -

248

come t h i s problem, Dean e t a1 .73 used electrophoreticdesorption o f t h e a f f i n i t y column a f t e r l o a d i n g and washing. E a r l i e r we performed a few e x p l o r a t o r y experiments on t h e p o s s i b l e p r e p a r a t i v e use o f t h e p r i n c i p l e o f a f f i n i t y e l e c t r o p h o r e s i s when we performed p r e p a r a t i v e i s o t a c h o p h o r e s i s i n a c r y l a m i d e g e l w i t h conA, b u t we d i d n o t c o n t i n u e t h i s approach because t h e a n a l y t i c a l aspects appeared more p r o m i s i n g (Bplg-Hansen e t a l .

18

BIOMEDICAL APPLICATIONS The a n a l y t i c a l e l e c t r o p h o r e t i c approach seems t o be an a l t e r n a t i v e t o preparat i v e methods and may be used when o n l y a s m a l l amount o f b i o l o g i c a l specimen i s a v a i l a b l e i n s t e a d o f t h e more m a t e r i a l - r e q u i r i n g column and b a t c h procedures. T h e r e f o r e , i t c o u l d be used r o u t i n e l y f o r d i a g n o s i s and c h e c k i n g o f human d i s eases.

Diagnosis of malignant diseases and neural tube defects As s e v e r a l i m p o r t a n t marker p r o t e i n s i n human diseases a r e g l y c o p r o t e i n s , t h e i r i n t e r a c t i o n w i t h l e c t i n s may g i v e i m p o r t a n t c l u e s about t h e s t a t e o f t h e disease. a - F o e t o p r o t e i n (AFP) i s one such p r o t e i n . A m n i o t i c f l u i d AFP f r o m normal pregnancies between 15 and 33.5 weeks o f pregnancy c o n s i s t s o f 12-45% o f t h e 79 conA n o n - r e a c t i v e f o r m ( S m i t h and K e l l e h e r ) , whereas f o e t a l serum has been r e p o r t e d t o c o n t a i n a s i g n i f i c a n t l y l o w e r o r o p o r t i o n o f conA n o n - r e a c t i v e v a r i a n t s 79 (2-10%) ( R u o s l a h t i e t al."), Smith and K e l l e h e r ) . T h i s l e d t o t h e d e m o n s t r a t i o n t h a t t h e p a t t e r n o f c o n A - a f f i n i t y v a r i a n t s i n a m n i o t i c f l u i d seems t o be v a l u a b l e i n d i a g n o s i n g f o e t a l a b n o r m a l i t i e s (Smith e t a1 .81).

I n f o e t a l a b n o r m a l i t i e s , c h a r a c t e r i z e d by a change i n AFP compartmentalizat i o n , such as t r a n s u d a t i o n o f f o e t a l serum across exposed f o e t a l membranes i n t h e presence o f n e u r a l t u b e d e f e c t s , a s h i f t i n t h e o a t t e r n o f a m n i o t i c f l u i d AFP c o n A - a f f i n i t y v a r i a n t s t o resemble t h a t o f f o e t a l serum, has been observed 79 (Smith e t al.81, Smith and K e l l e h e r ) . The measurement o f c o n k a f f i n i t y v a r i a n t s i n a m n i o t i c f l u i d has r e c e n t l y been performed u s i n g a f f i n i t y e l e c t r o p h o r e s i s w i t h conA i n c l u d e d i n t h e f i r s t - d i m e n s i o n g e l (Hinderson e t a1

.**, Ndrgaard-Pedersen

.

e t a1 52, T o f t a g e r - L a r s e n and NfJrgaard-

P e d e r ~ e n ~ ~T h) i. s method has been found t o b e more s i m p l e and r e l i a b l e t h a n t h e 82 ),

p r e v i o u s chromatographic t e c h n i q u e s (Hinderson e t a l .

The a f f i n i t y e l e c t r o p h o r e s i s system has a l s o been used i n t h e comparison o f 46 f o e t a l and hepatoma AFP l e c t i n - a f f i n i t y v a r i a n t p a t t e r n s ( K e r c k a e r t e t a l . , Breborowics and M a c k e w i c ~ ~ ~ The ) . p r o p o r t i o n o f t h e c o n A - a f f i n i t y v a r i a n t s seem t o be s i m i l a r i n hepatoma s e r a and s i m i l a r t o t h e p r o p o r t i o n i n f o e t a l sera.

).

249

However, t h e L C A - a f f i n i t y

p a t t e r n s v a r y among hepatoma sera, b u t g e n e r a l l y t h e

percentage o f t h e LCA-reactive v a r i a n t i s h i g h e r i n hepatoma s e r a t h a n i n f o e t a l 79 s e r a ( S m i t h and K e l l e h e r ).

Generalizations: o t h e r i n t e r a c t i n g systems By p e r f o r m i n g c r o s s e d immunoelectrophoresis w i t h h e p a r i n i n t h e f i r s t - d i m e n s i o n

g e l , B l e y l and P e i ~ h were l ~ ~a b l e t o s e p a r a t e a c t i v e a n t i t h r o m b i n f r o m t h e i n a c t i v e complex and t o measure i t q u a n t i t a t i v e l y i n p a t i e n t s under h e p a r i n t h e r a p y . T h i s example shows t h a t i t i s p o s s i b l e t o d i s t i n g u i s h between v a r i o u s m o l e c u l a r forms o f a c t i v e macromolecules a n a l o g o u s l y t o t h e m i c r o h e t e r o g e n e i t y forms o f g l y c o p r o t e i n s . A l s o , t h i s o f f e r s an o p p o r t u n i t y t o d i s t i n g u i s h between a c t i v a t o r c o n t r o l ( c o n t r o l by i n h i b i t o r s and a c t i v a t o r s and by a c t i v a t i o n o f p r e c u r s o r forms and i n a c t i v a t i o n by p a r t i a l d e g r a d a t i o n ) f r o m c o n t r o l by de novo s y n t h e s i s and t o t a l degradation. H o r e j s i6 4 y 6 5 p r e s e n t e d some t h e o r e t i c a l c o n s i d e r a t i o n s on t h e g e n e r a l q u a n t i t a t i v e use o f a f f i n i t y e l e c t r o p h o r e s i s , some g e n e r a l i z a t i o n s o f t h e p r e s e n t approach f o r t h e d e s c r i p t i o n o f r e c e p t o r f u n c t i o n s o f membrane p r o t e i n s were d i s cussed r e c e n t l y by B j e r r u m e t a1 .77 and Ramlau and Bock3’ d e s c r i b e d some o t h e r i n t e r a c t i n g systems.

ConcZuding remarks I n g e n e r a l , any macromolecular o r p a r t i c l e - b o u n d l i g a n d o r i n t e r a c t i n g subs t a n c e may be i n c l u d e d d u r i n g e l e c t r o p h o r e s i s t o S i v e a l i g a n d - i n d u c e d r e a c t i o n , such as a change o f t h e e l e c t r o o h o r e t i c m o b i l i t y o r a change o f t h e morDhology o r p r o f i l e o f t h e p r o t e i n . As seen f r o m some o f t h e examples mentioned above, t h e l i g a n d need n o t be charged. The main advantages o f t h e a n a l y t i c a l e l e c t r o p h o r e s i s approach appear t o be:

(1) I t can s e p a r a t e macromolecules t h a t i n t e r a c t w i t h a s p e c i f i c l i g a n d f r o m those t h a t do n o t . ( 2 ) I t can b e used f o r s t u d i e s o f i n t e r a c t i n g macromolecules. ( 3 ) I t i s n o t necessary t o p u r i f y i n t e r a c t i n g components. ( 4 ) A m u l t i t u d e o f p r o t e i n s r e a c t i n g w i t h t h e same l i g a n d may be s t u d i e d s i mu1taneous l y

.

( 5 ) I t can be g e n e r a l i z e d t o i n t e r a c t i o n s o t h e r t h a n t h o s e between l e c t i n s and g l y c o p r o t e i ns

.

( 6 ) I t can b e used f o r t h e p r e d i c t i o n o f p r e p a r a t i v e experiments. ACKNOWLEDGEMENTS !Is. P i a Jensen i s thanked f o r h e r s k i l f u l h e l p . The s t u d i e s were s u p p o r t e d by

t h e Danish Medical Research C o u n c i l .

250

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253

Chapter 11

LIPOPROTEINS H. PEETERS GENERAL ASPECTS

All methods used f o r plasma protein f r a c t i o n a t i o n and a n a l y s i s have been applied o r extended t o lipooroteins. However, l i p o p r o t e i n s made t h e i r appearance as a nuisance and remained i n t r a c t a b l e f o r a long time. Indeed, they generate t u r b i d i t y in serum and plasma, they cause t u r b i d i t y and an unreadable zone i n t h e f r e e T i s e l i u s e l e c t r o p h o r e s i s , they remain o a r t i a l l y on the a o o l i c a t i o n l i n e as a non-migrating p a r t i c l e during paper and gel e l e c t r o p h o r e s i s , they tend t o clog separation columns and a r e denatured a f t e r s a l t Drecipitation. From being a nuisance and a troublesome agent they became a source of informat i o n through ul t r a c e n t r i f u g a l f l o t a t i o n a n a l y s i s , and t h e lipoprotein p a t t e r n obtained a f t e r electrophoresis on a c a r r i e r became one of the r e a l l y p r a c t i c a l applications o f paper electrophoresis. With t h e develooment of o t h e r c a r r i e r s , such as g e l s , t h e evaluation of t h e bands became e a s i e r and 1iDoDrotein o r o f i l e s a r e a constant f e a t u r e in our range of methods aimed a t 1ipoDrotein disorders'". I t would be unfair t o f o r g e t t h a t f r a c t i o n s obtained by Cohn's p r e c i p i t a t i o n and, l a t e r , the pure u l t r a c e n t r i f u g a l f r a c t i o n s have been the main purveyors of objective information about the q u a l i t i e s of t h e l i p o p r o t e i n s . I t i s t h i s informat i o n which s u s t a i n s the importance of routine e l e c t r o p h o r e t i c liooprotein p r o f i l e s in c l i n i c a l chemistry and a c t s as a guide d u r i n g the i n t e r p r e t a t i o n of these oatterns. SYNTHETIC DATA ON LIPOPROTEINS The ul t r a c e n t r i f u g a l lipoprotein c l a s s e s a r e the reference data f o r the plasma l i p o p r o t e i n s . Fortunately, t h e r e i s a good correspondence between the basic u l t r a centrifugal pattern and the e l e c t r o p h o r e t i c p r o f i l e 3 , as summarized i n Table 11.1. Pre-6-lipoprotein precedes t h e 6-lipoprotein in t h e e l e c t r o p h o r e t i c p a t t e r n and i f t h i s f r a c t i o n i s increased the CM band a t the application l i n e often becbmes v i s i b l e a l s o . The appearance of a double 6-lipoprotein band can be due t o t h e presence o f bivalent metal ions such a s calcium and i s not a regular f e a t u r e of the normal p a t t e r n .

264

TABLE 11.1 CORRELATION BETWEEN ULTRACENTRIFUGAL AND ELECTROPHORETIC PROFILES Gravity ( f l o t a t i o n )

Electrophoresis = m o b i l i t y

Chyl omi c r a Very l o w d e n s i t y l i p o p r o t e i n Low d e n s i t y l i p o p r o t e i n High d e n s i t y l i p o p r o t e i n

Chyl omi c r a Pre-6-lipoprotein B-Lipoprotein a - L i p o p r o t e in

CM VLDL LDL HDL

none

++

i +i+

Through a n a l y s i s o f t h e u l t r a c e n t r i f u g a l f r a c t i o n s , t h e p r o t e i n and l i p i d comp o s i t i o n s o f t h e two l i p o p r o t e i n m o i e t i e s have been e s t a b l i s h e d . The l i o i d m o i e t y contains phospholipids, cholesterol

, cholesterol

e s t e r s and t r i g l y c e r i d e s i n d i f -

f e r e n t p r o p o r t i o n s a c c o r d i n g t o t h e p a r t i c l e considered. The p r o t e i n m o i e t y i s c a l l e d a p o p r o t e i n when i t i s l i p i d f r e e and i s composed o f s e v e r a l p o l y p e p t i d e s i n each d e n s i t y o r m o b i l i t y c l a s s . The a v a i l a b l e i n f o r m a t i o n 4 i s c o l l e c t e d i n T a b l e 11.2. TABLE 11.2 PERCENTAGE LIPOPROTEIN COMPOS,ITION Lioida

HDL LDL VLDL Lp-x

Protein

PL

C

CE

TG

50 26 10 6

23 20 15 66

15 9 10 22

7 37 6 3

5 8 59 3

aPL = p h o s p h o l i p i d , C = c h o l e s t e r o l , CE = c h o l e s t e r o l e s t e r , TG = t r i g l y c e r i d e s . These d a t a a r e n o t r i g i d b u t g i v e a f a i r e s t i m a t e o f o u r a c t u a l knowledge o f normal plasma l i p o p r o t e i n s . They a l s o i n c l u d e Lp-X, a l i p c j p r o t e i n t o be encount e r e d l a t e r under t h e t o p i c o f l i v e r p a t h o l o g y . A s i m p l i f i e d v e r s i o n o f t h e percentage c o y p o s i t i o n d a t a i s g i v e n i n Table 11.3. TABLE 11.3 SIMPLIFIED LIPOPROTEIN COMPOSITION Lipoprotein

CM

VLDL

LDL

HDL

P r o t e i n (%) L i p i d (%)

1 95

12 60

25 75

50 50

255 The a p o p r o t e i n c o m p o s i t i o n o f t h e l i p o p r o t e i n s 4 i s summarized i n Table 11.4. I n s p i t e o f t h e c o m p l e x i t y o f t h e l i p o p r o t e i n c o m u o s i t i o n , t h e good correspondence between r e s u l t s o b t a i n e d by u l t r a c e n t r i f u g a t i o n and e l e c t r o p h o r e s i s , w h i c h a r e based on e n t i r e l y d i f f e r e n t physico-chemi c a l concepts charge

-

-

p a r t i c l e d e n s i t y and

p r o v i d e s i n d i r e c t p r o o f o f t h e v a l i d i t y and c o m p a r a b i l i t y o f b o t h methods.

These e l e c t r o p h o r e t i c l i p o p r o t e i n p r o f i l e s make a u s e f u l c o n t r i b u t i o n t o t h e c l i n i c a l e v a l u a t i o n o f d y s l i p o p r o t e i n a e m i a. METHODOLOGICAL PROBLEMS

V a l i d i t y o f t h e e l e c t r o p h o r e t i c method T h i s s e c t i o n i n d i c a t e s t h a t l i p o p r o t e i n t y p i n g a c c o r d i n g t o Lees and Hatch and t o Levy and F r e d r i c k s o n ( a s quoted i n r e f e r e n c e 6 ) Fredrickson types I t o V

-

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commonly c a l l e d

s t i l l i s and w i l l remain a v a l i d c l i n i c a l chemical

method. There have been many arguments a g a i n s t t h e e l e c t r o o h o r e t i c method, e s p e c i a l l y because i t i s o n l y s e m i - q u a n t i t a t i v e .

T h i s o f t e n o c c u r r e d as a consequence o f t h e

p r e c i s e a n a l y t i c a l d a t a t h a t c o u l d be o b t a i n e d f r o m an a n a l y s i s o f u l t r a c e t r i f u g a l f r a c t i o n s o r o f l i p o p r o t e i n c l a s s e s i s o l a t e d by e l e c t r o c h r o m a t o g r a p h i c seuaration7-’.

One s h o u l d n o t f o r g e t , however, t h a t a l t h o u g h these methods, b e l o n g i n g

t o the research laboratory, established t h e t r u t h about l i p o p r o t e i n s , they cannot be a p p l i e d t o r o u t i n e a p p l i c a t i o n s . R o u t i n e procedures r e q u i r e s i m p l e r and more e f f i c i e n t methods t h a n t h e a n a l y t i c a l c e n t r i f u g e , w i t h r e s p e c t t o t h e economics o f personnel and equipment o r r e a g e n t s and a l s o t o s c a l e i n terms o f s m a l l sample s i z e s and numbers o f samples i n a run. On t o p o f t h a t , t h e t i m e r e q u i r e d t o p r o v i d e a v a l i d answer s h o u l d be t a k e n i n t o c o n s i d e r a t i o n and c o u l d become t h e main a r g w n t i n f a v o u r o f a c l i n i c a l chemical t e c h n i q u e such as e l e c t r o p h o r e s i s , which needs o n l y micro-amounts o f plasma o r serum (and t h i s i s n o t one o f t h e l e a s t advantages o f t h e method). F o r a l l t h e s e reasons and w i t h o u t any d i s r e g a r d f o r p r e c i s e b i o p h y s i c a l and b i o c h e m i c a l methodologies, t h e c l i n i c a l l a b o r a t o r y r e q u i r e s a good r a n k i n g p r o cedure f o r l i p o p r o t e i n p a t t e r n s f o r d a i l y r o u t i n e . When abnormal e l e c t r o p h o r e t i c l i p o p r o t e i n p r o f i l e s a r e observed, newer methods can be a p p l i e d such as an e v a l u a t i o n o f t h e HDL c h o l e s t e r o l , b u t t h i s s h o u l d be r e s e r v e d f o r t h e m e a n i n g f u l cases when t h e p r o f i l e i s n o t w i t h i n normal l i m i t s . I t i s t i m e t o r e t u r n t o e l e c t r o p h o r e s i s and t o r e - s t a t e t h e s i g n i f i c a n c e and

importance o f p r o f i l e s t h a t y i e l d i n f o r m a t i o n which no o t h e r method can g i v e a t present. It i s d i f f i c u l t o r even i m p o s s i b l e t o d e c i d e on l i p o p r o t e i n anomalies f r o m

1ump plasma o r serum 1 ip i d v a l u e s a1 one, whereas no abnormal e l e c t r o p h o r e t i c p a t t e r n s h o u l d be l e f t w i t h o u t b i o c h e m i c a l c o n f i r m a t i o n by l i o i d a n a l y s i s . The

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shortcuts i n t h e diagnosis o f dyslipaemia using e i t h e r cholesterol o r t r i g l y c e r i d e l e v e l s a l o n e o r t o g e t h e r a r e t h e cause o f d i a g n o s t i c and t h u s t h e r a p e u t i c f a i l u r e s which d i s c r e d i t t h e e n t i r e f i e l d . E l e c t r o p h o r e t i c d a t a a l l o w a more s y s t e m a t i c i n i t i a l d i a g n o s i s , and t h u s e a r l i e r response t o t h e r a o y and b e t t e r p r o g n o s i s . The arguments a g a i n s t t h e t y p e s o f F r e d r i c k s o n , i n c l u d i n g t h e f a c t s t h a t t h e y a r e n o t c l e a r - c u t and f a i l t o cover a l l p a t i e n t s , a r e i n v a l i d because t h e t y p i n g e l i m i n a t e s an enormous percentage o f normals, i n c l u d e s a l a r g e D r o p o r t i o n o f c l e a r c u t t y p e s and i n d i c a t e s a t e n d e n c y t o w a r d s t h e b a s i c t y p e s I 1 o r I V i n t h e o t h e r cases. I n a c l i n i c a l chemical l a b o r a t o r y , t h e lump d e t e r m i n a t i o n o f serum o r plasma l i p i d s must be complemented by a l i p o p r o t e i n p r o f i l e i f t h e r e s u l t s a r e d o u b t f u l . The r e v e r s e approach, s t a r t i n g w i t h a l i p i d o g r a m , f o l l o w e d b y chemical c o n f i r m a t i o n i f abnormal, c o u l d a l s o be used. An o b j e c t i v e e p i d e m i o l o g i c a l s t u d y comparing e i t h e r o f t h e s e two approaches as r e g a r d s c l i n i c a l e f f i c i e n c y s h o u l d be performed. Me thodological developments The h i s t o r i c a l development o f l i p o p r o t e i n e l e c t r o p h o r e s i s f o l l o w e d two l i n e s . The f i r s t was t h e d i s c o v e r y o f t h e p r e - and p o s t - s t a i n i n g o f l i p o p r o t e i n s by l i p o p h i l i c dyes i n o r d e r t o r e v e a l t h e e l e c t r o p h o r e t i c p a t t e r n , w h i c h i n i t i a l l y was performed on paper. The second development was t h e passage f r o m paper t o c e l l u l o s e and g e l s . I t i s t h e p r i n c i p l e o f p o s t - s t a i n i n g 'OY1'

w h i c h had t h e g r e a t e s t i n f l u e n c e on

t h e development o f t h e l i p o p r o t e i n p a t t e r n , w h i c h p r e v i o u s l y had been o b t a i n e d by u l t r a c e n t r i f u g a t i o n , an e x c e l l e n t b u t cumbersome and expensive procedure. The s t a i n i n g o f l i p o p r o t e i n l i p i d s i n a separated f r a c t i o n i n s i t u i s a procedure t h a t r u n s p a r a l l e l t o many o t h e r procedures w h i c h d e t e c t , e.g.,

an enzyme a c t i v i t y

p a t t e r n a f t e r e l e c t r o p h o r e s i s . The " s t a i n i n g " p r i n c i p l e covers a wide range o f r e a c t i o n s , such as t h e f i x a t i o n o f a r a d i o i s o t o p e o r o f an i n v i s i b l e l i g a n d t o be d e t e c t e d under UV l i g h t and t h e c l a s s i c a l p r o t e i n - d y e i n t e r a c t i o n w h i c h can r e a d i l y be s t u d i e d b y p h o t o m e t r i c procedures.

O b v i o u s l y a g r e a t problem w i t h any o f t h e s e r e c e p t o r - l i g a n d t y p e i n t e r a c t i o n s i s t o e s t a b l i s h t h e c o r r e c t s t o i c h i o m e t r i c r e l a t i o n s h i p between t h e r e c e p t o r m o l e c u l e and t h e l i g a n d . Obviously, when s t a i n i n g c o n s i s t s o f a l i p o p h i l i c dye d i s s o l v i n g i n t h e hydrop h o b i c m o i e t y o f a macromolecular l i p o p r o t e i n p a r t i c l e , s e v e r a l q u a n t i t a t i o n problems occur, and t h e s e a r e c o n s i d e r e d below. ( a ) Owing t o p h y s i o p a t h o l o g i c a l v a r i a t i o n s , t h e amount o f l i p i d i s n o t i n c o n s t a n t p r o p o r t i o n t o t h e amount o f a p o p r o t e i n and t h u s t h e amount o f dye b e i n g taken up by t h e p a r t i c l e i s n o t a c o r r e c t r e a d i n g f o r t h e p o l y D e p t i d e m o i e t y o f a l i p o p r o t e j n . E v i d e n t l y t h e same remark i s a l s o v a l i d f o r t h e d e t e r m i n a t i o n o f any l i p i d , such as c h o l e s t e r o l , used f o r t h e i n d i r e c t q u a n t i t a t i o n o f a l i p o p r o t e i n ,

268

w i t h a p o s s i b l e e x c e p t i o n f o r t h e p h o s p h o l i p i d c o n t e n t because p h o s p h o l i p i d s a r e s t r u c t u r a l l y r e l a t e d t o t h e p o l y p e p t i d e backbone o f t h e a p o p r o t e i n . ( b ) As t h e l i p i d d i s t r i b u t i o n o f t h e l i p i d m o i e t y d i f f e r s f o r each l i p o p r o t e i n c l a s s (see t h e s y n t h e t i c d a t a on l i p o p r o t e i n s ) s t a i n a b i l i t y by l i p i d - s o l u b l e dyes i s n o t s t r i c t l y p r o p o r t i o n a l t o t h e t o t a l amount o f l i p i d on t h e l i p o p r o t e i n . I ndeed, a g i v e n dye has d i f f e r e n t s o l u b i l i t y c o e f f i c i e n t s i n d i f f e r e n t l i p i d s and t h u s t h e r e l a t i v e r e a d i n g w i l l be d i f f e r e n t depending on t h e l i p i d r a t i o and d i s t r i b u t i o n i n t h e l i p o p r o t e i n p a r t i c l e . T h i s i n d i c a t e s why a method t h a t i s r e p r o d u c i b l e and p r e c i s e f o r normal samples may become d i f f i c u l t t o a p p l y t o p a t h o l o g i c a l samples i n which t h e l i p i d d i s t r i b u t i o n can be v e r y d i f f e r e n t f r o m t h a t o f a normal l i p o p r o t e i n o f t h e same c a t e g o r y . ( c ) As s t a i n i n g procedures w i t h l i p o p h i l i c dyes i n c l u d e t h e use o f non-aqueous s o l v e n t s , p q r t o f t h e l i p i d leaches i n t o t h e s t a i n i n g s o l u t i o n d u r i n g t h e s t a i n i n g procedure12. It i s obvious t h a t t h e l a r g e s t l i p o p r o t e i n p a r t i c l e s w i t h t h e l o o s e s t physico-chemical bond between l i p i d and a p o p r o t e i n a r e most s u b j e c t t o t h i s t y o e o f l i p i d l o s s t h r o u g h l e a c h i n g , T h i s a g a i n means t h a t o a t h o l o g i c a l samples w i l l be more d i f f i c u l t t h a n t h e normal ones t o e v a l u a t e c o r r e c t l y . I t a l s o i n d i c a t e s why p h o s p h o l i p i d s which a r e s t r o n g l y bonded t o t h e a p o p o l y p e p t i d e s p r o g r e s s i v e l y emerge as t h e more c o n s i s t e n t o f a l l l i p i d parameters i n b o t h plasma and l i p o p r o t e i n s 13-15

.

(d) Evidently t h e photometric q u a n t i t a t i o n o f l i p o p r o t e i n o r o f i l e s i s subject t o t h e same g e n e r a l problems o f photometry as any o t h e r s t a i n i n g procedure f o r h o l o p r o t e i n s , g l y c o p r o t e i n s o r enzymatic a c t i v i t y 16

.

From t h e s e remarks, t h e f o l l o w i n g c o n c l u s i o n s can be drawn: 1. L i p o p r o t e i n s t a i n i n g i s a good p r a c t i c a l procedure f o r t h e c l i n i c a l e v a l u a -

t i o n o f a plasma sample. The s c r e e n i n g o f p a t i e n t s i n t o normals and abnormals w i l l c o n t i n u e by one o f t h e s i m p l e l i p o p r o t e i n s t a i n i n g procedures now i n e x i s t e n c e .

2. The problems mentioned above mean t h a t broad c u t - o f f zones have t o be c o n s i d e r e d when c l a s s i f y i n g t h e p a t t e r n s i n t o d i f f e r e n t types. C o n s i d e r a t i o n o f t h e l i p i d values o f t o t a l plasma and c h a r a c t e r i z a t i o n o f a s p e c i f i c HDL phosphol i p i d o r c h o l e s t e r o l v a l u e a r e r e q u i r e d i n o r d e r t o improve t h e p l a u s a b i l i t y o f a lipoprotein pattern w i t h a pathological p r o f i l e . 3. The d e t e r m i n a t i o n o f a p o p r o t e i n l e v e l s i n t h e e l e c t r o p h o r e t i c a l l y s e p a r a t e d f r a c t i o n s w i l l become an i m p o r t a n t s t e p f o r t h e f u t u r e o f l i p o p r o t e i n e v a l u a t i o n , e s p e c i a l l y under p a t h o l o g i c a l circumstances. Selected methodologies

I n s t e a d o f summarizing t h e a l m o s t endless v a r i a n t s o f e l e c t r o p h o r e t i c l i p o p r o t e i n s e p a r a t i o n , l e t us c o n s i d e r t h r e e v a l i d a t e d procedures f o r q u a n t i t a t i v e e l e c t r o p h o r e t i c l i p o p r o t e i n separation.

259

Q u a n t i t a t i v e l i p o p r o t e i n s t a i n i n g according t o Lindgren. L i ndgren17 d e s c r i b e d an a u t o m a t i c system f o r t h e m i c r o d e n s i t o m e t r y and c a l c u l a t i o n s o f l i n o o r o t e i n p r o f i l e s as p a r t o f a q u a n t i t a t i v e agarose g e l e l e c t r o p h o r e s i s system. I t s advantage, as w i t h a l l e l e c t r o p h o r e t i c methods, i s t h a t i t l e n d s i t s e l f t o l a r g e s c a l e a p p l i c a t i o n a t r e a s o n a b l e c o s t . Only t h o s e p a t i e n t s who appear abnormal a r e s u b j e c t e d t o more r e f i n e d and o b j e c t i v e procedures such as u l t r a c e n t r i f u g a t i o n . The method i s e n t i r e l y d e s c r i b e d i n r e f . 17. The key t o t h e q u a n t i t a t i o n i s a m i c r o d e n s i t o m e t e r , an a n a l o g u e - t o - d i g i t a l c o n v e r t e r , a c a t h o d e - r a y tube t e r m i n a l , a t e l e p r i n t e r and a PDP 8/e computer equipped w i t h a d i s k . The scan i s c o r r e c t e d f o r b a s e l i n e d r i f t and pre-B asymmetry and w i l l p r o p e r l y i d e n t i f y t h e amounts o f HDL, LDL and VLDL. C o r r e c t i o n s f o r " s i n k i n g pre-B" and " f l o a t i n g 6" i n LDL and VLDL, r e s p e c t i v e l y , r e q u i r e t h e use o f an u l t r a c e n t r i f u g e . TABLE 11.5 L I P I D AND LIPOPROTEIN DISTRIBUTIONS ( g / l ) I N TWO PATIENTS Case 7184 C h o l es t e r o 1 Triglycerides HDL LDL VLDL

Case 7288 2.84 1.08 5.74 5.41 0.94

Cholesterol Triglycerides HDL LDL VLDL

2.92 1.11 1.79 7.50 0.94

Table 11.5 demonstrates t h e u s e f u l n e s s o f t h i s procedure f o r two p a t i e n t s o f t h e same age and sex w i t h i d e n t i c a l lump v a l u e s f o r plasma t o t a l c h o l e s t e r o l and t r i g l y c e r i d e s . There i s a s u b s t a n t i a l d i f f e r e n c e i n l i p o p r o t e i n d i s t r i b u t i o n i n s p i t e o f i d e n t i c a l lump serum l i p i d s . The d a t a a r e t a k e n f r o m r e f . 17. The a u t h o r compared h i s q u a n t i t a t i v e p h o t o m e t r i c d a t a w i t h u l t r a c e n t r i f u g a l d a t a and showed how t h e agarose method i s e q u i v a l e n t . A comparison w i t h a h e p a r i n MnC12 p r e c i p i t a t i o n method such as t h a t u t i l i z e d by t h e L i p i d Research C l i n i c s 18 t h a t moreover r e q u i r e s more serum were p o o r e r

17,19

.

I n c o n c l u s i o n , l i p o p r o t e i n t y p i n g , c e r t a i n l y i f q u a n t i t a t e d , i s much more i n f o r m a t i v e t h a n t h e lump d a t a o f c h o l e s t e r o l and t r i g l y c e r i d e s on serum o r p l asma.

Q u a n t i t a t i v e l i p o p r o t e i n p r e c i p i t a t i o n according t o Wieland and S e i d e l . T h i s t e s t uses two c o m m e r c i a l l y a v a i l a b l e p o l y a n i o n i c r e a g e n t s ( L i p i d o D h o r system) which p r e c i p i t a t e t h e l i p o p r o t e i n bands a f t e r an e l e c t r o p h o r e t i c run i n agarose Q u a n t i t a t i o n c o r r e l a t e s w i t h r = 0.96 a t n = 20 w i t h t h e c o n c e n t r a t i o n o f c h o l e s t e r o l i n each f r a c t i o n . I t i s assumed t h a t t h e c h o l e s t e r o l l e v e l remains

.

20

260

c o n s t a n t f o r each o f t h e l i p o p r o t e i n s . The c h o l e s t e r o l c o n c e n t r a t i o n s a - l i p o p r o t e i n 18%, B - l i p o p r o t e i n 55% and p r e - 8 - l i p o p r o t e i n 15% o c c u r w i t h o n l y m i n o r modi f i c a t i o n s under normal o r abnormal l i p o p r o t e i n metabolism. From t h i s s t a t e m e n t i t a l s o f o l l o w s t h a t l i p o p r o t e i n s can be e v a l u a t e d f r o m t h e r e l a t i v e c h o l e s t e r o l

percentage if t o t a l c h o l e s t e r o l i s determined s i m u l t a n e o u s l y . W i t h t h i s method t h e c o e f f i c i e n t s o f v a r i a t i o n i n a s e r i e s o f 20 cases were,

2% f o r B - l i p o p r o t e i n s , 2% f o r p r e - 6 - l i p o p r o t e i n s ,

2.3% f o r a - l i p o p r o t e i n s and

4.1% f o r t h e a:B r a t i o . T y p i c a l normal v a l u e s a r e a - l i p o p r o t e i n 53%, p r e - B - l i p o p r o t e i n 10%'and 8 - l i p o p r o t e i n 37%. The main advantage o f t h i s method i s t h e p o s s i b i l i t y o f u s i n g many commercial d e n s i t o m e t e r s , whereas i t c o u l d be c o n s i d e r e d a drawback t h a t t h e p r e c i n i t a t i n g r e a g e n t s a r e prepared a c c o r d i n g t o a p a t e n t f o r m u l a . An advantage o f t h i s method l i e s i n t h e s t a i n i n g procedure, which i n v o l v e s a two-step p r e c i p i t a t i o n o f t h e l i p o p r o t e i n s as t u r b i d bands on a n e a r l y t r a n s p a r e n t background, w h i c h i s favour a b l e f o r s a t i s f a c t o r y p h o t o m e t r i c scanning. The assumption o f a c o n s t a n t c h o l e s t e r o l r a t i o i n l i p o p r o t e i n s c a n n o t e a s i l y be accepted f o r p a t h o l o g i c a l sera. I n o u r hands, however, a comparison w i t h u l t r a c e n t r i f u g a l HDL c h o l e s t e r o l d a t a o b t a i n e d by Beckman M i c r o f u g e c e n t r i f u g a t i o n was s a t i s f a c t o r y . The advantage o f a r e a d i l y a v a i l a b l e

8- t o a - l i p o p r o t e i n r a t i o i s

n o t matched as e a s i l y by t h e u l t r a c e n t r i f u g e .

Lipoprotein Zipid determinations according t o Kupke. L i p o p r o t e i n 1 ip i d s a r e determined a c c o r d i n g t o Kupke 21y22

a f t e r agarose g e l e l e c t r o p h o r e s i s . The o r i g i -

n a l i t y o f t h e method l i e s i n t h e d e s t r u c t i o n o f t h e agarose s t r u c t u r e o f t h e g e l by means o f HC1 i n o r d e r t o be a b l e t o d e t e r m i n e c h o l e s t e r o l o r plasma l i p i d s by means o f c l a s s i c a l procedures such as an enzymatic d e t e r m i n a t i o n f o r c h o l e s t e r o l and t h i n - l a y e r chromatographic scanning f o r l i p i d s . The f r a c t i o n s a r e r e c o g n i z e d by t h e i r opalescence, c u t o u t w i t h a s c a l p e l , d r i e d , t r e a t e d w i t h HC1, which d e s t r o y s t h e agarose, t h e HC1 i s evaporated i n an a i r stream and t h e c h o l e s t e r o l o r l i p i d d e t e r m i n a t i o n s a r e performed. F u l l d e t a i l s f o r c h o l e s t e r o l a r e g i v e n i n r e f . 2 1 and f o r c h o l e s t e r o l e s t e r , c h o l e s t e r o l and t r i a c y l g l y c e r o l Dhosphol i p i d s i n r e f . 22. Q u a n t i t a t i o n i s e f f e c t e d by comparison w i t h r e f e r e n c e samples, i n t h i s case P r e c i l i p . Reference methods u s i n g r a d i o a c t i v e c h o l e s t e r o l o r s t a n d a r d l i p i d s demonstrated t h e v a l i d i t y o f t h i s approach. The i n t e r e s t i n g aspect o f Kupke's approach i s t h a t t h e s t a i n e d g e l band i s c u t o u t and d i s s o l v e d b e f o r e photometry. A f t e r t h i s step, r e g u l a r b i o c h e m i c a l p r o c e dures can be a p p l i e d . T h i s u n d o u b t e d l y has a t h e o r e t i c a l advantage o v e r methods u s i n g r e a c t i o n s i n s i t u i n s i d e t h e g e l , which i n e v i t a b l y i n c l u d e q u a n t i t a t i v e e r r o r s i n r e a d i n g s t a i n e d bands w i t h a g r a d i e n t o f c o n c e n t r a t i o n o v e r t h e s u r f a c e . I t has been shown t h a t no p e r f e c t c o r r e c t i o n f a c t o r can be a p p l i e d t o such a

situation

16

.

261 It could be claimed, however, t h a t f o r p r a c t i c a l purposes such a h i g h p r e c i s i o n

i s n o t r e q u i r e d and t h a t a d i r e c t r e a d i n g o f bands on a s t r i p g i v e s s u f f i c i e n t accuracy f o r c l i n i c a l m o n i t o r i n g o f disease o r t h e r a n k i n g o f p a t i e n t s . When several l i p i d s a r e t o be determined, however, i t i s c l e a r t h a t t h i s cannot be done by a p p l y i n g s t a i n i n g methods on t h e gel i n s i t u b u t t h a t t h i n - l a y e r o r o t h e r a n a l y t i c a l methods a r e r e q u i r e d . Kupke's methods s t r e s s t h e i n d i v i d u a l l i p o p r o t e i n l i p i d d a t a and thus open u? t h e p o s s i b i l i t y o f screening micro-amounts o f blood f o r i n d i v i d u a l l i p o p r o t e i n l i p i d s . H i t h e r t o t h i s was r e s t r i c t e d t o l a r g e samples and r e q u i r e d p r e p a r a t i v e u l t r a c e n t r i f u g a t i o n o r electrochromatography. A t t h i s p o i n t , we should r e p e a t o u r c a u t i o n about t h e q u a n t i t a t i o n o f l i p o p r o t e i n s by means o f a d e t e r m i n a t i o n o f l i p o p h i l i c dyes. H o p e f u l l y t h e procedures i n use i n t h e r e a d e r ' s own l a b o r a t o r y w i l l be checked w i t h r e s p e c t t o t h e preceding remarks, and t h i s may have a u n i f y i n g tendency on t h e a l l t o o numerous methodologies i n t h e f i e l d .

Apoprotein determinations An i d e n t i f i c a t i o n o f t h e l i p o p r o t e i n s a f t e r agar e l e c t r o o h o r e s i s by means o f anti-cr and anti-f3 a n t i s e r a was pioneered by Grabar's group. The a n t i s e r a i n use were a g a i n s t t o t a l serum and p r a c t i c a l c l i n i c a l conclusions were n o t obtained. Ageing o f l i p o p r o t e i n s i n v i t r o w i t h l i b e r a t i o n and subsequent uptake o f f r e e f a t t y a c i d s on t h e a - l i p o p r o t e i n s leads t o t h e s o - c a l l e d ralpid o r r h o - f r a c t i o n , which precedes albumin, on agarose g e l s , b u t t h i s i s n o t o f g r e a t c l i n i c a l i n t e r e s t . Immunoelectrophoresis has a l s o been h e l p f u l i n t h e d e t e c t i o n o f Lp-X, as shown f u r t h e r . T u r b i d i m e t r i c o r nephelometric q u a n t i t a t i o n o f l i p o p r o t e i n by means o f a n t i sera i s used as a c l i n i c a l chemical method. F o r 6 - l i p o p r o t e i n these data c o r r e l a t e w i t h t h e t r i g l y c e r i d e d a t a and f o r t h i s reason a r e s u p e r f l u ~ u s ~T~h i. s r e s u l t s from t h e f a c t t h a t apo B i s n o t o n l y present i n LDL b u t a l s o represents

25% o f t h e VLDL p o l y p e p t i d e s and thus b o t h f r a c t i o n s (LDL and VLDL) r e a c t s i m u l taneously. Such d e t e r m i n a t i o n i s , o f course, l e s s i n t e r e s t i n g than t h e B - l i p o protein-pre-8-1 i p o p r o t e i n s e p a r a t i o n obtained i n t h e e l e c t r o p h o r e t i c p a t t e r n . More r e c e n t l y (although t h i s l i e s o u t s i d e t h e scope o f a d i s c u s s i o n about e l e c t r o p h o r e s i s ) , s p e c i f i c a n t i s e r a a g a i n s t apopolypeptides have appeared and i t i s our o p i n i o n t h a t they w i l l be u s e f u l f o r t h e c l i n i c a l d i a g n o s i s o f d y s l i p o proteinaemia. The r a t i o o f A I t o A I 1 i n HDL c o u l d become an e x c e l l e n t i n d i c a t o r o f a - l i p o p r o t e i n q u a l i t y and thus d i f f e r e n t i a t e type I 1 and type I V HDL a g a i n s t HDL o f normal r e f e r e n c e persons. This would a l s o mean t h a t an HDL d e t e r m i n a t i o n and c l a s s i f i c a t i o n c o u l d have a tendency t o supersede t h e f3-lipoprotein-pre-Bl i p o p r o t e i n c l a s s i f i c a t i o n on which t h e Fredrickson p a t t e r n s were o r i g i n a l l y based.

262

THE LIPOPROTEIN PATTERN The reference p a t t e r n

I t i s useful t o elaborate on t h e so-called normal reference p a t t e r n and t o draw a parallel with other methodologies i n order t o e s t a b l i s h a c l e a r baseline before s t a r t i n g t h e discussion of physiological, genetic and acquired pathological deviations. The mobility of a lipoprotein r e s u l t s mainly from t h e mosaic of apoproteins t h a t i t contains and a d d i t i o n a l l y from adsorbed metal ions, f a t t y acid anions and maybesome phospholipids t h a t do not e x i s t in t h e form of zwitterions a t e l e c t r o phoretic pH values 3 . In g e l s , and formerly on paper, the migration due t o the net e l e c t r i c charge i s modified by t h e pore s i z e of the medium, which e s p e c i a l l y a f f e c t s t h e l a r g e s t p a r t i c l e s such as the chylomicra. There a r e a l s o s l i g h t i n t e r actions with t h e chemicals of the medium which a r e corrected by EDTA, eliminating divalent cations, and by albumin. These a r e constant a d d i t i v e s t o buffers adapted t o a clean e l e c t r o p h o r e t i c separation of lipoprotein bands with l i t t l e t r a i l i n g . The basic correspondence of the e l e c t r o p h o r e t i c p a t t e r n , r e s u l t i n g from net charge and gel f i l t r a t i o n , compared with u l t r a c e n t r i f u g a l lipoprotein data was described above. Electrophoresis can a l s o be used as a good check f o r the q u a l i t y of ul t r a c e n t r i f u g a l procedures now t h a t the Microfuge has introduced practical semimicro f l o t a t i o n proceduresz4. However, some refinements of the u l t r a c e n t r i f ugal separation, such a s HDLz and HDL3 f r a c t i o n a t i o n , a r e not within reach of t h e gel e l e c t r o p h o r e t i c methods. For c l a r i t y , Table 11.6 supplements the previous t a b l e of correspondence (Table 11.1) between the e l e c t r o p h o r e t i c bands and t h e u l t r a c e n t r i f u g a l f r a c t i o n s . TABLE 11.6

CORRESPONDENCE BETWEEN ULTRACENTRIFUGAL FRACTION AND ELECTROPHORETIC ZONE U1 tracentrifugal fraction Sf

s? s; SP

FQ

1.20

> 400

20-400 (when markedly elevated) 0-20

20-400 0-9

Electrophoretic zone Chyl omicrons Trai

Origin

f3-1Li poproteins Pre-6-1 ipoproteins a-Lipoproteins

Anode

263

A t t h i s p o i n t i t i s u s e f u l t o p r e s e n t a schematic o v e r v i e w o f " s t a n d a r d " abnormal l i p o p r o t e i n p a t t e r n s - t y p e s I - V o f F r e d r i c k s o n - a g a i n s t t h e r e f e r e n c e p a t t e r n ( F i g . 11.1).

Iipoproteins

f redr ickson

N

I

II

t y pes Ill

IV

V

alpha pre beta beta chylomicra

lipoproteins estimated

from electrophoretic

patterns

F i g . 11.1. Schematic r e p r e s e n t a t i o n o f F r e d r i c k s o n Types. Many i n t e r m e d i a t e p a t t e r n s a r e observed i n p a t i e n t s , b u t i n t h e a c t u a l s t a t e o f t h e a r t we s h o u l d channel o u r i n f o r m a t i o n i n t o t h e f i v e b a s i c p a t t e r n s . B e f o r e l e a v i n g t h e r e f e r e n c e p a t t e r n we s h o u l d m e n t i o n a s t u d y i n which u l t r a c e n t r i f u g a l and e l e c t r o p h o r e t i c p a t t e r n s were compared and w h i c h demonstrates t h e c r e d i b i l i t y o f a good zone e l e c t r o p h o r e t i c s e p a r a t i o n 2 5 y 2 6 . The d a t a c o v e r 20 sel e c t e d i n d i v i d u a l s : 4 normal, 6 w i t h t y p e 11, 3 w i t h t y p e I V and 5 w i t h t y p e V. C o r r e l a t i o n s a t p < 0.1 were 0.87 f o r HDL, 0.99 f o r p r e - 8 - l i p o p r o t e i n , 0.98 f o r 8 - l i p o p r o t e i n and 0.85 f o r c h y l o m i c r a . Thus we can a c c e p t t h a t e l e c t r o p h o r e t i c l i p o p r o t e i n p a t t e r n s g i v e a v a l i d percentage e v a l u a t i o n o f t h e l i p o p r o t e i n d i s t r i b u t i o n i n h e a l t h and disease.

PhysioZogicaZ v a ria tio n s NutritionaZ effects. ( a ) The most obvious d e v i a t i o n i s due t o t h e p o s t - p r a n d i a l t r i g l y c e r i d e t i d e i n t h e plasma. By analogy, t h e endogenic m o b i l i z a t i o n , i n t h e f a s t i n g s t a t e , o f f a t f r o m t h e f a t s t o r e s t o reach t h e l i v e r a l s o r e s u l t s i n an i n c r e a s e i n plasma t r i g l y c e r i d e . The most o b v i o u s r e s u l t i s i n c r e a s e d and v i s i b l e t u r b i d i t y as a r e s u l t o f i n c r e a s e d c h y l o m i c r o n and VLDL c o n c e n t r a t i o n . T h i s means t h a t e l e c t r o p h o r e s i s s h o u l d b e performed i n t h e f a s t i n g s t a t e t h e previous evening

-

-

a f t e r a l i g h t meal

and n o t performed on t u r b i d serum u n l e s s t h e b l o o d was

drawn w i t h t h e mentioned p r e c a u t i o n . A f t e r t a k i n g t h i s p r o p e r p r e c a u t i o n , t u r b i d i t y o b v i o u s l y becomes a symptom and t h e abnormal p a t t e r n becomes i n f o r m a t i v e .

264

( b ) A l c o h o l i s one o f t h e causes o f h y p e r t r i g l y c e r i d a e m i a t h r o u g h l i v e r i m p a i r ment o r damage and s i m u l a t e s t y p e I V o f F r e d r i ~ k s o n ~The ~ . inducement o f a t y p e I V o f t r a n s i t o r y t y p e i s n o t i n f r e q u e n t a f t e r heavy d r i n k i n g b o u t s and any t y p e

I V p a t t e r n must be c h a l l e n g e d on t h e b a s i s o f a n o n - f a s t i n g o r a p o s t - a l c o h o l i c state. ( c ) A deeper, more permanent e f f e c t on t h e p a t t e r n i s t h e r e s u l t o f t h e o v e r a l l e a t i n g h a b i t s and d i e t c o m p o s i t i o n o f t h e i n d i v i d u a l lipids

-

-

o r o t e i n s , g l u c i d e s and

t h e amount o f c a l o r i e s he absorbs and t h e t y p e o f f a t t y a c i d s i n t h e f a t .

These d a t a can a l l be c o n s i d e r e d under t h e one h e a d i n g o f o v e r - n u t r i t i o n .

It i s

d i f f i c u l t t o draw t h e l i n e between o v e r - n u t r i t i o n and o a t h o l o g y o f t y p e I 1 w h i c h we w i l l d e s c r i b e l a t e r . I n Western p o p u l a t i o n s o v e r e a t i n g i s a common f a u l t and a number o f t y p e I 1 p a t i e n t s a r e r e a d i l y improved o n l y b y r e d u c i n g t h e c a l o r i e i n t a k e . Here again, t h e b o r d e r l i n e between d i e t - i n d u c e d and h e r i d i t y - b a s e d e r r o r s o f @ - l i p o p r o t e i n metabolism i s d i f f i c u l t t o draw. ( d ) A d e v i a t i o n due t o u n d e r - n u t r i t i o n i s r a r e i n Europe, b u t u n f o r t u n a t e l y i t i s s e r i o u s and f r e q u e n t i n some p a r t s o f t h e w o r l d where i t i s o f t e n combined

w i t h worm i n f e s t a t i o n , which e l i c i t s I g E and o t h e r I g l e v e l s o u t o f p r o p o r t i o n w i t h those known elsewhere. I t seems t h a t k w a s h i o r k o r does n o t r e s u l t f r o m o r o t e i n m a l n u t r i t i o n alone, b u t a l s o f r o m simultaneous c a l o r i e d e f i c i t s , p o o r - q u a l i t y l i p i d s and p a r a s i t i c i n f e s t a t i o n . I n t h e samples we examined t h e h o l o o r o t e i n and l i p o p r o t e i n p a t t e r n s were low, Moreover, t h e f a t t y a c i d d i s t r i b u t i o n was more abnormal t h a n a n y t h i n g observed i n e x p e r i m e n t a l animals under t h e most e x o t i c diets. I n t h e l i t e r a t u r e 2 8 t h e l o w e r i n g o f a- o r HDL l i p o o r o t e i n i s c o n s t a n t and t h i s shows t h a t t h e b a s i c mechanism o f k w a s h i o r k o r i s a d e f e c t i n D r o t e i n s y n t h e s i s . I t i s indeed w e l l known t h a t HDL i s t h e p r i m e l i p o u r o t e i n and t h e predominant

f r a c t i o n i n a l l animals, i n c l u d i n g newborn humans. B - L i p o p r o t e i n w i t h human p e r centages i s found o n l y i n o v e r f e d chimpanzees and i n man and may t h u s r e s u l t f r o m our n u t r i t i o n a l

habit^^'-^'.

Normal c a l o r i e p o o r

-

not calorie deficient

-

people

show h i g h HDL and low LDL as t h e i r normal p a t t e r n . T h i s h y p o t h e s i s does n o t c o n t r a d i c t a simultaneous d e f i c i e n c y o f apo B and apo C p o l y o e o t i d e s y n t h e s i s which i n t e r p r e t s t h e simultaneous l o w e r i n g o f t h e B - f r a c t i o n s . Age and sex related variations

( a ) Data i n children It i s a c o n s t a n t o b s e r v a t i o f i i n c h i l d r e n t h a t t h e @ - l i p o p r o t e i n l e v e l s a r e

l o w e r t h a n i n a d u l t s . The p r e - $ band i s e a s i l y e l e v a t e d , p r o b a b l y i n r e l a t i o n t o d i e t a r y i n t a k e o f t r i g l y c e r i d e s . W i t h i n c r e a s i n g age t h e B - l i p o p r o t e i n s i n c r e a s e . The 1 i t e r a t u r e d a t a f o r u l t r a c e n t r i f u g a t i o n a r e e x t e n s i v e 2 ’ b u t do n o t correspond e x a c t l y w i t h e l e c t r o p h o r e t i c data. C o n s i d e r i n g t h e m u l t i p l e m e t h o d o l o g i c a l p r o b -

265

lems and v a r i a t i o n s i n c l u d e d i n t h e q u a n t i t a t i o n o f l i p o p r o t e i n bands t h i s i s n o t s u r p r i z i n g . E v i d e n t l y t h e range o f n o r m a l i t y i s v e r y wide and r e s p o n s i b l e f o r considerable overlapping. ( b ) Age and s e x r e l a t e d adult ranges

I n t h i s s e c t i o n we s h a l l , on purpose, n o t r e f e r t o t h e l i t e r a t u r e , w h i c h i s r e p l e t e w i t h data

t h a t a r e d i f f i c u l t t o g r a s p o r even i m p o s s i b l e t o remember i n

a p r a c t i c a l s i t u a t i o n . Long e x p e r i e n c e i n t h e f i e l d has shown how v e r y few p e o p l e a r e a b l e t o memorize t h e age and sex dependent l i p o p r o t e i n values and l i p i d d a t a which, t o make t h i n g s worse, have a v e r y b r o a d range. T h i s range d i s c o u r a g e s workers who a r e n o t s p e c i a l i s t s i n t h i s f i e l d and, as a r e s u l t , l i p o p r o t e i n s and l i p i d s remain a p o o r l y understood and " t u r b i d " f i e l d . We s h a l l t r y t o make p r o g r e s s b y keeping t h e d i s c u s s i o n s i m p l e and e f f e c t i v e , i . e . ,

we

s h a l l g i v e f e w e r d a t a and p r e s e n t a l o g i c a l s i m p l i f i c a t i o n which c o u l d tempt t h e c l i n i c i a n t o g i v e more a t t e n t i o n t o t h e l i p o p r o t e i n d a t a he r e c e i v e s f r o m t h e l a b o r a t o r y . A l t h o u g h some may b e l i e v e t h a t t h i s approach i s o v e r s i m p l i f i e d , we s h o u l d m e n t i o n t h a t o f t h e f i v e t y p e s o f F r e d r i c k s o n o n l y two t y p e s , namely I 1 and I V , a r e encountered i n p r a c t i c e , and i n any e v e n t r e p r e s e n t s a l l t h e i n f o r m a t i o n most c l i n i c i ans have about 1ip o p r o t e i ns. I n Table 11.7 we g i v e l i p o p r o t e i n d a t a f o r males and females aged 30, 40, 50 and 60 y e a r s . TABLE 11.7 LIPOPROTEIN DATA ( g / l ) ACCORDING

TO

AGE I N YEARS

Males

30

40

50

60

Range

a-Lipoprotein B-Li p o p r o t e i n Pre-8-1 i p o p r o t e i n

2.30 4.00 1.10

2.50 4.25 1.25

2.50 5.00 1.50

2.50 5.20 1.30

+O .80 t o . 90 t0.70

Females

30

40

50

60

Range

3.20 4.00 1.10

3.75 4.30 1.25

3.75 5.00 1.50

4.00 5.20 1.30

t1.00 to. 90 t0 .80

________~

~~

a-Li poprotein 8-Lipoprotein Pre-8-1 i p o p r o t e i n

We know t h a t t h e d a t a g i v e n i n t h i s and l a t e r t a b l e s a r e s u b j e c t t o v a r i a t i o n s a c c o r d i n g t o t h e method employed b u t we b e l i e v e t h a t t h e y a r e a v a l i d g u i d e l i n e f o r medical p r a c t i c e . I t i s a s t o n i s h i n g t h a t a f t e r r e a d i n g many papers on l i p o p r o t e i n p r o f i l e s , so

few c o n t a i n t r u l y p r a c t i c a l data. T h i s i s one o f t h e reasons why l i p o p r o t e i n s remain t o o obscure t o many and somehow d i s t a n t f r o m r e a l i t y . W ! e be1 i e v e t h a t

266

p r o g r e s s w i t h and u n d e r s t a n d i n g o f l i p o p r o t e i n d i s o r d e r s c o u l d p r o f i t f r o m f u r t h e r s t r e a m l i n i n g . We t o o k 4 5 - y e a r - o l d males and females as average and c o n s i d e r e d younger and o l d e r males and females f o r l o w e r and h i g h e r data. !Je a l s o added t h e

8- t o o r - l i p o p r o t e i n r a t i o ( T a b l e 11.8). TABLE 11.8 STREAMLINED LIPOPROTEIN DATA ( g / l )

or-Li p o p r o t e i n 8- L ip o p r o t e in Pre- 8- 1i p o p r o t e in B-:a-Lipoprotei n r a t i o

a-Lipoprotein 8-Li p o p r o t e i n Pre-8-1 i p o p r o t e i n 8- :a- L ipop r ot e i n r a t io

Younger males

Males 45 y e a r s

O l d e r males

2.25 4.25 1.00 1.75

2.50 4.50 1.50 1.80

2.50 5.00 1.25 2.00

Younger females

Females 45 y e a r s

O l d e r females

3.00

3.75 5.00 1.50 1.2

4.00

4.00

1.00 1.3

lipoproteins

ow%) 500

0 female

-

tmale

5.25 1.25 1.3

/*

beta

400.

300

-

200

-

100

-

01

d’

pre beta

0 / la---* 0

03’

I

younger *years

I

older

s t reamlined lipoprotein data F i g . 11.2. S t r e a m l i n e d l i p o p r o t e i n data.

c

age

267

0 female

+ male 2

1

younger

&years

older

age

streamlined lipoprotein r a t i o s

F i g . 11.3. S t r e a m l i n e d b e t a : a l p h a r a t i o . S c i e n t i f i c a l l y , t h e proposed s i m p l i f i c a t i o n s a r e sound because t h e normal ranges a r e so broad. I t i s u s e l e s s t o overburden t h e c l i n i c i a n ' s memory as i t has been p r o v e d r e p e a t e d l y t h a t nobody i s a b l e t o keep v e r y complex t a b l e s i n mind.

A g r a p h i c a l r e p r e s e n t a t i o n o f t h e d a t a g i v e n above c o u l d a l s o be h e l p f u l f o r r e p o r t i n g , as shown i n F i g s . 11.2 and 11.3. It i s easy t o s t e n c i l o r t o p r i n t t h e s e curves on a l a b o r a t o r y answering sheet and t o p l o t t h e d a t a found f o r t h e p a t i e n t e i t h e r m a n u a l l y o r by computer.

(el Progestagens, estrogens and l i p o p r o t e i n s The i n f l u e n c e o f e s t r o g e n s on t h e l i p o p r o t e i n p a t t e r n i s t y p i c a l i n women d u r i n g t h e i r r e p r o d u c t i v e age. They have more a - l i p o p r o t e i n and l e s s 6 - l i p o p r o t e i n t h a n men o f t h e same age and l e s s m y o c a r d i a l i n f a r c t i o n , as shown by t h e d a t a i n t h e p r e v i o u s s e c t i o n . The b i o c h e m i c a l s i d e o f t h i s e p i d e m i o l o g i c a l o b s e r v a t i o n was demonstrated by Gofman e t a l . i n 194g3'

i n h i s ultracentrifugal population

surveys. T h i s c l i n i c a l advantage o f a l o w e r r i s k o f c a r d i o v a s c u l a r a c c i d e n t s has

268 t o be c o n s i d e r e d as one o f t h e o l d e s t and s a f e s t o b s e r v a t i o n s c o r r e l a t i n g l i p o p r o t e i n s , a r t e r i a l p o l l u t i o n and i t s a c c i d e n t s . More r e c e n t l y , o r a l c o n t r a c e p t i v e s g e n e r a t e d an i a t r o g e n i c i n c r e a s e i n t h e c h o l e s t e r o l and 6 - l i p o p r o t e i n l e v e l s i n n o r m a l f e m a l e s and t h i s i n c r e a s e d t h e r i s k o f c a r d i o v a s c u l a r a c c i d e n t s 3 3 . An o v e r d o s a g e o f e s t r o g e n s such as d u r i n g o r a l c ~ n t r a c e p t i o ni n~c ~r e a s e s HDL and HDL c h o l e s t e r o l and d e c r e a s e s LDL l e v e l s . However, u n d e r t h e i n f l u e n c e o f p r o g e s t o g e n s t r i g l y c e r i d e s t e n d t o i n c r e a s e and t h i s may r e f l e c t i t s e l f a t t h e l e v e l o f VLDL and i n d i r e c t l y g e n e r a t e more LDL. P r o g e s t o g e n s a l s o r e d u c e t h e HDL l e v e l . As a g l o b a l i z e d r e s u l t t h e LDL t o HDL r a t i o i s h i g h e r than normal, t h u s g e n e r a t i n g a m o r t a l i t y r a t e o f i n f a r c t i o n which i s i d e n t i c a l w i t h t h a t f o r t h e normal male o f t h e same age. I t i s w e l l known t h a t r i s k f a c t o r s , such as an i n c r e a s e i n c h o l e s t e r o l ( i . e . ,

i n t h e 0- t o a - l i p o p r o t e i n r a t i o and i n t r i g l y c e r i d e s ( i . e . ,

i n @-lipoprotein), i n pre-@-lipoproteins)

have a c u m u l a t i v e e f f e c t t h a t superimposes i t s e l f on o t h e r r i s k f a c t o r s such as c i g a r e t t e smoking, o v e r e a t i n g and u n d e r e x e r c i s i n g . I n i n d i v i d u a l s w i t h t h e t r a i t o f g e n e t i c t y p e I 1 such permanent hormonal t r e a t m e n t may augment t h e r i s k o f a r t e r i a l p o l l u t i o n f o l l o w e d b y a t h e r o s c l e r o t i c accidents o f t h e coronaries. For t h i s reason lipidograms a r e i n d i c a t e d before ( a s a p r e c a u t i o n ) and d u r i n g c o n t r a c e p t i o n ( a s a c h e c k ) . The v a l u e o f e s t r o g e n s f o r i n c r e a s i n g a- o r HDL l i p o p r o t e i n l e v e l s has been a p p l i e d t o men a f t e r c o r o n a r y i n f a r c t i o n 3 4 w i t h i n c r e a s i n g HDL a - l i p o p r o t e i n l e v e l s and t h i s " f e m i n i z a t i o n " induced l o n g e r s u r v i v a l times.

( d l Data i n pregnancy Opalescence o f serum i n p r e g n a n c y i s one o f t h e o l d e s t c l i n i c a l c h e m i c a l obs e r v a t i o n s , d a t i n g back t o 184535. We s h a l l n o t go i n t o a l l t h e plasma p r o t e i n changes i n t h e m o t h e r , r e l a t e d t o t h e hormone l e v e l s and t o t h e p r e s e n c e o f t h e f o e t u s . Her serum l i p i d s i n c r e a s e t o 1.5 t i m e s t h e normal l e v e l s , t h e C:PL r a t i o i s l o w e r and t h e @ - l i p o p r o t e i n i s i n c r e a s e d , e s p e c i a l l y i n t h e t h i r d t r i m e s t e r o f pregnancy. O t h e r e s t r o g e n - s e n s i t i v e p r o t e i n s , such as t h y r o x i n e - b i n d i n g g l o b u l i n and c e r u l o p l a s m i n , f o l l o w t h e same l i n e . T h i s i s t h e p l a c e t o m e n t i o n p r a e e c l a m p s i a and h y p e r t e n s i o n , w h i c h a r e accompanied b y p r o f o u n d l i p o p r o t e i n

disturbance^^^.

The B- t o a - l i p o p r o t e i n r a t i o ,

w h i c h i n c r e a s e s f r o m 2 t o 3.5 i n normal p r e g n a n c y , goes up t o 4 . 5 i n p r a e e c l a m p s i a . As u s u a l w i t h l i p o p r o t e i n s t h e r e i s a c e r t a i n o v e r l a p p i n g o f h i g h n o r m a l and l o w p a t h o l o g i c a l d a t a . However, t h e f o l l o w - u p o f a case and t h e e f f e c t o f t h e r a p e u t i c measures c a n be m o n i t o r e d w i t h t h e 0- t o a - l i p o p r o t e i n r a t i o . T h e r e i s n o m a t h e m a t i c a l r e l a t i o n s h i p between t h i s r a t i o and t h e s e v e r i t y o f t h e d i s e a s e .

Genetic errors Under g e n e t i c e r r o r s we i n c l u d e some p r o f o u n d d e f i c i e n c i e s i n a p o p r o t e i n s y n t h e s i s and a l s o a m i s s i n g enzyme a t t h e m e t a b o l i c l e v e l . D e f i c i e n c i e s i n l i p o -

269

p r o t e i n t u r n o v e r a r e a l s o g e n e t i c a l l y induced, i n some i n s t a n c e s a t l e a s t , b u t a r e c o n s i d e r e d under t h e heading o f p a t h o l o g i c a l p a t t e r n s because a l l l i p o p r o t e i n s are present. The r a r i t y o f t h e s e d e f i c i e n c i e s i s compensated f o r by t h e i r i m p o r t a n c e i n t h e u n d e r s t a n d i n g o f normal l i p o p r o t e i n metabolism and t h e i n t e r c o n v e r s i o n between l i p o p r o t e i n s . They were d i s c o v e r e d t h r o u g h t h e i r c l i n i c a l m a n i f e s t a t i o n s , which r e q u i r e d l a b o r a t o r y i n v e s t i g a t i o n s , l e a d i n g i n t u r n t o t h e d i s c o v e r y o f t h e d e f i c i e n t l i p o p r o t e i n pattern. Apoprotein d e f i c i e n c i e s

( a ) Analphalipoproteinaemia (Tangier's d i s e a s e ) The more c o r r e c t t e r m i n o l o g y i s hypoalphalipoproteinaemia. The absence o f a - l i p o p r o t e i n s and abnormal c o n c e n t r a t i o n s o f t h e o t h e r plasma l i p o p r o t e i n s a r e t h e e l e c t r o p h o r e t i c i n d i c a t i o n s o f t h e syndrome. There i s a v e r y l o w t o t a l c h o l e s t e r o l and a normal o r e l e v a t e d t r i g l y c e r i d e c o n c e n t r a t i o n . C1 i n i c a l l y t h e l a r g e and y e l l o w t o n s i l s due t o c h o l e s t e r o l e s t e r d e p o s i t s a r e obvious. The same a l s o causes enlargement o f t h e l i v e r , s p l e e n and lymph nodes, and a l t e r a t i o n o f t h e cornea, i n t e s t i n a l mucosa and o t h e r t i s s u e s . T h i s d i s e a s e was d i s c o v e r e d i n 1961 b y F r e d r i c k s o n e t a l . 3 7 ,

t h e l i p o p r o t e i n anomaly was d e s c r i b e d by Lux e t 39 a l . 3 8 and an e x c e l l e n t paper on t h e p a t h o p h y s i o l o g y was p u b l i s h e d by Assman

.

( b l Abetalipoproteinaemia T h i s i s a d e f i c i e n c y o f t h e apo B p o l y p e p t i d e , which l e a d s t o t h e absence o f c h y l o m i c r a , VLDL and LDL. C l i n i c a l l y t h e r e i s f a t m a l a b s o r p t i o n , r e t i n i t i s p i g mentosa and a c a n t h o s i s o f t h e e r y t h r o c y t e s . The d i s e a s e had been d i s c o v e r e d i n 1950 and c a l l e d t h e Bassen-Kornzweig syndrome, b u t o n l y i n 1960 was t h e l i o o p r o t e i n anomaly d e t e c t e d . A good p a t h o p h y s i o l o g i c a l paper was D u b l i s h e d by Kostner 40

.

( c ) Hypobetalipoproteinaemia

T h i s d i s e a s e i s n o t a m i l d t y p e o f t h e p r e v i o u s one b u t an e n t i t y o f i t s own w i t h l o w apo B l e v e l s , f o r t u n a t e l y w i t h o u t t h e s e r i o u s c l i n i c a l syndrome des c r i b e d f o r t h e a b e t a l i p o p r o t e i n a e m i a . However, t h e homozygotes D r e s e n t complete a b e t a l i p o p r o t e i n a e m i a w i t h t h e same c l i n i c a l syndrome.

A f i r s t d e s c r i p t i o n was g i v e n i n 19604'.

The l i p o p r o t e i n p a t t e r n s have been

d e s c r i b e d by N a i t o and Lewis4*. An i n t e r e s t i n g comment on t h e advantage o f l o w

8- and h i g h a - l i p o p r o t e i n c o n c e n t r a t i o n s was made b y Glueck e t a l . 43

.

L e c i t h i n cholesterol a c y l transferase (LCAT) deficiency. I n t h i s syndrome,

l e c i t h i n c h o l e s t e r o l a c y l t r a n s f e r a s e , t h e enzyme w h i c h t r a n s f e r s a f a t t y a c i d f r o m l e c i t h i n t o c h o l e s t e r o l , t h u s g e n e r a t i n g a c h o l e s t e r o l e s t e r , i s absent. P a t i e n t s p r e s e n t v e r y h i g h plasma l e v e l s o f u n e s t e r i f i e d c h o l e s t e r o l and l e c i t h i n

270

and v e r y low c h o l e s t e r o l e s t e r and l y s o l e c i t h i n l e v e l s . T h e i r e r y t h r o c y t e s a r e b i o c h e m i c a l l y and m o r p h o l o g i c a l l y abnormal ( t a r g e t c e l l s ) . O t h e r t i s s u e s , such as t h e cornea, bone marrow and k i d n e y , accumulate l i p i d s , and t h i s generates c o r n e a l o p a c i t y , anaemia and p r o t e i n u r i a . I t seemed t h a t a - l i p o p r o t e i n was t o t a l l y absent i n c e l l u l o s e a c e t a t e s t r i p e l e c t r o p h o r e s i s , b u t i t s presence was demonstrated by ul t r a c e n t r i f u g a t i o n . Hence

t h i s i s n o t an a n a l p h a l i p o p r o t e i n e m i c d i s o r d e r . Abnormal f r a c t i o n s m i g r a t e as prealbumin and a l s o i n t h e a l p h a 2 r e g i o n . The p r e - 8 - l i p o p r o t e i n m a t e r i a l m i g r a t e s w i t h t h e 8 - l i p o p r o t e i n s , and t h i s i s p a r t i a l l y r e l a t e d t o a f a u l t y s y n t h e s i s o f apo B and apo C p o l y p e p t i d e s . The m o r p h o l o g i c a l aspect o f l i p o p r o t e i n p a r t i c l e s s e p a r a t e d by u l t r a c e n t r i f u g a t i o n i s c o m p l e t e l y abnormal: on t h e one hand t h e h i g h - d e n s i t y l i p o p r o t e i n s appear as d i s c s and on t h e o t h e r t h e s o - c a l l e d LDL f r a c t i o n c o n t a i n s a b n o r m a l l y l a r g e p a r t i c l e s which p r e s e n t m y e l i n f i g u r e s under t h e e l e c t r o n microscope.

A l l o f t h e s e symptoms o f an a b e r r a n t h y p e r l i p a e m i a o c c u r i n t h e course of t h e disease and may be p a r t i a l l y secondary t o t h e f a t d e n o s i t i n t h e t i s s u e s . A good i n t r o d u c t o r y s u r v e y o f t h e p h y s i o p h a t h o l o g y o f t h e disease, which

S

v e r y r a r e , was w r i t t e n by Glomset i n 197944 and i s s t i l l u s e f u l . Undoubtedly t h e s t u d y o f t h i s syndrome has added much t o o u r knowledge o f c h o l e s t e r o l metabo i s m and e s p e c i a l l y i t s e s t e r i f i c a t i o n . T h i s i s t h e main s i g n i f i c a n c e o f t h i s r a r e genetic abnormality. Patterns i n disease We observe d y s l i p o p r o t e i n a e m i a as a d e v i a t i o n o f t h e l i p o p r o t e i n p a t t e r n expressed b y combined l i p i d and a p o p r o t e i n anomalies. The m e t a b o l i c h i n g e of these a b n o r m a l i t i e s i s n o t always c l e a r o r single-handed. I t c o u l d i n d e e d l i e a t d i f f e r e n t l e v e l s : a d e f i c i t o r a d e v i a t i o n o f t h e synthesis o f apoprotein, o f l i p i d uptake by a p o p r o t e i n , a f a u l t y s u p p l y o f l i p i d components as a r e s u l t of d i e t a r y o r m e t a b o l i c e r r o r s o r l a s t l y a wrong m e t a b o l i c t u r n o v e r o f l i o o p r o t e i n s , be t h e y normal o r abnormal i n composition. I n l i p o p r o t e i n m a t t e r s , i n s p i t e of a l l t h e work i n t h e f i e l d , t h e r e a r e s t i l l c o u n t l e s s unknowns and many developments can be expected. I n t h e meantime, we a r e o b l i g e d t o s u b d i v i d e most d i s o r d e r s a c c o r d i n g t o disease, r a t h e r t h a n under causal headings as one would wish. The l i p o p r o t e i n anomalies have been s t r e a m l i n e d i n a p r a c t i c a l way i n t o t h e b a s i c F r e d r i c k s o n p a t t e r n s I t o V which r e p r e s e n t f i v e t y p i c a l p r o f i l e s , and t h i s nomenclature i s used i n d e p e n d e n t l y o f t h e cause o f t h e p r o f i l e . Arterial potZution and t y p e 11. F a u l t y a r t e r i a l w a l l metabolism induced t h e development o f atheroma l e s i o n s l o c a l i z e d i n some, i f n o t i n a l l , a r t e r i e s of

271

organism. S e l e c t i v e l o c a t i o n s w i t h dangerous anatomical consequences i n case o f subsequent o c c l u s i o n a r e t h e c o r o n a r i e s , t h e a r t e r i a l foramina a l o n g t h e a o r t i c stem, t h e r e n a l a r t e r i e s , t h e m e s e n t e r i a l a r t e r i e s , t h e l a r g e a r t e r i e s o f t h e l e g and o f t h e s a c r o i l i a c r e g i o n and t h e c e r e b r a l and r e t i n a l a r t e r i e s . An involvement of f a u l t y l i p o p r o t e i n metabolism i n a t h e r o s c l e r o t i c d i s e a s e i s c e r t a i n , b u t i s by no means t h e o n l y cause and even n o t t h e main cause o f a r t e r i a l p o l l u t i o n i n many cases. However, d i s o r d e r s o f t h e c h o l e s t e r o l l e v e l and l a t e r o f t h e l i p o p r o t e i n p a t t e r n have been e x t e n s i v e l y documented, e s p e c i a l l y i n c o r o n a r y 45-48 h e a r t d i s e a s e as we1 1 a f t e r u l t r a c e n t r i f u g a l 45 as e l e c t r o p h o r e t i c a n a l y s i s . The main l i p o p r o t e i n d i s t u r b a n c e observed i n a t h e r o s c l e r o s i s i s an abnormal p r o f i l e c h a r a c t e r i z e d b y a r e l a t i v e i n c r e a s e o f B- o r

LDL l i o o p r o t e i n and c a l l e d

t y p e I 1 o f F r e d r i c k s o n . I n o u r o p i n i o n t h e r e i s no reason t o e v a l u a t e t h e s e r i o u s ness o f an a r t e r i a l a c c i d e n t o r o f a r t e r i a l r i s k as a f u n c t i o n o f l i p o o r o t e i n o r l i p i d f i g u r e s . P s y c h o l o g i c a l l y , any r e a s o n a b l e i n v e s t i g a t o r o r p a t i e n t w i l l t r y t o do t h i s , b u t t h e f a c t t h a t t h e r e i s no c l e a r imiprovement i n r e i n f a r c t i o n r i s k a f t e r improvement o f t h e l i p o p r o t e i n and l i p i d p a t t e r n i s a s e r i o u s arqument f o r questioning t h e l o g i c s o f equating h i g h cholesterol w i t h h i g h r i s k . This l a s t sentence, t a k e n o u t o f c o n t e x t , i s o b v i o u s l y wrong because l o w c h o l e s t e r o l data, r e l a t i v e l y h i g h HDL c h o l e s t e r o l , and t h u s h i g h a - l i o o i p r o t e i n and l o w B - l i p o p r o t e i n , i.e.,

i n g e n e r a l t h e o p p o s i t e o f t y p e 11, a r e e v i d e n t l y b e t t e r i n d i c a t o r s

o f good h e a l t h and o f a v o i d i n g a r t e r i a l problems t h a n t h e o p p o s i t e . However, t o c o r r e l a t e t h e i n c r e a s e above normal w i t h l e v e l o f r i s k i s n o t p o s s i b l e a t p r e s e n t . There i s one e x c e p t i o n w i t h t h e t r u l y h e r e d i t a r y c o n g e n i t a l t y p e 11, a p p e a r i n g f r o m c h i l d h o o d , and accompanied by a v e r y h i g h c h o l e s t e r o l l e v e l . These o a t i e n t s have a v e r y h i g h r i s k o f a r t e r i a l p o l l u t i o n and a c c i d e n t s a r e a c o n s t a n t f e a t u r e o f t h i s disease. However, t h e t y p e I 1 a p p e a r i n g w i t h age i s n o t a p r e r e q u i s i t e f o r a r t e r i a l accidents, nor i s a r t e r i a l p o l l u t i o n o n l y confined t o these acquired type I 1 patients.

A good paper i n s i s t i n g on t h e i n y t o f a - l i p o p r o t e i n l e v e l s , and w h i c h r e q u e s t s 49 measures i n o r d e r t o a v o i d t y p e I 1 p a t t e r n s , was p u b l i s h e d b y R i f k i n d e t a l .

.

I t c o n t a i n s good d a t a on c h o l e s t e r o l l e v e l s as w e l l i n h i g h as i n l o w d e n s i t y

lipoproteins.

A newer approach t o l i p o p r o t e i n e v a l u a t i o n i s t o e v a l u a t e p h o s o h o l i o i d d a t a i n High r a t i o s mean h i g h c h o l e s t e r o l

terms o f t h e c h o l e s t e r o l t o p h o s p h o l i p i d r a t i o 2 ’ .

and l o w e r r a t i o s mean h i g h e r amounts o f p h o s o h o l i p i d s comoared w i t h c h o l e s t e r o l . T h i s o b v i o u s l y corresponds t o a h i g h l e v e l o f HDL l i p o p r o t e i n , w h i c h is t h e main p h o s p h o l i p i d c a r r i e r i n t h e organism. F o r t h i s reason a low t o t a l serum C:PL r a t i o i s indicative o f high a-lipoprotein levels. Another approach towards more r e f i n e d d i a g n o s i s i s t h e e v a l u a t i o n o f t h e phospholipid pattern, e s p e c i a l l y o f t h e l e c i t h i n o r phosphatidylcholine concentra-

212

t i o n s i n plasma and e v e n t u a l l y i n a - l i p o p r o t e i n s .

Work on human-like e x p e r i m e n t a l

animals, such as t h e chimpanzee, and on man has been performed and s t r e s s e s t h e 49 importance o f h i g h l e c i t h i n l e v e l s i n plasma and i n a - l i p o p r o t e i n

.

R e c e n t l y t h e d e t e r m i n a t i o n o f p h o s p h o l i p i d s i n a-1 i p o p r o t e i n o r HDL s u b f r a c 50

t i o n s was shown t o be an i m p o r t a n t parameter i n l i p o p r o t e i n d e t e r m i n a t i o n s

.

It i s i m p o r t a n t t o observe how t y p e 11, which o r i g i n a l l y s t a r t e d o u t as a h i g h

@ - l i p o p r o t e i n p a t t e r n and was f e a r e d as such has t o be l o o k e d a t i n a d i f f e r e n t way Nowadays t h e a - l i p o p r o t e i n l e v e l and a s t u d y o f a - l i p o p r o t e i n c o n t e n t such as t h e d e t e r m i n a t i o n o f HDL p h o s p h o l i p i d s and HDL c h o l e s t e r o l have tended t o supersede t h e n e g a t i v e aspect o f an e l e v a t e d @ - l i p o p r o t e i n c o n c e n t r a t i o n . It would be wrong t o l e a v e t h i s s e c t i o n w i t h o u t m e n t i o n i n g t y p e s I 1 A and I 1 B,

which d i f f e r i n t r i g l y c e r i d e c o n c e n t r a t i o n . Whether t h i s b i o c h e m i c a l d i f f e r e n c e has any r e a l c l i n i c a l meaning i s q u e s t i o n a b l e . Type I 1 B may be an i n t e r e s t i n g barometer f o r some c a r b o h y d r a t e o r a l c o h o l excess i n t h e d i e t on t o p o f e x c e s s i v e c a l o r i e i n t a k e and wrong l i p i d d i s t r i b u t i o n . Some s t r e a m l i n e d d a t a ( T a b l e 11.9) i l l u s t r a t i n g t h e b l o o d c h e m i s t r y o f t y p e I 1 and t y p e I V i s a f i t t i n g c o n c l u s i o n and an i n t r o d u c t i o n t o t h e f o l l o w i n g sec29

tion

.

