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Studies on the structure of human haptoglobin chain Hew, Choy-Leong 1970

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STUDIES ON THE STRUCTURE OF HUMAN HAPTOGLOBIN 6 CHAIN by CHOY-LEONG HEW B.Sc. Nanyang U n i v e r s i t y , S i n g a p o r e , 1963 M.Sc. Simon F r a s e r U n i v e r s i t y , B u r n a b y , 1966 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f B i o c h e m i s t r y F a c u l t y o f M e d i c i n e We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d May, 1970 U n i v e r s i t y o f B r i t i s h C o l u m b i a In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ivers i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying o f th i s thes is for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t i on of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of B i o c h e m i s t r y The Univers i ty o f B r i t i s h Columbia Vancouver 8, Canada Date 2 9 t h May 1970 ABSTRACT Haptoglobins are a group of glycoproteins found i n normal human serum and are capable of binding hemoglobin both i n vivo and i n v i t r o . Haptoglobin 2-1, one of the common haptoglobin phenotypes was i s o l a t e d i n large quantity from^the/ascites f l u i d . Upon reduction and a l k y l a t i o n i n the presence of 8 M urea, haptoglobin dissociates into two d i f f e r e n t classes of polypeptide chains, the a and the 3 chains. The haptoglobin 3 chain, which i s the larger and the only component that contains carbohydrate has been studied i n the present i n v e s t i g a t i o n . The homogeneity of t h i s polypeptide chain was examined using d i f f e r e n t c r i t e r i a , such as DEAE-cellulose chromatography, gel f i l t r a t i o n chromatography, urea starch gel and SDS d i s c gel electrophoresis. The 3 chain was found to be homogeneous as judged by the above d i f f e r e n t c r i t e r i a . The amino acid composition and carbohydrate composition as well as the N-terminal amino acid were determined. The 3 chain i s o l a t e d i n the -S-methyl-carboxyamide form was extensively aggregated. However, upon chemical modification using s u c c i n i c anhydride, the modified 3 chain was completely disaggregated, and i t was possible to determine the accurate molecular weight of the 3 chain using t h i s succinyl 3 chain. In order to elucidate the primary structure of the 3 chain, a cyanogen bromide reaction, which cleaved s e l e c t i v e l y at the m e t h i o n i n e r e s i d u e s was p e r f o r m e d . T h o u g h t h i s c l e a v a g e was i n c o m p l e t e i n 3 c h a i n , t h r e e f r a g m e n t s w e r e i s o l a t e d a n d c h a r a c -t e r i z e d . The a m ino a c i d s e q u e n c e o f t h e N - t e r m i n a l r e g i o n , t h e a , 3 i n t e r c h a i n d i s u l f i d e r e g i o n a n d t h e c a r b o h y d r a t e a t t a c h m e n t s i t e r e g i o n w e r e d e t e r m i n e d . The a l i g n m e n t o f some o f t h e c y a n -UN-? o g e n b r o m i d e c l e a v a g e f r a g m e n t s w e r e a c h i e v e d u s i n g t h e m e t h i o -n i n e p e p t i d e s f r o m a t r y p t i c d i g e s t o f t h e 3 c h a i n t o o v e r l a p t h e s e f r a g m e n t s . The m e t h i o n i n e p e p t i d e s w e r e i s o l a t e d b y a s e l e c t i v e d i a g o n a l p a p 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 . The l i n k a g e b e t w e e n t h e p r o t e i n a n d t h e c a r b o h y d r a t e i n h a p t o g l o b i n was e s t a b l i s h e d t o be an a s p a r t a m i d o - g l y c o s y l l i n k a g e T h i s was d e m o n s t r a t e d by t h e r e s i s t a n c e o f t h e l i n k a g e u n d e r m i l d a l k a l i t r e a t m e n t a n d t h e a c t u a l i s o l a t i o n o f an a s p a r t i c - c a r b o -h y d r a t e c o m p l e x . A d i f f e r e n t a p p r o a c h was t a k e n t o d e t e r m i n e t h e p r i m a r y s t r u c t u r e o f t h e 3 c h a i n . The 3 c h a i n was m a l e y l a t e d a t i t s e -amino l y s y l g r o u p s w i t h m a l e i c a n h y d r i d e a n d l a t e r d i g e s t e d w i t h t r y p s i n . The m a l e y l a t e d f r a g m e n t s w e r e i s o l a t e d a n d p a r -t i a l l y c h a r a c t e r i z e d . F u r t h e r m o r e , f i v e g r o u p s o f a r g i n i n e p e p t i d e s w e r e i s o l a t e d . An o v e r a l l s t r u c t u r e o f t h e 3 c h a i n was d e d u c e d f r o m t h e s t u d i e s o f t h e c y a n o g e n b r o m i d e p e p t i d e s , m e t h i o n i n e p e p t i d e s , t r y p t i c m a l e y l a t e d p e p t i d e s a n d t h e a r g i n i n e p e p t i d e s . ACKNOWLEDGMENT The a u t h o r i s g r e a t l y i n d e b t e d t o D r . G. H. D i x o n f o r h i s e x c e l l e n t g u i d a n c e , e n c o u r a g e m e n t a n d v a l u a b l e a s s i s t a n c e d u r i n g t h e c o u r s e o f t h i s s t u d y a n d t h e p r e p a r a t i o n o f t h i s t h e s i s . To D r . J . L e v y , t h e a u t h o r w i s h e s t o e x t e n d h i s t h a n k s f o r h e r c o n s t r u c t i v e c r i t i c i s m o f t h i s m a n u s c r i p t . T h a n k s a r e a l s o due t o M i s s D. K a u f f m a n a n d a l l o t h e r members i n t h e l a b o r a t o r y f o r d i s c u s s i o n s a n d s u g g e s t i o n s a n d t o Mr. J . Durgo f o r s k i l f u l t e c h n i c a l a s s i s t a n c e . The s t u d e n t s h i p s a w a r d e d b y t h e N a t i o n a l R e s e a r c h C o u n c i l a n d t h e M e d i c a l R e s e a r c h C o u n c i l a r e g r e a t l y a p p r e c i a t e d . DEDICATION To My W i f e Peak-Choo V TABLE OF CONTENTS ABSTRACT i ACKNOWLEDGMENT i i i DEDICATION i v LIST OF TABLES v i i i LIST OF FIGURES X LIST OF ABBREVIATIONS x i v INTRODUCTION 1 B i o c h e m i s t r y o f H a p t o g l o b i n s 1 G e n e r a l C h a r a c t e r i s t i c o f H a p t o g l o b i n 1 M o l e c u l a r S t r u c u t r e o f Hp 1-1 9 B i n d i n g o f H a p t o g l o b i n t o Hemoglobin 12 R e c o n s t i t u t i o n S t u d i e s on H a p t o g l o b i n 18 The R o l e o f H a p t o g l o b i n 19 S t u d i e s on the 3 C h a i n o f H a p t o g l o b i n 21 MATERIAL AND METHODS 2 5 P r e p a r a t i o n o f H a p t o g l o b i n 3 C h a i n 25 P o l y a c r y l a m i d e D i s c G e l E l e c t r o p h o r e s i s . . . 1 26 Amino A c i d A n a l y s i s 28 C a r b o h y d r a t e A n a l y s i s . 29 High V o l t a g e Paper E l e c t r o p h o r e s i s and Paper Chromatography . . 33 Use o f S p e c i f i c S t a i n s f o r P e p t i d e s and Amino A c i d s . . . 33 N - T e r m i n a l D e t e r m i n a t i o n 35 Edman D e g r a d a t i o n '. . - 40 Use o f L e u c i n e A m i n o p e p t i d a s e and C a r b o x y p e p t i d a s e A f o r Amino A c i d Sequence S t u d i e s 41 Amide D e t e r m i n a t i o n 42 P u r i f i c a t i o n o f Reagents 42 CHAPTER I : THE GENERAL CHARACTERIZATION OF HAPTOGLOBIN 3 CHAIN • 44 I n t r o d u c t i o n 44 E x p e r i m e n t a l 4 4 R e s u l t s 47 The Homogeneity o f H a p t o g l o b i n 3 C h a i n v i S u c c i n y l a t i o n S t u d i e s The Amino A c i d C o m p o s i t i o n 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 H a p t o g l o b i n 2-1 and I t s S e p a r a t e d C h a i n s CHAPTER I I : THE FRACTIONATION OF CYANOGEN BROMIDE PEPTIDES OF HAPTOGLOBIN 3 CHAIN 68 I n t r o d u c t i o n 68 E x p e r i m e n t a l 70 R e s u l t s 71 Chromatography o f Cyanogen Bromide C l e a v a g e Fragments E s t i m a t i o n o f the M o l e c u l a r Weight o f t h e Cyanogen Bromide P e p t i d e s S t u d i e s on 3-CNBr-8-A S t u d i e s on 3-CNBr-8-B S t u d i e s on 3-CNBr-8-C and" 3"CNBr-8-D S t u d i e s on 3-CNBr-8-E and 3~CNBr-8-F Amino A c i d A n a l y s i s The E q u i l i b r i u m between Homoserine - Homoserine L a c t o n e and P r e s e n c e o f Homoserine Amide CHAPTER I I I : STUDIES ON CYANOGEN BROMIDE CLEAVAGE FRAGMENTS 3-CNBr-8-E and 3-CNBr-8-F 109 I n t r o d u c t i o n 109 E x p e r i m e n t a l 109 R e s u l t s 110 S t u d i e s on 3-CNBr-8-F S t u d i e s on 3~CNBr-8-E CHAPTER IV: THE ISOLATION OF METHIONINE PEPTIDES 129 I n t r o d u c t i o n 12 9 E x p e r i m e n t a l 131 R e s u l t s 132 The M e t h i o n i n e Paper D i a g o n a l Amino A c i d Sequence S t u d i e s CHAPTER V: STUDIES ON THE GLYCOPEPTIDES OF HAPTOGLOBIN 3 CHAIN 140 I n t r o d u c t i o n 140 E x p e r i m e n t a l 141 R e s u l t s 146 The T r y p t i c G l y c o p e p t i d e s o f H a p t o g l o b i n 3 C h a i n The Nature o f t h e L i n k a g e The Amino A c i d Sequence o f G l y c o p e p t i d e s V 1 X CHAPTER V I : THE FRACTIONATION OF TRYPTIC MALEYLATED S t u d i e s on the A r g i n i n e P e p t i d e s from T r y p t i c D i g e s t S t u d i e s on t h e C h y m o t r y p t i c A r g i n i n e P e p t i d e s S t u d i e s on t h e T h e r m o l y s i n A r g i n i n e P e p t i d e s S t u d i e s on t h e S u b t i l i s i n A r g i n i n e P e p t i d e o f 3-CNBr-8-B The Amino A c i d Sequence o f the A r g i n i n e P e p t i d e s D a n s y l a t i o n S t u d i e s o f t h e T r y p t i c M a l e y l a t e d P e p t i d e s C h a r a c t e r i z a t i o n o f M a l e y l a t e d P e p t i d e s CHAPTER V I I : THE PRIMARY STRUCTURE OF THE HAPTOGLOBIN PEPTIDES AND THE ISOLATION, CHARACTERIZATION OF ARGININE PEPTIDES 175 I n t r o d u c t i o n E x p e r i m e n t a l R e s u l t s . . . 175 176 179 3 CHAIN 207 BIBLIOGRAPHY 214 v i i i L IST OF TABLES T a b l e Page I . The M o b i l i t i e s o f C o l o u r e d Markers on Paper 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 34 I I . M o l e c u l a r Weights o f H a p t o g l o b i n 1-1, H a p t o g l o b i n C h a i n and H a p t o g l o b i n 3- C h a i n 60 I I I . Amino A c i d A n a l y s e s o f Whole H a p t o g l o b i n 2-1 and t h e I s o l a t e d C h a i n s 66 IV. Observed and P r e d i c t e d Amino A c i d C o m p o s i t i o n o f H a p t o g l o b i n 2-1 67 V. Amino A c i d C o m p o s i t i o n o f Cyanogen Bromide C l e a v a g e Fragments 101 V I . C a r b o x y p e p t i d a s e A D i g e s t o f 3~CNBr-8-F 113 V I I . Amino A c i d C o m p o s i t i o n o f T r y p t i c P e p t i d e s o f B-CNBr-8-E 115 V I I I . Amino A c i d A n a l y s i s o f M a j o r T r y p t i c P e p t i d e s o f 3-CNBr-8-E A f t e r Edman D e g r a d a t i o n P r o c e d u r e . . . 118 IX. L e u c i n e A m i n o p e p t i d a s e D i g e s t s o f t h e T r y p t i c P e p t i d e s o f 3~CNBr-8-E 121 X. A Comparison Between t h e Amino A c i d C o m p o s i t i o n o f 3-CNBr-8-E and t h e Sum. o f I t s T r y p t i c P e p t i d e s . 124 X I . Amino A c i d C o m p o s i t i o n o f C h y m o t r y p t i c P e p t i d e s o f 3-CNBr-8-E 125 X I I . C a r b o x y p e p t i d a s e A D i g e s t s o f the T r y p t i c and C h y m o t r y p t i c P e p t i d e s o f 3~CNBr-8-E 127 X I I I . Amino A c i d C o m p o s i t i o n o f M e t h i o n i n e P e p t i d e s . . . 136 XIV. Amino A c i d A n a l y s i s o f T r y p t i c G l y c o p e p t i d e s . . . . 151 XV. Amino A c i d C o m p o s i t i o n o f Pronase P e p t i d e s from T-G I I 156 XVI. Amino A c i d C o m p o s i t i o n o f T r y p t i c G l y c o p e p t i d e s on A l k a l i Treatment 159 i x T a b l e Page X V I I . Amino A c i d C o m p o s i t i o n o f G l y c o p e p t i d e a f t e r P r onase and P a r t i a l A c i d H y d r o l y s i s Treatment 162 X V I I I . Amino A c i d Sequence o f G l y c o p e p t i d e s 164 XIX. Amino A c i d C o m p o s i t i o n o f T h e r m o l y s i n G l y c o p e p t i d e s 166 XX. Amino A c i d C o m p o s i t i o n o f Th-g-CNBr-8-B and I t s S u b t i l i s i n P e p t i d e s 169 XXI. Amino A c i d C o m p o s i t i o n o f T r y p t i c A r g i n i n e P e p t i d e s 180 X X I I . Amino A c i d A n a l y s i s o f T-Arg^ and T - A r g 2 a f t e r Edman D e g r a d a t i o n 185 X X I I I . L e u c i n e A m i n o p e p t i d a s e D i g e s t o f A r g i n i n e P e p t i d e s . . 186 XXIV. Amino A c i d C o m p o s i t i o n o f C h y m o t r y p t i c A r g i n i n e P e p t i d e s 188 XXV. C a r b o x y p e p t i d a s e A D i g e s t o f A r g i n i n e P e p t i d e s . . . . 