TABLE 11.9 PLASMA LIPIDS AND LIPID RATIOS I N NORMAL AND HYPERLIPOPROTEINAEMIC PATIENTS C/PL i s t h e c h o l e s t e r o l : p h o s p h o l i p i d r a t i o . L/S i s t h e l e c i t h i n : s p h i n g o m y e l i n ratio.

Normal Type I 1 A Type I 1 B Type I V

Total c h o l e s t e r o l (C) (g/l)

Phospholipid (PL) (g/l)

C/PL ratio

L/S ratio

TG (g/l)

1.90 3.00 3.00 2.70

2.30 2.80 2.80 3.00

0.85 1.10 1.10 0.90

4.0 3.1 3.3 5.0

0.90 1.00 1.80 3.00

Here again, o v e r s i m p l i f i e d d a t a focus a t t e n t i o n on t h e e s s e n t i a l s and f a v o u r t h e c l i n i c i a n ' s i n t e r e s t i n t h e meaning o f t h e l i p o p r o t e i n p a t t e r n r a t h e r t h a n

i n t h e f o r m a l types. A l a s t remark i s concerned w i t h t h e r a p e u t i c measures. It seems c l e a r t h a t a t r e a t m e n t - i n d u c e d i n c r e a s e i n t h e a - l i p o p r o t e i n l e v e l i s f a v o u r a b l e as a p r o t e c t i v e agent a g a i n s t v a s c u l a r a c c i d e n t s . T h i s a g a i n uroves t h e i m p o r t a n c e o f good a - l i p o p r o t e i n l e v e l s w i t h a l o w 6- t o a - l i p o p r o t e i n r a t i o above t h e i n creased 6 - l i p o p r o t e i n which s t a r t e d a l l t h e fuss.

273 Diabetes and t y p e IV. A d i s t u r b a n c e o f t h e m e t a b o l i c pathway f o r glucose, i n c l u d i n g an excess o f c a r b o h y d r a t e i n t a k e i n p e o p l e w i t h low r e s i l i e n c e t o h i g h charges o f c a r b o h y d r a t e s i n t h e i r d i e t , i s accompanied by a t y p i c a l i n c r e a s e i n t r i g l y c e r i d e s as t h e main l i p i d d i s t u r b a n c e . T h i s expresses i t s e l f i n h i g h pre-8and c o m p a r a t i v e l y h i g h 8 - l i p o p r o t e i n l e v e l s . The a - l i p o p r o t e i n and, i n f a c t , t h e c h o l e s t e r o l mechanism o f t h e plasma serum seem n o t t o be p r i m a r i l y i m p a i r e d . As a r e s u l t o f t h i s s i t u a t i o n a t y p e I V o f F r e d r i c k s o n a p p e a r s , a p r o f i l e w i t h conspicuous i n c r e a s e s i n pre-6- and 8 - l i p o p r o t e i n s . We r e f e r t o Table 11.9 f o r t h e l i p i d data. An e x t e n s i v e s t u d y o f d y s l i p o p r o t e i n a e m i a i n d i a b e t e s has been made by O p p l t

51

.

One o f t h e i m p o r t a n t c o n c l u s i o n s was t h a t d i a b e t i c s do n o t o n l y p r e s e n t t y p e IV b u t f r e q u e n t l y a l s o t y p e s I1 A and I 1 6. I n p r a c t i c e , most d i a b e t i c s (54%) p r e s e n t t y p e I 1 A, 24% a r e t y p e I 1 B and o n l y 22% t y p e IV51. T h i s o b s e r v a t i o n i s o f g r e a t p r a c t i c a l importance t o t h e c l i n i c i a n . Moreover, t h e h y p e r t r i g l y c e r i d a e m i a o f decompensated d i a b e t e s , w h i c h was a t t h e o r i g i n o f t h e o b s e r v a t i o n s on t h e l i p i d d i s t u r b a n c e i n t h i s disease, i s f a r l e s s common t o d a y t h a n i t used t o be. I t can be q u e s t i o n e d whether t y p e IV, i n b o t h d i a b e t i c and n o n - d i a b e t i c p a t i e n t s , i s t h e e x p r e s s i o n o f l i v e r impairment r a t h e r t h a n o f d i a b e t e s as such. Even d i a b e t i c s w i t h v a s c u l a r d i s e a s e do n o t p r e s e n t more l i p o p r o t e i n p a t h o l o g y t h a n t h o s e w i t h o u t c o m p l i c a t i o n s such as t h e K i m m e l s t i e l - W i l s o n syndrome o r c a r d i o v a s c u l a r a c c i d e n t s . I n g e n e r a l , d i a b e t i c s have a much h i g h e r i n c i d e n c e o f c a r d i o v a s c u l a r l e s i o n s , and t h i s o b v i o u s l y i n c l u d e s t h e 75% w i t h t y p e I 1 A and B i n t h i s disease. These o b s e r v a t i o n s demonstrate t h a t l i p o p r o t e i n p a t t e r n s such as t y p e I 1 o r

IV s h o u l d be r e l a t e d t o l i p o p r o t e i n metabolism and t h a t t h e d i a g n o s i s and t r e a t ment o f d y s l i p o p r o t e i n a e m i a a r e a s e p a r a t e t o p i c w i t h i n t h e p a t h o l o g y o f any g i v e n disease. F o r t h i s reason t h e r e i s even no d i r e c t correspondence between t y p e I1 and t h e v a s c u l a r a c c i d e n t s , and t y p e IV i s c e r t a i n l y n o t t h e s i g n a t u r e o f d i a b e t e s b u t o n l y an i n d i c a t i o n o f t h e p o s s i b i l i t y o f t h i s d i a g n o s i s . O n l y

t h e metabolism o f l i p o p r o t e i n l i p i d s and a p o p r o t e i n s w i l l e n a b l e us t o s e p a r a t e b i o c h e m i c a l d i s o r d e r s o r p a t h o p h y s i o l o g i c a l e n t i t i e s which can c o m p l i c a t e a l l kinds o f c l i n i c a l s i t u a t i o n s .

It i s f o r t h e same reason t h a t d i e t a r y excesses

generate F r e d r i c k s o n t y p e s i n normals and t h e s e t y p e s a r e i n d i s t i n g u i s h a b l e from t h e r a r e r c o n g e n i t a l d i s t u r b a n c e s such as t h e t r u l y h e r e d i t a r y t y p e I 1 disease.

Rarer hyperZipoproteinaemias: t y p e I, t y p e III and t y p e IV. The p a t t e r n s I ,

I11 and V have been compared w i t h t h e normal r e f e r e n c e p a t t e r n as a t r a n s i t i o n towards abnormal l i p o p r o t e i n p r o f i l e s a t t h e end o f t h e s e c t i o n on t h e r e f e r e n c e p a t t e r n (p. 263).

214 ( a ) Type

I

The b a s i c d e s c r i p t i o n o f t y p e I b y F r e d r i c k s o n and Lees6 c h a r a c t e r i z e s t h i s p a t t e r n as an excessive, f a t - i n d u c e d hyDerchylomicronaemia. C i r c u l a t i n g trig l y c e r i d e s , which a r e t h e main components o f t h e c h y l o m i c r a , seem t o accumulate i n t h e p e r i p h e r a l b l o o d and a r e a l s o s t o r e d i n t h e f a t c e l l s as combustion i n t h e l i v e r seems t o be impaired. T h i s r e s u l t s f r o m an enzymatic d e f i c i e n c y i n t h e normal l i p o l y s i s induced b y h e p a r i n , i . e . ,

t h e post-heparin l i p o l y t i c a c t i v i t y . This

disease i s n o t accompanied by i n t o l e r a n c e t o glucose and i s a r a r e syndrome.

( b ) Type 111 I n t y p e I 1 1 t h e main c u l p r i t i s a VLDL i n c r e a s e t o g e t h e r w i t h a " b r o a d 8 - l i p o p r o t e i n " band as a t y p i c a l p a t t e r n . The p a t i e n t s p r e s e n t p l a n a r and tuberous xanthomata and a r t e r i a l p o l l u t i o n i s a common c o m p l i c a t i o n . The LDL d a t a a r e a l s o i n c r e a s e d and u l t r a c e n t r i f u g a l a n a l y s i s shows t h e f l o a t i n g 8 - l i p o o r o t e i n s . P o l y a c r y l a m i d e g e l e l e c t r o D h o r e s i s (PAGE) was recommedded by Masket5* f o r a c o r r e c t d i a g n o s i s based on t h e v e r y l o w m o b i l i t y o f t h e p r e - 8 - p a r t i c l e s . A p o p r o t e i n s t u d i e s have demonstrated t h e e x i s t e n c e o f a p o p r o t e i n r i c h a p o p r o t e i n i n VLDL, and one o f these p o l y p e p t i d e s F r e d r i c k s o n t y p e I 1 1 p a t i e n t s which a r e

E I1

(E

E, t h e a r g i n i n e -

111) i s absent i n

homo zygote^^^.

T h i s i n d i c a t e s a spe-

c i f i c b i o c h e m i c a l d e f i c i e n c y i n t h i s disease and c o u l d a l s o i n t e r p r e t t h e d e t e c t i o n

54

.

o f a d o u b l e 8 - l i p o p r o t e i n band i n agarose p a t t e r n s (el Type V The t y p e V p a t t e r n i s i n some way a sum o f a l l o f t h e p r e v i o u s m e t a b o l i c e r r o r s , i n c l u d i n g an i n c r e a s e i n

CM, pre-6- and 8 - l i p o D r o t e i n . There i s a simultaneous

e r r o r o f endogenous pre-8-1 ip o p r o t e i n and exogenous c h y l omi c r o n p a r t i c l e s . The b u i l d - u p o f t h i s s i t u a t i o n p r o b a b l y r e s u l t s f r o m a l a c k o f c l e a r i n g o f VLDL. There a r e no xanthomata, b u t v a s c u l a r a c c i d e n t s a r e f r e q u e n t . The a - l i p o p r o t e i n band i s l o w e r t h a n normal. The d i s e a s e i s r a r e b u t can be mimicked b y extreme cases o f d i a b e t e s , h y p o t h y r o i d i s m , n e p h r o s i s o r a l c o h o l i s m 5 5 y 5 6 . The n a t i e n t s a r e s e n s i t i v e t o h e p a r i n and t u r b i d i t y c l e a r s up w e l l w i t h i n 15 min, which does n o t occur i n t y p e

I.

Liver disease. The l i v e r i s one o f t h e main chemical energy t r a n s d u c e r s o f t h e organism and o b v i o u s l y a s i t e o f i n t e n s e a p o p r o t e i n , l i p i d and l i p o p r o t e i n metabolism. A c l e a r l i p i d disturbance i s t h e hypercholesterolaemia o f cholestasis accompanied by a p r o f o u n d d i s t u r b a n c e o f t h e l i p o p r o t e i n p a t t e r n and t h e appearance o f l i p o p r o t e i n X. The c h o l e s t a s i s may f i n d i t s o r i g i n i n e x t r a - o r i n t r a h e p a t i c causes w h i c h have t h e same p h y s i o p a t h o l o g i c a l e f f e c t . There a l s o e x i s t l e s s c l e a r anomalies which appear d u r i n g g e n e r a l l i v e r d i s t u r b a n c e , and t h e s e i n v o l v e LDL and VLDL i n c r e a s e s , an HDL decrease and i n g e n e r a l h y p e r t r i g l y c e r i d a e m i a . These p a t t e r n s have a tendency t o s i m u l a t e t y p e I V o r t y p e V i n v e r y s e r i o u s cases.

275

l a ) Lp-X i n c h o l e s t a s i s H y p e r c h o l e s t e r o l a e m i a of c h o l e s t a s i s , accompanied by v e r y h i g h l e v e l s o f phosphol i p i d s and normal t r i g l y c e r i d e s , c h a r a c t e r i z e s t h e chemical c o m o o s i t i o n o f t h e plasma which on e l e c t r o p h o r e s i s seems t o l a c k o r a t l e a s t t o be v e r y Door i n a - l i p o p r o t e i n s b u t p r e s e n t s a s t r o n g 8 - l i p o p r o t e i n band. T h i s B - l i n o n r o t e i n r e a c t s t o HDL a n t i s e r u m and t h u s t h e s o - c a l l e d 8 - l i p o p r o t e i n band a l s o c o n t a i n s abnormal a-lipoproteins.

The c o m p o s i t i o n of t h i s p r o t e i n was g i v e n i n t h e f i r s t s e c t i o n .

The p r o t e i n c o m p o s i t i o n i s m a i n l y a l b u m i n , t o g e t h e r w i t h C I, C 11, C I11 and D a p o p o l y p e p t i d e s . Under m i g r a t i o n i n a g a r g e l t h e f r a c t i o n moves s l i g h t l y s l o w e r t h a n t h e 8 - l i p o p r o t e i n band, and t h i s i s t y p i c a l f o r Lp-X ( r e f . 20). A n t i s e r u m a g a i n s t Lp-X r e a c t s n o t o n l y w i t h Lp-X b u t of course a l s o w i t h a l b u m i n , as Lp-X c o n t a i n s l a r g e amounts o f albumin i n i t s p r o t e i n m o i e t y . A p r o t e i n p a r a l l e l t o Lp-X i s p r e s e n t i n p a t i e n t s w i t h LCAT d e f i c i e n c y 57,58

,

b u t whether Lp-X p h o s p h o l i p i d i s a v a l i d s u b s t r a t e f o r t h e LCAT enzyme i s n o t c l e a r . I t i s i m p o r t a n t t o n o t e t h a t t h e d e m o n s t r a t i o n o f Lp-X i s pathognomonic f o r c h o l e s t a s i s , a p a r t f r o m t h e minimal chance o f an LCAT d e f i c i e n c y .

A s i m p l e e l e c t r o p h o r e t i c t e s t k i t (Rapidophor) f o r d e m o n s t r a t i n g t h e nresence o f Lp-X based on t h e work o f Wieland and S e i d e l "

i s a v a i l a b l e (Immuno, Vienna).

Ibl Hypertriglyceridaemia A v e r y d i f f e r e n t t y p e o f d i s t u r b a n c e i s t h e i n c r e a s e i n c i r c u l a t i n g LDL i n

l i v e r d i s e a s e s i m u l a t i n g t y p e I V and a l s o found i n a l c o h o l i c l i v e r s . T h i s f r a c t i o n has more a 8 - l i p o p r o t e i n t h a n a p r e - 8 - l i p o p r o t e i n m o b i l i t y due t o an enormous t r i g l y c e r i d e l o a d . These anomalies a r e n o t c l e a r b u t h e l p t o i n t e r D r e t t h e f u z z y images found i n some l i p o p r o t e i n p a t t e r n s i n l i v e r disease.

I c ) HDL-related problems The r e l a t i v e absence o f HDL i n l i v e r d i s e a s e i s n o t due t o t h e absence o f t h e apo A p o l y p e p t i d e s b u t t o some problems i n t h e b i n d i n g mechanism f o r n e u t r a l l i p i d s . The apo A 1 1 - p h o s p h o l i p i d complex, v e r y poor i n o t h e r 1 i D i d s and o r e s e n t i n l i v e r disease, i s p o o r l y d e t e c t a b l e by e l e c t r o o h o r e s i s " .

Possibly a lack o f synthesis

o f apo C i s r e s p o n s i b l e f o r t h e s e abnormal HDL p a r t i c l e s .

Renal disease. The n e p h r o t i c syndrome was d e s c r i b e d as one o f t h e f i r s t s e r i ous p r o t e i n anomalies

-

and l a t e r l i p o p r o t e i n anomalies

-

detected by u l t r a c e n t r i -

f u g a t i o n and by f r e e e l e c t r o p h o r e s i s . These anomalies a r e accompanied by albumin d e f i c i e n c y , a v e r y h i g h 8 - l i p o p r o t e i n l e v e l and is r e m i n i s c e n t o f t h e m a l n u t r i t i o n p a t t e r n i n kwashiorkor. Here again a l l t y p e s o f l i p o p r o t e i n anomaly, e x c e p t t y p e I , have been d e t e c t e d . 59 Hyperbetalipoproteinaemia o f t y p e s I 1 B, I V and V i s t h e most f r e q u e n t . I t i s w e l l known t h a t a r t e r i a l problems and a c c i d e n t s c o m p l i c a t e t h e e v o l u t i o n o f r e n a l f a i l u r e even under d i a l y s i s . P a t t e r n s under d i a l y s i s have a tendency t o

276

n o r m a l i z e i f t h e uraemia i s w e l l regulated6'.

A f t e r t r a n s p l a n t a t i o n t h e administra-

t i o n o f immunosuppressive drugs t o g e t h e r w i t h a h i g h c a r b o h y d r a t e i n t a k e t e n d t o support high l e v e l s o f pre-B-lipoproteins 61

.

A v e r y r a r e syndrome, t h e f a m i l i a l LCAT d e f i c i e n c y w i t h p r o t e i n u r i a , was ment i o n e d i n t h e s e c t i o n on g e n e t i c d i s o r d e r s . Gout. An o l d c l i n i c a l o b s e r v a t i o n i s t h e l i n k between g o u t and d i a b e t e s as

b e l o n g i n g w i t h i n t h e same h e r e d i t a r y p a t t e r n . There i s a l s o a h i g h i n c i d e n c e o f a t h e r o s c l e r o t i c l e s i o n s i n c l u d i n g n e p h r o s c l e r o s i s as a c o m p l i c a t i o n o f t h e i n h e r i t e d d i s o r d e r . However, t h e e x a c t r e l a t i o n s h i p between l i p i d s and l i p o p r o t e i n s and t h e u r i c a c i d metabolism i s n o t c l e a r 62,63

.

Anyhow, gout i s accompanied by a h i g h i n c i d e n c e o f abnormal l i p o p r o t e i n p a t t e r n s even i n t h e absence o f r e n a l impairment o r v a s c u l a r a c c i d e n t s . D y s l i p o p r o teinaemia, however, cannot be used f o r t h e d i a g n o s i s o f g o u t n o r mentioned

-

-

as a l r e a d y

o f d i a b e t e s , whereas i n b o t h diseases t h e l i p o p r o t e i n d i s o r d e r s a r e

v e r y f r e q u e n t . C l i n i c a l r e a d e r s s h o u l d bear i n mind t h a t many papers a r e based on s t a t i s t i c a l evidence w h i c h has no b e a r i n g on t h e d i a g n o s i s o f an i s o l a t e d case. L i p o p r o t e i n p a t h o l o g y i n d i c a t e s t h e danger o f a r t e r i a l p o l l u t i o n i n a g i v e n d i s e a s e s t a t e b u t i s n o t a t o o l f o r d i a g n o s i n g a d i s e a s e as such.

Thyroid disorders. The c o r r e l a t i o n between c h o l e s t e r o l l e v e l and m e t a b o l i c r a t e i s n e a r l y as o l d as c l i n i c a l b i o c h e m i s t r y . The t h y r o i d f u n c t i o n i s one o f t h e c l e a r e s t a c t i v a t o r s o f l i p i d metabolism even i f t h e mechanism o f t h e a c c e l e r a t i o n r e s u l t s from e f f e c t s a t m u l t i p l e l e v e l s . T r i g l y c e r i d e s a r e a l o g i c a l r e s u l t o f t h e i n c r e a s e d t u r n o v e r o f f a t t y a c i d s i n t h e l i v e r under h y p e r t h y r o i d i s m . These changes s h o u l d o b v i o u s l y r e f l e c t themselves i n t h e l i p o p r o t e i n p a t t e r n

64

.

A c o n s t a n t f e a t u r e o f h y p e r t h y r o i d i s m i s t h e low c h o l e s t e r o l l e v e l w h i c h f o l l o w s t h e l o s s of LDL and a c t s as i n d i r e c t p r o t e c t i o n a g a i n s t a r t e r i a l p o l l u t i o n . However, t r e a t m e n t o f i n f a r c t i o n w i t h t h y r o i d hormones o r t h e i r d e r i v a t i v e s had t o be abandoned because h y p e r t r i g l y c e r i d a e m i a o c c u r r e d and t h e c l i n i c a l symptoms, such as angor, were n o t r e l i e v e d . Treatment o f h y p e r l i p i d a e m i a by means o f a crude mechanism such as t h e t h y r o i d hormone c h o l e s t e r o l l o w e r i n g e f f e c t i s an example o f i n c o r r e c t l o g i c . The l o w e r i n g o f c h o l e s t e r o l i n i t s e l f i s n o t a l o g i c a l aim f o r a t h e r a p y a i m i n g a t a r e d u c t i o n o f a r t e r i a l p o l l u t i o n . Any good t h e r a p e u t i c management w i l l i n c l u d e t h e i d e a o f a l t e r i n g o r i m p r o v i n g t h e d i s t r i b u t i o n o f plasma l i p i d s o v e r t h e l i p o p r o t e i n s and f o r t h i s reason p h y s i o l o g i c a l means, such as e a t i n g l e s s o r t a k i n g e x e r c i s e , n o t t o f o r g e t t h e e s t r o g e n l e v e l s i n t h e normal female, f a v o u r a- o v e r B - l i p o p r o t e i n s . Such measures and f a c t s go t o t h e r o o t o f t h e problem, have a chance o f b e i n g u s e f u l and a r e w i t h o u t danger.

The opposite s i t u a t i o n , thyroid deficiency, gives r i s e t o high cholesterol and lipoprotein l e v e l s which r e a c t p e r f e c t l y t o thyroid hormone administration. The level of hormone required can be derived from the normality of t h e plasma lipoprotein and l i p i d l e v e l s . Care should be taken t o e s t a b l i s h only progressive r e s t o r a t i o n t o normality because the h e a r t muscle i s severely impaired by t h e anoxaemia of hypothyroidism and angor pains can a r i s e when t h e heart r a t e has t o increase too rapidly i n the f i r s t weeks of a too energetic thyroid therapy. In thyroid syndromes lipoprotein p a t t e r n s a r e useful , even necessary, f o r evaluating the seriousness of t h e disease and the e f f e c t of s u b s t i t u t i o n therapy. NEW DEVELOPMENTS A discussion about the lipoprotein p a t t e r n s which have been with c l i n i c a l chemists and c l i n i c i a n s f o r many years requires a conclusion f o r each of the two groups i nvol ved. The problem r e s t i n g with t h e laboratory i s t o achieve b e t t e r q u a n t i t a t i o n of lipoprotein r a t i o s , including an evaluation of t h e apoprotein and phospholipid pattern e x i s t i n g in a-lipoproteins. The importance o f a-lipoprotein follows from physico-chemical s t u d i e s on apoprotein - phospholipid binding, together w i t h epidemiological data confirmed by c l i n i c a l experience. To j o i n t h e laboratory and t h e c l i n i c an e f f i c i e n t and automated lipoprotein l i p i d evaluation system i s needed. This will require, unfortunately, more sophist i c a t e d equipment than i s in use today. Modern biochemical methodology and data acquisition will be needed i n order t o produce data useful t o the c l i n i c i a n . I t can be foreseen t h a t e l e c t r o p h o r e t i c techniques will be adaoted t o and combined with subsequent chemical determination of both apoproteins and l i p i d s . The mul-

t i p l e data obtained w i l l have t o be evaluated by an e f f e c t i v e software i n o r d e r t o draw p r a c t i c a l and useful conclusions from complex biochemical data. REFERENCES 1 H. Peeters, i n H. Peeters ( E d i t o r ) , Proc. t u t e on the Lipoprotein Molecule, Bruges, York, 1978, pp. 3-8. 2 H. Peeters, i n H. Peeters ( E d i t o r ) , Proc. t u t e on the Lipoprotein Molecule, Bruges, York, 1978, pp. 295-298. 3 F.T. Lindgren, L.C.Jensen and F.T. Hatch,

of the Nato Advanced Study I n s t i May 8-20, 1977, Plenum Press, New

of the Nato Advanced S t u d y . I n s t i May 8-20, 1977, Plenum Press, New

i n G.J. Nelson ( E d i t o r ) , Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism., R. E . Krieger Publishing, Huntingdon, New York, 1979, Ch. 5, pp. 181-274. 4 P.N. Herbert, L.L. Bausserman, L . O . Henderson, R.J. Heinen, M.J. LaPiana, E.C. Church and R.S. Schulman, in H. Peeters ( E d i t o r ) , Proc. of the Nato Advanced Study I n s t i t u t e on the Lipoprotein Molecule, Bruges, May 8-10, 1977, Plenum Press, New York, 1978, pp. 35-56.

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5 6 7 8

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-

219

39 G. Assmann, i n H. P e e t e r s ( E d i t o r ) , PhosphatidyZchoZine, Proc. of a Symposiwn, Bmgge, Non. 25-18, 1975, S p r i n g e r Verlag, B e r l i n , H e i d e l b e r g , 1976, PO. 3446. 40 G. Kostner, I n K. Rommel , H. Goebell and R. Bohmer ( E d i t o r s ) , Lipid Absorption: Biochemical and CZinicat Aspects, MTP Press , 1976. 4 1 H.B. S a l t , O.H. W o l f f , J.K. L l o y d , A.S. Fosbrooke, A.H. Cameron and D.V. Hubble, Lancet, ii (1960) 325. 42 H.K. N a i t o and L.A. Lewis, CZin. Chem., 2 1 (1975) 990. 43 C.J. Glueck, P. G a r t s i d e , R.W. F a l l a t , J. S i e l s k i and P.M. S t e i n e r , J . Lab. CZin. Med., 88 (1976) 941. 44 J.A. Glomset, i n G.J. Nelson ( E d i t o r ) , Hood L i p i d s and Lipoproteins: Quantitation, Composition and Metabolism, R.E. K r i e g e r P u b l i s h i n g , Huntingdon, New York, 1979, Ch. 14, pp. 745-787. 45 J. Gofman, F.T. L i n d g r e n , H. E l l i o t t , W. Mantz, J . H e w i t t , B. S t r i s o w e r , V. H e r r i n g and T.P. Lyon, Science, 111 (1960) 166. 46 D.S. F r e d r i c k s o n , R . I . Levy and R.S. Lees, N . EngZ. J . Ned., 276 (1967) 34-43, 94-103, 148-155, 215-224, 273-281. 47 L.W. K i n s e l l , G. S c h l i e r f , W. Kahlke and G. S c h e t t l e r , i n G.J. S c h e t t l e r ( E d i t o r ) , L i p i d s and Lipidoses, Vol 1, S p r i n g e r V e r l a g , New York, 1967, pp. 446-489. 48 G. S c h e t t l e r , W . Kahlke and G. S c h l i e r f , i n G. S c h e t t l e r ( E d i t o r ) , Lipids and Lipidoses, S p r i n g e r Verlag, New York, 1967, pp. 412-445. 49 B.M. R i f k i n d , I. Tamir and G. Heiss, i n A.M. Gotto, Jr., N.E. M i l l e r and M.F. 01 iv e r ( E d i t o r s ) , Proc. 3rd ArgenteuiZ Symposiwn, High Density Lipoproteins and AtheroscZerosis, WaterZoo, Nov. 7-8, 1977, E l s e v i e r , Amsterdam, 1978, pp. 109- 119. 50 A. Kuksis, J.J. Myher, W.C. B r e c k r i d g e , J.A. L i t t l e , i n K. L i o p e l ( E d i t o r ) , Report of the High Density Lipoprotein MethodoZogy Workshop, Sun Francisco, March 12-14, 1979, N I H P u b l i c a t i o n , 1979, pp. 142-163. 5 1 J.J. O p p l t , i n L.A. Lewis and J.J. O p p l t ( E d i t o r s ) , CRC Handbook of EZmtrophoresis, VoZ. 11, Lipoproteins in Disease, CRC Press, Boca Raton, 1980, pp. 121-184. 52 B.H. Masket, i n L.A. Lewis and J.J. O p p l t ( E d i t o r s ) , CRC Handbook of EZectrophoresis, VoZ. 11, Lipoproteins in Disease, CRC Press, Boca Raton, 1980, pp. 79-102. 53 G. Uterman, M. Jaeschke and J. Menzel, FEBS Lett., 56 (1975) 352. 54 N.M. Papadopoulos and P.N. H e r b e r t , CZin. Chem., 2 3 (1977) 978. 55 W.V. Brown, M.L. Baginsky and C. Enholm, i n B.M. R i f k i n d and R . I . Levy ( E d i t o r s ) , HyperZipiduemia: Diagnosis and Therapy, Grune and S t r a t t o n , New York, 1977, p. 93. 56 L.A. Simons and P.F. W i l l i a m s , C Z i n . Chim. Acta, 6 1 (1975) 341. 57 D. S e i d e l , E. Gjone, J.P. Blomhoff and H.P. Geisen, I n t . Symp. Lipid Metabolism, Obesity and Diabetes Me ZZitus: Impact upon AtheroscZerosis, Wiesbaden, A p r i l , 1972.

58 H. T o r s v i k , K. Berg, H.N. Magnani, W.J. McConathy, P. A l a u p o v i c and E. Gjone, FEBS L e t t . , 24 (1973) 165. 59 S.R. Newmark, C.F. Anderson, J.V. Donadid and R.D. E l l e f s o n , Mayo CZinic h o e . , 50 (1975) 359. 60 J.D. Bagdade, D. P o r t e and C.L. Bierman, N . EngZ. J. Med., 279 (1968) 181. 6 1 P. Glosh, D.B. Evans, S.A. Tomlinson and R.Y. Calne, Transplantation, 15 (1973) 521. 62 Y. N i s h i d a , I. Akala, T. Nishizawa and T. Yoshimura, CZin. Chim. A c t a , 62 (1975) 103. 63 H.K. N a i t o and A.M. Mackenzie, CZin. Chem., 25 (1979) 371. 64 B. S t r i s o w e r , J.W. Gofman, E.F. G a l i o n i , J.H. Rubinger, J. Pouteau and P. Guzvich, Lancet, i (1957) 120.

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281

Chapter 1 2 LIPOPOLYSACCHARIDES PAUL F. COLEMAN and OTHMAR GABRIEL L i p o p o l y s a c c h a r i d e s (LPS) a r e complex molecules f o u n d i n t h e o u t e r membrane o f gram n e g a t i v e b a c t e r i a . A r e p r e s e n t a t i v e s t r u c t u r e f o r t h i s t y p e o f m o l e c u l e i s shown i n F i g . 12.1.

L i p i d A, c o n t a i n i n g t h e e s t e r i f i e d f a t t y a c i d s shown i n t h e

f i g u r e i n v a r i o u s p r o p o r t i o n s , c o n f e r s n e t hydrophobic p r o p e r t i e s t o t h e m o l e c u l e which cause a g g r e g a t i o n i n aqueous media and make i t necessary t o conduct e l e c t r o p h o r e t i c f r a c t i o n a t i o n s i n t h e presence o f d e t e r g e n t . The e x a c t p o s i t i o n and

qp-EtNH2-

Gal ‘Glc-Hep-Hep-~I(DO--KOO GlcFAc

(*re

Man-Rha-Gal

L

f

-o-antigen&. poLyrner

-Gal

/

12-

I HeD

H

R

4

0

!

BHMA

KDO P

BHMA

I

A”

Carbohydrate

F

I

E ~ N H ~

moiety

-

--Lipid

core

A moiety--

3- M y r i s t o x y m y r i s t a t e

F i g . 12.1. The g e n e r a l l y accepted chemical s t r u c t u r e o f SuZrnoneZZu typhimuriurn LT2 i s g i v e n above. The v a r i a b l e s u b s t i t u t i o n of f a t t y a c i d s on t h e L i p i d A m o i e t y i s i n d i c a t e d . BHMA = B - h y d r o x y m y r i s t i c a c i d , KDO = 2-keto-3-deoxyoctonate (3-deoxyD-mannooctulosonate), Hep = L-glycero-D-mannoheptose, Abe = Abequose, Rha = L-rhamnose, AraNH2 = 4-amino-4-deoxy-L-arabinose, E t N H 2 = ethanolamine.

d i s t r i b u t i o n of f a t t y a c i d residues e s t e r i f i e d t o t h e L i p i d A moiety are a t pres e n t unknown. N a t i v e LPS e x h i b i t s a s i n g l e s t a i n a b l e zone i n g e l e l e c t r o f o c u s i n g i n t h e presence o f n o n - i o n i c detergent’”.

S i m i l a r l y , g e l e l e c t r o p h o r e s i s o f LPS

i n n o n - i o n i c d e t e r g e n t y i e l d s a broad smear w h i c h does n o t appear u s e f u l f o r r e s o l v i n g LPS. Therefore, a l l f r a c t i o n a t i o n s o f LPS c o n s i d e r e d i n t h i s r e v i e w

282

F i g . 12.2. D i s c o n t i n u o u s SDS-PAGE o f (SaZrnoneZZa typhirnuriurn LT2) - LPS ( r e f . 1 2 ) . Gel c o n c e n t r a t i o n : 10% T , 2.5% C. The bands a r e v i s u a l i z e d by t h e p e r i o d a t e S c h i f f s t a i n (see r e f . 3 ) . L e f t : 0.2 mg LPS. Center: 0.5 mg LPS. R i g h t : 0 . 2 mg LPS. The e l e c t r o p h o r e t i c c o n d i t i o n s a r e t h e same as above b u t i n c l u d e 46 m~ HC1 i n t h e r e s o l v i n g g e l b u f f e r and 27 mM HC1 i n t h e anodic e l e c t r o l y t e . These cond i t i o n s r e s o l v e t h e t o p 20 0-Antigen c o n t a i n i n g bands as shown. The bands a r e numbered f r o m t h e t o p o f t h e p a t t e r n down, d i s r e g a r d i n g t h e uppermost 2 d i f f u s e bands. r e l a t e t o t h e sodium dodecyl s u l f a t e (SDS) d e r i v a t i v e o f LPS. Continuous SDS-PAGE f r a c t i o n a t i o n o f LPS d e r i v e d f r o m e i t h e r I?.

c o l i o r SaZmoneZla y i e l d s d i f f u s e

zones3, w h i l e d i s c o n t i n u o u s SDS-PAGE systems r e s o l v e LPS i n t o a m u l t i p l i c i t y o f w e l l d e f i n e d bands w i t h t h e o r d e r l y appearance o f an o l i g o m e r i c s e r i e s l Y 4 ( F i g .

- 1 . 2 ) . E l u t e d g e l f r a c t i o n s s u b j e c t e d t o r e - e l e c t r o p h o r e s i s produced t h e same bands w i t h i d e n t i c a l e l e c t r o p h o r e t i c m o b i l i t i e s . LPS from o t h e r organisms such as Neisseria appears t o y i e l d d i f f u s e bands i n a d i s c o n t i n u o u s PAGE system as

283 w e l l 5 . To e x p l a i n t h e band m u l t i p l i c i t y ( F i g . 12.2) i t has been assumed t h a t SDS binds e x c l u s i v e l y t o t h e L i p i d A p o r t i o n o f t h e

Based on t h a t as-

sumption, LPS species d i f f e r i n e l e c t r o p h o r e t i c m o b i l i t y s o l e l y due t o s i z e f r a c t i o n a t i o n o f t h e c a r b o h y d r a t e moiety, as has been shown f o r c o r e mutants ( F i g . 6 i n ref.

3, F i g . 4 i n r e f . 6 ) . Making t h e same assumption, b u t u s i n g c i r c u i t o u s

reasoning, t h e numbers o f bands i n SDS-PAGE have been c o r r e l a t e d w i t h v a r i a b l e c h a i n l e n g t h o f t h e 0 - a n t i g e n polymer4 Double l a b e l i n g experiments, i n w h i c h t h e

.

0-Antigen and L i p i d A/core m o i e t i e s ( F i g . 12.1) were d i f f e r e n t i a l l y l a b e l l e d by r a d i o i s o t o p e p r e c u r s o r s , a l l o w e d one t o a r r i v e a t a r e l a t i o n between c h a i n l e n g t h of t h e c a r b o h y d r a t e m o i e t y and m o b i l i t y . C l e a r l y , t h e c o r r e l a t i o n h o l d s f o r t h e h i g h m o b i l i t y bands ( t h e l i n e a r p o r t i o n o f F i g . 68 i n r e f . 1 and F i g . 3C i n r e f . 6 ) I t does n o t seem t o h o l d f o r t h e l o w e r m o b i l i t y bands which c o n t a i n t h e m a j o r i t y

o f the 0-Antigen.

To f u r t h e r i n v e s t i g a t e t h e r e l a t i o n between m o b i l i t y , SDS c o n t e n t and c h a i n l e n g t h , Fergusonl p l o t s 7 were c o n s t r u c t e d f o r t h e LPS components d e p i c t e d i n F i g . 12.1.

There was no change i n t h e o r d e r o f bands a t t h e v a r i o u s g e l c o n c e n t r a -

t i o n s as determined by t r a n s v e r s e p o r e g r a d i e n t g e l a n a l y s i s 8 ”

(unpublished data).

C l e a r l y , t h e j o i n t 95% c o n f i d e n c e enveloDes o f KR ( m o l e c u l a r s i z e ) and Yo (molecu l a r n e t charge) ( F i g . 12.3) i n d i c a t e t h a t t h e SDS d e r i v a t i v e s o f LPS c o n s i s t s o f 2 p o p u l a t i o n s , one b e i n g v e r t i c a l l y d i s p l a c e d and t h e o t h e r h o r i z o n t a l l y d i s The b u l k o f t h e 0-Antigen c o n t a i n i n g LPS s p e c i e s f a l l s i n t o t h e f o r m e r c a t e g o r y . The v e r t i c a l l y d i s p l a c e d p o p u l a t i o n of e l e c t r o p h o r e t i c bands ( t h e t o p 18 bands o f F i g . 12.2) a l l have t h e same m o l e c u l a r s i z e (KR) w h i l e v a r y i n g i n t h e m o l e c u l a r n e t charge ( y o ) . A p o s s i b l e e x p l a n a t i o n f o r t h e o r i g i n

3-

1.8 -

i 6

0

2

4

6

8 %T

1 0 1 2 1 4

1.2-

1.04.2

’

’

4.4

4.6

, 4.8

5.0

,/I,

5.2 5.4 KRxlOZ

-

5.6

,

,

5.8

6.0

F i g . 12.3. The Ferguson p l o t f o r (SuZmoneZZa typhimuriurn LT2) LPS i s shown on the. l e f t . The j o i n t 95% c o n f i d e n c e envelopes o f K~ and YO generated f r o m t h i s p l o t a r e shown a t t h e r i g h t . The p l o t r e p r e s e n t i n g t h e b e s t - f i t l i n e s and t h e c o n f i d e n c e envelopes were d e r i v e d by computer a n a l y s i s , u s i n g t h e PAGE-PACK p r o grams o f Rodbard and Chrambach7.

284

o f t h i s p o p u l a t i o n would be t h a t t h e L i p i d A m o i e t y o f these m o l e c u l e s d i f f e r s i n f a t t y a c i d c o m p o s i t i o n and/or conformation, r e s u l t i n g i n d i f f e r e n t i a l b i n d i n g o f SDS which l e a d s t o charge h e t e r o g e n e i t y . The h o r i z o n t a l l y d i s p l a c e d p o p u l a t i o n ( t h e bands f r o m 18-23 i n F i g . 12.2) a l l have t h e same charge d e n s i t y ( Y o ) b u t v a r y i n m o l e c u l a r s i z e ( K ~ )(see F i g . 12.3). The d o u b l e - l a b e l r a t i o d a t a c i t e d above would i n d i c a t e t h a t t h e s e LPS s p e c i e s w i t h h i g h m o b i l i t y d e r i v e f r o m a v a r i a b l e degree o f 0-Antigen p o l y m e r i z a t i o n . However, a decrease i n t h e s i z e o f t h e carboh y d r a t e m o i e t y a t c o n s t a n t SDS c o n t e n t o f t h e L i p i d A m o i e t y would be exoected t o l e a d t o an i n c r e a s e i n m o l e c u l a r charge d e n s i t y and t h e r e f o r e Y o . No such i n c r e a s e i n Yo i s seen among s p e c i e s 18-24 on F i g . 12.3. T h e r e f o r e , t h e f i n d i n g s r e p o r t e d h e r e a p p a r e n t l y c o n t r a d i c t t h e e a r l i e r h y p ~ t h e s i s ~t h’ a~t SDS b i n d s e x c l u s i v e l y and t o t h e same degree i n each L i p i d A m o i e t y . An a l t e r n a t i v e explanat i o n f o r t h e h o r i z o n t a l d i s t r i b u t i o n o f t h e h i g h m o b i l i t y s p e c i e s ( F i g . 12.3, numbers 18-24) i s t h a t t h e i r common f r e e m o b i l i t y (charge d e n s i t y , Y o ) d e r i v e s from a compensatory mechanism, whereby p r o g r e s s i v e s h o r t e n i n g o f t h e c a r b o h y d r a t e m o i e t y would l e a d t o ( a ) an i n c r e a s e i n Y o , due t o an i n c r e a s e d charge/mass r a t i o , assuming SDS b i n d i n g i n t h e L i p i d A m o i e t y ~ n l y and ~ ’ (~b ) a decrease i n y o , due t o an i n c r e a s e d p a r t i a l s p e c i f i c volume, 7 , s i n c e t h e L i p i d A m o i e t y has a o f a p p r o x i m a t e l y 0.9, w h i l e t h e carbohydrate m o i e t y has a 0.6,

7 value

7 value

o f approximately

i n t h e absence o f SDS. I n i t s presence, t h e d i f f e r e n c e i n 7 v a l u e s i s exac-

erbated. An i n c r e a s e d

7 indicates

an i n c r e a s e d e f f e c t i v e , h y d r a t e d m o l e c u l a r

volume and t h e r e f o r e a decreased f r e e e l e c t r o p h o r e t i c m o b i l i t y ( Y o ) . These app a r e n t l y c o n t r a d i c t o r y ways t o i n t e r p r e t t h e p r e s e n t l y a v a i l a b l e d a t a make f u r t h e r s t u d i e s on t h e i n t e r a c t i o n between SDS and LPS necessary. ACKNOWLEDGEMENTS The a u t h o r s w i s h t o thank D r . L.M. H j e l m e l a n d f o r v a l u a b l e h e l p i n t h e i n t e r p r e t a t i o n o f t h e data, D r . D. Rodbard f o r s u g g e s t i n g t h e t r a n s v e r s e p o r e g r a d i e n t g e l a n a l y s i s o f LPS, and D r . A. Chrambach f o r d i s c u s s i o n s and e d i t o r i a l h e l p . The o r i g i n a l m a t e r i a l r e p o r t e d i n t h i s r e v i e w comprises work done i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e Ph. D. degree a t Georgetown U n i v e r s i t y f o r P. F. C. REFERENCES

1 2 3 4 5

R.C. Goldman and L. L e i v e , Eur. J . Biochem., 107 (1980) 145-153. P. Coleman, unpublished r e s u l t s . B. Jann, K. Reske and K. Jann, Eur. J . Biochem., 60 (1975) 239-246. E.T. Palva and P.H. Makela, Eur. J . Biochem., 107 (1980) 137-143. R.R. R u s s e l l and K.G. Johnson, Can. J . MicrobioZ., 2 1 (1975) 2013-2018.

285

6 R.S. Munford, C . L . Hall and P. Rick, J . Bacteriol., 144 (1980) 630-640. 7 D. Rodbard a n d A. Chrambach, Anal. Biochem., 40 (1971) 95-134. 8 G. Kapadia, A. Chrambach and D. Rodbard, Electrophoresis and I s o e Z e c t r i c Focusing on polyacrglamide Gel, W. de Gruyter, Berlin, 1974, p . 115. 9 J . Margolis and K.G. Kenrick, Nature Ilondonl, 214 (1967) 1334-1336. 10 0. Rodbard a n d A. Chrambach, EZectrophoresis and I s o e l e c t r i c Focusing on Polyacrylamide Gel, W. de Gruyter, Berlin, 1974, p. 28. 11 P . F . Coleman and 0. Gabriel, Fed. Proe., Fed. Amer. Soc. E q . BioZ., 40 (1981) 1846. 12 U . K . Laemmli and M. Favre, J. Mol. BioZ., 80 (1973) 575-599.

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287

Chapter 13

ELECTROPHORETIC EXAMINATION OF ENZYMES

W. OSTROWSKI GENERAL ASPECTS Nearly a l l enzymes are globular p r o t e i n s w i t h spherical o r e l l i p s o i d a l molecular shapes. Their s t r u c t u r e s are s u i t a b l e f o r c a t a l y s i n g various degradative, s y n t h e t i c and transformations reactions i n b i o l o g i c a l systems. Compared w i t h o t h e r proteins, enzyme p r o t e i n s have some c h a r a c t e r i s t i c s t r u c t u r a l features t h a t may be important f o r t h e i r e l e c t r o p h o r e t i c separation and detection. A l l o f the p o l a r amino acids are on the o u t e r surface o f t h e enzyme molecule,

and non-polar s i d e groups are i n the i n t e r i o r o f the molecule and thus hidden from exposure t o water molecules. The compactness o f enzymic p r o t e i n s leaves l i t t l e room f o r water molecules i n s i d e t h e p r o t e i n molecule. Enzymic p r o t e i n s have a much small e r proportion o f t h e i r secondary s t r u c t u r e , i.e.,

t h e a - h e l i c a l form, u s u a l l y l e s s

than 30%, and show a tendency towards the use o f the B-conformation. Many enzymes contain more than one polypeptide chain. Such enzymes are c a l l e d oligomeric and can be dissociated i n t o f u n c t i o n a l o r r e g u l a t o r y subunits. Enzymes c o n s i s t i n g o f more than one type o f subunit can produce isoenzymes (see below) as a r e s u l t o f v a r i a t i o n s i n the combination o f the subunits i n t h e p r o t e i n molecule. I n some instances enzymes composed o f more than one i d e n t i c a l subunit can a l s o produce isoenzymes. The enzymes are simple and ,conjugated p r o t e i n s . Simple enzyme p r o t e i n s ( t r y p s i n , chymotrypsin, pepsin) g i v e o n l y amino acids when subjected t o degradative hydrol y s i s . Conjugated enzymes a l s o give o t h e r organic compounds, such as carbohydrates,

1i p i d s o r polynucleotides. Many enzymes, p a r t i c u l a r l y those i n v o l v e d i n o x i d a t i o n reduction reactions, c o n t a i n non-amino a c i d components which can be organic o r i n organic, o r both. These components are c a l l e d p r o s t h e t i c groups, c o f a c t o r s o r

CO-

enzymes, depending on t h e i r nature, f u n c t i o n and strength o f attachment. I n t h i s instance t h e p r o t e i n moiety, which by i t s e l f i s n o t an a c t i v e enzyme, i s c e l l e d an apoenzyme. The e n t i r e a c t i v e enzyme molecule c o n s i s t i n g o f t h e c o f a c t o r p l u s the p r o t e i n moiety i s named a holoenzyme. Because t h e number of newly discovered enzymes i s increasing r a p i d l y and they catalyse a l a r g e number o f reactians; a systematic c l a s s i f i c a t i o n o f enzymes has been compiled on t h e recommendation o f an I n t e r n a t i o n a l Union o f Biochemistry (IUB).

288

The new system

1

divides a l l enzymes i n t o s i x major classes and several subclasses,

according t o t h e type o f r e a c t i o n catalysed (Table 13.1). This system o f c l a s s i f i c a t i o n w i l l serve here as a guide f o r the d e t e c t i o n o f d i f f e r e n t groups o f enzymes on various supporting matrices. TABLE 13.1 KEY TO NUMBERING AND CLASSIFICATION OF ENZYMES (ACCORDING TO ENZYME NOMENCLATURE, IUB, 1978)

Name

E.C.

Oxidoreductases 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.97 Transf erases

Hydro1ases

2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11

Number Type o f r e a c t i o n

Acting on t h e CH-OH group o f donors Acting on t h e aldehyde o r 0x0 group o f donors Acting on t h e CH-CH group o f donors Acting on t h e CH-NH, group o f donor Acting on t h e CH-NH group o f donor Acting on NADH o r NADPH Acting on t h e nitrogenous compounds Acting on a sulphur group o f donors Acting on a haem group o f donors Acting on diphenols and r e l a t e d substances Acting on hydrogen peroxide as acceptor Acting on hydrogen as donor Acting on s i n g l e donors w i t h i n c o r p o r a t i o n o f molecu l a r oxygen (oxygenases) Acting on paired donors w i t h i n c o r p o r a t i o n o f molecu l a r oxygen Acting on superoxide r a d i c a l s as acceptor Oxidizing metals ions Acting on -CH2 groups Other oxidoreductases Transferring one-carbon groups Transferring aldehyde o r ketonic residues Acyltransferases Glycosyl transferases Transferring a1 k y l o r a r y l groups , o t h e r than methyl groups Transferring nitrogenous groups T r a n s f e r r i n g phosphorus-containing groups Transferring sulphur-containing groups Acting Acting Acting Acting Acting Acting Acting Acting Acting Acting Acting

on on on on on on on on on on on

e s t e r bonds glycosyl compounds ether bonds peptide bonds C-N bonds other than peptides a c i d anhydrides C-C bonds h a l i d e bonds P-N bonds S-N bonds C-P bonds

289

TABLE 13.1 (continued) ~~

~

Name

E.C.

Lyases

4 4.1 4.2 4.3 4.4 4.5 4.6 4.99

Isomerases

Ligases (Synthetases)

Number Type o f r e a c t i o n Carbon-carbon lyases Carbon-oxygen l y a s e s Carbon-nitrogen l y a s e s Carbon-sul phur lyases Carbon-ha1 i d e l y a s e s P hos pho r us -oxy gen 1ya s es Other l y a s e s

5 5.1 5.2 5.3 5.4 5.5 5.99

Racemases and epimerases Cis-trans isomerases I n t r a m o l e c u l a r oxidoreductases Intramolecular transferases Intramolecular lyases Other isomerases

6 6.1 6.2 6.3 6.4 6.5

Forming Forming Forming Forming Forming

C-0 bonds C-S bonds C-N bonds C-C bonds phosphate e s t e r bonds

ELECTROPHORETIC SEPARATION OF ENZYMES The a b i l i t y o f p r o t e i n molecules, i n c l u d i n g enzymes, t o m i g r a t e i n an e l e c t r i c f i e l d depends p r i m a r i l y on t h e i r n e t e l e c t r i c charge. With some g e l s as s u p p o r t i n g media ( s t a r c h gel

, polyacrylamide

gel)

, the

s i z e and shape o f t h e p r o t e i n molecule

a r e a l s o i m p o r t a n t f a c t o r s i n f l u e n c i n g t h e e l e c t r o p h o r e t i c m i g r a t i o n because of molecular s i e v i n g and r e t a r d a t i o n e f f e c t s . The t y p e and pH o f t h e b u f f e r may have a profound e f f e c t on t h e r a t e o f m i g r a t i o n and a c t i v i t y o f t h e enzyme under study. Some enzymes l o s e t h e i r c a t a l y t i c a c t i v i t y a t lower o r h i g h e r pHs, so t h a t t h e separation must be c a r r i e d o u t a t a pH c l o s e t o t h e i r o p t i m a l s t a b i l i t y . B u f f e r i o n s may r e a c t w i t h uncharged groups o f t h e p r o t e i n m o i e t y t o form charged d e r i v a t i v e s which move w i t h d i f f e r e n t v e l o c i t i e s . For example, b o r a t e i o n s can combine w i t h sugar r e s i d u e s o f t h e g l y c o p r o t e i n s , and heavy metals can codbine w i t h SH groups. The e l e c t r o p h o r e t i c m i g r a t i o n w i l l a l s o depend on t h e i o n i c s t r e n g t h of an e l e c t r o l y t e , t h e v i s c o s i t y o f t h e s o l u t i o n and t h e s t a b i l i t y o f t h e sample. Some enzymes when e x c e s s i v e l y d i l u t e d undergo d e n a t u r a t i o n ; a d d i t i o n t o t h e b u f f e r sol u t i o n o f e n z y m a t i c a l l y i n e r t p r o t e i n (serum albumin) o r coenzymes i n t h e case of some dehydrogenases may s t a b i l i z e t h e enzymes2. To a v o i d o v e r h e a t i n g and thermal i n a c t i v a t i o n o f t h e enzymes, s e p a r a t i o n should be performed a t low temperatures (about 4OC) by u s i n g e f f i c i e n t c o o l i n g systems d u r i n g t h e e l e c t r o p h o r e t i c run. The v e l o c i t y o f m i g r a t i o n o f p r o t e i n molecules may be m o d i f i e d by t h e e l e c t r o osmotic and hydrodynamic f l o w o f t h e solvent3. The e l e c t r o o s m o t i c f l o w i s most pronounced i n a l k a l i n e s o l u t i o n , and v a r i e s w i t h t h e t y p e o f s u p p o r t i n g medium used.

290

The e x t e n t o f e l e c t r o o s m o t i c and hydrodynamic replacement o f t h e s o l u t e ("back wash") d u r i n g e l e c t r o p h o r e s i s can be measured e x p e r i m e n t a l l y and, i f necessary, 4 t h e c o r r e s p o n d i n g values can be c a l c u l a t e d

.

A d s o r p t i o n o f t h e sample on t h e m a t r i x i s a n o t h e r f a c t o r t h a t i n f l u e n c e s t h e r e s o l u t i o n o f enzymes d u r i n g e l e c t r o p h o r e s i s on s o l i d media. The a d s o r p t i o n o f enzyme p r o t e i n on t h e s u p p o r t i n g medium i s m a n i f e s t e d by " t a i l i n g " , by t h e format i o n o f "comets" and/or b y " s e l f - s l o w i n g ' ' o f t h e p r o t e i n band. The a d s o r p t i o n and r e l a t e d s i d e - e f f e c t s can a l m o s t be e l i m i n a t e d by t h e c a r e f u l s e l e c t i o n o f t h e supp o r t i n g medium, a d d i t i o n o f d e t e r g e n t s t o t h e b u f f e r s o l u t i o n o r t h e use o f o t h e r substances a p p r o p r i a t e t o t h e p r o p e r t i e s o f t h e s e p a r a t e d enzyme. PREPARATIVE ELECTROPHORESIS D i f f e r e n t e l e c t r o p h o r e t i c t e c h n i q u e s a r e w i d e l y used i n t h e p u r i f i c a t i o n and i s o l a t i o n o f enzymes f r o m a n i m a l , p l a n t and b a c t e r i a l sources. The enzymes can be separated p r e p a r a t i v e l y i n f r e e s o l u t i o n , w i t h o u t a s u p p o r t i n g medium o r s t a b i l i z i n g substances, and b y d i f f e r e n t t e c h n i q u e s o f zone e l e c t r o p h o r e s i s . P r e p a r a t i v e separat i o n s do n o t need s p e c i f i c h i s t o c h e m i c a l i d e n t i f i c a t i o n o f an enzyme, because i n f r e e s o l u t i o n o r when enzymes a r e e l u t e d f r o m t h e b l o c k , column, t h i n l a y e r , e t c . , c o n v e n t i o n a l assay methods f o r i d e n t i f i c a t i o n and d e t e r m i n a t i o n o f t h e enzyme a c t i v i t y can be used.

Free-soZution techniques Moving-boundow electrophoresis. T h i s c l a s s i c a l t e c h n i q u e has been a p p l i e d i n 5

enzyme b i o c h e m i s t r y f o r more t h a n 40 y e a r s . Several p u r e enzymes were o b t a i n e d

d u r i n g t h e e a r l y stages o f i t s development, b u t t h e r e c o v e r y o f t h e sample components from t h e channel o f t h e e l e c t r o p h o r e t i c c e l l proved d i f f i c u l t . M o d i f i c a t i o n s o f t h e T i s e l i u s apparatus have been d e s c r i b e d

6 t h a t p e r m i t t h e continuous

removal o f a f r a c t i o n when i t i s separated by t h e a p p l i e d e l e c t r i c f i e l d . Other l i m i t a t i o n s o f t h e t e c h n i q u e a r e t h a t complete s e p a r a t i o n o f t h e components i n t o d i s t i n c t zones i s p o s s i b l e o n l y f o r t h e f a s t e s t and s l o w e s t moving boundaries; and t h e s t a b i l i t y o f t h e boundaries i s l i m i t e d and depends on t h e d e n s i t y o f t h e s o l u t i o n and t h e temperature. An advantage o f t h e T i s e l i u s method i s t h a t t h e c o m p o s i t i o n o f t h e m i x t u r e can be r e g i s t e r e d o p t i c a l l y d u r i n g t h e run and t h u s removal o f t h e samples can be c o n t r o l l e d . The T i s e l i u s apparatus has been used f o r t h e p u r i f i c a t i o n o f L-amino a c i d o x i d a s e (E.C.

8 p e r o x i d a s e and myeloperoxidase (E.C.

1.11.1.7)

1.4.3.2)

f r o m snake venom7, p l a n t

and f o r t h e s e p a r a t i o n o f p o l y -

saccharides f r o m o l d y e l l o w enzyme (E.C. 1.6.99.1)

9

.

EZectrodecuntation. E l e c t r o d e c a n t a t i o n i s by f a r t h e l a r g e s t s c a l e a p p l i c a t i o n o f e l e c t r o p h o r e s i s t o date, i n c l u d i n g i t s a d a p t a t i o n f o r i n d u s t r i a l purposes. I n

291 t h i s method t h e e l e c t r o d e s a r e s e p a r a t e d f r o m t h e b u l k o f t h e s o l u t e by a membrane ( F i g 13.1) which a l l o w s t h e m i g r a t i n g s p e c i e s t o b u i l d up on t h e membrane, r a t h e r t h a n on t h e e l e c t r o d e s . I f t h e c o n c e n t r a t e d p r o t e i n i s denser t h a n t h e remainder o f t h e s o l u t i o n , i t w i l l f r e q u e n t l y s i n k t o t h e b o t t o m i n a f a i r l y dense l a y e r , which can be removed f r o m t h e system. The molecules t h a t a r e i s o e l e c t r i c a t t h e p a r t i c u l a r pH w i l l n o t m i g r a t e and t h i s i s o e l e c t r i c f r a c t i o n w i l l t e n d t o b e d i s p l a c e d upwards. I n t h i s manner i t i s p o s s i b l e t o s e p a r a t e t h e i s o e l e c t r i c f r o m t h e charged s p e c i e s . Thus, an i n i t i a l f r a c t i o n a t i o n can b e e f f e c t e d i f t h e p I o f t h e enzyme which i t i s d e s i r e d t o p u r i f y i s known. When t h e c o n c e n t r a t e d m a t e r i a l has a tendency t o s t i c k t o t h e membrane i t can u s u a l l y b e r e l e a s e d by a momentary r e v e r s a l o f t h e c u r r e n t . A m u l t i - c o m p a r t m e n t c e l l w i t h s e v e r a l membranes can be used t o i n c r e a s e t h e e f f i c i e n c y o f t h e s.eparation 1 0 , l l

Semi

- Permeable

Membranes

1

Sample buller mixture

-

buller

F i g . 13.1. Schematic r e p r e s e n t a t i o n o f e l e c t r o d e c a n t a t i o n c e l l and b a s i c p r i n c i p l e s o f t h e m e t h o d . B , S t a t i o n a r y component;=, m i g r a t i n g component. E l e c t r o d e c a n t a t i o n has been used f o r t h e p u r i f i c a t i o n o f t r y p s i n (E.C. 3.4.21.4) and deoxyribonuclease (E.C. k i d n e y B-0-glucosidase (E.C.

3.2.1.50)

(E.C.

3.1.21.1)12 3.2.1.21),

10

and f o r t h e i n i t i a l f r a c t i o n a t i o n o f p i g sheep t e s t i c u l a r a-N-acetyl-D-glucosaminidase 13 3.4.24.4)

.

and e x t r a c e l l u l a r BaciZZus subtiZis p r o t e a s e (E.C.

A f t e r a s i n g l e cycle o f electrodecantation a s i x - t o t e n - f o l d increase i n s p e c i f i c a c t i v i t y was achieved w i t h a b o u t 70% r e c o v e r y . Posner14 d e s c r i b e d a t e c h n i q u e c a l l e d e l e c t r o p h o r e s i s - s e d i m e n t a t i o n

, which

dif-

f e r s f r o m c o n v e n t i o n a l e l e c t r o d e c a n t a t i o n i n t h a t i t employs semipermeable membranes

292

o f reduced area i n o r d e r t o m i n i m i z e t h e a d s o r p t i o n o f macromolecules and t h e e l e c t r o d e s a r e f i x e d h o r i z o n t a l l y . D u r i n g t h e s e p a r a t i o n , p r o t e i n descends u n i f o r m l y t o t h e b o t t o m o f t h e chamber w i t h a concomitant c l e a r i n g o f t h e upper p r o t e i n - f r e e s o l u t i o n of l o w e r d e n s i t y . T h i s t e c h n i q u e can be p a r t i c u l a r l y u s e f u l f o r v e r y d i l u t e s o l u t i o n s o f p r o t e i n s i n a very s h o r t time. Blectrophoresis-convection. T h i s t e c h n i q u e i s a f u r t h e r development o f e l e c t r o d e c a n t a t i o n and t h e apparatus and t h e o r y were m o s t l y developed b y K i rkwood and cow o r k e r ~ ~The~ a~p ~p l i~c a. t i o n o f e l e c t r o p h o r e s i s - c o n v e c t i o n t o t h e f i n e r e s o l u t i o n o f d i f f e r e n t p r o t e i n m i x t u r e s was d e s c r i b e d i n d e t a i l by B i e r 1 7 . U s i n g t h i s method, k i d n e y a l k a l i n e phosphatase (E.C. 19

were p u r i f i e d

3.1.3.1)18

and D-amino a c i d o x i d a s e (E.C. 1.4.3.3)

.

Forced-flow electrophoresis. T h i s i s a f u r t h e r r e f i n e m e n t o f t h e e l e c t r o p h o r e s i s -

convection technique t h a t allows t h e continuous i n p u t o r withdrawal o f t h e f r a c t i o n a t e d s o l u t i o n . O r i g i n a l l y d e v i s e d b y Bier2',

i t u t i l i z e s simultaneously differences

i n t h e r a t e s o f b o t h e l e c t r o p h o r e t i c m i g r a t i o n and p e r p e n d i c u l a r s e d i m e n t a t i o n o f t h e c o n c e n t r a t e d zones. I t was a p p l i e d t o t h e i s o l a t i o n and p u r i f i c a t i o n o f e l a s t a s e (E.C. 3.4.21.11) and h o r s e - r a d i s h peroxidase" and t o t h e s e p a r a t i o n o f b a c t e r i a l 22 c e l l s f r o m t h e i r e x t r a c e l l u l a r enzymes Free-fZow electrophoresis. F r e e - f l o w e l e c t r o p h o r e s i s has found widespread a p p l i c a t i o n i n t h e p r e p a r a t i v e s e p a r a t i o n o f s o l u b l e and i n s o l u b l e m a t e r i a l s such as p r o t e i n s , v i r u s e s , b a c t e r i a l c e l l s , c e l l o r g a n e l l e s and even v i a b l e c e l l s 23 ,24

.

.

The m i x t u r e o f substances t o be separated i s i n t r o d u c e d as a f i n e j e t i n t o t h e s e p a r a t i o n b u f f e r which i s moving across t h e f i e l d l i n e s o f an e l e c t r i c f i e l d . Charged m o l e c u l e s ( p a r t i c l e s ) a r e t h e n d e f l e c t e d f r o m t h e d i r e c t i o n o f f l o w o f t h e s u p p o r t i n g e l e c t r o l y t e a t an a n g l e r e s u l t i n g from t h e f l o w v e l o c i t y and t h e e l e c t r o p h o r e t i c m o b i l i t y . The a n g l e

a

i s then given by

rate o f electrophoretic migration tan

a =

rate o f buffer flow As t h e r a t e of m i g r a t i o n ,

v , o f a substance i s d i r e c t l y p r o p o r t i o n a l t o t h e c u r r e n t ,

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

c r o s s - s e c t i o n a l a r e a o f t h e e l e c t r o p h o r e s i s channel, q , and t h e s p e c i f i c conductance o f the b u f f e r solution,

K ,

w i l l b e d e f i n e d as:

Ui

v=-

qK

S u b s t i t u t i n g t h i s v a l u e i n t h e e q u a t i o n f o r t a n a , we o b t a i n an e x p r e s s i o n f o r t h e tangent o f t h e angle o f d e f l e c t i o n o f t h e given p r o t e i n :

ui

tan

a

=

q K w

293

where w i s a r a t e o f b u f f e r f l o w . I f t h e e l e c t r o p h o r e t i c m o b i l i t y ,

U ,

o f the

substance t o be separated i s known, t h i s equation can be used t o f i n d s u i t a b l e cond i t i o n s f o r t h e separation. The p o p u l a t i o n o f molecules w i t h i d e n t i c a l e l e c t r o p h o r e t i c m o b i l i t i e s m i g r a t e s as a narrow zone through t h e e l e c t r o p h o r e t i c chamber and f r a c t i o n s can be c o n t i n u o u s l y c o l l e c t e d . The s e p a r a t i o n chamber c o n s i s t s o f two r e c t a n g u l a r glass p l a t e s f i x e d p a r a l l e l t o t h e frame, so t h a t t o g e t h e r t h e y form a narrow channel w i t h a gap approximately 0.3-1.0

mm wide f o r t h e f l o w i n g f i l m o f b u f f e r . On t h e upper p l a t e o f t h e separa-

t i o n chamber i s t h e i n l e t f o r t h e b u f f e r and t h e i n j e c t i o n s l i t f o r t h e sample. Both g l a s s p l a t e s a r e cooled and maintained a t a constant temperature by an e l e c t r o n i c r e g u l a t i o n system. The e l e c t r ' i c a l c o n t a c t w i t h t h e f i l m o f f l o w i n g b u f f e r and e l e c t r o d e compartments occurs through ion-exchange membranes, which were developed e s p e c i a l l y f o r t h i s purpose. The emerging e l e c t r o p h o r e t i c f r a c t i o n s a r e c o l l e c t e d i n t e s t - t u b e s which can be kept i n a c o o l i n g c a b i n e t d u r i n g t h e separation. Using a commercial instrument, Evans25 separated tomato peroxidase isozymes. The b e s t r e s o l u t i o n was obtained a t pH 8.8 i n b o r a t e b u f f e r (0.025 m o l / l b o r i c acid

+

0.0092 m o l / l NaOH) c o n t a i n i n g 10% o f sucrose. A d d i t i o n o f sucrose increases

t h e d e n s i t y o f t h e b u f f e r and reduces t h e d i f f u s i o n o f t h e isozymes d u r i n g e l e c t r o p h o r e s i s . Grassmann e t a1 .26 p u r i f i e d c o l lagenase (E.C. 3.4.24.3)

from C l o s t r i d i m

h i s t o Z y t i c m f o r t h e f i r s t time. E l e c t r o p h o r e s i s was acomplished i n 0.05 m o l / l Tris-HC1 b u f f e r (pH 9.1) c o n t a i n i n g 0.01 m o l / l calcium a c e t a t e . Mehl and J a t z k e w i t z ( r e f . 27) p u r i f i e d a r y l s u l p h a t a s e A (E.C. 3.1.6.1),

type 2, which hydrolyses cere-

b r o s i d e sulphates i n t o cerebrosides and sulphate, 6000-fold.

Separation was c a r r i e d

o u t i n 0.025 m o l / l ammonium a c e t a t e b u f f e r (pH 5.1). A c t i v e haem-containing f r a g ments o f cytochrome c were e a s i l y i s o l a t e d i n t h e pure s t a t e by u s i n g t h e same 28 commercial apparatus

.

The technique o f continuous e l e c t r o p h o r e s i s i n f l u i d endless b e l t s was described by K o l i n and Luner".

A sheet o f e l e c t r o l y t e s o l u t i o n i s p r o p e l l e d past an i n l e t

where t h e sample i s i n j e c t e d t o form a s t r e a k . An e l e c t r i c f i e l d p e r p e n d i c u l a r t o t h e d i r e c t i o n o f f l u i d motion causes e l e c t r o p h o r e t i c m i g r a t i o n o f t h e charged components o f t h e s t r e a k and d e f l e c t s them from t h e o r i g i n a l course. The s t r e a k s can be d i r e c t e d o u t o f t h e apparatus where f r a c t i o n s can be c o l l e c t e d . The method can be a p p l i e d t o t h e s e p a r a t i o n and c h a r a c t e r i z a t i o n o f enzymes, s u b c e l l u l a r p a r t i c l e s 30

and even l a r g e mammalian c e l l s

.

Density gradient electrophoresis. This method i s a l o g i c a l m o d i f i c a t i o n o f electrophoresis i n f r e e solution. I n density gradient electrophoresis the solution i s s t a b i l i z e d a g a i n s t convection by a d e n s i t y g r a d i e n t o f sucrose, g l y c e r o l , ethylene g l y c o l , heavy water31 o r F i c 0 1 1 ~ ~S .o l i d s u p p o r t i n g media, e s p e c i a l l y g e l s r a t h e r than d e n s i t y g r a d i e n t s o l u t i o n s , have some l i m i t a t i o n s such as t h e p o s s i b i l i t y o f a s i e v i n g e f f e c t and t h e n e c e s s i t y f o r s p e c i a l e l u t i o n procedures. The a d d i t i o n a l

294

advantage o f d e n s i t y g r a d i e n t columns i s t h a t i t a l l o w s o p t i c a l v i s u a l i z a t i o n of t h e separated zones by u s i n g v i s i b l e and/or UV o p t i c s 3 3 y 3 4 and t h e p u r e f r a c t i o n s can be r e c e i v e d s i m p l y i n a f r a c t i o n c o l l e c t o r . The most c o n v e n i e n t and s i m p l e s t apparatus i s a l o n g j a c k e t e d g l a s s column w h i c h a l l o w s s e p a r a t i o n s a t l o w t e m p e r a t u r e and which can be e a s i l y s e t up f o r e l u t i o n o f t h e s e p a r a t e d zones34. A l i n e a r d e n s i t y g r a d i e n t o f 5-50% sucrose w i t h s u p p o r t i n g e l e c t r o l y t e a t a u n i f o r m c o n c e n t r a t i o n i s produced b y a s u i t a b l e d e v i c e used f o r column chromatography o r d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n . Another approach was d e s c r i b e d by K 0 1 i n ~and ~ t h e n adapted by Choules and B a l l e n t i n e 3 6 f o r p r e p a r a t i v e purposes, i n which s h o r t columns w i t h v e r y s t e e p dens i t y g r a d i e n t s a r e used. The h i g h s t a b i l i t y towards thermal c o n v e c t i o n p e r m i t s t h e use o f r e l a t i v e l y h i g h e l e c t r i c a l f i e l d s t r e n g t h s f o r b r i e f t i m e p e r i o d s . However, d e n s i t y g r a d i e n t e l e c t r o p h o r e s i s does n o t appear t o have found widespread u t i l i t y , because o f t h e g r e a t expansion o f i s o e l e c t r i c f o c u s i n g t e c h n i q u e s , and i t i s used s p o r a d i c a l l y f o r t h e s e p a r a t i o n and c h a r a c t e r i z a t i o n o f d i f f e r e n t 37-40 enzymes Preparative isoeZectric focusing i n density gradients. T h i s t e c h n i q u e g i v e s

b e t t e r r e s u l t s t h a n c l a s s i c a l d e n s i t y g r a d i e n t e l e c t r o p h o r e s i s owing t o i t s h i g h r e s o l v i n g power, and i t i s t h e most f r e q u e n t l y used e l e c t r o p h o r e t i c method f o r t h e p u r i f i c a t i o n o f enzymes and o t h e r substances. Sucrose i s t h e m a t e r i a l u s u a l l y used f o r p r e p a r i n g t h e d e n s i t y g r a d i e n t , b u t , a t h i g h e r pHs (above pH 8 ) sucrose undergoes d i s s o c i a t i o n and g l y c e r o l s h o u l d be used i n t h e s e i n s t a n c e s . F o r c r e a t i n g a pH g r a d i e n t i n t h e column, 1-2% (w/v) Ampholine c a r r i e r ampholytes o f t h e d e s i r e d pH range a r e most f r e q u e n t l y used. A f t e r t h e e l e c t r o p h o r e t i c r u n , c a r r i e r ampholytes from t h e enzyme s o l u t i o n can be separated by g e l f i l t r a t i o n on Sephadex 6-50 ( r e f . 41) , s a l t i n g - o u t 4 '

o r ion-exchange c h r ~ m a t o g r a p h y ~The ~ . t e c h n i q u e was v e r y e f f e c -

t i v e i n t h e separation o f pancreatic

enzyme^^^-^^,

the fractionation o f prostatic

a c i d phosphatase isozymes (E.C. 3 . 1 . 3 . 2 ) ~ ~g l ~y c~o l~y t~i c enzymes f r o m r a b b i t muscle49 4.2.1.1)",

u r o k i n a s e f r o m human u r i n e (E.C.

3.4.21.31)50,

c a r b o n i c anhydrase (E.C. 54

r i b o n u c l e a s e (E.C. 3.1.27.5) 52y53 and o t h e r enzymes

.

Continuous-fZow isoeZectric focusing. T h i s t e c h n i q u e i s s i m i l a r t o t h e c o n t i n -

uous-flow e l e c t r o p h o r e s i s method o f Hannig etl a1 .23. The s t a b i l i t y o f t h e r u n n i n g space may be achieved by a l a m i n a r f l o w o f t h e ampholytes i n t h e n a r r o w space between two g l a s s p l a t e s 5 5 o r by c a p i l l a r y o r d e n s i t y g r a d i e n t systems56. I n cont r a s t t o c o n t i n u o u s - f l o w e l e c t r o p h o r e s i s , t h e p r o t e i n sample i n t h i s t e c h n i q u e can be s u p p l i e d o v e r t h e whole w i d t h o f t h e s e p a r a t i o n c e l l and t h e o u t l e t p o s i t i o n s o f t h e separated zones a r e n o t changed f o r s e v e r a l days. N e v e r t h e l e s s , i s o e l e c t r i c f o c u s i n g i n a s i m p l e column s t a b i l i z e d w i t h a sucrose d e n s i t y g r a d i e n t i s t h e most f r e q u e n t l y used f o r t h e p r e p a r a t i v e s e p a r a t i o n o f enzymes. By t h i s t e c h n i q u e enzymes such as c y c l i c n u c l e o t i d e phosphodiesterase (E.C.

3.1.4.17)57,

p o l y n u c l e o t i d e phos-

295

phorylase (E.C. 2.7.7.8) mitochondria5',

from bacteria58, l o n g c h a i n acyl-CoA hydrolase from r a t

a c i d p r o t e i n a s e from r a t s p l een6',

t r a n s f e r a s e (E.C. 2.7.4.8)61,

mitochondria1 GTP-AMP phospho-

s e r i n e hydrolases (E.C.

microsomes62, c e l l u l a s e s (E.C.

3.2.1.4)

4.2.1.13)

from r a t l i v e r

~ ~ many from P e n i c i Z Z i m f ~ ? i c u Z o s w n and

o t h e r s were p u r i f i e d . P r e p a r a t i v e isotachophoresis. I s o t a c h o p h o r e t i c s e p a r a t i o n has been found t o have g r e a t p o t e n t i a l f o r t h e p u r i f i c a t i o n o f enzymes. Using a m i c r o s c a l e preparat i v e c a p i l l a r y system, sample c o l l e c t i o n must be e f f e c t e d b y a s p e c i a l construct i o n o f t h e apparatus, shown s c h e m a t i c a l l y i n F i g . 13.2. The separated f r a c t i o n s a r e c o l l e c t e d on c e l l u l o s e a c e t a t e s t r i p s 6 4 by means o f a T-tube mounted i n t h e c a p i l l a r y by which t h e l e a d i n g e l e c t r o l y t e i s pumped and c o l l e c t e d by a s u i t a b l e f r a c t i o n c o l l e c t o r . A f t e r c o l l e c t i o n o f the fractions, the c e l l u l o s e acetate s t r i p i s t r e a t e d w i t h a reagent s o l u t i o n s p e c i f i c t o t h e separated enzyme. C e l l u l o s e a c e t a t e s t r i p s w i t h t h e c o l l e c t e d enzyme can a l s o be used t o c a r r y o u t t h e enzymic r e a c t i o n i n t h e t e s t - t u b e o r can be e l u t e d w i t h b u f f e r t o o b t a i n an enzyme s o l u t i o n o f h i g h p u r i t y and h i g h s p e c i f i c a c t i v i t y i n microgram amounts. Using t h e above technique, commercial c h o l i n e s t e r a s e (E.C.

3.1.1.8)

was f u r t h e r p u r i f i e d and

i d e n t i f i e d 6 5 . T h e o r e t i c a l and p r a c t i c a l aspects o f t h e technique were described by A r l i n g e r 6 6 and Moberg e t a1 67

. .

E

4' fL

F i g . 13.2. P r i n c i p l e o f p r e p a r a t i v e c a p i l l a r y isotachophoresis. E = p l a t i n u m e l e c t r o d e s , L = l e a d i n g e l e c t r o l y t e , T = t e r m i n a t i n g e l e c t r o l y t e , D = UV d e t e c t o r , S = sample components, m = semipermeable membranes, f - f l o w o f l e a d i n g e l e c t r o l y t e f o r e l u t i n g sample zones, V = d i r e c t i o n o f e l e c t b o p h o r e t i c m i g r a t i o n . Flat-bed techniques

.

These techniques a r e f r e q u e n t j y used when working w i t h crude m a t e r i a l . A l l arrangements f o r zone e l e c t r o p h o r e s i s i n a h o r i z o n t a l d i r e c t i o n have i n common a

296

t r a y t o h o l d t h e s u p p o r t i n g medium b o t a t o s t a r c h , c e l l u l o s e powder, Sephadex, Bio-Gel, Pevikon ( a copolymer o f p o l y ( v i n y 1 c h l o r i d e ) and p o l y ( v i n y 1 a c e t a t e f l , two b u f f e r v e s s e l s w i t h immersed e l e c t r o d e s and a r e g u l a t e d d.c. v o l t a g e power supply. The e n t i r e apparatus s h o u l d be p l a c e d i n a r e f r i g e r a t o r o r a c o l d room t o p r e v e n t o v e r h e a t i n g o f t h e sample and i n h i b i t t h e growth o f microorganisms. A f t e r e l e c t r o p h o r e s i s , t h e b l o c k i s a l l o w e d t o d r y p a r t i a l l y and i s c u t i n t o 1-cm segments. Each segment i s p l a c e d i n a c e n t r i f u g e t u b e t o which a s u i t a b l e b u f f e r i s added and, a f t e r s t i r r i n g t h o r o u g h l y , t h e tubes a r e c e n t r i f u g e d . I n t h e s u p e r n a t a n t s o l u t i o n s assays f o r p r o t e i n c o n t e n t and enzymatic a c t i v i t y a r e performed. Owing t o t h e h i g h l o a d i n g c a p a c i t y o f t h e b l o c k , up t o 10 g o f p r o t e i n m i x t u r e can be applied. Segmented foam rubber68 and p o l y u r e t h a n e foam6’ have been used as v e r y conv e n i e n t s u p p o r t i n g media (segmented-sponge e l e c t r o p h o r e s i s ) i n f l a t - b e d techniques. The f r a c t i o n s can be recovered a f t e r an e l e c t r o p h o r e t i c r u n s i m p l y by squeezing t h e p a r t i c u l a r segments. T h i s t e c h n i q u e a l s o f a c i l i t a t e s t h e e s t a b l i s h m e n t o f a pH g r a d i e n t w i t h i n t h e s u p p o r t i n g bed. T h i s method has been used w i t h success f o r t h e p r e p a r a t i o n and c h a r a c t e r i z a t i o n o f p r o s t a t i c a c i d phosphatase and u r i n a r y B-glucuronidase (E.C. 2.7.1.43) 69

.

F l a t - b e d i s o e l e c t r i c f o c u s i n g i n a g r a n u l a t e d g e l (Sephadex 6-75 and Sephadex 6-200 S u p e r f i n e , Bio-Gels) i s a n o t h e r t e c h n i q u e f r e q u e n t l y used f o r t h e p u r i f i c a t i o n o f enzymes. When t h e e x p e r i m e n t a l c o n d i t i o n s a r e c a r e f u l l y s t a n d a r d i z e d , t h e method i s v e r y u s e f u l f o r f r a c t i o n a t i o n o f c r u d e m a t e r i a l . The equipment f o r f l a t bed i s o e l e c t r i c f o c u s i n g , p r e p a r a t i o n o f t h e g e l w i t h t h e c a r r i e r ampholytes s o l u 70 t i o n , sample i n t r o d u c t i o n and p r o t e i n e l u t i o n has been d e s c r i b e d by B l a n i c k g

.

The s t a r c h b l o c k t e c h n i q u e has been used f o r t h e p u r i f i c a t i o n o f d e o x y r i b o cell^^^^^^, hexoseaminidase C from human b r a i n 73 ,

nucleases f r o m b a c t e r i a l

B - g a l -a c t o s i d a s e (E.C. 3.2.1.23) cells”

f r o m E. coZi e x t r a c t s 7 4 , s o l u b l e enzymes f r o m r e d

and many o t h e r s .

The s u p p o r t i n g medium Pevikon i n v o l v e s a s i m p l e r p r e p a r a t i o n o f t h e bed, can e a s i l y be washed and recovered and t h e sample f r o m t h e segments can be s i m p l y e l u t e d w i t h a 60-70% y i e l d 76,77

.

F l a t - b e d i s o e l e c t r i c f o c u s i n g was a p p l i e d f o r t h e f r a c t i o n a t i o n on p o l y a c r y l amide g e l o f h o r s e - r a d i s h peroxidase7* and t h e c h a r a c t e r i z a t i o n o f glucose-6-phos phate dehydrogenase (E.C. 1.1.1.49) f r o m human erythrocyte^^^. D i f f e r e n t p l a n t enzymes such as phenolases, p e r o x i d a s e , e s t e r a s e and m a l a t e dehydrogenase have a l s o been s t u d i e d b y i s o e l e c t r i c f o c u s i n g i n p o l y a c r y l a m i d e g e l

80 .

Colwrm techniques i n porous or g e l media The d i s t i n c t i o n between b l o c k and column e l e c t r o p h o r e s i s has u s u a l l y been made because o f d i f f e r e n c e s i n t h e o p e r a t i o n s t h a t a r e performed a f t e r e l e c t r o p h o r e s i s i n each t e c h n i q u e . A b l o c k i s s l i c e d i n t o equal segments and s e p a r a t e d m a t e r i a l

297

i s e x t r a c t e d i n t e s t - t u b e s , whereas a column i s e l u t e d w i t h b u f f e r and t h e separated zones a r e c o l l e c t e d w i t h a f r a c t i o n c o l l e c t o r . The column must be homogeneously packed w i t h powdered m a t e r i a l s such as c e l l u l o s e powder, i n s o l u b l e s t a r c h , Pevikon, g l a s s powder o r gel m a t e r i a l s such as p o l y a c r y l amide gel, s t a r c h gel and dextran g e l . A d e s c r i p t i o n o f s u i t a b l e equipment, sample l o a d i n g and o t h e r p r a c t i c a l i n s t r u c t i o n s was g i v e n by Svendsen81. A u n i v e r s a l cons t r u c t i o n o f t h e column, adaptable t o a l l k i n d s o f s u p p o r t i n g media, was described 82 by Bergrahm

.

Columns f i l l e d w i t h porous media have been used f o r t h e s e p a r a t i o n and p u r i f i c a t i o n o f many enzymes. Pancreatic l i p a s e (E.C.

3.1.1.3)

was p u r i f i e d 1 3 5 - f o l d by

employing i n s o l u b l e starch83, and crude e l a s t a s e p r e p a r a t i o n s were p u r i f i e d by L ~ e v e nu s~i n~g t h e same m a t r i x . C e l l u l o s e powder was a p p l i e d t o t h e s e p a r a t i o n o f enolase (E.C. 4.2.1.11)85,

glutamine transaminase (E.C. 2.6.1.15)86

g l y c e r a t e kinase (E.C. 2.7.2.3)87, o f c h o l i n e s t e r a s e 88

and phospho-

and Pevikon has been used f o r t h e p u r i f i c a t i o n

.

Column e l e c t r o p h o r e s i s on polyacrylamide gel has r e c e n t l y been employed f o r t h e p u r i f i c a t i o n o f enzymes and t h e p r e p a r a t i v e i s o l a t i o n o f isozymes because o f i t s g r e a t r e s o l v i n g power. Human l i v e r and i n t e s t i n e a l k a l i n e phosphatase were prepat i v e l y separated by Smith and Moss8’,

p u r i f i c a t i o n o f n i t r i t e reductase (E.C.

1.7.99.3)

was described by Husain and Sadanago, a l k a l i n e proteases from a BaciZZus s t r a i n by Van Welzen and Zuidweg”,

and cytochrome P-450 from l i v e r microsomesg2, isozymes o f

l a c t a t e dehydrogenase (E.C.

1.1.1.27)93

and ATP-ase (E.C. 3.6.1.8)

from M. Zyso-

d e i c t i c u s g 4 have been described. Gros e t a1 .95 p u r i f i e d s o l u b i l i z e d adenylate c y c l a s e (E.C. 4.6.1.1)

by p r e p a r a t i v e i s o e l e c t r i c f o c u s i n g on a column f i l l e d w i t h Sephadex

F i g . 13.3. E l u t i o n p r o f i l e o f baker’s y e a s t i n o r g a n i c pyrophosphatase isozymes ( I , 11, 111) separated by p r e p a r a t i v e isotachophoresis on a p l y a c r y l a m i d e gel P r o t e i n ; dashed area represents enzymic a c t i v i t y g 6 . column. -,

298 G-15 as t h e s o l i d s u p p o r t . The column was e q u i l i b r a t e d w i t h 10% g l y c e r o l c o n t a i n i n g 2% Ampholine and 1%o f L u b r o l Px. P r e p a r a t i v e i s o t a c h o p h o r e s i s i n a column c o n t a i n i n g p o l y a c r y l a m i d e g e l was used f o r t h e p u r i f i c a t i o n o f t h e isozymes o f i n o r g a n i c p y r o phosphatase (E.C.

3.6.1.1)

f r o m b r e w e r ' s y e a s t g 6 ( F i g . 13.3). S e p a r a t i o n was c a r r i e d

o u t i n an LKB apparatus w i t h phosphate as t h e l e a d i n g i o n and E-aminocaproic a c i d as t h e t e r m i n a t i n g e l e c t r o l y t e ; t h e c o u n t e r i o n was T r i s whereas Ampholines (pH 5-8) were used as spacer i o n s . Column e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e g e l w i t h c o n t i n uous e l e c t r o e l u t i o n o f t h e separated zones was a p p l i e d t o t h e p u r i f i c a t i o n o f hypox a n t i n e phosphoribosyl t r a n s f e r a s e (E.C. 2.4.2.8)

f r o m human e r y t h r o c y t e s g 7 . A f t e r

a s i n g l e r u n a complex p r o t e i n m i x t u r e y i e l d e d a h i g h l y e n r i c h e d p r e p a r a t i o n o f t h e enzyme ( F i g . 13.4).

F i g . 13.4. P r e p a r a t i v e i s o l a t i o n o f hypoxanthine-guanine phosphoribosyl t r a n s f e r a s e by column e l e c t r o p h o r e s i s on 8% p o l y a c r y l a m i d e g e l . S e p a r a t i o n was conducted i n T r i s - g l y c i n e b u f f e r (pH 8.3) a t 12 W. -, P r o t e i n ; dashed area i s enzymic a c t i v i t y ( r e f . 97). ANALYTICAL SEPARATION OF ENZYMES The main groups o f procedures t h a t have r e c e n t l y become a v a i l a b l e f o r t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f enzymes a r e t h o s e u t i l i z i n g as t h e s u p p o r t i n g medium f i l t e r - p a p e r , c e l l u l o s e a c e t a t e membranes, agar/agarose g e l

, starch

g e l and p o l y -

a c r y l a m i d e g e l . For o t h e r t h a n s t r i c t l y r o u t i n e a p p l i c a t i o n s , one o f t h e h i g h r e s o l u t i o n t e c h n i q u e s , w i t h e i t h e r s t a r c h g e l o r p o l y a c r y l a m i d e g e l , s h o u l d be t h e p r i m a r y method. A l l o f t h e above t e c h n i q u e s have been d e s c r i b e d i n d e t a i l elsewhere ( r e f . 98-105),

and t h e r e f o r e i n t h i s s e c t i o n we s h a l l c o n c e n t r a t e m o s t l y on t h e

p r e p a r a t i o n o f t h e sample f o r t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f enzymes i n crude m a t e r i a l s , on methods f o r t h e i r d e t e c t i o n and on t h e s e p a r a t i o n of isozymes.

299

Preparation of samples Enzymes can be s e p a r a t e d as p u r e p r o t e i n s , as m i x t u r e s o f p r o t e i n s d u r i n g t h e p u r i f i c a t i o n o f enzymes and as c r u d e e x t r a c t s o f c e l l s , t i s s u e s o r body f l u i d s . The methods p r e s e n t e d below a r e g e n e r a l d e s c r i p t i o n s o f t h e p r e p a r a t i o n o f d i f f e r e n t samples i s o l a t e d f r o m c e l l s , t i s s u e s and body f l u i d s w h i c h would be r e a d y f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f enzymes and t h e i r isozymes d i r e c t l y on t h e mat r i x. Red cells. Red c e l l s c o l l e c t e d b y c e n t r i f u g a t i o n a t 2000 g f o r 10 m i n a t 4OC from a n t i c o a g u l a t e d b l o o d a r e washed t h r e e t i m e s w i t h 0.15 - m o l / l NaCl. The packed c e l l s a r e l y s e d by f r e e z i n g and t h a w i n g and t h i s m a t e r i a l can be used f o r e l e c t r o p h o r e s i s . An a l t e r n a t i v e method i s haemolysis by adding 1 volume o f d i s t i l l e d w a t e r and 0.4 volume o f t o l u e n e . A f t e r s h a k i n g t h e m i x t u r e f o r 20-30 min, t h e t o l u e n e and l i p i d l a y e r i s a s p i r a t e d and d i s c a r d e d and t h e r e m a i n i n g m a t e r i a l i s c e n t r i f u g e d a t 2000 g f o r 20 min; t h e c l e a r s u p p e r n a t a n t i s used f o r e l e c t r o p h o r e t i c examination. White ceZZs. To t h e a n t i c o a g u l a t e d blood, d e x t r a n w i t h an average m o l e c u l a r w e i g h t o f about 235,000 i s added t o make a 3% s o l u t i o n and t h e m i x t u r e i s a l l o w e d t o s t a n d u n d i s t u r b e d i n a t e s t - t u b e f o r 30 rnin. The s u p e r n a t a n t c o n t a i n i n g l e u c o c y t e s i s removed by u s i n g a P a s t e u r p i p e t t e . I f t h e s e p a r a t i o n o f lymphocytes f r o m g r a n u l o c y t e s i s d e s i r e d , t h i s can be a c h i e v e d by d e n s i t y g r a d i n e t c e n t r i f u g a t i o n i n a m i x t u r e o f F i c o l l and T r i o s i l l o 6 . To 2.5 m l o f F i c o l l - T r i o s i l m i x t u r e (5 m l o f a 34% aqueous s o l u t i o n o f T r i o s i l and 12 m l o f a 9% aqueous s o l u t i o n o f F i c o l l ) i n a c e n t r i f u g e t u b e add c a r e f u l l y 2 m l o f f r e s h h e p a r i n i z e d b l o o d and c e n t r i f u g e a t 3000 g f o r 10 min a t 4OC. The lymphocytes s e p a r a t e i n t h e upper p a r t o f t h e t u b e as a l a y e r between t h e s u p e r n a t a n t plasma and t h e F i c o l l - T r i o s i l m i x t u r e . The polymorphs form a l a y e r a t t h e i n t e r f a c e between t h e F i c o l l - T r i o s i l and t h e r e d c e l l s which a r e packed a t t h e b o t t o m o f t h e tube. The lymphocytes and g r a n u l o c y t e s a r e removed s e p a r a t e l y by means o f a P a s t e u r p i p e t t e , washed w i t h 0.2% NaC1, d i s r u p t e d by s o n i c a t i o n ( p r o b e s o n i c a t o r , two o r t h r e e 10-s b u r s t s i n an i c e - b a t h ) o r ground w i t h g l a s s powder and c l a r i f i e d b y c e n t r i f u g a t i o n a t 20,000 g f o r 60 min a t 4'~. CeZZ cultures. C u l t u r e d c e l l s ( f i b r o b l a s t s ) a r e washed t h r e e t i m e s w i t h 0.15

m o l / l NaCl and c e n t r i f u g e d a t 2000 g f o r 10 min a t 4OC. Add an equal volume o f d i s t i l l e d w a t e r and s o n i c a t e (see White c e l l s ) , keeping t h e specimen c o o l w i t h i c e . The d e b r i s i s removed b y high-speed c e n t r i f u g a t i o n .

Tissue extracts. To about 1 g o f washed t i s s u e add 1 m l o f b u f f e r e d s a l i n e s o l u t i o n and g r i n d t h e m i x t u r e i n a s u i t a b l e homogenizer f o r 1 min. The c o n t e n t s o f t h e tube a r e c e n t r i f u g e d a t h i g h speed. When p l a n t t i s s u e s a r e used, t h e m a t e r i a l i s ground w i t h p u r i f i e d sea sand and small amounts o f b u f f e r s o l u t i o n u n t i l t h e sample i s c o m p l e t e l y homogeneous. The m i x t u r e i s t r a n s f e r r e d i n t o a c e n t r i f u g e t u b e and t h e d e b r i s i s spun down b y h i g h -

300

speed c e n t r i f u g a t i o n . S i m i l a r procedure can b e used when microorganism c e l l s ( b a c t e r i a , a l g a e , moulds, y e a s t s ) a r e used as t h e s o u r c e o f enzymes.

Principles o f enzgne detection Several d i f f e r e n t approaches have been developed f o r i d e n t i f y i n g t h e enzymes and t h e i r isozymes on t h e electropherograms. I n t h i s s e c t i o n we s h a l l d e a l w i t h t h e general procedures o f enzyme s t a i n i n g and d e t e c t i o n methods. The enzyme a c t i v i t y on t h e e l e c t r o p h e r o g r a m can be d e t e c t e d by c u t t i n g up t h e s u p p o r t i n g m a t r i x , e l u t i n g t h e p r o t e i n and measuring t h e enzymic a c t i v i t y o f t h e r e s u l t i n g f r a c t i o n s . Using t h i s procedure a g r e a t d e a l o f work has been c a r r i e d o u t on t h e e l e c t r o p h o r e t i c c h a r a c t e r i z a t i o n o f d i f f e r e n t enzymes. P r a c t i c a l d e t a i l s 108 o f t h e methods were g i v e n i n books by W i l k i n s o n l o 7 and Brewer and S i n g

.

However, t h e above approach was r a p i d l y superseded b y u s i n g s p e c i f i c enzymes t a i n i n g t e c h n i q u e s f o r r e v e a l i n g t h e enzyme a c t i v i t y d i r e c t l y i n t h e e l e c t r o p h o r e t i c m a t r i x . Several methods have been d e v i s e d t h a t can be c o n s i d e r e d under s e v e r a l main groups o f procedures: s i m p l e c o l o u r development methods, methods u s i n g s p e c i a l chromogenic coup1 i n g substances, e l e c t r o n - t r a n s f e r d y e - s t a i n i n g methods , f l u o r e s c e n t s t a i n i n g , r a d i o c h e m i c a l t e c h n i q u e s , enzyme-linked s t a i n i n g methods, immunological methods and m i s c e l l a n e o u s t e c h n i q u e s . SimpZe colour deveZoprnent methods. These procedures u t i l i z e a c o l o u r l e s s sub-

s t r a t e , which i s t r a n s f o r m e d by t h e enzymic r e a c t i o n i n t o a c o l o u r e d p r o d u c t . Many h y d r o l a s e s a r e d e t e c t e d by t h i s t e c h n i q u e . As an example, a c i d phosphatase can b e d e t e c t e d b y u s i n g p h e n o l p h t h a l e i n phosphate and p - n i t r o p h e n y l phosphate as sub109 strates

.

P h e n o l p h t h a l e i n diphosphate (Nag s a l t ) (275 mg) i s d i s s o l v e d i n 100 m l 0.05 m o l / l c i t r a t e b u f f e r (pH 5-6). A f i l t e r - p a p e r s t r i p i s soaked w i t h t h e s o l u t i o n and o v e r l a y e d on a g e l s u r f a c e c o n t a i n i n g t h e s e p a r a t e d enzyme. A f t e r a b o u t 4 h o f i n c u b a t i o n a t 37OC, t h e f i l t e r - p a p e r i s removed and t h e g e l i s made a l k a l i n e w i t h s m a l l amounts o f ammonia s o l u t i o n . E n z y m a t i c a l l y a c t i v e zones a r e seen as a pink colour. A c i d and a l k a l i n e phosphatases can be s t a i n e d b y u s i n g p - n i t r o p h e n y l phosphate as a s u b s t r a t e . The s t r i p o f g e l i s i n c u b a t e d d i r e c t l y i n a 20 mmol/-1 s o l u t i o n o f t h e s u b s t r a t e a t t h e a p p r o p r i a t e pH f o r a p p r o x i m a t e l y 30 min. I n t e n s e y e l l o w bands o f p - n i t r o p h e n o l appearing i n t h e g e l s h o u l d b e photographed i m m e d i a t e l y a f t e r s t o p p i n g t h e r e a c t i o n w i t h 1 m o l / l NaOH t o p r e v e n t d i f f u s i o n o f t h e dye. Proteases can be r e v e a l e d by u s i n g a c o l o u r e d s u b s t r a t e , e.g.,

cytochrome c

( r e f . 110, 111). A f t e r e l e c t r o p h o r e t i c s e p a r a t i o n t h e g e l i s immersed i n a s o l u t i o n c o n t a i n i n g cytochrome c and i n c u b a t e d under s u i t a b l e c o n d i t i o n s . The g e l i s t h e n f i x e d i n 12.5% t r i c h l o r o a c e t i c a c i d t o d e n a t u r e t h e u n d i g e s t e d absorbed p r o t e i n s . P r o t e i n a s e bands appear as t r a n s p a r e n t zones a g a i n s t an e v e n l y p r e c i p i t a t e d brown background.

301 A1 t e r n a t i v e l y , p r o t e a s e s can be s t a i n e d b y i n c o r p o r a t i n g t h e s u b s t r a t e i n t o t h e g e l . F i b r i n o g e n i s a good s u b s t r a t e f o r t h i s purpose because i t s h i g h m o l e c u l a r w e i g h t (340,000) p r e v e n t s i t s movement i n t h e g e l '12,113. F i b r i n o g e n (0.1% s o l u t i o n i n 0.001 m o l / l NaOH) i s added p r i o r t o t h e p o l y m e r i z a t i o n o f t h e r u n n i n g g e l t o g i v e a f i n a l c o n c e n t r a t i o n o f about 170 pg/ml. A f t e r e l e c t r o p h o r e s i s and washing, t h e g e l i s i n c u b a t e d o v e r n i g h t i n 0.04 m o l / l T r i s b u f f e r o f s u i t a b l e pH i n a m o i s t chamber a t 37OC. F u l l y developed bands a r e u s u a l l y observed a f t e r 15-20 h. The g e l s can be photographed a g a i n s t a d a r k f i e l d o r a r e s t a i n e d w i t h Coomassie b l u e o r another reagent. A c t i v a t o r s o r i n h i b i t o r s o f p r o t e a s e s can be r e v e a l e d a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n by u s i n g t h e method d e s c r i b e d by Henssen and Dowdle114.

I n t h i s method

t h e enzyme and s u b s t r a t e a r e copolymerized i n t o a m a t r i x o f SDS-polyacrylamide g e l a t t h e t i m e o f c a s t i n g . A f t e r e l e c t r o p h o r e s i s of h i g h - m o l e c u l a r - w e i g h t a c t i v a t o r s o r i n h i b i t o r s , t h e g e l i s washed w i t h T r i t o n X-100 t o remove SDS and r e s t o r e enzyme a c t i v i t y , t h e n i s i n c u b a t e d t o r e v e a l t h e c o n c e n t r a t e d a c t i v a t o r o r i n h i b i t o r bands. Developed zones can b e t r e a t e d as f o r f i b r i n o g e n g e l . Plasminogen a c t i v a t o r s were 114

analysed by t h i s method

.

CoupZing to chromogenic substances. T h i s t e c h n i q u e employs d i f f e r e n t chemical r e a g e n t s which f o r m c o l o u r e d d e r i v a t i v e s by c o u p l i n g w i t h t h e p r o d u c t o f t h e enzyme r e a c t i o n o r b y r e a c t i o n w i t h untransformed s u b s t r a t e . The l o c a l i z a t i o n o f e s t e r a s e s i n e l e c t r o p h o r e t i c media can be achieved b y t h e use of a - o r B-naphthyl e s t e r s . The a- o r 8-naphthol l i b e r a t e d d u r i n g t h e enzymatic r e a c t i o n i s coupled w i t h t e t r a z o t i z e d o - d i a n i s i d i n e t o produce an i n t e n s e p u r p l e dye. Several o t h e r compounds can be a p p l i e d as t h e diazonium s a l t s f o r c o u p l i n g r e a c t i o n s w i t h n a p h t h o l , e.g.,

F a s t B l u e RR, F a s t Red TRY F a s t B l u e BB, F a s t B l u e

B

and F a s t Garnet GBC. The above t e c h n i q u e can b e e a s i l y adapted f o r t h e d e t e c t i o n o f d i f f e r e n t groups o f e s t e r a s e s such as lipases115, phosphatases116,

c h o l i n e ~ t e r a s e sand ~ ~ ~o t h e r

h y d r o l a s e s , and s a t i s f a c t o r y r e s u l t s have been o b t a i n e d when paper, agar g e l , s t a r c h g e l and p o l y a c r y l a m i d e g e l have been used as s u p p o r t i n g media. F i g . 13.5 shows an example o f t h e c o u p l i n g r e a c t i o n f o r t h e d e t e c t i o n o f B-N-acetylglucosaminidase (E.C.

3.2.1.30)lI8.

The s e p a r a t i o n and d e t e c t i o n o f t h i s enzyme and i t s

isozymes i n d i f f e r e n t organs a r e an i m p o r t a n t t e s t f o r Tay-Sachs disease'''. same t e c h n i q u e can be a p p l i e d t o d e t e c t sphingomyelinase D (E.C.

3.1.4.41)120

The and

Amylases can be d e t e c t e d b y r e a c t i o n of t h e u n h y d r o l y s e d 125 substrate (starch) w i t h iodine

many o t h e r enzymes

.

The p o l y a c r y l a m i d e g e l s t r i p s a r e i n c u b a t e d f o r 2 h i n a s o l u t i o n c o n t a i n i n g 0.1 m o l / l T r i s (pH 7 . 6 ) , 2 mmol/l CaC12 and 0.5% s o l u b l e s t a r c h . The g e l i s r i n s e d w i t h w a t e r and t r e a t e d w i t h a s o l u t i o n o f 1.3% 12/3% KI t o s t o p t h e r e a c t i o n and s t a i n t h e u n r e a c t e d s t a r c h background. Zones o f a-amylases a c t i v i t y appear as l i g h t bands a g a i n s t a d a r k background.

3 02

p - N-Acetylglucosarnine

Naphthol A S B I 2 - a c e t a m i d o -2 deoxy - D- glucopyranoside

-

-p

CHZOH

$H20H

B ra

o

G

NH-COCH3 O H

CONH

cH30fJ

NZN

c; CH3

Fast G a r n e t GE

Coloured

diazo compound

Fig. 13.5. Coup1 ing reactions in the staining method for B-N-acetylglucosaminidase The iodine-starch reaction is also used for the localization of glutathione S-transferase (E.C. 2.5.1.18) isozymes after separation in starch The two-stage staining procedure utilizes l-chloro-2,4-dinitrobenzene and reduced glutathione as substrates. Stage 1. l-Chloro-2,4-dinitrobenzene (8 mg in 0.8 ml of ethanol) is mixed with 14 mg of reduced glutathione and dissolved in 20 ml of 0.1 mol/l potassium phosphate (pH 6.5). The solution is used for saturating a sheet of filter-paper which is overlayed on the cut surface of the gel and incubated at 37OC for 40 min. Stage 2. The filter-paper from stage 1 is removed and the gel is overlayed with a developing solution prepared by combining 30 ml of iodine solution (0.9 ml of 1% I2 in KI diluted in 30 ml of water) with an equal volume of 2% molten agar. The agar sets on the gel surface and an intense blue starch-iodine colour appears immediately in areas where reduced glutathione has been conjugated with l-chloro2,4-dinitrobenzene by the action of glutathione S-transferase. The method is specific for the above enzyme; however, l-chloro-2,4-dinitrobenzene can be used as a substrate for other enzymes, e.g., glyoxalase (E.S. 4.4.1.5) 127. EZectron-transfer duestaining methods. The most widely used reagents for the detection of oxidoreductases are tetrazolium salts and carbazole derivatives.

3 03 Methyl t h i a z o l y l t e t r a z o l i u m (MTT), i n a d d i t i o n t o m e t h y l t h i a z o l y l b l u e (NBT) , i s commonly used as an acceptor f o r dehydrogenase r e a c t i o n s and 3-amino-9-ethylcarbazole f o r oxidases and peroxidases. MTT undergoes e a s i l y r e d u c t i o n t o i n s o l u b l e , i n t e n s e l y coloured formazans ( F i g . 13.6). t

Br-

I 'ZH5

3-Amino- 9- ethylcarbozole

aND

MTT

\I;

\

CH3S04

I CH 3

N - Methylphenazinium methylsulphate (PMS)

Fig. 13.6. Methylthiazolyltetrazolium (MTT), 3-amino-9-ethylcarbazole and N-methylphenazinium methylsulphate (PMS) used as e l e c t r o n acceptors f o r t h e d e t e c t i o n o f dehydrogenases, oxidases and peroxidases.

3-Amino-9-ethylcarbazole i s a y e l l o w s o l u b l e dye which can be o x i d i z e d by oxidases o r peroxidases t o a dark-brown i n s o l u b l e product. The r e a c t i o n w i t h MTT proceeds most r a p i d l y i n t h e presence o f N-methylphenazinium methylsulphate (phenazine methosulphate, PMS) as an i n t e r m e d i a t e f o r electron transfer: Substrate t NAD(P) = Product NAD(P)H

+

H+

+

PMS

+

+

NAD(P)H

+

Ht

MTT = Formazan ( p u r p l e , i n s o l u b l e )

+

NAD(P)

Thus, both MTT and PMS a t a c o n c e n t r a t i o n o f about 0.1 mg/ml must be present i n t h e s t a i n i n g s o l u t i o n f o r t h e d e t e c t i o n o f enzymes t h a t c a t a l y s e t h e r e d u c t i o n o f p y r i d i n e coenzymes, NAD and NADP. As t h e MTT-PMS m i x t u r e i s l i g h t - s e n s i t i v e ,

the

i n c u b a t i o n must be performed i n t h e dark. S o l u t i o n s o f MTT-PMS a r e u n s t a b l e i n a1 k a l i n e b u f f e r s . Sul f h y d r y l compounds (2-mercaproethanol

, dithiothreitol )

cause

immediate p r e c i p i t a t i o n o f formazan owing t o r a p i d r e d u c t i o n o'f MTT by t h i o l groups i n t h e presence o f PMS. Much b e t t e r r e s u l t s a r e obtained when s t a i n s a r e a p p l i e d t o t h e gel i n t h e form

o f f i l t e r - p a p e r s t r i p s o r agar gel o v e r l a y s r a t h e s than f r e e s o l u t i o n s t a i n s . The o v e r l a y technique i s more economic:l

because l e s s s t a i n i n g s o l u t i o n can be used.

304 Example 1. S t a i n i n g system f o r l a c t a t e dehydrogenase (LDH) can be accomplished according t o t h e enzymic r e a c t i o n L-lactate

t

NAD

JJ3 Pyruvate

t

NADH t H+

Several systems f o r LDH s t a i n i n g have been developed and c r i t i c a l l y reviewed 128

.

A f t e r completing t h e e l e c t r o p h o r e t i c separation, t h e electrophorogram i s overlayed w i t h an agar gel s t r i p c o n t a i n i n g NAD and calcium l a c t a t e i n an optimal concentrat i o n , u s u a l l y a f i n a l c o n c e n t r a t i o n o f 8 mmol/l, and b u f f e r s o l u t i o n (0.05 m o l / l Tris-HC1, pH 8.0). MTT and PMS ( 5 mg/ml) a r e i n c l u d e d i n t h e s t a i n i n g s o l u t i o n and trapped i n 2% agar gel. I n c u b a t i o n should be performed i n a h u m i d i t y chamber a t 37OC f o r about 2 h. The formazan p r e c i p i t a t e s a r e formed o n l y a t t h e p o i n t s o f LDH a c t i v i t y . S i m i l a r s t a i n i n g systems can be employed t o r e v e a l malate dehydrogenase (E.C.

, i s o c i t r a t e dehydrogenase (E. C. 1.1.1.41 ; 1.1.1 .42)130, phosphogl u1.1.1.37) lZ9 conate dehydrogenase (E.C.

1.1. 1.49)13',

1. 1.1.43)131

, glucose-6-phosphate

dehydrogenase (E.C.

glyceraldehyde-phosphate dehydrogenase (E.C. 1.2.1.9)133

u r a c i l dehydrogenase (E.C.

1.3.1.1) 134

.

Instead o f PMS, 2,6-dichlorophenol-indophenol

(DCIP) can be used as an i n t e r -

mediary c a t a l y s t . By t h i s system g l u t a t h i o n e reductase (E.C. 1.6.4.2)135, diaphorase (E.C. 1.6.99.1)136

and d i h y d r o

NADH-

and g l y o x a l a s e 1137 have been l o c a l i z e d on s t a r c h

and polyacrylamide gels. As an example, s t a i n i n g o f g l u t a t h i o n e reductase i s described below. Example 2 . The s t a i n i n g m i x t u r e c o n t a i n s i n 10 m l o f 0.25 m o l / l Tris-HC1 b u f f e r (pH 8.4),

20 mg o f o x i d i z e d g l u t a t h i o n e , 5 mg o f NADPH, 0.1 mg o f D C I P and

5 mg o f MTT. A f t e r completion o f e l e c t r o p h o r e s i s t h e g e l i s s t a i n e d i n t h e usual

manner. The r e a c t i o n s d u r i n g s t a i n i n g proceed according t o t h e scheme

reductase

MTT

-

\Formazan

Example 3. D e t e c t i o n o f c a t a l a s e (E.C. 1.11.1.6). sium hexacyanoferrate( 111) reagent*% H O CatalaseI

+

Potassium hexacyanoferrate( 111) reagent

I

H,O

according t o t h e r e a c t i o n

Dark green dye

-2,2

t 02N -o

Catalase i s d e t e c t e d by potas-

c o l o u r change

305

After electrophoresis incubate t h e gel f o r 15 min in d i l u t e (1 : 1000) hydrogen peroxide. Pour off t h e hydrogen peroxide, r i n s e t h e gel with water and immerse i t i n a f r e s h l y prepared 1 : 1 mixture of 2% potassium hexacyanoferrate(II1) and 2 % i r o n ( I I 1 ) chloride. Mix gently f o r a few minutes, then remove t h e s o l u t i o n . Catalase appears as yellow bands a g a i n s t a blue-green background 139 . Example 4. Another example of e l e c t r o n - t r a n s f e r s t a i n i n g system i s t h e detection of i s o c i t r a t e lyase (E.C. 4.1.3.1) on polyacrylamide gel140. The g e l s a r e pre incubated a t 25OC f o r 5 min i n a Petri dish w i t h a 10 mmol/l solution of morpholinopropanesulphonic acid containing 5.3 mmol/l MgC12, 4.2 mmol/l d i t h i o t h r e i t o l and 9.8 m o l / l c i t r i c acid (pH 7 . 5 ) . I s o c i t r i c acid i s omitted from c o n t r o l s . Then 0.2 m l of S c h i f f ' s reagent (fuchsin decolorized by sulphurous a c i d ) i s added f o r each 4.8 ml of t h e above reaction mixture and s t a i n i n g i s c o n t r o l l e d v i s u a l l y f o r 20-60 min u n t i l optimal banding p a t t e r n s of t h e red Schiff-aldehyde complex a r e developed. The gels a r e rinsed w i t h d i s t i l l e d water. Detection by fluorogenic staining. A non-fluorescent s u b s t r a t e i s transformed by an enzyme on t h e highly f l u o r e s c e n t product o r from the f l u o r e s c e n t s u b s t r a t e a non-fluorescent product i s formed. For the f i r s t p o s s i b i l i t y , d e r i v a t i v e s of 4-methylumbelliferone (Fig. 13.7), a r e employed as s u b s t r a t e s , being very sensit i v e f o r the detection of many hydrolases, such as phosphatases, sulphatases, carbohydrases and l i p a s e s . Methylumbelliferone fluoresces optimally a t a l k a l i n e pH, so i t i s common p r a c t i c e t o make t h e s u r f a c e of t h e electropherogram a l k a l i n e with ammonia vapour o r a s u i t a b l e buffer s o l u t i o n . Applying the same technique, d e r i v a t i v e s of fluorescein can a l s o be used a s s u b s t r a t e s f o r the detection of d i f f e r e n t enzymes.

Fluorescein

Fig. 13.7. 4-Methylumbelliferone and fluorescein used in fluorogenic s t a i n s .

As an example of so-called negative fluorescent s t a i n i n g a r e those reactions i n which reduced pyridine nucleotides a s the fluorescent cofactors a r e oxidized t o nonfluorescent species. Employing t h i s technique several enzymes and t h e i r isozymes can be detected on various supporting media. Glutathione peroxidase (E.C. 1.11.1.9) can 141 : be detected as follows

306 tert.-Butyl

alcohol

Glutathione peroxidase

NADP (non-fluorescent)

NADPH (fluorescent)

The above method needs e x t e r n a l enzyme g l u t a t h i o n e r e d u c t a s e t o reduce GSSG i n t h e presence o f NADPH. S i m i l a r t e c h n i q u e s a r e used t o s t a i n g l u t a m a t e - o x a l o a c e t a t e transaminase ( E . C. 2.6.1. 1)14', g l u t a m a t e - p y r u v a t e transaminase ( E . C. 2.6.1.2) 143 and many o t h e r s , b u t t h e above method u s u a l l y r e q u i r e s t h e a d d i t i o n o f exogenous enzymes t o t h e s t a i n i n g m i x t u r e and t h i s w i l l be d i s c u s s e d l a t e r . 4-Methylumbelliferyl d e r i v a t i v e s f o r p o s i t i v e fluorescent s t a i n i n g are widely used f o r d e t e c t i n g a c i d p h o s p h a t a s e ~ ' ~ ~a, r y l ~ u l p h a t a s e s ~a -~g~l u,c o s i d a s e s 146 , 8 - g a l a c t o ~ i d a s e land ~ ~ 8 - N - a c e t y l g l u c o ~ a m i n i d a s e s ~F1 ~ u~o. r e s c e i n d i a c e t a t e was used f o r s t a i n i n g c a r b o n i c anhydrase i s o z y ~ n e sand ~ ~ d~i f f e r e n t e s t e r a s e s l 5 O . The

-

scheme o f t h e r e a c t i o n s i s as f o l l o w s :

phosphatase

4-methylumbelliferyl

phosphate

-

Pi

esterase

fluorescein acetate

acetate

+

+

4-methylumbelliferone

(fluorescent

fluorescein (fluorescent)

Nucleases can be s t a i n e d b y t h e f l u o r e s c e n c e o f e t h i d i u m bromide which b i n d s t o h i g h - m o l e c u l a r - w e i g h t DNA and DNA f r a g m e n t s l 5 l . DNA a t 10 ug/ml i s i n c o r p o r a t e d i n t o t h e g e l ( p o l y a c r y l a m i d e , agarose g e l ) . A f t e r e l e c t r o p h o r e t i c s e p a r a t i o n o f enzymes, t h e enzymic a c t i o n i s i n i t i a t e d by p l a c i n g t h e g e l i n 0.04 m o l / l Tris-HC1 b u f f e r (pH 7.6) c o n t a i n i n g 2 mmol/l MgC12 and 2 mmol/l CaC12. Sodium a z i d e (0.02%) i s added t o p r e v e n t m i c r o b i a l growth. The g e l s s h o u l d be i n c u b a t e d a t 3OoC f o r about 2 h. Then e t h i d i u m bromide a t 2 ug/ml i s added t o t h e s o l u t i o n t o s t a i n t h e

DNA and i n c u b a t e d f o r 2 h b e f o r e photographing t h e f l u o r e s c e n c e o f e t h i d i u m bromide bound t o DNA i n t h e g e l . DNase g i v e s r i s e t o d a r k bands on a f l u o r e s c e n t background.

A s i m i l a r procedure was a p p l i e d t o t h e d e t e c t i o n o f r e s t r i c t i o n endonucleases i n HuernophiZus p u ~ a i n f Z u e n z a e and ~ ~ ~t o examine t h e s p e c i f i c i t y o f endodeoxyribon u c l e a ~ e and l ~ ~ P1 n u c l e a ~ e (l F~i g~. 13.8). U r o p o r p h i r i n o g e n I s y n t h e t a s e (E.C.

4.3.1.8)

was d e t e c t e d on t h e b a s i s o f t h e 155

r e d f l u o r e s c e n c e r e s u l t i n g f r o m t h e enzymatic p r o d u c t i o n o f u r o p o r p h i r i n o g e n

.

A f t e r i s o e l e c t r i c f o c u s i n g o f t h e enzyme on t h e g e l p l a t e s i n pH g r a d i e n t o f 4.0-6.5,

t h e g e l s were i n c u b a t e d f o r 45-90 min a t 45OC i n t h e presence o f 90 mmol/l

307

F i g . 13.8. R e s t r i c t i o n e n d o n u c l e a s e a n a l y s i s o f m t DNA f r o m d e l e t i o n m u t a n t s o f Sncchurornydcs ccrevisiae. E l e c t r o p h o r e s i s o f t h e d i g e s t s was c a r r i e d o u t o n 0.7% a g a r o s e g e l , t h e n s t a i n e d w i t h e t h i d i u n i b r o m i d e and p h o t o g r a p h e d u n d e r UV i l l u m i n a t i o n . Lanes 3, 5, 7 and 9 a r e r e s t r i c t i o n p a t t e r n s o f m t DNA f r o m m i t - A 101 m u t a n t d i g e s t e d w i t h Hha, H i n c 11, Eco R I and Bam H I r e s t r i c t a s e s , r e s p e t t i v e l y . Lanes 2, 4, 6 and 8 w e r e o b t a i n e d w i t h t h e same enzymes i n t h e c a s e o f r h o I D 4 1 - 6 / 1 6 1 s t r a i n . Lanes 1 and 10 a r e f r o m phage A DNA d i g e s t o b t a i n e d w i t h H i n d I 1 1 r e s t r i c t ase ( f r o n i N.J. A l e x a n d e r , R.D. V i n c e n t and P.S. Perlnian, J . B i a l . Chem., 254 ( 1 9 7 9 ) 2471). of p o r p h o b i l i n o g e n i n 0.3 m o l / l Tris-HC1

(pH 8 . 2 ) . The r e d f l u o r e s c e n t bands t h a t

a r o s e f r o m spntaneous o x i d a t i o n o f u r o p o r p h i r i n o g e n t o u r o p o r p h i r i n were p h o t o g r a p h e d w i t h UV i l l u m i n a t i o n . L-Gulono-y-lactone

oxidase

(E.C. 1 . 1 . 3 . 8 ) , an enzyme p o s s e s s i n g a c o v a l e n t l y

bound f l a v i n p r o s t h e t i c g r o u p , was r e v e a l e d o n S D S - p o l y a c r y l a m i d e g e l b y i t s i n t r i n s i c f l u o r e s c e n c e 1 5 6 . The method can be a p p l i e d t o t h e d e t e c t i o n o f o t h e r enzynies w i t h c o v a l e n t l y bound f l a v i n s as t h e p r o s t h e t i c g r o u p s .

RuciioclhemicaZ rneLhods. I n g e n e r a l , t h e s e methods depend on t h e d e t e c t i o n o f a r a d i o a c t i v e product b y autoradiographic techniques o r scanning o r counting o f t h e s l i c e d e l e c t r o p h e r o g r a m i n t h e s c i n t i l l a t i o n c o u n t e r . The m o s t d i f f i c u l t s t e p i s t o remove t h e e x c e s s o f r a d i o a c t i v e s u b s t r a t e f r o m t h e e l e c t r o p h o r e t i c medium.

308 One o f t h e most e f f e c t i v e approaches i s t o p r e c i p i t a t e t h e r a d i o a c t i v e p r o d u c t and wash o u t t h e excess o f s u b s t r a t e . T h i s t e c h n i q u e was a p p l i e d t o t h e l o c a l i z a t i o n o f g a l a c t o k i n a s e (E.C. ( r e f . 157), t r y p t o p h y l - t R N A s y n t h e t a s e (E.C.

6.1.1.2)15*

2.7.1.6)

and o t h e r enzymes. The

r e a c t i o n s w i t h t h e p r e c i p i t a t i o n o f a r a d i o a c t i v e p r o d u c t were c a r r i e d o u t f o r g a l a c t o k i n a s e as f o l l o w s :

c4clGalactose

\ TATP gal actokinase

c"1

L

galactose-

A

D

,

1-phosphate ( p r e c i p i t a t e d by lanthanum c h l o r i d e and a u t o r a d i o g r a p h e d ) and f o r tryptophan-tRNA s y n t h e t a s e as f o l l o w s :

[_' c] Tryptophan I'

-

tryptophan t R N A synthetase

AMP

+

PPi

(precipitated with cold 5% TCA, washed w i t h 95% e t h a n o l i n 1 m o l / l acet a t e b u f f e r , pH 5.0, and autoradiographed) R a d i o a c t i v e d e t e c t i o n has been a p p l i e d

o t h e determination o f base-specific

r i b o n u c l e a s e s i n d i f f e r e n t tissues159. 32P-Labelled rRNA was added t o l i m i t d i g e s t i o n under s t a n d a r d c o n d i t i o n s (2.5 min a t 37OC) and t h e d i g e s t s were separated on p o l y a c r y l a m i d e

t h e n a u t o r a d i o g r a p h e d by o v e r l a y i n g on X-ray f i l m ,

developed, scanned and analysed ( F i g . 13.9). Because o f t h e importance o f t h e phosphorylation-dephosphorylation r e a c t i o n s r e c e n t l y s t u d i e d i n many l a b o r a t o r i e s , t h e d e t e c t i o n o f p r o t e i n k i n a s e s o r phosp h o p r o t e i n phosphatases i n t h e e l e c t r o p h o r e t i c m a t r i x has r e c e i v e d i n c r e a s i n g a t t e n t i o n . C o n d i t i o n s f o r t h e assay o f p r o t e i n k i n a s e a c t i v i t y on p o l y a c r y l a m i d e g e l may be used as f o l l o w s 161y162:

a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n o f t h e enzymes

t h e g e l has t o be a d j u s t e d t o an a p p r o p r i a t e pH ( e q u i l i b r a t i o n phase). The g e l i s

309

F i g . 13.9. S p e c i f i c i t y o f c h t c k e n l i v e r RNase CL3 w i t h y e a s t 5.8 S r R N A as t h e s u b s t r a t e . Samples o f [5t-32P_l r R N A were i n c u b a t e d w i t h each enzyme i n d i c a t e d a t 37OC f o r 2.5 min, t h e n s u b j e c t e d t o p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and r a d i o autographed. -ENZ, c o n t r o l (no enzyme i n t h e sample). The numbers a l o n g t h e l e f t hand s i d e r e f e r t o t h e p o s i t i o n s o f n u c l e o t i d e r e s i d u e s i n y e a s t 5 . 8 S rRNA; t h e l e t t e r s on t h e r i g h t - h a n d s i d e r e f e r t r e s i d u e s a t t h e 5 ' - e n d o f t h e s p l i t o l i g o n u c l e o t i d e s by t h e c o r r e s p o n d i n g RNasePSg. then soaked w i t h s u b s t r a t e [histone,

protamine, e t c . ,

5 mg/ml i n 50 mmol/l mor-

p h o l i n o p r o p a n e s u l p h o n i c a c i d b u f f e r (pH 6.8) c o n t a i n i n g 1 mmol/l d i t h i o t h r e i t o l and 10 mmol/l magnesium a c e t a t e 1 f o r 30 min a t 3OoC ( p r e - i n c u b a t i o n phase). The 32 phosphoryl t r a n s f e r a s e r e a c t i o n i s i n i t i a t e d by t h e a d d i t i o n o f [ g y - A T P (0.1 p m o l / l , s p e c i f i c a c t i v i t y ca. 30 Ci/mmol ) and i n c u b a t e d f o r 30 m i n ( i n c u b a -

310 t i o n phase). Following t h e incubation, the gels are t r a n s f e r r e d i n t o Erlenmeyer f l a s k s containing a mixture o f 25% methanol and 15% TCA i n order t o p r e c i p i t a t e t h e p r o t e i n . For removal o f non-covalently bound r a d i o a c t i v i t y , each gel i s sepa r a t e l y extracted w i t h 3 x 80 m l o f t h e above s o l u t i o n a t 4OC f o r a t o t a l 20 h . TCA can be removed w i t h 3 x 80 m l washes w i t h 25% methanol. For r a d i o a c t i v i t y measurements the g e l s are autoradiographed o r are frozen i n d r y - i c e and c u t i n t o 1-mm s l i c e s w i t h a gel c u t t e r . The s l i c e s are d r i e d overnight a t 6OoC and counted

i n a l i q u i d s c i n t i l l a t i o n counter. The s e n s i t i v i t y o f the above method was suff i c i e n t t o detect about 150 u n i t s o f p r o t e i n kinase from r a t l i v e r c e l l s when histone was used as the substrate. The r e l a t i o n s h i p between t h e amount o f enzyme and 32P incorporated i n t h e p r o t e i n acceptor showed good l i n e a r i t y (Fig. 13.10).

4

8

12

16

20

24

28

Protein kinare (units x 10-2 per gel)

Fig. 13.10. Phosphorylation o f histone i n r e l a t i o n t o d i f f e r e n t amounts o f p r o t e i n kinase. The enzyme was separated by i s o e l e c t r i c focusing i n polyacrylamide g e l s i n t h e presence o f 0.87% Ampholine and 20% g l y c e r o l . Focusing was c a r r i e d o u t a t 4OC and 100 V f o r 12-16 h. The gels were c u t i n t o 5-mm s l i c e s pre-soaked w i t h substrate r o t e i n and assay s o l u t i o n containing P'tlATP f o r 30 min and t h e r a d i o a c t i v i t y o f g2P bound t o p r o t e i n was measured f o l l o w i n g p r e c i p i t a t i o n w i t h metbanol-TCA solut i o n (adapted from r e f . 162). Dephosphorylation k i n e t i c s o f t h e membrane p r o t e i n s catalysed by phosphoprotein phosphatase were studied by using SDS-polyacrylamide gel electrophoresis and autoradi~graphy'~ (Fig. ~ 13.11).

311 A

111

E,

E,

5: In

0

In

In

4

U

. . +

’-

0

0.5

1 0 0.5 Relative migration

1

F i g . 13.11. Scanning o f radioautograms a f t e r dephosphorylation o f 3 2 P - l a b e l l e d e r y t h r o c y t e p r o t e i n s by membrane phosphoprotein phosphatase. E r y t h r o c y t e p r o t e i n s were phosphorylated (A) w i t h o u t o r ( 6 ) w i t h t h e presence o f CAMP, washed f r e e o f ATP and used f o r phosphatase r e a c t i o n . A l i q u o t s were removed a t v a r i o u s times, r e s o l v e d by SDS-polyacryl amide gel e l e c t r o p h o r e s i s and t h e radioautograms scanned. a, Zero-time phosphatase r e a c t i o n ; b-d, 30-, 60- and 180-min r e a c t i o n time, r e s pect ive1y163. Autoradiographic techniques f o r t h e d e t e c t i o n o f phosphoryl t r a n s f e r a s e s were used by T i s c h f i e l d e t a1.164,

f o r DNase i n b l o o d serum by Zb’llner e t a1.165 and 166

f o r p o l y n u c l e o t i d e kinase by Szekely and Sanger

.

P r o t e i n s y n t h e s i s a c t i v i t y was detected on e l e c t r o p h o r e t i c m a t r i c e s by u s i n g r a d i o a c t i v e substrates16’.

t 5 g M e t h i o n i n e i s added t o t h e complete system f o r

p r o t e i n s y n t h e s i s and, a t t h e end o f i n c u b a t i o n , a l i q u o t s a r e removed and heated a t 90°C f o r 2 min i n 0.7 mol/l 2-mercaptoethanol-3% SDS-10% g l y c e r o l s o l u t i o n . Samples a r e then electrophoresed on a 10-20% g r a d i e n t polyacrylamide g e l i n t h e presence o f 0.1% SDS. The gel s l a b s a r e d r i e d under vaccum and autoradiographed u s i n g X-ray f i l m (Kodak b l u e f i l m ) . The enzymes can a l s o be s p e c i f i c a l l y r a d i o l a b e l l e d i n v i v o I 6 * o r r a d i o i o d i n a t e d i n v i t r o w i t h a l a c t o p e r o x i d a s e system16’.

The method a l l o w s t h e d e t e c t i o n o f t r a c e

amounts o f enzyme p r o t e i n t h a t are u s u a l l y n o t d e t e c t a b l e w i t h common enzyme s t a i n s ; i t i s about 1 5 0 - f o l d more s e n s i t i v e than c l a s s i c a l methods.

ImunoehernieaZ procedures. Enzymes and t h e i r m u l t i p l e forms can be detected i n electropherogram g e l s o r membranes b y immunochemical methods. N a t i v e enzymes can be separated i n agarose g e l c o n t a i n i n g a n t i b o d i e s l 7 O o r t h e immunoprecipitate f i r s t

312 formed i s s o l u b i l i z e d and s e p a r a t e d by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . When t h e enzyme is r a d i o a c t i v e i t s s u b u n i t c o m p o s i t i o n can be e a s i l y determined 171. I n t h e f i r s t technique17'

d u r i n g e l e c t r o p h o r e s i s t h e enzyme-antigens a r e t r a n s -

p o r t e d downwards i n t h e d i r e c t i o n o f t h e anode, t h e y e n t e r t h e g e l c o n t a i n i n g s p e c i f i c a n t i b o d i e s and f o r m complexes which f i n a l l y g i v e i m m o b i l e a n t i g e n - a n t i b o d y p r e c i p i t a t e s i n t h e g e l . The l a r g e r t h e amount o f a n t i g e n i n t h e sample, t h e l o n g e r t h e p r e c i p i t a t e extends i n t o t h e g e l phase. I m m u n o p r e c i p i t a t e s may be r e a d d i r e c t l y i n o b l i q u e l i g h t i f t h e y c o n t a i n enough p r o t e i n o r t h e y must be r e v e a l e d by h i s t o chemical s t a i n s f o r enzyme a c t i v i t y o r f o r p r o t e i n . The t e c h n i q u e d e s c r i b e d i s v e r y s e n s i t i v e and can a l s o be used f o r t h e q u a n t i t a t i v e e v a l u a t i o n o f t h e s e p a r a t e d enzymes. C l a s s i c a l t e c h n i q u e s o f immunoelectrophoresis have been used f o r t h e d e t e c t i o n o f human c a r b o n i c anhydrase isozymes CA1 and CA2172

, and

c r o s s e d immuno-affino-

e l e c t r o p h o r e s i s w i t h c o n c a n a v a l i n A i n t h e g e l was used f o r t h e s e p a r a t i o n o f human u r i n a r y c a r b o x y l i c e s t e r h y d r o l a s e and t o i d e n t i f y them as g l y c o p r o t e i n s

173

.

Immunoelectrophoresis has been used i n a s t u d y o f human serum a r y l e s t e r a s e and chol i n e ~ t e r a s e ' ~ f~o;l l o w i n g i m m u n o p r e c i p i t a t i o n t h e enzymes were s t a i n e d by t h e double s u b s t r a t e t e c h n i q u e u t i l i z i n g a m i x t u r e o f a- and 8 - n a p h t h y l a c e t a t e and Fast Red TR s a l t as c o u p l i n g agent. Crossed immunoelectrophoresis was used t o d e t e c t e l a s t a s e 2 i n b l o o d serum and p a n c r e a t i c j u i c e 1 7 5 , a l k a l i n e phosphodiesterase i n plasma membranes o f r a t l i v e r 1 7 6 , carbamoyl-phosphate s y n t h e t a s e (E.C. 6.3.4.16) 178,179 ( r e f . 177) and plasma p r o t e a s e i n h i b i t o r s o f human p a n c r e a t i c e l a s t a s e Plasma membrane a l k a l i n e phosphodiesterase f r o m r a t l i v e r c e l l s was s e p a r a t e d by fused r o c k e t immunoelectrophoresis i n agarose g e l c o n t a i n i n g Sepharose 46-conc a n a v a l i n A. The i m m u n o p r e c i p i t a t e was s t a i n e d f o r enzymic a c t i v i t y w i t h t h y m i d i n e

5'-monophospho-l-naphthyl

e s t e r and F a s t B l u e 8. By t h e above procedure i t was

shown t h a t t h r e e d i f f e r e n t a n t i g e n s possessing a c t i v i t y o f a l k a l i n e phosphodie s t e r a s e a r e p r e s e n t i n plasma membranes and t h a t a l l enzyme a n t i g e n s a r e g l y c o p r o t e i n s ( F i g . 13.12).

A c o m b i n a t i o n o f i m m u n o p r e c i p i t a t i o n and SDS-electrophoresis on a c r y l a m i d e g e l 171

i s v e r y u s e f u l f o r d e t e c t i n g t h e s u b u n i t c o m p o s i t i o n o f an o l i g o m e r i c enzyme

.

A n t i b o d i e s a r e r a i s e d i n r a b b i t s f o r t h e g i v e n holoenzyme. From t h e e x t r a c t i n which t h e enzyme p r e s e n t i s p r e c i p i t a t e d by s p e c i f i c a n t i b o d i e s , t h e immunoprecipi t a t e i s c o l l e c t e d by c e n t r i f u g a t i o n and washed w i t h b u f f e r s o l u t i o n c o n t a i n i n g

1%T r i t o n X-100. The i m m u n o p r e c i p i t a t e i s t h e n d i s s o l v e d by h e a t i n g a t 100°C f o r 5 min i n 2.5% SDS s o l u t i o n c o n t a i n i n g 0.1% o f 2-mercaptoethanol

and e l e c t r o -

phoresed i n an SDS-polyacrylamide g e l system. When t h e enzyme i n t h e e x t r a c t i s radioactive a f t e r e l e c t r o p h o r e t i c separation t h e gels a r e autoradiographed o r s l i c e d , d i g e s t e d w i t h hydrogen p e r o x i d e and t h e r a d i o a c t i v i t y i s measured i n a s c i n t i l l a t i o n c o u n t e r . The above procedure has been used t o f o l l o w t h e b i o s y n t h e s i s

313 o f m i t o c h o n d r i a 1 cytochrome c o x i d a s e ( E . C . 181 polymerase (E.C. 2.7.7.6)

1.9.3.1) 171y180 and DNA-dependent RNA

.

F i g . 13.12. Fused r o c k e t immunoelectrophoresis o f plasma membrane a l k a l i n e phosp h o d i e s t e r a s e w i t h an i n t e r m e d i a t e g e l c o n t a i n i n g c o n c a n a v a l i n A-Sepharose ( r i g h t ) o r Sepharose o n l y ( l e f t ) . Separated a n t i g e n s i n t h r e e d i l u t i o n s (2.5, 5 and 10 mg/ml of p r o t e i n ) were r e a c t e d a g a i n s t a n t i p l a s m a membrane a n t i s e r u m and t h e p l a t e was s t a i n e d f o r a l k a l i n e phosphodiesterase a c t i v i t y . A l l t h r e e o f t h e a c t i v e a n t i g e n s were r e t a i n e d i n t h e c o n c a n a v a l i n A - c o n t a i n i n g g e l , as seen f r o m t h e decreased h e i g h t s o f t h e p r e c i p i t a t e s ( r i g h t ) compared w i t h t h e c o n t r o l ( l e f t ) 1 7 6 .

Exogenous enzyme-Zinked staining me*hods. I n t h i s group o f methods, t h e p r o d u c t o f t h e enzymic r e a c t i o n t s t r e a t e d w i t h exogenous enzymes added i n s t a i n i n g m i x t u r e s t o o b t a i n a substance t h a t c o u l d be d e t e c t e d by i t s c o l o u r , f l u o r e s c e n c e o r r a d i o a c t i v i t y . The most common t e c h n i q u e u t i l i z e s exogenous dehydrogenase w i t h i t s c o r r e s p o n d i n g coenzymes as t h e l i n k i n g system t o f o r m a formazan a t t h e s i t e s o f t h e enzyme which c a t a l y s e s t h e p r i m a r y r e a c t i o n . F o r i n s t a n c e , hexokinase can b e det e c t e d i n t h e presence o f glucose-6-phosphate dehydrogenase as t h e l i n k i n g enzyme

182.

.

314

AT P

ADP hexokinase

G1 ucose

Glucose-6phosphate

G1 uconate-6-P

g l ucose-6-P dehydrogenase

NADPH

Formazan

MTT

As an example o f a f l u o r e s c e n t end r e a c t i o n p r o d u c t t h e d e t e c t i o n o f p y r u v a t e k i n a s e (E.C.

2.7.1.40)

can be considered183.

L a c t a t e dehydrogenase i s t h e l i n k i n g

enzyme and t h e s i t e o f p y r u v a t e k i n a s e a c t i v i t y i s d e t e c t e d as t h e n o n - f l u o r e s c e n t zones caused by NAD a g a i n s t a f l u o r e s c e n t background o f NADH: Phosphoenol pyruvate

>I(

pyruvate kinase ADPP AT

Pyruvate

Lactate

1a c t a t e dehydrogenase

NADH (fluorescent)

NAD (non-fluorescent)

A r e a c t i o n m i x t u r e c o n t a i n i n g phosphoenol p y r u v a t e , ADP, NADH, s a l t s and l a c t a t e dehydrogenase i s a p p l i e d t o t h e g e l on a f i l t e r - p a p e r o v e r l a y and, a f t e r incubat i o n under o p t i m a l c o n d i t i o n s , i s viewed under UV i l l u m i n a t i o n . Enzyme-linked s t a i n i n g systems f o r t h e l o c a l i z a t i o n o f some enzymes a r e more complex and can a l s o be used f o r t h e q u a n t i t a t i v e e v a l u a t i o n o f t h e enzymic a c t i v ity. Localization o f galactosyl transferase

i s an example184.

(E.C. 2.4.1.23)

i n polyacrylamide gel

U t i l i z i n g t h r e e i n t e r m e d i a t e enzymes i n t h e r e a c t i o n m i x t u r e ,

g a l a c t o s y l t r a n s f e r a s e a c t i v i t y has been coupled t o t h e p r o d u c t i o n o f NDAH w i t h s t o i c h i o m e t r y o f 2 mol o f NADH produced f o r each mole o f g a l a c t o s e t r a n s f e r r e d t o N-acetylglucosamine: gal actosyl UDP-galactose t a c c e p t o r

-

a c c e p t o r - g a l a c t o s e t UDP

transferase nucleotidase-5'-di

UDP t ATP

UTP t ADP

phosphate k i n a s e

315 UDP-g1 ucose UTP t glucose-1-phosphate

-

*

UDP-glucose t PPi

pyrophosphoryl ase

UDP-glucose

UDP-glucose + 2 NAD

UDP-glucuronic a c i d t 2 NADH ( f l u o r e s c e n t )

dehydrogenase

A s o l u t i o n o f c o u p l i n g enzymes and c o f a c t o r s i s mixed w i t h agarose s o l u t i o n t o

g i v e a f i n a l agarose c o n c e n t r a t i o n o f 2%. A f t e r e l e c t r o p h o r e s i s , acrylamide s l a b g e l s a r e incubated w i t h agarose i n d i c a t o r gel c o n t a i n i n g t h e c o u p l i n g enzyme system and viewed under UV l i g h t and photographed. The i n t e n s i t y o f fluorescence increases w i t h i n c r e a s i n g g a l a c t o s y l t r a n s f e r a s e a c t i v i t y a p p l i e d t o t h e g e l . Employing t h e above procedures, many enzymes have been l o c a l i z e d and evaluated i n d i f f e r e n t s u p p o r t i n g media. The most commonly used a r e t e s t s f o r phosphofructokinase (E. C. 2.7.1.11)

phosphoglycerate kinase (E. C. 2.7.2 .3)186,

creatine

kinase (E.C. 2.7.3.2) 187y188, nucleoside t r i p h o s p h a t e adenylate kinase (E.C. 2.7.4. phosphogl ucomutase (E.C. 2.7.5. , phosphoglyceromutase (E. C. 2.7.5.3)lg1,

c y c l i c AMP phosphodiesterase (E.C. 3.1.4.17)192,

a l d o l a s e l g 3 , arginase

(E.C. 3.5.3.1) 194y195, a r g i n i n o s u c c i n a s e (E.C. 4.3.2.1) 194y196, D-amino a c i d oxidase (E.C. 1.4.3.3)lg7 and triosephosphate isomerase (E.C. 5.3.1.1) 198 ,199 MisceZZaneous detection techniques. Dehydrogenases, such as l a c t a t e dehydro-

genase isozymes, have been detected and determined d i r e c t l y i n s i t u a f t e r e l e c t r o 200,201. phoreses i n agar gel by t h e technique c a l l e d enzymoelectrophoresis

*

second agar gel s t r i p c o n t a i n i n g t h e s u b s t r a t e ( p y r u v a t e ) and NADH a t t h e same pH and i o n i c s t r e n g t h o f t h e b u f f e r as those used f o r e l e c t r o p h o r e s i s i s prepared. The e l e c t r o p h o r e s i s g e l (on a g l a s s s l i d e ) i s superimposed on t h e s u b s t r a t e g e l and t h e p a i r e d s l i d e s a r e t r a n s f e r r e d t o a spectrophotometer i n a s p e c i a l r e c t a n g u l a r frame and t h e molar a b s o r p t i v i t y i s read a t 340 nm a t 0.25-mm i n t e r v a l s . As t h e enzymic r e a c t i o n proceeds, NADH i s o x i d i z e d and t h e absorbance decreases, so t h a t 99,202 by repeated scanning t h e k i n e t i c s o f t h e c a t a l y s e d r e a c t i o n can be f o l l o w e d Enzymes r e l e a s i n g orthophosphate can be revealed i n t h e s u p p o r t i n g medium by phosphomolybdate formation, which i n t h e presence o f a s c o r b i c a c i d i n s t r o n g l y a c i d i c s o l u t i o n g i v e s a b l u e dye. U s u a l l y a two-stage procedure i s c a r r i e d o u t , as f o l l o w s . Stage 1. A 2% agar gel c o n t a i n i n g t h e s u b s t r a t e and t h e b u f f e r s o l u t i o n i s overl a y e d t o t h e s e p a r a t i o n support and incubated f o r about 2 h a t 37OC i n t h e case o f d i f f e r e n t phosphatases. Stage 2. The agar o v e r l a y i s removed and r e p l a c e d w i t h another agar gel o v e r l a y c o n t a i n i n g 2.5% ammonium molybdate i n 8 m o l / l H2S04 and a s c o r b i c a c i d (1.25 g i n 25 m l ) ; b l u e spots t h e n appear. The above s t a i n i n g procedure was a p p l i e d t o t h e d e t e c t i o n o f p h o s p h o g l y c o l l a t e phosphatase (E.C. 3.1.3.18)'03,

i n o r g a n i c pyrophosphatase o f r e d c e l l s (E.C.

3.6.1.1)

3 16

( r e f . 204) , i n o s i n e t r i p h o s p h a t a s e Z o 5 and ATPase (E.C. 3.6.1.8)

199

.

I n o r g a n i c phosphate can be a l s o d e t e c t e d w i t h l e a d s a l t s i n t h e presence o f s u l p h i d e . T h i s m o d i f i c a t i o n has been used t o s t a i n o r n i t h i n e carbamoyl t r a n s f e r a s e 206 (E.C. 2.1.3.3) a c t i v i t y i n t h e f o r m o f b l a c k zones :

-enzyme

L - o r n i t h i n e t carbamoyl phosphate

L-citrul ine t Pi

P i t l e a d n i t r a t e t ammonium s u l p h i d e

black precipitate

L o c a l i z a t i o n o f enzymes by t h e f o r m a t i o n o f w h i t e p r e c i p i t a t e zones has been used t o mark t r e h a l a s e (E.C. 3.2.1.28)207.

The t r e h a l o s e h y d r o l y s i s i s coupled t o

t h e o x i d a t i o n o f t h e p e r o x i d a s e s u b s t r a t e , eugenol (2-methoxy-4-allylphenol), by

-

t h e use o f g l u c o s e o x i d a s e and p e r o x i d a s e as a n c i l l a r y enzymes: t r e h a l ase Trehalose t H20

2 Glucose

glucose

Glucose t H20 t O2

g l u c o n i c a c i d t H202

oxidase

p e r o x i dase Eugenol t H202

eugenol polymer ( w h i t e p r e c i p i t a t e ) t H20

The procedure i s based on t h e f a c t t h a t f r e e r a d i c a l s o f eugenol which a r e generated d u r i n g t h e coupled t r e h a l a s e assay condense t o f o r m a w h i t e p r e c i p i t a t e whose l o c a t i o n i n t h e g e l may be determined b y d e n s i t o m e t r i c scanning and whose s u r f a c e area

i s a l i n e a r f u n c t i o n o f t h e amount o f enzyme s u b j e c t e d t o e l e c t r o p h o r e s i s . A w h i t e p r e c i p i t a t e can a l s o be formed w i t h p u r e enzymes and t a n n i c a c i d . T h i s method was a p p l i e d t o t h e d e t e c t i o n o f lysozyme (E.C. A208

3.2.1.17)

and Taka-amylase

Some enzymes e l u t e d f r o m t h e s u p p o r t i n g medium can be i d e n t i f i e d and assayed b y u s i n g p h y s i c a l methods. Phospholipase A2 f r o m snake venom was measured p o l a r o g r a p h i c a l l y as 1 i p o x i d a s e - c a t a l y s e d oxygen i n c o r p o r a t i o n i n t o t h e e n s u i n g u n s a t u r a t e d f a t t y acidszo9. UDP-glucuronosyl t r a n s f e r a s e (E.C. 2.4.1.17) determined i s o t a c h o p h o r e t i c a l l y ' " .

a c t i v i t y was

The enzyme i s r e s p o n s i b l e f o r t h e c o n j u g a t i o n

o f a wide range o f substances, v i z . , exogeneous o r endogenous t o x i n s w i t h f u n c t i o n a l groups such as aryl-OH, -NH2 o r -SH.

G l u c u r o n i d a t i o n o f paracetamol (4-acetamido-

p h e n o l ) was f o l l o w e d b y c a p i l l a r y i s o t a c h o p h o r e s i s a t pH 3.89.

Isotachophoresis

was used a l s o f o r t h e i d e n t i f i c a t i o n and d e t e r m i n a t i o n o f p i c o m o l e amounts o f ADP and ATP formed e n z y m a t i c a l l y i n b i o l o g i c a l systems2''.

The above t e c h n i q u e s can

e a s i l y b e adapted t o t h e i d e n t i f i c a t i o n and d e t e r m i n a t i o n o f t h e p r o d u c t s o f erlz y m a t i c r e a c t i o n s performed i n t e s t - t u b e s f o l l o w i n g e l e c t r o p h o r e t i c s e p a r a t i o n o f t h e p a r t i c u l a r enzymes. The method needs o n l y picogram amounts o f t h e sample t o be analysed.

317 I n d i v i d u a l t e s t s have been d e s c r i b e d f o r h i s t o c h e m i c a l l o c a l i z a t i o n o f h i s t i d a s e (E.C. 4.3.1.3) (E.C.

and urocanase (E.C.

4 . 2 . 1 .49)'12,

r i b o n u ~ l e a s e rhodanase ~ ~ ~ ~ ~ ~ ~ ,

2.8. 1.1)'15,

n i t r i t e reductase216, h y a l u r o n i d a s e (E.C. 3 . ~ ! . 1 . 3 5 ) ' ~ ~ , n i t r o 219 genase (E.C. 1.18.2.1)'18 and a c r o s i n (E.C. 3.4.21.10) . The o r i g i n a l method f o r t h e l o c a l i z a t i o n o f r i b o n u c l e a s e s was developed b y K a r p e t s k y e t al.'14.

Yeast RNA o r p o l y - U (0.03-0.05%) were p o l y m e r i z e d i n a c r y l -

amide g e l . F o l l o w i n g e l e c t r o p h o r e s i s t h e g e l s were i n c u b a t e d o v e r n i g h t a t 24OC i n 3.5 1 o f 0.1 m o l / l Tris-HC1 b u f f e r (pH 7.6) c o n t a i n i n g 10 mmol/l NaCl. D u r i n g t h i s p e r i o d h y d r o l y s i s o f t h e polynucleotide took place i n t h e regions o f t h e gel c o n t a i n i n g enzyme. The g e l was t h e n s t a i n e d w i t h p o l y n u c l e o t i d e - s p e c i f i c dye (1% p y r o n i n Y i n 7% a c e t i c a c i d ) . The t e c h n i q u e i s h i g h l y s p e c i f i c and s e n s i t i v e ; 10 pg o f b o v i n e p a n c r e a t i c RNase can b e d e t e c t e d . Polynucleotide-polyacrylamide gel electrophoresis gives t h e p o s s i b i l i t y both o f d e f i n i n g t h e p o s i t i o n o f t h e enzyme i n t h e g e l and g i v i n g an i n d i c a t i o n o f t h e n a t u r e o f t h e c a t a l y t i c a c t i v i t y p r e s e n t i n t h e sample, i . e . ,

t h e s p e c i f i c i t y o f t h e enzyme.

Separation and d e t e c t i o n of isoenzymes A n a l y s i s o f t h e e l e c t r o p h o r e t i c p a t t e r n o f isoenzymes o f a p a r t i c u l a r enzyme as t h e p r o d u c t o f t h e genes has a fundamental v a l u e i n s t u d i e s o f g e n e t i c p o l y morphism and i n h e r i t e d v a r i a t i o n s , d e f e c t s o f chromosome a c t i v i t y , q u a n t i t a t i o n o f m u t a t i o n , t h e m o l e c u l a r b a s i s o f c e l l u l a r d i f f e r e n t i a t i o n and morphog'enesis. Isozymes s e r v e as a markers i n t h e s t u d y o f c e l l c u l t u r e l i n k a g e and p o p u l a t i o n studies. The isozymes a r e m u l t i p l e m o l e c u l a r forms o f a g i v e n enzyme which c a t a l y s e t h e same r e a c t i o n and o c c u r w i t h i n a s i n g l e s p e c i e s , i n an organism o r even i n a s i n g l e c e l l . They d i f f e r from each o t h e r i n e l e c t r o p h o r e t i c m o b i l i t y , chromatog r a p h i c p r o p e r t i e s , amino a c i d c o m p o s i t i o n and o t h e r p r o p e r t i e s . E l e c t r o p h o r e t i c h e t e r o g e n e i t y o f some enzymes i s due t o t h e o l i g o m e r i c s t r u c t u r e o f t h e i r molec u l e s . Enzymes c o n s i s t i n g o f more t h a n one t y p e o f s u b u n i t can produce isozymes as a r e s u l t o f v a r i a t i o n i n c o m b i n a t i o n o f t h e s u b u n i t s . The number o f isozymes can be c a l c u l a t e d f o r any enzyme i f t h e number o f s u b u n i t s and t h e number o f t y p e s o f s u b u n i t s i n t h e m o l e c u l e a r e known ( c f . , F i g . 13.13).

The s u b u n i t com-

p o s i t i o n o f an enzyme can be f o u n d by p u r i f i c a t i o n o f t h e enzyme and s t u d i e d by d i s s o c i a t i o n and r e a s s o c i a t i o n t e c h n i q u e s . An a l t e r n a t i v e method i s t o f i n d a g e n e t i c v a r i a n t o f t h e enzyme, i f one e x i s t s , and t o o b t a i n i n f o r m a t i o n about i t s i s o z y m i c p a t t e r n . F o r example, i n t h e LDH system a m u t a t i o n p r o d u c i n g a h e t e r o zygous f o r m i n one o f two d i f f e r e n t p o l y p e p t i d e c h a i n s p r e s e n t i n t h e enzyme molec u l e would g i v e 15 p o s s i b l e c o m b i n a t i o n s o f i s o z y m i c bands a s v a r i a n t s o f a t e t r a m e r i c m o l e c u l e ( T a b l e 13.2)220.

I n some i n s t a n c e s enzymes composed o f more

t h a n one i d e n t i c a l s u b u n i t can a l s o produce isozynie p a t t e r n s . T h i s i s a t t r i b u t e d

318

F i g . 13.13. Schematic r e p r e s e n t a t i o n o f p o s s i b l e isozyme p a t t e r n s expected i n h e t e r o z y g o t e s i n monomeric, d i m e r i c , t r i m e r i c and t e t r a m e r i c enzymes. t o a v a r i a t i o n i n t h e number o f s u b u n i t s i n t h e p r o t e i n m o l e c u l e o r t o conformat i o n a l d i f f e r e n c e s i n i t s t e r t i a r y s t r u c t u r e ( m i c r o h e t e r o g e n e i t y 221 y222). Another p o s s i b i l i t y i s t h a t d u r i n g e x t r a c t i o n of an enzyme f r o m t h e t i s s u e s i t may b e m o d i f i e d , l e a d i n g t o e l e c t r o p h o r e t i c a l l y s e p a r a b l e forms, f o r example, s p l i t t i n g by p r o t e o l y t i c enzymes, removal o f s i a l i c a c i d r e s i d u e s f r o m c a r b o h y d r a t e c h a i n s , o x i d a t i o n o f SH groups o r a g g r e g a t i o n . Hence t h e d i f f e r e n t isozymes can be genera t e d i n d i f f e r e n t ways and can be c l a s s i f i e d i n t o t h r e e main groups: 1. The isozymes which o c c u r o f m u l t i p l e gene l o c i c o d i n g s t r u c t u r a l l y d i f f e r e n t p o l y p e p t i d e c h a i n s o f t h e enzyme.

2. The isozymes which o c c u r o f m u l t i p l e a l l e l i s m a t a s i n g l e l o c u s d e t e r m i n i n g s t r u c t u r a l l y d i s t i n c t polypeptide chain. 3. The "secondary" isozymes due t o p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n o f t h e en223 , zyme molecule I t i s necessary t o p o i n t o u t t h a t sometime two o r more causes a r e i n v o l v e d i n

t h e f o r m a t i o n o f a s e t o f isozymes. Thus, t h e isozyme p a t t e r n s observed may be v e r y complex and t h e i r e l u c i d a t i o n w i l l need b o t h g e n e t i c and b i o c h e m i c a l approaches The s u b u n i t s i n any one o l i g o m e r i c isozyme may be i d e n t i c a l i n p r i m a r y s t r u c t u r e and such isozymes a r e c a l l e d homomeric; i f t h e s u b u n i t s a r e n o n - i d e n t i c a l , t h e isozymes a r e h e t e r o m e r i c . I f t h e organism i s homozygous, i n w h i c h two a l l e l e s p r e s e n t a r e t h e same, i t w i l l s y n t h e s i z e o n l y one f o r m o f p o l y p e p t i d e c h a i n . I n heterozygous i n d i v i d u a l s t h e r e a r e two d i f f e r e n t a l l e l e s and two s t r u c t u r a l l y

319 TABLE 13.2 SUBUNIT COMPOSITION OF LDH ISOZYMES I N NORMAL AND HETEROZYGOUS ORGANISMS FOR MUTANT GENES A T E I T H E R THE "H" OR "M" L O C I

Note f o r m a t i o n of 15 bands when a m u t a t i o n i s superimposed on t h e one o f two LDH loci. Isozyme Normal subunit composition

Subunit c o n s t i t u t i o n i n a heterozygote f o r an LDHH locus varianta

Subunit c o n s t i t u t i o n i n a heterozygote f o r an LDHM locus varianta

LDHl

MMMM

MMMM

MMMM MMMW MMM* W MWWW MkM*WW

LDH2

MMMH

MMMH MMMH*

MMMH MMW H MWM*H M*WMXH

LDH3

MMHH

MMHH MMHH* MMH* H*

MMHH MM* HH M*M*HH

LDH4

MHHH

MHHH MHHH* MHH*H* MH*H*H*

MHHH M*HHH

LDH5

HHHH

HHHH HHHH* HHH*H* HH* H* H* H* H* H* H*

HHHH

aM and H r e p r e s e n t t h e normal s u b u n i t s , M* and H* r e p r e s e n t t h e v a r i a n t subunits. d i s t i n c t p o l y p e p t i d e chains w i l l be formed. The r a t i o o f t h e a c t i v i t i e s f o r p r i m a r y isozymes formed i n heterozygotes w i l l be 1 : 1 f o r monomers, 1 : 2 : 1 f o r dimers, i n t h e 3-band system, 1 : 3 : 3 : 1 f o r t r i m e r s , 1 : 4 : 6 : 4 : 1 f o r t e t r a m e r s , and so on. From t h i s one can see t h a t i n t h e dimers 50% o f t h e t o t a l a c t i v i t y w i l l be obtained from t h e homomeric forms, i n t r i m e r s 25% and i n t e t r a m e r s 12.5% 106

.

CZinicaZ uppZication of isozymes. Some heteromeric isozymes c o n t a i n s u b u n i t s d e r i v e d from d i f f e r e n t gene l o c i . I n l a c t a t e dehydrogenase, which i s a t e t r a m e r i c enzyme, subunits H and M y d e r i v e d from t h e l o c i LDHH and LDHM, forlm n o t o n l y homomeric isozymes w i t h t h e composition HHHH and MMMM b u t a l s o t h e heteromeric i s o zymes HHHM, HHMM and HMMM. The l a c t a t e dehydrogenase isozymes vary i n t h e i r r e -

320 l a t i v e p r o p o r t i o n s i n d i f f e r e n t tissues''*

and t h i s p r o p e r t y i s e x p l o i t e d i n t h e

medical d i a g n o s i s o f h e a r t and l i v e r diseases ( F i g . 13.14).

LDHl isozyme i s e l e v a t e d

i n m y o c a r d i a l i n f a r c t i o n , m y o c a r d i t i s and shock, LDHP i n m y o c a r d i a l and pulmonary i n f a r c t i o n , some anaemias and malignancy, LDH3 i n leukaemias, pulmonary i n f a r c t i o n and malignancy, LDH4 i n i n f e c t i o u s mononucleosis and malignancy and LDH5 i n cong e s t i v e h e a r t f a i l u r e , h e p a t i t i s , h e p a t i c c i r r h o s i s , d e r m a t o m y o s i t i s , shock and malignancy.

F i g . 13.14. LDH isozymes (A) i n d i f f e r e n t human t i s s u e s and ( 6 ) i n d i f f e r e n t diseases as found i n b l o o d serum. A: 1, h e a r t ; 2, l i v e r ; 3, t e s t i s . B: 1, normal; 2, m y o c a r d i a l i n f a r c t i o n ; 3, a c t i v e l i v e r disease.

F i g . 13.15. Isozymes o f a l k a l i n e phosphatase i n human b l o o d serum s e p a r a t e d on c e l l u l o s e a c e t a t e . L1 and L2, f a s t and s l o w isozymes, r e s p e c t i v e l y , d e r i v e d f o r m l i v e r ; I , isozyme f r o m i n t e s t i n e ; 6, f r o m bone; P, f r o m p l a c e n t a .

321 Serum a l k a l i n e phosphatase isozymes ( F i g . 13.15) may be e l e v a t e d i n b i l i a r y o b s t r u c t i o n , h e p a t i c neoplasm and bone d i s e a s e s 2 2 4 y 2 2 5 . 8-N-Acetylglucosaminidase isozymes A and B a r e used i n t h e d i a g n o s i s o f Tay-Sachs and S a n d h o f f ' s diseases 226.