189 XXVI. Amino A c i d C o m p o s i t i o n o f T h e r m o l y s i n A r g i n i n e P e p t i d e s and S u b t i l i s i n P e p t i d e 191 :XXVII. Amino A c i d C o m p o s i t i o n o f M a l e y l a t e d P e p t i d e s . . . . 202 X LIST OF FIGURES Figure Page 1. Electrophoretic Patterns of Haptoglobin i n Starch 3 Gel Electrophoresis 2. Electrophoretic Separations of Six Haptoglobin Preparation of Different Types After Reduction and A l k y l a t i o n of 8 M Urea - Formate Starch Gel . . 5 1 2 3. Amino Acid Sequence of Haptoglobin a and a Chain. . 7 4. Amino Acid Sequence of the N-Terminal, C-Terminal, and Junction of Haptoglobin Chain Showing the Po s i t i o n of Crossover Which Led to the Observed Sequence for the a Chain 8 5. The D i s u l f i d e Peptides of Haptoglobin 13 6 . The Structure of Haptoglobin 1-1 ^4 7. Separation of the Polypeptide Chains of Reduced and Alkylated Haptoglobin 2-1 on a Sephadex G-75 . . . 27 8a. Standard Amino Acid Run - Homoserine System 30 8b. Standard Amino Acid Run - Hexosamine System . . . . . . 31 8c. Standard Amino Acid Run - Single Column System. . . . 32 9. Separation of Dansyl-Amino Acid by Thin Layer Chromatography 37 10a. Chromatography of Haptoglobin B Chain on 6 M Urea -DEAE Cellulose Column 4 8 10b. Chromatography of Haptoglobin 3 Chain on G-200 Column 4 8 11. Urea - Starch Gel Electrophoresis of Various Fraction of Haptoglobin 3 Chain from DEAE -Cellulose Chromatography 4 9 12. SDS Disc Gel Electrophoresis of Haptoglobin 3 Chain 51 13. N-Terminal Determination of Haptoglobin 3 Chain . . . 53 x i Figure Page 14a. Chromatography of Succinyl 6 Chain on a Sephadex G-200 Column 55 2 14b. Chromatography of Succinyl a Chain on a Sephadex G-75 Column 5 5 15. Sedimentation Veloc i t y Studies on Succinyl 3 Chain 56 2 16. Sedimentation V e l o c i t y Studies on Succinyl a Chain 57 17. Sedimentation Equilibrium Studies on Succinyl 6 Chain Using Archibald Approach to Equilibrium Method 58 2 18. Molecular Weight Determination of Succinyl a Chain Using Interference Optics Method Described by Van Holde . . 61 19. Chromatography of Cyanogen Bromide Cleavage Peptide of 3 Chain (3-CNBr-6) on a Sephadex G-100 Column . 72 20. Chromatography of Cyanogen Bromide Cleavage Peptide (3-CNBr-8) on a Sephadex G-100 Column 73 21a. Molecular Weight Estimation of CNBr Fragments (3-CNBr-5) on a Sephadex G-100 Column 76 21b. Molecular Weight Estimation of CNBr Fragments (3~CNBr-8) on SDS Disc Gel Electrophoresis (10% Gel) 76 22. SDS Disc Gel Electrophoresis of 3~CNBr-8 Fragments i n 10% Gel 77 23. Chromatography of 3~CNBr-8-A on a Sephadex G-200 Column 80 24a. Chromatography of Succinyl 3~CNBr-8-A on a Sephadex G-200 Column 82 24b. Chromatography of Maleyl 3-CNBr-8-A on a Sephadex G-200 Column 82 25a. Chromatography of M-3-CNBr-8-A I on a Sephadex G-200 Column 83 25b. Chromatography of M-3-CNBr-8-A II on a Sephadex G-200 Column 83 26a. Chromatography of M-3~CNBr-8-A III on a Sephadex G-100 Column 84 26b. Chromatography of M-3-CNBr--8-A IV on a Sephadex G-100 Column 84 x i i F i g u r e Page 27. D i s c G e l E l e c t r o p h o r e s i s o f M a l e y l a t e d Components o f B-CNBr-8-A. 85 28a. Chromatography o f 3~CNBr-8-B on a Sephadex G-200 Column 87 28b. Chromatography o f S u c c i n y l 3-CNBr-8-B on a Sephadex G-2 00 Column 87 29. 8 M Urea D i s c G e l E l e c t r o p h o r e s i s o f B-CNBr-8-B.. . . . 88 30. Chromatography o f 3~CNBr-8-B on a Sephadex G-75 Column . 8 9 31. Chromatography o f 3-CNBr-6a-D on Ca r b o x y m e t h y l -C e l l u l o s e Column 94 32. D a n s y l a t i o n S t u d i e s o f Cyanogen Bromide C l e a v a g e P e p t i d e s o f 3 C h a i n 96 33. The A l i g n m e n t o f Cyanogen Bromide C l e a v a g e o f P e p t i d e s 104 34. The I s o l a t i o n o f Homoserine Amide from t h e A c i d H y d r o l y s a t e o f Homoserine 108 35. L e u c i n e A m i n o p e p t i d a s e D i g e s t on 3 -CNBr-8-F 112 36. P r o p o s e d Amino A c i d Sequence o f 3-CNBr-8-E 123 37. A n a l y t i c a l M e t h i o n i n e Paper D i a g o n a l o f T r y p t i c D i g e s t o f 3 C h a i n at pH 4.0 133 38. S e p a r a t i o n o f T r y p t i c P e p t i d e s o f H a p t o g l o b i n 3 C h a i n on Dowex 50 x 2 Ion Exchange Chromatog-raphy 14 3 39. Chromatography o f T r y p t i c G l y c o p e p t i d e s on Dowex 1 x 4 Ion Exchange Chromatography ' 1 4 5 40. Chromatography o f D e s i a l i z e d T r y p t i c G l y c o p e p t i d e s on a BioRad P-10 Column • 148 41. D a n s y l a t i o n S t u d i e s o f T r y p t i c G l y c o p e p t i d e s 150 42. 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 T-G I I a t pH 1.9 . . • 152 43a. Chromatography o f Pronase D i g e s t o f T-G I I on a BioR a d P-4 Column 155 43b. Chromatography o f T h e r m o l y s i n D i g e s t o f 3~CNBr-8-B on a BioRad P-10 Column 155 44a. Pronase D i g e s t o f T r y p t i c 3 _CNBr-8-B Chromato-graphed on a P-4 Column 161 x i i i F i g u r e Page 44b. Second Pronase D i g e s t on A Chromatographed on a P-4 Column 161 44c. P a r t i a l A c i d H y d r o l y s i s on B Chromatographed on a P-4 Column 161 45. D a n s y l a t i o n S t u d i e s o f Th - 3-CNBr-8-B 170 46. S e p a r a t i o n o f S u b t i l i s i n P e p t i d e s o f Th - 3-CNBr-8-B on pH 1.9 High V o l t a g e E l e c t r o p h o r e s i s 172 47. The Amino A c i d Sequence of the C a r b o h y d r a t e Attachment S i t e o f H a p t o g l o b i n 3 C h a i n 174 48. N - T e r m i n a l Amino A c i d o f T r y p t i c A r g i n i n e P e p t i d e s . 181 49. D a n s y l a t i o n S t u d i e s o f T r y p t i c A r g i n i n e P e p t i d e s a f t e r 1 s t and 2nd Edman D e g r a d a t i o n 182 50. D a n s y l a t i o n S t u d i e s o f T r y p t i c A r g i n i n e P e p t i d e s a f t e r 3 r d and 4 t h Edman D e g r a d a t i o n 183 51. The Amino A c i d Sequence o f A r g i n i n e P e p t i d e s o f H a p t o g l o b i n 3 C h a i n 194 52. Chromatography o f T r y p t i c M a l e y l a t e d P e p t i d e s o f H a p t o g l o b i n 3 C h a i n on a Sephadex G-75 Column . 196 53. D a n s y l a t i o n o f T r y p t i c D i g e s t o f M a l e y l a t e d 3 C h a i n 197 54. D a n s y l a t i o n o f T r y p t i c M a l e y l a t e d Fragments o f 3 C h a i n . 200 55. 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 T-M-3 V I I on pH 6.5 199 56. E n l a r g e d Sequence o f 3-CNBr-8-E 203 57. The P r i m a r y S t r u c t u r e o f H a p t o g l o b i n 3 C h a i n . . . . 213 x i v LIST OF ABBREVIATION Hp h a p t o g l o b i n Hb hemoglobin DEAE d i e t h y l a m i n o e t h y l CNBr cyanogen bromide DNSC1 1 d i m e t h y l a m i n o n a p t h a l e n e - 5 -s u l f o n i c c h l o r i d e DNS d a n s y l CHO c a r b o h y d r a t e PITC p h e n y l i s o t h i o c y a n a t e 1 INTRODUCTION Biochemistry of Haptoglobins Haptoglobins (Hp) are a group of glycoproteins found i n normal human serum and capable of binding hemoglobin (Hb) both i n vivo and i n v i t r o . These proteins migrate with the a 2 f r a c t i o n of serum proteins by the c l a s s i c a l electrophoretic technique and contain approximately 15 - 20% of carbohydrate by •weight. Since t h e i r discovery by Polonovski and Jayle (1) i n the l a t e 1930's, haptoglobins have been the subject of many i n -vestigations designed to determine t h e i r molecular structure and p h y s i o l o g i c a l r o l e , as well as the population d i s t r i b u t i o n of i t s phenotypic variants. Since these variants represent such genetic phenomena as point mutations, p a r t i a l gene d u p l i c a t i o n and intragenic crossing over, haptoglobin provides an important subject f o r studies of protein molecular evolution. General C h a r a c t e r i s t i c of Haptoglobin Polonovski and Jayle (1) f i r s t observed that the peroxidase a c t i v i t y of Hb was enhanced by the addition of serum. The bind-ing substance i n the serum was l a t e r characterized by these workers and given the name of Haptoglobin. Further i n v e s t i g a t i o n showed that haptoglobin i s not a single molecular species, but a group of c l o s e l y r e l a t e d molecules. With the introduction and use of starch gel electrophoresis, Smithies (2) demonstrated that human haptoglobin f e l l into three classes, representing phenotypic variants of a ge n e t i c a l l y polymorphic protein. Family studies 2 by S m i t h i e s and Walker (3) showed t h a t t h e t h r e e e l e c t r o p h o r e t i c p a t t e r n s were i n h e r i t e d i n a manner c o n s i s t e n t w i t h t h e p r e s e n c e o f two a l l e l i c a u tosomal genes, c a l l e d Hp^ and Hp 2; t h e t h r e e r e s u l t a n t phenotypes were c a l l e d Hp 1-1, Hp 2-2 and Hp 2-1. The 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 t h e s e t h r e e t y p e s a r e shown i n F i g . 1. As can be seen i n t h e f i g u r e , Hp 1-1 behaves as a s i n g l e p r o t e i n , whereas t h e Hp 2-2 and Hp 2-1 show more t h a n t e n bands o f d e c r e a s i n g c o n c e n t r a t i o n and m o b i l i t y . T h i s phenonomena was c o n f i r m e d by t h e u l t r a c e n t r i f u g a t i o n s t u d i e s o f B e a m and F r a n k l i n ( 4 ) . Hp 1-1 shows a s i n g l e boundary c h a r a c t e r i s t i c o f a s i n g l e homogeneous m o l e c u l e i n t h e u l t r a c e n t r i f u g e whereas t h e m u l t i p l e -banded Hp 2-2 and Hp 2-1 show c o n s i d e r a b l e asymmetry. F u r t h e r m o r e , a s i m p l e m i x t u r e o f Hp 1-1 and Hp 2-2 does n o t g i v e r i s e t o a p a t t e r n r e s e m b l i n g t h a t o f t h e h e t e r o z y g o t e Hp 2-1. C o n n e l l and S m i t h i e s (5) showed t h a t t h e m u l t i p l e components o f t h e h e t e r o -geneous h a p t o g l o b i n 2-1 and 2-2 were n o t a r t i f a c t s , b u t r e p r e s e n t -ed d i s t i n c t p r o t e i n components, even though t h e i r p r e s e n c e appear-ed i n c o n s i s t e n t w i t h t h e g e n e t i c d o c t r i n e o f one gene, one p o l y -p e p t i d e . S m i t h i e s and C o n n e l l (6) , A l l i s o n (7) and P a r k e r and B e a m (8) p o s t u l a t e d t h a t t h e Hp 2-2 phenotype r e p r e s e n t s a s e r i e s 2 of s t a b l e p o l y m e r s c o n t a i n i n g t h e Hp gene p r o d u c t , whxle t h e Hp 2-1 phenotype r e p r e s e n t s a s i m i l a r p o l y m e r i c s e r i e s , each member 2 1 of w h i c h c o n t a i n s p r o d u c t s o f b o t h Hp and Hp genes, e x c e p t f o r 2 t h e f a s t e s t - m i g r a t i n g band, w h i c h does n o t have any Hp gene p r o d u c t . F u r t h e r c h a r a c t e r i z a t i o n o f t h e h a p t o g l o b i n m o l e c u l e by C o n n e l l , D i x o n and S m i t h i e s (9) on 8 M u r e a 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 f t e r r e d u c t i v e c l e a v a g e i n u r e a and 3 _ m e r c a p t o e t h a n o l Figure 1. Electrophoretic patterns of haptoglobin i n starch gel electrophoresis. 4 i l l u s t r a t e d the presence of two kinds of polypeptide chains, the a and 3 i n a l l three types of haptoglobin. No genetic polymorphism was noted i n the 3 chain which i s the slowest migrat-ing component. However, three d i f f e r e n t a chains could be d i s t i n -guished under these conditions. Two a l t e r n a t i v e Hp a 1 chains IF IS were detected, namely Hp (F for fast) and Hp (S for slow). The difference i n mobility was l a t e r shown to be due to the IF s u b s t i t u t i o n of a single l y s i n e residue at p o s i t i o n 54 i n a IS by a glutamic acid residue i n a (9, 10). This s u b s t i t u t i o n i s consistent with a single base change of the codon for glutamic A A acid GA^ to lys i n e AA^. Thus the haptoglobin 1-1 phenotype can be subtyped as either 1F-1F, 1F-1S, or 1S-1S. The Hp a 2 chain stained more heavily and migrated more slowly than either of the Hp a 1 chains. The heterozygote Hp 2-1 phenotype could be sub-typed as either Hp 2-1S or 2-1F. Hp 2-2 has only one subtype 2-2. Obviously, six common haptoglobin phenotypes would be predicted and a l l have been found as shown in F i g . 