,

isozymes A and B a l s o change i n l i v e r t i s s u e i n d i f f e r e n t l i v e r l e s i o n s 2 2 7 , which was demonstrated by i s o e l e c t r i c f o c u s i n g i n t h i n l a y e r s . Serum isoamylases a r e e l e v a t e d i n a c u t e p a n c r e a t i t i s whereas i n l i v e r d i s e a s e s t h e amylase a c t i v i t y i s u s u a l l y l o w e r t h a n normalzz8. D e t e r m i n a t i o n o f d i f f e r e n t g e n e t i c v a r i a n t s o f al-antitrypsin such analyses2".

i n h i b i t o r i s a n o t h e r example o f t h e g r e a t c l i n i c a l i m p o r t a n c e o f S u b j e c t s w i t h t h e d e f i c i e n t phenotypes o f a l - a n t i t r y p s i n

can,

f o r i n s t a n c e , d e v e l o p emphysema as e a r l y as 35-40 y e a r s o f age and j u v e n i l e c i r rhosis.

a1

a2-a1 a2

a4-al

a4-a2 a3-aZ a3 a3-a1 a4-a3 a4

F i g . 13.16. Isozymes o f phosphoglucomutase f r o m t e n human phenotypes. D i f f e r e n $ 2 9 l e u c o c y t e l y s a t e s were s e p a r a t e d by i s o e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e g e l Phosphoglucomutase i s p r e s e n t i n most human organs i n d i f f e r e n t i s o z y m i c forms ( F i g . 13.16) and i t s polymorphism has been used i n b l o o d t y p i n g f o r t r a n s f u s i o n s ( r e f . 2 3 0 ) . C l i n i c a l a p p l i c a t i o n s o f isozymes and t h e i r e l e c t r o p h o r e t i c examina106 t i o n were reviewed by W i l k i n s o n l o 7 , Brewer and S i n g l o 8 , H a r r i s and Hopkinson 231 and Holvey and T a l b o t t

.

Phenotype studies. Many enzymes a r e p r e s e n t i n animal and human t i s s u e s i n several d i f f e r e n t p h e n o t y p i c forms. T h i s polymorphism o f v a r i o u s enzymes can be s t u d i e d by s t a r c h g e l and p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and by i s o e l e c t r i c f o c u s i n g i n t h i n l a y e r s f o l l o w e d by s p e c i f i c s t a i n i n g o f t h e enzymes. I n t h e l a s t i n s t a n c e , t h e i s o e l e c t r i c p o i n t s o f each enzyme band can be d e t e r m i n e d by u s i n g a surface electrode. As an example, t h e a n a l y s i s o f a l c o h o l dehydrogenase isozymes (ADH) i n human a d u l t s and f o e t a l l i v e r w i l l be considered232. The enzyme i s c o n t r o l l e d b y t h r e e autosomal l o c i , ADH1,

ADH2 and ADH3, d e t e r m i n i n g a - , 8- and y - p o l y p e p t i d e c h a i n s ,

r e s p e c t i v e l y , and a l l isozymes a r e d i m e r s . A t pH 8 . 6 i n s t a r c h g e l c o n t a i n i n g NAD, a l l o f t h e ADH isozymes m i g r a t e c a t h o d a l l y . They can be s t a i n e d by t h e formazan technique:

.

322

Ethanol

rmazan

ADH

Acetaldehyde

NADHtH'

MTT

I n e a r l y f o e t a l l i f e ( l e s s t h a n 20 weeks o f g e s t a t i o n ) o n l y ADHl

a c t i v i t y i s pres-

e n t . I n l a t e r f o e t a l l i f e ADH2 a c t i v i t y i n c r e a s e s p r o g r e s s i v e l y . I n e a r l y p o s t n a t a l l i f e ADH3 isozyme a c t i v i t i e s appear, and i n a d u l t s marked a c t i v i t y a t t r i b u t a b l e t o a l l t h r e e l o c i can be d e t e c t e d . F i g . 13.17 shows a diagrammatic r e p r e s e n t a t i o n o f t h e ADH isozymes i n l i v e r f r o m a d u l t s o f t h e ADH3-1,

ADH3-2 and ADH3-2-1 phenotypes. The s u b u n i t c o m p o s i t i o n o f each isozyme dimer i s i n d i c a t e d 233

.

-

."

I

PP

ADH3 21

AD%1

ADHj 2

F i g . 13.17. A l c o h o l dehydrogenase isozyme p a t t e r n s separated b y s t a r c h g e l e l e c t r o p h o r e s i s a t pH 8.6 i n l i v e r samples f r o m a d u l t s o f t h e ADH31, ADH 2-1 and ADH32 phenotypes. S u b u n i t c o m p o s i t i o n o f each isozyme i s p o s t u l a t e d ? r e f . 233).

t 1 2 oripin

3

4

5

F i g . 13.18. Guanylate k i n a s e isozyme p a t t e r n s i n d i f f e r e n t human t i s s u e s . Fibrob l a s t s ; 2 , lymphocytes; 3, e r y t h r o c y t e s ; 4, f o e t a l l i v e r ; 5, a d u l t k i d n e y 2 j 4 .

323 As was shown e a r l i e r (Fig. 13.14),

t h e t i s s u e d i s t r i b u t i o n s o f t h e isozymes o f

some enzymes may d i f f e r . Another example i s guanylate kinase ( A T P

+

GMP = ADP

+

GDP).

The enzyme possesses seven isozymes i n various t i s s u e s , represented by d i f f e r e n t e l e c t r o p h o r e t i c patterns (Fig. 13.18) 234

.

Somatic c e t l hybrid studies. Cel loge1 electrophoresis o f d i f f e r e n t enzymes was employed i n t h e study o f man-mouse and man-Chinese hamster somatic c e l l hybrids 235 Because o f t h e occurrence o f p r e f e r e n t i a l l o s s o f human chromosomes and the e x i s tence o f b u i l t - i n enzyme markers during linkage, the above somatic c e l l h y b r i d systems became important t o o l s f o r human genetic analysis236. Hybrids lead t o the formation o f c e l l s w i t h varying numbers and combinations o f human chromosomes i n a d d i t i o n t o t h e complete s e t o f mouse o r Chinese hamster chromosomes. Thus, phenot y p i c expressions o f l i n k e d o r non-linked genes products may be analysed e l e c t r o phoretical l y

0

cm

- BBBB

8

A'B B B

6 -

ABBE A'A'B B A'A B B A A B B t A'A'A'B A'A'A B A A A A + A' A A B

4 -

m

m

2 -

0

=.

---

A'A A A AAAA

2 -

# 1

2

3

4

5

6

Fig. 13.19. Zymogram p a t t e r n s o f LDH isozymes on Cellogel showing human ( l ) , Chinese-hamster DON (2), human + DON ( 3 ) and t h r e e d i f f e r e n t cloned man-Chinese hamster somatic c e l l hybrids (4, 5, 6 ) . The DON o r any other Chinese hamster f i b r o b l a s t i c c e l l l i n e has o n l y one band, A ' A ' A ' A ' . The h y b r i d c e l l s (channels 4, 5 and 6 ) appear t o have r e t a i n e d t h e human LDHA human LDH and human LDHA and LDHB l o c i , r e s p e c t i v e l y , i n t h e i r genomes i n a d d i t i o n t o &he Chinese hamster locus f o r LDHA. The probable subunit s t r u c t u r e o f each band i s i n d i c a t e d (adapted from r e f . 235). Fig. 13.19 shows t h e e l e c t r o p h o r e t i c p a t t e r n s o f LDH isozymes on Cellogel found i n human, Chinese hamster and t h e i r somatic h y b r i d c e l l s . As can be seen, t h e h y b r i d c e l l s i n channiels 4, 5 and 6 appear t o have r e t a i n e d t h e human LDHA, LDHB and human LDHA and B l o c i , r e s p e c t i v e l y , i n t h e i r genomes, i n a d d i t i o n t o t h e

.

324

Chinese hamster l o c u s f o r LOHA. O t h e r enzymes such as glucose-6-phosphate genase, NADP-dependent i s o c i t r a t e dehydrogenase (E.C. genase (E.C.

1.1.1.37),

indophenol o x i d a s e (E.C.

1.1.1.42),

dehydro-

m a l a t e dehydro-

1.9.3.1),

phosphoglucomutase and 235,236 3-phosphoglycerate k i n a s e were a l s o s t u d i e d i n somatic h y b r i d c e l l s e x t r a c t s S i m i l a r work w i t h p l a n t isozymes was reviewed by Shannon237 and S c a n d a l i o ~and ~~~ 239,240

others

PopuZation and taxonomic studies. The s t u d y o f t h e polymorphism o f d i f f e r e n t enzymes i s o f g r e a t v a l u e i n c l i n i c a l medicine229 and i n t h e i d e n t i f i c a t i o n o f 242 p l a n t v a r i e t i e s 2 4 1 and s p e c i e s s p e c i f i c i t y among d i f f e r e n t groups o f animals

.

Also, s e v e r a l b a c t e r i a l s p e c i e s have been i d e n t i f i e d b y i s o e l e c t r i c f o c u s i n g on t h i n l a y e r s f o l l o w e d by s p e c i f i c s t a i n i n g f o r p r o t e a s e s , e ~ t e r a s e so r~ B-lactam~~ 244 ase (E.C. 3.5.2.6) isozymes

.

The e l e c t r o p h o r e t i c e x a m i n a t i o n o f human, animal and p l a n t p o p u l a t i o n s i s an i m p o r t a n t t o o l i n s e a r c h i n g f o r a l l e l i c f r e q u e n c i e s and v a r i a n t s expressed by d i f f e r e n c e s i n i s o z y m i c p a t t e r n s . A c o n s i d e r a b l e number o f m u t a t i o n s r e s u l t i n g i n s i n g l e amino a c i d s u b s t i t u t i o n s can be d e t e c t e d by e l e c t r o p h o r e t i c e x a m i n a t i o n , b u t i t must be k e p t i n mind t h a t n o t a l l m u t a t i o n s cause a l t e r a t i o n s i n t h e charge o f t h e p o l y p e p t i d e chain. A g e n e t i c v a r i a n t o f an isozyme may be d e t e c t e d i n heterozygous i n d i v i d u a l s o n l y i f i t has r e t a i n e d t h e s p e c i f i c c a t a l y t i c a c t i v i t y c h a r a c t e r i s t i c f o r t h e g i v e n enzyme. Seed plants

1

2

Mosses

3

Ferns

4

5

6

Eukaryotic algae

7

8

9

Prokaryolic algae

13

14

10 11 Photosynlhetic bacteria

15

12

16

F i g . 13.20. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f e x t r a c t s o f p h o t o s y n t h e t i c o r g a nisms and a c t i v i t y p a t t e r n s o f superoxide dismutase i n t h e presence and absence o f cyanide. F o r each p a i r , t h e l e f t - h a n d s t r i p s a r e c o n t r o l s and t h e r i g h t - h a n d s t r i p s were immersed i n 5 mmol/l KCN b e f o r e s t a i n i n g f o r enzyme a c t i v i t y . Top o f each p a i r o f g e l s i t h e cathode; b o t t o m i s t h e anode. The achromatic zones i n d i c a t e enzymatic a c t iv i ty215.

325

I n t e r e s t i n g s t u d i e s on s u p e r o x i d e d i s m u t a s e (E.C.

1.15.1.1)

isozymes i n p h o t o -

s y n t h e t i c organisms a t d i f f e r e n t e v o l u t i o n a r y l e v e l s were c a r r i e d o u t by Asade e t a1.245. The l e v e l o f t h e isozymes v a r i e d w i t h t h e s p e c i e s and growth c o n d i t i o n s ( F i g . 13.20). The r e s u l t s showed t h a t t h o s e p h o t o s y n t h e t i c organisms which appeared up t o t h e l a t e r S i l u r i a n age do n o t c o n t a i n t h e Cu,Zn-superoxide dismutase ( s e n s i t i v e t o c y a n i d e ) and have o n l y t h e cyanide i n s e n s i t i v e Fe- and Mn-isozymes. EXAMINATION

OF SOME PHYSICO-CHEMICAL PROPERTIES OF

ENZYMES

D i f f e r e n t e l e c t r o p h o r e t i c t e c h n i q u e s a r e f r e q u e n t l y used f o r t h e d e t e r m i n a t i o n

of t h e homogeneity o f enzymes, t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s , s u b u n i t s t r u c t u r e s and i s o e l e c t r i c p o i n t s , f o r t h e s t u d y o f t h e i r i n t e r a c t i o n s w i t h v a r i o u s l i g a n d s such as s u b s t r a t e s , i n h i b i t o r s , coenzymes and o t h e r f a c t o r s , f o r t h e s t u d y o f t h e mechanisms o f enzymic r e a c t i o n s and f o r g e n e t i c s t u d i e s . Examples o f a p p l i c a t i o n s o f e l e c t r o p h o r e t i c methods t o t h e s t u d y o f t h e mentioned c h a r a c t e r i s t i c s

w i l l be c o n s i d e r e d b r i e f l y . Determination of subunit nwnber There a r e s e v e r a l approaches by w h i c h t h e s u b u n i t c o m p o s i t i o n o f most o l i g o m e r i c enzymes can be e s t a b l i s h e d . The most common method i s S D S - e l e c t r o p h o r e s i s i n p o l y a c r y l a m i d e g e l and g e l f i l t r a t i o n , b u t o t h e r methods such as isozyme p a t t e r n s observed i n h e t e r o z y g o t e s , isozyme p a t t e r n s i n somatic c e l l h y b r i d s o r i n v i t r o d i s s o c i a t i o n - r e c o m b i n a t i o n experiments a r e f r e q u e n t l y used. Enolase i s t h e enzyme t h a t has been analysed by a l l p o s s i b l e methods 246-253. SDS-polyacrylamide 248, 254-256: rapid g e l e l e c t r o p h o r e s i s o f f e r s a number o f a n a l y t i c a l advantages d e t e r m i n a t i o n o f enzyme m o l e c u l a r w e i g h t , i d e n t i f i c a t i o n o f s u b u n i t a f t e r complete d i s s o c i a t i o n o f t h e enzyme m o l e c u l e and a p p l i c a b i l i t y t o i n s o l u b l e enzymes, i . e . , membrane-bound and aggregated enzymes. The method i s e s p e c i a l l y u s e f u l when, a f t e r SDS-electrophoresis,

t h e d e t e c t i o n of enzymic a c t i v i t y i s p o s s i b l e . Experiments

on t h e r e n a t u r a t i o n o f enzymes a f t e r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e presence o f SDS showed t h a t most monomeric enzymes c o u l d be r e n a t u r a t e d even a f t e r d i s r u p t i o n o f t h e i r d i s u l p h i d e bonds257. Proteases and 01 i g o m e r i c enzymes composed o f i d e n t i c a l subunits a r e p o o r l y renaturable. The r e n a t u r a t i o n o f enzyme a c t i v i t y a f t e r SDS-polyacrylamide g e l e l e c t r o p h o r e s i s can be a c h i e v e d as f o l l o w s 257y258: t h e g e l s a r e r i n s e d w i t h w a t e r , t h e n washed by shaking g e n t l y i n a t l e a s t 10 t i m e s t h e i r volume o f 0.04 m o l / l T r i s b u f f e r (pH 7.6) a t room t e m p e r a t u r e f o r s e v e r a l h o u r s w i t h p e r i o d i c b u f f e r changes. Three washes o f 1 h each a d e q u a t e l y removed t h e SDS. The h i g h e r a l k y l s u l p h a t e s appear t o b i n d more t e n a c i o u s l y t o enzyme p r o t e i n s i n t h e g e l . F o l l o w i n g t h e washing, t h e g e l s can be s t a i n e d f o r enzymic a c t i v i t y dr p r o t e i n o r can be s l i c e d , e x t r a c t e d and 259 p a r t i c u l a r enzymes determined by c o n v e n t i o n a l methods

.

326

Determination of isoeZectr4c point Zone e l e c t r o p h o r e s i s i s w i d e l y used f o r t h e d e t e r m i n a t i o n o f t h e i s o e l e c t r i c p o i n t s o f enzymes, even when t h e enzyme i s i n a m i x t u r e w i t h o t h e r p r o t e i n s . When f i l t e r - p a p e r s , c e l l u l o s e a c e t a t e membranes and o t h e r s u p p o r t i n g media a r e used i t i s necessary t o prepare b u f f e r s o l u t i o n s o f c o n s t a n t i o n i c s t r e n g t h , which i s n o t easy. An a d d i t i o n a l d i f f i c u l t y i s t h e n e c e s s i t y t o c a l c u l a t e e l e c t r o o s m o t i c and hydrodynamic f l o w values. From t h i s p o i n t o f view, r e c e n t l y t h e b e s t method f o r t h e d e t e r m i n a t i o n o f t h e IP v a l u e o f an enzyme has been found t o be column i s o e l e c t r i c f o c u s i n g i n sucrose d e n s i t y g r a d i e n t s o r on t h i n - l a y e r p o l y a c r y l a m i d e g e l . I n t h e column t e c h n i q u e t h e i s o e l e c t r i c p o i n t s o f t h e s e p a r a t e d s p e c i e s a r e determined b y measuring t h e pHs and enzymic a c t i v i t i e s o f t h e e l u t e d f r a c t i o n s ( r e f . 260-263). The e l u t i o n p a t t e r n s o f 'enzymic a c t i v i t y and t h e pH g r a d i e n t c u r v e i s p l o t t e d on t h e same graph, t h e n t h e P v a l uI e o f each component o f t h e sample can e a s i l y be determined. The a p p l i c a t i o n o f t h i n - l a y e r p o l y a c r y l a m i d e g e l s t o s t a b i l i z e pH g r a d i e n t s has been adapted t o PI d e t e r m i n a t i o n s as an easy and r e p r o d u c i b l e t e c h n i q u e , e s p e c i a l l y when o n l y m i n u t e amounts o f an enzyme a r e a v a i l a b l e . The sample i s focused i s o e l e c t r i c a l l y on 4-5% p o l y a c r y l a m i d e gel s t r i p s , 1-mm t h i c k , c o n t a i n i n g a f i n a l c o n c e n t r a t i o n o f 2% Amphol i n e c a r r i e r ampholytes c o v e r i n g a s u i t a b l e pH range. F o l l o w i n g i s o e l e c t r i c f o c u s i n g ( a b o u t 24 h ) , g e l s t r i p s a r e c u t i n t o 5-mm s e c t i o n s and e l u t e d w i t h 25-50 ~l o f t h e s o l v e n t . I n each f r a c t i o n t h e enzymic a c t i v i t y and pH a r e determined and p l o t t e d on t h e graph. When a p u r e enzyme i s analysed a f t e r t h e i s o e l e c t r i c r u n t h e g e l can b e s t a i n e d f o r p r o t e i n bands i n a s o l u t i o n o f Coomassie B r i l l i a n t B l u e o r Amino B l a c k 10B. The pH g r a d i e n t can a l s o be measured w i t h a s u i t a b l e m i c r o s u r f a c e e l e c t r o d e a t p o i n t s 5-10 mm a p a r t a l o n g t h e g e l s t r i p . With p u r e o l i g o m e r i c enzymes, one g e l s t r i p a f t e r i s o e l e c t r i c f o c u s i n g i s e l e c t r o phoresed i n a second dimension i n an SDS system t o c o n f i r m t h e presence o f a l l s u b u n i t s i n t h e analysed p r o t e i n molecule. The r e s u l t s o f such experiment a r e shown i n F i g . 13.21264. Using t h i s t e c h n i q u e t h e IP values o f numerous enzymes have been determined w i t h g r e a t accuracy. I s o e l e c t r i c f o c u s i n g i s a l s o v a l u a b l e f o r c o n f o r m a t i o n a l s t u d i e s o f enzymes, e s p e c i a l l y t h o s e w i t h known chemical s t r u c t u r e s . The comparison o f t h e i s o e l e c t r i c f o c u s i n g p r o f i l e s o f n a t i v e and denatured enzymes i s a u s e f u l way o f a s s e s s i n g t h e s t a t e o f d i s s o c i a b l e groups i n n a t i v e p r o t e i n m o l e c u l e s . To p r e v e n t o x i d a t i o n o f SH groups i n p r o t e i n s , r e d u c i n g agents a r e added d u r i n g t h e i s o e l e c t r i c f o c u s i n g

run52. The m i c r o h e t e r o g e n e i t y o f h i g h l y p u r i f i e d g l y c o l y t i c enzymes was s t u d i e d by Susor e t a1.49.

The h e t e r o g e n e i t y was p a r t l y dependent on t h e s u b u n i t composi-

t i o n and t h e c o n f o r m a t i o n a l v a r i a t i o n o f t h e enzymes s t u d i e d .

327

"H -0

0 0

I

0

e 01 01

I

60 000

38 000

F i g . 13.21. Determination o f t h e i s o e l e c t r i c p o i n t o f phosphoprotein phosphatase s p e c i f i c t o i n i t i a t i o n f a c t o r eIF-2. Focusing i n 4% polyacrylamide g e l . The IP o f t h e n a t i v e enzyme was found t o be 6.1. Another g e l was electrophoresed i n t h e second dimension (bottom) i n an SDS-polyacrylamide g e l system t o c o n f i r m t h e presence o f b o t h s n i t s i n t h e enzyme w i t h estimated molecular weights o f 60,000 and 38,000 d a l t o n s

YkY .

10.5 10.0

9.5

0.10 9.0 0.05

P

8.5

I I I

0

b

B.

1

E

Eo,

6rnol/l Urea

110.5

=

0.15

(Ye4 4

0.10 0.05 0

Fraction Numbec

F i g . 13.22. I s o e l e c t r i c f o c u s i n g p r o f i l e s o f bovine p a n c r e a t i c RNase ( A ) i n t h e absence and ( B ) i n t h e presence o f 6 m o l / l urea. The P rI t h e enzyme i n ureaf r e e medium was 9.33,and i n urea-containing medium 9.58

&.

328

F i g . 13.22 shows t h e i s o e l e c t r i c f o c u s i n g p r o f i l e s of p a n c r e a t i c r i b o n u c l e a s e i n t h e absence and presence o f 6 m o l / l urea265. I t can b e seen t h a t u r e a s h i f t e d the P o f tI h e enzyme from pH 9.33 t o 9.58. T h e r e f o r e , t h e P o f aIn a t i v e p r o t e i n c o u l d be m o d i f i e d b y i t s t h r e e - d i m e n s i o n a l c o n f i g u r a t i o n and i s d i f f e r e n t i n t h e unfolded molecule. The n a t i v e c o n f i g u r a t i o n keeps some d i s s o c i a b l e groups w i t h i n t h e i n t e r i o r o f t h e m o l e c u l e and causes a v a r i a t i o n i n t h e n e t charge i n i t s i s o e l e c t r i c r e g i o n . S i m i l a r experiments w i t h a s p a r a g i n e a m i n o t r a n s f e r a s e (E.C. 266 f r o m p i g h e a r t i n 8 m o l / l u r e a were c a r r i e d o u t b y B a r r a e t a l .

2.6.1.14)

.

SimuZtaneous determination of mobiZity and d i f f u s i o n c o e f f i c i e n t by eZectrophoretic Zight scattering

The t e c h n i q u e o f e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g i s a method a p p l i c a b l e m o s t l y t o t h e s e p a r a t i o n o f charged p a r t i c l e s and macromolecules i n s o l u t i o n s 2 6 7 . I n enzymology, t h i s t e c h n i q u e can be u t i l i z e d f o r t h e c h a r a c t e r i z a t i o n o f s u r f a c e r e a c t i o n s , d i s s o c i a t i o n r e a c t i o n s on t o s u b u n i t s and s t a t e o f a g g r e g a t i o n and f o r det e r m i n i n g t h e e l e c t r o p h o r e t i c m o b i l i t y and d i f f u s i o n c o e f f i c i e n t i n a s i n g l e exp e r i m e n t268-270

SCATTERING CELL

I , ELECTRODES < I

OPTICS

REAL- TIME SPECTRUM ANALYSER

DATA DISPLAY

F i g . 13.23. Diagram o f t h e apparatus f o r e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g . The sample is i l l u m i n a t e d w i t h a He-Ne l a s e r and t h e l i g h t s c a t t e r e d a t a p a r t i c u l a r angle, o , i s d e t e c t e d . A t i m e r c o n t r o l s t h e a p p l i c a t i o n o f an e l e c t r i c f i e l d t o t h e samp7e and synchronizes t h e d a t a c o l l e c t i o n w i t h t h e f i e l d p u l s e s . The power spectrum o f t h e s &yJ7f;om t h e p h o t o d e t e c t o r i s c a l c u l a t e d and d i s p l a y e d by t h e spectrum a n a l y s e r

329

The method i s a c o m b i n a t i o n o f e l e c t r o p h o r e s i s and l a s e r l i g h t s c a t t e r i n g spectroscopy. A schematic diagram o f t h e a p p a r a t u s i s shown i n F i g . 13.23. The average m o b i l i t y o f m o l e c u l a r p o p u l a t i o n s i n t h e e l e c t r o p h o r e t i c c e l l i s deduced f r o m t h e d i s t a n c e o f t h e s c a t t e r e d l a s e r l i g h t measured a t t h e o r i g i n o f f r e q u e n c y on t h e a b s c i s s a t o t h e c e n t e r o f t h e s p e c t r a l peak and can be c a l c u l a t e d f r o m t h e equation

where M i s a p p a r e n t m o l e c u l a r w e i g h t , vD t h e f r e q u e n c y s h i f t , E t h e e l e c t r i c f i e l d along t h e d i r e c t i o n o f t h e s c a t t e r i n g v e c t o r , A t h e wavelength, n t h e r e f r a c t i v e i n d e x o f t h e medium and 0 t h e s c a t t e r i n g a n g l e i n t h e medium. As t h e r e s o l u t i o n o f electrophoretic l i g h t scattering i s proportional t o the electrophoretic m o b i l i t y d i v i d e d by t h e d i f f u s i o n c o e f f i c i e n t , D , i t i s p o s s i b l e t o measure s i m u l t a n e o u s l y b o t h U and D f r o m a s i n g l e experiment, because R E A,o

R=-

2~rnD0

where R i s t h e r a d i u s o f t h e s p h e r i c a l macromolecule, U t h e e l e c t r o p h o r e t i c m o b i l i t y and 2an and A.

a r e t h e components o f t h e wave v e c t o r f o r t h e i n c i d e n t l i g h t ,

k o , which has a magnitude 2nn/Ao ( r e f . 271).

Data o b t a i n e d f r o m t h e above e q u a t i o n can a l s o be used t o c a l c u l a t e t h e charge on t h e s u r f a c e o f t h e macromolecule. Moreover, as t h e measuring process does n o t r e q u i r e t h e s e p a r a t i o n o f t h e d i f f e r e n t s p e c i e s i n s o l u t i o n and no c o n c e n t r a t i o n g r a d i e n t s a r e e s t a b l i s h e d , i t i s p o s s i b l e t o s t u d y i n t e r a c t i n g systems w i t h u n i form c o n c e n t r a t i o n s , e.g.,

d i m e r i z a t i o n r e a c t i o n s , s t a t e o f a g g r e g a t i o n or f u s i o n ,

a n t i g e n - a n t i b o d y r e a c t i o n s and o t h e r r e a c t i o n k i n e t i c s . Isotachophoretic monitoring of enzymic reactions

When i s o t a c h o p h o r e s i s i s a p p l i e d t o examine m e t a b o l i t e s i n enzymic r e a c t i o n s , b o t h t h e s u b s t r a t e s and p r o d u c t s o f r e a c t i o n can be assayed a t t h e same t i m e . T h i s can be achieved w i t h o u t c o m p l i c a t e d assay systems or r a d i o a c t i v e compounds. Some examples w i l l g i v e an i d e a o f t h e a p p l i c a b i l i t y o f t h e method. Conversion o f p y r u v a t e t o l a c t a t e by LDH i n t h e presence o f NADH can be monit o r e d as f o l l o w s 2 7 2 . P y r u v a t e ( 4 0 m m o l / l ) and NADH (11 m m o l / l ) were d i s s o l v e d i n water and b u f f e r e d w i t h T r i s t o pH 7.6. The m i x t u r e was e q u i l i b r a t e d a t 5OC and t h e r e a c t i o n was s t a r t e d b y a d d i n g 2.5 111 o f LDH suspension. Every 10 min, a 0.3-ul

sample was removed and analysed by i s o t a c h o p h o r e s i s a t pH 4.75 under t h e

c o n d i t i o n s g i v e n i n t h e l e g e n d t o F i g . 13.24. I t can be seen t h a t a l l o f t h e react a n t s and p r o d u c t s formed a r e c l e a r l y separated, g i v i n g q u a l i t a t i v e and q u a n t i t a -

330

t i v e information about a l l i o n species involved i n t h e enzymatic r e a c t i o n . Nanomole amounts o f the sample were analysed i n about 15 min and no special p r e - t r e a t ment o f t h e sample ( d e p r o t e i n i z a t i o n , concentration o r d e i o n i z a t i o n ) was necessary.

r -A

-B

Fig. 13.24. Isotachophoretic monitoring o f enzymatic conversion o f p y r u v i c a c i d i n t o l a c t i c a c i d by p i g h e a r t LDH. A, Reaction m i x t u r e a f t e r conversion; B, before conversion. Top, UV absorption a t 254 nm; bottom, c o n d u c t i v i t y ( c ) . Time o f analy s i s , 12 min; current, 70-100 v l ; recorder c h a r t speed, 6 cm/min; temperature, 22OC. 1 = chloride, 2 = sulphate, 3 = u n i d e n t i f i e d , 4 = pyruvate, 5 = l a c t a t e , 6 = NADH, 7 NAD, 8 = MES (morpholinoethanesulphonic acid, terminator). Leading e l e c t r o l y t e , 0.01 mol/l HC1 , containing E-aminocaproic acid, h i s t i d i n e and poly( v i n y l a l c o h o l ) (pH 4.75)272. The analysis o f a complex mixture o f keto and hydroxy acids as t h e t r a n s i e n t metabolites o f t h e t r i c a r b o x y l i c a c i d cycle i s very d i f f i c u l t even when using t h e 2 73 gas and l i q u i d chromatography. Bozek e t a l .

most promising methods, i.e.,

c a r r i e d o u t systematic studies i n t h i s f i e l d using isotachophoresis. They obtained good r e s u l t s i n the separation o f t e n a c i d i c species w i t h 0.011 mol/l HC1 t 8-alan i n e (pH 3.8) as the leading e l e c t r o l y t e and 0.004 m o l / l a c e t i c a c i d as t h e t e r minating e l e c t r o l y t e . C a p i l l a r y isotachophoretic a n a l y s i s o f reactants and products o f glucuronidation was performed by Brunner and Holloway2 10

,

Study of enzyme i n t e r a c t i o n s with d i f f e r e n t Zigands The e l e c t r o p h o r e t i c behaviour o f enzymes may be a f f e c t e d i n a s p e c i f i c way by i n t e r a c t i o n w i t h electricably'charged substrates, cofactors and i n h i b i t o r s . They u s u a l l y a c t a t very l o w concentrations and do n o t a f f e c t p r o t e i n s t h a t l a c k spe-

331 c i f i c binding s i t e s . The s p e c i f i c changes i n e l e c t r o p h o r e t i c m o b i l i t y a f t e r binding t o the l i g a n d may be e x p l o i t e d f o r the d e t e c t i o n o f p r o t e i n zones corresponding t o p a r t i c u l a r enzymes without a c t i v i t y s t a i n i n g procedures and f o r the c o n t r o l o f t h e p u r i t y o f enzyme preparations. Therefore, t h e use o f e l e c t r o p h o r e t i c techniques may provide i n f o r m a t i o n on t h e i n t e r a c t i o n between enzymes and t h e i r e f f e c t o r s , on the strength o f t h e forces involved i n t h e binding, on t h e amount o f b i n d i n g s i t e s , e t c . The e l e c t r o p h o r e t i c m o b i l i t y o f c r y s t a l l i n e aldolase from r a b b i t muscle was g r e a t l y a f f e c t e d i n t h e presence o f f r u c t o s e diphosphate and hexitol-1,6-diphosphate as the s p e c i f i c substrate and competitive i n h i b i t o r , respectively274. I n t h e presence o f low concentrations o f substrate o r i n h i b i t o r , t h e m o b i l i t y towards t h e cathode was s i g n i f i c a n t l y retarded (Fig. 13.25) and a t concentrations above 5

-

mol/l no f u r t h e r change i n m o b i l i t y was observed. De R i e l and P a ~ l u s ' studied ~~ the change i n e l e c t r o p h o r e t i c m o b i l i t y o f g l y c e r o l kinase i n the presence and absence o f f r u c t o s e diphosphate. The change i n e l e c t r o p h o r e t i c m o b i l i t y i n t h i s instance was r e l a t e d t o a r e v e r s i b l e d i s s o c i a t i o n o f t h e enzyme subunits caused by i n t e r a c t i o n w i t h t h e ligand.

I

origin

Fig. 13. 25. E f f e c t o f fructose-1,6-diphosphate on e l e c t r o p h o r e t i c m o b i l i t y o f r a b b i t 1i v e r aldolase on c e l l u l o s e acetate. Electrophoresis was c a r r i e d o u t i n 0.09 mol /1 T r i s - b o r a t e b u f f e r (pH 7.3) containing 2.5 mmol/l EDTA and 2-mercaptomol/l ethanol. P r t e i n was stained w ' t h Ponceau S. a, without251P added; b y FDP; c, 10- m o l / l ; d, 5 * 10- mol/l and e, 10-3 m o l / l

4

4

.

The formation o f enzyme-substrate complexes can e a s i l y be demonstrated by crosselectrophoresis. Fig. 13.26 shows t h e r e s u l t o f t h e cross-electrophoresis on f i l t e r - p a p e r o f t r y p s i n and ovoalbumin as i t s substrate276. Under t h e experimental conditions used, t r y p s i n migrated towards t h e cathode and ovoalbumin towards t h e anode. The l i n e o f ovoalbumin formed the groove a t t h e crossing w i t h t h e l i n e o f t r y p s i n Mhich was caused by t h e i n t e r a c t i o n o f the enzyme w i t h i t s substrate. S i m i l a r experiments have been performed w i t h o t h e r enzymes and t h e i r subs t r a t e s 276,277

332

F i g . 13.26. Cross-electrophoresis on f i l t e r - p a p e r o f t r y p s i n w i t h ovalbumin as i t s substrate. Trypsin (40 p l y 2% s o l u t i o n ) on t h e l i n e EL, ovalbumin (10 111, 5% s o l u t i o n ) on he l i n e SL. Phosphate b u f f e r (pH 6.0, p = 0 . 0 5 ) , s t a i n e d w i t h bromophenol b l ueh76. Crossed immunoelectrophoretic a n a l y s i s was a p p l i e d t o t h e s t u d y o f p a n c r e a t i c e l a s t a s e i n h i b i t o r s i n human b l o o d serum178. a 1 : 1 molar complex w i t h al-antitrypsin

I t was found t h a t t h e e l a s t a s e forms

o f blood serum and t h e complex i s en-

z y m a t i c a l l y i n a c t i v e . When p o r c i n e t r y p s i n was used i n s t e a d o f e l a s t a s e , a 1 : 1 complex was a l s o formed b u t p o r c i n e t r y p s i n was n o t i n h i b i t e d by human al-antit r y p s i n i n h i b i t o r 179

.

Changes i n e l e c t r o p h o r e t i c m o b i l i t y have been used as a c r i t e r i o n f o r t h e de278 t e c t i o n o f a b i o t i n - c o n t a i n i n g s u b u n i t i n wheat germ acetyl-CoA carboxylase E l e c t r o p h o r e t i c m i g r a t i o n o f t h e subunit combined w i t h b i o t i n was g r e a t l y reduced

.

i n t h e presence o f a v i d i n i n comparison w i t h u n t r e a t e d carboxylase w i t h b i o t i n i n h i b i t o r . Complex formation between propionyl-CoA carboxylase and a v i d i n i n 4 m o l / l urea-polyacrylamide gel has been s t u d i e d by Gravel e t a1.279. The b i n d i n g o f methotrexate t o d i h y d r o f o l a t e reductase (E.C.

1.5.1.3)

was s t u d i e d by Henderson

e t a1 .280 on polyacrylamide g e l . They obtained a f l u o r e s c e n t d e r i v a t i v e o f t h e i n h i b i t o r ( f l uoresceindiaminopentane methotrexate) which, a f t e r combining w i t h enzyme, gave a f l u o r e s c e n t product e a s i l y d e t e c t a b l e on t h e g e l . Methotrexate t i g h t l y bound t o t h e enzyme g r e a t l y a c c e l e r a t e d i t s anodal m i g r a t i o n a t pH 8.3. D i s t i n c t changes i n e l e c t r o p h o r e t i c m o b i l i t y o f enzymes a r e observed d u r i n g t h e i r 281-283 i n t e r a c t i o n w i t h metals and c h e l a t i n g agents

333

The c o m b i n a t i o n o f e l e c t r o p h o r e s i s and b i o s p e c i f i c i n t e r a c t i o n s has r e c e n t l y been e x p l o i t e d i n which a f f i n i t y m a t r i c e s have been suggested as e l e c t r o p h o r e t i c media284y285. O p t i m i z a t i o n o f t h e a f f i n i t y e l e c t r o p h o r e s i s o r e l e c t r o p h o r e t i c d e s o r p t i o n t e c h n i q u e was d e s c r i b e d 2 8 6 y 2 8 7 and i n t e r e s t i n g r e s u l t s , f o r example on t h e k i n e t i c s o f t h e i n t e r a c t i o n o f glycogen w i t h v a r i o u s t i s s u e phosphorylases, were obtained288. The advantage o f t h e method i s t h a t i t a v o i d s c h a o t r o p i c r e agents, which may n o t be a c c e p t a b l e when enzymatic a c t i v i t y must be preserved. Another advantage i s t h a t i t overcomes even t h e s t r o n g i n t e r a c t i o n s between an enzyme m o l e c u l e and s p e c i f i c l i g a n d s , which i s i m p o r t a n t i n r e v e r s i b l e b i n d i n g studies. Neuhoff e t a1 .289 used m i c r o - d i s c e l e c t r o p h o r e s i s and m i c r o - d i f f u s i o n t e c h n i q u e s f o r t h e s t u d y o f enzyme-template complex f o r m a t i o n between t h e DNA-dependent RNA polymerase from E.coZi and p o l y - d ( A - T ) .

The mechanism o f t h e i n f l u e n c e o f

h e p a r i n and r i f a m p i c i n as i n h i b i t o r s o f t h e complex f o r m a t i o n was suggested. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s has a l s o been used t o demonstrate t h e s p e c i f i c i n t e r a c t i o n o f RNA polymerase f r o m E.coZi w i t h tRNA2” sarcoma virus2g1.

and t h e genome o f a v i a n

I s o e l e c t r i c f o c u s i n g has been used t o f o l l o w t h e b i n d i n g o f

c y c l i c AMP t o d i f f e r e n t p r o t e i n k i n a s e s 2 9 2 y 2 9 3 and c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s has been a p p l i e d t o i n v i t r o s t u d i e s o f t h e b i n d i n g o f c r e a t i n e k i n a s e 294

t o h e a r t and l i v e r m i t o c h o n d r i a

.

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341

Chapter 1 4 NUCLEOTIDES, NUCLEOSIDES, NITROGENOUS CONSTITUENTS

s.

OF NUCLEIC A C I D S

ZADRA~IL

GENERAL ASPECTS H e t e r o c y c l i c n i t r o g e n o u s bases d e r i v e d f r o m p u r i n e s and p y r i m i d i n e s

-

adenine,

guanine o r c y t o s i n e , u r a c i l and thymine, and t h e c o r r e s p o n d i n g n u c l e o s i d e s and nucleotides

-

a r e t h e o n l y , though d e c i s i v e , c e l l c o n s t i t u e n t s i n l i v i n g organisms

bound t o n u c l e i c a c i d s . I n a d d i t i o n t h e r e a r e

t h e i r polyphosphate d e r i v a t i v e s

(immediate p r e c u r s o r s o f n u c l e i c a c i d b i o s y n t h e s i s and n a t u r a l sources o f energy)

1-3

01 i g o n u c l e o t i d e s and m o d i f i e d n u c l e o s i d e s ( m o s t l y m e t h y l a t e d d e r i v a t i v e s a r i s i n g 4 a t t h e l e v e l o f macromolecules) as a d d i t i o n a l c o n s t i t u e n t s o f t h e a n a b o l i c and c a t a b o l i c processes o f n u c l e i c a c i d s i n t h e c e l l , o r n u c l e o t i d e coenzymes and 5 as f u r t h e r c o n s t i t u e n t s o f t h e m e t a b o l i c processes, Large numbers o f

antibiotics

d e r i v a t i v e s and analogues of t h e n u c l e i c a c i d c o n s t i t u e n t s r e s u l t f r o m i n v i t r o s y n t h e t i c a c t i v i t y i n o r g a n i c c h e m i s t r y l a b o r a t o r i e s where, i n a d d i t i o n t o t h e i n c r e a s i n g l y used n u c l e o t i d e s and 01 i g o n u c l e o t i d e s 6 , i n b i o c h e m i s t r y and molec u l a r b i o l o g y ( s y n t h e t i c l i n k e r s , p r i m e r s , genes and t h e i r p o r t i o n s ) t h e number o f analogues and a n t i m e t a b o l i t e s 7 , w h i c h have been s y n t h e s i z e d , t e s t e d and used as p o t e n t i a l and r e a l v i r o s t a t i c s , b a c t e r i o s t a t i c s , c y t o s t a t i c s

, carcinostatics

o r o t h e r t h e r a p e u t i c s i s extended. The study, a p p l i c a t i o n and u t i l i z a t i o n o f a l l o f t h e s e n a t u r a l and s y n t h e t i c substances need a v a r i e t y of a n a l y t i c a l methods and approaches p e r m i t t i n g t h e i r s e p a r a t i o n , f r a c t i o n a t i o n and c h a r a c t e r i z a t i o n , as w e l l as t h e i d e n t i f i c a t i o n o f i n d i v i d u a l compounds which a r e p r e s e n t i n more o r l e s s complex m i x t u r e s o f chem4,7-11 i c a l l y c l o s e l y r e l a t e d and s i m i l a r m a t e r i a l s . A1 though chromatographic methods a r e m a i n l y a p p l i e d f o r b a s i c s e p a r a t i o n , a l l o f t h e compounds c o n s i d e r e d c o n t a i n many i o n i z a b l e groups ( T a b l e 14.1) and t h e r e f o r e e l e c t r o p h o r e t i c methods can a l s o be used. A p a r t f r o m zone e l e c t r o p h o r e s i s on paper 12-14 and t h i n l a y e r s 15 I 16 and in

i n b o t h one- and two-dimensional arrangements, t h e more r e c e n t l y

developed method of i s o t a c h o p h o r e s i s 2 0 has proved u s e f u l . The apparatus employed f o r t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f n u c l e i c a c i d c o n s t i t u e n t s does n o t need any s p e c i a l a c c e s s o r i e s ; hence a1 1 commercial a p p a i a t u s and apparatus m o d i f i e d i n t h e l a b o r a t o r y can be used f o r paper and g e l e l e c t r o l p h o r e s i s , f o r example, t h e c o o l e d f l a t p l a t e apparatus2’,

an a p p a r a t u s w i t h 1 i q u i d -

,

342

TABLE 14.1 PK;

VALUES OF PURINES,

PYRIMIDINES,

NUCLEOSIDES

AND NUCLEOTIDES~

Data from r e f . 13. Compound Adenine 6-Methyl aminopurine 6-Dimethylaminopurine Adenosine 2 ' -phosphate 3 ' -phosphate 5 ' -phosphate 5 ' -pyrophosphate 5' - t r i p h o s p h a t e Guanine Guanosine 2'-phosphate 3'-phosphate 5 ' -phosphate 5 ' -pyrophosphate 5 ' - t r iphosphate Hypoxanthine Inosine 5 ' -phosphate Xanthi ne Xanthosine Cytosine 5-Methyl c y t o s i n e 5-Methylcytidine Cyt id ine 2'-phosphate 3 ' -phosphate 5 ' -phosphate 5 ' - pyro pho s phate 5 ' - t r iphosphate Uracil Uridine 2 ' -phosphate 3 ' -phosphate 5'-phosphate 5'-pyrophosphate 5'-triphosphate Thymine Thymidine-5'-phosphate

P r ima ry phosphate

Amino (basic)

Secondary phosphate

Enol (acidic)

-

4.22 4. 18 3.87 3.45 3.80 3.65 3.74 3.95 4.0 3.3 1.6 2.3 2.3 2.4 2.9 3.3 1.98

-

9.8 9.99 10.5

0.89 0.89

0.7 0.7

1.54

-

-

-

0.8 0.8

-

4.45 4.6 4.28 4.22 4.36 4.28 4.5 4.6 4.8

6.15 5-88 6.05 6.26 6.48

-

5.9 5.9 6.1 6.3 6.5

-

6.0

-

-

-

9.2, 12.3 9.16 9.7 9.7 9.4 8.9, 12.1 8.75 a. 9 7.4, 11.1 5.75 12.2 12.4

-

6.17 6.0 6.3 6.4 6.6

-

5.9 5.9 6.4 6.5 6.6

-

6.5

9.5 9.17 9.4 9.4 9.5 9.4 9.5 9.8 10.0

aThe deoxyribonucleotides have pK values s i m i l a r t o those o f t h e r i b o n u c l e o t i d e s .

343

immersed paper a c c o r d i n g t o Markham and Smith12 o r o t h e r m o d i f i c a t i o n s o f i t acc o r d i n g t o Sanger and c o - ~ o r k e r s ~t h~e ~Rushizky ~ ~ , apparatus designed f o r twod i m e n s i o n a l c o m b i n a t i o n s o f e l e c t r o p h o r e s i s and chromatography 2 3 y 2 4 on paper and t h e S t u d i e r g e l e l e c t r o p h o r e s i s apparatus o r i t s m o d i f i c a t i o n s25,26

.

D e t e c t i o n o f s e p a r a t e d c o n s t i t u e n t s , as i n most methods w o r k i n g w i t h n u c l e i c a c i d c o n s t i t u e n t s , i s based on a d s o r p t i o n o f s h o r t - w a v e l e n g t h UV l i g h t (254 nm), which p e r m i t s r e g i s t r a t i o n o f t h e s e p a r a t i o n process u s i n g a c o n t a c t p h o t o p r i n t p l a c e d on t h e paper and p a s s i n g UV l i g h t on t o t h e l i g h t - s e n s i t i v e Autoradiographs o f r a d i o a c t i v e l y l a b e l l e d ( 32 P, 14C, 35S) s e p a r a t e d m a t e r i a l s a r e p r e p a r e d i n a s i m i l a r manner14. I n t h i s case, q u a n t i f i c a t i o n i s performed by d i r e c t measurements i n a s c i n t i l l a t i o n c o u n t e r on areas c u t o u t f r o m t h e paper u s i n g t h e a u t o r a d i o g r a p h as a guide. I n o t h e r cases, t h e m a t e r i a l i s e l u t e d f r o m t h e s u p p o r t ( w i t h w a t e r , ammonia s o l u t i o n , 0.1 m o l / l h y d r o c h l o r i c a c i d , t r i e t h y l a m m o n i u m carbonate, e t c . ) and 30 f u r t h e r analysed , NUCLEOSIDES AND NUCLEOTIDES S e p a r a t i o n of b a s i c n u c l e i c a c i d c o n s t i t u e n t s i n a m i x t u r e o f n u c l e o t i d e s ( a f t e r a l k a l i n e RNA h y d r o l y s i s and enzymatic h y d r o l y s i s o f b o t h RNA and DNA) o r n u c l e o s i d e s and bases ( a f t e r a c i d RNA o r DNA h y d r o l y s i s ) i s r e q u i r e d l a r g e l y w h e n t h e base c o m p o s i t i o n o f n u c l e i c a c i d s o r t h e i r fragments i s t o be determined. For t h e s e p a r a t i o n o f m i x t u r e s o f n u c l e o t i d e s d e r i v e d f r o m RNA and DNA, s i m p l e paper e l e c t r o p h ~ r e s i 14, s ~ ~which ~ adequately f u l f i l s t h e c r i t e r i a o f r a p i d i t y , p r e c i s i o n and s e n s i t i v i t y , i s used i n a d d i t i o n t o chromatographic methods. The pKa v a l u e s

+ , L 2 4 6 8 1 0

U

PH

F i g . 14.1. Curves d e m o n s t r a t i n g a n e t charge o f t h e 3 ' - r i b o n u c l e o t i d e s as a f u n c t i o n o f pH ( c a l c u l a t e d f r o m PKa d a t a i n Table 14.1). ( A c c o r d i n g t o r e f . 13)

W

P P

TABLE 14.2 ELECTROPHORETIC BUFFERS FOR SEPARATION OF NUCLEIC ACID CONSTITUENTS Data m a i n l y f r o m r e f . 13. pH range

General c o m p o s i t i o n a

Compounds separated

2.5-6 3.5 3-4.5 4-5.5 6.5-8 8.5-10 9.2

Sodium c i t r a t e Pyridine-acetic acid Ammonium f o r m a t e Ammonium a c e t a t e Sodium phosphate Glycine o r bicarbonate Borate

Nucl eosides, n u c l e o t i d e s , polyphosphates Mononucleotides, s m a l l o l i g o n u c l e o t i d e s Nucleosides, m o d i f i e d n u c l e o s i d e s , mono- and o l i g o n u c l e o t i d e s N u c l e o t i d e s and c y c l i c n u c l e o t i d e s S y n t h e t i c analogues o f Up, Gp, Tp 2 ' , - 3 ' - u n s u b s t i t u t e d n u c l e o s i d e s and n u c l e o t i d e s

a S u i t a b l e c o n c e n t r a t i o n s o f b u f f e r s i n t h e r a n g e 0.05-0.1

mol/l.

345

Fig. 14.2. Separation o f an alkaline hydrolysate o f rat liver rRNA by paper electrophoresis in 0.05 mol/l citrate buffer pH 3.831. A contact photoprint o f t h e electropherogram in UV light according t o ref. 27. Order o f the 3'-ribonucleotides (from top to bottom): Cp, Ap, Gp and Up.

346

F i g . 14.3. R a d i o a c t i v i t y t r a c i n g on t h e e l e c t r o p h e r o g r a m o f an enz m a t i c hydrol y s a t e ( b y DNaseI and snake venom phosphodiesterase) o f phage 629 33P-DNA ( r e f . 3 2 ) . S e p a r a t i o n c o n d i t i o n s as i n F i g . 2. Upper p a r t : s e p a r a t i o n o f t h e 5'-mononucleotides d e t e c t e d i n UV l i g h t ; l o w e r p a r t : r a d i o a c t i v i t y measurement. Order o f components ( f r o m l e f t t o r i g h t ) : dpC, dpA, dpG and pT. i n Table 14.1 and t h e d i s s o c i a t i o n curves i n F i g . 14.1 show t h a t t h e m i x t u r e o f b a s i c r i b o n u c l e o t i d e s can be separated o p t i m a l l y i n t h e pH range 3-4, when each c o n s t i t u e n t has a s u f f i c i e n t l y d i f f e r e n t n e g a t i v e charge t o m i g r a t e a t a d i f f e r e n t v e l o c i t y t o t h e anode, i . e . , and Up -1.0

f o r pH 3.5 a p p r o x i m a t e l y Cp -0.16,

Ap -0.46,

Gp -0.92

(as a r e s u l t o f complete d i s s o c i a t i o n o f t h e p r i m a r y phosphate group

and p a r t i a l d i s s o c i a t i o n o f t h e amino group). An example o f such a s e p a r a t i o n o f an a l k a l i n e r R N A h y d r o l y s a t e f r o m r a t l i v e r i n 0.05 m o l / l c i t r a t e b u f f e r (pH 3.8) i s shown i n F i g . 14.Z3'.

S i m i l a r s e p a r a t i o n s can be used f o r a p r e c i s e determina-

t i o n o f t h e base c o m p o s i t i o n i n an enzyme h y d r o l y s a t e o f 3 2 P - l a b e l l e d DNA, where t h e r a d i o a c t i v i t y o f d i f f e r e n t i n d i v i d u a l n u c l e o t i d e s can be measured d i r e c t l y on t h e e l e c t r o p h e r o g r a m ( F i g . 14.3: 629 DNA h y d r o l y s a t e by DNase I and snake venom 32 phosphodiesterase

.

TABLE 14.3 RELATIVE MOBILITIES OF I S O M E R I C NUCLEOTIDES (Up = 1) I n 0.05 m o l / l sodium c i t r a t e (pH 3.8) a t 30 V/cm on Whatman 3 MM paper.

3' -Phosphates

5'-Phosphates

2 ' -Deoxy-3 -phosphates

CP AP

AMP ADP ATP U DP UTP

dAP dGo dCp TD

GP UP

0.23 0.52 0.83 1.0

0.44 1.16 1.34 1.73 1.85

0.40 0.80 0.23 0.98

347

I t i s e v i d e n t f r o m t h e pKa v a l u e s i n T a b l e 14.1 t h a t f o r t h e s e p a r a t i o n o f some groups of substances, a c o r r e s p o n d i n g pH r e g i o n can be found w h i c h s u i t s b e s t t h e s e p a r a t i o n . The b u f f e r s most w i d e l y used f o r such s e p a r a t i o n s and groups o f substances t o be separated a r e shown i n Table 14.2.

E l e c t r o p h o r e s i s i s seldom

used f o r t h e s e p a r a t i o n o f bases. S i m i l a r l y m o d i f i e d n u c l e o t i d e s o f any t R N A can h a r d l y be separated f r o m o t h e r n u c l e o t i d e s b y paper e l e c t r o p h o r e s i s 4,13922 and a d d i t i o n a l s e p a r a t i o n s a r e needed . ( m a i n l y by chromatography) f o r t h e complete a n a l y s i s o f t h e s e RNAs4’11.

The m o b i l i t i e s o f some i s o m e r i c n u c l e o t i d e s and p r e -

c u r s o r s o f RNA and DNA i n t h e system o f 0.05 m o l / l c i t r a t e (pH 3.8) i n t h i s a r rangement a r e shown i n Table 14.3. S i m i l a r e l e c t r o p h o r e s i s on a t h i n c e l l u l o s e l a y e r proved most s u i t a b l e f o r t h e d e t e r m i n a t i o n o f t h e 5 ’ - t e r m i n a l sequences o f

f2P]DNA

by a l i m i t e d enzymatic method, r e q u i r i n g t h e s e p a r a t i o n o f mono-tetra-

n u ~ l e o t i d e s ~T~h i.s s i m p l e system o f paper and t h i n - l a y e r e l e c t r o p h o r e s i s i s a l s o u s e f u l f o r t h e s e p a r a t i o n o f some l o w e r o l i g o n u c l e o t i d e s ( T a b l e 14.4: simultaneous i n f l u e n c e o f t h e charge and c h a i n lencjth on m o b i l i t y , even though, a p a r t f r o m ion-exchange chromatography’’,

e l e c t r o p h o r e s i s on DEAE-paper 1 4 3 2 2

o r separation

w i t h t h e combined systems o f e l e c t r o p h o r e s i s and chromatography 22-24 used f o r complex m i x t u r e s o f o l i g o n u c l e o t i d e s o b t a i n e d a f t e r cleavage o f n u c l e i c a c i d s by endonucleases, a r e more a p p r o p r i a t e f o r t h i s purpose. TABLE 14.4 RELATIVE MOBILITIES OF SMALL OLIGONUCLEOTIDES (Up = 1 ) I n 0 . 1 m o l / l ammonium f o r m a t e . Data f r o m r e f . 13 0.13 0.16 0.26 0.28 0.38 0.68 0.74

GPC UPC GPA APG APU GPU UPU

APAPAP (APCP )GP APAPGP APAPUP (CPUP )GP (APUP )GP UPUPGP

0.57 0.64 0.72 0.82 0.93 1.02 1.31

Although n a t u r a l complex m i x t u r e s o f l o w - m o l e c u l a r - w e i g h t c o n s t i t u e n t s (nuc l e o s i d e and n u c l e o t i d e pool o r e x t r a c t o f l i v i n g c e l l s ) a r e n o t a t t a c k a b l e e l e c t r o p h o r e t i c a l l y f r o m t h e p o i n t o f view o f s e p a r a t i o n , an i s o t a c h o p h o r e t i c meth0d20’34 was a p p l i e d t o t h e s e p a r a t i o n o f an a r t i f i c i a l model m i x t u r e o f p u r i n e and p y r i m i d i n e bases and n u c l e o s i d e s ( 1 6 components) and n u c l e o t i d e s (ATP, ADP,

NADH,

IMP, CAMP, NAD), as w e l l as UV-absorbing substances i n t h e u r i n e and muscle

e x t r a c t s o f p a t i e n t s w i t h Lesch-Nyhan syndrome, o r o t h e r i n b o r n e r r o r s i n p u r i n e metabolism35y36; 12 components were i d e n t i f i e d by t h i s method. The c o n d i t i o n s f o r

348 TABLE 14.5 ELECTROLYTE SYSTEMS FOR ISOTACHOPHORETIC SEPARATIONS OF PURINE AND P Y R I M I D I N E BASES, NUCLEOTIDESS5 AND NUCLEOTIDES36 C u r r e n t 40 PA; UV d e t e c t i o n a t 254 nm.

Anion Concentration Counter i o n PH Additive

Leading e l e c t r o l y t e

Terminating e l e c t r o l y t e

Ref. 35

Ref. 35

Ref. 36

8-Alanine 0.02 m o l / l Ba2+ 10.4-10.5 None

Caproate 0.05 m o l / l Na+ 6.0

Ref. 36

c10.005 m o l / l Ammediol 8-A1 an ine 8.57t0.02 3.89 0.3% H y d r o x y e t h y l c e l l u l o s e

s e p a r a t i o n s o f t h i s t y p e a r e l i s t e d i n Table 14.5. The q u o t e d papers and t h e poss i b i l i t y o f f o l l o w i n g s i m p l y , f o r example, t h e g l u c u r o n i d a t i o n process i n v o l v i n g

UDP37-40,

t h e s e p a r a t i o n o f i s o m e r i c adenosine o r u r i d i n e n u c l e o t i d e s and t h e i r 1 6 p o l y p h ~ s p h a t e s ~ ' ,t h e s y n t h e s i s o f N - and N - m o d i f i e d ATP o r NAD4 2 3 4 3 o r conc e n t r a t i o n s o f 5 - f l u o r o u r a c i 144, have c o n f i r m e d unequivocal l y t h a t is o t a c h o p h o r e t i c methods a r e s u i t a b l e f o r t h e c h a r a c t e r i z a t i o n and a n a l y s i s o f n u c l e i c a c i d cons t i t u e n t s i n b i o c h e m i c a l r e a c t i o n s and b i o l o g i c a l environments. OLIGONUCLEOTIDES The most i m p o r t a n t problem i n t h e s e p a r a t i o n o f n u c l e i c a c i d c o n s t i t u e n t s i s t h e f r a c t i o n a t i o n o f v e r y complex o l i g o n u c l e o t i d e m i x t u r e s a r i s i n g by f r a g m e n t a t i o n o f n u c l e i c a c i d s d u r i n g t h e i r sequence a n a l y s i s . A l t h o u g h sequence a n a l y s i s i s a t p r e s e n t o r i e n t e d m a i n l y t o r a p i d DNA o r RNA sequencing methods ( r e f s . 45-47;

see Chapter 1 5 ) ,

u t i l i z i n g o n l y p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , t h e f i n g e r p r i n t t e c h n i q u e s o f twodimensional e l e c t r o p h o r e t i c s e p a r a t i o n o f o l i g o n u c l e o t i d e s d e r i v e d f r o m u n i f o r m l y 3 2 P - l a b e l l e d RNA14y22,

which r e p r e s e n t a c o n t i n u a t i o n 2 3 y 2 4 y 3 8o f o r i g i n a l s e p a r a t i o n

e f f o r t s w i t h e l e c t r o p h o r e t i c and chromatographic f i n g e r p r i n t s 2 3 y 2 4 y 4 8 and t h e d e v e l opment o f more advanced

method^^'-^^

32P-labelling o f oligonucleotides i n

combined w i t h homochromatography* a f t e r p r e v i o u s remain t h e i m p o r t a n t approaches t o

t h e s o l u t i o n o f s p e c i f i c problems i n t h e s t u d y o f t h e s t r u c t u r e s o f n u c l e i c a c i d s , i n c l u d i n g p o l y n u c l e o t i d e s f u l l y s y n t h e s i z e d i n v i t r o 6,59-61 The Sanger f i n g e r p r i n t method13y22, w h i c h was d e v i s e d f o f t h e f r a c t i o n a t i o n o f 32 PlRNA, i s o f g r e a t importance. I n t h e f i r s t dimension

ribonuclease digests o f

L

h i g h - v o l t a g e e l e c t r o p h o r e s i s i s r u n on c e l l u l o s e a c e t a t e i n p y r i d i n e - a c e t a t e b u f f e r (pH 3.5);

a s m a l l amount o f 3 2 P - l a b e l l e d m a t e r i a l ( l o w c a p a c i t y o f t h e

s u p p o r t ) m i g r a t e s b e t t e r on t h i ' s i u r f a c e and forms s h a r p e r s p o t s . I n t h e second *Homochromatography = an ascending DEAE-paper o f t h i n - l a y e r chromatographic separat i o n o f 32P-labelled oligonucleotides w i t h t h e b u f f e r e d 7 mol/l urea containing c o l d o l i g o n u c l e o t i d e m i x t u r e as t h e d e v e l o p i n g e l u e n t (homomixture).

349 Cellulose acetate pH 3.5

F i g . 14.4.Diagrams i l l u s t r a t i n g t h e r e l a t i o n s h i p between t h e base c o m p o s i t i o n o f o l i g o n u c l e o t i d e s and t h e i r p o s i t i o n on f i n g e r p r i n t u s i n g e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e a t pH 3 . 5 and i n 7% f o r m i c a c i d f o r t h e DEAE-paper dimension ( t h e Sanger method14922. Each s p o t r e p r e s e n t s t h e 01 i g o n u c l e o t i d e c o m p o s i t i o n c o r r e sponding t o t h e q u a n t i t y and q u a l i t y o f bases as marked w i t h numbers ( 1 - 3 ) and symbols (A, C ) on b a s i c l i n e s ( G - C o r G - A, UG - C o r A, e t c . ) . The l e f t diagram f o r t h e o l i g o n u c l e o t i d e s f r o m a h y p o t h e t i c a l RNA d i g e s t by RNase T1; t h e r i g h t d i a gram f o r dephosphorylated 01 i g o n u c l e o t i d e s of t h i s d i g e s t ( b y a1 k a l i n e phosphatase).

Cellulose

PH 3.5

acetate pH 2.8

n + T

I

A

Fi

0

+

14.5. F i n g e r p r i n t s e p a r a t i o n (Sanger method) o f a RNase T1 d i g e s t o f S U I I I y%']tRNAprQli ( r e f . 6 2 ) . Autoradiogram and diagram show t h e p o s i t i o n of t h e o i g o n u c l e o f i d e s w i t h t h e i r i d e n t i f i c a t i o n numbers and sequences. B i s t h e b l u e dye marker. 8.

dimension, a f t e r t r a n s f e r o f t h e m a t e r i a l t o DEAE-paper, t h e s e p a r a t i o n i s a g a i n r u n e l e c t r o p h o r e t i c a l l y a t an a c i d i c pH ( m o s t l y u s i n g 7% f o r m i c a c i d ) u s i n g s i m u l t a n e o u s l y ion-exchange chromatography ( t h r o u g h an e l e c t r o o s m o t i c f l o w o f b u f f e r c a r r y i n g t h e n u c l e o t i d e s t o t h e anode). The p r i n c i p l e s o f s e p a r a t i o n a r e e v i -

360

c -Cellulose acetate 3 5

I

17

178

7OCAG

'2

6OCCG 20CG

Fig. 14.6. Fingerprint separation (Sanger method) o f a pancreatic RNase digest of the same material as in Fig. 14.5 (ref. 62).

-

Cellulose acetate 3 5

Fig. 14.7. Fingerprint separation (Sanger method) o f a combined RNase T1 and a]kaline phosphatase digest o f the same material as in Fig. 14.5 (ref. 62). The smaller oligonucleotides were allowed t o run o f f the paper.

361 ceiiuiose acetate 3 5

I 17

17A

AAKC wCCCCCACCCCCA,

15 @ 14

130U”CCCG

F i g . 14.8. Sequence a n a l y s i s o f o l i g o n u c l e o t i d e s Nos. 13, 14 and 15 f r o m t h e 62 f i n g e r p r i n t i n F i g . 14.7 by p a r t i a l d i g e s t i o n w i t h snake venom p h o s p h o d i e s t e r a s e S e p a r a t i o n by e l e c t r o p h o r e s i s on DEAE-paper i n p y r i d i n e - a c e t a t e b u f f e r (pH 3.5). C, 3, 2 and 1 a r e a l i q u o t s o f t h e m a t e r i a l d i g e s t e d f o r 0, 15, 10 and 5 min, r e s p e c t i v e l y . B i s t h e b l u e dye marker.

.

d e n t from t h e schemes shown i n F i g . 14.4 and from u s i n g t h e method i n t h e sequence a n a l y s i s o f E. c o l i tRNAs Tyr62 a f t e r d i g e s t i o n w i t h RNase T1 and RNase A ( F i g s . 14.5-14.7).

F i g . 14.8 i l l u s t r a t e s t h e a p p l i c a t i o n o f one-dimensional

electro-

p h o r e s i s o f p a r t i a l d i g e s t s o f v e r y s i m i l a r h e x a n u c l e o t i d e s on DEAE-paper f o r t h e d i r e c t e l u c i d a t i o n o f t h e i r sequences a f t e r e l u t i o n f r o m t h e f i n g e r p r i n t ( i n F i g . 14.7). To o b t a i n l a r g e r t R N A fragments, w h i c h would a l l o w t h e p r e c i s e l o c a l i z a t i o n o f a l l o l i g o n u c l e o t i d e s f r o m complete h y d r o l y s a t e s , a second e l e c t r o p h o r e t i c separat i o n was s u b s t i t u t e d f o r h o m o ~ h r o m a t o g r a p h yy64,~ ~ c a p a b l e o f s e p a r a t i n g much l a r g e r fragments ( u p t o 50 n u c l e o t i d e s ) i n p a r t i a l RNase T1 d i g e s t ( F i g . 14.9). T h i s sep a r a t i o n system i n a t h i n - l a y e r arrangement4’

has become t h e b a s i s f o r t h e so-

c a l l e d m o b i l i t y s h i f t method f o r t h e d i r e c t sequence a n a l y s i s o f o l i g o n u c l e o t i d e s up t o 20 n u c l e o t i d e s i n l e n g t h . A v a r i a t i o n o f t h i s p r o c e d u r e i n v o l v e s a p o l y e t h y l e n e i m i n e ( P E I ) t h i n - l a y e r p l a t e r a t h e r t h a n a DEAE.plate i n t h e second d i m e n ~ i o n ~T h~ i .s s u b s t i t u t i o n r e s u l t e d i n s m a l l , compact f i n g e r p r i n t s p o t s . B o t h o f t h e s e procedures were used t o f i n g e r p r i n t l a r g e 01 i g o n u c l e o t i d e s such as t h o s e r e s u l t i n g f r o m a complete RNase T1 d i g e s t i o n o f R17 o r MS2 phage RNAs65,66 as w e l l

352

C321 15

C321 C321 -

14

13

Fig. 14.9. Homochromatography separation (according to the method o f Brownlee et a1 - 5 4 ) o f a RNase I T1 partial digest o f the same material as in Fig. 14.5 (ref, 62) for the isolation o f larger oligonucleotides a, b, c and d containing 21, 13, 27 and 40 nucleotides, respectively.

353 as of RSV v i r i o n 60-70s RNA67. Another method of f i n g e r p r i n t i n g RNA d i g e s t s cont a i n i n g v e r y l a r g e o l i g o n u c l e o t i d e s (up t o 80 r e s i d u e s ) u t i l i z e s two-dimenional p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s under d e n a t u r i n g c o n d i t i o n s 6 8 y 6 9 . The g e l t e c h n i q u e on 10% s l a b g e l a t pH 3.5 i n 6 m o l / l urea ( i n t h e f i r s t dimension) and on 20% g e l a t pH 8 ( i n t h e second d i m e n s i o n ) has t h e advantage o f b e i n g h i g h l y r e p r o d u c i b l e w i t h s m a l l e r s p o t s i n comparison w i t h homochromatography, as has been p r o v e d many t i m e s 70,71 by f i n g e r p r i n t i n g , f o r example, RNAs o f d i f f e r e n t RNA tumour v i r u s e s

.

A p r e c i s e , q u a n t i t a t i v e e v a l u a t i o n o f t h e r e s o l v i n g power o f t h e s e g e l s under den a t u r i n g and n o n - d e n a t u r i n g c o n d i t i o n s has been completed u s i n g homooligomers f r o m t h e f o u r d e o x y r i b o n u c l e o t i d e s as c h a i n - l e n g t h s t a n d a r d s 7 2 . These i n v e s t i g a t i o n s exh i b i t e d remarkable d i f f e r e n c e s i n m o b i l i t y depending m a i n l y on base c o m p o s i t i o n b u t n o t on sequence. T h i s adds t o an e x a c t c h a i n - l e n g t h d e t e r m i n a t i o n w i t h r e s p e c t t o base c o m p o s i t i o n and t o a d e t a i l e d i n t e r p r e t a t i o n o f complex o l i g o n u c l e o t i d e m i x t u r e s . The above methods and t h e i r m o d i f i c a t i o n s were f o r a l o n g p e r i o d t h e b a s i c approaches t o t h e s e p a r a t i o n o f complete o r p a r t i a l o l i g o n u c l e o t i d e s i n s p e c i f i c t R N A h y d r o l y s a t e s 62'73-75, 5S, 15s and 23s rRNA76'77 79-81 M d 5 y66 y 7 8 and v i r o i ds

and phage RNA R17, Q and

OLIGONUCLEOTIDE SEOUENCE ANALYSIS On t h e b a s i s o f t h e s e p a r a t i o n o f t h e c o n s t i t u e n t s o f an a l k a l i n e o r enzymic h y d r o l y s a t e o f t h e t r i n u c l e o t i d e , f o r example, pApCpG

-f

pAp t Cp t G, t h e s i m p l e s t

paper e l e c t r o p h o r e s i s can p r o v i d e a1 1 t h e i n f o r m a t i o n r e q u i r e d f o r d e t e r m i n i n g t h i s s i m p l e sequence. S i m i l a r l y , t h e whole s t r u c t u r e can be c l e a r l y deduced f r o m a p a r t i a l h y d r o l y s a t e o f h e x a n u c l e o t i d e s w i t h snake venom phosphodiesterase, as shown i n F i g . 14.8, when f r a c t i o n a t e d by e l e c t r o p h o r e s i s on DEAE-paper under t h e c o n d i t i o n s o f s e p a r a t i o n a c c o r d i n g t o charge and l e n g t h o f t h e fragment. Twodimensional s e p a r a t i o n t e c h n i q u e s s h o u l d be a p p l i e d i n more c o m p l i c a t e d p a r t i a l d i g e s t s o f l o n g e r o l i g o n u c l e o t i d e s . Most s u i t a b l e f o r t h i s purpose i s t h e m o d i f i e d Sanger f i n g e r p r i n t technique4'

u s i n g e l e c t r o p h o r e s i s on a c e l l u l o s e a c e t a t e s t r i p

a t pH 3.5 ( s e p a r a t i o n a c c o r d i n g t o base c o m p o s i t i o n ) , f o l l o w e d by homochromatography on a DEAE-cellulose t h i n - l a y e r p l a t e ( s e p a r a t i o n m a i n l y a c c o r d i n g t o l e n g t h o f f r a g m e n t s ) . Ling8' f i r s t observed a s p e c i f i c m o b i l i t y s h i f t caused by a d d i t i o n o f dpC ( o r dCp) o r dpT d u r i n g a n a l y s i s o f DNA p o l y p y r i m i d i n e t r a c t s . By m o d i f y i n g t h e chromatographic homomixture" i t was p o s s i b l e t o c r e a t e c o n d i t i o n s f o r a m o b i l i t y s h i f t d i f f e r e n t i a t i o n between t h e a d d i t i o n o f p u r i n e and p y r i m i d i n e n u c l e o t i d e s t o b o t h p h o s p h o r y l a t e d and dephosphorylated 01 i g o d e o x y r i b o n u c l e o t i d e ,~ and ~ ~ an independent method f o r t h e i r sequencing was t h u s proposed50y83. The observed *Homomixture = t h e b u f f e r e d m i x t u r e o f o l i g o n u c l e o t i d e s o b t a i n e d under t h e c o n t r o l l e d a l k a l i n e h y d r o l y s i s o f RNAs c o n t a i n i n g 7 m o l / l urea and used as t h e e l u e n t d u r i n g homochromatographic s e p a r a t i o n s o f 32P-label l e d 01 i g o n u c l e o t i d e s .

354

TABLE 14.6 EXPERIMENTALLY DETERMINED CHARGE VALUES OF 5 I -MONODEOXYRIBONUCLEOTIDES AT pH 2.8

AND 3.5 Data from r e f . 53.

5 I -dNMP

dTMP dGMP dAMP dCMP

pH 3.5

pH 2.8

Pyr id i ne acetatea

Trimeth lamine acetate

Pyr id ine format ec

T r i m e t h y l ami ne formated

-1.0 -0.83 -0.40 -0.15

-1.0 -0.83 -0.40 -0.14

-1.0 -0.54 -0.10 -0.06

-0.57

i

;5% g l a c i a l a c e t i c acid, 5 mmol/l 5% q l a c i a l a c e t i c acid, 5 mmol/l :1.25% f o r m i c acid, 5 mmol/l EDTA 1.25% f o r m i c acid, 5 mmol/l EDTA

-1.0 -0.12 -0.06

EDTA t i t r a t e d t o pH 3.5 EDTA t i t r a t e d t o pH 3 . 5 t i t r a t e d t o pH 2.8 w i t h t i t r a t e d t o pH 2.8 w i t h

-

with pyridine. w i t h 20% trimethylamine. pyridine. 20% trimethylamine.

Cellulose acetate 3.5

Fig. 14.10. Diagrams o f o l i g o n u c l e o t i d e ( n ) s h i f t s on two-dimensional e l e c t r o phoresis-homochromatography a f t e r extension o f i t s c h a i n by a s i n g l e nucleotide53. The f i r s t dimension i s e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e a t pH 3 . 5 ( a ) o r 2.8 ( b ) , t h e second dimension i s homochromatography on t h e DEAE-cellulose t h i n l a y e r .

355

5' 32pTpCpGpTpApCpCpGpTpAo,, 3'

1

P a r t i a l venom phosphodiesterase

dtgestion 1.

+

5' HoTpCpGpTpApCpCpGplpAP32 3'

32 pTpCpGpTpApCpCpGpTpAoH

2. 32 PT~CPGPTPAPCPCPGPT~~

P a r t i a l spleen phosphodiesterase digestion HOTpCpGpTpApCpCpGpTpAp32

+

HoGpTpApCpCpG~T~A~32 3.

+

+ 4.

+

32pTpCpGpTpApCpCoH

HoTpA ~ C ~ C P G P T ~ A4.P ~ ~

etc.

+

etc.

I t

+

H o C p G ~ T ~ A ~ C ~ C ~ G ~2.T ~ A ~ 3 2

+ 3. 3 2 p T p C ~ G p T p A ~ C ~ C ~ G O H

1.

Analysis b y t w o - d i m e n s i o n a l homochromatography

t

I

Fig. a par i a

Scl.-mes f o r sequencing t e r m i r I l y l a b e l l e d DNA o l i g o n u c . ? o t i d e s u s i n g phosphodiesterase d i e s t i o n and t h e s e p a r a t i o n on two-dimensional e l e c -

trophoresis-homochromatography~3. change i n t h e p o s i t i o n o f t h e o l i g o n u c l e o t i d e a f t e r p r o l o n g a t i o n by one s p e c i f i c n u c l e o t i d e f o r two o f t h e most w i d e l y used systems i s i l l u s t r a t e d i n F i g . 14.10. Table 14.6 summarizes t h e r e l a t i v e c h a r g e v a l u e s of t h e r e s p e c t i v e d e o x y n u c l e o t i d e i n t h e most commonly used e l e c t r o p h o r e t i c b u f f e r s . The m o b i l i t y s h i f t method, a l s o c a l l e d two-dimensional homochromatography, p e r m i t s t h e d e t e r m i n a t i o n o f a c o m p l e t e n u c l e o t i d e sequence o f o l i g o d e o x y r i b o n u c l e o t i d e ~o~r ~01 igoribonucleotides50y52y58 l a b e l l e d w i t h 32P a t one o f t h e c h a i n ends ( w i t h p o l y n u c l e o t i d e k i n a s e f o r t h e 5 ' - e n d and w i t h d e o x y n u c l e o t i d y l t e r m i n a l t r a n s f e r a s e , t R N A - n u c l e o t i d y l t r a n s f e r a s e o f T4 RNA 1 i g a s e f o r t h e 3 ' - e n d ) a f t e r p a r t i a l h y d r o l y s i s w i t h nucleases o f t h e t y p e o f E. coZi exonuclease 111, snake venom o r spleen phosphodiesterase and nuclease P1. The schemes i n F i g . 14.11 i l l u s t r a t e t h e i n d i v i d u a l d i f f e r e n t Drocedures.

366

5' p5' -p

P

p

5'HO

(OH)

5' HO

P

5' P

p 3' (OH)

P 5'HO

5' P

3'

5'HO

(;HI P

3'

P (OH) 3'

etc.

Label 5 ' - O H ends w i t h 32P and separate m i x t u r e according t o size on polyacrylamide gel

+ 2 00

75

60

55

Transfer 5'-32P oligonucleotides on to a s t r i p o f polyethyleneimine cellulose digest in s i t u w i t h RNase T 2 and ; d e n t i f ~ [ ~ ~ P ] by X pthin-layer chromatography

F i g . 14.12. Scheme f o r t R N A sequencing u s i n g a p a r t i a l a l k a l i n e d i g e s t i o n , a n e l e c t r o p h o r e t i c s e p a r a t i o n o f t h e o b t a i n e d m i x t u r e on p o l y a c r y l a m i d e g e l and f u r t h e r l a b e l l i n g , u n s p e c i f i c d i g e s t i o n t o mononucleotides and a chromatographic s e p a r a t i o n on p o l y e t h y l e n e i m i n e c e l l u l o s e t h i n l a y e r 4 5 .