2. The chemical structure of a 1 and a 2 chains were further investigated by Dixon and co-workers, and more recently by Black and Dixon who determined the amino acid sequence of both the a 1 and a 2 chains (11). Smithies et a l . (12) and Connell et a l . (13) determined the molecular weight of a 1 and a 2 as 8,900 and 17,300 + 1,400 respectively. The molecular weight of a 2 chain being nearly twice as much as the a 1 chain. Peptide finger p r i n t analysis of the chymotryptic digest of a 2 chain revealed the presence of one add i t i o n a l peptide (J) when compared with the a 1 chain digest. On the other hand, the N and C-terminal of the a 1 chain was decreased i n amount i n the a 2 digest. The 5 F i g u r e 2. 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 x h a p t o g l o b i n p r e p a r a t i o n o f d i f f e r e n t t y p e s a f t e r r e d u c t i o n and a l k y l a t i o n i n 8 M u r e a - f o r m a t e s t a r c h g e l . 6 amino acid sequence of a 1 and a 2 chains, and the peptides N, C and J are shown i n F i g . 3 and F i g . 4 r e s p e c t i v e l y . The junction peptide J can be seen to represent the fusion of the C-terminal portion of one haptoglobin a 1 chain (A) at residue A 70 or 71 with the N-terminal portion of a second haptoglobin a 1 chain (B) at residue B 12 or 13. An a l a n y l residue occurs both at A 71 and B 12, and so i t i s not possible to d i s t i n g u i s h whether the fusion was from A 71 to B 13 or from A 70 to B 12. In other words, Hp a 2 chain can be considered as a fusion of the two a 1 chains with a short sequence of amino acids (12 + 1) missing at the point of t h e i r junction. If the a 1 chain had been completely duplicated to produce two i d e n t i c a l repeated sequences joined end to end, the r e s u l t i n g a 2 chain should have an amino acid composition i d e n t i c a l to a 1 chain with a chain length of 166 residues. How-ever, the amino acid analysis of a 2 chain d i f f e r s from a 1 and corresponds to that for the 142 residue polypeptide which would be expected from a p a r t i a l gene d u p l i c a t i o n (14). Smithies, Connell and Dixon (10) proposed a mechanism of unequal crossing over between asymmetrically paired a l l e l e s oc-1F IS curing i n a Hp Hp heterozygote, with a resultant p a r t i a l gene dupl i c a t i o n i n one chromosome and a corresponding deletion i n the 2 other to explain the evolution of the Hp locus. The occurence of s i m i l a r p a r t i a l gene duplications during evolution has also been suggested a f t e r the discovery of i n t e r n a l homologies of amino acid sequence i n ferredoxin (15), cytochrome c (16), the haemo-globin chains (17), the l i g h t and heavy chains of the immunoglo-bulins (18) and herring protamine (19). This p a r t i a l gene dupl i c a t i o n i n Hp appears to be r e l a t i v e l y recent evolutionary Haptoglobin i cx1 83 residues a IF Lys at position 54 a IS Glu at position 54 1 10 20 •Val--Asn--Asp--Ser-•Gly-Asn--Asp-•Val--Thr-•Asp--He- -Ala -Asp-•Asp-•Gly -Gln-Pro-•Pro-•Pro-•Lys 30 40 •Cys -He- -Ala--His-•Gly-Tyr--Val-•Glu' -His--Ser--Val--Arg -Tyr-•Gln-•Cys -Lys-Asn-•Tyr-•Tyr-•Lys 50 60 •Leu' -Arg' -Thr--Gln-•Gly-Asp--Gly-•Val--Tyr-•Thr--Leu--Asn -Asn-•Glu-•Lys -Gln-Trp-•Ile-•Asn-•Lys 70 80 •Ala--Val--Gly-•Asp-P. A •Lys-Leu--Pro-•Glu--Cys-•Glu--Ala--Val -Cys-•Gly-•Lys -Pro-Lys-•Asn-•Pro-•Ala Asn--Pro--Val- O * i •Gln-•COOH 10 20 Val--Asn--Asp-•Ser-•Gly-Asn--Asp-•Val--Thr-•Asp-- l i e --Ala' -Asp-•Asp-•Gly -Gln-Pro-•Pro-•Pro-•Lys 30 40 Cys-- l i e --Ala-•His-•Gly-Tyr--Val-•Glu--His-•Ser--Val--Arg' -Tyr-•Gln-•Cys -Lys-Asn-•Tyr-•Tyr-•Lys 50 •Lys- 60 Leu--Arg--Thr-•Gln-•Gly-Asp--Gly-•Val--Tyr-•Thr--Leu--Asn' -Asn-•Glu-•Lys -Gln-Trp-•Ile-•Asn-•Lys 70 80 Ala--Val--Gly-•Asp-•Lys-Leu--Pro-•Glu--Cys-•Glu--Ala--Asp--Asp-•Gly-•Gin' -Pro-Pro-•Pro-•Lys-•Cys 90 100 l i e --Ala--His-•Gly-•Tyr-Val--Glu-•His--Ser-•Val--Arg--Tyr--Gln-•Cys-•Lys -Asn-Tyr-•Tyr-•Lys-•Leu 110 -Lys- 120 Arg--Thr--Gln-•Gly-•Asp-Gly--Val-•Tyr--Thr-•Leu--Asn--Asn--Glu-•Lys-•Gin' -Trp-Ile-•Asn-•Lys-•Ala 130 140 Val--Gly--Asp-•Lys-•Leu-Pro--Glu-•Cys--Glu-•Ala--Val--Cys' -Gly-•Lys-•Pro' -Lys-Asn-•Pro-•Ala-•Asn 143 Pro--Val--Gln-•COOH Figure 3. Amino acid sequence of haptoglobin a 1 and a 2 chains. a1 chain B • N peptide -1 10 ' < 20 XH, - Val - Asn - Asp - Ser - Gly - Asn - Asp - Val - Thr • Asp He - Ala - Asp - Asp - Gly • Gin - Pro - Pro - Pro • Lys - Cys - He - Ala - His - Gly - Tyr i l chain A cli I 00 70 I 80 ' He - Asn - Lys - Ala - Val - Gly - Asp - Lys - Leu - Pro - Glu - Cys - Glu - Ala • Val - Gly • Lys - Pro - Lys - Asn - Pro - Ala - Asn • Pro - Val - Gin - COOH I I - • I • ch. C peptide ch. a' chain 60 70 80 He - Asn - Lys - Ala - Val - Gly - Asp - Lys - Leu - Pro • Glu - Cys.- Glu - Ala - Asp - Asp - Gly - Gin - Pro - Pro - Pro - Lys • Cys - He - Ala - His - Gly - Tyr Junction peptide • ch. Figure 4. Amino-acid sequences of the N-terminal> C-terminal and junction of haptoglobin a chain showing the p o s i t i o n of crossover (A 70 - 71 to B 12 - 13) which led to the observed sequence for the a 2 chain, ch. = P o s i t i o n of chymotryptic attacks. 9 e v e n t i n t h e human l i n e ( 11). T h i s i s s u p p o r t e d by t h e o b s e r v a t -i o n t h a t a l l p r i m a t e h a p t o g l o b i n s so f a r examined appear s i m i l a r t o Hp 1-1 and f a i l t o show t h e p o l y m e r i c bands on s t a r c h g e l e l e c t r o p h o r e s i s w h i c h appear c h a r a c t e r i s t i c o f t h e p a r t i a l d u p l i -c a t e d a 2 genome. The d u p l i c a t i o n t h e r e f o r e appears t o be a more r e c e n t e vent t h a n t h e e v o l u t i o n a r y d i v e r g e n c e between t h e human p r e c u r s o r and t h e h i g h e r p r i m a t e l i n e . F u r t h e r m o r e , i t was p r e -d i c t e d t h a t by u n e q u a l b u t homologous c r o s s i n g o v e r i n t h e o r i g i n a l 2FS 2FF 2SS Hp a gene, new gene p r o d u c t s , Hp a and Hp a c o u l d be formed. T h i s was i n f a c t v e r i f i e d s u b s e q u e n t l y by Nance and S m i t h i e s (20) i n a B r a z i l i a n p o p u l a t i o n . S i m i l a r l y , by c r o s s i n g o v e r i n an a s y m m e t r i c a l l y p a i r e d chromosome i n t h e Hp 2-2 homo-z y g o t e , a t r i p l i c a t e a 3 c h a i n c o u l d r e s u l t . A r a r e h a p t o g l o b i n , t h e Johnson t y p e ( 2 1 ) , has been shown by B l a c k and D i x o n (22) t o have an a c h a i n w i t h a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 25,000, c l o s e t o t h a t e x p e c t e d f o r a t r i p l i c a t e d c h a i n . The 3 c h a i n o f h a p t o g l o b i n i s i d e n t i c a l i n a l l t h r e e pheno-t y p e s and i s a p p a r e n t l y c o n t r o l l e d by a d i f f e r e n t l o c u s (10, 1 2 ) . T h i s p o l y p e p t i d e c h a i n has n o t been s t u d i e d e x t e n s i v e l y and i s t h e s u b j e c t o f p r e s e n t i n v e s t i g a t i o n . A X M o l e c u l a r S t r u c t u r e o f Hp 1-1 The m o l e c u l a r w e i g h t o f Hp 1-1 has been e s t i m a t e d as about 100,000 by C h e f t e l and M o r e t t i ( 2 3 ) . An a c c u r a t e m o l e c u l a r w e i g h t was d e t e r m i n e d r e c e n t l y by B l a c k e_t a_l. (14) t o be 98, 000 + 1,000. Two N - t e r m i n a l amino a c i d s , v a l i n e and i s o l e u c i n e were found t o be p r e s e n t i n e q u a l c o n c e n t r a t i o n by Sm i t h e t a_l. ( 2 4 ) . S i n c e v a l i n e was found t o be t h e N - t e r m i n a l amino a c i d ^ o f t h e a y 10 chain, t h i s implied that isoleucine i s the N-terminal amino acid of the 3 chain and that the two chains are present i n equal amounts. The a 1 chain accounts for approximately 20% by weight of the whole molecule, and the t o t a l contribution for the a 1 chain w i l l give a value of 17,000 - 20,000. Since the molecular weight of the a 1 chain has been determined by Smithies et_ a l . (12) as 8 ,900, t h i s suggests the presence of two a 1 chains i n the whole hapto-globin molecule. The 3 chain which has a molecular weight of 0, 1 42,000 (14) accounts for 8 0% of the t o t a l weight, obviously two 3 chains are also present. This agrees well with the r e s u l t of the N-terminal determination. Thus, the structure of Hp 1-1, resembles the immunoglobulin IgG which contains two l i g h t chains and two heavy chains. Since d i s u l f i d e bonds are responsible for l i n k i n g the l i g h t and heavy chains together i n immunoglobulins, i t was necessary to f i n d out whether the same structure holds for haptoglobin. The reduction of Hp 1-1 and Hp 2-2 produces 18 and 24 free SH group res p e c t i v e l y (25). Since there are no free SH groups i n the haptoglobin (25, 26, 27), a l l SH groups are involved as d i - . s u l f i d e s , so that there are 9 and 12 d i s u l f i d e s i n the Hp 1-1 and Hp 2-2 res p e c t i v e l y . In the case of Hp 2-1, 21 half cystines have been determined as c y s t e i c acid by Black et a_l. (14) . The odd number of half cystine residues requires some explanation. E a r l i e r sequence determination by Black and Dixon (11) assigned 3 carboxymethylcysteine (CMC) residues to the a 1 chain, and 6 to the a 2 chain. However, these values have recently been correct-ed (28), so that there are 4 half cystines i n the a 1 chain, 7 i n the a 2 chain and 10 ( 5 X 2 ) i n the two 3 chains making a t o t a l of 21. This agrees with the values i n Hp 1-1 ( 2 X 4 + 5 X 2 = 18) and Hp 2-2 ( 7 X 2 + 5 X 2 = 24). Furthermore, there are only 7 half cystine residues instead of 8 i n a 2 chain, so that the half cystine at residue 73, which i s involved i n the aB interchain d i s u l f i d e i s l o s t i n the p a r t i a l l y duplicated a 2 chain. The re-s u l t i n g a 2 structure i s then responsible for the formation of polymer structure. I t has been suggested by Smithies and Connell (6) that a r e l a t i v e l y small a l t e r a t i o n i n the protein, such as the introduction of a cysteine residue into the molecule, might permit the formation of a series of polymers which could not be formed by the unaltered protein. Dixon and Connell (29) e a r l i e r demonstrated the presence of an aB "half molecule" i n haptoglobin by performing a s u l f i t e cleavage on the Hp 2-2 molecule. Hp 2-2 was reacted with 0.05 M sodium s u l f i t e i n the presence of p-hydroxymercuribenzoic acid. The reaction product, when examined on urea - starch gel e l e c t r o -phoresis, i s i d e n t i c a l with the reaction product derived from Hp 1-1, and was faster running than co n t r o l Hp 1-1. Since Hp 1-1 was t e n t a t i v e l y suggested as a 2 6 2 , t h i s species was designated as an aB half molecule. The presence of t h i s half molecule was l a t e r confirmed and characterized by Rorstad and Dixon (30). The "half molecule" had a molecular weight of 50,000, which was con-s i s t e n t with the sum of one a 1 and one B chain. Furthermore, the a and B chains were linked by an a,B interchain d i s u l f i d e , and i s s t i l l capable of binding with Hb. These studies demon-strated the importance of a,a d i s u l f i d e linkage i n constructing the molecule. Apparently, the Hp 1-1 i s constituted of 2 aB "half molecules" linked by a d i s u l f i d e which was r e a d i l y cleaved by s u l f i t e . Kauffman and Dixon (31) i s o l a t e d and characterized the d i s u l f i d e peptides from Hp 1-1. These are shown i n F i g . 5. The structure of Hp 1-1 recently elucidated by Malchy and Dixon (28) i s shown in F i g . 6. I t i s obvious that Hp 1-1 has a s i m i l a r structure to the IgG. However, a major diff e r e n c e between these two structure i s observed, namely that heavy - heavy interchain d i s u l f i d e ^ a r e y r e s -ponsible f o r holding the two half molecules together i n IgG, while a - a interchain d i s u l f i d e are.involved i n Hp 1-1. This •a - a d i s u l f i d e bond i s cleaved i n haptoglobin by s u l f i t e to give r i s e to the "half molecule" discussed e a r l i e r . Binding of Haptoglobin to Hemoglobin Haptoglobin r e a d i l y combines with hemoglobin or free globin, and the r e a c t i o n i s e s s e n t i a l l y i r r e v e r s i b l e . This i s shown by the f a c t that once the complex i s formed, there i s no exchange of the bound Hb with i s o t o p i c a l l y l a b e l l e d Hb (32). However, Hp does not bind to heme or myoglobin. The d e t a i l of the binding between Hp and Hb i s unknown, although i t has been shown that neither d i s u l f i d e bonds (33, 34) nor other covalent linkages are involved, nor does the carbohydrate moiety p a r t i c i p a t e i n the binding (35, 36). The studies of Chan and Dixon (37) on the binding of Hp and Hb under d i f f e r e n t environmental conditions showed that e l e c t r o -s t a t i c i n t e r a c t i o n s cannot be the sole intermolecular forces involved i n binding since masking e l e c t r i c a l l y charged groups with a high concentration of ions does not a f f e c t the i n t e r a c t i o n a - a interchain d i s u l f i d e Ileu-Ala-Asp-Asp-Gly-Glu-Pro-Pro-(Pro,Lys)Cys-Ileu-Ala-His-Gly I S I s Ileu-Ala-Asp-Asp-Gly-Glu-Pro-Pro-(Pro,Lys)Cys-Ileu-Ala-His-Gly a - a intrachain d i s u l f i d e Ala-Val-Gly-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala I S I s Val-Arg-Tyr-Gln-Cys-Cys-Asn-Tyr a - 3 intrachain d i s u l f i d e Ileu-Cys-Pro-Leu-Ser-Lys-Asp I f Val-Cys-Gly-Lys-Pro-(Pro,Asp) g - 3 .intrachain d i s u l f i d e Tyr-Gln-Glu-Asp-Thr-Cys-Tyr-(Gly,Asp,Ala,Gly,Ser,Ala) l ? S Phe-Asp-Lys-Cys-(Ser,Ala) 3 - 3 intrachain d i s u l f i d e Val-Ala-Asp-Gln-Asp-Glu-Cys I S I s Phe-Cys gure _5. The d i s u l f i d e peptides of haptoglobin. CHO N 1 3 chain N 2 1 3 5 6 9 7 3 1 I s— S J C a chain N 2 1 3 5 6 9 7 3 I S C a chain N CHO S I I f C 3 chain Figure 6. The structure of haptoglobin 1-1, nor does wide v a r i a t i o n i n t h e i o n i c s t a t e o f c h a r g e d groups on t h e two p r o t e i n s p r oduces any s i g n i f i c a n t e f f e c t on t h e b i n d i n g . However, i t i s s t i l l p o s s i b l e t h a t e l e c t r o s t a t i c f o r c e s t o g e t h e r w i t h a n o t h e r t y p e of i n t e r a c t i o n such as van de W a a l s 1 f o r c e s a c t i n g i n a c o - o p e r a t i v e manner may be r e s p o n s i b l e f o r t h e s t a -b i l i t y o f t h e Hp-Hb complex. Upon s u c c i n y l a t i o n , t h e Hp-Hb complex i s d i s s o c i a t e d (37) i n d i c a t i n g t h a t t h e s t r o n g e l e c t r o s t a t i c r e p u l s i o n f o r c e s e t up by c o n v e r t i n g p o s i t i v e t o n e g a t i v e c h a r g e s can cause such a p r o f o u n d c o n f o r m a t i o n a l change t h a t t h e t i g h t complex d i s s o c i a t e s . The v e r y f a c t t h a t t h e complex does d i s s o -c i a t e i n d i c a t e s t h a t c o v a l e n t l i n k a g e s a r e u n l i k e l y t o be i n v o l v e d i n t h e b i n d i n g , as such l i n k a g e s would be u n a f f e c t e d by s u c c i n y l -a t i o n . The v i s i b l e s p e c t r a o f oxyHb A and oxyHbHp a r e i d e n t i c a l , b u t some o t h e r p r o p e r t i e s o f Hb a r e c o n s i d e r a b l y a l t e r e d by c o m b i n a t i o n w i t h Hp. F o r example, t h e complex has a much h i g h e r oxygen a f f i n i t y t h a n Hb A, and b o t h t h e s i g m o i d and t h e Bohr e f f e c t a r e l o s t ( 3 8 ) . Deoxyhemoglobin does n o t combine w i t h hap-t o g l o b i n ( 3 9 ) . When oxyhemoglobin i s bound by h a p t o g l o b i n , sub-sequent d e o x y g e n a t i o n does n o t d i s r u p t HpHb, and t h e c o n f o r m a t i o n a l change c h a r a c t e r i s t i c o f t h e f o r m a t i o n o f deoxyhemoglobin i s n o t o b s e r v e d . However, when t h e hemoglobin i s t r e a t e d w i t h c a r b o x y -p e p t i d a s e A, w h i c h removes t h e l a s t two amino a c i d s from t h e $ c h a i n , b o t h i t s oxy and deoxy forms a r e bound by h a p t o g l o b i n ( 4 0 ) . Though c a r b o x y h e m o g l o b i n , methemoglobin and cyanmethemoglobin, as w e l l as a v a r i e t y o f a n i m a l hemoglobins and abnormal human hemo-g l o b i n s , form a s t a b l e complex w i t h h a p t o g l o b i n , t h e r e i s no d e m o n s t r a b l e b i n d i n g o f t h e 3 and y t e t r a m e r s , Hb H and Hb B a r t s . I t should be noted that both of these Hb's have a molecular configuration s i m i l a r to that of deoxyhemoglobin. The a b i l i t y of a and 3 chain monomers to combine with haptoglobin has been more d i f f i c u l t to determine. Nevertheless, recent studies by Nagel and Gibson (41) suggest that the s i t e on haptoglobin binds the a globin chain s p e c i f i c a l l y and once a chains have interacted, rapid binding of 3 chains follows. The normal binding of Hb involves either the consecutive binding of a and 3 monomers or attachment of a3 dimers through the a chain. The s t r u c t u r a l and functional resemblance of the Hp-Hb bind-ing to antibody - antigen reaction i n immunoglobulin i s s t r i k i n g . S t r u c t u r a l l y , both haptoglobin and immunoglobulin 7S molecules consist of two heavy and two light chains linked through d i s u l f i d e s The carbohydrate moiety i s found only i n the heavy chains i n both cases. Functionally, both have the properties of t i g h t and spe-c i f i c combination with proteins which are normally foreign to t h e i r environment. Though both Hp and Hb are blood proteins, Hb i s contained i n the red blood c e l l s . I t can be considered that when i t i s released by hemolysis into the plasma, the l o c a t i o n of haptoglobin, i t becomes a protein foreign to that p a r t i c u l a r compartment of the body. Thus haptoglobin, i n complex with Hb, acts i n a manner analogous to that of an antibody binding to a antigen. The common phenotypes of haptoglobin a l l combine s t o i c h i o -m e t r i c a l l y with Hb and i t has been established by Jayle and Moretti (4 2) and confirmed by others that 1.0 mg of Hb combines with 1.3 mg of Hp, which corresponds to an equimolar combination of Hb with a Hp 1-1 unit . The 1 : 1 stoichiometry of the HbHp complex would suggest at f i r s t sight that Hp possess only one 17 b i n d i n g s i t e f o r Hb, a s i t u a t i o n d i f f e r e n t from i m m u n o g l o b u l i n w h i c h i s b i v a l e n t and can b i n d two a n t i g e n m o l e c u l e s . However, L a u r e l l (43) found t h a t by a d d i n g l e s s t h a n s t i o c h i o m e t r i c amounts o f Hb t o Hp, an i n t e r m e d i a t e was found w h i c h behaved e l e c t r o -p h o r e t i c a l l y l i k e a complex between a h a l f m o l e c u l e o f Hp and one m o l e c u l e o f Hp, t h u s i n d i c a t i n g t h a t a$ dimer m i g h t be t h e sub-u n i t o f Hb b i n d i n g t o Hp. The i n t e r m e d i a t e complex has r e c e n t l y been i s o l a t e d and c h a r a c t e r i z e d by Hamaguchi (44). The m o l e c u l a r w e i g h t o f t h i s i n t e r m e d i a t e complex i s 140 ,000 , a v a l u e c o n s i s t e n t w i t h t h a t e x p e c t e d f o r a complex o f one h a l f hemoglobin and one h a p t o g l o b i n . Recent s t u d i e s o f Malchy and D i x o n (45) show t h a t c o v a l e n t l y - l i n k e d Hb d o u b l e m o l e c u l e s (a^Bi*) can a c t as c r o s s -l i n k e r s between Hp m o l e c u l e s t o produce a s e r i e s o f a g g r e g a t e s o f i n c r e a s i n g s i z e ; t h u s e s t a b l i s h i n g t h e b i v a l e n t n a t u r e o f h a p t o g l o b i n . B e s i d e s t h e s t r u c t u r a l and f u n c t i o n a l s i m i l a r i t i e s between h a p t o g l o b i n and i m m u n o g l o b u l i n , sequence homology has been found t o e x i s t between t h e a c h a i n o f h a p t o g l o b i n and t h e Kappa and Lambda c h a i n s o f Bence Jones p r o t e i n s , n o t a b l y around t h e d i s u l f i d e r e g i o n s (11). I t seems l i k e l y t h a t a common a n c e s t r a l gene f o r a c h a i n and i m m u n o g l o b u l i n might e x i s t w h i c h l a t e r d u p l i c a t e d i n t o a n c e s t r a l genes w h i c h d i v e r g e d t o code f o r t h e two f a m i l i e s o f p r o t e i n s . F u r t h e r m o r e , Hp 1-1 and i m m u n o g l o b u l i n may have a s i m i l a r s econdary s t r u c t u r e . E a r l y s t u d i e s by Waks and A l f s e n (46) i n -d i c a t e d a n e g l i g i b l e h e l i c a l c o n t e n t and t h e p o s s i b i l i t y o f t h e p r e s e n c e o f some b e t a s t r u c t u r e i n Hp. The c i r c u l a r d i c h r o i s m b e h a v i o u r i n t h e 200 - 240 nm r e g i o n o f t h e t h r e e common t y p e s of Hp s t u d i e s by Hamaguchi (47) i n d i c a t e s t h e p r e s e n c e o f , b e t a s t r u c t u r e , and a l m o s t no h e l i c a l c o n t e n t i n human Hp.( S i n c e V, S a r k a r and Doty (48) r e p o r t e d t h e same f i n d i n g on gamma g l o b u l i n so t h a l ^ i t i s c o n c e i v a b l e t h a t b o t h gamma g l o b u l i n and Hp 1-1 p o s s e s s a s i m i l a r s e c o n d a r y s t r u c t u r e . R e c o n s t i t u t i o n S t u d i e s on H a p t o g l o b i n The r e c o n s t i t u t i o n o f h a p t o g l o b i n has r e c e n t l y been r e p o r t -ed by B e r n i n i - V o l t a t t o r n i (25). E l e c t r o p h o r e t i c p a t t e r n , hemo-g l o b i n b i n d i n g , m o l e c u l a r s i z e and a n t i g e n i c p r o p e r t i e s were used as t h e c r i t e r i a f o r e s t i m a t i n g t h e degree o f r e n a t u r a t i o n and f o r comparing n a t i v e and r e a c t i v a t e d h a p t o g l o b i n . The r e c o n s t i t u t i o n was pe r f o r m e d under v a r i o u s c o n d i t i o n s b u t each gave e s s e n t i a l l y t h e same c o n c l u s i o n . H a p t o g l o b i n was re d u c e d by B-mercapto-e t h a n o l i n 8 M u r e a o r i n 6 M g u a n i d i n e H C l . A f t e r d e s a l t i n g on Sephadex G-25, t h e c h a i n s were a l l o w e d t o recombine i n t h e p r e -sence o f oxygen and a c a t a l y t i c amount o f t h i o l . The r e c o v e r y f o r Hp 1-1 was a p p r o x i m a t e l y 70 - 90% and t h o s e o f Hp 2-1 and Hp 2-2 f o r 60 - 70%. F u r t h e r m o r e , r e c o m b i n a t i o n was a l s o a c h i e v e d when an e q u i -m o l a r m i x t u r e o f i s o l a t e d and s e p a r a t e l y r e a c t i v a t e d a and B c h a i n s were i n c u b a t e d a t pH 8.15 and 20°C, i n t r i s - p h o s p h a t e -EDTA-3-mercaptoethanol b u f f e r . The y i e l d o f Hp 1-1 was com-p a r a b l e w i t h t h e r e s u l t s from e a r l i e r methods, however, t h e y i e l d o f Hp 2-2 was low. H a p t o g l o b i n 1-1 - Hb complex was a l s o o b t a i n e d i n h i g h y i e l d when i s o l a t e d a 1 c h a i n was i n c u b a t e d w i t h B-Hp-Hb complex. H a p t o g l o b i n B c h a i n i s known t o be c a p a b l e o f b i n d i n g Hb (49, 37). The r e a c t i v a t i o n o f Hp was found t o be v e r y s p e c i f i c 1 9 and was not i n h i b i t e d by the presence of other reduced proteins such as serum albumin i n the incubation mixture. These experiments demonstrate that haptoglobin, though i t possesses a complicated multichain structure, can be reconsitut-ed under suitable conditions. These r e s u l t s support the hypothe-s i s that only the unique amino acid sequence of a given polypeptide chain i s responsible for the stable, s p e c i f i c and b i o l o g i c a l active three dimension configuration. S i m i l a r l y , the successful r e c o n s t i t u t i o n of IgG which also has an o v e r a l l architecture l i k e haptoglobin has also been reported (50, 51, 52). The Role of Haptoglobin Haptoglobin i s synthesized i n the l i v e r (53, 54). Besides being present i n plasma, v a r i a b l e amounts of haptoglobin can be found i n lymph, p l e u r a l , a s c i t e s and sp i n a l f l u i d s and also i n the urine. The amount of haptoglobin produced i s subjected to a number of influences, such as inflammatory disease, t i s s u e ne-c r o s i s , various malignancies and may possibly be under hormoral t c o n t r o l . For example, androgen tends to increase and estrogen to decrease the haptoglobin l e v e l (55), adrenalectomy i n rat s i n t e r -feres with the increased production of haptoglobin which normally accompanies inflammation (56). Recent studies by John and M i l l e r (57) using perfused rat l i v e r shows that haptoglobin syn-thesis together with fibrinogen,ai acid glycoprotein were stimulated three f o l d by the presence of C o r t i s o l , and t h i s represents the f i r s t instance of i n v i t r o hormoral induction of rat plasma protein synthesis. It has been