A s i m i l a r method u t i l i z i n g t h e m o b i l i t y s h i f t i n p o l y a c r y l a m i d e g e l s , c a l l e d "wandering s p o t " a n a l y s i s , f o r o l i g o n u c l e o t i d e sequencing has a l s o been d e v i s e d

84,85

b u t i t s r e s o l v i n g power i s n o t comparable t o t h a t o f t h e p r e c e d i n g procedure. Another method f o r d e t e r m i n i n g t h e sequences o f RNA o l i g o n u c l e o t i d e s , p a r t i c u l a r l y t h o s e c o n t a i n i n g m o d i f i e d n u c l e o s i d e s (tRNA), i s t h e procedure shown i n F i g . 14.12, u t i l i z i n g c o n t r o l l e d a l k a l i n e h y d r o l y s i s when a homologous s e r i e s o f o l i g o n u c l e o t i d e s b e a r i n g t h e common 3 ' - h y d r o x y l end o f t h e RNA i n q u e s t i o n oc-

,

357

curs 54y56. Such o l i g o n u c l e o t i d e s a f t e r 32P l a b e l l i n g o f t h e 5'-end can be separ a t e d by polyacrylamide gel e l e c t r o p h ~ r e s i s ~Determination ~. o f the terminal f2P]nucleotide

o f each o l i g o n u c l e o t i d e then y i e l d s t h e complete sequence o f t h e

o r i g i n a l p o l y n u c l e o t i d e because s e p a r a t i o n i n t h e gel proceeded according t o l e n g t h o f homologous fragments. Such determinations can be made by t r a n s f e r o f 32 P - l a b e l l e d o l i g o n u c l e o t i d e s from t h e g e l on t o a polyethyleneimine (PEI)

5'-

c e l l u l o s e s t r i p and a f t e r h y d r o l y s i s i n s i t u w i t h RNase T2 by chromatographic i d e n t i f i c a t i o n o f t h e appearing f2P]Xp

on P E I c e l l u l o s e 5 5 y 5 6 y 8 6 . This method

a l s o y i e l d e d many new data i n t h e sequence a n a l y s i s o f n a t u r a l and s y n t h e t i c p o l y n u c l e o t i d e s and c l e a r l y c o n t r i b u t e d t o t h e demonstration t h a t a l l sequence analyses a r e dependent on a s u i t a b l e r a p i d , p r e c i s e and s e n s i t i v e f r a c t i o n a t i o n o f t h e c o n s t i t u e n t s o f p a r t i c u l a r m i x t u r e s . I n a l l o f t h e aforementioned cases, such a s e p a r a t i o n i s obtained by some o f t h e e l e c t r o p h o r e t i c techniques o r by a comb i n a t i o n o f e l e c t r o p h o r e s i s and chromatography. REFERENCES

1 J. M u s i l , 0. Novlkov5 and K. Kunz, Biochemistry i n Schematic Perspective, Avicenum, Prague, 1977. 2 P.R. Stewart and D.S. Letham ( E d i t o r s ) , The RibonucZeic Acids, Springer Verlag, New York, 1977. 3 A. Kornberg, DNA Synthesis, 2nd ed., Freeman, San Francisco, C A Y 1979. 4 R.S. H a l l , The Modified NucZeosides i n NucZeic Acids, Columbia U n i v e r s i t y Press, New York, 1971. 5 R.J. Suhadolnik, NucZeoside Antibiotics, W i l e y - I n t e r s c i e n c e , New York, 1970. 6 H. Kdster ( E d i t o r ) , NucZeic Acid Synthesis: AppZications to MoZeeuZur BioZogy and Genetic Engineering, N u c l e i c A c i d Symposia Series No. 7, IRL, Oxford, 1980. 7 M. Laskowski, S r . , Methods EnzymoZ., 12 (1967) 281. 8 K. Randerath and E. Randerath, Methods !JnzymoZ., 12 (1967) 323. 9 B.Z. Egan and A.D. Kelmers, Methods Enzymoz., 29 (1974) 469. 10 S. Z a d r a i i l , i n W.W. Zorbach and R.S. Tipson ( E d i t o r s ) , Synthetic Procedures i n NucZeic Acid Chemistry, Vol. 2, Wiley, New York, 1973, p. 533. 11 S. Z a d r a i i l , i n Z. Deyl , K. Macek and J . J a n l k ( E d i t o r s ) , Liquid C o Z m Chromatography,. E l s e v i e r , Amsterdam, 1975, p. 831. 12 R. Markham and J.D. Smith, Biochem. J . , 52 (1952) 552. 13 J.D. Smith. Methods EnzumoZ.. 12 (19671 350. 14 F. Sanger and G.G. Browilee,-Methdds E h q m o Z . , 12 (1967) 361. 15, K. Randerath, Thin-Layer Chromatography, Academic Press, New York, 1966. 16 H.O. Smith, T.J. K e l l y and P.H. Roy, Methods EnzymoZ., 29 (1974) 282. 17 T.M. Jovin, Methods EnzymoZ., 2 1 (1971) 179. 18 P. P h i l i p p s e n and H.G. Zachau, FEBS Lett., 15 (1971) 69. 19 P. P h i l i p p s e n and H.G. Zachau, Methods EnzymoZ., 29 (1974) 473. 20 H. Haglund, Sci. TooZs, 17 (1970) 2. 21 V.M. Ingram and A.O.W. S t r e t t o n , Biochim. Biophys. Acta, 62 (1962) 456. 22 S. Sanger, G.G. Brownlee and B.G. B a r r e l l , J . MoZ. BioZ., 13 (1965) 373. 23 G.W. Rushizky and C.A. Knight, ViroZogy, 11 (1960) 236. 24 G.W. Rushizky, Methods Enzymoz., 12 (1967) 395. 25 F.W. S t u d i e r , J . MoZ. BioZ., 79 (1973) 237. 26 M.W. McDonell, M.N. Simon and F.W. S t u d i e r , J . MoZ. BioZ., 110 (1977) 119. 27 R. Markham and J.D. Smith, Biochem. J . , 45 (1949) 294. 28 K.C. Smith and F.W. A l l e n , J . Amer. Chem. Soc., 75 (1953) 2131. I

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P

3 59

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

82 83 84 85

J. H o r s t , J. K e i t h and H. Fraenkel-Conrat, Nature (New BioZ.), 240 (1972) 105. R. De Wachter and W. F i e r s , A m Z . Biochern., 49 (1972) 184. J.M. C o f f i n and M.A. B i l l e t e r , J . MoZ. BioZ., 100 (1976) 293. K.L. Beemon, Curr. Top. Microbial. ImunoZ., 79 (1978) 73. J.-L. D a r l i x , M. Lavray, P.A. Bromley and P.-F. Spahr, NucZeic Acids Res., 6 (1979) 471. R. Frank and H. Kb'ster, Nucleic Acids Res., 6 (1979) 2069. M. S p r i n z l , F. G r i i t e r and D.H. Gauss, NucZeic Acids Res., 5 (1978) r15. P.R. Schimmel, D. S 6 l l and J.N. Abelson ( E d i t o r s ) , Transfer RNA: Structure, Properties and Recognition, Cold S p r i n g Harbor L a b o r a t o r y , Cold S p r i n g Harbor, 1979. J.E. C e l i s and J.D. Smith ( E d i t o r s ) , Nonsense Mutations and tRNA Suppressors, Academic Press, London, 1979. G.G. Brownlee, E . C a r t w r i g h t , T. McShane and R. W i l l i a m s o n , FEBS Lett., 25 (1972) 8. L. Magrum, L. Zablen, D. S t a h l and C. Voese, Nature (London), 257 (1975) 423. N.D. Z i n d e r ( E d i t o r ) , RNA Phages, Cold S p r i n g Harbor L a b o r a t o r y , Cold S p r i n g Harbor, 1975. H.J. Gross, H. Domdey, C. Lossow, P. Jank, M. Raba, H. A l b e r t y and H.L. Sanger, Nature (London), 273 (1978) 203. D. Riesner, K. Henco, V. Rokohl, G. K l o t z , A.K. K l e i n s c h m i d t , H. Domdey, P. Jank, H.J. Gross and H.L. Sanger, J . MoZ. BioZ., 133 (1979) 85. H.J. Gross, H. A l b e r t y , H. Domdey, G. Krupp, V. L i e b l , C. Lossow, K. Ramm and H.L. Sanger, i n J. Augustyniak ( E d i t o r ) , BioZogical ImpZications of ProteinNucZeic A c i d Interactions, E l s e v i e r / N o r t h H o l l a n d , Amsterdam and A. M i c k i e w i c z U n i v e r s i t y Press, Poznan, 1980, p . 251. V. L i n g , J . MoZ. BioZ., 6 4 (1972) 87. E. Jay, R.A. Bambara, R. Padmanabhan and R. Wu, Nucleic Acids Res., 1 (1974) 331. Y.F. Lee, N. Kitamura, A. Nomoto arid E. Wimmer, J . Gen. ViroZ., 44 ( 1 9 7 9 ) 311. A. Nomoto and N. Imura, Nucleic Acids Res., 7 (1979) 1233.

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361

Chapter 1 5

NUCLEIC ACIDS

s.

ZADRA~IL

GEFiERAL ASPECTS N u c l e i c a c i d s , t h e b a s i c g e n e t i c m a t e r i a l o f a l l organisms,

p l a y an i m p o r t a n t

r o l e i n fundamental b i o l o g i c a l processes w h i c h may b e encompassed i n t o t h e t e r m "gene expression"'.

S t u d i e s o f t h e i r s t r u c t u r e and o f t h e r e l a t i o n s h i p between

s t r u c t u r e and f u n c t i o n , as i n o t h e r b i o l o g i c a l l y a c t i v e s u b s t r a t e s , have always been dependent on t h e m e t h o d i c a l development and t h e l e v e l o f a g i v e n a r e a o f b i o l o g y and b i o c h e m i s t r y , as w e l l as p h y s i c a l c h e m i s t r y , and t h e i r a s s o c i a t e d apparatus and equipment. The same h o l d s f o r e l e c t r o p h o r e s i s ( t h e movement o f charged p a r t i c l e s under t h e i n f l u e n c e o f an e l e c t r i c f i e l d ) , t h e d i f f e r e n t t e c h niques and systems o f which (moving boundary e l e c t r o p h o r e s i s and zone e l e c t r o p h o r e s i s ) have a l s o been a p p l i e d t o t h e a n a l y s i s o f n u c l e i c a c i d s h a v i n g t h e nature o f polyanions. The general process o f n u c l e i c a c i d i s o l a t i o n which, a p a r t f r o m c e l l d i s r u p t i o n ( w h i l e m a i n t a i n i n g t h e c o n d i t i o n s p r e v e n t i n g mechanical d e g r a d a t i o n o f nuc l e i c a c i d macromolecules) and e f f e c t i v e i n h i b i t i o n o f n a t u r a l nucleases, i n c l u d e s v a r i o u s degrees o f s e p a r a t i o n and p u r i f i c a t i o n f r o m o t h e r c e l l u l a r low- and h i g h m o l e c u l a r - w e i g h t components and mutual f r a c t i o n a t i o n o f t h e i r own macromolecules, p r o v i d e s c o n s i d e r a b l e scope f o r a n a l y t i c a l and p r e p a r a t i v e a p p l i c a t i o n s o f e l e c t r o p h o r e t i c s e p a r a t i o n and f r a c t i o n a t i o n techniques. For example, t h e o r i g i n and t h e u n u s u a l l y r a p i d development o f methods o f gene m a n i p u l a t i o n (gene e n g i n e e r i n g ) and r a p i d DNA sequencing a r e c l o s e l y a s s o c i a t e d w i t h t h e f r a c t i o n a t i o n o f p o l y n u c l e o t i d e s and o l i g o n u c l e o t i d e s b y one- and two-dimensional

systems o f paper

o r g e l e l e c t r o p h o r e s i s w i t h t h e r e s o l v i n g power o f o n e ' n u c l e o t i d e i n many i n stances2-12.

A t p r e s e n t , w i t h t h e u s e o f p o l y a c r y l a m i d e o r agarose g e l s , g e l e l e c -

t r o p h o r e s i s i s most w i d e l y u s e d f o r t h e f r a c t i o n a t i o n o f n u c l e i c a c i d s and i s also convenient i n studies o f molecular conformation, n o t o n l y f o r t h e r e s o l u t i o n o f s i n g l e - s t r a n d e d and d o u b l e - s t r a n d e d s t r u c t u r e s 'lYl3 b u t a l s o o f l i n e a r and c i r c u l a r (open and c o v a l e n t l y c l o s e d ) molecules13 and t h e degree o f t h e i r superh e l i c i t y and ~ a t e n a t i o n l ~ - E~l e~ c. t r o p h o r e s i s on s o l i d and semi-sol i d g e l s , w i t h i t s advantageous p r o p e r t i e s , has supplanJed o t h e r e l e c t r o p h o r e t i c t e c h n i q u e s and s u p p o r t s and has become an a l m o s t u n i v e r s a l t e c h n i q u e f o r t h e s e p a r a t i o n , p u r i f i -

362 c a t i o n and f r a c t i o n a t i o n o f n u c l e i c acids and, together w i t h the p o s s i b l e h y b r i d i z a t i o n s e l e c t i o n o f analysed molecules (Southern17 and "Northern"18 b l o t t i n g techniques) , t h e i r e l u t i o n and f u r t h e r chemical o r enzymatic processing, has remained unequalled and completely i r r e p l a c e a b l e by o t h e r techniques. So f a r a l l well known supports used i n zone electrophoresis have been applied

t o n u c l e i c acids, i.e.,

paper and i t s ion-exchange

modification^^^^^-^^,

partic-

u l a r l y DEAE- and acetate-cellulose, c e l l u l o s e and s u b s t i t u t e d c e l l u l o s e s i n powder form f o r t h e t h i n - l a y e r systems 3y4y22, agar23s24, p ~ l y a c r y l a m i d e , ~ agarose'' ~-~~ and combinations o f the l a s t two substances i n composite gelsLu, i n c l u d i n g systems ,A

i n the form o f gradient

The choice o f the support i s d i c t a t e d by t h e

properties required f o r a p a r t i c u l a r separation. For example, acetate-cellulose and DEAE-substituted papers permitted a unique f i n g e r p r i n t technique t o be used 2 i n the sequence analysis o f r a d i o a c t i v e l y l a b e l l e d RNA DEAE-papers, which a r e

.

extremely f r a g i l e and d i f f i c u l t t o manipulate, were l a t e r replaced w i t h more advantageous DEAE-cel l u l o s e t h i n - l a y e r plates22, and a new technique, t h e m o b i l i t y s h i f t electrophoresis-homochromatography method, was developed f o r the sequence analysis o f oligonucleotides derived f r o m RNA and DNA3';

t h i s method i s very

s u i t a b l e f o r t h e a n a l y t i c a l c o n t r o l o f s y n t h e t i c a l l y prepared 01 igonucleotides. Polyacrylamide gels used i n many modifications o f f e r advantages o f chemical stab i l i t y and inertness, transparency and non-existent adsorption and electroosmosis, which f a c i l i t a t e t h e i r a p p l i c a t i o n i n m i c r ~ a n a l y s e s ~ I~n. addition, g e l s o f a desired pore s i z e (molecular s i e v i n g e f f e c t ) can be prepared, which i s o f d e c i s i v e importance f o r the r e s o l v i n g power o f the method and i t s adaptation t o t h e widely d i f f e r i n g sizes o f sample molecules. Many designs f o r electrophoresis apparatus have been developed; some o f them have been s p e c i a l l y constructed f o r t h e analysis, separation and f r a c t i o n a t i o n o f nucleic acids and t h e i r components2'33-39. Nevertheless, simple and cheap "homemade" ( d i r e c t l y i n biochemical and b i o l o g i c a l l a b o r a t o r i e s ) apparatus compares favourably w i t h commercial apparatus. For example, various m o d i f i c a t i o n s o f 2 klarkham and Smith's33 and Sanger e t a l . ' s paper electrophoresis apparatus, Studier and co-workers' gel electrophoresis34y35 and Sanger and Coulson's'O system f o r t h i n polyacrylamide gels belong t o t h e most widely used apparatus, Discussions over the advantages o f t h e horizontal o r v e r t i c a l arrangement o f gel electrophoresis stem mainly from t h e f a c t t h a t gels i n a horizontal p o s i t i o n (semi-solid agarose gels below 0.5%) are easier t o manipulate. V e r t i c a l gels provide a sharper and a more d i s t i n c t separation o f the bands and a r e therefore used e x c l u s i v e l y f o r r a p i d sequence analyses o f n u c l e i c acids, For t h e d e t e c t i o n o f t h e separated components, the f o l l o w i n g methods a r e used, as i n other n u c l e i c a c i d analyses: UV absorption a t 260 nm (contact photocopies o f electropherograms and f i n g e r p r i n t s i n UV l i g h t 3 3 , autoradiography o r f l u o r o -

363

graphy o f papers o r g e l s w i t h f r a c t i o n a t e d r a d i o a c t i v e m i x t u r e o f molecules, and fluorescence o f n u c l e i c a c i d s w i t h e t h i d i u m bromide o r a c r i d i n e orange i n UV l i g h t and s t a i n i n g w i t h methylene blue, b o t h p e r m i t t i n g simple photorecording on a sen. s i t i v e f i l m u s i n g f i l t e r s , which i s i n v a l u a b l e i n a l l gel t e c h t ~ i q u e s ~ ' - ~ ~Whenever short-wavelength UV l i g h t i s used, p o s s i b l e damage t o t h e DNA d u r i n g exposure should be taken i n t o account, p a r t i c u l a r l y i n e v a l u a t i n g i t s b i o l o g i c a l a c t i v i t y 4 6

.

For t h e d e t e c t i o n o f s p e c i f i c sequences, DNA-DNA o r DNA-RNA molecular h y b r i d i z a t i o n , w i t h t h e probe e x h i b i t i n g h i g h s p e c i f i c r a d i o a c t i v i t y , can be used a f t e r t r a n s f e r o f analysed n u c l e i c a c i d f r a c t i o n s from t h e gel o n t o t h e corresponding h y b r i d i z a t i o n support [ n i t r o c e l l u l o s e membrane according t o Southern17 o r diazobenzyloxymethyl (DMB) paper, denoted "Northern" by anaTogy 18] , o f f u r t h e r process i n g o f t h e f r a c t i o n e l u t e d from t h e gel u s i n g a s p e c i f i c enzyme restriction

-

endonuclease, nuclease S1 , e t c . Several methods have been proposed f o r t h e recovery o f n u c l e i c a c i d s from gel s l i c e s , examples o f which a r e e l e c t r o e l u t i o n 35,47-49 e l u t i o n by diffusion'',

e l u t i o n by mechanical o r c e n t r i f u g a l gel c o m p r e ~ s i o n ~ ~ - ~ ~

and g e l (agarose) d i s s o l ~ t i o n ~u s~i n- g~ c~h a o t r o p i c agents such as potassium i o d i d e o r sodium p e r c h l o r a t e . RIBONUCLEIC A C I D S C e l l r i b o n u c l e i c a c i d s show g r e a t e r h e t e r o g e n e i t y than t h e DNAs, and s e t s o f t h e i r molecules d i f f e r i n s i z e , s t r u c t u r e , composition and f u n c t i o n 5 7 . The e x i s tence o f low-molecular-weight cytoplasmic tRNAs, d i s t i n c t classes o f rRNAs (5S, 16s and 23S, o r 18s and 28s) and v a s t groups ( i n s i z e ) o f mRNAs and t h e i r precursors (premRNA) have c o n s t i t u t e d a h i s t o r i c a l l y s u i t a b l e area o f s u b s t r a t e s f o r a p p l i c a t i o n n o t o n l y o f d i f f e r e n t s e p a r a t i o n and f r a c t i o n a t i o n techniques b u t a l s o o f techniques f o r molecular s i z e determination.

I n comparison w i t h DNA, t h e

s u i t a b i l i t y o f sample m a t e r i a l s and t h e p o s s i b i l i t y o f an e f f e c t i v e study o f s t r u c t u r e and f u n c t i o n were augmented by t h e r e l a t i v e l y h i g h i n c i d e n c e o f m o d i f i e d ribonucleosides which increase t h e number o f d i f f e r i n g components i n polymerscomposed o f o n l y f o u r b a s i c b u i l d i n g u n i t s , and by t h e r e l a t i v e l y e a r l y d i s c o v e r y o f h i g h l y s p e c i f i c c a t a b o l i c enzymes (RNases) which f a c i l i t a t e d c o n t r o l l e d degrsd a t i o n and m o d i f i c a t i o n o f RNA molecules. Apart from these s i n g l e - s t r a n d e d c e l l RNAs w i t h c e r t a i n i r r e g u l a r secondary s t r u c t u r e s , t h e r e a r e o t h e r RNAs w i t h a c i r c u l a r s t r u c t u r e ( v i r o i d s , pathogens i n h i g h e r p l a n t s ) , homogeneous and heterogeneous s i n g l e - s t r a n d e d and double-stranded RNAs o f t h e phages, and p l a n t and animal v i r u s e s o f v a r i o u s c o m p l e x i t y where RNF. i s the basic genetic material. Although i n most o f these RNA m a t e r i a l s methods based on s e l e c t i v e p r e c i p i t a t i o n and e x t r a c t i o n , p a r t i t i o n i n g between aqueous and o r g a n i c phases and v a r i o u s

TABLE 15.1

THE MOST FREQUENT COMPOSITIONOF POLYACRYLAMIDE GELS AND THEIR SEPARATION P R O P E R T I E S ~ Gel type

Concentration of gels (%)

Ratio of acrylamide t o bisacrylamide

1 2 3 4

Q.5 2-5 5-15

ca. ca. ca. ca.

10-20

20 25 35

40

Number of nucl eotides i n polymers

100 10 1

-

-

>3000 3000 300

100

Mol ecul a r we i gh t ( Md )

Nucleic acid t o be separated

Mi gra t i on distance of t R N A

>1 0.030 0.003 0.0003-

DNA, v i r a l RNA rRNA, mRNA mRNA, t R N A 5s RNS t R N A o l i g o NT

Run off 32 cm 14 cm 4 cm

-

1 0.10 0.03

aElectrophoresis conditions: 40 m m o l / l T r i s - a c e t i c acid ( p H 8.0), gel s l a b 40 x 20 cm, 6- mm t h i c k , voltage between electrodes 250-500 V , c u r r e n t 25-40 mA. Data from r e f s . 26, 27 and 61.

365 c h r o m a t o g r a p h i c procedures i n c l u d i n g a f f i n i t y chromatography and sucrose g r a d i e n t v e l o c i t y s e d i m e n t a t i o n have been and a r e s t i l l used f o r s e p a r a t i o n , f r a c t i o n a t i o n and c h a r a c t e r i z a t i o n , e l e c t r o p h o r e t i c methods, of w h i c h g e l e l e c t r o p h o r e s i s occ u p i e s a c e n t r a l p o s i t i o n , have a l s o been w i d e l y a p p l i e d . Whereas t h e advantages o f s e p a r a t i o n on a paper s u p p o r t and c e l l u l o s e i n a oneand two-dimensional arrangement o r i n c o m b i n a t i o n w i t h chromatography a r e u t i l i z e d m a i n l y f o r low-molecular-weight

n u c l e o t i d e s and o l i g o n u c l e o t i d e s ( s e e t h e preced-

i n g c h a p t e r ) , p a r t i c u l a r l y i n t h e sequence a n a l y s i s o f o l i g o n u c l e o t i d e s and small

RNP. fragments (see a l s o t h e s e c t i o n on sequence a n a l y s i s ) , g e l e l e c t r o p h o r e s i s u s i n g p o l y a c r y l a m i d e , agarose o r m i x t u r e s t h e r e o f under v a r i o u s c o n d i t i o n s i s a t P r e s e n t e s s e n t i a l l y t h e o n l y t e c h n i q u e used f o r t h e e l e c t r o p h o r e t i c s e p a r a t i o n and f r a c t i o n a t i o n o f RNA. E l e c t r o p h o r e s i s on p o l y a c r y l a m i d e and i n a g a r and agarose g e l s i s more r a p i d and has a g r e a t e r r e s o l v i n g power than, f o r example, chromatography on MAK c o l u x n s o r sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n 18y24-28y58

The o r i g i n a l arrangement

of p o l y a c r y l a m i d e g e l was a t h r e e - l a y e r one: a l a r g e - p o r e sample g e l and spacer gel f o r i n i t i a t i o n and c o m p l e t i o n o f t h e e l e c t r o p h o r e t i c c o n c e n t r a t i o n o f t h e sample i o n s and t h e t h i r d r u n n i n g g e l w i t h s m a l l pores where t h e e l e c t r o p h o r e t i c s e p a r a t i o n t o o k place. The pH values o f t h e r u n n i n g g e l , spacer g e l and e l e c t r o p h o r e s i s b u f f e r were d i f f e r e n t and c r i t i c a l f o r s e p a r a t i o n

-

the discontinuous

e l e c t r o p h o r e t i c system59y60. Subsequently i t was found t h a t t h e r u n n i n g g e l i s s u f f i c i e n t f o r t h e r e q u i r e d s e p a r a t i o n when t h e n e c e s s i t y f o r v e r y s m a l l amounts o f a p p l i e d sample and i t s s u f f i c i e n t c o n c e n t r a t i o n i s adhered t o and EDTA i s emp l o y e d as t h e b a s i c component o f t h e b u f f e r so t h a t t h e d i s c o n t i n u o u s b u f f e r system need n o t be usedz6. Examples o f t h e c o m p o s i t i o n and uses o f a c r y l a m i d e g e l s a r e p r e s e n t e d i n Table 15.1.

I n a d d i t i o n t o t h e most w i d e l y used T r i s - a c e t a t e

b u f f e r , a c o m b i n a t i o n w i t h phosphate and b o r a t e i s u s e f u l , whereby t h e b u f f e r i n g c a p a c i t y i n c r e a s e s t o c o u n t e r b a l a n c e t h e r i s i n g a l k a l i n i t y o f t h e anode and t h e a c i d i t y of t h e cathode space d u r i n g e l e c t r o p h o r e s i s ( r e c i r c u l a t i o n o f t h e b u f f e r s i n t h e e l e c t r o d e spaces s h o u l d be ensured i n some i n s t a n c e s ) . The Tris-HC1 b u f f e r system i s n o t s u i t a b l e because i t has a l o w b u f f e r i n g c a p a c i t y and h y p o c h l o r i t e f o r m a t i o n m i g h t occur. The same b u f f e r s can be used f o r d i f f e r e n t l y c o n c e n t r a t e d agarose g e l s . The a c r y l a m i d e t o N,N'-methylenebisacrylamide r a t i o s h o u l d range f r o m 10 t o 100, o b s e r v i n g t h e r u l e t h a t t h e v a l u e o f t h e r a t i o s h o u l d n e c e s s a r i l y increase w i t h increasing acrylamide concentration i n order t o o b t a i n s u f f i c i e n t e l a s t i c i t y o f t h e gel i n r o d s o r s l a b s . Admixture o f agarose i n t h e f r e q u e n t l y In 28,42,62,63. used composite g e l s a f f e c t s f a v o u r a b l y t h e e l a s t i c i t y o f t h e g e l a d d i t i o n t o an a p p r o p r i a t e r a t i o o f t h e monomer t o a c r o s s - l i n k i n g a g e n t i n aqueous s o l u t i o n , o t h e r components a r e EDTA and, s h o r t l y b e f o r e p o u r i n g t h e m i x t u r e i n t o t h e prepared mould, a p o l y m e r i z a t i o n i n i t i a t o r N,N,N',N'-tetrarnethylethylene-

366

diamine; TEF4ED) and a p o l y m e r i z a t i o n c a t a l y s t (ammonium p e r s u l p h a t e ) . A l a y e r o f water i s u s u a l l y p i p e t t e d on t o t h e s u r f a c e o f t h e p o l y m e r i z i n g g e l i n o r d e r t o o b t a i n a f l a t surface. P o l y m e r i z a t i o n t a k e s p l a c e a t room t e m p e r a t u r e and t h e t o t a l p o l y m e r i z a t i o n t i m e i s dependent on t h e g e l c o n c e n t r a t i o n i t increases

-

w i t h i n c r e a s i n g a c r y l a m i d e c o n t e n t . A c o n s i d e r a b l e amount o f h e a t i s r e l e a s e d d u r i n g p o l y m e r i z a t i o n and t h e r e f o r e i t i s u s e f u l and sometimes necessary t o evacu a t e t h e s o l u t i o n p r i o r t o p o l y m e r i z a t i o n so as n o t t o g i v e r i s e t o a i r bubbles, p a r t i c u l a r l y i n t h e v i c i n i t y of t h e comb f o r t h e f o r m a t i o n o f t h e w e l l s f o r sample appl i c a t i on, The p r e p a r a t i o n o f agarose g e l s , a t c o n c e n t r a t i o n s o f 1-22 f o r RNA, i s much s i m p l e r . An a p p r o p r i a t e amount o f agarose i s m e l t e d i n water, a t e n - t i m e s conc e n t r a t e d e l e c t r o p h o r e s i s b u f f e r i s added, t h e m i x t u r e i s l e f t t o c o o l t o 50-60°C and t h e n poured i n t o t h e prepared mould, where i t i s a l l o w e d t o s o l i d i f y a t rooll: temperature o r i n a c o l d room. S i m i l a r l y , t h e prepared agarose s o l u t i o n i s added t o t h e a c r y l a m i d e p o l y m e r i z a t i o n m i x t u r e f o r p r e p a r a t i o n o f composite gels, and t h e m i x t u r e formed a f t e r s o l i d i f i c a t i o n o f agarose a t 4OoC p o l y m e r i z e s d u r i n g an extended p e r i o d a t room temperature. E l e c t r o p h o r e s i s i n v a r i o u s m o d i f i c a t i o n s o f t h e apparatus arrangement, shape and c o n c e n t r a t i o n o f t h e g e l s and e l e c t r o p h o r e t i c c o n d i t i o n s has been used f o r biochemical, m o l e c u l a r b i o l o g i c a l and g e n e t i c s t u d i e s o f t h e RNA o f v a r i o u s t y p e s and o r i g i n s . P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s has proved t o be t h e most s u i t a b l e method f o r t h e f r a c t i o n a t i o n o f rRNA and mRNA. T h i s may be e x e m p l i f i e d by a m i x t u r e o f c y t o p l a s m i c and m i t o c h o n d r i a l rRNAs e x t r a c t e d f r o m two y e a s t species, Candida parapsiZosis and Sachuromyses c e r e ~ i s i a e ~F~i g. 15.1 shows t h a t t h e two

p a i r s of m i t o c h o n d r i a l rRNAs (MIC,

M2C, MIS

and M2S) c o u l d be W e l l d i s t i n g u i s h e d

on a 2.4% g e l , whereas t h e c y t o p l a s m i c rRNAs have two common peaks, CISl

and

C2S2. The a b i l i t y o f t h e g e l t o separate t h e m a j o r c o n s t i t u e n t s o f c y t o p l a s m i c

c e l l u l a r RNA o f p r o k a r y o t i c and e u k a r y o t i c c e l l s has been c o n f i r m e d many times, even on a m i c r o - s c a l e i n t h e c a p i l l a r i e ~so~ t~h a t i n d i v i d u a l c o n s t i t u e n t s o f

r RNA and tRNA serve, t o g e t h e r w i t h some phage and v i r a l RNAs, as s i z e standards (see Table 15,265-67) w h i c h a r e u s e f u l e s p e c i a l l y i n t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f d i f f e r e n t I ~ R N A S ~ ~ - ~i nOc, l u d i n g mRNA f o r t h e l i p o p r o t e i n o f t h e E. c o Z i o u t e r membrane63. A s i m i l a r s e p a r a t i o n o f 28s and 18s rRNA, and 9s mRNA f o r c h i c k e n g l o b i n , can be o b t a i n e d on 1.1%agarose gel71. I n t h e comparisons o f t h e genomes o f F i j i v i r u s e s , which a r e r e p r e s e n t e d by segmented double-stranded RNA (dsRNA f r o m r i c e ragged s t u n t v i r u s , maize rough d w a r f v i r u s , pangola s t u n t v i r u s and o a t s t e r i l e d w a r f v i r u s ) , i t was p o s s i b l e t o r e v e a l d i f f e r e n c e s i n t h e number and amount o f i n d i v i d u a l dsRNA segments7' 0.2-2.9

i n t h e m o l e c u l a r w e i g h t range

Md by e l e c t r o p h o r e s i s on 5% p o l y a c r y l a m i d e g e l ,

367

Relative mobility, crn

Fig. 15.1. E l e c t r o p h o r e t i c s e p a r a t i o n o f a m i x t u r e o f cytoplasmic and mitochondria1 (M) high-molecular-weight rRNA species o f s charomyces c e r e v i s i a e ( S ) and Candida parapsilosis (C) on 2.4% polyacrylamide gel

84.

TABLE 15.2 S I Z E STANDARDS FOR RNA GEL ELECTROPHORESIS data from r e f s . 65-67.

RNA t y p e

Sedimentation coefficient (S)

M o l e c u l a r weight (Md 1

E. c o l i tRNA E. coZi rRNA

4 5 16 23 18 28 18 28

0.026 0.036 0.56 1.13 0.71 1.9 0.78 1.42 1.4 1.23 2.0

HeLa cells Yeast

rRNA rRNA

Phage Q Phage MS2

mv

The h i g h r e s o l v i n g power o f t h e gel f o r dsRNAs corresponds t o a previous obs e r v a t i o n t h a t conformation changes caused by p o i n t mutations can be e a s i l y d i s t i n g u i s h e d i n dsRNA73a74. T h i s f o l l o w s from t h e f a c t t h a t a more f l e x i b l e con-

368 f o r m a t i o n of ssRNA makes e l e c t r o p h o r e t i c bands d i f f u s i v e , which can a l s o be seen a f t e r t h e s e p a r a t i o n o f complementary s t r a n d s o f denatured DNA. Because, as has a l r e a d y been s t a t e d , t h e m o b i l i t i e s o f n u c l e i c a c i d molecules a r e i n v e r s e l y proportional t o t h e i r sedimentation

coefficient^^^

( F i g . 15.2) and

I

1c ln I

0 X

3

E

1

0.'

I

I

4 Mobility, cm

2

F i g . 15.2. L i n e a r r e l a t i o n s h i p between e l e c t r o p h o r e t i c m o b i l i t i e s nd sedimentat i o n c o e f f i c i e n t s o f c e l l u l a r RNAs o b t a i n e d on p o l y a c r y l a m i d e g e l 78

.

changes i n secondary and t e r t i a r y s t r u c t u r e s c o n s i d e r a b l y a f f e c t t h e m o b i l i t y , attempts were made t o f i n d more s u i t a b l e c o n d i t i o n s f o r a p r e c i s e m o l e c u l a r w e i g h t d e t e r m i n a t i o n , m o s t l y by g e l e l e c t r o p h o r e s i s under d e n a t u r i n g c o n d i t i o n s , which a l s o i n c r e a s e t h e r e s o l v i n g power i n h i g h e r p o l y n u c l e o t i d e s . P o l y a c r y l a m i d e g e l c o n t a i n i n g 7 m o l / l urea76 ensures i d e n t i c a l m o b i l i t i e s o f RNA and DNA molecules and fragments w i t h c h a i n l e n g t h s u p t o 150 n u c l e o t i d e s , b u t l o n g e r p o l y n u c l e o t i d e s a r e n o t c o m p l e t e l y denatured under these c o n d i t i o n s and t h e r e f o r e t h e i r m o b i l i t y , as i n normal g e l s , does n o t depend o n l y on c h a i n l e n g t h . Nevertheless, such d e n a t u r i n g g e l s ( F i g . 15.3) a r e f r e q u e n t l y used, n o t o n l y i n methods o f r a p i d n u c l e i c a c i d sequence a n a l y s i s b u t a l s o f o r f r a c t i o n a t i o n and m o l e c u l a r w e i g h t d e t e r m i n a t i o n o f b o t h t y p e s o f n u c l e i c a c i d s 7 7 , f o r example, fragments a f t e r s i t e s p e c i f i c cleavage o f v i r a l RNA ( S a t e l l i t e tobacco n e c r o s i s v i r u s ) o r 5.55 rRNA ( y e a s t ) by RNase H i n t h e presence o f sequence s p e c i f i c s y n t h e t i c o l i g o d e o x y n u ~ l e o t i d e s ~o ~r ,f o r t h e a n a l y s i s o f f i v e lambda phage s p e c i f i c t r a n s c r i p t s (5-12s) s y n t h e s i z e d i n v i t r o 7 ' .

A d e t a i l e d c h a r a c t e r i z a t i o n o f the tRNA mixtures

from E. c o l i , B. subtilis and B . s t e a r o t h e r m o p h i h s * O Y 8 '

was performed on dena-

369

t u r i n g 6.5% p o l y a c r y l amide-urea gels, where some i s o a c c e p t o r tRNAs c o u l d a1 so be separated.

1000

500

t

s

100

50

20 Relative mobility

Fig. 15.3. R e l a t i v e m o b i l i t i e s o f denatured DNA fragments on a 12% polyacrylamiae gel w i t h 7 m o l / l urea (lower l i n e ) and on a 5% poly7crylamide gel i n 98% formamide (upper l i n e ) used f o r t h e c h a i n l e n g t h c a l i b r a t i o n A = x y l e n e cyanol dye and B = bromophenol blue.

.

Gels c o n t a i n i n g formamide ( p r e f e r a b l y 98%) capable o f d e n a t u r i n g n u c l e i c a c i d s a t room temperature do n o t p e r m i t i d e n t i c a l m o b i l i t i e s o f RNA and DNA molecules o f t h e same l e n g t h 7 7 y 8 2 (RNA m i g r a t e s more r a p i d l y i n g e l s ) , b u t can o f t e n be used t o i s o l a t e and c h a r a c t e r i z e s p e c i f i c mRNAs83-91

.

Denaturing g e l s w i t h u r e a o r formamide a r e a l s o s u i t a b l e f o r t h e s e p a r a t i o n and c h a r a c t e r i z a t i o n o f RNA t r a n s c r i p t s prepared i n v i t r o f o r DNA h y b r i d i z a t i o n analysisg2. To improve t h e d e n a t u r i n g c o n d i t i o n s , one can add 1 m o l / l formalbehyde t o t h e prepared agarose gel c o n t a i n i n g formamide a t a l o w e r c o n c e n t r a t i o n (52%) and denature t h e RNA i n a s i m i l a r m i x t u r e p r i o r t o a p p l i c a t i o n , as has been t e s t e d i n t h e c h a r a c t e r i z a t i o n o f t o t a l RNA i s o l a t e d by t h e new method u s i n g p r o t e i n a s e K and sodium perchlorateg3. To determine t h e molecular weight o f t h e l o n g molecules

o f s i n g l e - s t r a n d e d RNAs, i t i s p o s s i b l e t o use low-percentage agarose g e l s cont a i n i n g methylmercury hydroxideg4, which a1 so denatures t h e samples p r i o r t o e l e c trophoresisg5. T h i s method has f r e q u e n s l y been used f o r t h e f r a c t i o n a t i o n o f mpNA,

370 where subsequent b l o t t i n g and h y b r i d i z a t i o n on DBM-paper i s a n t i c i p a t e d g 6 . Den a t u r i n g p o l y a c r y l a m i d e g e l w i t h t h e d e t e r g e n t sodium dodecyl s u l p h a t e (SDS) added has i n t e r e s t i n g l y been used f o r k i n e t i c s t u d i e s w i t h ppp(A2 ' p)nA-dependent RNaseg7, which e v i d e n t l y p l a y s a r o l e i n t h e mechanism o f a c t i o n o f i n t e r f e r o n . Whereas t h e e l e c t r o p h o r e t i c m o b i l i t y o f samples serves m a i n l y t o d e t e r m i n e p r e c i s e l y t h e m o l e c u l a r weight, i t i s p o s s i b l e t o s u b s t i t u t e t h e c o n t i n u a l presence o f d e n a t u r a n t s i n t h e g e l f o r an i r r e v e r s i b l e d e n a t u r a t i o n o f n u c l e i c a c i d molecules b e f o r e running, and t h u s m a i n t a i n t h e e l e c t r o p h o r e t i c m o b i l i t y as a s i r r p l e f u n c t i o n o f t h e p o l y n u c l e o t i d e l e n g t h , g e l c o m p o s i t i o n and v o l t a g e a p p l i e d . I n such i n s t a n c e s , p o l y a c r y l a m i d e and agarose gels, m o s t l y i n b u f f e r s o f low i o n i c s t r e n g t h (0.01 m o l / l ) , which h e l p t o m a i n t a i n c o m p l e t e l y denatured molecules i n an extended c o n f o r m a t i o n 28,42,98*99,

can be used f o r e l e c t r o p h o r e t i c separa-

t i o n s . W i t h b o t h t y p e s o f n u c l e i c a c i d s , 1 m o l / l g l y o x a l i n 50% d i m e t h y l s u l p h o x i d e ( t h e l a t t e r i s n o t necessary) c a n be used f o r d e n a t u r a t i o n . G l y o x a l can r e a c t r e v e r s i b l y w i t h a l l bases o f n u c l e i c a c i d s , b u t t h e r e a c t i o n w i t h guanine a t pH 7 i s i r r e v e r s i b l e . Electrophoresis c a r r i e d o u t immediately a f t e r t h e r e a c t i o n should be r u n a t n e u t r a l pH and w i t h r e c i r c u l a t i o n o f f r e q u e n t l y changed b u f f e r which i s exhausted i n t h e d i l u t e d s t a t e , D i s i n t e g r a t i o n o f t h e g u a n i n e - g l y o x a l adduct a t pH 8 and subsequent d i s i n t e g r a t i o n o f g l y o x a l p e r m i t t h e r e c o v e r y o f RNA i n t h e b i o l o g i c a l l y a c t i v e f o r m o r capable o f m o l e c u l a r h y b r i d i z a t i o n 1 8 . Table 15.3 TABLE 15.3 GELS USED FOR MOLECULAR WEIGHT DETERMINATIONS Data f r o m r e f s . 28 and 42. Gel

6% A c r y l ami de 2.5% Composite 2.5% Composite 2.5% Composite 2.0% Compostie 1.75% Composite 1.5% Composite 1.5% Agarose 1.0% Agarose

Running c o n d i t i o n s Time (min)

Voltage (V 1

195 105 180 300 300 180 115 120 120

100 130 130 130 130 140 130 100 100

Approximate l ai ln e a r r a n g e a a a (Md) 0.025-0.20 0.025-0.20 0.20-0.70 0.50-1.4 0.50-1.75 0.50-1.75 0.20-1.0 0.20-1.0 0.40-1.75

a W i t h i n t h i s l i n e a r range, t h e r e l a t i v e m o b i l i t i e s o f g l y o x a l - d e n a t u r e d n u c l e i c b a c i d s a r e d i r e c t l y p r o p o r t i o n a l t o t h e l o g a r i t h m s o f t h e i r m o l e c u l a r weights. Always c o n t a i n i n g 0.5% agarose.

371 shows examples o f e l e c t r o p h o r e t i c c o n d i t i o n s s u i t a b l e f o r molecular weight d e t e r minations, w i t h i n which t h e r e i s a l i n e a r r e l a t i o n s h i p w i t h t h e m o b i l i t y i n a given arrangement ( i n a semi-logarithmic p l o t ) . A 2.5% composite gel was used w i t h success f o r t h e i d e n t i f i c a t i o n , c h a r a c t e r i z a t i o n and mapping o f t h e products of n u c l e a r RNA processing from e a r l y and l a t e adenovirus-infected c e l l s 100,101 as w e l l as l a t e adenovirus cytoplasmic mRNA 102,103 and o t h e r ~ R N A s ~S~i m. i l a r l y , a composite gel (1.75% acrylamide and 0.7% agarose) has been used f o r t h e separat i o n o f i n v i t r o synthesized RNA t r a n s c r i p t s d u r i n g t h e study o f a s p e c i f i c u t i l i z a t i o n o f promoter s i t e s i n B. subtiZis phage d29 DNA by d i f f e r e n t b a c t e r i a l RNA polymerases 104

.

For t h e f r a c t i o n a t i o n o f complex n u c l e i c a c i d m i x t u r e s and t h e i r fragments w i t h a wide molecular weight range, an e l e c t r o p h o r e t i c technique has been developed, u s i n g a g r a d i e n t gel as a support i n which t h e gel c o n c e n t r a t i o n and thus i t s molecular s i e v i n g p r o p e r t i e s c o n t i n u a l l y change 29y105;

t h e l a t t e r i n homogeneous

gels w i t h one c o n c e n t r a t i o n cause a non-1 i n e a r r e l a t i o n s h i p between t h e m o b i l i t y and l o g Mu i n fragments and molecules which cannot e n t e r t h e gel w i t h one pore s i z e because o f a h i g h molecular weight, The use o f a g r a d i e n t gel-making device (analogous t o t h e p r e p a r a t i o n o f sucrose d e n s i t y g r a d i e n t s ) and t h e necessary a d a p t a t i o n o f t h e e l e c t r o p h o r e t i c apparatus f o r v e r t i c a l separation should f u l f i l t h e c o n d i t i o n s o f c o r r e c t m i x i n g o f t h e g r a d i e n t components, f i l l i n g o f t h e space f o r g e l f o r m a t i o n from t h e bottom and a s u f f i c i e n t v e l o c i t y o f f i l l i n g , which must be f i n i s h e d b e f o r e p o l y m e r i z a t i o n begins4'.

Apart from t h e s e p a r a t i n g g r a d i -

e n t polyacrylamide gel, t h e s t a r t i n g gel i s added i n t h e v i c i n i t y o f t h e cathode space (near t h e upper p a r t o f t h e g r a d i e n t ) , which p e r m i t s t h e c o n t i n u a l e n t r y o f t h e sample i n t o t h e gel and produces t h e c o n c e n t r a t i o n e f f e c t i n t h e f r a c t i o n ated m i x t u r e a t t h e i n t e r f a c e o f t h e s t a r t i n g and s e p a r a t i n g gels. Table 15.4 l i s t s t h e compositions o f t h e most f r e q u e n t l y used f r a d i e n t gels, o f which t h e 3.5-7.5%

c o n c e n t r a t i o n gel i s g e n e r a l l y used f o r t h e f r a c t i o n a t i o n o f uncharacter-

i z e d m i x t u r e s o f molecules. The i m p o r t a n t c o n d i t i o n s f o r g r a d i e n t p r e p a r a t i o n a r e t h e g e l a t i o n ( p o l y m e r i z a t i o n ) from t o p o f t h e g r a d i e n t ( f r o m t h e l o w e s t concent r a t i o n t h a t does n o t mix t h e g r a d i e n t s d u r i n g exothermic polymerization)- and an a p p r o p r i a t e r a t i o o f t h e monomer and c r o s s - l i n k e r c o n c e n t r a t i o n s i n a l l regions o f t h e c o n c e n t r a t i o n g r a d i e n t , which i s necessary f o r t h e p h y s i c a l p r o p e r t i e s o f t h e gel. The r a t i o o f t h e acrylamide and b i s a c r y l a m i d e c o n c e n t r a t i o n s should cor41,106 respond t o t h e r e l a t i o n s h i p Bisacrylamide conc. ( % ) =

acrylamide conc. ( % )

*

0.003

which i s a p p r o p r i a t e f o r t h e c o n c e n t r a t i o n range o f 2-40% acrylamide gels. A s u i t a b l e s t a b i l i z i n g element i n t h e p r e p a r a t i o n o f g r a d i e n t g e l s i s t h e s i m u l t a neous f o r m a t i o n o f a sucrose d e n s i t y g r a d i e n t ( m o s t l y 10-20%) which minimizes

TABLE 15.4 COMPOSITION OF POLYACRYLAMIDE CONCENTRATION GRADIENT GELS Data f r o m r e f . 41. Component

(%I Acrylamide Bisacrylamide Sucrose Ammonium p e r s u l phate TEMED~

Separating gel mixturea

Starting gel C

B

A

B

A

B

A

3.5 0.17 10

7.5 0.10 20

5.0 0.11 10 0.027 0.040

10.0 0.10 20

10.0 0.10 10

20.0 0.07 18.3

0.056

0.017

0.013

0.027

0.010

2.5 0.12

-

0.10

;The s o l u t i o n s i n c o r r e s p o n d i n g v e s s e l s o f t h e g r a d i e n t m i x e r (A i s always i n t h e m i x i n g v e s s e l ) . N, N, N' ,N ' -Tetramethyl e t h y l enediami ne.

373 v e r t i c a l m i x i n g and d i f f u s i o n i n t h e g r a d i e n t . A l t h o u g h t h e g r a d i e n t g e l s a r e c h i e f l y used f o r t h e f r a c t i o n a t i o n o f complex DNA f r a g m e n t m i x t u r e s a f t e r r e s t r i c t i o n , t h i s method can a l s o be a p p l i e d t o RNA; a good example i s t h e s e p a r a t i o n o f HlqA molecules i n t h e m o l e c u l a r w e i g h t range f r o m 2.5

DNA r e s t r i c t i o n fragment

*

lo4

lo7

to

5'

3'

3'

5'

mRNA ( a f t e r splicing of pre-mRNA w i t h one intron )

5'

3

.

2

I

-

3' High formamide hybridization

5' 3'

5'

'I

20 3'-

3'-

( r e f s . 29, 105).

20

5'-3'

5'

3' 5'

+

51 nuclease digestion

40

5'

40 3'

Gel electrophoresis RNA mapping

60

-

40

-

20

-

One- dimensional gel

Two-dimensional gel

-

F i g . 15.4. Scheme o f nuclease S1 g e l e l e c t r o p h o r e s i s mRNA mappinglo8 i l l u s t r a t i n g s t e p s o f DNA-RNA h y b r i d i z a t i o n , n u c l e a s e S1 d i g e s t i o n and one- o r two-dimensional e l e c t r o p h o r e s e s under n e u t r a l (N) and a1 k a l i n e (A) c o n d i t i o n s . Two-dimensional s e p a r a t i o n i n a g e l o f t h e same c o n c e n t r a t i o n b u t under d i f f e r e n t c o n d i t i o n s ( n e u t r a l and a l k a l i n e pH), which r e q u i r e s no t e c h n i c a l a d j u s t ment o f t h e apparatus, has been used f o r t h e c h a r a c t e r i z a t i o n o f s p e c i f i c RNA t r a n s c r i p t s o f v i r a l and c e l l u l a r DNA, e s p e c i a l l y f o r t h e c o n s t r u c t i o n o f t r a n s c r i p t i o n maps and t h e l o c a l i z a ' i i o n o f s p l i c i n g p o i n t s by two-dimensional

nuclease

S1 g e l rnappinglo6-lo8. The p r i n c i p l e s o f t h e method a r e shown i n F i g . 15.4;

the

method a l l o w s t h e r a p i d and unequivocal demonstration, a c c o r d i n g t o t h e charac-

374

t e r i s t i c l o c a l i z a t i o n o f t h e s p o t s on o r under t h e main diagonal o f t h e t w o - d i mensional electropherogram, o f whether t h e RNA t r a n s c r i p t (mRNA,

f o r example,

f o r g l o b i n ) o f a p u r i f i e d DNA i s s p l i c e d .

C+AE1

C+A= 2

U+G

U+G

F i g . 15.5. Diagram o f an o l i g o n u c l e o t i d e s e p a r a t i o n a c c o r d i n g t o t h e i r c h a i n l e n g t h and base c o m p o s i t i o n u s i n two-dimensional e l e c t r o p h o r e s i s w i t h d i f f e r e n t g e l c o n c e n t r a t i o n s and pH values

%.

The t e c h n i c a l l y more d i f f i c u l t two-dimensional

separation a t d i f f e r e n t gel

c o n c e n t r a t i o n s [ f o r example, (1) 10% PAG w i t h 6 m o l / l u r e a a t pH 3.5,

and ( 2 )

20% PAG a t pH 81 was used f o r t h e f r a c t i o n a t i o n o f a complex p o l y n u c l e o t i d e mixt u r e a f t e r h y d r o l y s i s o f RNAs o f d i f f e r e n t o r i g i n s , f r a c t i o n a t i o n a c c o r d i n g t o c o m p o s i t i o n o f bases

r a t i o [(C+A)/(U+G)] and number o f n u c l e o t i d e s i n fragments ( n = 20-50), see F i g . 15.5 ( r e f . 6). A m o d i f i c a t i o n o f t h i s f r a c t i o n a t i o n , used f o r d i a g n o s t i c purposes ( p o l i o RNA), was a d j u s t e d f o r t h e q u a n t i t a t i v e evaluat i o n o f t h e fragments u p t o 200 n u c l e o t i d e s l o n g79109 and can g e n e r a l l y be used t o c h a r a c t e r i z e any RNA. A s i m i l a r d e t a i l e d c h a r a c t e r i z a t i o n o f h i g h - m o l e c u l a r w e i g h t RNA o f r e t r o v i r u s e s 110-112,

a f t e r h y d r o l y s i s w i t h RNase T1 (50-80 d i s t i n -

g u i s h a b l e o l i g o n u c l e o t i d e s denoted 5'-32P a f t e r hydrolysis113),

was c a r r i e d o u t

on a m i c r o - s c a l e f o r 200-400 ng o f RNA and p e r m i t t e d sequence a n a l y s i s i n a t o t a l amount o f RNA o f l e s s t h a n 1 p g 112

.

E l e c t r o p h o r e s i s need n o t be used o n l y f o r t h e s e p a r a t i o n o f t h e components o f m i x t u r e s b u t can a l s o be used f o r t h e t r a n s f e r o f t h e m a t e r i a l f r o m one e n v i r o n ment ( g e l ) i n t o a n o t h e r ( f o r example, diazobenzyloxymethyl-paper). The m a t e r i a l o b t a i n e d i n t h i s manner i s n o t contaminated w i t h t h e o r i g i n a l s u p p o r t m a t e r i a l .

375

F o r example, e l e c t r o e l u t i o n i s performed f o r i s o l a t i o n o f s e p a r a t e d n u c l e i c a c i d molecules o r fragments f r o m t h e g e l s 35y47-49, o r general e l e c t r o p h o r e t i c t r a n s f e r o f RNA, DNA and p r o t e i n s f r o m g e l s on t o DBM-paper i n an a t t e m p t t o c h a r a c t e r i z e

-

an a n a l o g y o f t h e b l o t t i n g t e c h n i q u e which i s n o t so e f f e c t i v e . An e v a l u a t i o n o f [ 32 PImRNAs p o p u l a t i o n u s i n g a s i m u l them f u r t h e r b y m o l e c u l a r h y b r i d i z a t i o n

taneous s i z e e s t i m a t i o n o f mRNAs and t h e i r l o c a t i o n on DNA fragments ( p h y s i c a l map) i s p o s s i b l e by " c r i s s - c r o s s " DNA-RNA h y b r i d i z a t i o n o f m a t e r i a l s o f t h e

An e x p l a n a t o r y scheme i s shown i n Fig. 15.6.

s e p a r a t i n g gels115.

poly A - mRNA

A

B

D

285

185

I

I

45

M ,.,

markers

I I

DNA fragments

l

e

t

-

b

185

0

I

t

165

265

135

d

t

I

I

I

305

185

F i g . 15.6. Diagram o f c r i s s - c r o s s h y b r i d i z a t i o n t e c h n i q u e betw mRNAs and DNA r e s t r i c t i o n fragments separated by agarose g e l e l e c t r o p h o r e s i s f f ' . (A) Autoradiogram o f polyA-mRNA g e l w i t h Mu marker RNAs; ( 6 ) separated DNA fragments v i s u a l i z e d by e t h i d i u m bromide s t a i n i n g o f gel p r i t o Southern t r a n s f e r ; (C) a u t o r a d i o graphy r e s u l t i n g f r o m h y b r i d i z a t i o n o f g h P - l a b e l l e d polyA-mRNAs ( i n t h e g e l ) o n t o Southern b l o t o f t h e DNA r e s t r i c t i o n fragment p a t t e r n (on a n i t r o c e l l u l o s e s t r i p ) ; ( D ) i n t e r p r e t a t i o n o f h y b r i d i z e d s p o t p a t t e r n on a p h y s i c a l map o f DNA.

DEOXYRIBONUCLEIC A C I D S B a s i c g e n e t i c m a t e r i a l o f most o f t h e organisms, t h e o n l y f u n c t i o n o f which i s t h e t r a n s c r i p t i o n o f g e n e t i c i n f o r m a t i o n and i t s t r a n s f e r f r o m g e n e r a t i o n t o g e n e r a t i o n whereby r e p r o d u c t i o n and s t a b i l i t y o f t h e species a r e ensured, i s r e p r e s e n t e d c h i e f l y by l o n g p o l y d e o x y r i b o n u c l e o t i d e c h a i n s arranged i n a c i r c u l a r o r l i n e a r d o u b l e - h e l i c a l genome m o l e c u l e ( s e e Table 15.5) composed o f f o u r a l t e r n a t i n g basic b u i l d i n g u n i t s

-

d e o x y r i b o n u c l e o t i d e s . The h i g h m o l e c u l a r w e i g h t and

316

TAELE 15.5 CONTENT 3F NUCLEAR DNA I N CELLS OF DIFFERENT ORGANISMS Data m a i n l y from r e f . 116. ~

~

Organism Phase and v i r u s e s (human papovavirus) BK Simian v i r u s 40 PX 174 ( s i n g l e s t r a n d ) M13 ( s i n g l e s t r a n d ) Lambda T2 B a c t e r i a ( h a p l o i d genome) iiaemophilus influenzae Bacillus s u b t i l i s Escherichia c o l i Eu k a r y o t s Drosophilla melanogaster Sea u r c h i n ( h a p l o i d genome) Xenopus Carp Chic ken Mouse Man

~~

~

No1e c u l a r w e i g h t ( d )

Base p a i r s

lo6 lo6 lo6

4963 5226 5375 6407

1.5 1.6 1.7 2.0 30 130 0.7 1.3 2.6 0.1

-- ll oo 66 *

106

.. lo9 . ll oo 99 10;

28.0 0.5 ;32 2.0 * 1012 1.2 ' 10;; 10 3.0 3.6 lo1'

49

ZOO

.. ll oo 33

1 ' 106 2 10;

4

10

0.2.109 9 0.8*109 45.0*109 3.3*109

2.1 'lo9 4.7*109 6.6.10

t h e immense number o f combinations o f t h e a l t e r n a t i n g b u i l d i n g u n i t s n o t o n l y o f f e r s u f f i c i e n t numbers o f v a r i a n t s f o r c o d i n g a l l t h e p r o p e r t i e s o f p r o k a r y o t i c and e u k a r y o t i c organisms, b u t a l s o t h e y r e p r e s e n t e d f o r a l o n g t i m e an apparent s t r u c t u r a l homogeneity o f t h i s t y p e o f n u c l e i c a c i d s , i t s s t a b i l i t y and i n v a r i a b i l i t y , which stemmed t o a l a r g e e x t e n t from m e t h o d i c a l d i f f i c u l t i e s i n v o l v e d i n i s o l a t i n g and c h a r a c t e r i z i n g t h e DNA molecules o b t a i n e d m o s t l y i n t h e form o f random m i x t u r e s o f fragments o f t h e whole genomes. The concept o f t h e r i g i d i t y and i n v a r i a b i l i t y o f DNA molecules117 has been shaken m a i n l y by d e m o n s t r a t i n g t h e e x i s t e n c e o f v a r i o u s c o n f o r m a t i o n a l v a r i a n t s and t h e i r i n t e r m e d i a t e s i n chromosomal and extrachromosomal DNAs ( r i g h t - and l e f t - h a n d e d d o u b l e h e l i c e s ; d i f f e r e n t d o u b l e - h e l i x conformations; s i n g l e - s t r a n d e d r e g i o n s and genomes; s u p e r h e l i c e s , open c i r c l e s and l i n e a r molecules; b r e a t h i n g c o n f o r m a t i o n i n t e r m e d i a t e s ; DNA-protein r e c o g n i t i o n s i t e s ; e t c . ) ,

t h e existence

o f s p e c i f i c r e s t r i c t i o n m o d i f i c a t i o n systems and d i s c o n t i n u o u s s t r u c t u r e o f e u k a r y o t i c genes ( s p e c i f i c m e t h y l a t i o n , s e q u e n c e - s p e c i f i c cleavage and s p l i c i n g ) and t h e e x i s t e n c e o f moveable g e n e t i c elements ( t r a n s p o s a b l e elements i n maize, i n s e r t i o n o f temperate phages, s i t e - s p e c i f i c recombination, I S and Tn t r a n s p o s i tions, etc.).

A l l o f t h i s shows c o n v i n c i n g l y t h a t DNA p r o p e r t i e s and c o n f o r m a t i o n

377 p l a y a n i n t i m a t e r o l e i n gene r e g u l a t i o n and t h a t DNA does n o t have t h e same s t r u c t u r e and conformation throughout i t s e n t i r e l e n g t h (e.g.,

DNA rearrangements i n

d i f f e r e n t i a t i n g v e r t e b r a t e lymphocytes t o form s p e c i f i c types o f imnunoglobul i n s ) ( r e f . 118). Remarkable progress i n methodical approaches, which p e r m i t t e d t h e r e p r o d u c i b l e i s o l a t i o n , degradation and c h a r a c t e r i z a t i o n o f DNA and a l l o f i t s s t r u c t u r a l tyDes, and c l o n i n g o f t h e s p e c i f i c fragments o f i t s molecules i n c l u d i n g t h e r e g u l a t o r y regions, c o n t r i b u t e d t o changes i n o u r views on t h e s t r u c t u r e and f u n c t i o n o f DNA. An account o f t h e methodical approaches i n c l u d e s gel e l e c t r o p h o r e s i s which, w i t h t h e use o f agarose and polyacrylamide g e l s i n d i f f e r e n t concentrations,

permits

incomparable working p o s s i b i l i t i e s w i t h high- and low-molecular-weight

fragments

w i t h a s u f f i c i e n t r e s o l v i n g power f o r v e r y s i m i l a r components o f complex mixtures. The wide use and s u i t a b i l i t y o f these s e p a r a t i o n techniques i s a l s o confirmed by the f a c t t h a t many manufacturers of commercial enzyme p r e p a r a t i o n s ( r e s t r i c t a s e s ligases120,

etc.)

119

i n c l u d e i n t h e c h a r a c t e r i s t i c s o f t h e i r products p i c t u r e s o f gel

e l e c t r o p h o r e t i c separations o f s p e c i f i c d i g e s t s o f model substrates,

f o r example,

r e s t r i c t i o n p a t t e r n s o f DNA from lambda phage, SV40 o r pBR322 plasmid. Agarose and polyacrylamide gel e l e c t r o p h o r e s i s has been most f r e q u e n t l y used f o r t h e s e p a r a t i o n and c h a r a c t e r i z a t i o n o f DNA i s o l a t e s 121-125

and t h e i r d i g e s t s

a f t e r treatment w i t h v a r i o u s r e s t r i c t i o n endonucleases126, which i s p a r t o f t h e p h y s i c a l mapping of genomes and often a p r e r e q u i s i t e f o r t h e p r e p a r a t i o n o f r e combinant DNA molecules. The p r e p a r a t i o n o f g e l s and t h e uses o f many types o f e l e c t r o p h o r e t i c apparatus 39a127-129

do n o t d i f f e r from t h e c o n d i t i o n s d e s c r i b e d

i n t h e s e c t i o n on t h e RNA, b u t agarose i s more w i d e l y used w i t h DNA because o f t h e h i g h e r molecular weight and r e s i s t a n c e t o a l k a l i s 13,35Y130,131. Table 15.6

TABLE 15.6 BUFFERS FOR AGAROSE GEL ELECTROPHORESIS According t o Cold S p r i n g Harbor

B u f f e r s a r e u s u a l l y prepared as ten-

times concentrated s t o c k s o l u t i o n s . Component and p r o p e r t i e s

Tris-borate

Tris-phosphate

Tris-acetate

A1 k a l i n e

T r i s-OH Boric acid H PO4 EdTA (Na2) PH Ca pac it y

89 mmol/l 89 mmol/l

89 mmol/l

40 mmol/l

-

-

2.5 mmol/l 8.3 High

-

23 mmol/l 2.5 mmol-/l 8.3 High

-

2 mmol/l 8.1 Low

-2 ml/l

-

Very low

p r e s e n t s a s u r v e y and g i v e s t h e c h a r a c t e r i s t i c s o f t h e most f r e q u e n t l y used b u f f e r s . Bromophenol b l u e and x y l e n e c y a n o l e FF, which, however, decompose a t a n a l k a l i n e pH, a r e t h e most commonly used t r a c k i n g dyes. DNA bands t h a t have t h e same r n i o b i l i t y as t h e t r a c k i n g dyes cannot be observed because o f t h e f l u o r e s c e n c e a d s o r p t i o n f r o m DNA-bound e t h i d i u m bromide, which i s most o f t e n used f o r DNA det e c t i o n . I n many i n s t a n c e s i t i s a p p r o p r i a t e t o a p p l y t h e sample w i t h 5-10% g l y c e r o l o r sucrose t o t h e w e l l f i l l e d w i t h e l e c t r o p h o r e s i s b u f f e r . Recommended cond i t i o n s f o r s e p a r a t i o n a r e l i s t e d i n Table 15.7; t h e general r u l e i s t h a t a b e t t e r TABLE 15.7 SEPARATION OF DNA FRAGMENTS I N AGAROSE GELS I n T r i s - a c e t a t e b u f f e r . Data f r o m r e f . 132. Fragment duplex l e n g t h (bp)

Agarose

(%I

Voltage gradient (v/cm)

150-1000 300-2500 500-4000 700-6000 1000-9000

1.8 1.4 1.0 0.7 0.5

2-3 2-3 1-2 0.5-1 0.5-1

r e s o l u t i o n , e s p e c i a l l y i n l a r g e fragments (more t h a n 70 kb), i s o b t a i n e d a t a l o w v o l t a g e g r a d i e n t (0.5 V/cm). Double-stranded r e s t r i c t i o n fragments o f known l e n g t h o r sequence a r e most o f t e n used as s i z e standards; some o f them a r e shown i n Table 15.8. As c o n f o r m a t i o n and s i z e r e f e r e n c e molecules plasmids may a l s o be used, f o r example, t h o s e i s o l a t e d f r o m a m u l t i p l e p l a s m i d - c o n t a i n i n g E. coZi s t r a i n ( r e f . 133). E l e c t r o p h o r e t i c s e p a r a t i o n can a l s o be performed i n s p e c i a l p r e p a r a t i v e apparatus 479126a1289129w h i c h p e r m i t s t h e s e p a r a t i o n o f l a r g e amounts o f sample; t h e f r a c t i o n s o b t a i n e d c o n t a i n 30-100 pg/ml o f DNA, which corresponds t o t h e amount d e t e c t a b l e i n a spectrophotometer. Agarose e l e c t r o p h o r e s i s has proved t o be a r a p i d method f o r g e n e r a l s c r e e n i n g i n t h e i s o l a t i o n and a n a l y s i s o f extra-chromosomal elements ( p l a s m i d s ) , as has been demonstrated i n Escherichia coZi 122’133’134, BaciZZus subtiZis1359136and s t r e p t o cocci137. Rapid DNA i s o l a t i o n s , t o g e t h e r w i t h e l e c t r o p h o r e t i c a n a l y s i s , have been f u r t h e r proposed f o r o t h e r i m p o r t a n t b i o l o g i c a l sources, p a r t i c u l a r l y c l o n e d DNAs i n phages, b a c t e r i a , y e a s t and t i s s u e c u l t u r e c e l l s , t h e d e c i s i v e c r i t e r i o n b e i n g t h e u s a b i l i t y o f DNA i s o l a t e s f o r enzymatic ( r e s t r i c t i o n ) and h y b r i d i z a t i o n a n a l y s i s 138’139. F i g 15.7 g i v e s a n example o f a s i m p l e f r a c t i o n a t i o n o f phage DNA h y d r o l y s a t e ( f r o m B. subtiZis phages 829, PZA and PZE140-143)a f t e r d i g e s t i o n w i t h

319

TABLE 15.8 SOFIE RESTRICTION FRAGMENTS OF PHAGE LAMBDA AND pBR322 DNAs AS S I Z E STANDARDS FOR GEL ELECTROPHORESIS

Fragment

E F G H I

J

K L M N 0 P

Lambdaa

~BR322~

Eco R I

Hind I 1 1

Eco R I + Hind I11

Taq I

Hinf I

Hae I 1

Alu I

21.8 7.52 5.93 5.54 4.80 3.41

23.7 9.46 6.75 4.26 2.26 1.98 0.58

21.8 5.24 5.05 4.21 3.41 1.98 1.90 1.71 1.32 0.93 0.84 0.58

1444 1307 475 368 315 312 141

1631 517 506 3 96 344 298 221 220 154 75

1876 622 439 430 370 227 181 83 60 53 21

910 659 655 521 403 281 257 226 136 100 63 57 49 19 15 11

r e s t r i c t i o n enzyme E C O R I i~n~ 1% ~ agarose gel. I n a d d i t i o n t o f r a c t i o n a t i o n acc o r d i n g t o t h e molecular weight o f t h e fragments, t h e p o s s i b i l i t y o f a d i r e c t determination o f t h e t e r m i n a l fragments by comparing hydrolysates o f 1 i n e a r and c i r c u l a r DNA i s shown. The c i r c u l a r i z a t i o n o f t h e genome i n these phages r e s u l t s from u n i t i n g t h e DNA molecule ends through a 5 ' - c o v a l e n t l y bound p r o t e i n 1 i n k . This p r o t e i n t h e r e f o r e l i n k s i n t h e enzyme h y d r o l y s a t e o f c i r c u l a r DNA b o t h t e r m i n a l fragments which as a " n u c l e o p r o t e i n " do n o t e n t e r t h e e l e c t r o p h o r e s i s gel and t h e r e f o r e , i n comparison w i t h t h e r e s t r i c t i o n p a t t e r n o f l i n e a r DNA i n t h e f i g u r e , they a r e m i s s i n g ( t h e y remain a t t h e s t a r t i n t h e sample w e l l ) . The s i z e s o f t h e fragments o b t a i n e d were measured u s i n g r e s t r i c t i o n standard DNA fragments from t h e lambda phage and SV40 ( F i g . 15.8)

i n t h e same s l a b gel. Almost

none o f t h e present s t u d i e s f o r c h a r a c t e r i z i n g t h e DNA s t r u c t u r e can o m i t t h e study o f t h e r e s t r i c t i o n p a t t e r n s u s i n g gel e l e c t r o p h o r e s i s i n which a wide spectrum o f s p e c i f i c r e s t r i c t i o n endonucleases (more than 260 enzymes r e p o r t e d a t t h e beginning o f 1981) i s involved. Conversely, t h e search f o r t h e characteri z a t i o n and d e t e r m i n a t i o n o f sequence s p e c i f i c i t y o f t h e new r e s t r i c t a s e s i s a l s o n e c e s s a r i l y based on t h e use o f gel e l e c t r o p h o r e s i s ( i n general r e f s . 119 and 144 and as examples r e f s . 145-148).

The s i z e s o f t h e fragments o b t a i n e d ( t h e

380

o b t a i n e d by EcoRI d i g e s t i o n F i g . 15.7. E l e c t r o p h o r e t i c s e p a r a t i o n o f DNA f r a Fragments i n 1%agarose o f DllAs f r o m B. subtilis phages @29, PZE and PZA ?@;is3 g e l were v i s u a l i z e d by e t h i d i u m bromide s t a i n i n g and photographed i n U V l i g h t . Lane o r d e r ( f r o m t h e l e f t ) : 1, fi29 c i r c u l a r DNA; 2, d29 l i n e a r DNA; 3, PZE c i r c u l a r DNA; 4, PZE l i n e a r DNA; 5, PZA c i r c u l a r DNA; 6, PZA l i n e a r DNA. o f fragments ( f r o m t o p t o bottom) 6.1, 4.3, 3.8, 1.7, 1.0, 0.5 and 0.3 !4d.

.

range o f m o l e c u l a r w e i g h t v a l u e s ) , which a r e g e n e r a l l y a s s o c i a t e d w i t h t h e number o f n u c l e o t i d e s i n t h e t a r g e t sequence r e c o g n i z e d by t h e enzyme (4-7 bp), determines whether agarose o r p o l y a c r y l a m i d e w i l l be used as t h e support, o r whether g r a d i ent4’ o r two-dimensional

s e p a r a t i o n w i l l be a p p l i e d ; t h e l a t t e r p e r m i t s as many

as 1000 fragments t o be d i s t i n g u i s h e d i n t h e m i x t u r e , m o s t l y upon simultaneous use o f t h e g r a d i e n t o f d e n a t u r i n g agent i n t h e g e l .

381

105

L--.+2

Relative

6

mobility, cm

F i g . 15.8. C a l i b r a t i o n curve f o r t h e e s t i m a t i o r 4 8 f molecular weights o f DNA fragments by e l e c t r o p h o r e s i s i n 1%agarose gel DNA r e s t r i c t i o n fragments o f known molecular weight d e r i v e d from lambda, SV40 and 829 DNAs were used.

.

The h i g h p r e c i s i o n and resolv.ing power o f gel e l e c t r o p h o r e s i s i n a l l o f these arrangements were u s e f u l i n many experimental s t u d i e s o f t h e physical mapping o f

DNA o f v a r i o u s o r i g i n s u s i n g complete and p a r t i a l d i g e s t i o n s w i t h r e s t r i c t a s e s , f o r example, c o l i p h a g e 186 ( r e f . 150), B a c i l l u s phages 829, PZA, PZE, 815 ( r e f s . 140-143), and l y t i c and temperate forms o f t h e t a 1 5 1 and 14 ( r e f . 152), lambda phage153, and adenovirus t y p e 6 ( r e f . 154), i n comparative s t u d i e s o f t h e DNA o f r e l a t e d T r y p a n o ~ o m e s ~i ~n ~f ,o l l o w - u p s t u d i e s o f t h e p e r i o d i c i t y o f s a t e l l i t e

D H A s ~ ~i ~n , t h e i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f mitochondria1 DNAs i n c y b r i d s and r e c o n s t i t u t e d cells157 and p l a s t i d DNAs f o r gene m a n i p u l a t i o n i n plants158 and,

i n t h i s connection a1 so, i n t h e preparation, c h a r a c t e r i z a t i o n and

uses o f t h e vectors, vector-host systems and combinant DNA molecules i n gene engineering ( i n general r e f s . 159 and 160 and as examples r e f s . 161-167). For example, a c h a r a c t e r i z a t i o n o f cDNAs synthesized on t h e poly-A-mRNA p o p u l a t i o n and used f o r t h e c o n s t r u c t i o n o f a recombinant cDNA l i b r a r y r e p r e s e n t a t i v e o f t h e normal human lymphocytes has been c a r r i e d o u t w i t h t h e gel e l e c t r o p h o r e s i s on 1%agarose168.

Criss-cross DNA-RNA h y b r i d i z a t i o n , mentioned i n t h e RFIA sec-

t i o n (Fig. 15.6),

was o r i g i n a l l y developed t o f a c i l i t a t e t h e r a p i d c o n s t r u c t i o n

o f physical 1 inkage maps o f DNA fragments generated by r e s t r i c t i o n

endonucleases

( r e f s 169 and 170). Zone e l e c t r o p h o r e s i s i n agarose i s n o t o n l y a s u i t a b l e and important method

382 f o r t h e s e p a r a t i o n o f n u c l e i c a c i d fragments of v a r i o u s s i z e s b u t i s a l s o a method f o r d i s t i n g u i s h i n g t h e m a j o r c o n f o r m a t i o n isomers ( f o r m s 1-111) o f double-he1 i c a l DNA molecules and f o r t h e s e p a r a t i o n o f t h e complementary s t r a n d s a f t e r a1 k a l i n e denaturation13a35a133. T h i s has been confirmed, f o r example, i n d e t a i l e d s t u d i e s o f t h e c o n f o r m a t i o n forms c o n t a i n e d i n one sample

-

closed c i r c u l a r ( I ) , nicked 6 10 ), 0x174 r e p l i c a t i v e

c i r c u l a r (11) and l i n e a r (111) o f pM2 phage DNA (Mu 6.62 DNA (M, 3.51

-

l o 6 ) and r a t m i t o c h o n d r i a 1 DNA (Mu 10.6

dx

rnt

174 PM2 DNA

@X

@X

rnt

174 PM2 DNA

-

1 0 ~ ) ~ ~F i g, . ~ 15.9 ~ .

mt

174 PM2 DNA

U I

m

f U I

m

4.93 1.0%V lgel crn

0.6V. gel 4.93 Vlcrn

1.0 % gel 4.93 V / c m

t 2 5 r n M NaCl

F i g . 15.9. Diagrams o f e l e c t r o p h o r e t i c s e p a r a t i o n s o f DNA c o n f o r m a t i o n a l isomers as a f u n c t i o n o f agarose g e l c o n c e n t r a t i o n and i o n i c s t r e n g t h 1 3 . ( I ) Form I ( s u p e r c o i l e d c i r c l e s ) ; (11) f o r m I1 (open c i r c l e s o f n i c k e d m o l e c u l e s ) ; (111) ( l i n e a r m o l e c u l e s ) o f DNAs f r o m phages 0174 ( a r e p l i c a t i v e form) and PE12 and f r o m r a t l i v e r m i t o c h o n d r i a (mtDNA). summarizes t h e r e s u l t s o f t h e i r e l e c t r o p h o r e t i c s e p a r a t i o n as a f u n c t i o n o f g e l c o n c e n t r a t i o n , v o l t a g e g r a d i e n t and i o n i c s t r e n g t h , which a l l o w t h e o p t i m i z a t i o n o f t h e s e p a r a t i o n o f d o u b l e - h e l i c a l DNA w i t h t h e f o l l o w i n g c o n c l u s i o n s : DNA forms

I and I1 o f t h e same m o l e c u l a r w e i g h t can be s e p a r a t e d under any o f t h e c o n d i t i o n s used; and a l t e r a t i o n s i n v o l t a g e g r a d i e n t and i o n i c c o n d i t i o n s l e a d t o p r e d i c t a b l e changes i n t h e m o b i l i t i e s o f t h e d i f f e r e n t DNA forms, so t h e c o n d i t i o n s under which a l l t h r e e forms a r e separated can be found. A s i m i l a r s e p a r a t i o n o f t h e conf o r m a t i o n forms o f plasmid DNA PUB110 from B . subtiZis i n 1%agarose i s shown i n F i g . 15.10.

The a1 k a l i n e d e n a t u r a t i o n makes p o s s i b l e t h e s e p a r a t i o n o f complemen-

t a r y s t r a n d s i n t h e l i n e a r and n i c k e d c i r c u l a r DNA forms o f t h e above-mentioned phages, w h i l e t h e c l o s e d c i r z u l a r DNA i s s t a b l e 1 3 . The m o b i l i t y o f a l l o f t h e DF!A forms s t u d i e d can be a d e q u a t e l y i n f l u e n c e d by an i n c r e a s e d c o n c e n t r a t i o n o f e t h i dium bromide i n t h e gel. A s i m i l a r r e s o l v i n g power f o r c o n f o r m a t i o n isomers has a l s o been u t i l i z e d f o r t h e s t u d y o f c a t e n a t i o n and d e c a t e n a t i o n c a t a l y s e d by DNA

383

Fig. 15.10. E l e c t r o p h o r e t i c separation o f plasmid PUB110 conformational isomers i s o l a t e d from B. subtiZis i n 1%agarose gel. Lane order ( f r o m l e f t t o r i g h t ) : PUB110 DNA; and recombinant PUB110 DNA c o n t a i n i n g a phage DNA i n s e r t , DNA conformation forms (from t o p t o bottom): open c i r c l e s , l i n e a r molecules and superc o i l e d c i r c l e s . DNA was v i s u a l i z e d by ethidium bromide s t a i n i n g and photographed i n UV l i g h t . topoisomerases under d i f f e r e n t

condition^'^-^^.

The changes i n t h e m o b i l i t y o f

d i f f e r e n t conformation s t a t e s o f DNA served t o determine t h e s t r a n d breaks i n supercoiled DNA, f o r example, upon treatment w i t h a n i c k i n g - c l o s i n g enzyme171 on

PF12 DNA, a nuclease TT1 from Thermus t h e r m o p h i Z ~ s ~on ~ *gX174 DNA and an endonuclease V on UV-irradiated c o l E l DNA173,

o r i n t e s t i n g nuclease i m p u r i t i e s / c o n 174 taniination i n commercial samples o f E. coZi a1 kal i n e phosphatase

.

SEQUENCE ANALYSIS OF NUCLEIC ACIDS The determination o f t h e f i r s t complete n u c l e o t i d e sequence i n t h e polynu

-

c l e o t i d e chain o f n u c l e i c acids has been connected w i t h t h e a n a l y s i s o f tP.NA?h’&

384 (ref. 175) , through m o b i l i z a t i o n o f endo- and e x o n u c l e o l y t i c cleavage and t h e c l a s s i c a l chromatographic s e p a r a t i o n techniques and, i n p a r t i c u l a r , t h e i r q u a n t i t a t i v e development. An almost r o u t i n e a n a l y s i s o f n u c l e o t i d e sequences i n tRNAs and rRlvXs 176-178 was then p e r m i t t e d by a p p l y i n g a new two-dimensional ( f i n g e r p r i n t ) paper e l e c t r o p h o r e s i s technique2 and a combination o f paper e l e c t r o p h o r e s i s and homochromatography21a22 f o r t h e separation and f u r t h e r a n a l y s i s o f o l i g o n u c l e o t i d e s l a b e l l e d w i t h 32P i n v i v o o r a f t e r enzymic d i g e s t i o n o f n u c l e i c a c i d s i n v i t r o u s i n g a p o l y n u c l e o t i d e kinase 113a179.