suggested that hemoglobin bound to Hp does not t r a n s v e r s e t h e g l o m e r u l a r membrane. However, i f t h e amount o f Hb p r e s e n t i n t h e plasma exceeds t h e b i n d i n g c a p a c i t y o f Hp, i t c i r c u l a t e s i n t h e f r e e s t a t e and r e a d i l y p a s s e s i n t o t h e g l o -m e r u l a r f i l t r a t e . One p h y s i o l o g i c a l f u n c t i o n o f Hp, o r i g i n a l l y p r oposed by A l l i s o n (58) i s t o p r e v e n t t h e l o s s o f Hb from t h e body t h r o u g h t h e k i d n e y s and hence t o p l a y a r o l e i n i r o n r e -t e n t i o n and t o p r o t e c t t h e k i d n e y s from s i d e r o s i s produced by c h r o n i c f i l t r a t i o n o f Hb. Ano t h e r h a p t o g l o b i n f u n c t i o n has been s u g g e s t e d by Bunn and J a n d l ( 5 9 ) . These a u t h o r s o b s e r v e d t h a t t h e f r e e exchange o f heme between m o l e c u l e s o f methemoglobin and oxyhemoglobin d i d not o c c u r when t h e methemoglobin was f i r s t bound t o Hp. The t r a n s f e r o f heme from methemoglobin t o a l b u m i n was s i m i l a r l y p r e v e n t e d by h a p t o g l o b i n . I t i s t h e r e f o r e s u g g e s t e d t h a t Hp may h e l p t o p r e v e n t i n d i s c r i m i n a t e l o s s o f heme and i t s i r o n by b l o c k i n g t h e d i s s o c i a t i o n o f heme from methemoglobin. However, t h e r e p o r t e d exchanges o f heme were s t u d i e d i n a n o n - c e l l u l a r system, and i t seems v e r y l i k e l y t h a t heme i s f r e e l y exchanged between t h e i n t a c t r e d c e l l s and i t s plasma environment ( 6 0 ) . A f u r t h e r Hp f u n c t i o n , t h e f a c i l i t a t i o n o f heme c a t a b o l i s m was proposed by Nakajima e t a l . (61) who o b s e r v e d t h a t t h e l i v e r enzyme, heme a-methenyl oxygenase c o u l d c a t a l y s e an o x i d a t i v e c l e a v a g e o f t h e heme r i n g o n l y when t h e Hb m o l e c u l e was combined w i t h Hp. I t was i n f e r r e d t h a t t h e b i n d i n g o f Hb by Hp causes a d i s t o r t i o n i n t h e Hb c o n f o r m a t i o n such t h a t t h e haems n o r m a l l y p r e s e n t i n t h e h y d r o p h o b i c c r e v i c e s become exposed t o t h e enzyme. More r e c e n t l y , S nellman and S y l v i n (62) found t h a t h a p t o -g l o b i n a c t s as i n h i b i t o r o f c a t h e p s i n B a c t i v i t y , w h i c h i s r e -leased during various inflammatory processes, tumour diseases and tissue injury. However, the ph y s i o l o g i c a l s i g n i f i c a n c e of t h i s finding remains to be established. The possible s e l e c t i v e advantages of p a r t i a l gene dup l i c a t i o n can be deduced from the function of Hp, notably the retention of Hb and the f a c i l i t a t i o n of heme catabolism ( 1 1 ) . Since the function of the Hp i s to bind Hb and prevent i t s loss through the kidney, Hp 2-2 and 2-1 which contain polymers of higher molecular weight, w i l l be less l i k e l y to be f i l t e r e d through than Hp 1 - 1 , and yet s t i l l possess equal binding capacity for Hb. In the case of heme metabolism by a-methenyl oxygenase, the oxidation of complexes of methemoglobin with the three d i f f e r e n t genetic types of haptoglobin showed that the i n i t i a l v e l o c i t y of haem oxidation i s greater i n the complex with Hp 2-2 than i n that Hp 2-1 or Hp 1-1 ( 6 3 ) . Thus, i t i s possible that the s e l e c t i v e 2 advantage i n the Hp gene l i e s i n a more e f f i c i e n t metabolism of the Hb complex into b i l e pigments. A p o s i t i v e c o r r e l a t i o n be-2 tween the high frequency of Hp i n India and South East Asia having a high incidence of hemolytic disease i s consistent with the above arguments. Studies on the 6_ Chain of Haptoglobin The 6 chain of haptoglobin i s the heavier of the two chains of Hp. It has a molecular weight of approximately 40,000 - 45,000 and contains a l l the carbohydrate found i n haptoglobin. Unlike the a chain of haptoglobin, the 8 chain has not been subjected to detailed s t r u c t u r a l analysis. Peptide maps and amino acid analy-ses of the i s o l a t e d 6 chains from the three common Hp phenotypes appear to be very s i m i l a r (63). S i m i l a r l y , no difference was found i n the glycopeptides i s o l a t e d from Hp 2-1 and Hp 2-2 (64). I t has been shown by Shim and co-workers (65) that antibodies against the Hp 6 chain reacted with free, but not with Hb-complex Hp, while antibodies against the Hp a chain reacted with Hp re-gardless of whether or not Hb was present. Their conclusion that the 3 chain of Hp contains the Hb-binding s i t e was l a t e r supported by the studies of Gordon and Beam (49) and more recently by Chan and Dixon (37) who demonstrated that i s o l a t e d , reduced and alky-lated 3 chain binds s i g n i f i c a n t l y with the Hb while the a 1 and a 2 chains do not. Besides the normal 3 chain, two 3 chains variants have been reported. A 3 chain variant HpMb (for Marburg) has been found by Aly et a]L. (66) and studied by Cleve and Deicher (67) and Weerts et a l . (68). These authors reported that a l l of the elec-trophoretic components of Hp 2-1 Hb demonstrated a t y p i c a l immuno-l o g i c a l r e action, i n that antigenic determinant!^ normally blocked by Hb s t i l l reacts with i t s s p e c i f i c antibody when the Hp was f u l l y complexed with Hb. Furthermore, although on subtyping, the p u r i f i e d Hp contains no unusual a polypeptide components, there are s l i g h t differences i n the appearance of the 3 chain. Cleve and Deicher (67) concluded that these properties of the Marburg type were consistent with mutation at the locus determining the 3 rather than the a chain of Hp. Peptide map analysis of t h i s 3 chain variant by Bowman and Cleve (69) did i n fac t demonstrate a dif f e r e n c e between the normal and the variant 3 chain. Another 3 chain variant phenotype, Hp 2-1 Bellevue, has been reported by Javid (70). This phenotype Hp 2-1 Bellevue has the same kind of immunological behaviour as Hp 2-1 Mb, i n -dic a t i n g a v a r i a t i o n i n the 8 chain. However, the electrophoretic pattern was d i s t i n c t l y d i f f e r e n t . On subtyping i n urea starch r1 g e l , no difference was observed for a 1 and a 2 chain^ however, a fast e r moving component was present i n Hp 2-1 Bellevue i n addition to the usual d i f f u s e B-chain band. This component s t i l l migrated faster a f t e r treatment with s i a l i d a s e suggesting that the d i f f e r -ence observed i s not due to the heterogeneity of the carbohydrate moiety. Since the mobility of the anodal (Hp 1-1) band was not altered i n the Hp 2-1 Bellevue or the Hp 1-1 Bellevue phenotype, the altered mobility of the polymers' band compared to normal Hp 2-1 and Hp 2-2 phenotype might r e f l e c t the interference by the mutant chain with the regular polymerization of these molecules. More recently, the C-terminal amino acid sequence of the haptoglobin 8 chain has been determined by Barnett, Lee and Bowman (71). This i s established as Val-Glx-Lys-Thr-Ileu-Ala-Glu-AsnCOOH. In the present i n v e s t i g a t i o n , the primary structure of haptoglobin 8 chain has been studied. Haptoglobin 2-1 was i s o -lated i n large quantities from the as c i t e s f l u i d of a patient with a carcinoma of the ovary,, the p u r i f i e d Hp was reduced and alkylated and the chains separated on Sephadex G-7 5 column. The homo-geneity of the i s o l a t e d 8 chain was examined using d i f f e r e n t c r i t e r i a such as DEAE-cellulose chromatography, urea starch elec-trophoresis, and SDS disc g el electrophoresis. The N-terminal amino acid, amino acid composition as well as the carbohydrate compositions were determined. The physi^ochemical properties of the modified 8 chain a f t e r s u c cinylation were investigated and the sedimentation v e l o c i t y constant ( S o n ) and the molecular 2 4 weight of t h i s modified 3 chain were l a t e r determined. The primary structure of the 3 chain was further i n v e s t i -gated by performing a s p e c i f i c cyanogen bromide cleavage reaction at the four methionyl - residues of the chain. Three regions of p a r t i c u l a r i n t e r e s t were studied. The amino acid sequence of the N-terminal region, the interchain d i s u l f i d e region and the carbo-hydrate attachment s i t e have been determined. Furthermore, the nature of the linkage between the carbohydrate and protein moiety has been established as an aspartiamido-glycosyl linkage. Since the cyanogen bromide cleavage of 3 chain was incomplete, another approach was taken to determine the primary structure of t h i s polypeptide chain. The 3 chain was maleylated with maleic anhydride and l a t e r digested with t r y p s i n . The t r y p t i c maleylated peptides were i s o l a t e d and characterized. From the studies of these t r y p t i c maleylated peptides together with the cyanogen bromide cleavage peptides, an o v e r a l l structure of the haptoglobin 3 chain was elucidated. MATERIAL AND METHODS 1. P r e p a r a t i o n o f h a p t o g l o b i n 6_ c h a i n The h a p t o g l o b i n 8 c h a i n used i n t h e s e s t u d i e s was k i n d l y s u p p l i e d by Dr. J . B l a c k . I t i s , h o w e v e r , p e r t i n e n t t o g i v e a b r i e f o u t l i n e o f t h e p r e p a r a t i o n o f h a p t o g l o b i n and i t s a and 6 c h a i n s d e v i s e d i n t h i s l a b o r a t o r y (14). Hp 2-1 t y p e a s c i t e s f l u i d was o b t a i n e d from a female p a t i e n t w i t h a c a r c i n o m a o f t h e o v a r y . The cr u d e p r e p a r a t i o n was p r e c i -p i t a t e d w i t h ammonium s u l f a t e t o 55% s a t u r a t i o n and t h e pH m a i n t a i n e d a t 7.1. The p r e c i p i t a t e was washed once o r t w i c e w i t h 55% s a t u r a t e d ammonium s u l f a t e and l a t e r d i s s o l v e d i n 0.01 M sodium a c e t a t e b u f f e r , pH 4.7 and d i a l y z e d a g a i n s t t h e same b u f f e r u n t i l f r e e from ammonium s u l f a t e . The d i a l y s a t e was t h e n a p p l i e d t o a D E A E - c e l l u l o s e column (5.0 X 9.5 cm) e q u i l i b r a t e d w i t h 0.01 M sodium a c e t a t e b u f f e r , pH 4.7. The column was d e v e l o p e d w i t h a g r a d i e n t made up o f 300 ml o f 0.01 M sodium a c e t a t e , 0.01 M N a C l , pH 4.7, and 300 ml o f 0.01 M sodium a c e t a t e , 0.3 M N a C l , pH 4.7. The e l u a t e was m o n i t o r e d by s t a r c h g e l e l e c t r o p h o r e s i s . The h a p t o g l o b i n - c o n t a i n i n g f r a c t i o n s were p o o l -ed and p r e c i p i t a t e d a g a i n a t pH 7.1 by 55% ammonium s u l f a t e . The p r e c i p i t a t e was t h e n d i s s o l v e d i n 0.05 M NH^Ac and chromato-graphed on a Sephadex G-200 column (2.8 X 18 6 cm) u s i n g t h e same s o l v e n t . H a p t o g l o b i n - c o n t a i n i n g f r a c t i o n s were p o o l e d , d i a l y z e d a g a i n s t w a t e r and f r e e z e - d r i e d . F o r t h e r e d u c t i o n and a l k y l a t i o n o f h a p t o g l o b i n , Hp 2-1 (6.4 g). was d i s s o l v e d i n 120 ml o f s o l u t i o n w h i c h was 8 M i n u r e a , 0.1 M b o r i c a c i d , 0.04 M i n NaOH w i t h a f i n a l pH'of 8.6 and 36 ml o f 0.1 M B-mercaptoethanol were added. The r e a c t i o n m i x t u r e was k e p t a t room t e m p e r a t u r e f o r 1 hour^ 12 ml o f 0.6 M i o d o a c e t a m i d e w h i c h had been e x t r a c t e d w i t h c h l o r o f o r m t o remove any f r e e i o d i n e were t h e n added. A f t e r 15 min, 6.0 ml o f 0.1 M B-mercaptoethanol were added. The m i x t u r e was d e s a l t e d on a G-25 column (2 X 20 cm) w i t h 0.2 N a c e t i c a c i d . A f t e r l y o p h i l i z a t i o n , t h e c h a i n s were r e d i s s o l v e d i n 0.2 N a c e t i c a c i d and chromatographed on G-75 columns (3 X 2 00 cm) w i t h o.2 N a c e t i c a c i d as s o l v e n t . H a p t o g l o b i n 6 c h a i n , a 2 and a 1 c h a i n s were w e l l s e p a r a t e d from each o t h e r as seen i n F i g . 7. 2. P o l y a e r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s P o l y a c r y l a m i d e g e l s were p r e p a r e d a c c o r d i n g t o D a v i s ( 7 2 ) . The p r e p a r a t i o n o f a 5% a c r y l a m i d e - 8 M u r e a g e l , w h i c h was most f r e q u e n t l y u s e d , i s as f o l l o w s : G l a s s t u b i n g , 8 cm i n l e n g t h and 5 mm i n d i a m e t e r were used as moulds f o r t h e d i s c g e l . The bottom o f t h e tube was s t o p p e r e d w i t h a r u b b e r p l u g . 7.2 gm o f u r e a was added t o 10 ml o f T r i s - g l y c i n e b u f f e r [0.075 M g l y c i n e , 0.015 M t r i s (hydro-x y m e t h y l aminomethane)] g i v i n g a f i n a l volume o f 15 ml and f i n a l c o n c e n t r a t i o n o f 0.05 M g l y c i n e and 0.01 M t r i s . A c r y l a m i d e , 7.5 gm and 35 mg o f N, N' m e t hylene b i s a c r y l a m i d e were added. R i g h t b e f o r e u s e , 5.0 X o f t e t r a m e t h y l e t h y l e n e d i a m i n e and 6 mg o f ammonium p e r s u l f a t e were added. The g e l l i n g s o l u t i o n was i m m e d i a t e l y a p p l i e d t o t h e t u b e s t o e q u a l h e i g h t s . The t o p o f t h e g e l l i n g s o l u t i o n was c a r e f u l l y c o v e r e d w i t h d i s t i l l e d w a t e r . A f t e r g e l l i n g , t h e s u r f a c e was washed w i t h water t o remove any E c o 00 CN -P 1.6 -1.2 -0.8 " 0.4 60 70 Fraction Number Figure 7. Separation of the polypeptide chains, of. reduced .and alkylated haptoglobin 2-1 on a Sephadex G-75 column (3 x 200 cm) with 0.2 N acetic acid as solvent. ro u n g e l l e d m a t e r i a l , and t h e e l e c t r o p h o r e s i s was s e t up as d e s -cr i b e d ^ a & ~ D a v i s ( 7 2 ) . The t o p and bottom p a r t s o f t h e g e l were immersed i n 0.05 M t r i s g l y c i n e b u f f e r . The samples were d i s s o l v e d i n 8 M u r e a , and a p p l i e d g e n t l y on t o p o f t h e g e l . The e l e c t r o p h o r e s i s was per f o r m e d f o r 1 h r a t 200 - 300 v o l t s a t room t e m p e r a t u r e . The g e l was removed from t h e t u b i n g u s i n g a f i n e w i r e , s t a i n e d w i t h 0.5% Amido B l a c k s o l u t i o n f o r 45 min, and d e s t a i n e d w i t h 10% a c e t i c a c i d . 3. Amino a c i d a n a l y s i s Samples were h y d r o l y z e d i n 0.5 ml o f 6 N HC1 i n e v a c u a t e d , s e a l e d t u b e s a t 110°C f o r 18 - 24 h o u r s . G l y c o p e p t i d e s were h y d r o l y z e d i n a c o n c e n t r a t i o n o f 0.1% o r l e s s t o m i n i m i z e t h e amino a c i d - c a r b o h y d r a t e i n t e r a c t i o n ( 7 3 ) . A f t e r r e m o v a l o f HC1 by r a p i d e v a c u a t i o n o v e r NaOH, amino a c i d a n a l y s e s were p e r f o r m e d on a Beckman model 120C amino a c i d a n a l y z e r . F o r homoserine -c o n t a i n i n g p e p t i d e s , t h e d r i e d h y d r o l y s a t e s were i n c u b a t e d w i t h 100 A o f 2 N NH 4OH a t 37°C f o r 1 hour. The samples were d r i e d a g a i n , d i s s o l v e d i n 0.2 N sodium c i t r a t e b u f f e r , p H 2.2 and a n a l y z e d i m m e d i a t e l y u s i n g t h e 4 hour s t a n d a r d p r o c e d u r e (74) w i t h a s l i g h t m o d i f i c a t i o n as d e s c r i b e d by Tang and H a r t l e y ( 75). Due t o t h e t r e a t m e n t w i t h NH^OH, i t has been found advantageous t o a n a l y z e t h e b a s i c amino a c i d s from a homoserine p e p t i d e h y d r o -l y s a t e w i t h a 15 cm Aminex A-5 r e s i n bed (Bio-Rad L a b o r a t o r y , Richmond, C a l i f o r n i a ) u s i n g t h e s t a n d a r d pH 5.28 c i t r a t e b u f f e r f o r e l u t i o n ( 7 6 ) . T h i s same system was a l s o used f o r hexosamine a n a l y s i s . I n l a t e r work on amino a c i d sequence s t u d i e s , p e p t i d e h y d r o l y s a t e s were a n a l y z e d i n a s i n g l e column system as d e s c r i b e d by Devenyi (77) . A t y p i c a l s e p a r a t i o n o f s t a n d a r d amino a c i d s under t h e s e d i f f e r e n t s y s t e m s ^ a r e shown i n F i g . 8. 4. C a r b o h y d r a t e a n a l y s i s Fo r hexose and hexosamine a n a l y s i s , t h e samples were h y d r o -l y z e d w i t h 4 N HCl a t 100°C f o r 8 h o u r s . The h y d r o l y z e d samples were d r i e d o v e r sodium h y d r o x i d e . Hexosamine was a n a l y z e d on th e Beckman 120C amino a c i d a n a l y z e r u s i n g B i o - R a d A-5 r e s i n as d e s c r i b e d i n t h e p r e v i o u s s e c t i o n . Hexose was a s s a y e d by t h e p h e n o l - s u l f u r i c a c i d method o f Dubois e t a l . ( 7 8 ) . The p r o c e d u r e i n v o l v e s t h e a d d i t i o n o f 0.05 ml o f 8 0% p h e n o l t o 2.0 ml o f s o l u t i o n c o n t a i n i n g 10 - 70 yg o f hexose f o l l o w e d by t h e r a p i d d e l i v e r y o f 5.0 ml o f c o n c e n t r a t e d s u l f u r i c a c i d . A f t e r c o o l i n g t o room t e m p e r a t u r e , t h e o p t i c a l d e n s i t y a t 4 90 nm was measured. A l l samples were a s s a y e d i n d u p l i c a t e and r e a d a g a i n s t a b l a n k c o n t a i n i n g d i s t i l l e d w a t e r . The r e s u l t s were r e c o r d e d i n terms of t o t a l g l u c o s e c o n t e n t . T h i s method i s s i m p l e , and i s l a r g e l y u n a f f e c t e d by t h e p r e s e n c e o f p r o t e i n s and amino s u g a r s ( 7 9 ) . The s i a l i c a c i d c o n t e n t was measured by t h e t h i o b a r b i t u r i c a c i d method (8 0 ) . The samples were f i r s t h y d r o l y z e d w i t h 0.01 N HCl f o r 1/2 h r a t 100°C. To a sample c o n t a i n i n g 0.05 ymoles o f s i a l i c a c i d i n a volume o f 0.2 m l , 0.1 ml o f sodium p e r i o d a t e s o l u t i o n (0.2 M i n 9 M p h o s p h o r i c a c i d ) was added and k e p t a t room t e m p e r a t u r e f o r 20 m i n u t e s . One ml o f a r s e n i t e s o l u t i o n (sodium a r s e n i t e , 10% i n a s o l u t i o n o f 0.5 M sodium s u l f a t e , 0.1 N H 2 S O 4 ) was added, and t h e t u b e s were shaken u n t i l a y e l l o w brown c o l o r d i s a p p e a r e d . Three ml o f t h i o b a r b i t u r i c a c i d i • • Cytteic Acid Atpartic Acid oy Q •Threonine : '-Serin* - Homoserine 'Glutamic Acid >Proline 15. 3 3" o o a c 3 •Glycine 'Alanine Half Cyitine Valine "Methionine Isoleucine -Leucine Nor leucine OJ 09 OJ 5 3 5 : 'Tyrosine --Phenylalanine OJ 1» o Q 3 °J (M ~-Ol o O 3 3" o 3 o 2 *4 C CO cn rt P> CU H Cu C PJ o N 3 CD O cn to B H-0 CD Cfl cn r t 1 Lysine Glucosamine : -.-Galactosamine > Homoserine Lactone CD CD a: >< o CO Q 3 3' ro 4? 01 ~* 3 C O o o Q: J ) Arginine Standard Amino Acid Run Figure 8 c . Standard amino acid run Single Column System Buffer A PH 3 43 02N Sodium Citrate Buffer B pH 4-25 0 8N SodiumCifralo Buff or Chongo 70 m/nutos s i n g l e column system. 33 ( t h i o b a r b i t u r i c a c i d 0.6% i n 0.5 M sodium s u l f a t e ) were added and t h e samples were h e a t e d i n a b o i l i n g water b a t h f o r 15 m i n u t e s . The t o t a l aqueous s o l u t i o n was e x t r a c t e d w i t h 4.3 ml o f c y c l o h e x a n o n e . The o p t i c a l d e n s i t y a t 54 9 run was t h e n measured on t h e o r g a n i c phase. The OD v a l u e was c o n v e r t e d t o ymoles o f s i a l i c a c i d u s i n g a s t a n d a r d c u r v e f o r s i a l i c a c i d . 5. 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 and paper chromatography 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 was p e r f o r m e d on Whatman 3 MM paper i n f o u r d i f f e r e n t pH b u f f e r s . B u f f e r systems em-p l o y e d were pH 6.5 ( p y r i d i n e / a c e t i c a c i d / H 2 0 100:4:900), pH 4.0 ( p y r i d i n e / f o r m i c a c i d (90%)/H 2O 12:10.25:980), pH 3.6 ( p y r i d i n e / a c e t i c a c i d / H 2 0 1:10:89) and pH 1.9 (2% f o r m i c a c i d : 8 % a c e t i c a c i d ) . The t i m e o f each e l e c t r o p h o r e s i s r u n was judged by t h e m o b i l i t y o f a s e r i e s o f c o l o u r e d m a r k e r s . The m o b i l i t y o f t h e s e markers a t d i f f e r e n t pH's compared t o s t a n d a r d amino a c i d s i s t a b u l a t e d i n T a b l e I . D e s c e n d i n g paper chromatography w i t h n - b u t a n o l - a c e t i c a c i d -w a t e r - p y r i d i n e (15:3:12:10) (81) as s o l v e n t was used t h r o u g h o u t t h e s t u d i e s . The chromatography u s u a l l y t a k e s 16 - 18 h o u r s . 6. Use o f s p e c i f i c s t a i n s f o r p e p t i d e s and amino a c i d s Throughout t h e p r e s e n t s t u d i e s , c e r t a i n c o m b i n a t i o n s o f s p e c i f i c s t a i n s f o r amino a c i d s were v e r y u s e f u l i n p e p t i d e f i n g e r p r i n t i n g and i s o l a t i o n . The f o l l o w i n g c o m b i n a t i o n s were used most o f t e n . (a) Cadmium-ninhydrin (82) a f t e r p h e n a t h r o n e - q u i n o n e s t a i n (83) : 34 Table I The M o b i l i t i e s of Coloured Markers on paper High Voltage Electrophoresis Compound pH 2.1 pH 3.6 pH 6.5 DNP l y s i n e ser y= 0.56 y=0. 0 y=0. 0 DNP Armatine ser lys asp y= 1.1 y= 0.67 y= -.57 Xylene Cyanol FF ser lys asp y= -.29 y= -.43 y= .35 Orange G lys asp y= -.86 y= 0.92 Methyl green ser lys asp y= 2.0 y= 1.05 y= -.91 Cryst a l v i o l e t ser lys asp y= 0.82 y= 0.46 y= -.37 E q u a l volumes o f 0.02% phen a t h r o n e - q u i n o n e i n e t h a n o l and 10% NaOH i n 60% e t h a n o l were mixed and s p r a y e d . A f t e r d r y -i n g , t h e paper was viewed under UV l i g h t . A r g i n i n e - c o n t a i n i n g p e p t i d e s a r e s t r o n g l y f l u o r e s c e n t . The s p o t s were c i r c l e d , and t h e paper was washed 3 t i m e s w i t h e t h a n o l , once w i t h 5% a c e t i c a c i d i n a c e t o n e , and d r i e d . F i n a l l y , t h e paper was d i p p e d i n c a d m i u m - n i n h y d r i n r e a g e n t . (b) E h r l i c h a f t e r n i n h y d r i n (84); The paper was d i p p e d -wi-thr 0 . 5 % n i n h y d r i n ^ k e p t i n an / oven a t 50 - 60°C o v e r n i g h t . N i n h y d r i n s p o t s were c i r c l e d . The paper was t h e n d i p p e d i n 2% p - d i m e t h y l a m i n o b e n z a l d e h y d e i n a c e t o n e , d r i e d , and d i p p e d a g a i n i n 10% HC1 i n a c e t o n e . T r y p t o -phan p e p t i d e s w i l l t u r n p u r p l e , w h i l e o t h e r n i n h y d r i n s p o t s f a d e . (c) P l a t i n i c i o d i d e , n i n h y d r i n and P a u l y s t a i n (84)', The p l a t i n i c i o d i d e r e a g e n t was made by c o m b i n i n g 4 ml o f p l a t i n o c h l o r i c a c i d (1 mg/ml R"20) , 0.25 ml o f 1 N K I , 0.4 ml o f 2 N HC1 and 76 ml o f a c e t o n e . The paper was d i p p e d w-tfeh^ f r e s h l y made r e a g e n t . M e t h i o n i n e p e p t i d e s appear as w h i t e s p o t s o v e r a s l i g h t l y p i n k background. A f t e r c i r c l i n g t h e s p o t s , t h e paper was d i p p e d i n 0.5% n i n h y d r i n . L a s t l y , t h e paper was s p r a y -ed w i t h i c e c o l d P a u l y r e a g e n t (1 volume o f 1% s u l f a n i l i c a c i d i n 1.2 N HC1, 1 volume o f 5% NaN0 2 and 2 volumes o f 10% Na 2CC> 3). H i s t i d i n e s p o t s a r e p i n k o r o r a n g e - p i n k , t y r o s i n e s p o t s a r e d u l l p u r p l e . 7. N - t e r m i n a l d e t e r m i n a t i o n The N - t e r m i n a l amino a c i d s o f b o t h p e p t i d e s and p r o t e i n s was d e t e r m i n e d u s i n g t h e d a n s y l c h l o r i d e (DNS-C1) method f i r s t described by Gray (85). (a) Application to peptides ' An aliq u o t of solution containing 0.01 ymoles of pep-tid e was dried down i n a hydrolysis tube (Corning Cat. No. 99445, 10 X 75 mm), dissolved i n 25 A of 0.1 M NaHC03 and again dried down1 i n vacuo to remove NH^• An equal volume of d i s t i l l e d water and dansyl chloride (3 mg/ml i n acetone), usually 25 A S^ each was added, and the tube covered with parafilm and incubated at 37°C for 2 hours. Afte r drying the contents of the tube i n vacuo, the sample was then taken up i n 0.2 ml 6 N HC1 and hydro-lyzed i n vacuo at 110°C for 16 hours. If dansyl p r o l i n e was suspected as the N-terminal, a 6 hr hydrolysis as well, as the normal 16 hr hydrolysis was performed. The dried hydrolysate was dissolved i n 10 A of 2 M NH^OH and applied to two separate thi n - l a y e r plates. The thin-layer plates were prepared by spreading a s l u r r y (25 g i n 50 ml water) of s i l i c a g el G (accord-ing to Stahl, E.: Merck AG, Darmstadt) i n a 0.25 mm layer (Desaga spreader) on 20 X 20 cm glass plates, and the plates were activated at 120°C i n an oven for 30 minutes before use. The two solvent systems described by Black and Dixon (86) were used. The f i r s t system chloroform/methanol/acetic acid 95:10:1 by volume separates the dansyl derivatives of nonpolar amino acids and i t takes appro-ximately one and a half hours. The second system propanol/NH^OH 80:20 by volume separates the dansyl d e r i v a t i v e s of polar amino acids and takes 4 hours for the run. The chromatography was run at room temperature. F i g . 