S u b s t a n t i a l f u r t h e r progress i n

sequence a n a l y s i s was made by t r a n s f e r r i n g t h e f i n g e r p r i n t technique from t h e paper support t o t h e gel 6a7a112, where t h e h i g h r e s o l v i n g power c o n t r i b u t e d n o t o n l y t o t h e a n a l y s i s and screening o f complex m i x t u r e s o f fragments ( i n s p e c i f i c enzyme h y d r o l y s a t e s ) b u t a l s o t o t h e i s o l a t i o n o f pure m a t e r i a l f o r o l i g o n u c l e o t i d e sequencing. F i n a l l y , t h e d i s c o v e r y o f t h e so f a r most s p e c i f i c endonucleases

-

r e s t r i c t a s e s f o r r e p r o d u c i b l e DNA degradation 119a180, t o g e t h e r w i t h t h e a b i l i t y of t h i n d e n a t u r i n g polyacrylamide g e l s t o separate t h e complementary DNA strands i n e l e c t r o p h o r e s i s and t o d i s c e r n o l i g o - and p o l y n u c l e o t i d e s w i t h t h e p r e c i s i o n o f one n u c l e o t i d e d i f f e r e n c e 10-12a181, l e d t o s o - c a l l e d r a p i d DNA sequencing methods. These methods u t i l i z e base-specific chemical cleavage approaches ( r e f s . 11, 12, 182-184) f o r s p e c i f i c processing o f 32P-end l a b e l l e d fragments, l i m i t e d synthesis o f DNA i n v i t r o f o r t h e p r e p a r a t i o n o f analysable 32P-labelled fragments by DNA polymerases under c o n d i t i o n s o f l i m i t e d supply o f deoxynucleosidetri phosphates ( p l u s one o f f o u r needed o r minus one o f f o u r i n f o u r p a r a l l e l r e a c t i o n s ) , c a l l e d t h e "plus-minus" method8, o r i n t h e presence o f a n u c l e o t i d e - s p e c i f i c chain t e r m i n a t o r s ( 2 ' ,3'-dideoxynucleosidetriphosphates p a r a l l e l r e a c t i o n s ) , c a l l e d t h e c h a i n t e r m i n a t i o n method

-

one o f f o u r i n f o u r 9,185-187

A l l o f t h e above methods, t h e p r i n c i p l e s o f which a r e represented schematicala r e based d i r e c t l y on o r associated v e r y c l o s e l y w i t h modern

l y i n Fig. 15.11,

methods o f DNA m a n i p u l a t i o n and c l o n i n g 159~160~188~189, e s p e c i a l l y t h e l a s t one 190-192 o f Sanger e t a1.' i n combination w i t h DNA c l o n i n g i n M13 phage v e c t o r s and thus were m u t u a l l y and w i t h f u r t h e r experimental m o d i f i c a t i o n s used f o r comphage bX174 ( r e f s . 193, 194), p l e t e sequence e l u c i d a t i o n s o f many DNAs, e.g., G4 ( r e f . 195), f d ( r e f . 196), M13 ( r e f . 197), plasmid pBR322 ( r e f . 198), simian 201 v i r u s 40 ( r e f . 199), human papovavirus BK ( r e f . 2 0 0 ) and human mitochondria The p r i n c i p l e s o f chemical sequence a n a l y s i s " i n a m o d i f i e d form were a l s o used f o r RNA sequencing182, as i n instances where chemical agents were s u b s t i t u t e d f o r RNases 202-204, mostly w i t h a s i n g l e base s p e c i f i c i t y (RNase T1 f o r C, RNase U2 f o r A, Rliase A f o r U and C, and RNase PhyI from P h y s a m poZycephaZwn f o r A, G and U, o r a f t e r a m o d i f i c a t i o n o f C residues w i t h a methoxyamine-bisulphite

.

w i t h RNase T2 i n s t e a d o f RNase PhyI). An analogous a p p l i c a t i o n i n t h e RNA area i s p o s s i b l e f o r t h e "plus-minus" method i f a r e v e r s e t r a n s c r i p t a s e i s used t o

385 C f f i C T A C C A A C T 5'

3,-

Template DNA

Primer ( a restriction P \ \fragment ' ] / 1 DNA Polymerase

-ddATP

'qP8

+@'

Short time

GTP+ddGTP GTP GTP CTP CTPcddCTP CTP TTPtddTTP TTP TTP TTP

t

J

-CCGGTAGCAACTX -GGCC2TCGTTGA -GGCC$TCGTTC

4;;

Long

time

I 1

-CCGGTAGCAACT-

I

X

X

-GGCCeTCGTddT -GGCC2TCGddT - GGCCAddT

-GGCC$TCGTT -GGCC$TCGT -GGCCr$TCG -GGCC2TC

I t

I I

I

I

I

I I

I

I

I

I

I

Primers sdecificiollv cleaved o f f '

i

-GGCCeT

CGTCGTddT CC$CGddT CC$TCGTTddG

-GGCCA

CCAddT

-1

' /

CCATCddG

-GG + CCATCGTTCA\ CCATCGTTG\

I

I

I

I

\

cyTddc C&TcTCGTTGddA

A

CCATCGT, \ '\ CCATCG \ \ \\, CCATC '\ \ \ \ CCAT\ \ \ CCA \\'\ \ \ \

\ \

'

G

T

Polyocrylomide gel electrophoresis under denaturing condition

G

\ \

C T

\

F i g . 15.11. Diagrammatic i l l u s t r a t i o n o f DNA sequence a n a l y s i s by primed s ntheses under i n h i b i t i o n w i t h dideoxynucl e o s i d e t r i p h o s p h a t e as a c h a i n t e r m i n a t o r 1 9 7 . A c r o s s above t h e l e t t e r i n d i c a t e s 3 2 P - l a b e l l e d component o r p r e c u r s o r . T, G, C an6 A r e p r e s e n t diagrams o f e l e c t r o p h o r e t i c l a n e s f o r t h e s e p a r a t i o n o f o l i g o n u c l e o t i d e m i x t u r e o b t a i n e d w i t h c o r r e s p o n d i n g dideoxynucl eoside t r i p h o s p h s t e (ddTTP, ddGTP, ddCTP and ddATP) as t h e c h a i n t e r m i n a t o r . Analysed sequence i s t h e n r e a d a t t h e r i g h t s i d e o f t h e e l e c t r o p h o r e t i c diagrams s t a r t i n g f r o m t h e b o t t o m upwards (TCGTTGA).

mRNA

AAAAAA

TTTTT

+

cDNA

a'

b'

c'

d'

e'

CAP

5'

- - - --3'

ac

d

- -3'

F i g . 15.12. Diagram showing a s t e p w i s e sequence a n a l y s i s o f a e u k a r y o t i c mRNA t h r o u g h B i p e d cDNA syntheses u s i n g p r i m e r s complementary t o s p e c i f i c r e g i o n o f t h e mRNA

.

TABLE 15.9 GELS USED FOR SEQUENCE ANALYSIS OF NUCLEIC ACIDS

Components and conditions

Maxam-Gil b e r t 11,12 sequence a n a l y s i s

A c r y l ami de B i s a c r y l ami de Urea B u f fe r a

20% 0.067% 7 mol/l 50 mmol/l T r i s - b o r a t e pH 8.3, 1 mmol/l EDTA 0.07% 0.00007-0.0003 volume 0.15 x 20 x 40 cm 1 h, 1000 V 800-1OOOV

Ammonium p e r s u l phate TEMED Gel s i z e s Pre-electrophoresis Voltage Current T r a c k i n g dyes : Bromophenol blue, 0.05% Xylene cyanol , 0.05%

10 NT l e n g t h equiv. 28 NT l e n g t h equiv.

aThis i s a l s o t h e b u f f e r used i n t h e e l e c t r o p h o r e s i s r e s e r v o i r s .

10

Sanger-Coul son sequence a n a l y s i s

Maxam-Gil b e r t 11,12 strand separation

6% o r 8% 0.37% o r 0.42% 7 mol/l 89 mmol/l T r i s - b o r a t e pH 8.3, 2.5 m o l / l EDTA 0.05% 0.001 volume 0.035 x 20 x 40 cm

8% 0.27%

-

-

50 w o l / l T r i s - b o r a t e pH 8.3, 1 mnol/l EDTA 0.08% 0.00007-0.0003 volume 0.2 x 18 x 18 cm

-

1300-1700 V 30 mA

150 V

30 NT l e n g t h equiv, 80 NT l e n g t h equiv.

25 NT l e n g t h equiv. 105 NT l e n g t h equiv.

387

Fig. 15.13, Autoradiograms as examples o f sequence a n a l y s i s o f plasmid pBR322 and phage PZA DNA fragments u s i n g t h e Sanger dideoxy c h a i n - t e r m i n a t i n g method ( l e f t ) and Maxam-Gil b e r t chemical method ( r i g h t ) .

388

s y n t h e s i z e cDNA on a p p r o p r i a t e mRNA [ i n t h i s i n s t a n c e f o r g l o b i n , ovalbumin and immunoglobulin w i t h an o l i g o ( d T ) 1 0 as t h e p r i m e r ] . T h i s enzyme i s u t i l i z e d t o keep s y n t h e s i z i n g t h r o u g h o u t t h e "minus" system too205. U t i l i z a t i o n o f t h e t e r 9 n i i n a t i o n method f o r RNA sequencing i s a l s o based on s p e c i f i c t e r m i n a t i o n w i t h t r i p h o s p h a t e s analogues i n cDNA o r cRNA s y n t h e s i s , c a t a l y s e d a g a i n w i t h t h e r e v e r s e t r a n s c r i p t a s e 206-208 o r w i t h

Q B RNA r e p l i c a ~ e ~ ' ~Using , r e s t r i c t i o n frag-

ments o f t h e p a r e n t a l DNA as p r i m e r s f o r t h e r e v e r s e t r a n s c r i p t i o n o f even a l o n g mRNA, i t i s p o s s i b l e by s e q u e n t i a l b i n d i n g o f t h e s e p r i m e r s t o mRNA ( F i g . 15.12)

t o sequence t h e whole mRNA m o l e c u l e by t h e s e methods210. Any cDNA p r o d u c t s can thus be sequenced, f o r example, by t h e Maxam-Gilbert chemical method and t h e 211-217

o r i g i n a l mRNA molecule would be d e r i v e d f r o m o v e r l a p p i n g r e g i o n s

The p r a c t i c a l a p p l i c a t i o n o f a l l o f t h e aforementioned sequence methods and approaches i s c o m p l e t e l y dependent on t h e f i n a l f r a c t i o n a t i o n o f t h e r e a c t i o n m i x t u r e s b y e l e c t r o p h o r e s i s i n t h i n p o l y a c r y l a m i d e g e l (0.03-0.15

cm), which

ensures f i n a l r e a d a b i l i t y o f analysed sequence. The c o m p o s i t i o n and p r o c e s s i n g of sequence g e l s , prepared a c c o r d i n g t o t h e b a s i c p r e s c r i p t i o n s o f Maxam and G i l b e r t 1 l Y l 2 o r Sanger and Coulson p r a c t i c e , a r e summarized i n T a b l e 15.9. mentary DNA strands",

which a r e most w i d e l y used i n sequence C o n d i t i o n s f o r t h e s e p a r a t i o n o f comple-

which a r e g e n e r a l l y a p p l i c a b l e , a r e a l s o shown. Examples

o f b o t h sequencing g e l s used i n analyses o f DNA p o r t i o n s o f B. subtiZis phage PZP. a r e p r e s e n t e d i n F i g . 15.13.

Methods o f d i r e c t sequence r e a d i n g a r e most

a p p r o p r i a t e f o r DNA and t h o s e RNAs which do n o t c o n t a i n m o d i f i e d n u c l e o s i d e s i n which m o d i f i c a t i o n can d i s t u r b t h e r e g u l a r i t y and t h u s t h e p r e c i s i o n o f r e a d i n g . I n t h e case o f tRNAs o r n u c l e i c a c i d s , w i t h h i g h e r c o n t e n t s o f m o d i f i e d bases, i t i s advantageous t o use s i m u l t a n e o u s l y t h e methods sequencing small o l i g o n u -

c l e o t i d e s , o r s u i t a b l e chromatographic methods 218a219.

Because o f t h e r a p i d ac-

c u m u l a t i o n o f i n f o r m a t i o n on t h e sequence arrangement o f n u c l e i c a c i d s , i t i s h i g h l y d e s i r a b l e t o p r e p a r e and adopt c o m p u t e r - a s s i s t e d methods

w i t h programs 220,221

d i r e c t e d towards t h e m a n i p u l a t i o n and a n a l y s i s o f n u c l e i c a c i d sequences REFERENCES

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187 A.J.H. Smith, Methods EnzymoZ., 65 (1980) 560. 188 Recombinant DNA, Science, 196 (1977) N0.4286 ( a n e n t i r e i s s u e devoted t o recombinant DNA r e s e a r c h ) . 189 Recombinant DNA, Science, 209 (1980) N0.4463 (an e n t i r e i s s u e devoted t o recombinant DNA r e s e a r c h ) . 1% B. Gronenborn and J. Messing, Nature (London), 272 (1978) 375. 191 P.H. S c h r e i e r and R. Cortese, J . MoZ. B i d . , 122 (1979) 169. 192 G. Heidecker, J. Messing and B. Gronenborn, Gene, 10 (1980) 69. 193 F. Sanger, G.M. A i r , B.G. B a r r e l l , N.L. Brown, A.R. Coulson, J.C. Fiddes, C.A. Hutchinson, 111, P.14. Slocombe and M. Smith, Nature (London), 265 (1977) 687. 194 F. Sanger, G.M. A i r , B.G. B a r r e l l , N.L. Brown, A.R. Coulson, J.C. Fiddes, C.A. H u t c h i s o n , 111, P.M. Slocombe, M. Smith, J. Drouin, T. Friedmann and A.J.H. Smith, i n D.T. Denhardt, D. D r e s s l e r and D.S. Ray ( E d i t o r s ) , The SingZeStranded DNA Phages, Cold S p r i n g Harbor L a b o r a t o r y , Cold S p r i n g Harbor, 1978, p. 659. 195 G.N. Godson, B.G. B a r r e l l , R. Staden and J.C. Fiddes, Nature (London), 276 (1978) 236. 196 E. Beck, R. Sommer, E.A. Auerswald, C. Kurz, B. Zink, G. Osterburg, H. S c h a l l e r , K. Sugimoto, H. S u g i s a k i , T. Okamoto and M . Takanami, NucZeic Acids Res., 5 (1978) 4495. 197 P.M.G.F. van Wezenbeck, T.J.M. Hulsebos and J.G.G. Schoenmakers, Gene, 11 (1980) 129. 198 J.G. S u t c l i f f e , NucZeic Acids Res., 5 (1979) 2721. 195 V.B. Reddy, B. Thimmappaya, R. Dhar, K.N. Subramanian, B.S. Zain, J. Pan, P.K. Ghosh, M.L. Celma and S.M. Weisman, Science, 200 (1978) 494. 20G R.A.C. Yang and R. Wu, Science, 206 (1979) 456. 201 S. Anderson, A.T. Bankier, B.G. B a r r e l l , H.H.L. de B r u i j n , A.R. Coulson, J. Drouin, I.C. Eperon, D.P. N i e r l i c h , B.A. Roe, F. Sanger, P.H. S c h r e i e r , A.J.H. Smith, R. Staden and I.G. Young, Nature (London), 290 (1981) 457. 202 H. D o n i s - K e l l e r , H. Maxam and W. G i l b e r t , NucZeic Acids Res., 4 (1977) 2527. 203 A. Simoncsits, G.G. Brownlee, R.S. Brown, J.R. Rubin and H. G u i l l e y , Nature (London), 269 (1977) 833. 204 A.M. Mazo, T.D. Mashkova, T.A. Avdonina, N.S. Ambartsumyan and L.L. K i s e l e v , NucZeic Acids Res. , 7 (1979) 2467. 205 G.G. Brownlee and E.M. C a r t w r i g h t , J . MoZ. BioZ., 114 (1977) 93. 206 D. Zimmern and P. Kaesberg, Proc. Nat. Acad. S c i . U.S., 75 (1978) 4257. 207 P.H. Hamlyn, G.G. Brownlee, C.C. Chang, M.J. G a i t and C. H i l s t e i n , CeZZ, 15 (1978) 1067. 208 D.J. IlcGeoch and N.T. T u r n b u l l , NucZeic Acids Res., 5 (1978) 4007. 209 F.R. Kramer and D.R. ? l i l l s , Proc. Nat. Acad. Sci. U.S., 75 (1978) 5334. 210 P.K. Ghosh, V.B. Reddy, M. P i a t a k , P. L e b o w i t z and S.M. Weissman, Methods EnzymoZ., 65 (1980) 580. Bohl and R. Wu, Nature (London), 265 (1977) 211 J.W. Szostak, 3.1. S t i l e s , C.-P. 61. 212 L. McReynolds, B.W. O ' M a l l e y , A.D. N i s b e t , J.E. F o t h e r g i l l , D. G i v o l , S. F i e l d s , M. Robertson and G.G. Brownlee, Nature (Londonl, 273 (1978) 723. 213 D. Hagenbuchle, M . Santer, J. S t e i t z and R.T. Mans, CeZZ, 13 (1978) 551. 214 P.K. Ghosh, V.B. Reddy, J. Swinscoe, P.V. Choudary, P. L e b o v i t z and S.M. Weissman, J . B i d . Chem., 253 (1978) 3648. 215 P,K. Ghosh, V.B. Reddy, J. Swinscoe, P. L e b o v i t z and S.M. Weissman, J . MoZ. BioZ., 126 (1978) 813. 216 V.B. Reddy, P.K. Ghosh, P. L e b o v i t z and S.M. Weissman, NucZeic Acids Res., 5 (1978) 4195. 217 V.B. Reddy, P.K. Ghosh, P. L e b o v i t z , 11. P i a t a k and S.M. Weissman, J . ViroZ., 30 (1979) 279. 218 S, Z a d r a f i l , i n Z. Deyl, K. Macek and J. JanZk ( E d i t o r s ) , Liquid CoZwnn Chromatography, E l s e v i e r , Amsterdam, 1975, p. 858. 219 K. Randerath, R.C. Gupta and E. Randerath, Methods Enzymol., 65 (1980) 638. 220 T.R. Gingeras and R.J. Roberts, Science, 209 (1980) 1322. 221 C.L. Queen and L.J. Korn, Methods EnzymoZ., 65 (1980) 595.

This Page Intentionally Left Blank

395

Chapter 16 ALKALOIDS

2. DEYL A l k a l o i d s a r e n i t r o g e n o u s bases o f p l a n t o r i g i n and i n c l u d e a l m o s t a l l o f t h e known n i t r o g e n - c o n t a i n i n g r i n g systems. A g i v e n p l a n t s p e c i e s v e r y seldom c o n t a i n s a s i n g l e a l k a l o i d and u s u a l l y m i x t u r e s o f a l k a l o i d s w i t h s i m i l a r b i o l o g i c a l prope r t i e s a r e found t o g e t h e r . Commercial p r e p a r a t i o n s a r e u s u a l l y crude m i x t u r e s c0ntainin.g v a r y i n g amounts o f a l k a l o i d s and zone e l e c t r o p h o r e s i s o n paper has been w i d e l y a p p l i e d t o t h i s c a t e g o r y o f compounds. A1 k a l o i d s g e n e r a l l y m i g r a t e as c a t i o n s o v e r t h e pH range 1.8-12.5. TABLE 16.1 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS AT DIFFERENT pH VALUES (REF. 12)a A1 k a l o i d

A pomorph ine Atropine Caffeine Cocaine Ergotamine Heroin Lobe1 i n e Morphine Nicotine Papaveri ne Quinine Scopolamine Sparteine Strychnine Theobromine Tubocurarine a

M i g r a t i o n (cm) a t pH

1.8

2.1

-

8.4 12.4 - 3.9 - 14.0 - 7.5 - 13.7 - 12.5 - 13.8 - 24.3 - 11.4 - 17.9 - 11.9 - 20.1 - 11.8 - 4.4 - 15.0

10.0 14.1 - 5.9 14.6 6.8 - 13.6 - 12.6 - 14.0 27.8 - 12.6 - 20.2 - 14.0 - 22.1 - 13.4 - 4.3 - 14.9

-

-

-

-

-

4.2

-

7.2 14.0 - 5.1 - 16.3 - 7.2 - 15.2 - 14.2 - 15.3 - 18.3 - 12.1 - 14.9 - 13.0 - 18.8 - 12.6 - 5.2 15.3

-

.

6.4

-

5.2 14.3 - 6.4 14.5 - 4.5 - 12.7 - 12.7 - 13.1 17.2 - 7.6 - 11.5 - 14.6 - 15.8 - 12.1 - 6.7 - 12.7

-

-

-

8.6

-

4.2 15.2 8.0 - 14.7 - 6.2 12.0 - 13.2 - 12.0 - 13.0 - 5.0 10.2 - 11.6 - 16.8 - 10.5 - 8.4 11.8

-

-

-

-

-

11.2

-

2.0 13.7 - 8.5 9.9 - 4.4 - 7.0 - 4.4 - 7.2 - 7.5 - 5.1 - 7.2 8.7 - 16.7 - 8.0 - 6.1 - 8.3

-

-

-

12.5 1.0 - 6.0 - 3.5 - 10.1 - 6.3 - 7.6 - 7.4 - 7.9 - 3.5 - 3.0 - 3.0 - 4.0 - 9.5 - 3.0 - 6.5 - 9.0

B r i t t o n - R o b i n s o n phosphate-acetate-borate (0.04 m o l / l o f each) b u f f e r was used between pH 1.8 and 11.2 and a s o l u t i o n o f g l y c i n e a t pH 12.5; S c h l e i c h e r and S c h i i l l 2043 b paper ( 3 x 60 cm); 500 V ( 8 V/cm) f o r 3 h; movement i s towards t h e cathode except f o r apomorphine a t pH 12.5

396

TABLE 16.2 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS AT DIFFERENT pH VALUES (REF. 13)a ALKALOID

M i g r a t i o n (cm) i n b u f f e r b o f pH 2.3

Apomo r ph in e Arecol i n e Aspidospermine Atropine Berberine Bicuculline Bol d i n e Brucine Bul bocapnine Caffeine Cal ebassine Chel i d o n i n e C i n c h o n i d i ne Cinchonine Cocaine Col c h i c i n e Coniine C o r l umine Co r y d ine Curarine Cotarnine Dicentrine Dimet h y 1 b e r b e r ine Dinethyl tubocurarine Emetine Ephedrine Ergomet r ine Eserine F1 u o r o c u r i n e Gal eg ine Glaucine Harrnine Hero i n Homatropi ne Hydrast ine Hydrast in ine L-Hyoscyamine Jervine Lupan ine Lupinine Ma va c u r ine llescal i n e Mo r ph i ne Narceine N a r c o t i ne Nicotine Pa paver ine Pilocarpine P i per ine

- 4.2 - 8.8 - 5.24 - 6.3

- 1.5 - 5.1 - 2.2 - 4.5

-

- 3.7 - 0.3 - 6.5 - 4.95

10.4 10.2 6.1 0.2 8.9 4.95 4.6 10.6 8.4 1.5 6.6 6.8 7.5 8.0 3.5 6.15 7.7 9.1 2.8 @,.l 5.3 6.3 5.05 9.5 6.6 4.1 5.8 8.0 3.1 7.4 - 5.8 4.0 5.0 18.0 4.7 7.0 0

-

4.3

6.4

- 9.75 - 5.7 - 6.6 - 2.7 - 5.0 - 2.5 - 4.6 - 3.5 - 1.2 - 4.55 - 6.8 - 6.8 - 6.8 - 0.8 - 8.9 - 4.95 - 4.6 - 9.8 - 8.5 0 - 7.9 - 6.95 - 6.6 - 10.0 - 3.6 - 6.5 - 13.0 - 2.7 - 3.1 - 6.0 - 8.4 - 5.4 - 9.9 - 6.6 - 3.9 - 7.35 - 8.3 -

-

-

7.05 6.25 1.3 5.3 9.2 5.0 8.5

0

8.2

-. -13.5 8.7 10.7 - 2.0 6.2 3.0 6.9 3.3 2.35 8.0 5.8 9.3 8.0 - 11.0 2.1 13.0 5.9 7.1 11.0 12.5

-

-

-

0 - 10.4 - 9.9 - 8.5 - 10.0 - 3.8 - 10.5 - 8.0 - 11.8 - 3.8 2.6 - 9.5 - 10.4 - 6.1 - 13.2 - 10.3 - 5.3 - 10.6 - 12.1 - 2.4 - 10.2 - 10.0 - 2.6 - 5.4 - 12.3 - 3,o - 10.6 -

0.4

-

-

-

-

-

10.5 0 9.0 0 8.4 1.3 0 1.3 4.2 1.7 2.0

9.0

0 5.7 5.1 5.5 1.95 12.0 0 2.7 10.3 11.3 0 9.45 2.0 5.8 11.9 - 1.5 6.2 - 9.0 12.0 1.2 0 5.2 10.5 0 12.5 - 8.8 0 9.3 10.5 3.0 - 9.3 - 6.5 1.7 0 8.7

-

-

1.1 2.8 0 8.0 1.05 0 0 2.1 0.6 2.3 7.8 0 - 3.2 3.0 5.3 1.7 10.0 0 .- 1.4

-

-

-

-

-

-

-

10.8 0 7.8 8.7

--

10.6

- -1.3 - 5.0

- 10.0

4.0 0

10.8 - 0.3 0 2.85

0 - 11.3 6.9 0 8.7 10.4 2.4 8.2 2.85 - 1.65 0 - 4.5 0 1.95 0

-

0 1.5 0.9 0 3.8

-

-

-

0

-

-

-

-

-

-

-

-

-

-

-

11.4

-

-

-

-

-

1.3 0.3 5.0

0 0 0 1.5

-

2.1

-

0 6.4 4.5 0 5.3 4.9 0 2.2 1.5 0.9 or.2 6.8

0 0 0 1.5 0 4.5 1.8

r

1.8

- 1.3 - 1.9

-

1.2 1.3 1.5, 0 1.6 0 2.3 0

0.5

397 TABLE 16.2 (continued) ~~

.41 k a l o i d

Protopine P r o t o v e r a t r i ne Quinine Sanquinarine Scopol ami ne Sempervirine Sol an ine Sparteine S t r y c hn ine Tetra hydropal ma t i n e Theobromine Theophyll i n e Tropacocaine Tropine Tubocurarine V e r a t r i ne Yohimbine

~

H i g r a t i o n (cm) i n b u f f e r b o f pH

2.3

4.3

6.4

8.2

- 4.6 - 3.5 - 10.2 - 1.5 - 6.7 - 0.8 - 2.65 - 11.5 - 5.6 - 4.5 - 0.45 - 0.25 - 6.7 - 7.5 - 7.0 - 4.0 - 4.5

- 5.0 - 3.5 - 6.8 - 1.38 - 6.65 - 0.7 - 2.9 - 10.0 - 5.85 - 4.2 - 0.8 - 1.0 - 8.6 - 6.6 - 6.7 - 3.7 - 4.7

- 7.7 - 5.85 - 6.5 - 4.8 - 7.6 - 4.6 - 1.1 0 - 10.4 - 6.0 - 0.5 0 - 4.0 - 4.0 - 10.8 - 10.1 - 8.5 - 5,2 - 4.1 0 - 2.1 - 1.8 - 2.1 0.9 - 10.2 - 10.6 - 14.5 - 5,85 - 8.4 - 5.8 - 6.2 - 3.8 - 6.8

10.5

11.4

- 3.9 - 2.5 0 - 3.3 -

0 0 0

-

0 0 9.9 0

0

1.3 0 0 4.7

--

0

- 1.8 1.15 - 8,8 0 - 4,7 .4 -- 11.0

0

2.0 5.5 2.0 0 0.7 0 0

aWhatman No. 1 paper; 8 V/cm f o r 3 h. bUniversal Britton-Robinson b u f f e r d i l u t e d 1:4. Movement i s towards t h e cathode i n most instances, as i n d i c a t e d by t h e minus signs.

TABLE 16.3 ELECTROPHORETIC MOBILITIES OF ALKALOIDS AT pH 5.0 AND 9.2 (REF. 8 ) A1 k a l o i d

Mobilitya i n b u f f e r o f DH 5.0b

Anabasi ne Anatabine N-Methylanabasine N-Methyl anatabine Metani c o t i n e Methylmetanicotine Myosmi ne

A1 k a l o i d

9.2'

- 10.7 - 10.5 - 10.1 - 10.0 - 12.7 - 12.5 - 8.0 -

6.1 2.3 0.8 0.8 7.7 7.0 0.8

Mobilitya i n b u f f e r o f pH 5.0b

Nicotine Nicotone N i co t y r i ne Nornicotine Norni c o t y r i ne Oxy nic o t ine

9.2'

-

12.1 - 3.4 - 3.2 - 12.2 - 1.5 - 3.9 -

;Mobility x lo5 cm2 V - l sec-' a t 10°C towards t h e cathode. Potassium hydrogen p h t h a l a t e b u f f e r (pH 5.0). Although t h k c o n c e n t r a t i o n o f b u f f e r i s n o t g i v e n i n r e f . 8, i t i s thought t h a t i t was 0.05 mol/l. '0.025 m o l / l borax s o l u t i o n (pH 9.2).

2.5 0.7 0

7.0 0.7 0.7

398

TABLE 16.4 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS I N 5 mol/l ACETIC ACID' A1 k a l o i d

Migrationa (cm)

Aconi t i ne Atropine Bruci ne Caffeine Cinchoni ne Coni i n e Ephedrine

- 3.2 - 4.5 - 4.0 - 0.5

-

-

-

8.0 10.5 5.2

A1 k a l o i d

Migrationa (cm)

Hyoscyami ne Lobe1 ine N i co t i ne Quinine Sol ani ne Strychnine Sparteine

-

-

4.6 4.6 9.9 7.4 3.4 4.8 7.6

aMigration towards the cathode; 5 mol/l a c e t i c acid; 750 V f o r 2 h.

Universal s e r i e s o f Britton-Robinson b u f f e r s can be used f o r zone electropho1 r e s i s on paper. By aFplying a high voltage i t i s possible t o separate, e.g., candicine, tembetarine, N-methyl isocorydine and n i t i d i n e 2 Good separations a r e

.

obtained a t pH 8.0 w i t h l a u r i f o l i n e , magnoflorine, berberine, palmatine and t h e benzophenanthridines. A t t h i s pH i t i s also p o s s i b l e t o d i f f e r e n t i a t e between cuscohygri ne, a t r o p i n e , hyoscyami ne , apoatropi ne , 6-hydroxyhyoscyami ne , scopolamine, 3,6-ditigloyl-7-oxytropane and metcloidine3 Britton-Robinson b u f f e r s (pH

.

1.8-8.0)

have f u r t h e r been a p p l i e d t o the separation o f codeine, quinine, dibazole, p l a t y p h y l l ine, hydrotartarate, s a l s o l ine, pachycarpine and papaverine9 Acetic

.

a c i d s o l u t i o n s o f d i f f e r e n t concentrations (0.88, 1.0, 4.4 and 5 m o l / l ) can also be ~ s e d ~ -Potassium ~ . hydrogen phthalate (0.05-0.10 m o l / l , pH 5,O) gives equally good r e s u l t s 8 . F i n a l l y borax (0.025 mol/l ; pH 9.2) has been used s u c ~ e s s f u l l y ~ * ~ , e.g.,

f o r ephedrine. P y r i d i n e acetate b u f f e r s (pH around 3.5) o r aqueous ammonia

(pH 11.0) a r e preferred i n t h e electrochromatography (mapping) o f d i v e r s e a1 kaloidsl'.

I n a systematic study o f anions used i n t h e e l e c t r o p h o r e t i c b u f f e r s i t

was found t h a t p e r c h l o r i c a c i d and t r i c h l o r o a c e t a t e are the anions o f choice when possible''. Separations are c a r r i e d a t about 20-25 V/cm; a running time 2 h i s u s u a l l y s u f f i c i e n t t o o b t a i n good separations. Some idea o f t h e m i g r a t i o n o f d i verse a l k a l o i d s i n an e l e c t r i c a l f i e l d can be gained from Tables 16.1-16.4. I n a d d i t i o n t o the vast number of r e s u l t s obtained on the separation o f alkal o i d s by paper electrophoresis , o t h e r sorbents have occasionally been used. Thus, polyacrylamide gel e l e c t r o p h ~ r e s i s ' ~i n acetate b u f f e r s was a p p l i e d t o t h e separat i o n o f raunatin and r e l a t e d compounds. Thin-layer e l e c t r o p h ~ r e s i s ' ~was made use o f by Wan using three d i f f e r e n t running b u f f e r s (Table 16.5). S i m i l a r r e s u l t s can be obtained a t lower voltages (500 V per p l a t e ) .

399 TABLE 16.5 ELECTROPHORETIC MOBILITIES OF ALKALOIDS AT 3000 V 15 cm x 20 cm p l a t e s coated w i t h C e l l u l o s e MN 300, t h i c k n e s s 0.5 mm15

A1 k a l o i d

.

Electrolyte Formic a c i d - a c e t i c a c i d water (26: 120: 1000)

Ammonia so1n.water (2:98)

MQa Atropine Brucine Cocaine Codeine Colchicine Emeti ne Ephedrine Ergometri ne Homatropine Hyo s c ine Mo r ph ine Papaverine Pilocarpine Physostigmine Quinidine Quin i ne Reserpine Strychnine Tubocurarine

.-

Ammonia s o l n w a t e r (10:90)

MAb

0.68 0.50 0.42 0.66 0.12 0.73 0.78 0.41 0.71 0.66 0.66 0.51 0.82 0.64 0.98 1.00 0.27 0.59 0.73

1.00 0.41 0.89 0.78 0.74 0.09 1.18 0.39 1.07 0.81 0.60 0.14 0.85 0.81 0.23 0.23 0.02 0.45 0.39

1.oo 0.42 0.90 0.81 0.74 0.11 1.16 0.40 1.10 0.83 0.58 0.17 0.84 0.82 0.23 0.24 0.00 0.45 0.37

1.00 0.42 0.83 0.77 0.68 0.10 1.09 0.37 1.04 0.80

-

0.16 0.81 0.77 0.22 0.23 0.03 0.43 0.29

1.00 0.40 0.83 0.76 0.83 0.11 1.13 0.35 1.04 0.86 -0.68 0.17 0.85 0.74 0.27 0.23 0.00 0.49 0.30

: M o b i l i t y r e l a t i v e t o q u i n i n e . E l e c t r o p h o r e t i c r u n : 10 min. M o b i l i t y r e l a t i v e t o a t r o p i n e . E l e c t r o p h o r e t i c r u n : 20 min. A l k a l o i d s s p o t t e d 3 cm fr m end o f p l a t e . 'As f o r b u t a l k a l o i d s s p o t t e d mid-way between ends o f p l a t e .

A l k a l o i d s e i t h e r absorb o f f l u o r e s c e i n UV l i g h t , which can be used f o r t h e i r d e t e c t i o n . The most f r e q u e n t d e t e c t i o n r e a g e n t s a r e cerium( I V ) s u l p h a t e , cinnamaldehyde-HC1

, Dragendorf

r e a g e n t , i o d o p l a t i n i c a c i d r e a g e n t or phosphomolybdic

a c i d r e a g e n t ( f o r a r e v i e w , see r e f . 16). REFERENCES

1 H.T.S. B r i t t o n and R.A. Robinson, J . Chern. Soc., (1931) 1456. 2 J.M. Calderwood and F. F i s h , J . Phurm. P h m a c o Z . , 2 1 (1969) 126. 3 S. Kisgyorgy, Rev. Med. (Tirgu-Mures R o n . ) , 17 (1971) 87. 4 G.C. C a s i n o r i , M. L e d e r e r and G.B. M a r i n i - B e t t d o , Guzz. Chim. I t a Z . , 86 (1956) 342.

400

5 C.L. Winek, J.L. Beal and M.P. Cava, J . Chromatogr., 10 (1963) 246. 6 G.B. M a r i n i - B e t t t i l o and M. L e d e r e r , Nature (London), 174 (1954) 133. 7 T. Kariyone, Y . Hashimoto, I . M o r i and M. Kimura, J . Pharm. Soc. J u p . , 73 (1953) 805. 8 H. i9ich1, J . Chrornutogr., 1 (1958) 93. 9 Nguyen-Hoang-Narn, M. H e r b e r t , Nguyen-Dat-Xuong and L . P i c h a t , B U Z Z . SOC. Chim. F r . , (1968) 2667. 10 J. G i s b e r t - C a l a b u i g , E. V i l l a n e u v a and G. Rubin de C e l i s , Zacchia, 6 (1970) 386. 11 M. S i n i b a l d i and B. R i n a l d u z z i , A m Z . L e t t . , 4 (1971) 125. 12 F. I n t r o n a , Med. Leg. A s s i c u r . , 12 (1964) 3. 13 G.B. M a r i n i - B e t t t i l o and J.A. Coch-Frugoni, &ZZ. Chim. I t a Z . , 8 (1956) 1324. 14 A.G. V o l l o s o v i c h , V . I . Safonov and M.P. Safonova, T r . Lenivqr. Khim.-Fum. I n s t . , 2 1 (1972) 192. 15 A.S.C. Wan, J . Chrornatogr., 60 (1971) 371. 16 G. Zweig and J.R. Whitaker, Paper Chrcmatography and EZectrophoresis, Academic Press, New York, 1961, p . 314.

401

Chapter 17 VITAMINS Z. DEYL

AS w i t h many o t h e r l o w - m o l e c u l a r - w e i g h t compounds, paper o r c e l l u l o s e e l e c t r o phoresis i s g r e a t l y p r e f e r r e d f o r separations o f vitamins. Thiamine and i t s p h o s p h o r i c e s t e r s a r e r e a d i l y s e p a r a t e d by c o n v e n t i o n a l paper e l e c t r o p h o r e s i s ( a c e t a t e b u f f e r , p=0.05, pH 5 . 4 4 ) l . S e p a r a t i o n i s c a r r i e d o u t a t 3 mA f o r 6-7 h. Thiamine and i t s d e r i v a t i v e s c o u l d be d e t e c t e d under UV l i g h t e i t h e r d i r e c t l y o r a f t e r spraying t h e electropherogram w i t h a s o l u t i o n containing

96% e t h a n o l , 10% NaOH and 2.5% K3(CN)6 (200:100:5).

Thiamine and i t s e s t e r s a r e

c o n v e r t e d i n t o t h e c o r r e s p o n d i n g thiochromes and appear as green-blue s p o t s under UV l i g h t . I n t e r f e r e n c e s due t o s a l t s , b u f f e r s and contaminants were c o n s i d e r a b l e disadvant a g e s o f t h i s method i n t h e p a ~ t ~ R - e~c .e n t l y , however, t h e method was r e i n v e s t i g a t e d and i t was shown t h a t c i t r a t e b u f f e r s c o n t a i n i n g methanol, e t h a n o l o r propanol g i v e e x c e l l e n t r e s u l t s [50 n m o l / l sodium c i t r a t e (pH 5 . 6 ) c o n t a i n i n g 2.5% ( v / v ) of methanol-ethanol-propanol (1:l:lLl. The s e p a r a t i o n i s c a r r i e d o u t a t h i g h v o l t a g e s

(2-4 kV p e r 48-cm sheet f o r 45-75 m i n ) . F o r q u a n t i t a t i o n r e f e r e n c e samples a r e r u n ; t h e s e a r e sprayed w i t h a l k a l i n e h e x a c y a n o f e r r a t e ( I I 1 ) r e a g e n t and t h e f l u o r e s c e n t bands a r e r e v e a l e d w i t h UV l i g h t . The sample s t v i p i s t h e n c u t a c c o r d i n g t o t h e r e f e r e n c e sample e l u t e d w i t h 50% e t h a n o l and s u b j e c t e d t o f l u o r i m e t r i c det e r m i n a t i o n . When t h i a m i n e compounds a r e assayed i n b i o l o g i c a l m a t e r i a l , deprot e i n a t i o n w i t h t r i c h l o r o a c e t i c a c i d i s necessary. The sample i s n e u t r a l i z e d w i t h K2C03 and t h e n l y o p h i l i z e d i n o r d e r t o c o n c e n t r a t e t h e t h i a m i n e d e r i v a t i v e s . Benzenesul phony1 c h l o r i d e i s used t o p r e v e n t o x i d a t i o n o f t h i a m i n e t o thiochrome. C e l l u l o s e powder column e l e c t r o p h o r e s i s ' r e s u l t s i n a complete and q u a n t i t a t i v e s e p a r a t i o n o f t h i a m i n e and i t s phosphate e s t e r s ; however, i t i s u n l i k e l y t o be used much today. I n t h i s procedure c e l l u l o s e i s suspended i n pH 5.44 a c e t a t e b u f f e r (p=0.05! (see above) and poured i n t o t h e s e p a r a t i o n column (50 x 3 cm); p r i o r t o p a c k i n g t h e s l u r r y i s degassed by a p p l y i n g a vacuum. The m a t e r i a l i s moved h a l f way down t h e column by l e t t i n g 70 m l o f b u f f e r pass t h r o u g h t h e column ( v o i d volume 150 m l ) ; t h i s i s done because t h i a m i n e and t h i a m i n e monophosphate move towards t h e cathode whereas t h i a m i n e pyrophosphate and t r i p h o s p h a t e move towards t h e anode. E l e c t r o p h o r e s i s i s c a r r i e d o u t f o r 15 h a t 30 mA, t h e n t h e column i s disconnected from t h e e l e c t r o d e v e s s e l s , connected t o a f r a c t i o n c o l l e c t o r and e l u t e d w i t h t h e acetate b u f f e r .

402

Hokawa and Cooper

5

p u b l i s h e d a n o t h e r m o d i f i c a t i o n o f o l d e r methods' f o r t h e

e l e c t r o p h o r e t i c s e p a r a t i o n of t h i a m i n e and i t s phosphate e s t e r s and subsequent 6 S e p a r a t i o n i s c a r r i e d o u t on f l u o r i m e t r i c d e t e r m i n a t i o n of t h i a m i n e compounds

.

c e l l u l o s e p o l y a c e t a t e (Sepraphore I 1 o r I 1 1 s t r i p s ) i n a c e t a t e b u f f e r (0.05 m o l / l , pH 3.8). F o r q u a n t i t a t i o n , f l u o r e s c e n t s p o t s a r e r e v e a l e d w i t h long-wave UV l i g h t , c u t o u t from t h e s t r i p a l o n g w i t h a b l a n k a r e a o f c o r r e s p o n d i n g s i z e , and p l a c e d i n c u v e t t e s c o n t a i n i n g 1 m l o f 2% potassium h e x a c y a n o f e r r a t e ( I I 1 ) and 15 m l o f 15% NaOH i n 76 m l o f water. A f t e r 2 min, 0.01 m l o f 30% H202 i s added t o d e s t r o y t h e reagent and e l u t i o n o f t h e f l u o r o p h o r e i s a l l o w e d t o proceed f o r 20 min. Eval u a t i o n i s c a r r i e d o u t f l u o r i m e t r i c a l l y a t 365/430 nm (see a l s o r e f s . 7 and 8 ) . Compounds b e l o n g i n g t o t h e r i b o f l a v i n (B2), n i c o t i n a m i d e and v i t a m i n B6 groups a r e u s u a l l y separated a t l o w e r pH9, A c e t a t e b u f f e r (p=0.05) i s used as w i t h t h i a mine. A t 3.5 mA and d u r i n g a 12-h r u n t h e s e p a r a t i o n o f members o f each group and t h e s e p a r a t i o n o f a l l o f t h e combined compounds a r e r e a d i l y p o s s i b l e . i s today an e x t r e m e l y i m -

T h i n - l a y e r e l e c t r o p h o r e s i s on s i l i c a g e l plates''

p o r t a n t approach t o t h e s e p a r a t i o n o f v i t a m i n B6 compounds. A c e t a t e b u f f e r s o f l o w e r pH (3.95 and 4.53) a r e used. The a p p l i c a t i o n o f b u t y r a t e b u f f e r s , which were o r i g i n a l l y used f o r paper e l e c t r o p h o r e s i s , can a l s o be used ( T a b l e 17.1).

TABLE 17.1 MIGRATION OF VITAMIN B6 COI'IPOUNDS ON SILICA GEL THIN-LAYER PLATES Movements o f compounds i n m i l l i m e t r e s f r o m t h e o r i g i n . The r u n n i n g t i m e was 55 min a t 25 V/cm f o r pH 3.95 b u f f e r o r 40 m i n a t t h e same v o l t a g e f o r pH 4.53 buffer. Compound

Pyri doxol Pyri doxal Pyridoxamine P y r i d o x o l phosphate P y r i d o x a l phosphate Pyridoxamine phosphate

Buffer pH 3.95

pH 4.53

-

-

54 35 56 0 i.24 11

-

As r e p o r t e d b y Colombini and McCoy", p y r i d o x i n e 5'-phosphate,

19 9 34 t 12 t 21 - 3

p y r i d o x i n e , p y r i d o x a l , pyridoxamine,

p y r i d o x a l 5'-phosphate and pyridoxamine 5-phosphate can

be separated on c e l l u l o s e p l a t e s i n 0.05 m o l / l a c e t a t e b u f f e r (pH 4.9-5.1) a l s o r e f . 12).

(see

403 UV l i g h t can be recommended f o r d e t e c t i o n . R i b o f l a v i n , FPlN and FAD can be i d e n t i f i e d by t h e i r y e l l o w f l u o r e s c e n c e . Then t h e e l e c t r o p h e r o g r a m i s sprayed w i t h 2% Na2S203 and 4% NaHC03. I n short-wave UV l i g h t NAD and NADP a r e r e v e a l e d as f l u o r e s c e n t spots. F i n a l l y , t h e e l e c t r o p h e r o g r a m i s sprayed w i t h d i a z o t i z e d p - a c e t o phenone and heated f o r 10 min a t 50-60°C. pink

- yellow

The v i t a m i n B6 group o f compounds g i v e

spots.

The r e s u l t s of t h e paper e l e c t r o p h o r e t i c s e p a r a t i o n o f n i c o t i n i c a c i d and der i v a t i v e s a r e summarized i n Table 1 7 . 2 ~ ~ S~e p~ a.r a t i o n i s u s u a l l y c a r r i e d o u t a t 280 V w i t h i n 1-2 h. N i c o t i n i c a c i d , n i c o t i n a m i d e and n i c o t i n u r i c a c i d can be det e c t e d by f i r s t exposing t h e s t r i p t o cyanogen bromide vapour f o r 15 min and t h e n s p r a y i n g t h e chromatogram w i t h a 0.28% s o l u t i o n o f b e n z i d i n e i n e t h a n o l . The e l e c tropherogram i s t h e n exposed t o cyanogen bromide vapour again. N i c o t i n i c a c i d appears a s a d a r k r e d band, n i c o t i n a m i d e forms a brown s p o t and t h e s p o t o f n i c o -

NAD and NADP appear as b l u e f l u o -

t i n u r i c a c i d i s p u r p l e . N-Plethylnicotinamide,

r e s c e n t bands i n UV l i g h t a f t e r t h e e l e c t r o p h e r o g r a m has been t r e a t e d w i t h e t h y l methyl ketone-ammonia vapour. Zone e l e c t r o p h o r e s i s o f v i t a m i n B12 p r e p a r a t i o n s on paper u s u a l l y r e v e a l s f o u r o r f i v e bands17, 0.5 m o l / l a c e t i c a c i d c o n t a i n i n g 0.01% o f KCN i s used i n o r d e r t o keep t h e compounds i n t h e cyano form. The e l e c t r o p h e r o g r a m s h o u l d be p r o t e c t e d f r o m s t r o n g l i g h t d u r i n g t h e run. Adequate s e p a r a t i o n s a r e o b t a i n e d i n 16 h a t 8 V/cm. An e l e c t r o p h o r e t i c t e c h n i q u e u s i n g C e l l o g e l s t r i p s was d e v i s e d f o r t h e s e p a r a t i o n o f cyanocobalamin, hydroxocobalamin, methylcobalamin and cobamide by T o r t o l a n i and F e r r i 1 8 . The f o l l o w i n g b u f f e r s can be used: 0.5 m o l / l a c e t i c a c i d (pH 2.9);

0.1 m o l / l sodium c i t r a t e - 0 . 1

m o l / l h y d r o c h l o r i c a c i d (pH 4.0);

sodium c i t r a t e - 0 . 1 m o l / l sodium h y d r o x i d e (pH 5.0); m o l / l sodium h y d r o x i d e (pH 6.0),

0.1 m o l / l sodium c i t r a t e - 0 . 1

pH 7.0 Siirensen b u f f e r ; and 0.2 m o l / l sodium

borate-0.1 m o l / l h y d r o c h l o r i c a c i d (pH 8.0). l u s t r a t e d i n Table 17.3.

0.1 m o l / l

The e l e c t r o p h o r e t i c b e h a v i o u r i s il-

A f t e r t h e s e p a r a t i o n t h e cobalamins a r e d e t e c t e d v i s u a l -

l y . Cyanocobal amin i s v i o l e t and hydroxocobal amin, methylcobalamin and cobamide a r e red. I s o e l e c t r i c f o c u s i n g of v i t a m i n B12-protein range 2.0-5.0 10

.

complexes i s p o s s i b l e i n t h e pH

A s c o r b i c a c i d can be separated f r o m a v a s t number o f c a r b o x y l i c a c i d s and v a r i o u s sugar a c i d s and l a c t o n e s by zone paper e l e c t r o p h o r e s i s ; 0.75 m o l / l f o r m i c a c i d (pH 2.0) o r 0.5 m o l / l a c e t i c a c i d a d j u s t e d t o pH 4 a r e s u i t a b l e b u f f e r s . T h i n - l a y e r e l e c t r o p h o r e s i s on a l a y e r o f c e l l u l o s e powder a t 500 V ( 2 0 x 20 cm p l a t e ) can a l s o be recommended f o r t h e s e p a r a t i o n o f a s c o r b i c acid’.’, o x a l i c a c i d as t h e r u n n i n g buffer. f o r r e v e a l i n g c a r b o x y l i c a c i d s , e.g.

u s i n g 2%

D e t e c t i o n can be e f f e c t e d w i t h r e a g e n t s used

, bromophenol

b l u e , bromocresol p u r p l e , a n i -

l i n e g l u c o s e reagent, i r o n ( 111) c h l o r i d e r e a g e n t , 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e o r

TABLE 17.2 ELECTROPHORETIC IIIGRATION OF COENZYHES AND RELATED COFIPOUNDS Compound

llig r a t i on i n b u f f e r la(14)

31, Coenzyme A Creatine phosphate 72b F l a v i n mononucleotide (FNN) 3 7c F l a v i n adenine d i n u c l e o t i d e (FAD) 40d N i c o t i namide N I-Plethyl n i c o t i nami de Nicotinic acid Nicotinuric acid NI.1N NAD( DPN+) 17 NADH(DPNH) 53 NAD P( TPN+ ) 51 NADPH(TPNH) Pyridoxal phosphate 38 Pyridoxine phosphate Pyridoxamine phosphate -7 Thiamine Thiamine monophosphate Thiamine pyrophosphate 2 Thiamine t r i p h o s p h a t e

i

1

Ze(15) (flpi)

3f(4) 0lpi)

4h(l) (PAr)

5i(13) (cm/h)

6'(13) (cm/h)

7k(13) (cm/h)

8l(13) (cm/h)

9"(13) (cm/h)

10n(13) (cm/h)

-0.6 -4.6 2.7 2.7

-1.8 -9.0 5.1 3.7

-1.5 -6.8 3.6 2.4

-1.0 -6.9 4.0 2.8

-1.7 -8.3 5.3 4.6

-1.0 -6.6 3.8 2.8

61 29 36

2 17 42 44 63 39 100

36 36

13 489

4.7 5.5

32 -20

3

aRef. 2; 0.25 m o l / l amoniun? a c e t a t e (ptl 3.5); , niobil i t y r e l a t i v e t o orthophosphate. DConsiderabl e decomyosition. CS1 i g i i t deconi20si t i o n . dcompounci t r a i l s .

0

-100 - 1-9 40 63

S c h l e i c h e r and SchU11 5 T Green ?ibbon paper (unwashed); 600 V f o r 2.5 h;

'Ref. 15; pyridine-acetic acid-water (20:64:916) (pH 3.9); ldhatman 3 MM paper (washed; 7.5 x 22. 5 i n ) ; 35 V/cm f o r 2 h ; mobility r e l a t i v e t o orthophosphate. fRef. 16; 1 mol/l n-butyric acid-0.025 mol/l NaOH (pH 3.2); Whatman No. 3 paper (washed; 15.5 x 38 cm); 4CO V ?or 4.5 h ; 2OoC; mobility r e l a t i v e t o orthophosphate. goxidized slowly d u r i n g separation hAcetate buffer (pH 5.44, p = 0.05); Munktell No. 20 paper; 3.0 mA f o r 7.3 h ; mobility r e l a t i v e to thiamine. iRef. 13; 0.1 mol/l acetate buffer (pH 4.6); !,;hatman No. 1 cellulose paper ( 4 x 30 cm); 2SC! V f o r 1-2 h a t room temperature. i , except 0.033 mol/l borate buffer (pH 8.9). lAs i , except 0.033 mol/l phosphate buffer (pH 7.G). As i , except 0.033 mol/l phosphate buffer (pH 10.2). mAs i , except 0.05 mol/l Tris buffer (pH 9 . 2 ) . A ' s i , except 0.05 mol/l Verona1 buffer (pH 8. 6).

AS

P

0 UI

406

diazotized p - n i t r a n i l i n e . For q u a n t i t a t i o n the bands o f ascorbic a c i d are marked under UV l i g h t , scraped o f f and the colours are developed w i t h i r o n ( I I 1 ) ammonium sul phate and o-phenanthrol ine-HC1. TABLE 17.3 RELATIVE MOBILITIES OF VITAMIN B12 DERIVATIVES AT DIFFERENT pH VALUES For b u f f e r composition see t e x t . The data represent m o b i l i t y r e l a t i v e t o cobamide ( = 1.0).

PH

Cyanocobal amin

Methylcobalamin

Hydroxocobal ami n

2.9 4.0 5.0 6.0 7.0 8.0

0.22 1.10 1.10 1.02 1.02 0.75

0.36 0.83 0.90 0.77 0.78 0.60

0.92 1.20a 1.25a 1. 25a 1.05a 1.75a

a A t pH > 2.9 hydroxocobalamin gives a number o f d i f f e r e n t coloured spots (yellow, v i o l e t , pink, red).

A method f o r i s o l a t i n g and i d e n t i f y i n g b i o t i n peptides has been described by Swack e t a1.20.

The method e x p l o i t s the f a c t t h a t b i o t i n binds s t o i c h i o m e t r i c a l l y

t o a v i d i n t o form a s t a b l e b i o t i n - a v i d i n complex. The contaminating substances are removed by chromatography on a column o f a v i d i n-Sepharose. A f t e r removing nons p e c i f i c a l l y bound p r o t e i n bywashing, the b i o t i n enzymes a r e e l u t e d w i t h urea-SDS. Because b i o t i n proteins a r e f r e q u e n t l y mu1t i - s u b u n i t systems and c o n t a i n nonb i o t i n y l i n a d d i t i o n t o b i o t i n y l peptides, and as t h e n o n - b i o t i n y l peptides may contaminate the f r a c t i o n e l u t e d w i t h urea-SDS from the avidin-Sepharose column, Swack e t a l . suggested t h e f o l l o w i n g procedure f o r the i d e n t i f i c a t i o n o f ' b i o t i n y l polypeptides. The p a r t i c u l a r f r a c t i o n obtained from the avidin-Sepharose column i s run on 5 o r 10% SDS-polyacrylamide gels. Usually d u p l i c a t e s a r e used, one o f which i s stained w i t h Coomassie blue i n the usual way and the o t h e r i s soaked overnight i n fluorescent avidin. The a v i d i n - t r e a t e d g e l s are r i n s e d and the b i o t i n peptides a r e revealed under long-wave UV l i g h t as b r i g h t f l u o r e s c e n t zones. Fluorescent a v i d i n i s prepared from a v i d i n by mixing w i t h fluorescamine dissolved i n d r y acetone. A l t e r n a t i v e l y i t i s a l s o possible t o prepare I4C-labelled b i o t i n 21 samples and t o evaluate the electropherograms by r a d i o a c t i v i t y counting

.

B i o c y t i n (E-N-biotinyl-L-lysine) can be determined i n t h e hydrolysate o f b i o t i n p r o t e i n s by two-dimensional mapping on paper22. The hydrolysed sample i s applied

407

on paper and electrophoresed a t 3000 V f o r 1 h i n pH 1.9 b u f f e r [acetic acid-98% f o r m i c acid-water (87:25:88811.

On a separate sheet standards o f a - l c a r b o n y l - 1 4 g -

b i o t i n , b i o c y t i n , a l a n i n e and t y r o s i n e a r e a p p l i e d and subjected t o s i m i l a r e l e c t r o p h o r e s i s . A f t e r d r y i n g , b o t h sheets a r e subjected t o chromatography i n n-butan o l - g l a c i a l a c e t i c acid-water (80:20:20).

Detection i s effected with ninhydrin

and t h e standard spot o f b i o t i n i s measured by r a d i o a c t i v i t y scanning. TABLE 17.4 ELECTROPHORETIC NOBILITIES

OF PTERIDINES

Com pou nd

E l e c t r o p h o r e t i c m o b i l i t y t o anode

L-threo-Neopterin-2':3'-cyclic phosphate

33 3or - 3 - 3 35 - 3

D-erythro-Neopter in -3 ' -phosphate L-threo-Neopteri n D-erythro-Neopterin

2-Amino-6-carboxy-4-hydroxypteridine 2-Amino-4-hydroxy-6-methylpteridine

A number o f p t e r i d i n e s were s t u d i e d by Urushibara e t a1.23.

(mm)

Paper e l e c t r o p h o -

r e s i s and chromatographic methods were used f o r i d e n t i f i c a t i o n . E l e c t r o p h o r e s i s was c a r r i e d o u t i n pH 4.7 a c e t a t e b u f f e r a t 20 V/cm f o r 1 h (Table 17.4). e l e c t r i c f o c u s i n g i n t h e pH range 3.0-8.5

Iso-

and i n a saccharose g r a d i e n t was a l s o

found t o be s u i t a b l e f o r separating a number o f p t e r i d i n e d e r i v a t i v e s (Table 17 .s)*~. TABLE 17.5 ISOELECTRIC FOCUSING AND EXPERIMENTALLY DETERMINED P VALUES I OF PTERINS Compound

PI ~~

P t e r i n (2-amino-4-0xo-3~4-di h y d r o p t e r i d i n e ) 6-Nethyl i s o x a n t h o p t e r i n Rhodopterin ( p t e r o r h o d i n ) Xanthopter in 7-Kethyl x a n t h o p t e r i n Pterin-6-carboxyl i c a c i d P t e r i n-7-carboxyl ic a c i d

3.37 4.88 3.05 4.51 4.85 < 3 < 3

Tate and Johnson25 used simple paper e l e c t r o p h o r e s i s i n 0.1 m o l / l o x a l a t e (pH 1.5)

f o r t h e s e p a r a t i o n o f p h y t i c acids. I n a d d i t i o n t o several polyphosphate

f r a c t i o n s , several zones w i t h d e f i n i t e chemical composition were observed (Table

17.6) (see a l s o Table 3.12).

408 TABLE 17.6 ELECTROPHORETIC HOB1L I T 1ES OF llYOINOSITOL POLY PHOSPHATES RELATIVE TO INORGANIC PYROPHOSPHATE Compound

Relative m o b i l i t y

I n o r g a n i c pyrophosphate I n o r g a n i c orthophosphate Phytic a c i d (from cereal g r a i n ] Xy o inos it o 1 t r ipy r o p ho s p ha t e

1.00 0.32 1.26 1.65

REFERENCES

1 0. S i l i p r a n d i and N. S i l i p r a n d i , Biochirn. Biophys. Acta, 13 (1954) 52. 2 Y. Hokawa and J.R. Cooper, Methods Cnzyrnol., 18A (197C) 91. 3 H.K. P e n t t i n e n , Acta Chern. Scund., 832 (1978) 609. 4 H.K. P e n t t i n e n , Meshods EnzyrnoZ., 620 (1979) 6?. 5 Y. Hokawa and J.R. Cooper, Methods Enzyrnol., 18 (1970) 91. 6 L.W. Lewen and R. Uei, Anal. Biochern., 16 (1966) 29. 7 C. P a t r i n i and G. R i n d i , I n t . J . V i i t m . Nutr. R e s . , 50 (1350) 10. 8 H. Ahrens and W. Korytnyk, AnaZ. Biochern., 30 (1969) 413. 9 ti]. S i l i p r a n d i , D. S i l i p r a n d i and H. L i s , Biochirn. Biophys. A c t a , 13 (1W’) 1.75. 10 V.-H. Stenman and R. GrZsback, Biochirn. Biophys. Actu, 286 (1F72) 2 4 3 . 11 C.E. Colornbini and E.E. McCoy, Anal. Biochern., 3n (1970) 451. 12 E.E. ElcCoy, C. Colombini and K. Strynadka, Methods EnzyrnoZ., 62 (1979) 410. 13 T.K. Sundaram, K.V. Rajagopalan and P.S. Sarma, J . Chromatogr., 2 (1959) 531. 14 R.L. B i e l e s k i and R.E. Young, A m Z . Biochern., 6 (1953) 54. 15 B.S. Vanderheiden, Anal. Biochern., 8 (1964) 1. 16 H.E. Wade and O.H. llorgan, Biochern. J . , 60 (1955) 264. 17 E.S. Holdsworht, Nature (London), 171 (1953) 148. 18 G. T o r t o l a n i and P.G. F e r r i , J . Chromatogr. , 88 (1974) 430. 19 E. L e t z i g and K. Fuecker, Erntihrungsforschung, 15 (1970) 355. 20 J.A. Swack, G.L. Zander and M.F. U t t e r , Anal. Biochern., 87 (1978) 114. 21 R.R. Fow F a l l , Methods EnzymoZ. , 62 (1979) 390. 22 K. Dakshinamurti and P.N. G i l l e v e t , Methods Ennzymol., 62 (1979) 398. 23 T. Urushibara, H.S. F o r r e s t , D.S, Hoare and P.N. P a t e l , Biochern. d . , 125 (1971) 141. 24 C.H. Engster, E.F. F r a u e n f e l d e r and H. Koch, Helv. Chim. Acta, 53 (1970) 131. 25 M.E. T a t e and L.F. Johnson, Can. J . Chern., 47 (1969) 63.

409

Chapter 1 8

ANTIBIOTICS V . BETINA

GENERAL ASPECTS Electrophoresis has been applied i n the f i e l d of a n t i b i o t i c s s i n c e 1951, when a Japanese group used i t f o r t h e separation of carbohydrate antibiotics'". Of the various techniques, low- and high-vol tage paper e l e c t r o p h o r e s i s , high-vol tage thin-layer e l e c t r o p h o r e s i s , gel electrophoresis and c a r r i e r - f r e e continuous electrophoresis have been applied t o a n t i b i o t i c s t u d i e s 3 . Basic, a c i d i c and amphoteric a n t i b i o t i c s may be analysed by e l e c t r o p h o r e s i s . I t s main a p p l i c a t i o n s c o n s i s t of separation, i d e n t i f i c a t i o n , preparation and pur i t y control i n the search f o r new a n t i b i o t i c s and in t h e pharmaceutical industry. Electrophoretic techniques a r e a l s o used in s t r u c t u r a l s t u d i e s and i n attempts t o synthesize natural a n t i b i o t i c s and t h e i r d e r i v a t i v e s . A n t i b i o t i c s and t h e i r degradation or transformation products can be analysed in body f l u i d s and o t h e r biological materials. Various applications concerning t h e systematic a n a l y s i s o r i d e n t i f i c a t i o n of a n t i b i o t i c s in feeds and foods have been published. Two s p e c i f i c aspects need t o be emphasized: (1) t h e g r e a t chemical d i v e r s i t y of a n t i b i o t i c s and ( 2 ) t h e p o s s i b i l i t y of t h e i r detection by means of bioautography in addition t o other common detection techniques. The principal achievements of electrophoresis in t h e a n t i b i o t i c area u n t i l t h e mid-1970s were reviewed by Umezawa and Kondo 3 . In t h i s chapter, a s e l e c t i o n o f a p p l i c a t i o n s and r e s u l t s of older and more recent pub1 i c a t i o n s a r e given. BIOAUTOGRAPHY

Bioautography i s based on t h e biological a c t i v i t i e s of a n t i b i o t i c s . In a typical procedure f o r bioautographic detection of paper electropherograms, paper sheets o r s t r i p s a r e placed on t h e surface of l a r g e n u t r i e n t agar p l a t e s inocul a t e d with microorganisms t h a t a r e s e n s i t i v e t o the a n t i b i o t i c s under study. Antibiotics d i f f u s e from t h e i r positions on t h e paper i n t o t h e agar l a y e r and i n h i b i t the growth of t h e t e s t organism. After about 20-30 min t h e paper i s removed and the p l a t e i s incubated a t an appropriate temperature u n t i l a t h i n film of the growing organisms i s v i s i b l e on t h e agar surface. Clear zones of i n h i b i t i o n a r e seen in t h e areas where a n t i b i o t i c s a r e present.

410 I n general

, bioautography

i s v e r y s i m i l a r t o paper and t h i n - l a y e r chromato-

I n t h i s section a universal graphy. S p e c i a l techniques may be found t e c h n i q u e f o r paper e l e c t r o p h o r e s i s and two o t h e r s f o r gel e l e c t r o p h o r e s i s a r e described.

Bioautography f o r paper electrophoresis

3

A 120-ml p o r t i o n of n u t r i e n t agar c o n t a i n i n g (w/v) 1.0% o f meat e x t r a c t , 1.0% o f peptone, 0.2% o f sodium c h l o r i d e and 1.2% o f agar i n w a t e r (pH 7.0 a f t e r s t e r i l i z a t i o n ) i s poured a t 6OoC and d i s t r i b u t e d o v e r a s t e r i l i z e d g l a s s p l a t e w i t h a s t a i n l e s s - s t e e l frame (46 x 16 cm). A f t e r s o l i d i f i c a t i o n o f t h e a g a r l a y e r f o r

30 min, a second l a y e r (60 m l ) o f n u t r i e n t a g a r seeded w i t h a 0.5% ( v / v ) suspen6 s i o n o f a t e s t organism ( B a c i l l u s s u b t i l i s , a suspension o f a b o u t 50.10 spores p e r m i l i l i t r e ; Staphylococcus aureus and Escherichia c o l i , an o v e r n i g h t c u l t u r e i n a n u t r i e n t medium a t 37OC; Micrococcus f l a u u s , an o v e r n i g h t c u l t u r e i n a nut r i e n t medium a t 27OC) i s poured on t h e agar l a y e r a t 4OoC. The paper s t r i p i s superimposed on t h e upper l a y e r o f t h e a g a r f o r 20 min. (Caution: i f an e l e c t r o l y t e s o l u t i o n has been used t h a t c o u l d i n h i b i t t h e t e s t organism, such as f o r m i c a c i d o r a c e t i c a c i d , t h e s t r i p must be d r i e d w i t h a f l o w o f warm, d r y a i r t o r e move e l e c t r o l y t e s o l u t i o n ! ) A f t e r removal o f t h e paper s t r i p , t h e d o u b l e l a y e r o f t h e agar medium i s i n c u b a t e d a t 37OC f o r 18 h (M. f l a v u s a t 27-30°C).

After

i n c u b a t i o n , t h e p o s i t i o n s o f t h e a n t i b i o t i c s a r e d e t e c t e d by t r a n s p a r e n t i n h i b i t i o n zones.

Bioautography f o r agar ge 1 e lectrophoresis

6

When e l e c t r o p h o r e s i s i n a g a r g e l i s f i n i s h e d , a second l a y e r (400 m l ) o f a n u t r i e n t agar c o n t a i n i n g (w/v) 0.6% o f peptone, 0.15% o f b e e f e x t r a c t , 0.3% o f y e a s t e c t r a c t and 1.0% o f agar i n w a t e r (pH 7.9k0.1 a f t e r s t e r i l i z a t i o n ) i s seeded w i t h 2% ( v / v ) o f a suspension a f a t e s t organism ( B . s u b t i l i s , 20.10 6 -30.10 6 spores p e r m i l ili t r e ) and poured a t 7OoC on a n o t h e r sheet o f t h e l e v e l l e d p l a t e g l a s s w i t h t h e s t a i n l e s s - s t e e l frame. When t h e a g a r l a y e r has s o l i d i f i e d f o r 5 min, i t i s superimposed on t o p o f t h e b u f f e r e d a g a r g e l s l a b by g e n t l y s l i d i n g i t f r o m t h e p l a t e g l a s s . The double l a y e r o f agar i s i n c u b a t e d a t 39OC f o r 18 h

and t h e p o s i t i o n s o f t h e a n t i b i o t i c s a r e e v i d e n t as zones o f i n h i b i t i o n . By t h e measurement o f t h e diameters, t h e a n t i b i o t i c s can be assayed q u a n t i t a t i v e l y .

Bioautography for geZ e lectrophoresis 7 Microorganisms and media ( g / l ) a r e used as f o l l o w s . Sarcina Zutea ATCC 9341: peptone 5, b e e f e x t r a c t 1.5, y e a s t e x t r a c t 1.5, sodium c h l o r i d e 3.5, agar 10 (pH a f t e r s t e r i l i z a t i o n ) . Micrococcus fZavus ATCC 10240: t r y p t o n e 10, b e e f e x t r a c t

1.5, y e a s t e x t r a c t 3 , g l u c o s e 1, agar 10 (pH 8.4 a f t e r s t e r i l i z a t i o n ) . Bacillus

411

cereus ATCC 11778: t r y p t o n e 10, b e e f e x t r a c t 1.5, y e a s t e x t r a c t 3, g l u c o s e 1, agar 10 (pH 6.6 a f t e r s t e r i l i z a t i o n ) , t h e n 10 m l o f 0.5% methylene b l u e i n water a r e added. D e t e c t i o n . The g l a s s p l a t e w i t h t h e g e l i s removed f r o m t h e apparatus and framed w i t h a s t a i n l e s s - s t e e l frame which i s f i x e d b y p i p e t t i n g t h e a g a r i z e d medium a t i t s i n n e r edges, t h e n 300 m l o f one o f t h e f o u r media i n o c u l a t e d a t 48OC w i t h t h e c o r r e s p o n d i n g c u l t u r e a r e poured on t h e s u r f a c e . A f t e r c o o l i n g , t h e p l a t e i s i n c u b a t e d o v e r n i g h t a t 3OoC. The zones o f i n h i b i t i o n a r e b l u e on a p a l e b l u e background f o r B . cereus. With S . Zutea, t h e i r v i s i b i l i t y i s enhanced by s p r a y i n g w i t h a 0.05% s o l u t i o n o f t r i p h e n y l t e t r a z o l i u m c h l o r i d e i n 0.1% glucose, which makes t h e background r e d . PENICILLINS AND CEPHALOSPHRINS M a r k o v i z e t a1 .8 separated s e m i s y n t h e t i c p e n i c i l l i n s by e l e c t r o p h o r e s i s on paper, s t a r c h gel and t h i n l a y e r s o f s i l i c a g e l . The g e l was prepared by b o i l i n g 10 g o f h y d r o l y s e d s t a r c h , r e t i c u l a t e d a c c o r d i n g t o r e f . 9, i n 200 m l o f 0.03 m o l / l T r i s - h y d r o c h l o r i c a c i d b u f f e r (pH 7 . 5 ) . U s i n g t h e same b u f f e r , e l e c t r o p h o r e s i s was c a r r i e d o u t a t 3 V/cm f o r 20 h. H i g h - v o l t a g e e l e c t r o p h o r e s i s was c a r r i e d o u t on t h i n l a y e r s o f s i l i c a g e l on NM-Polygram SIL-NHR p l a t e s ( M a c h e r e y , Nagel & Co.) i n a h i g h - v o l t a g e apparatus w i t h p y r i d i n e - a c e t i c a c i d b u f f e r (pH 6.5) a t 90 V/cm f o r 20 min. Descending paper e l e c t r o p h o r e s i s a c c o r d i n g t o r e f . 10 was c a r r i e d o u t on Whatman 3 MM paper i n p y r i d i n e - a c e t i c a c i d b u f f e r (pH 5.6) a t 30 V/cm f o r 90 min. D e t e c t i o n : i s e f f e c t e d w i t h i o d i n e - a z i d e r e a g e n t o r i o d i n e vapour f o l l o w e d by n i n h y d r i n f o r d e g r a d a t i o n p r o d u c t s . B l o o d and u r i n e samples were a l s o analysed. The b e s t r e s u l t s were o b t a i n e d w i t h paper e l e c t r o p h o r e s i s i n w h i c h t h e f o l l o w i n g p e n i c i l l i n s moved t o t h e anode: c a r b e n i c i l l i n , s u n c i l l i n , p r o p i c i l l i n , pheneticill in, oxacill in, c l o x a c i l l i n , dicloxacill in, methicillin, n a f c i l l in, h e t a c i l l i n and c l o m e t o c i l l i n . A m p i c i l l i n and a m o x y c i l l i n m i g r a t e d t o t h e cathode. Jusko”

s u b m i t t e d t o agar g e l e l e c t r o p h o r e s i s serum samples o b t a i n e d a f t e r

i n t r a v e n o u s a d m i n i s t r a t i o n o f a m p i c i l l i n . A l i q u o t s o f 0.01 m l o f serum were app l i e d t o t h e a g a r g e l b u f f e r e d w i t h T r i s - m a l e a t e a t pH 5.6.

Electrophoresis o f

these, and a l s o o f a m p i c i l l i n standards, was c a r r i e d o u t a t a p p r o x i m a t e l y 240 V and 60 mA f o r 45 min. The a g a r g e l was t h e n o v e r l a y e d w i t h 1.5% n u t r i e n t agar ( D i f c o ) c o n t a i n i n g B . subtiZis spores. A f t e r i n c u b a t i o n a t 35OC f o r 16-20 h, t h e bioautogram was examined f o r i n h i b i t i o n zones. Agarose g e l o r h y d r o l y s e d s t a r c h was used i n e l e c t r o p h o r e t i c s e p a r a t i o n s o f c l i n i c a l l y used s e m i s y n t h e t i c p e n i c i l l i n s 1 2 . A method f o r l o w - v o l t a g e paper e l e c t r o p h o r e s i s , u s e f u l f o r t h e r a p i d d e t e c t i o n o f p e n i c i l l i n s and t h e i r d e g r a d a t i o n p r o d u c t s i n r a b b i t serum and u r i n e and f o r f o l l o w i n g t h e b a c t e r i a l h y d r o l y s i s o f p e n i c i l l i n s i n b r o t h c u l t u r e , was d e s c r i b e d b y t h e same a u t h o r s .

412 The e x c r e t i o n and m e t a b o l i c f a t e o f

[14CJcephachlomezine

i n r a t s was m o n i t o r e d

i n u r i n e and b i l e samples by measurement o f r a d i o a c t i v i t y and a n t i b i o t i c a c t i v i t y , and a l s o by paper chromatography and paper e l e c t r o p h ~ r e s i s l ~I .n t h e l a t t e r , 1 5 - ~ 1 p o r t i o n s o f s u i t a b l e u r i n e and b i l e d i l u t i o n s were s p o t t e d on s t r i p s o f Toyo No. 5 1 paper ( 2 x 40 cm). E l e c t r o p h o r e s i s was c a r r i e d o u t i n 0.05 m o l / l p y r i d i n e a c e t a t e b u f f e r (pH 4 . 5 ) a t 14 V/cm f o r 1.5 h. The r a d i o a c t i v i t y on t h e paper s t r i p s was examined w i t h a radio-chromato-scanner. Areas r e p r e s e n t i n g a n t i b i o t i c a c t i v i t y were l o c a t e d by b i o a u t o g r a p h y u s i n g Streptococcus kaemoZyticus S-23 as t h e t e s t organism. Under t h e c o n d i t i o n s used, t h e o r i g i n a l a n t i b i o t i c m i g r a t e d 2.0 cm t o wards t h e anode and two i t s m e t a b o l i t e s by 9.8 cm and 12.9 cm, r e s p e c t i v e l y , a l s o towards t h e anode. P r e p a r a t i v e paper e l e c t r o p h o r e s i s u s i n g 10% a c e t i c a c i d as b u f f e r a f f o r d e d p u r e 7a-methoxycephalosporin C, p a r t i a l l y s y n t h e s i z e d f r o m benzhydryl 7-amino-7cl-meth14

oxycephalosporanate by a c y l a t i o n

.

E l e c t r o p h o r e s i s was used t o c h a r a c t e r i z e v a r i o u s r e a c t i o n o r h y d r o l y s i s p r o d u c t s o f 8-lactam a n t i b i o t i c s . Products o f t h e h y d r o l y s i s o f f i v e p e n i c i l l i n s and t h e i r sulphoxides b e f o r e and a f t e r t i t r a t i o n w i t h p e r i o d a t e were c h a r a c t e r i z e d by h i g h v o l t a g e e l e c t r o p h o r e s i s and t h i n - l a y e r c h r ~ m a t o g r a p h y l ~R. e a c t i o n p r o d u c t s a f t e r t h e a c t i o n o f p r i m a r y and secondary a r o m a t i c amines on c e p h a l o s p o r a n i c a c i d s were s u b m i t t e d t o e l e c t r o p h o r e s i s a t pH 1.9 t o 7.0 t o d e t e c t t h e i r n e t charge and m i g r a t i o n 1 6 . Sabath e t a1.17 analysed t h e p r o d u c t s o f t h e h y d r o l y s i s o f benzylpen i c i l l i n , c e p h a l o s p o r i n C, cephaloram, c e p h a l o t i n and c e p h a l o r i d i n by B-lactams o f Pseudomonas aeruginosa. 7-Aminocephalosporanic a c i d and o t h e r p r o d u c t s o f c e p h a l o s p o r i n C h y d r o l y s i s were separated by paper e l e c t r o p h o r e s i s and paper c h r o 18 matography

.

AMINOGLYCOSIDE AND OTHER CARBOHYDRATE ANTIBIOTICS Hosoya and c o - w o r k e r s l Y 2 a p p l i e d e l e c t r o p h o r e s i s t o t h e s e p a r a t i o n o f roseot h r i c i n s and s t r e p t o c y m i n on f i l t e r - p a p e r ( 1 x 20 cm) i n 0 . 1 m o l / l a c e t a t e b u f f e r (pH 5.0) o r 0 . 1 m o l / l phosphate b u f f e r (pH 7.0) f o r 4-5 h. R o s e o t h r i c i n s were separated i n t o two components whereas s t r e p t o m y c i n behaved as one component. I n h i g h - v o l t a g e paper e l e c t r o p h o r e s i s u s i n g a c e t i c a c i d - f o r m i c a c i d - w a t e r (75:25:900) a t 3500 V f o r 15 min, a d e n y l y l s t r e p t o m y c i n ( a p r o d u c t o f s t r e p t o mycin i n a c t i v a t i o n by E. c o Z i ) moved 10.5 cm and s t r e p t o m y c i n 13.0 cm towards 19 t h e cathode

.

H i g h - v o l t a g e paper e l e c t r o p h o r e s i s o f kanamycin, t h e i n a c t i v a t e d kanamycins, paromamine and t h e i n a c t i v a t e d paromamine was c a r r i e d o u t on Toyo No. 5 1 paper ( 2 x 60 cm) i n a c e t i c a c i d - f o r m i c a c i d - w a t e r (75:25:900) a t 3500 V f o r 15 min. [3Hlkanamycins, a l s o by r a d i o a c -

D e t e c t i o n was e f f e c t e d w i t h n i n h y d r i n and, f o r

413 t i v i t y scanning. The t r a v e l l i n g d i s t a n c e s t o w a r d t h e cathode were as f o l l o w s : kanamycin 13.1-15.4 cm, i n a c t i v a t e d kanamycin-I 10.4-11.5 cm, i n a c t i v a t e d kanam y c i n - I 1 7.5-8.6 cm, paromamine 12.5-15.0 cm, i n a c t i v a t e d paromamine 7.8-9.8 cm and kanamycin-6'-phosphate 11.2 cm20

.

3',4'-Dideoxykanamycin

8 - 2 ' ' - a d e n y l a t e was p r e p a r e d by enzymic a d e n y l a t i o n o f

t h e p a r e n t a n t i b i o t i c . On h i g h - v o l t a g e paper e l e c t r o p h o r e s i s a t 3 5 0 0 V f o r 15 min u s i n g a c e t i c a c i d - f o r m i c a c i d - w a t e r (75:25:900),

t h e p a r e n t compound moved 16.8

cm towards t h e cathode whereas i t s a d e n y l a t e moved 13.4 cm2 1 3',4'-Dideoxykanamycin

.

B (DKB) and two o f i t s i n c a t i v e d e r i v a t i v e s , 3 ' , 4 ' - d i -

deoxykanamycin B - 2 " - g u a n y l a t e

(GDKB) and 2 " - a d e n y l a t e

(ADKB), were compared by

h i g h - v o l a t g e e l e c t r o p h o r e s i s a t 3500 V f o r 15 min u s i n g a c e t i c a c i d - f o r m i c a c i d w a t e r (75:25:900)

and were d e t e c t e d w i t h n i n h y d r i n . GDKB moved 9.5 cm, DKB 14.0

cm and ADKB 10.7 cm towards t h e cathode".

The same a u t h o r s 2 3 compared 3 ' , 4 ' - d i -

deoxykanamycin B and i t s i n a c t i v e 6 ' - N - a c e t y l

d e r i v a t i v e by t h e same t e c h n i q u e .

The i n a c t i v a t e d DKB moved 11.2 cm and DKB 13.5 cm towards t h e cathode. M o r i e t a l . 2 4 c h e m i c a l l y c o n v e r t e d l i v i d o m y c i n A i n t o l i v i d o m y c i n B by e l i m i n a t i n g t h e niannose moiety. The o r i g i n a l and c o n v e r t e d a n t i b i o t i c were compared by h i g n - v o l t a g e e l e c t r o p h o r e s i s a t 3000 V and 1 mA/cm, on Toyo No. 51 paper u s i n g a c e t i c acid-formic acid-water

(75:25:900)

(pH 1.8).

The m o b i l i t i e s , r e l a t i v e t o

a l a n i n e = 1.0, O F l i v i d o m y c i n A and B were 2.14 and 2.16,

respectively. Detection

was e f f e c t e d w i t h n i n h y d r i n and by b i o a u t o g r a p h y . Yamamoto e t a1.25 compared t h e m e t h a n o l y s i s p r o d u c t s o f l i v i d o m y c i n A and of e n z y m i c a l l y o r s y n t h e t i c a l l y p h o s p h o r y l a t e d l i v i d o m y c i n A 5 ' '-phosphate by h i g h v o l t a g e e l e c t r o p h o r e s i s u s i n g a c e t i c a c i d - f o r m i c a c i d - w a t e r (75:25:900)

a t 3000

V f o r 20 min. D e t e c t i o n w i t h n i n h y d r i n gave p o s i t i v e r e s u l t s w i t h t h e t h r e e p a r e n t

p r e p a r a t i o n s and a l s o w i t h t h e i r m e t h a n o l y s i s p r o d u c t s . P h o s p h a t e - c o n t a i n i n g p r o d u c t s and l i v i d o m y c i n A 5 ' I - p h o s p h a t e were a l s o d e t e c t e d w i t h Hanes and I s h e r wood'sz6 r e a g e n t . L i v i d o m y c i n A and B and two a n t i b i o t i c s of t h e paromomycin group, i s o l a t e d from Streptomyces Zividus, were compared w i t h known a m i n o g l y c o s i d e a n t i b i o t i c s by t h i n - l a y e r chromatography and e l e ~ t r o p h o r e s i s ~L~i v. i d o m y c i n s and N. 2230-C c o u l d be d i s t i n g u i s h e d f r o m paromomycin by b o t h methods, whereas a n t i b i o t i c N. 2230-D proved t o be i d e n t i c a l w i t h paromomycin ( T a b l e 18.1).

A phosphoaminosugar ( p o s s i b l y p h o s p h o r y l a t e d neomycin C), when s u b m i t t e d t o paper e l e c t r o p h o r e s i s a t 140 V f o r 2.5 h a t 4OC i n d i f f e r e n t b u f f e r s ( f o r m i c acid-0.2 m o l / l ammonia) a t pH 3.5, 7.0 and 9.0, m i g r a t e d 7.6, 3.1 and 0.8 cm, 28 r e s p e c t i v e l y , towards t h e cathode 3 r e p o r t e d a successful p r e p a r a t i v e s e p a r a t i o n o f kanamycin Umezawa and Kondo

.

and 6'-N-tert.-butyloxycarbonylkanamycin trophoresis.

by meansof c a r r i e r - f r e e c o n t i n u o u s e l e c -

414 TABLE 18.1 COMPARISON OF THIN-LAYER CHROMATOGRAPHY AND HIGH-VOLTAGE ELECTROPHORESIS OF

LIVIDOMYCIN A AN0 B, NO. 2230-C AND NO. 2230-D WITH OTHER AMINOGLYCOSICID ANTIBIOTICS (FROM REF. 27)

.,"

RFa

Lividomycin A Lividomycin B NO. 2230-C NO. 2 2 3 0 4 Kanamycin Neomyc in Paromomyc in Gentami c i n

A

B

C

0.64 0.65 0.57 0.58 0.64 0.46 0.58 0.71

0.36 0.73 0.27 0.64 0.62 0.65 0.64 0.89

1.78 2.10 1.76 2.06 1.94 2.05 2.06 2.14

aA: T h i n - l a y e r chromatography u s i n g s i l i c a gel D-5 (Camag) and t h e s o l v e n t system chloroform-methanol-17% ammonia ( 2 : l : l ) (upper l a y e r ) . B: T h i n - l a y e r chromatography u s i n g aluminium o x i d e G t y p e E (Merck) and t h e same s o l v e n t system as i n A. C : High-voltage e l e c t r o p h o r e s i s a t 3000 V (20 mA per 10 cm); e l e c t r o l y t e s o l u t i o n , f o r m i c a c i d - a c e t i c acid-water (22:75:900) (pH 1.8); Toyo No. 51 paper. RM=mobility r e l a t i v e t o alanine=l.O. Detection: n i n h y d r i n .

Vancomycin was l a b e l l e d w i t h t e r i a l cells2'.

125

I and used t o f o l l o w i t s d i s t r i b u t i o n i n bac-

The aqueous s o l u t i o n o f r a d i o a c t i v e vancomycin was p u r i f i e d by

continuous-flow paper e l e c t r o p h o r e s i s u s i n g 0.1 m o l / l f o r m i c a c i d as e l e c t r o l y t e .

A constant c u r r e n t o f 40 mA was passed and t h e a p p l i e d p o t e n t i a l was ca. 500 V. F r a c t i o n s were c o l l e c t e d , t h e tubes being changed a u t o m a t i c a l l y every 12 h. The p o s i t i o n o f t h e product was determined by measuring r a d i o a c t i v i t y and t h e molar a b s o r p t i v i t y a t 280 nm. A s i n g l e peak was o b t a i n e d i n a tube s l i g h t l y towards t h e cathode. High-voltage paper e l e c t r o p h o r e s i s was a p p l i e d t o t h e a n a l y s i s o f products o f a c i d h y d r o l y s i s o f bleomycin A2 on Toyo No. 51 paper u s i n g f o r m i c a c i d - a c e t i c acid-water (25:75:900)

as t h e b u f f e r . Seven products were d e t e c t e d w i t h n i n h y d r i n

(see r e f . 30). Paper e l e c t r o p h o r e s i s was a l s o used t o c h a r a c t e r i z e a peptide, 31

t e t r a p e p t i d e S, from bleomycin A2

.

PEPTIDE ANTIBIOTICS Amino a c i d s i n hydrolysates o f actinomycin m i x t u r e s were separated by highv o l t a g e e l e c t r o p h o r e s i s (4% formate b u f f e r , 4800 V, 3 h ) o r by h i g h - v o l t a g e e l e c -

415 t r o p h o r e s i s i n one dimension and ascending paper chromatography i n a n o t h e r ( b u t a n o l - a c e t i c a c i d - w a t e r , 4:1:2).

The e l e c t r o p h o r e t i c m o b i l i t i e s o f v a r i o u s amino

a c i d s r e l a t i v e t o t h a t o f s a r c o s i n e were determined. D e t e c t i o n was e f f e c t e d w i t h n i n h y d r i n and p - n i t r o b e n z o y l c h l o r i d e - p y r i d i n e ( f o r N-methylamino a c i d s ) 32

.

I n a s y n t h e t i c approach t o g r a m i c i d i n S, i n t e r m e d i a t e s were analysed by e l e c t r o p h o r e s i s on Toyo No. 52 paper w i t h f o r m i c a c i d - a c e t i c acid-methanol-water (1:3:6:10)

(pH 1.8) and a v o l t a g e g r a d i e n t o f 17 V/cm. Substances b e a r i n g f r e e

amino groups were d e t e c t e d w i t h n i n h y d r i n and h e a t i n g ; substances w i t h b l o c k e d amino groups were d e t e c t e d w i t h 47% hydrobromic a c i d f o l l o w e d by h e a t i n g and t h e n n inhyd r in

.

Syn t he t ic [4 ,5- 6-a m i nova 1e r ic a c id]

- gram ic id in

S wa s c ha r a c t e r ized

by paper e l e c t r o p h o r e s i s (15 x 40 cm) i n f o r m i c a c i d - a c e t i c a c i d - w a t e r (4:15:180) (pH 1.9) a t 600 V f o r 2 h. One n i n h y d r i n - p o s i t i v e s p o t was found (11.8 cm towards 34 t h e cathode) moving s l i g h t l y f a s t e r t h a n g r a m i c i d i n S 14 Crude [2g g l y c y l p o l y m y x i n B was p u r i f i e d by p r e p a r a t i v e paper e l e c t r o p h o -

.

r e s i s on Whatman M3 paper a t 22 V/cm i n a b u f f e r o f pH 5.7 f o r 2 h. A f t e r e l u t i o n 35

w i t h 0.05 mol/l a c e t i c a c i d , e v a p o r a t i o n and d r y i n g , a p u r e p r o d u c t was o b t a i n e d SDS-polyacrylamide gel e l e c t r o p h o r e s i s was used i n s t u d i e s o f p o l y m y x i n b i o s y n 36 t h e s i s b y Bacillus poZymyxa

.

The " c o r e h e x a p e p t i d e " o f amphomycin was c h a r a c t e r i z e d by h i g h - v o l t a g e e l e c t r o p h o r e s i s , c a r r i e d o u t on t h e f l a t p l a t e o f a Savant a p p a r a t u s . I n p y r i d i n e a c e t i c a c i d - w a t e r (25:1:225)

a t pH 6.5 and 2000 V f o r 1 h, t h e p e p t i d e moved 5.2

cm towards t h e cathode. I n 2 m o l / l a c e t i c a c i d a t 2200 V f o r 1.5 h, i t t r a v e l l e d 16.3 cm towards t h e cathode. I n 0.05 mol/l b o r a t e b u f f e r , a d j u s t e d w i t h 2 m o l / l NaOH t o pH 10, a t 3000 V f o r 45 min, t h e c o r e p e p t i d e moved 2 cm towards t h e c a t h ode 37

.

H i g h - v o l t a g e e l e c t r o p h o r e s i s o f capreomycidine ( a d e g r a d a t i o n p r o d u c t o f v i o mycin) was performed on Whatman No. 3 paper w i t h a c e t i c a c i d - f o r m i c a c i d b u f f e r (pH 1.9)38.

I n s i m i l a r s t u d i e s 3 ' Whatman No. 3 MM paper, a c e t i c a c i d - f o r m i c a c i d -

water (20:2:78)

and a p o t e n t i a l o f 35 V/cm and 50 mA f o r 2 h were used. D e t e c t i o n

was e f f e c t e d w i t h n i n h y d r i n i n 1 - b u t a n o l . A i r d r y i n g r e v e a l e d most o f t h e h y d r o l y s a t e components f r o m viomycin, and h e a t i n g b r i e f l y a t l l O ° C

was r e q u i r e d i n o r d e r

t o develop t h e s p o t c o r r e s p o n d i n g t o v i o m y c i d i n e . Urea was d e t e c t e d as a b r i g h t y e l l o w s p o t w i t h E h r l i c h r e a g e n t . I n f e r m e n t a t i o n samples40, i n t e r m e d i a t e s and v i o m y c i n were determined on Whatman No. 1 paper impregnated w i t h a s o l u t i o n

Of

6.23 g o f Verona1 and 4.11 g o f sodium a c e t a t e i n 1 1 o f w a t e r a d j u s t e d t o pH 8 w i t h 1 m o l / l HC1, a t 3000 V and a p o t e n t i a l g r a d i e n t o f 115 V/cm f o r 3 h. Detect i o n was e f f e c t e d w i t h n i n h y d r i n . The amounts o f v i o m y c i n were e s t i m a t e d by t h e spot-area method. Under h i g h - v o l t a g e paper e l e c t r o p h o r e s i s a t 3300 V f o r 20 m i n i n f o r m i c a c i d a c e t i c a c i d - w a t e r (25:75:900),

cyclamidomycin (a pro1 i n e d e r i v a t i v e ) moved 12.9

.

416 cm towards t h e cathode w i t h RM = 1.29 ( L - a l a n i n e = 1 . 0 ) . It was d e t e c t e d by b i o 41 autography w i t h KZebsieZZa pnewnociae . E l e c t r o p h o r e s i s has a l s o been a p p l i e d i n s t u d i e s o f p e p t i d e s such as edeines

42

,

t y r ~ c i d i n e ~e ~t a, m y ~ i n ~and ~ , many o t h e r s . ivlISCELLANEOUS I n s c r e e n i n g new a n t i b i o t i c s , h i g h - v o l t a g e paper e l e c t r o p h o r e s i s proved t o be a u s e f u l t o o l f o r t h e s e p a r a t i o n and c h a r a c t e r i z a t i o n of a n t i b i o t i c s . The r e l a t i v e m o b i l i t i e s o f 92 a n t i b i o t i c s were e s t i m a t e d by Maeda e t a1 .45. The a n t i b i o t i c s were d e t e c t e d by bioautography (B. s u b t i l i s , S. aureus o r E. c o Z i ) , n i n h y d r i n , t h e Sakaguchi r e a c t i o n and UV l i g h t . The compounds s t u d i e d i n c l u d e d s e v e r a l macro1 i d e s , t e t r a c y c l i n e s , polyenes, aminoglycosides and n u c l e o s i d e s . Lightbown and De Rossi‘

s t u d i e d e x t e n s i v e l y 2 1 a n t i b i o t i c s by agar gel e l e c -

t r o p h o r e s i s and bioautography. TABLE 18.2 MIGRATION OF ANTIBIOTICS AS A FUNCTION OF THE ELECTROLYTE (ADAPTED FROM REF. 7 ) Anti b i o t i c

D i s t a n c e (cm) i n e l e c t r o l y t e

Aa

Detection‘

Bb

M i g r a t i o n towards t h e cathode Streptomycin Erythromycin 01 eandomycin Spiramyci n T y l o s in Baci t r a c i n Chlortetracycl ine Oxytetracycl i n e Tetracycline Virginiamycin Framycetin Neomyc in

20 20 20

ia 14 13 13 13 9

-

29 17 17 17 9 13

-

13 7-20 7-20

Bs Bc, Bc, Bc, Bc, Bc, Bc, Bc, Bc, Mf, Bs Bc

Mf, Mf, Mf, Ilf, Mf, Bs,

S1 S1 S1 S1 S1 S1 Bs, S1 Bs, S1 S1

M i g r a t i o n towards t h e anode F1avomyc in Penicillin

9 13

6 10

Bc Bs

a0.075 m o l / l Tris(hydroxymethyl)aminomethane, 0.075 m o l / l m a l e i c a c i d and 0.023 bmol/l NaOH (pH 5.6). 0.10 m o l / l Sodium d i e t h y l b a r b i t u r a t e and 0.02 m o l / l d i e t h y l b a r b i t u r i c a c i d (pH 8.6). ‘Bc=BaciZZus cereus ; Bs=BaciZZus s u b t i l i s ; Mf=Micrococcus f l a m s ; S1 =Sarcina Zutea.

417

0-Demethyl puromycin was p u r i f i e d by p r e p a r a t i v e paper e l e c t r o p h ~ r e s i s ~Puro~. 47 mycin and N - f o r m y l m e t h i o n y l puromycin were c h a r a c t e r i z e d by paper e l e c t r o p h o r e s i s R e c e n t l y , t h e i n t e r a c t i o n s o f DNA f r o m t h e b a c t e r i o p h a g e PM-2 w i t h 20 known a n t i n e o p l a s t i c a n t i b i o t i c s were s t u d i e d u s i n g agarose g e l e l e c t r o p h o r e s i s and f l u o r e s c e n c e assay systems48. A new method was t h u s e l a b o r a t e d f o r s c r e e n i n g a n t i tumour compounds i n samples f r o m m i c r o b i a l f e r m e n t a t i o n s . Ochab4’ compared t h e e l e c t r o p h o r e t i c m o b i l i t i e s on paper o f t e t r a c y c l i n e , oxyt e t r a c y c l i n e , c h l o r t e t r a c y c l ine, demethylchlortetracycl ine, r o l i t e t r a c y c l ine, m e t a c y c l i n e , v i b r a m y c i n , n y s t a t i n , t r i c h o m y c i n , natamycin and a m p h o t e r i c i n s A and B. Agar gel e l e c t r o p h o r e s i s , f o l l o w e d by b i o a u t o g r a p h y , was used t o i d e n t i f y 14 a n t i b i o t i c s i n f e e d samples’.

The s e p a r a t i o n was c a r r i e d o u t a t two d i f f e r e n t pH

v a l u e s u s i n g 1000 V and 250 mA f o r 2 h. M i g r a t i o n s o f a n t i b i o t i c s as a f u n c t i o n of t h e e l e c t r o l y t e a r e p r e s e n t e d i n T a b l e 18.2. H o r n i n g e t a1 .50 d e s c r i b e d t h e s y s t e m a t i c a n a l y s i s o f r e s i d u a l a n t i b i o t i c s i n foods u s i n g a g a r gel e l e c t r o p h o r e s i s and a n t i m i c r o b i a l s p e c t r a a g a i n s t s i x m i c r o b i a l species. REFERENCES 1 S. Hosoya, M. Soeda, N. Komatsu, N. Hara, Y . Sonoda and R. Arai; J . Antibiot., 4 (1951) 314. 2 S . Hosoya, M. Soeda, N. Komatsu, Y . Sonoda and R. A r a i , J . Antibiot., 4 (1951) 317. 3 H. Umezawa and S . Kondo, Methods EnzymoZ., 43 (1975) 279. 4 V. B e t i n a , J . Chrornatogr., 78 (1973) 41. 5 V. B e t i n a , Methods EnzymoZ.., 43 (1975) 100. 6 J.W. Lightbown and P. de Rossi, Analyst (London), 90 (1965) 89. 7 F. Bozzi and P. Valdebouze, J . Chromatogr., 87 (1973) 305. P h a m a z i e , 32 (1977) 594. 8 0. MarkoviC, L. F a i t h and L. Nov-ikovi, 9 0. Markovic‘ and L. Kuniak, J . Chrornatogr., 91 (1974) 873. 10 0. MikeS, CoZZect. Czech. Chem. Cormnun., 22 (1957) 831. 11 W.J. Jusko, J . Pharm. S c i . , 60 (1971) 728. 12 A.H. Thomas and R.A. Broadbridge, AnaZyst (London), 95 (1970) 459. 13 M. O k u i , K. H a t t o r i and M. N i s h i d a , J . A n t i b i o t . , 24 (1971) 667. 14 R.W. R a t c l i f f e and B.G. C h r i s t e n s e n , Tetrahedron L e t t . , (1972) 2907. 15 H. Moehrle and G. L u t h e r , Deut. Apoth.-Ztg., 111 (1971) 1937. 16 J. Bradshaw, S. E a r d l e y and A.G. Long, J . Chem. Soc. C , (1968) 801. 17 L.D. Sabath, M. Jag0 and E.P. Abraham, Biochem. J . , 96 (1965) 739. 18 E.P. Abraham and G.G.F. Newton, Biochem. J . , 79 (1961) 377. 19 H. Umezawa, S. Takasawa, M. O k a n i s h i and R. Utahara, J . A n t i b i o t . , 2 1 (1968) 81. 20 S. Kondo, M. Okanishi, R. Utahara, K. Maeda and H. Umezawa, J . Antibiot., 21 (1968) 22. 21 M. Yagisawa, H. Naganawa, S. Kondo, M. Hamada, T. Takeuchi and H. Umezawa, J . A n t i b i o t . , 24 (1971) 911. 22 M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi and H. Umezawa, J . A n t i b i o t . , 25 (1972) 492. 23 M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi and H. Umezawa, J . A n t i b i o t . , 25 (1972) 495.

.

418

24 25 26 27 28 29 30 31 32 33

T. M o r i , Y. K y o t a n i , I. Watanabe and T. Oda, J . A n t i b i o t . , 25 (1972) 149. H. Yamamoto, S. Kondo, K. Maeda and H. Umezawa, J . A n t i b i o t . , 25 (1972) 485. C.S. Hanes and F.A. Isherwood, Nature (London), 164 (1949) 1107. T. M o r i , T. I c h i y a n a q i , H. Kondo, K. Tunaka, T. Oda and K. Munakata, J . A n t i b i o t . , 24 ( f i 7 1 ) - 3 3 9 . M.K. Majumdar and S.K. Majumdar, J . Antibiot., 22 (1969) 174. H.R. P e r k i n s and M. N i e t o , Biochem. J . , 116 (1970) 8 3 . T. T a k i t a , Y. Muraoka, K. Maeda and H. Umezawa, J . A n t i b i o t . , 21 (1968 79 * Y. Muraoka, T. T a k i t a , K. Maeda and H. Umezawa, J . A n t i b i o t . , 25 (1972 185. T. Yajima, M.A. G r i g g and E . Katz, Arch. Biochem. Biophys., 151 (1972) 565. M. Ohno, K. Kuromizu, H. Ogawa and N. Izumiya, J . Amer. Chern. Soc., 93 (1971)

5251. 34 I . Muramatsu, S. Sofuku and A. H a g i t a n i , J . A n t i b i o t . , 25 d1972) 189. 35 M. Havranek and K. Vere;, 2. Natur.forsch. B , 26 (1971) 451. 36 R. B a l a k r i s h n a n , S. K a u r i A.K. G o d , S. Padmavathi and K. Jayaraman, Arch. Biochem. Biophys., 200 (1980) 45. 37 M. Bodanszky, A.A. Bodanszky, C.A. R a l o f s k y , R.C. S t r o n g and R.L. F o l t z , J . A n t i b i o t . , 24 (1971) 294. 38 B.W. B y c r o f t , L.R. C r o f t , A.W. Johnson and T. Webb, J . Chem. Soc. Perkin I, (1972) 820. 39 G . Biichi and J.A. Raleigh, J . Org. Chem., 36 (1971) 873. 40 Z . K o t u l a , P. Bukowski and Z. Kowszyk-Glndifer, Med. Dosw. MikrobioZ., 22 (1970) 95. 4 1 S. Takahashi, M. Nakajima, Y. Ikeda, S. Kondo, M. Hamada, K. Maeda and H. Umezawa, J . A n t i b i o t . , 24 (1971) 902. 42 T.P. M e t t i n g e r and L.C. C r a i g , Biochemistry, 9 (1970) 1224. 43 K. Kuromizu and N. Izumiya, B U Z Z . Chem. Soc. J a p . , 43 (1970) 2944. 44 J.E. Walker, M. Bodanszky and D. Perlman, J . A n t i b i o t . , 23 (1970) 255. 45 K . Maeda, A. Yagi, H. Naganawa, S . - I . Kondo and H. Umezawa, J . A n t i b i o t . , 22 (1969) 635. 46 M.M. Rao, P.R. Rebello and B.M. Pogell , J . BioZ. Chem., 244 (1969) 112. 47 H. Bachmayer and G. K r e i l , Biochim. Biophys. A c t a , 169 (1968) 95. 48 S. Mong, J.E. Strong, J.A. Bush and S.T. Crooke, Antimicrob. A g . Chemother., 16 (1979) 398. 49 S. Ochab, Diss. Pharm. PharmacoZ., 24 (1972) 205. 50 C.-B. Horng, J.-T. Hsieh, H.-C KO, R.-H. Jan and J.H. L i , .?roc. Nut. S c i . Counc. Repub. China, 3 (1979) 382.

419

Chapter 19 DYES AND PIGMENTS

2 . DEYL W a t e r - s o l u b l e dyes, e s p e c i a l l y t h o s e used i n f o o d c h e m i s t r y , were one o f t h e f i r s t c a t e g o r i e s of compounds t o be used t o demonstrate e l e c t r o p h o r e t i c separat i o n s . A g r e a t v a r i e t y o f e l e c t r o l y t e s have been used: 1 m o l / l ,

5.2 m o l / l a c e t i c acid'",

1.75 m o l / l and

p h o s p h a t e - c i t r a t e b u f f e r s (0.2 m o l / l disodium phosphate

and 0.1 m o l / l c i t r i c a c i d a d j u s t e d t o pH 3.0, 4.0 and 5.0 w i t h sodium h y d r o x i d e ) '3 , p y r i d i n e - a c e t i c a c i d - w a t e r (pH 3.5)3, phosphate b u f f e r s 3 , 0.1 m o l / l sodium a c e t a t e and i s o p r o p a n o l

(pH a d j u s t e d t o 4.6 w i t h g l a c i a l a c e t i c a c i d ) 4 , b o r a t e b u f f e r s

(0.2 m o l / l b o r i c a c i d , 0.2 n o l / l potassium c h l o r i d e , pH a d j u s t e d t o 6.9 o r 10.1) ammonia (0.06-0.1

3,

m o l / l ) o r sodium hydrogen c a r b o n a t e (0.06 m o l / l ) ? . T h i s l i s t i s

f a r f r o m e x h a u s t i v e ; a number o f v a r i a t i o n s o f these b u f f e r systems have been r e p o r t e d ( f o r a r e v i e w , see r e f . 5 ) . There a r e two f a c t s t h a t have t o b e remembered w i t h r e g a r d t o t h e s e p a r a t i o n o f s y n t h e t i c dyes. Some o f t h e dyes a r e i n d i c a t o r s and may b e c o l o u r l e s s below o r above c e r t a i n pH values, w h i c h n i i g h t cause problems i n d e t e c t i o n and t h e conc e n t r a t i o n o f b u f f e r s must be h i g h enough t o p r e v e n t t h e zone spreading t h a t occ u r s i n d i l u t e e l e c t r o l y t e s o l u t i o n s . Some s y n t h e t i c dyes, such as Congo r e d dyes, a r e s t r o n g l y absorbed on paper and would n o t move i n t h e usual b u f f e r system under t h e i n f l u e n c e o f an e l e c t r i c f i e l d . I n o r d e r t o overcome t h i s d i f f i c u l t y , K i t a o k a e t a1 .6 used aqueous dimethylformamide s o l u t i o n c o n t a i n i n g sodium c h l o r i d e as r u n n i n g b u f f e r ( d i m e t h y l formamide-0.1 m o l / l NaCl, 1:l o r 5 : l ) . E l e c t r o p h o r e t i c m o b i l i t i e s o f a number o f dyes a r e g i v e n i n Tables 19.1 and 19.2. D e t e c t i o n , q u a n t i t a t i o n and t h e procedure a t b o t h l o w and h i g h v o l t a g e f o l l o w t h e convent i o n a l procedure w i t h paper as s o r b e n t . Tewari e t a1.7 used paper e l e c t r o p h o r e s i s i n K o l t h o f f b u f f e r o f pH 11 (50 m l o f 0.1 m o l / l sodium carbonate, 3 m l o f 0.1 m o l / l h y d r o c h l o r i c a c i d ) f o r t h e separ a t i o n o f a number o f dyes used i n l i q u o r s and beverages ( T a b l e 19.3). I n s t e a d

5% a c e t i c 10% f o r m i c a c i d ( b u f f e r B), M c I l v a i n e b u f f e r (pH 3 ) (411 m l o f

o f paper, s i l i c a g e l G can be made use o f w i t h e q u a l l y good r e s u l t s : acid (buffer A),

0.2 m o l / l Na2HP04

+

1589 m l o f 0.1 m o l / l c i t r i c a c i d , b u f f e r C ) , M c I l v a i n e b u f f e r

(pH 7 ) (1647 m l o f 0.2 m o l / l 14a2HP04

+

353 m l of 0.1 m o l / l c i t r i c a c i d , b u f f e r

and Sb'rensen b u f f e r (pH 10) (1200 m l o f 0.5 m o l / l borax

+

000 m l o f 0.1 m o l / l

sodium h y d r o x i d e b u f f e r E ) a r e used i n t h e s e s e p a r a t i o n s (300 V, 60 min, 20-cm p l a t e ) ( T a b l e 19.4).

D)

TABLE 19.1 ELECTROPHORETIC MIGRATIONS ON CELLULOSE PAPER OF SOME WATEE-SOLUBLE FOOD DYES (REF. 3 ) a ~~

~~

M i g r a t i o n (cm) i n e l e c t r o l y t e b o f pH

Red No.1 (Ponceau 32) Red 140.2 (Amaranth) Red No.4 (Ponceau S X ) Red No. 101 (Ponceau R ) Red No.102 (New Coccine) Orange 140.1 (Orange I ) Yellow No.5 ( T a r t r a z i n e ) Yellow No.6 (Sunset Yellow FCF) Yellow No.1 (Naphthol Yellow) Blue No.2 ( I n d i g o Carmine) Red No.3 ( E r y t h r o s i n e ) Red No.103 (Eosine) Red IJo.104 ( P h l o x i n e ) Red No.105 (Rose Bengal) Red No.106 ( A c i d Red) Green No.1 (Guinea green B) Green No.2 ( L i g h t Green SF) Green 140.3 ( F a s t Green FCF) Blue No.1 ( B r i l l i a n t Blue FCF) V i o l e t No.1 ( A c i d V i o l e t 68)

3.0

3.5

4.0

4.5

5.0

5.7

2.1 3.6 2.4 2.9 9.0 1.1 6 .4d 4.4 8.3

5.0 6.9 G.7 7.2 13.2 3.6 15. 5d 12.3 13.8 9.5

1.3 3.7 2.8 2.3 8.7 0.3 7Jd 5.2 8.3 3.9

0.7 3.2 2.3 l.G 9.0 1.4 7.1d 6.0 6.7 2.7

1.4 3.0 1.9 2.0 7.4 1.0 7.Zd 5.1 8.6 4.0 9.6 1.0

1.c 3.6 2.6 3.0 10.3 1.7 8.2d 6.5 9Io 1.5 00.5 1.7 2.Sf 2.6 6.19 5.0 10.2 9.3 9.8 2.69

_3e6

6.0f 7.3f 6.8 6.1f 8.7f

-

6.7 13.3 11.9 12.5d 7.5

-

-

-

-

4.5 8.0f f

4.99 4.7

5.0 5.3f

8.0 7.9d 7.8

3.0 8.9 8.6 3.89

6.8d 7.7 7.7

-

-

-

5.8 1.0 2.3 2.4 2.2

lo.oc 1.3 6.gd 5.2 8.3 2.G 0.4 1.2 2.8 2.0 4..4 5.8; 8.0 8.2d 9.1 5.0g

6.9

7.3

3.1

10.1

11.6

1.2 3.8 3.2 3.0 ll.OC 1.2 8.0 6.5d 3.1 3.5d 0.5 1.1 3.2 1.7 a.5

2.5 6.4 5.5 5.1 12.5 2.7 ll.Od 9.4 13.8 7.0 1.0 2.8 4.7 2.8 6.2

1.7 4.5 3.7 3.0 9.7 1.7 7.4d 6.2 10; 6 4 0

2.2 7.2 8.0 4.8 15.0 6.2' 16.4d 11.7 15.2 7.0d 1.5 3.7 5.8 3.9

3.0 7.2 6.1 5.6 13.5 2.7' 15.0d 12.5 15I2 6.8

-f

-f

13.5 9.2d 10.5 -g

15.5f 14.2 13.8 5.8

-

.

-P3

9.Of 9.4d 9.1 4.8s

5.5f 15.2c 11.8d 7.59

-

$.6 - 1

13.0f 12.0c 11.3 5.8

abWhatman No.1 (4x60 cm) filter-paper; 50 V/cm; 30 min. Buffers: pH 3.0, 0.2 mol/l disodium phosphate-0.1 mol/l citric acid adjusted with sodium hydroxide; pH 3.5, pyridineacetic acid-water (concentration not given); pH 4.0, 0.2 mol/l disodium phosphate-0.1 mol/l citric acid adjusted with sodium hydroxide; pH 4.5, 0.2 mol/l potassium acid phthalate-0.2 mol/l sodium hydroxide; pH 5.0, 0.2 mol/l disodium phosphate-0.1 mol/l citric acid adjusted with sodium hydroxide; pH 5.7, 0.067 mol/l monopotassium phosphate-0.067 mol/l disodium phosphate; pH 5.8, 0.2 mol/l potassium acid phthalate-0.2 mol/l sodium hydroxide; pH 6.9 (0.2 mol/l boric acid-0.2 mol/l potassium chloride)-0.2 mol/l sodium hydroxide; pH 7.3, 0.2 mol/l monopotassium phosphate-0.2 mol/l sodium hydroxide; pH 8.1, 0.2 mol/l monopotassium phosphate-0.2 mol/l sodium hydroxide; pH 10.1 (0.2 mol/l boric acid-0.2 mol/l potassium chloride)-0.2 mol/l sodium hydroxide; pH 11.6, 0.1 mol/l sodium tetraborate-0.1 mol/l sodium hydroxide. :Changed colour. Gave more than one zone. e- indicates colour disappeared under conditions used. fColours are diluted and faded. gCglours spread.

P

f!

TABLE 19.2 ELECTROPHORETIC MIGRATIONS ON CELLULOSE PAPER AND THIN LAYERS OF SOME WATER-SOLUBLE FOOD DYES (REF. l)a

Su p po rt

Ponceau 4R

Pmaranth (Fd and C Red 140.2) F a s t Red E

P K A S P K A S P K

A Carmo is i ne

S P K

A Black PN

Ponceau SX (FD and C Red No.4)

S P K A S P K

A Ponceau M X

S P K A S

M i g r a t i o n (cm/h) i n e l e c t r o l y t e ‘

1

2

3

4

5

6

4.7e 3.9 2.8 4.1 l.ge 3.5f 2.7 4.2 1.1 4.1 2. l e a f 3.7 1.4 3.4f 2.3 3.8 0.6f 3.3 0.1 3.5f 2.1 3.2h 2.5 3.6f 1.3 3.gh 2,6 , 1.7g 3.4

3.Geyf 3.7 4.4 3.9 1.5 3.2f 3.3 4.0 0.9f 3.3 0.5 3.8 0.8 3.2f 3.1 3.4f 0.2 3.3 0.4 3Af 1.2 3.4h 2.5 3.3h 0.9 3.2f 2.2 3.5

2.8e’f 4.4 5.3 4.7 0.8 4.6 4.3f 4.7

1.6f 4.1 4.6 3.9 0.7 4.2 3.7eyf 3.9 0.3 3.4 2.6e5f 3.2h 0.3 3.4 3.4 3.4 0.1 4.4 0.2 3.gf 0.4 3.6 3.7 3.5f 0.2 3.5 2.8e*f 3.4

4.1 5.6 5.3 5.2 1.5 5.8

7.9 2.9 3.2 2.6f 4.6 3.1 1.9eyf 2.3; 2.1 2.4 0.1 2.Ph 3.8f 2.9 2.7 2.1; 2.1 1.6 0 2.3g 5.9f 2.7h 2.4 2.0; 3.0 2.1 2.1eyf 1.9gyh

0.5f

4.5 0.5f 4.2h 0.5 3.8f 4.7 3.4 0.1 4.5

0.lf 4.2f 0.7 3Sf 4.3 3.6f 0.5f 4.2 4.3e.f 3.7

5.1g 4.7 0.4 5.5 2.4e’f 4.7 1.8 :;e,f 4.4 0.4 5.0f 0.3 4.8f 2.6 5.2 3.9eJ 4.6h 1.8 5.1 3.4e,f 4.4

Orange G

P K

A Orange RN

S P K A

S Red 26

P K

A

S Sunset Yellow FCF (FC and C Yellow No.6)

P

K A

S Red GB

Red 10B

P K A S P

K. A

S Chocolate Brown HT

P K A S

Yellow RFS

P K A

S Yellow RY

Brown

P K A

4.4 3.5 3.3 4.0 1.1 2.5 2.4 2.3 2.2 3.4 3.1 3.3 2.2 3.5 3.4 4.0 1.3 3.5 2.7 3.2, 2.3 4.5 3.5 3’6d e 2.5 ’ 2.1d9e 0 2.6e 3.7 4.2 3.8 4.1 4.2

4.0

A

3.4 4.2 0.4 0.2 1. Ed ,-0.4 0.1 1.4

S

2 .l$ ,0.2

S P K

3.0 3.3 4.3 3.4 0.7 2.3f

1.2 2.1, 3.2i 2.1 2.7 4.3 3.1f 1.7 3.0f 3.2 3.gf 1.0 2.7 3.2 3.P 1.3 3.1 4.0 3.4 3.OeYf 2.4eyf 0 l.ge 3.0 3.0 4.7 3.9 2.7 3.6 3.7 4.0 0.3,0.3,1.4 2.5 2.8‘ ,O .gd

2.6e 3.8 5.2 3.4f 0.5 2.6 4.7e, l.gh 1.3 3.2 4.9 3.0h 1.7 3. 5h 5.0 4.3f 0.4 3.6” 4.5 3.4h 0.9 4.1 5.2 3.9

3.Ze

Q.2dse

O1.3hf 4.1 4.2 5 .O 4.3

2.09 4.4 5.3 4.5 0.1,

0.3

2.2,-1.0 4.7,4.1 0.6

1.4e 3.6 3.4 3.6 0.2 2.7 2.0h 2.5 0.8 3.1 3.1 2.3 0.9 3.7 3.6 3.5 0.1 3.2 3.4 3.3h 0.5 3.0 3.3 313

1.0;’e 1.8 0 2.8d 2.4 2.9 3.5 2.9, 0.7 4.0 4.0 3.9 0.1, 3.6, 3.6, 2.9,

0.4 1.6 2.8 2.1

4.2 4.6 4.7 4.4 0.7 4.0 2.8e9f 2.4esf 1.3 4.6 4.7 4.0 1.9 5.2 5.1 4.4 1.0 4.9 4.5 4.2 1.7 4.9 4.9 4.3 4.3dye 5.3d,e 0 e 2.9 5.0 5.1 4.7 3.2 5.4 5.1 5.1 0.2, 0.7 4.8, 3.6 4.0, 3.0 4.1, 2.5

6.7f 2.0 2.7 1.8‘ 1.2 l.Zf 1.6 LOf 5.0 1.9 2.7 1.5; 4.5 3.0 2.8 1.@,h 3.4 1.8f 2.5f 1.5 y g 2.8 2.0 3.2

2.09 6.5ddYe 1.3 0 1.6dye 6.8 2.3 3.1d 1.8, 6.1 2.9 2.4, 2.8‘ 0.4, 1.1 2 . 0 , 1.1 2.7d 2.4 1.9 , l . l d y e

(Continued on p. 424)

A

N w

Table 19. 2 (continued)

P

N Ip

Dye

Blue VRS

Support

P K

A Violet

S P

K A Green S

S P K

A S

Red FB

P K

A S

Yellow 26

P K

A S

Tartrazine (FD and C Yellow No.5)

P

K

A Indigo Carmine (FD and C Blue No.2)

L i g r a t i o n (cm/h) i n e l e c t r o l y t e '

1

2

3

1.2 0.4 2.1 0.2

1.7 0.7 1.8 0.1

2.2

0.2 0.7e 0.3 1.89 1.0 2.4 0.6 0 2.7 0 2.6e 4.8 3.2 3.5 3.8 3.2 3.3 3.9

1.2 0.2 0 0.1 1.9 1.6 0.8 0.4 0 2.3e 0 1.7dye 3.6 3.0 4.5 3.2 3.8 3.6 4.2

3.1 5.3 3.6 2.5 5.1 4.9

'3.6 0.1, 0 2.0, 0 1.3, 2.9 0.7, -

4.4 0.1, 0.3 2.5,-1.0 4.G, 4.1 0.4, -

l.ze

4 h

0.6 2.6 0.1 0.8e 0.1 0.8e 0.1 0.5e 1.o 3.2 0.2 0 2.7e 0.1 1.3e 2.2

S P K

A Chocolate Brown FB

S P

K

A S

3.7 0.4, 0.2 1.5,-0.7 0 , 2.0

0, -

2.2f -0.1 1.2 0.1 0.4e ;:;dye

0 1.1 2.oe 2.L?

0.8 0 0.1 0.1 1.7e 2.5 3.4 4.2 3.4 1.4 4.9 4.9 4.1 0.6e 3.3 3.5d9e 3.5

0, -

3.6, 2.0 2 . 8 , 2.0 3.2, 2.4

5

6

0.4 2.4h 2.5 0.1 1.4e 3.3e 0.6g 0.1, 2.5d 3.5 2.4 0.3 0 4.2

2.6 0.8 1.6 0 l.ge 0.6eyf

0.2 1.9dye 413 5.4 4.3 4.1 4.0 5.9 5.2 4.7 2.1 5.2 4.8 4.7 0.2, 0.9 4.3, 3.3 3.8, 3 . 2 4.0, 2 . 5

O.!je

0.1 4.29 0.9 1.8 0.1 0.1 1.9 0.2

l.oe

7.9 2.5 3.1f 1.9 7.6 3.5 3Ad 2.1 ,g 3.6 2.Zd 3.0

2.29 0.3, 0.9 2 . 0 , 0.8

1.9, 1.5 1.3, -

Erythrosine BS

P K A S

0

0

0.1

0 0

0.1

0.13.0; 9

n

Y

0.2

0.5

3.7

^

^

9 c

a

1 h a t 200 V. bP = paper; K = Kieselguhr; A = alumina; S = s i l i c a g e l .

‘Buffers: 1, 1 M a c e t i c a c i d ; 2 , pH 4.0 ( 6 ml of 0.1 M sodium hydroxide, 750 ml of 0 1 mol/l potassium hydrogen phthalate d i l u t e d t o 1.5 1 ) ; 3 , pH 3.0 (85.5 ml o f 0.1 M sodium hydroxide, 750 ml of 0.1 mol/ potassium dihydrogen orthophosphate d i l u t e d t o 1.5 1 ) ; 4 , pH 8.0 (702 ml of 0.1 M sodium hydroxide, 750 ml of 0.1 mol/l potassium dihydrogen orthophosphate d i l u t e d t n 1.5 1 ) ; 5, 0.05 mol/l aqueous solution of borax (pH 9 . 2 ) ; 6 , 0.1 M amon a . dFaded. :Tailed. Faded gS1 i g h t l y faded. hFaint. 1 Composed of two components, brown ( l i s t e d f i r s t ) , yellow ( l i s t e d second).

426

TABLE 19.3 MIGRATION DISTANCES OF CONSTITUENTS ON CONTROL SYNTHETIC DYESTUFFS7 A d d i t i o n a l coloured spots were a l s o observed t o a r i s e from spots 13, 14, 15, 16, 17, 19, 2 1 and 22 when t h e chromatographic s t r i p was viewed under UV l i g h t (254 nm) a f t e r separation. I n a d d i t i o n t o t h e e l e c t r o l y t e s o l u t i o n o f pH 11.0 ( K o l t h o f f b u f f e r ) , two o t h e r e l e c t r o l y t e s , ( a ) 5.0% a c e t i c a c i d and ( b ) P a l i t z s c h b u f f e r solut i o n o f pH 8.0 (0.2 m o l / l b o r i c a c i d and 0.05 m o l / l borax), were a l s o t e s t e d f o r t h e d y e s t u f f s under s i m i l a r c o n d i t i o n s , b u t t h e K o l t h o f f b u f f e r produced b e t t e r separat i o n s and was t h e e l e c t r o l y t e o f choice. No.

Control d y e s t u f f s used i n 1 i q u o r s and beverages

Migration d i s t a n c e (cm)

Colour o f spot

"Bush" Dark Brown

1.3 2.6 4.3 5.6 6.0 8.4 4.3 8.4 1.0 1.7 2.6 4.3 6.0 8.4 4.4 4.9 8.4 2.6 4.3 6.0 7.9 1.0 1.4 2.6 4.3 6.0 8.4 1.0 2.5 3.5 4.9 8.4 1.4 4.3 8.4 2.5 4.9 5.6 8.4 3.6 8.4

pink red scarlet green orange lemon y e l l o w scarl e t 1 emon ye1 low l i g h t blue pink red scarl e t orange 1emon ye1 1ow l i g h t blue red 1 emon ye1 low red scarlet orange red ( f a i n t ) l i g h t blue red ( f a i n t ) red scarl e t orange lemon y e l l o w l i g h t blue pink l i g h t blue red 1 emon ye1 1 ow pink scarl e t lemon y e l l o w pink red green lemon y e l l o w blue 1emon ye1 l o w

"Bush" Lemon Red "Bush" Brown Dye

"Bush" Permical Lemon Yellow "Bush" Permical Orange-Red Powder 6

"Bush" Permical Vino Powder Dye

7

"Bush" Permi c a l Caramal i n e Powder Dye

"Bush" Caramal i n e Chocolate Brown "Bush" Caramal i n e Powder 10

Caramal ine I n d i g o " T a r t r a z ine"

427

Table 19.3 (Continued)

No.

Control dyestuffs used i n 1 iquors and beverages

Mi g ra t i o n d i s t a n c e (cm)

Colour of spot

11

8.4

lemon yellow

12

H.D.Tartrazine ( s y n t h e t i c organic food colour) Abracs Raspberry Red

13

Colour Rose "Bush"

14

LR0/6356 Rose Colour

1.4 4.3 8.2 9.5 4.5 6.7 8.2 9.5 0.6 4.3 6.3 7.3 8.3 0.6 1.9 3.2 4.3 5.5 6.7 7.3 0.6 4.3 6.3 7.3 8.3 3.4 6.8 0.6 1.8 4.3 6.0 7.0 7.3 8.3 2.6 6.0 7.3 1.4 4.3 6.7 8.4 1.1 1.9 2.8 4.3 5.6 6.5 7.3 7.8 8.4 4.3 6.0

red scarl e t red green (UV) pink blue (UV)

15

16

Raspberry Colour "Bush" I SI / 11378

Raspberry Colour "Bush" MN/24035

17

Raspberry Colour "Bush" I s/ 2342 9

18

Raspberry Col our "Bush" I W/5989 Raspberry Colour "Bush" I RO/ 1242

19

20

Colour Orange "Bush" NP/25257

21

Pine Appl e Colour

22

Vino Colour "Bush" KT/ 15 532

23

Vino Colour Powder "Bush"

red green (UV) grey scarl e t yellow ( U V ) pink red ( f a i n t ) grey

red (UV)

violet scarlet red blue (UV) pink grey scarl e t yellow (UV) pink red ( f a i n t ) pink v i o l e t brown grey violet scarl e t ye1 1 ow pink ( f a i n t ) greyish brown red ( f a i n t ) red orange pink red scarl e t blue (UV) lemon yellow greyish v i o l e t yellow ( U V ) blue scarlet green 1 i g h t green pink red-orange lemon yellow scarl e t orange (Continued on p. 428)

428

Table 19.3 (continued) No.

24 25 26

27

Control dyestuffs used i n 1 iquours and beverages

Migration distance (cm)

Colour of spot

AP0/5240 Raspberry Powder Red "Bush" MX/5901 Orange Powder Red "Bush" AW0/5964 Caramal ine Powder "Bush" x/779

8.4 1.4 4.3

1 emon ye1 low

4.3 6.0 7.3 1.4 4.3 5.6 6.0 8.4 3.0 a .4

scarl e t orange pink ( f a i n t ) red scarl e t green orange lemon yellow blue 1 emon ye1 low

Apple Green Colour 'I Bush" AP/ 112G4

red scarl e t

Additional data regarding t h e electrophoretic migration of s y n t h e t i c dyes in the above buffers a r e given in Table 19.5. The behaviour o f a number of anthraquinone dyes during electrophoresis on paper a t pH 9.0 (518 ml of 0.1 mol/l KH2P04 in 50% ethanol + 482 ml of 0.1 mol/l sodium hydroxide in 50% ethanol) was surveyed by Gasparic4 (Table 19.6). Paper e l e c t r o phoresis a1 so appears s u i t a b l e f o r t h e separation of sulphophthalein indicators: i t i s possible to distinguish between o-cresol red, rn-cresol purple, thymol blue, xylenol blue, bromocresol purple, bromophenol blue, bromocresol green, bromothymol blue and bromoxylenol blue by using t h e mobility of phenol red a s a standard 10

.

Eosin and dibromofluorescein a r e a l s o amenable t o paper electrophoretic separat i o n (Negri e t a1.l'). Water-soluble coal t a r dyes (Acid Red, B r i l l i a n t Blue FCT, Indigotine, New Coccine and Tartrazine) a r e e a s i l y separated i n 10% polyacrylamide gel using T r i s glycinate (pH 8.0, 0.01 mol/l, containing 0.1% of sodium c h l o r i d e ) a s running 12 buffer Of n a t u r a l l y occurring pigments, red beet pigments13 and p o r p h y r i n ~ l ~were ~'~ t h e f i r s t to be subjected t o electrophoretic separations. Separation of t h e former was effected o n paper in 0.1 mol/l c i t r a t e buffer (pH 5.5). Seven d i s t i n c t zones were separated under these conditions,

.

The separation of uroporphyrin, coproporhyrin and protoporphyrin i s amenable t o paper electrophoretic separations in 0.05 mol/l b a r b i t u r a t e buffer (pH 8 . 6 ) . A t 8 V/cm and a f t e r a running time of 3 h t h e following migration distances were found: uroporphyrin 10-15 cm, coproporphyrin 0.5-2 cm and protoporphyrin 0.2-0.3 cm. Separation i s also possible a t pH 11 (Larsen e t a 1 . l 5 ) , b u t isomeric compounds

429

TABLE 19.4 THIN-LAYER ELECTROPHORETIC MOBILITIES OF SEPARATED COLOUR COMPONENTS OF DYESTUFFS8 The m i g r a t i o n d i s t a n c e o f each c o l o u r e d s p o t measured f r o m c e n t r e o f s p o t t o p o i n t o f a p p l i c a t i o n . Together w i t h t h e d y e s t u f f s , t a r t r a z i n e was a p p l i e d on each e l e c t r o p h o r e t i c p l a t e as a marker r e f e r e n c e dye, t o check t h e r e p r o d u c i b i l i t y o f s e p a r a t i o n o f o t h e r c o l o u r zones.

No.

Dyestuff

Migration distance o f colour component (mm) e l e c t r o l y t e A

B

C

D

E

Colour o f spot

1

"Bush" Dark Brown

11 76 92 104

3 41 74 93

4 80 99 105

5 42 69 81

6 31 75 79

greenish blue scarl e t orange 1 emon-ye1 1ow

2

"Bush" Brown Dye

76 87 92 104

41 61 74 93

80 92 99 105

42 65 69 a1

41 69 75 73

scarl e t blue (faint)a orange lemon y e l l o w

3

I' Bu s h" Permi c a l Lemon Yellow

-

-

38 104

70 93

56 90 105

62 G9 81

58 65 79

bluish violet red lemon y e l l o w

'I Bu s h " Permic a 1 Orange Red

76 92

41 74

80 99

42 69

31 75

scarl e t orange

"Bush" Permi ca 1 Carmal i n e

95 104

96 105

GG

93

8:

65 79

violet lemon y e l l o w

"Bush" (Carmal i n e ) C h o c o l a t e Brown

-

76

41

-

-

104

93

105

42 50 81

31 60 79

scarl e t red lemon y e l l o w

93

105

a1

79

lemon y e l l o w

50

42 77

31 73

scarlet brown ( f a i n t )

4 5 6

-

7

H.D.Tartrazine ( s y n t h e t i c organic food colour)

104

a

Abracs Raspberry Red

- 76 41 -

9

Orange Powder Red ( " B u s h " )

10

Apple Green Sun Brand (Anant Chemicals)

a s p o t f a d e s on d r y i n g .

80

-

16 76 92

13 41 74

6 80 99

12 42 69

6 31 75

1i g h t p i n k scarl e t orange

a7 104

61 93

92 105

65 81

69 79

blue ( f a i n t ) lemon y e l l o w

430 TABLE 19.5 THIN-LAYER No.

ELECTROPHORETIC MOBILITIES

Synthetic colour

1 2 3 4 5 6 7

Carmo is ine Tartrazine Sunset ye1 l o w I n d i g o carmine Amar a n t h B1 ue-VRS Ponceau-4R

OF SYNTHETIC

COLOURS~

Migration distance o f colour component (mm), e l e c t r o l y t e A

B

C

D

E

76 104 92 87 16

41 93 74 61 13

DO 105 99 92 G 56 90

42 81 69 65 12 62 69

31 79 75 69

-

88

-

70

G 50 65

Colour o f spot

scar1 e t 1 emon ye1 1ow orange blue (faint)a l i g h t pink bluish violet red

a s p o t fades on d r y i n g . a r e n o t s e p a r a t e d a t pH 8.6 o r 11. I n 0.04 m o l / l sodium carbonate c o n t a i n i n g m o l / l o f EDTA i t i s p o s s i b l e t o o b t a i n zones o f u r o p o r p h y r i n , h e p a t o p o r p h y r i n , hexaporphyrin, pentacarboxyl i c a c i d p o r p h y r i n and c o p r o p o r p h y r i n s i d e by side. The zones a r e r e v e a l e d under UV l i g h t ; f o r g u a n t i t a t i o n t h e f l u o r e s c e n t m a t e r i a l can be e x t r a c t e d w i t h methanol-concentrated s u l p h u r i c a c i d (95:5). Zweig and 5 Whitaker i n d i c a t e d t h e n e c e s s i t y f o r complete h y d r o l y s i s o f p o r p h y r i n e s t e r s b e f o r e e l e c t r o p h o r e s i s i f an e s t e r i f i c a t i o n s t e p i s used t o i s o l a t e t h e s e compounds f r o m b i o l o g i c a l m a t e r i a l . An i n c o m p l e t e l y h y d r o l y s e d sample o f u r o p o r p h y r i n g i v e s f o u r bands, c o r r e s p o n d i n g t o f r e e p o r p h y r i n and mono-, d i and t r i e s t e r . t o paper e l e c t r o p h o r e s i s ,

the separation o f octa-,

hexa-, penta-,

I n addition

t e t r a - and t r i -

c a r b o x y l i c a c i d p o r p h y r i n s can be e f f e c t e d i n 0.5-1% a g a r g e l s i n Verona1 b u f f e r (pH 8.6,

~ = 0 . 0 5 ) a t G V/cm w i t h i n 10-12 h. 16,17

, The g r e a t e s t d i f f i c u l t y w i t h t h e s e p a r a t i o n o f i n d i v i d u a l humic a c i d s p e c i e s i s Several procedures have been d e v i s e d f o r t h e s e p a r a t i o n o f humic a c i d s

t h e i r p o l y m e r i c s t r u c t u r e , which c h e l a t e s m e t a l l i c i o n s and i n t e r a c t s w i t h b o t h i n o r g a n i c c o l l o i d s and o r g a n i c macromolecules. R e c e n t l y Castagnola e t a1 .I8 were a b l e t o d i s t i n g u i s h t h r e e components o f humates i n v a r i o u s s o i l e x t r a c t s u s i n g SDS-polyacrylamide g e l s and d i f f e r e n t c o n c e n t r a t i o n s o f u r e a i n t h e r u n n i n g b u f f e r (2-6 m o l / l ). S e p a r a t i o n s were c a r r i e d o u t i n T r i s - g l y c i n a t e (pH 8.3); t h e p o l a r i t y a p p l i e d s h o u l d be n e g a t i v e a t t h e bottom o f t h e g e l ( s e e a l s o r e f . 19). The separat i o n o f humic a c i d s from non-humic substances i n b l o o d can be e f f e c t e d b y p o l y a c r y l amide g e l e l e c t r o p h o r e s i s i n 7.5% g e l (pH 8.9).

A f t e r f i x a t i o n w i t h 10% l e a d a c e t a t e

t h e humic a c i d s can be s t a i n e d w i t h 0.2% A l c i a n B l u e 8GS. A q u a n t i t a t i v e e v a l u a t i o n o f d i s c e l e c t r o p h o r e s i s i s p o s s i b l e by d i r e c t p h o t o m e t r i c measurement o f t h e g e l s a t 560 nm. F o r d i s c r i m i n a t i n g between h e p a r i n and humic-type substances, r e a c t i o n

431 TABLE 19.6 ELECTROPHORETIC MOBILITIES OF A C I D ANTHRAQUINONE DYES RELATIVE TO COMPOUND I E l e c t r o l y t e : b u f f e r s o l u t i o n , pH 9; p o t e n t i a l , 10 V/cn; Compound

time, 6 h. Mo b i 1 ity

0

W

NH2

”

0

I

H

1.00

R=H

0.91

NH-R

0.99 1.04 1.04 1.04

1.46 1.60

1.51 1.66 1.66 1.66 1.65 0.70

0.82 Xqr

R=Q

0.86 COOC2H5

XXLL

R = -&02CH2CHpOH

S~NCCI$CH,OHC,,

XIX

R=Q

0.82 0.72

0.86 NHp

( C o n t i n u e d on p. 432)

432 Table 19.6 ( c o n t i n u e d ) Compound

Mobi 1 i t y

1.51 XXI

0.22

xx I I -xxv I

1.32

1.34

1.38

1.49

XXVII-XXVIII

0.73

XXIX-XXXI

0.75

1.08 1.22 1.22 1.42

433 Table 19.6 ( c o n t i n u e d ) Coinpou n d

Mobi 1 it y

434

w i t h i r o n ( I I 1 ) chloride-potassium hexacyanoferrate(II1) o r pre-treatment o f t h e sample w i t h 1 mol/1 potassium permanganate can be recommended".

Isotachophoresis

and i s o e l e c t r i c f o c u s i n g have a l s o been a t t e m p t e d w i t h t h i s c a t e g o r y o f compounds

21

:

t h r e e bands were observed w i t h humic a c i d s and two w i t h f u l v i c a c i d ( s e e a l s o r e f . 23 22). The term i s o e l e c t r i c f o c u s i n g o f Iiumic substances was q u e s t i o n e d by Thornton , however, who observed f r a c t i o n a t i o n o f humic substances on p o l y a c r y l a m i d e g e l s w i t h c a r r i e r ampholytes, b u t t h e m i g r a t i o n was c o n t i n u a l l y p r o c e e d i n g towards t h e anode r e g a r d l e s s o f t h e placement o f t h e sample. T h i s would suggest t h a t t r u e i s o e l e c t r i c f o c u s i n g i s n o t a p r e v a i l i n g phenomenon here. Another c a t e g o r y o f n a t u r a l l y o c c u r i n g pigments t h a t a t t r a c t s a l o t o f a t t e n t i o n i s c h l o r o p h y l l - p r o t e i n complexes and v i s u a l pigments. The s e p a r a t i o n p r o p e r t i e s o f b o t h a r e governed by t h e methods used g e n e r a l l y f o r p r o t e i n s e p a r a t i o n s . Thus, f o r i n s t a n c e , i n 9% p o l y a c r y l a m i d e g e l s e v e r a l bands o f c h l o r o p h y l l - p r o t e i n complexes were v i s u a l i z e d by Henriques and Park24y25. Bar Nun e t a1.26 adapted t h e method f o r t h i s purpose. P h o t o s y n t h e t i c pigments o f maize t h y l a k o i d s (Zea o f Laemml i27

mays L.) can be separated on 5% p o l y a c r y l a m i d e g e l i n 12 mmol/l Tris-9.6 glycine-0.2% D e r i p h a t 160 (pH = 8.3).

mmol/l

During t h i s separation t h e formation o f f r e e

o r detergent-complexed c h l o r o p h y l l i s n e g l i g i b l e . E l e c t r o p h o r e t i c s e p a r a t i o n he1 ped h e r e t o p r o v e t h a t a l l o f t h e c h l o r o p h y l l i n maize t h y l a k o i d s e x i s t s as c h l o r o p h y l l 28 p r o t e i n complexes

.

P u r i f i e d proteosyntheticmembranes o f Rhodopseudomonas spheroides when t r e a t e d w i t h 1 i t h i u m dodecyl s u l p h a t e and s u b j e c t e d t o p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (7.5% g e l ) g i v e u p t o e l e v e n pigment p r o t e i n complexes29. D e t a i l s o f t h e s e p a r a t i o n procedure, which sometimes i n v o l v e s two subsequent e l e c t r o p h o r e t i c steps, were p u b l i s h e d by D e l l e p e l a i r e and Chua3O. I n a s i m i l a r way i t i s p o s s i b l e t o f r a c t i o n a t e 31

t h e photosystem I p r o t e i n complex f r o m Anabaena fZos aquae

.

C l e a r l y , t h e above-mentioned a p p l i c a t i o n s o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s t o p r o t e i n - p i g m e n t complexes a r e o n l y o f an i n f o r m a t i v e n a t u r e . A d e t a i l e d d e s c r i p t i o n o f a l l o f t h e d i v e r s e s e p a r a t i o n s i n t h i s f i e l d i s beyond t h e scope o f t h i s review. P o l y a c r y l a m i d e g e l s e p a r a t i o n s can be a p p l i e d t o r h o d o p s i n and r e t i n o c h r o m e (7.5 o r 10% g e l , 0.5% SDS, 0.075 m o l / l sodium phosphate, pH ~ . 2 ) ~ ' , p h y c o b i l i p r o t e i n s (10% p o l y a c r y l a m i d e s l a b g e l i n 0.97 g o f NaH2P04-H20 t 4.82 g o f Na2HP04. 7 H20 t 0.25% SDS per l i t r e ) 33-35 and o t h e r p r o t e i n - p i g m e n t complexes. Sometimes paper e l e c t r o p h o r e t i c s e p a r a t i o n s can a l s o be made use o f ; i t i s p o s s i b l e t o sepa r a t e serum p r o t e i n - b o u n d b i l i r u b i n i n t h i s way 36

.

REFERENCES

1 W.J. C r i d d l e , G.J. Moody and J.D.R. Thomas, J . Chromatogr., 16 (1964) 350. 2 I.M o r i and 14. Kimura, J . P h a m . SOC. J a p . , 74 (1954) 179. 3 Y. N i i t s u , Jap. AnaZyst, 13 (1964) 1239.

435

4 f4.H. Anwar, S. Norman, B. Anwar and P. Laplaca, J . Chem. Ed., 40 (1963) 537. 5 G. Zweig and J.R. Whitaker, Paper Chromatography and Electrophoresis, Academic Press, New York, London 1967, p. 328. 6 Y. K i t a o k a , T. Yamase, T. Fukunura and T. K i t a v , J . Chromatogr., 132 (1977) 175. 7 S.N. Tewari, V.K. Sharma and I . C . Sharma, Chraatographia, 7 (1974) 84. 3 S.N. Tewari, I . C . Sharma and V.K. Sharma, Chromatographia, 9 (1976) 405. 9 J. G a s p a r i t , J . Chromatogr., 54 (1971) 436. 10 E.P. KrysFn, A.P. Boldyreva and G.I. M i k h a i l o v , T r . Vses. Nauchno-IssZed. Inst Khim. Reakt. Osobo Christ. Khim. Veshchestu., 35 (1973) 75; C.A. , 82 (1975) 7a53g. 11 A.S. N e g r i , N.R. Bannerjee and V. Sarda, J . Chem., 13 (1975) 274; C.A., 83 ( 1975) 57G05u. 12 D.B. Yeh, J . Chromatogr., 132 (1977) 5G6. 13 G. L i n d s t e d t , Acta Chem. Scand., 10 (1956) 698. 14 T.K. With, Scand. J . Clin. Lab. I n v e s t . . 8 (1956) 113. 15 E.G. Larsen, I. M e l c e r and J.M. Osten, &ed.'Proc:, Fed. Soc. Erp. B i o l . , 14 (1955) 440. 16 fyi. S c h n i t z e r and S.U. Kahn. Hwnic Substances in t h e Enviroment, Marcel Dekker, New Yark, 1972. 17 J.N. E l o f f and F.W. P a u l i , Plant S o i l , 42 (1975) 413. 18 M. Castagnola, R.G. de l a Heras and G.B. M a r i n i - B e t t d l o , J . Chromatogr., 147 (1978) 438. 19 M. Castagnola, C. N i g r o , G.B. M a r i n i - B e t t d l o , A. M i l a n a and R.G. de l a Heras, J . Chromatogr., 177 (1979) 130. 20 R. K l o e c k i n g , V. E i c h h o r n and T. Blumoehr, Z. Anal. Chem., 292 (1978) 408. 21 N.R. C u r v e t t o , N.A. Balmaceda and G.A. O r i o l i , J . Chromatogr., 93 (1974) 248, 22 R. K l o e c k i n g , J . Chromatogr., 73 (1973) 409. 23 3. Thornton, J . Chromatogr., 103 (1975) 402. 24 S.T. Henriques and R.B. Park, Biochem. Biophys. Res. Comun., 8 1 (1978) 1113. 25 S.F. Henriques and R.B. Park, Plant Physiol., 60 (1977) 64. 26 S. Bar Nun, R. Schantz and I.Ohad, Biochim. Biophys. Acta, 459 (1977) 451. 27 U.K. Laemmli, Nature (London), 227 (1970) 680. 28 J.P. Markwell , J.P. Thornber and R.T. Boggs, Proc. Nut. Acad. S c i . U.S. , 76 (1979) 1233. 29 R.M. B r o g l i e , C.N. Hunter and P. D e l l e p e l a i r e , Proc. Nut. Acad. S c i . U.S., 77 (1980) 87. 30 P. D e l l e p e l a i r e and N.H. Chua, Proc. Nut. Acad. S c i . U.S., 76 (1979) 111. 31 S.M. K l e i n and L.P. Vernon, Biochim. Biophys. Acta, 459 (1977) 364. 32 K. Nashima, f.1. M i t s u d o and Y. K i t o , Biochim. Biophys. Acta, 356 (1978) 78. 33 J.G. Cosner and R.F. T r o x l e y , Biochim. Biophys. Acta, 519 (1978) 474. 34 A.S. Brown and R.F. T r o x l e r , Biochern. J . , 163 (1977) 571. 35 M. Makino, T. Mamanaka, Y. O r i i and Y. K i t o , Biochim. Biophys. Acta, 495 (1977) 299. 36 A.Y. Sweet, Pediatr. Res., 11 (1977) 808.

This Page Intentionally Left Blank

437

Chapter 20 I N O R G A N I C COMPOUNDS F.M. EVERAERTS and Th.P.E.M.

VERHEGGEN

GENERAL ASPECTS I n o r g a n i c i o n s g e n e r a l l y have h i g h e r m o b i l i t i e s t h a n t h o s e f r o m p r o t e i n s . I n t h e past, e l e c t r o p h o r e t i c techniques have been found u n s u i t a b l e f o r t h e separat i o n o f i n o r g a n i c i o n s . Many s t u d i e s on t h e d e t e r m i n a t i o n o f ( i n ) s t a b i l i t y cons t a n t s and ( e f f e c t i v e ) m o b i l i t i e s have been r e p o r t e d and reasons f o r t h i s can e a s i l y be found. Moving boundary e l e c t r o p h o r e s i s i s an ” i n c o m p l e t e ” a n a l y t i c a l technique’’*

and t h e r e s u l t o f t h e s e p a r a t i o n i s almost always d i f f i c u l t t o

i n t e r p r e t . F o r i s o e l e c t r i c f o c u s i n g t h e i o n i c compound needs t o be a m p h o l y t i c . 2 The drawback i n u s i n g zone e l e c t r o o h o r e s i s i s i t s d e t e c t i o n I n isotachophoresis

.

small * d i f f e r e n c e s i n e f f e c t i v e mobi 1 ity a l m o s t always guarantee a complete separat i o n i f t h e o p e r a t i n g c o n d i t i o n s a r e chosen c o r r e c t l y . I o n f o c u s i n g s t i l l must prove i t s a n a l y t i c a l v a l u e

5.

SEPARATIONS V I A ZONE ELECTROPHORESIS As usual i n zone e l e c t r o p h o r e s i s 6 ,

s t a b i l i z i n g media such as ( s i l i c a ) g e l ,

paper and c e l l u l o s e a c e t a t e have t o b e a p p l i e d . I n Chapter 5 t h e s t a b i l i z i n g e f f e c t o f a narrow-bore t u b e i s demonstrated. Because t h e s e s u b j e c t s have been e x t e n s i v e l y d i s c u s s e d i n books by E v e r a e r t s e t al.’,

Dey12 and Sargent and George

6

,

s t a b i l i z i n g media w i l l be c o n s i d e r e d o n l y b r i e f l y . A l s o , t h e d e t e c t i o n methods ( g e n e r a l l y s p e c i f i c chemical r e a c t i o n s a f t e r t h e s e p a r a t i o n ) w i l l n o t be discussed. A l b e r t i e t a1.’

and Q u r e s h i and c o - ~ o r k e r s ~r e’ ~p o r t e d t h e a n a l y s i s o f metal

i o n s u s i n g papers impregnated w i t h i n o r g a n i c i o n exchangers. A l a r g e number o f s e p a r a t i o n s o f i m p o r t a n t b i n a r y and t e r n a r y m i x t u r e s have been achieved. . 10 i n v e s t i g a t e d t h e e l e c t r o p h o r e t i c m o b i l i t i e s C v j e t i E a n i n and JovanoviE-Kovatovic

-

o f d i v a l e n t mercury, cadmium, l e a d and copper i n v a r i o u s i n o r g a n i c and o r g a n i c a c i d s . The f o r m a t i o n of complexes as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f t h e a c i d s

* I n t h e steady s t a t e even no d i f f e r e n ~ e ~i n’ ~e f f e c t i v e m o b i l i t y i s p r e s e n t i n some i n s t a n c e s o r i o n s m i g r a t e i n t h e o p p o s i t e o r d e r t o t h e i r e f f e c t i v e m o b i l it i es334.

438

a p p l i e d and e x p e r i m e n t a l c o n d i t i o n s f o r o p t i m a l s e p a r a t i o n s were d e s c r i b e d . Pol a n s k j and Bdr"

d e s c r i b e d t h e d e t e r m i n a t i o n o f t r a c e amount o f y t t r i u m (lo-"-

m o l / l ) i n t h e pH range 1.6-11.6,

u s i n g unscreened e l e c t r o d e s and e l e c t r o d e s

screened w i t h a c e l l o p h a n e membrane. The f o r m a t i o n o f a n e g a t i v e l y charged c o l l o i d o f y t t r i u m h y d r o x i d e was d i s c u s s e d . B e l l and MatyeviE"

investigated the

f o r m a t i o n and growth o f hydrous c h r o m i u m ( I I 1 ) o x i d e p a r t i c l e s , p r e p a r i n g t h e s o l s by h e a t i n g d i l u t e s o l u t i o n s o f c h r o m i u m ( I I 1 ) s a l t s i n t h e presence o f s u l phate i o n s . B l a s i u m and Neumann13 r e p o r t e d on t h e i s o t o p e e f f e c t s t h a t o c c u r r e d d u r i n g e l e c t r o p h o r e s i s i n aqueous s o l u t i o n s o f v a r i o u s complex i o n s o f c o b a l t and i r o n . The change i n t h e c o m p o s i t i o n o f t h e i s o t o p e s was found t o decrease when t h e i s o t o p e s were used i n t h e f o r m o f h i g h l y charged c a t i o n s o r anions. W r i g h t and F r i e d e n 1 4 s t u d i e d t h e r e a c t i o n o f r e d - v i o l e t complexes o f copper and p e n i c i l l a m i n e u s i n g zone e l e c t r o p h o r e s i s . C h o u l i s e t a1 .15 examined t h e degree of m i g r a t i o n o f o p t i c a l l y a c t i v e , racemic and n o n - o p t i c a l l y a c t i v e a c i d s and bases on c e l l u l o s e , s i l i c a g e l a n d a l u m i n a t h i n l a y e r s as a f u n c t i o n o f pH. Dumont e t a l . l6 determined t h e c o a g u l a t i o n values, e l e c t r o k i n e t i c p o t e n t i a l s and p e p t i z a t i o n p r o p e r t i e s o f i r o n ( I 1 ) o x i d e h y d r o s o l s i n t h e pH range 2-12 i n t h e presence o f monovalent i o n s . Magdelenat e t a1 . I 7 s t u d i e d t h e e l e c t r o p h o r e t i c m o b i l i t i e s o f b i v a l e n t c a t i o n s i n t h e presence o f a l i n e a r a n i o n i c p o l y s a c c h a r i d e e x t r a c t e d from c a r t i l a g e c o n d r o i t i n sulphate.

A comparison was made between t h e e l e c t r o p h o r e t i c m o b i l i t y and t h e s e l f - d i f s t u d i e d t h e m i g r a t i o n o f halogeno complexes f u s i o n c o e f f i c i e n t . Meier e t a l . l8 of t h e p l a t i n u m m e t a l s and v a r i o u s m e t a l i o n s i n i n o r g a n i c s a l t s o l u t i o n s . Poss i b l e reasons f o r t h e complex f o r m a t i o n on t h e m o b i l i t y b e h a v i o u r were discussed. O s s i n i and B a l z o n i l '

d e s c r i b e d t h e i n t e r a c t i o n o f c o b a l t ( I I 1 ) complexes and s o l u -

t i o n s o f orthophosphate, pyrophosphate and metaphosphate. P r e e t z and KUhl 2o r e p o r t e d on t h e m i g r a t i o n b e h a v i o u r and f o r m a t i o n o f mixed f l u o r o - c h l o r o complexes o f p l a t i n u m ( 1 V ) and i r i d i u m ( 1 V ) . C a s i l l o e t a1.21 surveyed aspects o f t h e i n t e r a c t i o n between t r i v a l e n t m e t a l complexes and anions, u s i n g paper as t h e s t a b i l i z i n g medium. Kasabov and Genov22 s t u d i e d t h e m o b i l i t y b e h a v i o u r o f thorium(1V) i o n s as a f u n c t i o n o f v i s c o s i t y and pH. The v i s c o s i t y was i n v e r s e l y p r o p o r t i o n a l t o pH, and t h e p r o d u c t o f v i s c o s i t y and m o b i l i t y was c o n s t a n t . N a ~ s ei p a~r a t~e d f i v e a l k a l i and a l k a l i n e e a r t h m e t a l s on paper i n ammonia s o l u t i o n . N a ~ a lis o~ r e~p o r t e d on t h e s e p a r a b i l i t y o f cadmium(I1) and m e r c u r y ( I 1 ) i n aqueous l a c t i c a c i d s o l u t i o n s o f s e v e r a l c o n c e n t r a t i o n s . Murata e t a1 .25 s t u d i e d t h e e l e c t r o p h o r e t i c m o b i l i t y o f g r a p h i t e i n a l k a l i n e s o l u t i o n s and t h e e f f e c t s t

o f L i t , Na', Kt, NH4 and Cst i o n s . B r u y n i n c k x e t a1.26 d e s c r i b e d a t e s t f o r t h e l o c a l i z a t i o n o f copper on p o l y a c r y l a m i d e g e l s , based on quenching o f t h e f l u o r e s c e n c e o f b a t h o c u p r o i n e s u l p h o n a t e by Cut, w h i c h i s s e n s i t i v e t o 0.1 nmol o f f r e e o r p r o t e i n - b o u n d copper. No f a l s e r e a c t i o n s were observed w i t h haeme

439 p r o t e i n , f r e e Fe3+, Fe2+, Co2+ o r Mn2+. C u r r e r i e t a1.27 s t u d i e d t h e e l e c t r o p h o r e t i c b e h a v i o u r o f c a l c i u m o x a l a t e monohydrate i n terms o f a s i m p l e doublel a y e r model. The r e s u l t s w i t h a v a r i e t y o f e l e c t r o l y t e s where i n good agreement w i t h t h e NerstlGouylChapmanlStern d o u b l e - l a y e r model. SEPARATION V I A I O N FOCUSING

Schonhofer and Grass2* r e p o r t e d on t h e e f f e c t o f e l e c t r o p h o r e t i c i o n f o c u s i n g i n i n s t a n c e s where t h e c o n d i t i o n s f o r t h e u s u a l e x p l a n a t i o n a r e n o t f u l f i l l e d .

A q u a l i t a t i v e e x p l a n a t i o n i s g i v e n t h a t t a k e s i n t o account t h e inhomogeneity o f t h e e l e c t r i c f i e l d and e x p l a i n s t h e s e p a r a t i o n i n t o v e r y narrow a d j a c e n t zones by t h e p r i n c i p l e o f i s o t a c h o p h o r e s i s ' .

To s e p a r a t e l a r g e r amounts o f i n o r g a n i c

i o n s on p o l y a c r y l a m i d e and s i l i c a g e l , Schonhofer and Grass"

recommended e l e c -

t r o p h o r e t i c i o n focusing. Wagner and Neupert 30y31 r e p o r t e d t h e use o f c o n t i n u o u s e l e c t r o p h o r e t i c i o n f o c u s i n g f o r t h e s e p a r a t i o n and e n r i c h m e n t o f m e t a l i o n s . F r e e - f l o w e l e c t r o p h o r e t i c i o n f o c u s i n g was c a r r i e d o u t c o n t i n u o u s l y i n a s u i t a b l e a p p a r a t u s . U s i n g complexing agents d i f f e r e n t m e t a l i o n s c o u l d be s e p a r a t e d p u r e f r o m m i x t u r e s . Simultaneous e n r i c h m e n t t a k e s p l a c e d u r i n g t h e e l e c t r o p h o r e s i s , r e s u l t i n g i n an i n c r e a s e i n t h e d e t e c t i o n and d e t e r m i n a t i o n l i m i t s . V a r i b u s p o s s i b i l i t i e s f o r t h e s e p a r a t i o n of metal i o n s b y means o f c o n t i n u o u s e l e c t r o p h o r e t i c i o n f o c u s i n g have been demonstrated3'

i n a p r a c t i c a l approach.

SEPARATIONS V I A ISOTACHOPHORESIS I t w i l l be c l e a r f r o m r e f s . 1 and 2 t h a t i n o r g a n i c i o n s have f r e q u e n t l y been

used i n model experiments t o demonstrate t h e r e p r o d u c i b i l i t y and h i g h r e s o l u t i o n o f t h i s s e p a r a t i o n t e c h n i q u e and t h e t h e o r y and t h e f u n c t i o n o f t h e apparatus. I t has been f o u n d l Y 2 t h a t a l m o s t always c o n d i t i o n s c o u l d be found f o r a complete

q u a l i t a t i v e and q u a n t i t a t i v e s e p a r a t i o n o f any g i v e n p a i r o f ( i n ) o r g a n i c i o n i c compounds. The work o f E v e r a e r t s and co-workers has been c o m p i l e d i n r e f . 1, i n which an a l m o s t complete s u r v e y i s g i v e n Appendix C, p. 379 o f work on i s o t a c h o p h o r e s i s pub1 ished up t o 1976. The h a l i d e s have a l m o s t i d e n t i c a l m o b i l i t i e s i n w a t e r and a r e d i f f i c u l t * ' t o 1 I t i s p o s s i b l e , however,

s e p a r a t e u s i n g t h e g e n e r a l l y a p p l i e d o p e r a t i o n a l system

.

t o s e p a r a t e t h e h a l i d e s p e r f e c t l y by u s i n g methanol as t h e s o l v e n t f o r t h e e l e c t r o l y t e s . Because of i t s i m p o r t a n c e i n b i o l o g i c a l r e a c t i o n s , o r t h o p h o s p h a t e has demonstrated t h e been d e t e r m i n e d b y s e v e r a l workers y32-37 E v e r a e r t s e t a1

.'