9 shows the separations of the dansyl amino acids i n these two solvent systems. Figure 9_. (a) Separation of dansyl-amino acids by thin-layer chromatography i n solvent A (chloroform/methanol/acetic acid, 95:10:1 by volume).— Origin at the bottom and development from ^ bottom to top. Order of the dansyl d e r i v a t i v e s from l e f t to r i g h t : NH^, l i e , Val, Pro, Leu, di-Tyr, di-Lys, Phe, Met, Ala, di- H i s , Try, mono-Tyr (in t h i s preparation almost a l l i s di-Tyr; when present i t runs alongside Gly), Gly, Met sulfone, Thr, Ser, Met (to show the po s i t i o n of the slow-running s u l f o x i d e ) , Glu, Lys (prepared to contain mainly the mono-substituted derivatives) His (as previous Lys), di-Cys, Arg, Cys(Cm), Cys (prepared to contain mainly the mono-substituted d e r i v a t i v e ) , c y s t e i c acid, Asp. The l i n e of spots close to the o r i g i n i s due to dansyl-acid (b) Separation of dansyl-amino acids by thin - l a y e r chromatography i n solvent B (propanol/concentrated NH^ ,- 80:20 by volume)^ Order of the dansyl d e r i v a t i v e s as i n F i g . 9a. In t h i s case dansyl-acid gives a l i n e of fast-moving spots. Di-Tyr runs with the dansyl-acid. The separate, slower running spot i s the mono-substituted d e r i v a t i v e . a Occasionally, when only l i m i t e d amounts of a peptide •was- ava i l a b l e , the peptide hydrolysate was applied on one plate, and chromatographed i n the f i r s t system. After drying, the same plate was rechromatographed i n the second solvent system i n the same d i r e c t i o n . Photography i s the best and most convenient method of recording the separations obtained. In t h i s work, a Polaroid Model 16 0 camera used was f i t t e d with a copy lens and UV f i l t e r ( T i f f e n photar UV-16 series 7), using polaroid Land Picture R o l l Type 47, 3000 speed and the number 10 aperture on the camera. When the plate was illuminated with the UV l i g h t from a Mineralight UVS 11 obtained from U l t r a v i o l e t Products Inc., San Gabriel, C a l i f o r n i a , a 45-second exposure gave a s a t i s f a c t o r y p r i n t . I t i s desirable to examine and record the separations as soon as possible a f t e r development of the t h i n -layer plates as the fluorescence of the dansyl amino acids fades with time. (b) Application to proteins : To i d e n t i f y the N-terminal amino acid of protein or large peptides, the above method for the dansylation of the N-terminus of peptides was modified. About 2.0 mg of protein was dissolved i n 0.5 ml of 8 M urea solution, buffered with NaHCO^ (0.5 M). To t h i s was added 0.5 ml of dansyl chloride solution (20 mg/ml acetone). The reaction was kept at 37°C for 2 hours and desalted on a G-25 column (0.9 X 90 cm) using 0.1 M NH^OH as solvent. The e l u t i o n was monitored with a UV lamp. The dansyl protein moved r a p i d l y through the column as a yellow fluorescent band, ahead of the massive amount of dansyl s u l f o n i c acid and s a l t . A f t e r lyo-p h i l i z m g t h e sample, i t was h y d r o l y z e d i n 6 N HCl and i d e n t i f i e d as i n t h e e a r l i e r s e c t i o n . (c) P r e p a r a t i o n o f s t a n d a r d d a n s y l amino a c i d s The d a n s y l d e r i v a t i v e s o f s t a n d a r d amino a c i d s were p r e p a r e d a c c o r d i n g t o Gray ( 8 7 ) . To 1.0 ml o f each s t a n d a r d amino a c i d s o l u t i o n (6.5 ymoles i n 0.1 M NaHC0 3) was added an e q u a l volume o f d a n s y l c h l o r i d e s o l u t i o n (6 mg/ml i n acetone) and t h e m i x t u r e was l e f t a t room t e m p e r a t u r e over n i g h t . The NaHCO^ was p r e c i p i t a t e d by a d d i n g 8 ml o f a c e t o n e . The s u p e r -n a t a n t , w h i c h c o n t a i n s t h e d a n s y l amino a c i d , was k e p t a t 4°C. 8. Edman d e g r a d a t i o n (88) S e q u e n t i a l d e g r a d a t i o n from t h e amino t e r m i n a l o f p e p t i d e was p e r f o r m e d by t h e Dansyl-Edman p r o c e d u r e d e s c r i b e d by Gray (89) . The p e p t i d e s o l u t i o n (0.01 umole - 0.1 ymole) was t r a n s -f e r r e d t o a s m a l l tube ( C o r n i n g C a t . 99445, 13 X 100 mm), and d r i e d i n vacuo. The sample was r e d i s s o l v e d i n 100 X o f 50% aqueous p y r i d i n e (V/V, r e d i s t i l l e d p y r i d i n e ) . On a d d i t i o n o f 100 X p h e n y l i s o t h i o c y a n a t e (PITC) s o l u t i o n , a one phase r e a c t i o n m i x t u r e was o b t a i n e d . The PITC s o l u t i o n c o n s i s t s o f 1 ml o f r e d i s t i l l e d N-methyl m o r p h o l i n e and 100 X o f PITC. The r e a c t i o n m i x t u r e was t h e n f l u s h e d w i t h N 2 , a n& c o v e r e d w i t h p a r a f i l m . The c o u p l i n g was a l l o w e d t o p r o c e e d f o r 2 1/2 hours a t 37°C. E x c e s s r e a g e n t s , w a t e r , and p y r i d i n e were moved by r a p i d e v a c u a t i o n o v e r NaOH. The d r i e d r e s i d u e c o n t a i n i n g t h e PTC p e p t i d e was d i s s o l v e d i n 200 A o f anhydrous t r i f l u o r o a c e t i c a c i d (Matheson, Coleman & B e l l , Norwood, Ohio, TFA 7454. TX1275), f l u s h e d w i t h N 2, covered and incubated at 37°C for one hour. TFA was then removed i_n vacuo. 500 X of water was added to the dried residue and extracted 4 times with 1 ml of reagent grade ethyl acetate. The aqueous phase was dried, dissolved i n a small volume'of water, and an aliquot corresponding to 0.01 ymole was taken for dansyl-ation. The remainder was again dried and subjected to another cycle of the degradation procedure. A subtractive Edman procedure (90) was also performed a f t e r every two Edman cycles to confirm the r e s u l t s from dansylation studies. 9. Use of leucine aminopeptidase (91) and carboxypeptidase A (92) for amino acid sequence studies Leucine aminopeptidase, DFP-treated, 61C, 9 mg/ml was pur-chased from Worthington Biochemical Corp., Freehold, New Jersey. A t y p i c a l incubation mixture consisted of 0.05 ymole of peptide i n 200 X of 0.2 M NH^Ac buffer containing 0.005 M MgCl 2, pH 8.5, 2.0 X of enzyme. The mixture was incubated at 37°C for various periods of time up to 24 hours. For sjamll peptides, aliquots taken at d i f f e r e n t times were dried, dissolved i n 0.2 N sodium c i t r a t e buffer pH 2.2, and analyzed on the amino acid analyzer. For larger peptides, the amino acids released during the d i g e s t i f were absorbed on Dowex 50-X8 (20 - 50 mesh) r e s i n , H + form, the peptides and enzymes were removed with water washes, and the absorbed amino acids were then eluted with 5 M NH^OH. Carboxypeptidase A, 3 X c r y s t a l l i z e d , DFP-treated, 50 mg/ml aqueous suspension was also a product of Worthington Biochemical Corp. Aliquots (5.0 X) of enzyme c r y s t a l s were washed 3 - 4 times with 200 X of H„0, centrifuged, and the enzyme c r y s t a l s were dissolved i n 2 M NH^HCO^, pH 8.0. Aliquots were transferred to the peptide solution (0.02 - 0.1 ymole) so that the f i n a l concentration was about 0.2 M NH^HCO^. The digest was kept at 37°C for various period time. The resultant digests from both small and large peptides were worked up as i n the leucine aminopeptidase studies. The a c t i v i t y of the carboxypeptidase was measured with synthetic substrate benzoylglycyl-L-phenyalanine. 10. Amide determination The mobility of peptides containing aspartic acid or glu-tamic acid r e l a t i v e to aspartic acid was determined by high voltage electrophoresis at pH 6.5. I t was then possible to assign the charge of the peptide from i t s amino acid composition using the chart described by Offord (93). Another approach, using leucine aminopeptidase (91) to hydrolyze the peptide was also taken to confirm the assignment by the mobility studies. Under these conditions, asparagine and glutamine eluted separately from aspartic acid and glutamic acid on the amino acid analyzer. 11. P u r i f i c a t i o n of reagents Pyridine (Fisher, reagent grade, P. 368) was r e d i s t i l l e d over ninhydrin to react any amines and was stored at -20°C to prevent decomposition. a-Picoline (Matheson, Coleman & B e l l , p r a c t i c a l grade, P. 27 05) and N-methylmorpholine (Eastman organic chemical, Rochester, New York, p r a c t i c a l grade, P. 6760) were r e d i s t i l l e d d i r e c t l y and stored at -20°C. Phenylisothiocyanate (PITC) (Eastman organic chemical, 43 1484) was vacuum d i s t i l l e d and stored at -20°C. In l a t e r studies, highly p u r i f i e d reagent (Sequanal grade, No. 27500) was supplied by Pierce Chemical Company, Rockford, I l l i n o i s and used without further p u r i f i c a t i o n . Hexafluoroacetone trihydrate was a g i f t from Dr. P. E. Wilcox, Department of Biochemistry, University of Washington, Seattle. The reagent was l a t e r purchased as hexafluoroacetone sesquihydrate (CF^)2 C 0*!• 5 H2° f r o m E- D u Pont De Nemours & Co., Wilmington, Delaware. The trihydrate (CF 3) 2CO*3H 20, b o i l i n g point 105°C, was f r a c t i o n a l l y d i s t i l l e d , and kept at room temperature (94). CHAPTER I: THE GENERAL CHARACTERIZATION OF HAPTOGLOBIN 8 CHAIN Introduction Prior to the amino acid sequence studies on the 6 chain of haptoglobin,the homogeneity of the chain was examined using d i f f e r e n t c r i t e r i a . In the present chapter, the 8 chain i s o l a t e d from hapto-globin 2-1 was further characterized by ion exchange chromato-graphy, gel f i l t r a t i o n chromatography, u l t r a c e n t r i f u g a t i o n studies and sodium dodecyi su l f a t e d i s c gel electrophoresis. The amino acid composition, carbohydrate composition, N-terminal amino acid as well as the accurate molecular weight of the 8 chain was determined. Studies oh the 8 chain have met with some d i f f i c u l t i e s . The chain, i s o l a t e d i n the carboxymethylamido form, i s extensively aggregated i n solution. Chemical modification using succinic anhydride was undertaken to overcome these d i f f i c u l t i e s . The succinyl 8 chain was completely disaggregated, and was soluble at neutrai pH. Experimental 1• DEAE-cellulose chromatography The 8 chain (58.2 mg i n 2 ml s t a r t i n g buffer) was chromato-graphed on a DEAE-cellulose column (1.2 X 46 cm) (Whatman 32, microgranular grade) i n the presence of 6 M deionized urea, using 45 a gradient of 300 ml of 0.01 M t r i s - HCl pH 8.0 and 300 ml of 0.15 M t r i s - H C l pH 8.0. The e l u t i o n was monitored at 280 nm. The e l u t i o n p r o f i l e i s shown i n F i g . 10a. The f r a c t i o n s , A, B, C, and D, were pooled, dialyzed and l y o p h i l i z e d . These fracti o n s were analyzed on urea - starch formate g e l , 0.05 M formic acid, 0.01 M NaOH, 8 M urea, pH 4.0 (12) run for 25 hours at 160 v o l t s . The gel was stained with Amido Black, and l a t e r destained with 10% acetic acid. The r e s u l t i s shown i n Fig.11. 2. Gel f i l t r a t i o n chromatography on Sephadex G-200 The 3 chain (166 mg i n 7 ml) was chromatographed on Sephadex G-200 column (1.2 X 186 cm) using 0.2 N acetic acid as eluant at room temperature. The e l u t i o n p r o f i l e i s shown i n F i g . 10b. Recovery was calculated to be 95% on the basis of O.D. units at 280 nm. 3. N-terminal determination Dansylation of the a 1 and 3 chain was performed according to•the method described i n the general method section. 