*An approach i s g i v e n i n r e f . 42; t h e c o r r e c t l y chosen o p e r a t i n g c o n d i t i o n s show the f l e x i b i l i t y o f isotachophoresis.

440

q u a n t i f i c a t i o n o f pyrophosphate and orthophosphate t o g e t h e r w i t h dexamethasonesodium phosphate. Gower and Woledge"

s t u d i e d t h e p o s s i b i l i t y o f measuring o r t h o -

phosphate i n muscle e x t r a c t s , b u t t h e y found t h a t t h e orthophosphate zone a l s o c o n t a i n e d u n i d e n t i f i e d compounds o r i g i n a t i n g f r o m t h e e x t r a c t . Van d e r Hoeven e t a1.37 determined orthophosphate i n d e n t a l p l a q u e w i t h o t h e r a c i d s . BoEek and co-workers 38y39 used i s o t a c h o p h o r e s i s t o d e t e r m i n e o r t h o - and pyrophosphate i n a r t i f i c i a l f e r t i l i z e r s a f t e r 1 0 0 0 - f o l d d i l u t i o n . H i g h e r condensed phosphates d i d n o t i n t e r f e r e . BoEek and co-workers 40y41 determined n i t r a t e and s u l p h a t e i n p r a c t i c a l samples. I n q u a l i t y c o n t r o l d u r i n g t h e manufacture o f a - o l e f i n monosulphonates and d i s u l p h o n a t e s , t h e s e i o n s have been determined q u a n t i t a t i v e l y t o g e t h e r w i t h i n o r g a n i c s u l p h a t e s . The s e p a r a t i o n was performed i n water-acetone m i x t u r e s . N i t r a t e , hypophosphi t e , phosphite, orthophosphate and l a c t a t e i n baths 41 f r o m n i c k e l p l a t i n g processes have been measured I n aqueous systems BoEek e t a1 ,42 separated t h e h a l i d e s q u a n t i t a t i v e l y t o g e t h e r

.

w i t h carbonate and s u l p h a t e , u s i n g cadmium(II1) as t h e c o u n t e r i o n and n i t r a t e as t h e l e a d i n g i o n . P r a c t i c a l l y t h i s s o l u t i o n has been a p p l i e d t o t h e determinat i o n o f s a l t anions i n m i n e r a l w a t e r . A k i ~ a m ameasured ~~ t h i o s u l p h a t e , s u l p h a t e and s u l p h i t e i n f a c t o r y waste w a t e r . Using h y p o s u l p h i t e as t h e l e a d i n g i o n , c h l o r i d e and s u l p h a t e were determined i n waste w a t e r f r o m a cardboard f a c t o r y * . SEPARATIONS I N NON-AQUEOUS MEDIA So f a r e l e c t r o p h o r e t i c s e p a r a t i o n s i n non-aqueous media have r e c e i v e d l i t t l e a t t e n t i o n . One o f t h e m a j o r problems i n u s i n g s o l v e n t s o t h e r t h a n w a t e r i s t h e i r g e n e r a l l y l o w b o i l i n g p o i n t s and t h e f a c t t h a t d u r i n g e l e c t r o p h o r e s i s h e a t i s produced. I f we c o n s i d e r t h e s t a b i l i z i n g e f f e c t s ' o f narrow-bore t u b e s and t h e f a c t t h a t i n c l o s e d systems' e v a p o r a t i o n i s prevented, t h e way i s open t o p e r f o r m e l e c t r o p h o r e t i c analyses i n s o l v e n t s * * i n which t h e compounds under i n v e s t i g a t i o n a r e p r e s e n t i n i o n i c form. S u i t a b l e s o l v e n t s f o r e l e c t r o p h o r e s i s a r e , f o r example, water, methanol, dioxane, methyl C e l l o s o l v e and m i x t u r e s o f w a t e r w i t h a l c o h o l s and/or ketones; more s o l v e n t s c o u l d e a s i l y be recommended. I n r e f . 1 analyses i n methanol and methanol-water m i x t u r e s a r e shown, b u t o t h e r combinations o f s o l v e n t s can g i v e t h e expected d i f f e r e n c e i n e f f e c t i v e m o b i l i t i e s necessary f o r a s e p a r a t i o n a l o n g t h e l i n e s o f any e l e c t r o p h o r e t i c p r i n c i p l e .

*Both LKB (Bromma, Sweden) and Shimadzu (Kyoto, Japan) w i l l p r o v i d e f u l l i n f o r m a t i o n on r e q u e s t about a p p l i c a t i o n s and o t h e r t e c h n i c a l i n f o r m a t i o n w i t h r e s p e c t t o isotachophoresis. **Mixed s o l v e n t s .

441 B l a s i u s and K l e r n ~ nr e~ p~o r t e d on an improved chamber f o r h i g h - v o l t a g e e l e c t r o p h o r e s i s t h a t u t i l i z e s a t e m p e r a t u r e g r a d i e n t o f 3'C/cm.

An improvement o f separa-

t i o n e f f i c i e n c y o f up t o 50% compared w i t h s e p a r a t i o n s a t O°C was found. I t must be b o r n e i n mind t h a t a t l o w e r temperatures t h e a b s o l u t e m o b i l i t i e s i n c r e a s e d r a s t i c a l l y . A d d i t i o n a l advantages a r e achieved w i t h non-aqueous systems. B l a s i u s and A ~ g u s t i nr e~p ~o r t e d on t h e d i f f e r e n c e i n m o b i l i ty o f cyanothiocyanochromate( 111) i n w a t e r and a c e t o n i t r i l e . The t e m p e r a t u r e c o e f f i c i e n t s o f t h e two hexapseudohalegenochromates( 111) were determined. J o k l e t a1 .46 r e p o r t e d t h e zone e l e c t r o p h o r e t i c s e p a r a t i o n o f o r g a n i c a c i d s and bases i n w a t e r - a l c o h o l s o l v e n t s . The e l e c t r o p h o r e t i c b e h a v i o u r o f t e n o r g a n i c a c i d s and f o u r bases as model samples i n water-methanol and w a t e r - e t h a n o l s o l v e n t s was s t u d i e d w i t h r e s p e c t t o t h e o r g a n i c s o l v e n t c o n c e n t r a t i o n i n t h e s u p p o r t i n g e l e c t r o l y t e and pH. The changes i n t h e p e r m i t t i v i t y o f t h e s o l v e n t i n f l u e n c e t h e r e l a t i v e m o b i l i t y ( v i a changes i n t h e pKa). From t h e e l e c t r o p h o r e t i c d a t a t h e approximate pKa v a l u e s o f t h e observed substances i n mixed s o l v e n t s were c a l c u l a t e d . REFERENCES

1 F.M. E v e r a e r t s , J.L. Beckers and Th.P.E.M. Verheggen, Isotachophoresis. Theory, I n s t r u m e n t a t i o n and A p p l i c a t i o n s , E l s e v i e r , Amsterdam, Oxford, New York, 1976. 2 Z. Deyl ( E d i t o r ) , BZectrophoresis. A Survey of Techniques and AppZications. Part A : Techniques, E l s e v i e r , Amsterdam, Oxford, New York, 1979. 3 F.E.P. M i k k e r s , F.M. E v e r a e r t s and J.A.F. Peek, J . Chromatogr., 168 (1979) 293. 4 F.E.P. M i k k e r s , F.M. E v e r a e r t s and J.A.F. Peek, J . Chromatogr., 168 (1979) 317. 5 F. Schumacher and H.J. S t r e i f f , HeZv. Chim. Acta, 40 (1957) 228. 6 J.R. Sargent and S.G. George, Methods i n Zone EZectrophoresis, BDH Chemicals, Poole, 3 r d Ed., 1975. 7 G. A l b e r t i , A. Conte, G. G r a s s i n i and M. Lederer, EZectroanaZ. Chem. InterfaciaZ. EZectrochem., 4 ( 1962) 301. 8 M. Q u r e s h i , K.G. Varshney and R.P.S. R a j p u t , Ann. Chirn. Rome, 66 (1976) 337. 9 M. Q u r e s h i and S.D. Sharma, Separ. S c i . TechnoZ., 13 (1978) 723. 10 N.M. C v j e t i E a n i n and 0. JovanoviE-KovaEeviE, J . Chrornatogr., 94 (1974) 349. 11 P . P o l a n s k j and J. Bar; CoZZect. Czech. Chem. Commun., 39 (1974) 1025. 12 A. B e l l and E. MatyeviE, J . Inorg. NucZ. Chem., 37 (1975) 907. 13 E. B l a s i u s and W. Neumann, J . Chromatogr., 110 (1975) 273. 14 J.R. W r i g h t and E. F r i e d e n , Bioinorg. Chem., 4 (1975) 163. 15 N.H. C h o u l i s , M . S . McQuade and M. C h o u l i s , Pharmazie, 3 1 (1976) 381. 16 F. Dumont, Dang Van Tan and A. W a t i l l o n , J . CoZZoid I n t e r f a c e S c i . , 55 (1976) 678. 17 H. Magdeignat, P. Turq, M. Chemla and B. Para, BiopoZymers, 15 (1976) 175. 18 H. M e i e r , E. Zimmerhack, W. A l b r e c h t , D. Bijsche, W. Hecker, P. Menge, E. Unger and G. Z e i t l e r , Microchirn. Acta, (1976) 181. 19 L. O s s i c i n i and M. B a l z o n i , J . Chromatogr., 117 (1976) 464. 20 W . P r e e t z and H. KUhl, 2. Anorg. A L l g . Chern., (1976) 97. 2 1 M. C a s i l l o , M. L e d e r e r and L. O s s i c i n i , J . Chrornatogr., 135 (1977) 256. 22 G. Kasabov and L. Genov, God. V i s s h . Khimikotekhnol. I n s t . , 22 (1977) 143. 23 J.C. Nasi, C i e n t i f i c a , 5 (1977) 11. 24 J.C. Nasi, C i e n t i f i c a , 5 (1977) 7 . 25 T. Murata, Y . Matsuda and H. Imagawa, Tanso, 89 (1977) 55. 26 W.J.B. Bruyninckx, S. G u t t e r i d g e and H.S. Mason, Anal. Biochern., 80 (1978) 174. 27 P. C u r r e r i , G . Y . Onoda and B. F i n l a y s o n , J . CoZZoid Interface Sci., 69 (1979) 170.

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28 29 30 31 32 33 34 35 36

F. Schiinhofer and F. Grass, J . Chromatogr., 110 (1975) 265. F. Schonhofer and F. Grass, Microchim. Acta, (1975) 89. H. Wagner and D. Neupert, J . Chromatogr., 147 (1978) 281. H. Wagner and D. Neupert, J . Chromatogr., 156 (1978) 219. D.C. Gower and R . C . Woledge, S c i . TooZs, 24 (1977) 2. A. Kopwillem, J . Chromatogr., 82 (1973) 407. J.P.D. Dunn and R.B. Kemp, Protides BioZ. FZuids, 22 (1975) 727. B. S j o d i n , A. Kopwillem and J. Karlsson, Frotides BioZ. FZuids, 22 (1975) 733. B. S j o d i n , A . Kopwillem and A. K a r l s s o n , Scand. J . CZin. Lab. Invest., 35

(1975) 699. 37 J.S. Van d e r Hoeven, H.C.M. Franken, P.J.M. Camp and C.A. D e l l a b a r r e , A p p Z . Environ. MicrobioZ., 35 (1978) 17. 38 P. BoEek, M. Deml and J. J a n i k , J . Chromatogr., 156 (1978) 323. 39 P. BoEek, 6. Kaplanovb, M. Deml and J . J a n i k , J . Chromatogr., 153 (1978) 287. 40 P. BoEek, B. Kaplanovi, M. Deml and J . J a n i k , CoZZect. Czech. Chem. C o m n . , 43 (1978) 2707 41 P. BoEek, S . Pavelka, M. Deml and J. J a n i k , J . Chromatogr., 151 (1978) 436. 42 P. BoEek, I . Miedzak, M. Deml and J . J a n i k , J . Chromatogr., 137 (1977) 83. 43 J. Akiyama, Dev. Biochem., 2 (1978) 109. 44 E. B l a s i u s and G. Klemm, J . Chromatogr., 135 (1977) 323. 45 E . B l a s i u s and H. Augustin, J . Chromatogr., 109 (1975) 107. 46 V. J o k l , J. D o l e j S o v i and M. MatuSovi, J . Chromatogr., 172 (1979) 239.

443

.

CONTENTS OF ELECTROPHORESIS PART A: TECHNIQUES

............................................................ P r e f a c e ................................................................. I n t r o d u c t i o n ............................................................

Contributors

.

. V a c i k ) ...................... ....................................................... E q u i l i b r i a i n e l e c t r o p h o r e t i c systems .............................. Processes i n e l e c t r o p h o r e t i c systems ............................... Mathematical d e s c r i p t i o n o f t h e e l e c t r o p h o r e t i c process ............ References .........................................................

1 Theory o f e l e c t r o m i g r a t i o n processes ( 3 Introduction

2 . C l a s s i f i c a t i o n o f e l e c t r o r n i g r a t i o n methods (3 Introduction

.......................................................

............................................... The moving boundary method ......................................... I s o t a c h o p h o r e s i s ................................................... Focusing methods ................................................... Combined methods ................................................... References ........................................................ . Vacik .. ................................................... ... ... D e t e r m i n a t i o n o f m o b i l i t y ...................................... P r i n c i p l e s o f q u a n t i t a t i v e e v a l u a t i o n ..............................

. Evaluation

o f t h e r e s u l t s o f e l e c t r o p h o r e t i c separations ( J

Introduction

4

. Molecular s i z e

. D e y l ) ................ ....................................................... E l i m i n a t i o n o f charge d i f f e r e n c e s .................................. Choice of s t a n d a r d s e r i e s .......................................... M o l e c u l a r s i z e d e t e r m i n a t i o n by p o r e 1 i m i t e l e c t r o p h o r e s i s ......... D e t e r m i n a t i o n o f Stokes r a d i i by r h e o p h o r e s i s ...................... and shape i n e l e c t r o p h o r e s i s (Z

Introduction

.........................................................

References

5

. Zone

XI XV

1 1

2 8 18 20

. V a c i k ) ................. 23

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

3

IX

23 24 26

27 29 33

37 39 39 40

42 45 45 49 61 62 63 66

e l e c t r o p h o r e s i s ( e x c e p t g e l - t y p e t e c h n i q u e s and immunoelectro-

............................................. Paper t e c h n i q u e s ................................................... T h i n - l a y e r e l e c t r o p h o r e s i s ......................................... References ..........................................................

phoresis) (W

.

Ostrowski)

69 70 96 103

444

6

. Gel-type

. H r k a l ) ........................................ ........................................................

techniques ( Z

Introduction

113 113

....................................... 114 S t a r c h g e l e l e c t r o p h o r e s i s .......................................... 116 Acrylamide g e l e l e c t r o p h o r e s i s ...................................... 117 Agarose g e l e l e c t r o p h o r e s i s ......................................... 128 Agarose-acrylamide composite g e l s ................................... 129 References .......................................................... 130 Theory o f g e l e l e c t r o p h o r e s i s

7

(P.J. Svendsen) .................... 133 ........................................................ 133 Chemicals and s o l u t i o n s .............................................134 Apparatus and a c c e s s o r i e s ........................................... 136 P r a c t i c a l a p p l i c a t i o n ............................................... 139 153 References ..........................................................

. Q u a n t i t a t i v e immunoelectrophoresis Introduction

8

. Moving boundary e l e c t r o p h o r e s i s

i n narrow-bore t u b e s (F.M.

Everaerts

..................................................... ........................................................ P r i n c i p l e s o f moving boundary e l e c t r o p h o r e s i s ....................... P r a c t i c a l a p p l i c a t i o n ............................................... S t a t e o f t h e a r t and comparison w i t h o t h e r t e c h n i q u e s ............... References ..........................................................

and J.L.

Beckers)

Introduction

9

. Isoelectric

focusing (N

. Catsimpoolas) ................................

155 155 156 160 162 165 167

........................................................... 167 ................................................. 168 C a r r i e r ampholytes .................................................. 170 Support media .......................................................171 Gel tubes ........................................................... 172 T h i n l a y e r s ......................................................... 173 175 D e n s i t y g r a d i e n t .................................................... 176 Free s o l u t i o n ....................................................... Two-dimensional methods ............................................. 176 176 I m m u n o i s o e l e c t r i c f o c u s i n g .......................................... T r a n s i e n t s t a t e i s o e l e c t r i c f o c u s i n g ................................ 179 190 Conclusions ......................................................... 190 References .......................................................... Principle

T h e o r e t i c a l aspects

445

.

. Vacik and F.M. E v e r a e r t s ) ............ 193 194 I n t r o d u c t i o n ....................................................... T h e o r e t i c a l ........................................................ 195 Q u a l i t y and q u a n t i t y i n i s o t a c h o p h o r e s i s ........................... 206

10 A n a l y t i c a l i s o t a c h o p h a r e s i s ( J

.

.................................................... .............................................. .........................................................

Instrumentation

208

Practical application

218

References

224

12

.

.

.................. 229 229 I n t r o d u c t i o n ....................................................... General aspects .................................................... 231 E l e c t r o p h o r e t i c c e l l s o f o t h e r shapes .............................. 235 F a c t o r s a f f e c t i n g zone w i d t h i n f l o w - t h r o u g h c e l l s ................. 235 References ......................................................... 250

11 Continuous f l o w - t h r o u g h e l e c t r o p h o r e s i s ( Z

Continuous f l o w d e v i a t i o n e l e c t r o p h o r e s i s (A

Prusik)

.

Kolin)

.................

.......................................................

Introduction

T h e o r e t i c a l aspects o f c o n t i n u o u s f l o w d e v i a t i o n e l e c t r o p h o r e s i s

253 253

...

255

Modes o f i m p l e m e n t a t i o n o f continuous f l o w d e v i a t i o n e l e c t r o p h o r e s i s 261 F l a t f l u i d band e l e c t r o p h o r e s i s ( " f r e e - f l o w " e l e c t r o p h o r e s i s )

......

................................. ......................................................... Acknowledgements ................................................... Symbols and u n i t s .................................................. References ......................................................... 13

.

Endless f l u i d b e l t e l e c t r o p h o r e s i s

267

Conclusion

295

P r e p a r a t i v e e l e c t r o p h o r e s i s i n g e l media ( Z . H r k a l )

..................

296 296 299 299

Gel

...................................................... c o m p o s i t i o n and c o n c e n t r a t i o n ................................. dimensions and sample l o a d ....................................

299

..................... D u r a t i o n o f e l e c t r o p h o r e s i s and f l o w - r a t e ......................... References ........................................................

300

Voltage g r a d i e n t and t h e e l e c t r o p h o r e s i s t i m e

.

295

Introduction Gel

14

261

P r e p a r a t i v e e l e c t r o p h o r e s i s i n columns (P.J.

...............

307 307

......................................................... ..............................

315

........................................................

324

P r a c t i c a l a p p l i c a t i o n and procedures References

301 305

......................................................

Introduction Apparatus

Svendsen)

300

308

446

15

16

. Preparative

. B l a n i c k g ) ...................... I n t r o d u c t i o n ...................................................... General aspects ................................................... P r e p a r a t i v e i s o e l e c t r i c f o c u s i n g t e c h n i q u e ........................ Conclusion ........................................................ References ........................................................ i s o e l e c t r i c f o c u s i n g (P

328 328 332 341 342

........................345 I n t r o d u c t i o n ...................................................... 345 Chemicals and s o l u t i o n s ........................................... 346

. Preparative

i s o t a c h o p h o r e s i s (P.J. Svendsen)

......................................................... P r a c t i c a l a p p l i c a t i o n ............................................. References ........................................................

Apparatus

17

327

348 355 362

....... 363 ...................................................... 363 S m a l l - s c a l e p r e p a r a t i v e i s o t a c h o p h o r e t i c methods .................. 364 P r i n c i p l e o f p r e p a r a t i v e c a p i l l a r y method ......................... 365 370 A p p l i c a t i o n s o f p r e p a r a t i v e c a p i l l a r y method ...................... 376 References ........................................................

. Preparative

i s o t a c h o p h o r e s i s on t h e m i c r o s c a l e ( L . A r l i n g e r )

Introduction

L i s t o f f r e q u e n t l y o c c u r r i n g symbols Subject index

....................................

...........................................................

379 385

447

SUBJECT I N D E X Adsorption dyes, d e t e c t i o n 82 see aZso i n d i v i d u a l t r a d e names A c e t o n e - s i l v e r nitrate-Na0H reagent 15 A f f i n i t y , d e t e r m i n a t i o n 227 e l e c t r o p h o r e s i s 219-252 -, combined w i t h SDS PAGE 247 -, m o d i f i c a t i o n s 244, 245, 246 -, one dimensional 241-243 -, p r e p a r a t i v e 247 Agar 362, 411 Agarose 20, 22, 202, 213, 260, 296, 362, 378, 382 -, d e s t r u c t i o n o f t h e gel 260 d i s c g e l s 22 A l c i a n Blue 25 A l c o h o l i c s i l v e r n i t r a t e reagent 30 A l k a l i n e s i l v e r n i t r a t e reagent 15, 30 N-Alkylsulphobetaines as d i s aggregating agent 118 Ammonium molybdate as d e t e c t i o n reagent 7, 30 Amphol ines 23 -, s t r o n g i n t e r a c t i o n w i t h t h e s o l u t e 23 A n i l i n e diphenylamine phosphoric a c i d reagent 15 hydrogen p h t h a l a t e reagent 31 phosphate reagent 15 p - A n i s i d i n e reagent 15, 30 A n o l y t e 115 A r t i f a c t s i n i s o e l e c t r i c focusing 23 Autoradiography 311 BASO-DALT technique 83 Benzenesul phohydroxami c a c i d as d e r i va t i z i ng reagent 9 Benzidine reagent 103 Bioautography 409 f o r agar g e l e l e c t r o p h o r e s i s 410 paper e l e c t r o p h o r e s i s 410 Bio-Gels 296 B i s d i a z o t i z e d b e n z i d i n e as detect i o n reagent 7 Borate complexes 13 Britton-Robinson b u f f e r s 398 B u f f e r s f o r agarose g e l e l e c t r o phoresis 377 free flow electrophoresis 89

-

-

-

--

--

Capi 11a r y isotachophoresi s 83 (6-Carboxyethyl) xanthydrazide 12 Carboxymethylation o f p r o t e i n s 187 C a t h o l y t e 115 C e l l u l o s e a c e t a t e 19, 20, 22, 23, 35, 57, 83, 201, 202, 362 m i c r o e l e c t r o p h o r e s i s 201 l a y e r 57, 73, 103 paper 19 powder 296 column 402 C h l o r i n a t i o n d e t e c t i o n procedure 94, 103 Cholate 118 Chromatoelectrophoresis 58, 73 Chromium r e l a t e d reagents 15, 30 Chromogenic substances, c o u p l i n g 301, 302 Col umn e l e c t r o p h o r e s i s 297 Complex f o r m a t i o n d u r i n g s e p a r a t i o n 13, 36, 42 Composite g e l s 20, 182, 362 Compound g e l method 243 Continyous e l e c t r o p h o r e s i s 293 Convection, b l o c k i n g 35 Coomassie B r i l i a n t Blue s t a i n 103, 104, 226 Cross-electrophoresis 332 Crossed immunoelectrophoresi s 157, 213 , 215, 222 D a l t o n 110 DEAE c e l l u l o s e 362 D e n s i t y g r a d i e n t electrophores'is 293 Deoxycholate 118, 121, 143 Derivatization before electrophoresis 1, 2, 9, 13, 24, 90, 91, 187 D e t e c t i o n o f enzymic a c t i v i t i e s 300 D e t e r g e n t ( s ) c o n t a i n i n g b u f f e r s 112 -, non-denaturing 118 -, n o n - i o n i c (NP-40) 126, 143 Diagonal e l e c t r o p h o r e s i s 86 N,N'-Diallyl t a r t a r d i a m i d e as crossl i n k e r 134 D i a z o s u l p h a n i l i c a c i d as d e t e c t i o n reagent 103 D i a z o t i z e d p - n i t r a n i l i n e 405 D i f f u s i o n c o e f f i c i e n t d e t e r m i n a t i o n 328 p-Dimethylaminobenzaldehyde reagent 103 Discontinuous SDS-PAGE 282 D i s s o c i a t i o n constant 230, 231 e v a l u a t i o n 234 D r a g e n d o r f f ' s reagent 1

---

- -,

448

Edman d e g r a d a t i o n 87 E h r l i c h ' s r e a g e n t 46, 103 E l e c t r i c charge c r i t e r i o n 83 E l e c t r o e l u t i o n 82 Electroendoosmotic f l o w 289 Electroimmunoprecipi t a t i o n 219 Electron acceptors i n detection r e a c t i o n s 303 E l e c t r o p h o r e s i s , c o n v e c t i o n 292 E l e c t r o p h o r e t i c l i g h t s c a t t e r i n g 328 m o b i l i t y 81, 328 E l u t i o n o f g e l s 282 Exogenous enzyme s t a i n i n g methods

-

313 Ferguson's p l o t

112, 113, 126, 194,

283

F i b r o u s c a r r i e r s , c h o i c e 82 F i n g e r p r i n t i n g 93, 350 F i x i n g o f g e l s 25 Fluorescamine as l a b e l 103, 104 Fluorescence d e t e c t i o n 19, 73, 74,

91, 92, 103, 104, 399 - l a b e l i n g 19, 73, 74, 91, 92, 103, 104 Foam r u b b e r as s o r b e n t 296 F o l i n ' s r e a g e n t 46 Free s o l u t i o n techniques 290 Fused r o c k e t irnmunoelectrophoresi s

313 260

-,

used f o r m o l e c u l a r w e i g h t d e t e r m i n a t i o n 370 G i r a r d T r e a g e n t 12 Glass paper 19 G r a d i e n t g e l s 202, 311, 372 Homochromatography 352 Hydrogen p e r o x i d e - a c e t i c a c i d r e a g e n t

30 Immunochemical d e t e c t i o n 311 Immunoelectrophoresis 216 I n d i c a t o r s o l u t i o n s 31, 69 I o n f o c u s i n g 35 Iron(II1)chloride sulphosalicylic a c i d r e a g e n t 30 I r o n ( I I 1 ) r e a g e n t 7, 9 I s a t i n as d e t e c t i o n r e a g e n t 103 ISO-DALT t e c h n i q u e 83 I s o e l e c t r i c f o c u s i n g 66, 81, 94, 95,

-

113, 114, 117, 121, 141, 143, 161163, 166- 169, 173 - 175, 188, 194, 202, 213, 214, 326, 327, 403 - -, c o n t i n u o u s f l o w 294 , f l a t bed 296 - - i n Sephadex 166 - -, p r e p a r a t i v e 214 - - , -, i n d e n s i t y g r a d i e n t 294 p o i n t s 81, 132, 326 I s o t a c h o p h o r e s i s 35, 36, 42, 48, 49, 63, 81, 97, 202, 329

-

p r e p a r a t i v e 295 I s o t a c h o p h o r e t i c b u f f e r s 98-100 5 - I s o t h i o c y a n a t o f l u o r e s c e i n 19 J o u l e ' s h e a t 84 Kupke's method 261 L e c t i n i n t e r a c t i o n immunoelectrophoresis

221, 224- 226, 232

- i n t h e f i r s t dimension g e l 223 - - - i n t e r m e d i a t e g e l 223 - r e a c t i o n before electrophoresis - Sepharose 221

220

L i n d g r e n ' s s t a i n i n g procedure 259 L i p i d o p h o r system 259 L i p o p h i l i c dyes 257 Lowry d e t e c t i o n procedure 94 L u b r o l PX 117, 118 Methoxydiphenylfuranone as f l u o r e s c e n t l a b e l 91, 104 M o b i l i t y , d e t e r m i n a t i o n 81, 328 M o l e c u l a r s i e v i n g 362 w e i g h t c r i t e r i o n 82 d e t e r m i n a t i o n 22, 88, 91, 110,

-

--

136, 145, 156, 362, 370 381 , standards 367, 379

- - -, c a l i b r a t i o n

--- -, s t a t i s t i c a l

d e f i n i t i o n 113 Moving boundary e l e c t r o p h o r e s i s 35, 290 M u l t i e l e c t r o p h o r e t i c s e p a r a t i o n s 55, 62,

85

Gel ( s ) d i s s o l u t i o n b e f o r e photometry

--

-,

M u l t i p h a s i c zone e l e c t r o p h o r e s i s 82 M u l t i p l e bands 283 N e g a t i v e f l u o r e s c e n t s t a i n i n g 305 Nephelometric q u a n t i t a t i o n 261 Net charge 88, 113, 343 N i n h y d r i n 30, 46, 94 p-Nitrobenzenediazonium f l u o r o b o r a t e 46 a-Nitroso-6-Naphthol as d e t e c t i o n r e a g e n t

103 NM-Polygram S I L NHR p l a t e s 411 O f f o r d ' s r e l a t i o n s h i p 87 Optimal pH f o r g e l e l e c t r o p h o r e s i s 111 pore s i z e f o r gel electrophoresis

112 - solvents

f o r g e l e l e c t r o p h o r e s i s 111 O p t i m i z a t i o n o f SDS-PAGE 111 PAGE-Pack computer progrgm 112, 113 Pauly r e a g e n t 103 P e p t i d e mapping i n g e l s 93 Pevi kon as s o r b e n t 296 Phosphorescence d e t e c t i o n 103 o-Phthalaldehyde as f l u o r e s c e n t l a b e l

104 pK v a l u e , ranges 84 P o l y a c r y l a m i d e g e l ( s ) , c o m p o s i t i o n 366 e l e c t r o p h o r e s i s , p r e p a r a t i v e 114 used i n s t r u c t u r e s t u d i e s 387 P o l y u r e t h a n e foam 296 Potassium i o d i d e - s t a r c h r e a g e n t 103 P r e - f o c u s i n g 23 P r e p a r a t i v e e l e c t r o p h o r e s i s 290

---

449

Procion Blue 3GS 25 - dyes, d e r i v a t i z a t i o n before e l e c t r o phoresis 24 - Red 2BS 25 Protein-SDS-complexes 91 Q u a n t i t a t i o n 133, 236- 238, 259, by the intermediate gel technique 239 - - - f i r s t dimension gel technique 240 -, photometric 258 Radioactive deteciton 308 Radioimmunoelectrophoresis 213, 215 Radiolabeling before electrophoresis 311 Relation between pK and PI 83 Retardation c o e f f i c i e n t 112, 228 -, concentration dependent 228 Sakaguchi reagent 103 S c h i f f ' s periodate reagent 25 SDS-polyacrylamide gel 90, 91, 110 - - , continuous 282 - -urea-polyacrylamide gel 90, 91 Separation s t a r t e g y 113 Separax, see Cell ul ose a c e t a t e Sephadex 82, 85, 103, 166, 296, 297 S i l i c a gel t h i n l a y e r 35, 103, 402, 411, 414 Silk thread 19 Si 1 ver ni trate-NaOH-pentaerythri to1 reagent 15 - - -sodium ethoxide reagent 10, 15

-

Stacking gel 93, 94 Starch gel 19, 289, 411 (4-Sulphobenzyl) xanthydrazide 12 Takeo-Nakamura p l o t 229 Temperature of polyacrylamide polymer i za t i on 112 Thin l a y e r electrophoresis 296 see a l s o S i l i c a gel t h i n l a y e r Three dimensional separations 58, 59 o-Tolidine reagent 103 Toluidine Blue 25 Transverse pore gradient gel 283 Trimethylaminoacetohydrazide chloride reagent 12 Triton X-100 1 1 7 , 121, 125 Turbidimetric q u a n t i t a t i o n 261 Two dimensional electrophoresis 20, 85, 121, 1 2 2 , 141, 146, 149, 1 7 7 , 188, 190, 373 Urea-phosphoric acid reagent 15 - - t a r t r a t e reagent 15 UV detection 7 , 10, 1 2 , 15, 42, 46, 102, 343, 362, 399, 403, 416 V o l a t i l e buffers f o r zone e l e c t r o phoresis 85 Wieland-Seidel detection procedure 259 Xylene B r i l l i a n t Cyanine G 104 Zimmerman reagent 41, 42

460

INDEX OF COMPOUNDS SEPARATED

A

-,

Abequose 281 Acacetin 7 Acetaldehyde 11 -, phenyl- 51 A c e t i c a c i d 36, 38 Acetone 11 Acetophenone 11 -, bromo- 11 Acetylacetone 11 A c e t y l -CoA carboxylase (E.C. 6.4.1.2) 332 N-Acetyl gal actosami ne 6-sul phatase 151 a- N- Acetyl - D-g 1ucosami n idase ( E C 3.2.1.50) 291 8-N-Acetyl g l ucosaminidase (E. C. 3.2.1.30) 301, 321 a-N-Acetylhexosaminidase 151 6-N-Acetyl hexosaminidase (E. C. 3.2.1.30) 149, 153 a-1-Acid g l y c o p r o t e i n 229 A c i d phosphatase isozymes (E.C. 3.1.3.2) 227, 294, 300 p r o t e i n a s e from r a t spleen 295 Red 420, 428 - V i o l e t 6B 420 Aconi t i ne 398 Acrosin (E.C. 3.4.21.10) 317 ACTH 89, 92, 95 A c t i n 174 B-Actin, non muscle 173 y - A c t i n , non muscle 173 a - A c t i n i n 172 Acyl-CoA hydrolase 295 Adenine 342 Adenosine 2'-phosphate 342 3'-phosphate 342 5'-phosphate 342 - 5'-pyrophosphate 342 5 ' - t r i p h o s p h a t e 342 Adenylyl streptomycin 412 Adipate 36 Agar 25 Agaropectin 26 Agarose 26 Agmatine 50 -, N2-benzoyl- 74 a-Alanine 56, 57, 60 -, 9-amino- 68 -, B-amylamino- 69 -, 6-butylamino- 69 -, e t h y l - 47

..

-

-

B-ethylamino- 69 B-oxalylamino- 77 , 8-propyl amino- 69 B-Alanine 56, 57, 60 Albumin 207,-210Alcohol dehydrogenase (E.C. 1.1.1.1.) 235, 321. 322 A l d o l a s e (E.C. 4.1.2.-) 235, 315, 331 A1 dosterone 44 A l k a l i n e e a r t h metals 437 metals 437 phosphatase (E.C. 3.1.3.1) 292, 312, 313, 320 A l k a l o i d s 395 A l l e r g e n s 213, 214, 215 A l l i t o l 28 A l l o i s o l e u c i n e , N-methyl- 76 D-A1 losamine, N-acetyl- 31 D-Allose 16 A l l y l a l c o h o l 1, 2 A l l y l a m i n e 50 D-A1 t r i t o 1 28 0 - A l t r o s e 16 Amaranth 420, 422, 430 Amino a c i d s 55-79 -, dansyl d e r i v a t i v e s 74 - - , DNP-derivatives 70, 71, 73 , f l uoresceinthiocarbamyl d e r i v a t i v e s 73 , PTH d e r i v a t i v e s 73 , r a r e , s y n t h e t i c , aromatic 64 D-Amino a c i d oxidase (E.C. 1.4.3.3) 292, 315 Ammonia 437 A m o x y c i l l i n 411 Amphetamine 50 Amphomyci n 415 Amphotericin A 416 B 416 A m p i c i l l i n 411 n-Amy1 ami ne 50 Amylase 301, 321 Anabasi ne 397 -, N-methyl- 397 Anatabine 397 -, N-methyl- 397 Androgen r e c e p t o r s 132 Androst-5-en-17-oneY 3a-hydroxy- 44 Androsterone s u l p h a t e 42 A n g i o t e n s i n 89, 92, 99 -, ~ ~ 1 595A n i l i n e 47, 53 -, o - c h l o r o - 47, 53

-,

-

-

--

----

-

451

-,

m-chloro- 47, 53 p - c h l o r o - 47, 53 d i e t h y l - 47 dimethyl- 47, 53 4 - n i t r o - 47 A n k y r i n 172 Anthiol 7 A n t h r a n i l i c a c i d 47 Anthraquinone, 1-hydroxy- 4, 7 -, 1,2-dihydroxy- 7 -, l Y 4 - d i h y d r o x y - 7 dyes 431 A n t i b i o t i c s 409 Antigen 214 0 283 A n t i haemophil i c g l o b u l i n 207 A n t i t r y p s i n 207, 208, 210, 225, 230, 233 Apigenin 7 Apomorphine 395, 396 Apoprotein(s) 261, 269, 274 - a r g i n i n e r i c h 274 - E 274 D - A r a b i n i t o l 28 L - A r a b i n i t o l 28 D-Arabinose 16 -, 2-deoxy- 16 1-Arabinose 16 -, 4-amino-4-deoxy- 231 A r a b i n o i c a c i d 32 A r e c o l i n e 396 Arginase (E.C. 3.5.3.1) 315 A r g i n i n e 50, 56, 57, 60 -, N2-p-aminobenzoyl - 75 -, d i m e t h y l - 76 -, N2-benzoyl- 74 -, monomethyl- 47, 77 Argininosuccinase (E.C. 4.3.2.1) 315 Arylesterase, a i l i p o p r o t e i n associated ( E . C . 3.1.1.2) 230 Arylsulphatase (E.C. 3.1.6.1) 152, 153 Arcaine 50 Ascorbate-2-sul phatase 152 Asparagine 56, 57, 60 - aminotransferase (E.C. 2.6.1.14) 328 A s p a r t i c a c i d 56, 57, 60 - -, benzyl- 36 Aspergi 11 us e x t r a c t s 215 Aspidospermine 396 Astralgin 7 ATP-ase (E.C. 3.6.1.8) 297, 316 Atranol 10 A t r o p i n e 395, 396, 398, 399

-, -, -, -,

-

-

B

BacilZus phages - subtilis protease (E.C. 3.4.24.4) 291

B a c i t r a c i n 92, 99, 100, 416 Bence Jones p r o t e i n s 204 Benzaldehyde 9, 10, 11, 12 -, p-amino- 11 -, p - c h l o r o - 11 -, 2,4-di hydroxy- 10 - , 3,4-di hydroxy- 10 -, 3,4-dimethoxy- 10 -, p-dimethylamino- 11 -, 2-hydroxy- 4, 10 -, 2-hydroxy-3-methoxy- 10 -, 3-hydroxy- 10 -, 4-hydroxy- 5 -, 4-hydroxy-3-methoxy- 5, 10, 11 -, 4-hydroxy-3,5-dimethoxy10 -, 4-methoxy- 11 -, 4 - n i t r o - 11 -, 3,4,5-trimethoxy10 Benzidine 47 Benzoic a c i d 36, 74 - -, p-amino- 47, 53, 75 - - , 2-hydroxy- 4, 5 - - , 3-hydroxy- 5 - - , 4-hydroxy- 5 Benzophenone 11 Benzylacetone 11 Benzylalcohol 1 , 2 -, 2-hydroxy- 4 Benzylideneacetone 11 Benzylideneacetophenone 11 B e n z y l p e n i c i l l i n 412 B e r b e r i ne 396 -, d i m e t h y l - 396 B i cucul 1ine 396 8 i 1 ir u b i n 434 B i o c y t i n 406 B i o t i n 405, 406 Biphenyl, 2-hydroxy- 4, 7 , 2,2 ' -di.hydroxy- 4 -, 4-hydroxy- 7 Bleomycin A 2 414 Blue-VRS 430 B o l d i n e 396 B r a d y k i n i n 89, 100 -, met-lys- 100 B r i l l i a n t b l u e 420, 428 Bromocresol green 428 p u r p l e 428 Bromophenol b l u e 428 Bromothymol b l u e 428 Bromoxyi enol b l ue 428 Brucine 396, 398. 399 Bul bocapni ne 396 1-Butanol 1, 2 -, 3-methyl- 1, 2 2-Butanol 1 -, 3-methyl- 1, 2 Butane, 1,4-diamino50, 51 n-Butylamine 48, 50 n-Butyraldehyde 9, 11

-

-

452

-, 2 - e t h y l - 9 B u t y r i c a c i d 38 , a-amino- 56, 57, 60 , y-ami no- 50, 60

--

--

- - , d,Z-erythro-4-amino-ZY3-dihydroxy-3-methyl-

75

C

C-proteins 207, 208, 209 Cadaveri ne 50 Cadmi um( II ) 436 , 437 Caerul opl asmin 207 Caesium 437 Caffeic acid 5 Caffeine 395, 396, 398 Cal c i t o n i n 95 Calebassine 396 Canavani ne 76 -, desami.no- 76 Capreomycidine 415 Caprylaldehyde 9, 11 Carbamoyl-phosphate synthetase (E.C. 6.3.4.16) 312 - t r a n s f e r a s e (E.C. 2.1.3.3) 316 Carbazole, 2-hydroxy- 7 C a r b e n i c i l l i n 411 Carbonate 439 Carbonic anhydrase (E.C. 4.2.1.1) 294, 312 Carboxylesterase (E.C. 3.1.1.1) 230, 2 34 Carmoisine 422, 430 Carnosine 76 Catalase (E.C. 1.11.1.6) 304 Catechol 5 Cathepsin D 153 C e l l o b i i t o 1 (@-1:4) 28 Cellobiose 17 C e l l s u r f a c e antigens 141-148 C e l l u l a s e (E.C. 3.2.1.4) 295 Cephachlomezine 412 Cephaloram 412 Cephaloridin 412 Cephalosporanic a c i d 412 - - , 7-amino- 412 Cephalosporin(s) 411, 412 C, 7a-methoxy- 412 Cephalotin 412 Chelidonine 396 Chlorate 36 Chloride 36 C h l o r o c r u o r i n 156 Chlorogenic a c i d 5 Chl orophyl 1- p r o t e i n complexes 434 C h l o r t e t r a c y c l i n e 416 Cholecystokinin 98, 99 Cholesterol 254, 259, 271 - e s t e r s 254

-

Choline 50 295, Cholinesterase (E.D. 3.1.1.8) 297, 301 C h o n d r o i t i n sulphates 21, 22, 23, 183 D-Chondrosamine, N-acetyl- 31 Chorionic gonadotropin (CG) 194, 198 Chromate 36 Chromium(II1) oxide 437 Chrysene, 6-hydroxy- 7 Chrysin 7 Chyl omi crons 262 , 263 Cinchonidine 396 Cinchonine 396, 398 Cinnamaldehyde 9, 10, 11 C i t r a l 9, 10 C itronel la1 9 C i t r u l l i n e 60 -, N2-p-aminobenzoyl- 74, 75 C l o m e t o c i l l i n 411 C l o x a c i l l i n 411 Cobalamin, methyl- 403, 405 Cobalt 437, 438 - complexes 437 Cobamide 403 Cocaine 395, 396, 399 Codeine 399 Coenzyme A 404 C o l c h i c i n e 396, 399 Coliphage 186 381 C o l l agen , a-chains 177 -, aA c h a i n 178, 180 -, aB c h a i n 178, 180 -, aC c h a i n 178, 180 -, type I 178, 179 - , type I, procol lagen 181 -, t y p e I 1 178 -, t y p e I 1 1 178, 179 -, type 111, procollagen 181 -, type I V 178, 180 -, type V 178, 180 -, methylated 177 Collagenase (E.C. 3.4.24.3) 293 Complement C3 226 Coniine 396, 398 C o n t r a c t i l e p r o t e i n s 172-176 Copper 436 peni c i 11ami ne complexes 437 Coproporhyrin 428 Corl umine 396 C o r t i c o s t e r o n e 44 C o r t i s o l 44 Cortisone 44 g l y c i n a t e 42 Corydine 396 Cotarnine 396 p-Coumaric a c i d 5 Coumari n , 7- hydroxy- 5 - , 7-hydroxy-4-methyl- 7 Creatine 50

-

-

453

- phosphate

404 k i n a s e (E.C. 2.7.3.2) 235, 315, 333 C r e a t i n i n e 50 C r e s o l , 0-, m-, p- 4 rn-Cresol p u r p l e 428 o-Cresol r e d 428 Crotonaldehyde 9 C u r a r i n e 396 Cyanocobalarnin 403, 405 Cyclamidomycin 415 C y c l i c AMP phosphodiesterase (E.C. 3.1.4.17) 315 I-Cyclohexanol 1, 2 Cyclohexane-l,3-dione 10 Cyclohexanone 10, 11 Cystathione 68 C y s t a t h i o n i n e 63 -, N - a c e t y l - 75 C y s t e i c a c i d 56, 60, 63, 6 8 C y s t e i n e 56, 63, 68 -, S - b u t y l - 63 -, S-carboxymethyl- 60, 66, 68 -, S-(1,2-dicarboxyethy1)66

-

-,

-

S-(1,2-dimethyl-2-carboxyethyl)-

76

-,

S-ethyl-

-,

S-methyl- 63, 63, 76 sulphone 63, 68 s u l p h o x i d e 63, 68 S-( 1-methyl -2-carboxyethy1)-

63, 68

-, S-(2-hydroxy-2-carboxyethy1)-

-,

-,

-

-, -, S-(2-methyl-2-carboxyethy1)-

Dexarnethasone-sodium phosphate 439 D i a c e t y l 10, 12 D i a1 a n i ne 100 D i - n - b u t y l ketone 11 D i benzofuran, 2-hydroxy- 7 D i c e n t r i n e 396 D i c l o x a c i 11 i n 411 D i c o r t i s o l e phosphate 42 Diethanolamine 50 Diethyldithiophosphate 3 D i e t h y l 'ketone 11 D i g a l a c t u r o n i c a c i d 32 D i g l y c i ne 100 D i h y d r o f o l a t e r e d u c t a s e (E.C. 1.5.1.3) 332 D i h y d r o u r a c i l dehydrogenase (E.C. 1.3.1.1) 304 Dimethylamine 50 D i s a r c o s i n e 100 D j e n k o l i c a c i d 63, 68 DNA 361-393 - @ X 174 382 , c i r c u l a r 381, 382, 388 -, c o n f o r m a t i o n a l isomers 382 - dependent RNA polymerase (E.C. 2.7.7.6) 313 -, fragments 378, 380 - l i n e a r 379, 382 -, m i t o c h o n d r i a 1 382 -, n u c l e a r 376 - - RNA h y b r i d s 373, 375, 381 -, sequence a n a l y s i s 384, 385 Dodecyl ami ne 48 Dopamine 51 Dyes, s y n t h e t i c 422, 424, 426 Dyneins 190

76

76 66

C y s t e i n e s u l p h i n i c a c i d 63, 68 C y s t i n e 56, 60, 63, 68 C y t i d i n e , 5-methyl- 342 - 2'-phosphate 342 3'-phosphate 342 - 5'-phosphate 342 5'-pyrophosphate 342 - 5 ' - t r i p h o s p h a t e 342 Cytochrome C 92 c o x i d a s e (E.C. 1.9.3.1) 313 C y t o s i n e 50, 342 -, 5-methyl- 342 C y t o s k e l e t a l p r o t e i n s 172

-

D

1-Decanol 1, 2 DecanaI 0 , 11 Decyl amine 48 Dehydroepiandrosterone s u l p h a t e 42 - - , 7-hydroxy- 42 - - , 7-0x0- 42 Deoxyribonuclease (E.C. 3.1.21.1) 291 Desmin 173, 175 D e s o x y c o r t i c o s t e r o n e 44

E Edeines 416 E l a s t a s e (E.C. 3.4.21.11) 292, 332 i n h i b i t o r 332 E l a s t i n 182 Emetine 396, 399 Enanthaldehyde 9, 10, 11 E n a n t h i c a c i d 36 297 Enolase (E.C. 4.2.1.11) Enzyme(s) 287-339 -, m i c r o h e t e r o g e n e i t y 151 Eosine 420, 428 Ephedrine 51, 396, 398, 399 E p i n e p h r i n e 51 Ergometrine 396, 399 Ergotamine 395 E r y t h r i t o l 28 E r y t h r o c r u o r i n 156 E r y t h r o m y c i n 416 E r y t h r o n i c a c i d 32 D-Ervthrose 16

-

464

-, 2-deoxy- 16 -, 1,2-dideoxy- 16 - 4-phosphate 26 E r y t h r o s i ne 420, 425 Esculin 7 Eserine 396, 399 Estamycin 416 Estrogen r e c e p t o r (ER) 130, 132 Ethanol 1, 2 -, 2-ethoxy- 1, 2 -, 2-( 2-ethoxyethoxy)- 2 -, 2-methoxy- 2 -, 2-(2-methoxyethoxy)- 2 -, 2-phenyl- 1 Ethanol amine 51, 281 Ethylamine 51, 281 -, 2-phenyl- 51 Ethyl c a r b i t o l 2 C e l l o s o l v e 1, 2 Ethylenediami ne 51

-

F Fast Red E 422 - Green 420 Ferrihaemoglobin 163 Ferulic acid 5 F i b r i n 99 F i b r i n o g e n 206, 207, 208, 210 F l a v i n adenine d i n u c l e o t i d e (FAD) 403, 404 mononucleotide (FMN) 403, 404 Flavomycin 416 Flavonoids 5, 6 Fluorene, 2-hydroxy- 7 -, 3-hydroxy- 7 F1uorescein , d i bromo- 428 F1 u o r o c u r i ne 396 Foetal haemoglobin 161 a-Foetoprotein (AFP) 228, 229, 230, 231, 232 Food dyes 427 Formaldehyde 9 Framyceti n 416 D-Fructose 17 - 1-phosphate 26, 30 - 6-phosphate 26, 30 1,6-diphosphate 26, 30 D-Fucosami ne , N-acetyl - 31 L-Fucose 16 a-L-Fucosidase 152 F u l v i c a c i d 434 F u r f u r a l 10 , w-hydroxymethyl- 10 a - F u r f u r y l a1 coho1 2

-

-

G

Gal a c t i t o 1 28 Galactokinase (E.C.

2.7.1.6)

308

Gal actomannans 24 G a l a c t o n i c a c i d 32 D-Galactosamine 31, 5 1 -, N-acetyl- 31 D-Gal actose 16 1-phosphate 26 6-phosphate 26 B-Galactosidase (E.C. 3.2.1.23) 152, 296 Galactosyl t r a n s f e r a s e (E.C. 2.4.1.23) 314 Gal a c t u r o n i c a c i d 32 1-phosphate 32 Galegine 396 Gallic acid 5 Gentamicin 414 Gentiobiitol(6-1:6) 28 Gentiobiose 17 Glaucine 396 Glucagon 92 Glucans 24 G l u c a r i c a c i d 32 D-GI u c i t o 1 28 G1 u c o c o r t i c o i d r e c e p t o r s (GR) 132, 133, 136 Gluconic a c i d 32 6-phosphate 26 , 2-0x0- 32 -, 5-0x0- 32 D-Glucosamine 31, 51 6-phosphate 26 -, N-acetyl- 31 Glucosaminic a c i d 31 D-Glucose 16 - 1-phosphate 26, 30 6-phosphate 26, 30 - 1,6-di phosphate 26 G1 ucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) 296, 304, 313, 324 a-Glucosidase (E.C. 3.2.1.20) 153 6-Glucosidase (E.C. 3.2.1.21) 150, 291 D-Glucuronic a c i d 32 1-phosphate 32 , 4-0-methyl- 32 6-Glucuronidase (E.C. 3.2.1.31) 151, 152, 296 Glutamate-oxaloacetate transaminase (E.C. 2.6.1.1) 306 G1 utamate-pyruvate transaminase (E. C. 2.6.1.2) 306 Glutamic a c i d 36, 56, 57, 60 -, p-aminobenzioyl- 75 -, benzoyl- 14 -, y-hydroxy-y-methyl- 76 -, y-methyl- 76 , y-methylene- 76 Glutamine 56, 57 -, N2-p-aminobenzoyl- 75 -, N2-benzoyl- 74

-

-

---

-

---

--

455

-, y-methylene- 76 Glutamine transaminase (E.C. 2.6.1.15) 297 Glutaric acid, 3-(S-cysteinyl)76 G l u t a t h i o n e 99, 100, 302 G l u t a t h i o n e peroxidase (E.C. 1.11.1.9) 305 - reductase (E.C. 1.6.4.2) 304 DL-Glyceraldehyde 16 Glyceraldehyde-phosphate dehydrogenase (E.C. 1.2.1.9) 304 Glycerol 28 Glycinamide 51 Glycine 51, 56, 57, 60 , p-aminobenzoyl- 75 -, benzoyl- 74 - , cis-a- (carboxycycl opropy1)- 76 -, trans-a-(carboxycyclopropyl 76 -, trans-a-2-(carboxymethylcyc opropy1)- 76 Glycopeptides 219-252 G l y c o p r o t e i n ( s ) 219-252 -, a2-HS 230 G1ycosami nogl ycans 19 Glycosidases lysosomal 149- 155 Glyoxal 12 Glyoxalase (E.C. 4.4.1.5) 302, 304 Glyoxyl ic a c i d 32 Gramicidin S 415 -, ~4,5-s-arninovaleric acid] 415 Gramine 5 1 Growth hormone (GH) 99, 194, 196 GTP-AMP phosphotransferase (E. C. 2.7.4.8) 295 Guanine 342 Guanosine 2'-phosphate 342 3'-phosphate 342 - 5'-phosphate 342 - 5'-pyrophosphate 342 5'-triphosphate 342 Guanylate kinase 322, 323 L-Gulono-y-lactone oxidase (E.C. 1.1.3.8) 307 D-Gulose 16

-

.I-

-

-

H

Haemerythrin 156 Haemocyanins 156-160 Haemogl o b i n ( s ) 161-165 - 9 "282 163 -, a2+82 163 -, A 161 -, A l c 162 -, A2 161 -, A, c h a i n 161 -, canine 162 -, F 161 -, Gower 1 161

-, -, -, -,

Gg c h a i n 161 162 mutants 162 P o r t l a n d 161 S 161 Haemopexi n 99 Halides 438 Haptoglobin 207, 208, 226, 230 Harmine 396 Hemicelluloses 26 Heparan sulphate 21, 23 Heparin 19, 21, 22, 23, 430 -, N-(2,4-dinitrophenyl)19 1-Heptanol 2 n-Heptanal 9, 10, 11 H e r o i n 395, 396 H e t a c i l l i n 411 Hexadecylamine 48 1-Hexanol 1, 2 -, 2 - e t h y l 1 n-Hexanal 9, 11 -, 2 - e t h y l - 9 4-Hexenoi c a c i d , 5-methyl -2-ami no- 76 Hexokinase (E.C. 2.7.1.1) 313 Hexosaminidase C 296 Hexylamine 48 High d e n s i t y l i p o p r o t e i n s (HDL) 254, 256, 259, 263, 264, 272, 275 H i p p u r i c a c i d 74 Histamine 51 H i s t i d a s e (E.C. 4.3.1.3) 317 H i s t i d i n e 51, 56, 57, 60, 66, 67 -, N2-p-aminobenzoyl 75 -, N2-benzoyl- 74 -, 1-methyl- 66, 67, 77 -, 3-methyl- 66, 67 HLA antigens 142, 144 , p r e c u r s o r s 145 Homatropi ne 396 , 399 Homocysteic a c i d 63, 68 Homocysteine, S - ( l Y 2 - d i c a r b o x y e t h y 1 ) 76 Homocystine 63, 68 L-Homomethi o n i ne 76 Homoserine, N-amidino- 75 -, anhydride 75 Horseradish peroxidase (E.C. 1.11.1.7) 292 House d u s t m i t e a l l e r g e n s 214 Human serum p r o t e i n s 224 Humic a c i d s 430, 434 H y a l u r o n i c a c i d 21, 23 Hyaluronidase (E.C. 3.2.1.25) 317 H y d r a s t i n e 396 H y d r a s t i n i n e 396 Hydroquinone 4, 5 -, hydroxy- 4, 5 Hydroxocobal ami ne 433, 405 Hydroxylamine 5 1

- , Malmo

-

--

-

456

Hydroxylysine

56

Kanamycin 6, 3',4'-dideoxy-, 2 ' I - a d e n y l a t e 413 - y 2"-guanylate 413 Keratan sulphate 21, 22, 23 K i n i n s 89 Kynurenine 51

-, g a l a c t o s y l - 69 - , g l ucosyl gal a c t o s y l -

-

69 Hydroxyprol i n e 56, 57, 60 Hyoscyami ne 396, 398 Hyoscine 399 Hypophosphite 439 Hypoxanthine 342 - phosphoribosyl t r a n s f e r a s e (E.C. 2.4.2.8) 298 Hypusi ne ' 76

L

-

I

I a - l i k e antigens 145, 146 L - I d i t o l 28 L-Idose 28 a-L-Iduronidase 152 Immunoglobulins 166-171, 203, 206, 210, 226, 264 , paraproteins, monoclonal 203 I n d i g o carmine 420, 430 I n d i g o t i n e 428 Indophenol oxidase (E.C. 1.9.3.1) 324 I n o s i n e 5'-phosphate 342 triphosphatase 316 I n o s i t o l polyphosphates 26 I n s e c t venoms 214 I n s u l i n 95 I n u l i n 18 I r i d i u m ( 1 V ) 437 Iron 437, 438 Isoamyl amine 50 I s o b u t e i n e 66 I s o b u t y l ami ne 50 I s o c i t r a t e dehydrogenase (E. C. 1.1.1.41: 1.1.1.42) 304 lyase (E.C. 4.1.3:l) 305 Isododecanal 9 Isoenzymes 235 I s o l e u c i n e 56 -, N-methyl- 77 aZZo- I s o l e u c i n e 56 I s o m a l t i t o l ( ~ . - l : 6 ) 28 Isomal t o s e 17 oligosaccharides 235 Isomaltulose 17 Isopentanal 9 I s o q u i no1 i n e , 5-hydroxy7 a-Isosaccharinic a c i d 32 I s o x a n t h o p t e r i n , 6-methyl- 406

-

-

-

-

J Jervine

396

K K a l l i d i n 100 Kanamycin 412, 413, 414

-,

6'-N-tert.-butyloxycarbamoyl-

8-Lactamase (E.C. 3.5.2.6) 324 L a c t a t e 38, 330, 439 dehydrogenase (E.C. 1.1.1.27) 235, 297, 314, 319, 323, 330 L a c t i c a c i d 38, 330, 439 L a c t i t o 1 (6-1 :4) 28 8 - L a c t o g l o b u l i n A 95 B 95 Lactose 17 Lambda phage 381 Laminaribiitol(8-1:3) 28 Lami n a r i b i ose 17 L a n t h i o n i n e 68 Lead 436 L e c t i n s 244-246 Leucine 56, 57, 60 Leucrose 17 L e v u l i n i c a c i d , 5-amino- 75 Lima-bean t r y p s i n i n h i b i t o r 92 L i n k p r o t e i n 183 Lipase 297, 331 Lipopolysacchari des 281-285 L i p o p r o t e i n s 253-279 -, low d e n s i t y 226, 254, 256, 259, 274 -, p r o f i l e s 270 very low d e n s i t y 254, 256, 263, i74 -, X 254, 256, 275 a - L i p o p r o t e i ns 262, 264, 265 , 266 , 272 B-Lipoproteins 210, 262, 264, 265, 266, 268, 272 L i t h i u m 437 Lividomycin A 413, 414 - A 5"-phosphate 413 - B 413, 414 L o b e l i n e 395, 398 Lupanine 396 L u p i n i n e 396 Lysine 51, 56, 57, 60 gal a c t o s y l -hydroxy- 69 g l ucosyl g a l a c t o s y l -hydroxy- 69 -, hydroxy- 56 , N6-monomethyl 77 -, N 6 - t r i m e t h y l - 66 L y s i n o a l a n i n e 68 Lysosomal hydro1 ases 152 Lysozyme (E.C. 3.2.1.17) 92, 316 D-Lyxose 16 -, 2-deoxy- 16

413

-

-

-

-

457

M M - l i n e p r o t e i n s 172 a ? - M a c r o q l o b u l i n 210, 226 M a l a t e dehydrogenase (E.C. 1.1.1.37) 304. 324 M a l o n i c a c i d 36 Maltitol(a-l:4) 28 M a l t o s e 17 M a l t u l o s e 17 Malvinon 7 Manganese 438 Mannans 18 D-Manni t o 1 2 8 D-Mannooctul osonate 281 Mannosamine 3 1 -, N - a c e t y l - 31 D-Mannose 16 - 1-phosphate 26 - 6-phosphate 26 Mavacurine 396 Melanocyte s t i m u l a t i n g hormone 89 M e l i b i i t o l ( ~ - l : 6 ) 28 Me1 ib i o s e 17 Membrane p r o t e i n s , denatured 120-124 - - , n a t i v e 117-119 - r e c e p t o r s 125-128 Mercury 436, 437 Mescaline 51, 396 Metacycl i n e 416 M e t a n i c o t i n e 397 -, methyl- 397 Met a p hos p h a t e 437 Methaemoglobin 163 Methanol 1, 2 -, phenyl- 1, 2 em-3,4-Methanoprol i n e 76 M e t h i c i l l i n 411 M e t h i o n i n e 56, 57, 60, 63, 68, 76 methylsulphonium c h l o r i d e 63, 68 sulphone 60, 63, 68, 76 s u l p h o x i d e 63 s u l p h o x i m i n e 63, 68 -, N - a c e t y l - 75 -, N-formyl- 76 D-B-Methionine 76 M e t h o t r e x a t e 332 Methylamine 48, 51 Methyl n-amyl ketone 11 n - b u t y l ketone 11 - carbitol 2 - Cellosolve 2 - e t h y l ketone 11 - n - h e x y l ketone 11 - n - p r o p y l k e t o n e 11 a2-Mi c r o g l obul in 204 B 2 - M i c r o g l o b u l i n 144, 204 M i c r o t u b u l a r p r o t e i n s 185

-

-

M i c r o t u b u l e a s s o c i a t e d p r o t e i n s 190 M i t o c h o n d r i a 295 Mold a l l e r g e n s 215 Morin 7 Morphine 395, 396, 399 Morphol i n o e t h a n e s u l p h o n i c a c i d 330 Mouse u r i n e a l l e r g e n s 214 Muramic a c i d 31 Myeloma p r o t e i n s 235 M y o i n o s i t o l t r i p y r o p h o s p h a t e 407 Myosin 172 Myosmine 397 M y r i s t i c a c i d , 3-hydroxy- 281 N

NAD 330, 403, 404 NADH 330, 404 NADH-diaphorase (E.C. 1.6.99.1) 304 NADP 403 NADP-dependent i s o c i t r a t e dehydrogenase (E.C. 1.1.1.42) 324 NADPH 404 N a f c i l l i n 411 Naphthacenequinone, 6 , l l - d i h y d r o x y 7 l a p h t h a l e n e , 2,3-dihydroxy4 -, 2,6-dihydroxy- 4 -, 2,7-dihydroxy- 4 Naphthalene-1-carboxylic a c i d , 3-methoxy5-methyl- 76 1-Naphthol 4, 7 2-Naphthol 4, 7 s u l p h a t e 36 -, 1 - n i t r o s o - 47 Naphthol Yellow 420 1-Naphthylamine 47 -, d i e t h y l - 47 2-Naphthylamine 47 N a r c e i n e 396 N a r c o t i n e 396 Natamycin 416 Neomycin 413, 414, 416 0-erythro-Neopterin 406 - 3'-phosphate 406 L - t h r e o - N e o p t e r i n 406 - 2 ' : 3 ' - c y c l i c phosphate 406 Neurine 5 1 New Coccine 420, 428 N i c o t i n a m i d e 402, 403, 404 -, N-methyl- 403, 404 N i c o t i n e 51, 395, 396, 397, 398 N i c o t i n i c a c i d 403, 404 N i c o t i n u r i c a c i d 403, 404 N i c o t o n e 397 N i c o t y r i n e 397 N i g e r i t o l ( a - 1 : 3 ) 28 N i g e r o s e 17 N i t r a t e 439 N i t r i t e r e d u c t a s e 317

-

468

N i t r o g e n a s e (E.C. 1.18.2.1) 317 1-Nonanol 1, 2 Nonyl amine 48 Norepinephrine 51 N o r n i c o t i n e 397 N u c l e i c a c i d s , sequence a n a l y s i s 383 see aZso DNA, RNA N u c l e o p r o t e i n s 379 Nucleoside t r i p h o s p h a t e adenyl a t e k i n a s e (E.C. 2.7.4.10) 315 Nucleosides 341-359 N u l c e o t i d e s 341-359 N y s a t i n 416 0 Octadecylamine 48 1-Octanol 2 2-Octanol 2 n-Octanal 9 O c t u l o s e I y 8 - d i p h o s p h a t e 26 O c t y l ami ne 48 Oleandomycin 416 O r n i t h i n e 51, 56, 60 -, N5-p-aminobenzoyl- 75 -, N5-benzoyl- 74 -, N2 ,N5-di-p-aminobenzoy175 , N2 ,N5-di b e n z o y l - 74 -, N5-methyl- 67 Orosomucoid 207, 208, 229, 230 Orthophosphate 30, 407, 437, 439 O r t h o v a n i l l i n 10 Ovalbumin 92 O x a c i l l i n 411 O x a l i c a c i d 37 1,2-0xazin-3-oneY 4-amino-2-amidinoperhydro- 75 Oxyhaemoglobi n 163 O x y n i c o t i n e 397 O x y t e t r a c y c l ine 416 O x y t o c i n 99 -, 0-methyl- 98

-

P Palmatine, t e t r a h y d r o - 397 P a n c r e a t i c l i p a s e (E.C. 3.1.1.3) 30 1 t r y p s i n i n h i b i t o r 98 Papaverine 395, 396, 399 Paromamine 412 Paromomycin 413, 414 Peanut a1 l e r g e n s 214 P e c t i n s 18 Peni c i 1 1ami ne , copper complexes P e n i c i l l i n 411, 416 -, b e n z y l - 412 P e n t a e r y t h r i t o 1 28 n-Pentanal 9, 11

297,

-

437

Pentane, 1,5-diamino50 1-Pentanol 1, 2 0-eqthro-Pentulose 16 D-threo-Pentulose 16 P e p t i d e s 81-107 Phage DNA 383 P h e n e t i c i l l i n 411 Phenol 4, 47 -, 2-amino- 47 -, 3-amino- 47 -, 4-bromo- 4 -s 2-chloro- 4 -, 3 - c h l o r o - 4 -, 4 - c h l o r o - 4 -, 4-iOdO- 4 -, 2 - n i t r o - 4 -, 3 - n i t r o - 4 -s 4-nitro- 4 P h e n y l a l a n i n e 56, 57, 60 -, 3,4-dihydroxy- 5, 60 m-Phenylenediamine 47 p-Phenyl enedi ami ne 47 P h e n y l g l y o x a l 12 P h l o r o g l u c i n o l 4, 5 P h l o x i n e 420 Phosphatase(s) 298, 301, 315 P h o s p h i t e 439 Phosphodiesterase (E.C. 3.1.4.17) 294 0-Phosphoethanolamine 60 P h o s p h o f r u c t o k i n a s e (E.C. 2.7.1.11) 315 Phosphoglucomutase (E.C. 2.7.5.1) 315, 321, 324 Phosphogl uconate dehydrogenase (E.C. 1.1.1.43) 304 Phosphoglycerate k i n a s e (E.C. 2.7.2.3) 315, 324, 397 Phosphoglyceromutase (E.C. 2.7.5.3) 315 Phosphoglycol l a t e 315 Phosphol i p a s e A2 316 P h o s p h o l i p i d s 254, 271, 272 Phosphoprotein phosphatase 311 Phosphoryl ase-g1 ycogen complex 235 Phosphoserine 60, 77 Phosphothreonine 77 Photosystem I p r o t e i n complex 434 P h y s o s t i gmi ne 396, 399 P h y t i c a c i d 407 P i c r a m i c a c i d 47 P i l o c a r p i n e 396, 399 P i p e c o l i c a c i d 77 P i p e r a z i n e 51 - y N-(2-aminoethy1)50 -, N-(2-hydroxyethy1)- 5 1 P i p e r i d i n e 51 -, 3-hydroxy- 51 P i p e r i n e 396 Plasma p r o t e i n s 201-212 p r o f i l i n g 211 P l a s m i d ( s ) 378 pBR322 DNA, sequence 386

-

-)

459

P l a s t i d DNA 381 P1 atinurn( I V ) 437 pM2 Phage DNA 382 P o l l e n e x t r a c t s 215 Poplar t r e e p o l l e n , a l l e r g e n s 214 P o l y g l y c i n e 98 P o l y l y s i n e 92 Polymyxin B 415 P o l y n u c l e o t i d e phosphorylase (E.C. 2.7.7.8) 294, 295 Polysaccharides 24, 26 -, n e u t r a l 18 Ponceau MX 422 R 420 3R 420 4R 422, 430 SX 420 P o r p h y r i n ( s ) 428 e s t e r s 430 Potassium 437 Preal bumi n 207 Prednisolone, hemisuccinate 42 phosphate 42 Prednisone, g l y c i n a t e 42 Pregnancy-associated p r o t e i n s 207 44 Pregnane-3a, 116 ,17a,ZOa-tetrol Pregnane-3a, 17a ,206-trio1 44 Pregnan-11-one , 3 a , l l ~,20a , Z l - t e t r a hydroxy- 44 Pregnan-20-oneY 3 a , l l ~ , 1 7 ~ , 2 1 - t e t r a hydroxy- 44 Pregn-5-ene-3cr ,17cr , 2 0 a - t r i 01 44 Pre-6-lipoproteins 262, 263, 265, 266, 2747276 Procollagens 177, 180, 181 Progesterone r e c e p t o r (PR) 130, 132, 136 , s u b u n i t s 130, 131, 133 P r o l a c t i n 194, 195 P r o l i n e 56, 57, 60, 67 -, hydroxy- 56, 57, 60 -, trans-3-methyl- 67 -, cis-4-methyl- 67 , trans-4-methyl 67 -, cis-5-methyl- 67 -, trans-5-methyl- 67 50 Propane, 1,3-diaminolY3-Propanedi01 , 2-ami no-2-hydroxymethyl- 50 1-Propanol 1, 2 -, 3-amino- 50 -, 2-methyl- 1, 2 2-Propanol 1, 2 -, I-amino- 50 1, 2 2-Propen-1-01 P r o p i c i l l i n 411 Propionaldehyde 11 P r o p i o n i c a c i d 36 , 6-chloro- 36

-

-

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--

--

-

n-Propylamine 48, 51 -, 3-dimethylamino- 51 P r o t e i n ( s ) , CNBr fragments 96 P r o t e i n kinase 310, 333 Protein-pigment complexes 434 Proteoglycans 182 Protocatechualdehyde 10 Protocatechuic a c i d 5 Protopine 397 P r o t o p o r p h y r i n 428 P r o t o v e r a t r i n e 397 D-Psicose 17 P t e r i d i n e , 2-amino-4-hydroxy-6-methyl406 P t e r i d i n e - 6 - c a r b o x y l i c a c i d , 2-amino4-hydroxy406 P u r i ne , 6-dimethyl ami no- 342 , 6-methyl ami no- 342 Puromycin 416 -, 0-demethyl- 416 -, N-formylmethionyl- 416 Putreanine 77 P u t r e s c i n e 50, 51 Pyrazole-3,5-dicarboxylic a c i d 36 Pyrene, 1-hydroxy- 7 P y r i d o x a l 401, 402 phosphate 401, 402, 404 Pyri doxamine 401, 402 phosphate 401, 402, 404 P y r i d o x i n e see Pyridoxol Pyridoxol 401, 402 phosphate 401, 402, 404 Pyrocatechol 4 P y r o g a l l o l 4, 5, 47 Pyrophosphatase (E.C. 3.6.1.1) 315 Pyrophosphate 407, 434, 439 P y r r o l i d i n e 51 Pyrrolidonecarboxyl i c a c i d 77 Pyruvate kinase (E.C. 2.7.1.40) 314 Pyruvaldehyde 12 P y r u v i c a c i d 330 PZA phage DNA, fragments 386

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Q Q u i n i d i n e 399 Q u i n i n e 395, 397-399 Q u ino1 ine , 5-hydroxy-, 8-hydroxy- 7

7

R

R a f f i n o s e 17 Ragweed p o l l e n a l l e r g e n s 214 Receptors f o r s t e r o i d hormones Red beet pigments 428 Reserpine 399 Resorcinol 4, 5

129

460

R e s t r i c t i o n endonuclease 307 fragments 379 Retinochrome 434 L-Rhamnose 16, 281 Rhodanase (E.C. 2.8.1.1) 317 Rhodopsin 434 Rhodopterin 406 R i b i t o l 28 R i b o f l a v i n (B2) 402, 403 Ribonuclease 92, 294, 309, 317, 328 D-Ribo-hexose, 2-deoxy16 -, 3-deoxy- 16 Ribose 16 I y 5 - d i p h o s p h a t e 26 diphosphate, c y c l i c 26 5-phosphate 26 1-pyrophosphate 26 -, deoxy-, 1-phosphate 26 R i b u l ose, 1,5-di phosphate 26 5-phosphate 26 RNA 361-393 -, s t r u c t u r e s t u d i e s 388 dsRNA 365 rRNA 365, 367 mRNA 365, 371, 374, 388 -, s t r u c t u r e s t u d i e s 373, 385 ssRNA 368 t R N A 368, 369 Robinin 7 Rol it e t r a c y c l i n e 416 Rose Bengal 420 R o s e o t h r i c i n s 412 Ryegrass p o l l e n a l l e r g e n s 214

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-

S i a l i c a c i d 31 Sodium 437 S o l a n i n e 397, 398 S o m a t o s t a t i n 99 Sophorose 17 L-Sorbose 17 S o r b i t o l 28 S o r p h o r i t o 1 ( 6 - 1 :2) 28 Soybean t r y p s i n i n h i b i t o r 92 S p a r t e i n e 395, 397, 398 S p e c t r i n 172 Spermidine 51 Spermine 52 Sphingomyelinase D (E.C. 3.1.4.41) 301 Spi ramycin 416 S t a r c h 26 S t e r o i d ( s ) 41-44 oxygenated 43 - r e c e p t o r s , m u l t i p l e forms 136 S t r e p t o m y c i n 412, 416 -, a d e n y l y l - 412 S t r y c h n i n e 395, 397,-399 Sucrose 17 S u l p h a n i l i c a c i d 47 Sul phates , lysosomal 149-155 S u l p h a t e 36, 330, 439 S u l p h i t e 439 S-Sul phocys t e i ne 68 S-Sul phogl u t a t h i o n e 68 S u l p h o p h t h a l e i n i n d i c a t o r s 428 S - S u l p h o t h i o c y s t e i n e 68 S u n c i l l i n 411 Sunset y e l l o w 430 Superoxide dismutase (E.C. 1.15.1.1) 325 Syri n g a l dehyde 5, 10

-

S

T a - S a c c h a r i n i c a c i d 32 Saccharose 17 S a l i c y l a l d e h y d e 4, 10 S a l i c y l i c a c i d 4, 5 S a n q u i n a r i n e 397 Sarcosine 100, 415 S a t e l l i t e DNA 381 Scopolamine 395, 397 S e c r e t i n 98 Sedoheptulose, 1,7-diphosphate 26 7-phosphate 26 Sedulose 7-phosphate 26 Selenocysteine, Se-methyl- 77 Sempervirine 397 S e r i n e 56, 57, 60 -, a c e t y l - 69 -, f o r m y l - 69 -, N-malonyl- 76 S e r i n e h y d r o l a s e s (E.C. 4.2.1.13) 295 Serotonin 51 Serum c h o l i n e s t e r a s e 227

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D-Tagatose 17 Taka-amylase A 316 D-Talosamine, N-acetyl31 D-Talose 16 TA-receptor complex 134 T a r t r a z i n e 420, 428-430 T a u r i n e 52, 56, 61, 63, 68 Techtochrysin 7 T e s t o s t e r o n e g l y c i n a t e 42 T e t r a c y c l i n e 416 T e t r a d e c y l a m i n e 48 Tetraethylenepentamine 52 T e t r a g l y c i n e 100 Theobromine 395, 397 T h e o p h y l l i n e 397 Theta phage 381 Thiamine 401, 402, 404 monophosphate 404 pyrophosphate 404 - t r i p h o s p h a t e 404 T h i a l y s i n e 77

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461 4-Thia-1-piperideine-4-carboxy1.i~ a c i d 77 p-Thiocresol 4 Thiols 3 Thiophenol , 4 - n i t r o - 4 Thiosulphate 439 Thorium( I V ) 437 L-Threi t o 1 28 Threonine 56, 57, 6 1 L-Threose 16 Thymidine-5'-phosphate 342 Thymine 342 Thymol b l u e 428 o-Toluidine 47 m-Toluidine 47 -, d i e t h y l - 47 p-To1 u i d i ne 47 S-Transferase ( E . C . 2.5.1.18) 302 T r a n s f e r r i n 207, 210, 230, 235 Trehalase ( E . C . 3.2.1.28) 316 a,a-Trehalose 17 T r i a l a n i n e 100 Trichomycin 416 T r i e t h y l a m i n e 52 T r i g l y c e r i d e s 254, 259 T r i g l y c i n e 100 Trimethylamine 52 Triosephosphate isomerase ( E . C . 5.3.1.1) 315 Tropacocaine 397 Tropine 397 T r o p o e l a s t i n 182 -, dansyl- 182 Tropomyosin 172, 174, 175 Troponin 172, 174 T r y p s i n (E.C. 3.4.21.4) 291 Tryptamine 52 -, 3-hydroxy- 51 -, 5-hydroxy- 51 Tryptophan 56, 57, 6 1 Tryptophyl - t R N A synthetase (E.C. 6.1.1.2) 308 Tubocurarine 395, 397, 399 -, dimethyl- 396 T u b u l i n 172, 173, 185-187 S-carboxyamidomethylated 187 S-carboxymethylated 187 m u l t i p l e forms 188, 189 Turanose 17 T y l o s i n 416 Tyramine 52 -, 3-hydroxy- 51 T y r o c i d i n e 416 Tyrosine 56, 5.7, 6 1 -, d i i o d o - 67 -, monoiodo- 67

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U UDP-g1 ucuronosyl t r a n s f e r a s e ( E . C . 2.4.1.17) 316 Umbel 1 iferone 5 -, 4-methyl- 5 U r a c i l 342 Urea 52 U r i c a c i d 38 U r i d i n e 2'-phosphate 342 3'-phosphate 342 5'-phosphate 342 - 5'-pyrophosphate 342 5 ' - t r i p h o s p h a t e 342 Urinary glycoproteins, heterogeneity 240 Urocanase ( E . C . 4.2.1.49) 317 294 Urokinase ( E . C . 3.4.21.31) Urophorphyrin 428 Uroporphyrinogen I synthetase (E.C. 4.3.1.8) 306

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V n-Valeraldehyde 9, 11 V a l e r i c a c i d , 5-amino-4-hydroxyV a l i n e 56, 57, 6 1 -, N-methyl- 77 Vancomycin 414 V a n i l l i n 5, 10, 11 -, f o r m y l - 10 Vasopressin 89 , arg- 99, 100 -, des-NH,-D-a1 a- 98 -, l y s - 98, 100 -, t r i g l y c y l - 98 Veratraldehyde 10 V e r a t r i ne 397 Vespid venoms 216 Vibramycin 416 Viomycidine 415 Viomycin 415 V i r g i n i a m y c i n 416 Visual pigments 434 Vitamins 401

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X

Xanthine 342 Xanthone, 4-hydroxyX a n t h o p t e r i n 406 -, 7-methyl- 406 Xanthosine 342 lY2,5-Xyl en01 4 lY3,5-Xyl en01 4 1,4,5-Xyl en01 4 Xylenol b l u e 428 m-Xylidine 47

7

75

239,

462

Xylitol 28 Xylonic acid 32 D-Xylosamine, N-acetyl- 31 Xylose 16 - 1-phosphate 26 2-0-(4-0-methyl-a-glucuronosyl)j2 Xylulose 5-phosphate 26

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Y Yohimbine 397 Yttrium 437