4. Succinylation (95) To 60 mg of protein i n 30 ml of 0.1 M sodium phosphate buffer (pH 7.0) were added i n aliquots with magnetic s t i r r i n g at 4°C, 200 mg of succ i n i c anhydride (approximately 50 f o l d molar excess over ly s i n e content). The pH of the reaction mixture was maintained at pH 7.0 - 8.5 by the addition of 1.0. M NaOH from a syringe. After 2 hr, the succinylated protein was dialyzed against 0.005 M phosphate buffer pH 7.0 at 4°C (five changes of 1 l i t e r ) and f i n a l l y l y o p h i l i z e d . 5. Ul t r a c e n t r i f u g a t i o n studies U l t r a c e n t r i f u g a t i o n studies were performed with Beckman model E a n a l y t i c a l u l t r a c e n t r i f u g e . Sedimentation v e l o c i t y and the Archibald approach to equilibrium method (96) was performed with the usual a n a l y t i c a l D rotor with a Schlieren o p t i c a l system. The sedimentation equilibrium method described by Van Holde (97) was performed using the double sectored c e l l with the interference o p t i c a l system. Succinyl g chain and succi n y l a 2 chain used for c e n t r i -fugation were prepared as follows: about 40 mg of the samples were dissolved i n 3ml of 0.1 N phosphate buffer pH 7.0, and dialyzed against the same buffer f o r 48 hours, centrifuged at 40,000 rpm for 2 hours i n Beckman L2 65 u l t r a c e n t r i f u g e . Portions of 0.4 ml y,&s- used for studies. 6. Amino acid anlysis and carbohydrate analysis Amino acid analysis of haptoglobin 2-1 and i t s separated chains was performed.by Dr. J. Black. Aliquots of the protein and separated chains were hydrolyzed i n 6 N HC1 i n vacuo at 105°C f o r 16, 40 and 73 hours. Duplicate amino acid analyses at each time i n t e r v a l were performed on a Beckman 120 B amino acid analyzer. Tryptophan was determined separately by the method of Beaven and Holiday (98). Cysteic acid was determined following performic acid oxidation (99, 100) of the protein. In t h i s case, amino acid analysis was performed on the Beckman 47 120C amino a c i d a n a l y z e r u s i n g t h e a c c e l e r a t e d system. C a r b o h y d r a t e a n a l y s i s was p e r f o r m e d as d e s c r i b e d i n t h e g e n e r a l methods. R e s u l t s The Homogeneity o f H a p t o g l o b i n 3 C h a i n When t h e 3 c h a i n was chromatographed by 6 M u r e a D E A E - c e l l u l o s e chromatography, a s i n g l e peak was o b t a i n e d ( F i g . 1 0a). However, v a r i o u s f r a c t i o n s o f t h e peak show^a s l i g h t d i f f e r e n c e i n m o b i l i t y as seen i n u r e a - s t a r c h - g e l e l e c t r o p h o r e s i s ( F i g . 1 1 ) . E a r l i e r s t u d i e s by S m i t h i e s e_t a l . (12) showed t h a t 3 c h a i n gave r i s e t o m u l t i p l e bands when examined by u r e a - f o r m a t e - s t a r c h g e l e l e c t r o -p h o r e s i s . The s e p a r a t i o n a c h i e v e d from D E A E - c e l l u l o s e m i g h t r e p r e s e n t t h e p a r t i a l f r a c t i o n a t i o n o f t h e s e components. These m u l t i p l e bands may a r i s e as a r e s u l t o f t h e r e m o v a l o f v a r y i n g amounts o f a c i d - l a b i l e s i a l i c a c i d r e s i d u e s p r e s e n t i n t h e mole-c u l e . T h i s argument was s u p p o r t e d by l a t e r s t u d i e s on CNBr c l e a v a g e p e p t i d e s ^ one o f t h e s e f r a g m e n t s , a g l y c o p e p t i d e , g i v e r i s e t o m u l t i p l e bands on d i s c g e l e l e c t r o p h o r e s i s . However, upon m i l d a c i d h y d r o l y s i s w h i c h removed t h e s i a l i c a c i d , 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 was s i m p l i f i e d . On G-200 chromatography ( F i g . 1 0 a ) , a minor component was e l u t e d l a t e r t h a n t h e major component. U l t r a c e n t r i f u g a t i o n s t u d i e s o f t h e 3 c h a i n i n 0.2 N a c e t i c a c i d a l s o r e v e a l e d t h e p r e s e n c e o f a s l o w moving minor component. The S 2 Q w v a l u e f o r t h e major component was 3.6 S, t h e minor component about 2.0 S. 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 u s i n g t h e A r c h i b a l d approach t o e q u i l i b r i u m 48 30 40 50 60 70 80 90 Fraction Number Figure 10a. Chromatography of haptoglobin g chain on 6M urea-DEAE-c e l l u l o s e column using a t r i s - H C l gradient, pH 8.0. 10b. Chromatography of haptoglobin 6 chain on Sephadex G-200 column using 0.2 N acetic acid as solvent. c B A D C B A F i g u r e 11. Urea - s t a r c h g e l e l e c t r o p h o r e s i s o f v a r i o u s f r a c t i o n s o f h a p t o g l o b i n B c h a i n from DEAE-c e l l u l o s e chromatography. procedure on the major peak give a value of 6.7 X 10 . This indicates that the 3 chain i s extensively aggregated under these conditions. Black et a l . (14) also observed a f a i n t band in 8 M urea-formate-starch gel i n between the a 2 chain and the 3 chain using the same preparation of separated chains. This was subsequently found to elute on Sephadex G-7 5 i n 0.2 N a c e t i c acid between the 3 chain and a 2 chain. However, t r y p t i c digest peptide maps show that t h i s band gave a v i r t u a l l y iden-t i c a l pattern to the 3 chain (J. Black, unpublished observation), and probably represents 3 chain from which s i a l i c acid residues have been removed. Later carbohydrate analysis did, i n f a c t , demonstrate differences between t h i s component and the major component. To investigate the i d e n t i t y of these two components further, they were subjected to SDS disc gel electrophoresis as described by Weber and Osborn (101). In t h i s experiment, 7% cross - l i n k -ed gels were used and the electrophoresis was run at approxi-mately 8 mA per gel for 4 hours. The gels were stained with Comassie Blue for 2 hours and l a t e r destained with methanol/ acetic acid/water ( 50:75:875 ). As can be seen i n F i g . 12, both the major and minor components of haptoglobin 3 chain has the same mobility under these conditions. The s l i g h t d i f f e r -ence i n the carbohydrate content would have l i t t l e e f f e c t on t h e i r mobility i n the presence of SDS. Carbohydrate analysis showed that the minor component contained less s i a l i c acid than the major component. 5 1 F i g u r e 1 2 . SDS d i s c g e l e l e c t r o p h o r e s i s o f h a p t o g l o b i n 8 c h a i n . From l e f t t o r i g h t : 1. i n s u l i n ; 2. lysozyme; 3. b o v i n e a l b u m i n ; 4. a 1 c h a i n ; 5. a 2 c h a i n ; 6. 6 c h a i n ( m a j o r ) ; 7. 6 c h a i n ( m i n o r ) . Dansylation studies of the 3 chain revealed only isoleucine as the N-terminal amino acid (Fig. 13). E a r l i e r studies by Smith et a_l. (24) showed that haptoglobin 2-1 contained equi-molar amounts of valine and isoleucine as the N-terminal amino acids. This suggests a^empirical composition 2 3 + a 1 + a 2 for haptoglobin 2-1. Since both a 1 and a 2 have valine as the N-terminal, i t was implied that isoleucine i s the N-terminal of the 3 chain. Present studies confirm the above assignment, and indicate that the 3 chain i s r e l a t i v e l y free of other con-taminants. Succinylation Studies As discussed e a r l i e r , the 3 chain was extensively aggre-gated i n 0.2 N acetic acid. The S o n was determined as 3.6 S, 3 z U , w which was simiar to that of i n t a c t Hp 1-1 (4.3 S) which i s known to consist of two a 1 chains and two 3 chains. Furthermore, the molecular weight of the 3 chain was estimated as 6.7 X 10^ by the Archibald approach to equilibrium procedure. The S 2 Q ^ and molecular weight determined under these conditions, therefore, did not represent the true physiochemical properties of the A 3 chain. In order to characterize the 3 chain further, i t was found necessary to modify i t chemically. Succinylation using succinic anhydride was selected for various reasons. The chemical modifi-. .. Q «1 W fS 9 * ^ 53 1 5 9 Figure 13. N-terminal determination of haptoglobin 3 chain 1. Isoleucine. 2. Isoleucine + haptoglobin 8 chain. 3. Haptoglobin 8 chain. 4. Isoleucine + haptoglobin 8 chain. 5. Haptoglobin 8 chain. 6. Valine. 7. Valine + haptoglobin a 1chain. 8. Haptoglobin a 1 chain. 9. Valine + Isoleucine. 54 cation reaction has been reported to attack the l y s y l amino group s p e c i f i c a l l y under very mild conditions (95) and has been used extensively and successfully i n modifying various proteins (102). The reagent, being a s o l i d , i s stable and easy to handle experi-mentally. Gounaris and Perlmann (103) found that reaction can also occur with tyrosine and hydroxy amino acid residues, although these O^succinyl groups would be r a p i d l y hydrolyzed under the extensive d i a l y s i s conditions employed here. Since the a 2 chain was known to aggregate i n neutral or a l k a l i n e s o l u t i o n (13), s u c c i n y l a t i o n of a 2 chain was performed together with the modification of the 3 chain as a control for the chemical modification studies. As can be seen i n F i g . 14, s u c c i n y l 3 chain and succinyl a 2 chain were homogeneous on Sephadex G-200 and G-75 r e s p e c t i v e l y . Sedimentation v e l o c i t y studies (Fig. 15 and F i g . 16) demonstrated that both succinyl 3 chain, s u c c i n y l a 2 chain appear as single symmetrical boundary at a rotor speed of 56,000 rpm. The' S~n value for succinyl 3 chain and s u c c i n y l a 2 chain 20,w J are 1.86 and 1.45 r e s p e c t i v e l y . The S~ n of s u c c i n y l 3 chain was found to be independent of concentration ranging from 1% -0*,25%, and has the same value i n 0.2 N NaCl solu t i o n ( S o n = 2.0 S) . It i s i n t e r e s t i n g to notice that the succinyl a 2 chain has a same S„„ value as the unmodified a 2 chain i n aqueous 20 ,w a c i d i c buffer (13). Molecular weight determination were performed on these succinyl chains. Archibald approach to equilibrium technique was performed on succinyl 3 chain at 8,000 rpm. F i g . 17 shows aK ^ u l t r a c e n t r i f u g a t i o n pattern obtained under these conditions. 0.3 e c o 00 CN 4J 0.2 Q 6 o . i -Figure 14 a. Figure 14 b. 10 20 30 40 10 Fraction Number 50 Chromatography, of. succinyl 6 chain on. a Sephadex G-200 column. 2 Chromatography of succinyl a chain on a .Sephadex. G-75 column. CJ1 160 192 224 256 Figure 15. Sedimentation v e l o c i t y studies on succinyl @ chain i n 0.1 N Phosphate buffer PH 7.0 temperature 9.5°C, K.F. c e l l , 32 min i n t e r v a l , 35' 43* 5 1 ' 59' Sedimentation v e l o c i t y studies on succinyl a 2 chain i n 0.1 N phosphate buffer pH 7.0, temperature 13°C, synthetic boundary c e l l , 8 min i n t e r v a l . Figure 17. Sedimentation equilibrium studies on su c c i n y l B chain using Archibald approach to equilibrium method. Succinyl B chain was dissolved i n 0.1 N phosphate buffer PH 7.0, temperature 9.5°C, 8,000 rpm. 59 A sample c a l c u l a t i o n based on the measurement of the u l t r a -centrifuge pattern i n F i g . 17 was the following: 1 dx n x „ dc C = C - — T — E x Z ; — — =0.60 m ° 2 „ .n n , x F n dx m o m 1 0.1 C = 0.322 - . : 104.95 m 34.9251 10 = 0.292 RT (dc/dx) M = — w (1 - Vp) CO2 X c . mm 8.314 • 10 7 • 280.5 0.6 0.2648 • 7.015 • 10 5 0.292 * 5.91 = 43,700 Values obtained from the sedimentation equilibrium using interference optics were i n excellent agreement with those obtained above. These r e s u l t s are shown i n Table I I . The molecular weight of succinyl a 2 chain was determined using the interference method described by Van Holde. F i g . 18 i l l u s t r a t e d the u l t r a c e n t r i f u g a t i o n pattern obtained under these conditions for succinyl a 2 chain. TABLE II 2 Molecular Weights of Haptoglobin 1-1, Haptoglobin a Chain, and Haptoglobin 6 Chain Protein sample 20,w Method and conditions Molecular weights Mean molecular weight Correction for Corrected su c c i n y l molecular groups weight Haptoglobin 1-1 Succinylated 120 min, 1% i n 0.05 M KC1 - 3.18 0.01 M Na phosphate, pH 7.0 2 Haptoglobin a chain succinylated f o r 120 min, 0.5% i n 0.1 M 1.40 sodium phosphate,pH 7.0 Haptoglobin 3 chain succinylated f o r 1.84 120 min Archiblad (96) 13,000 r.p.m. 22°C Sedimentation equilibrium (97) 13,000 r.p.m. 7°C Archibald (96) 12,000 r.p.m. 24°C 0.5% protein Archibald (96) 8,000 r.p.m. 9. 5°C 1.0% protein Sedimentation equilibrium (97) 10,000 r.p.m. 22.2°C 0.5% protein i n 0.1 M Na phos-hate - 0.2 M KC1 103,105 (222') 103,475 (258') 104,804 (288') 103,795+890 7000 (70)* 96,795 19,153 18,980 18,952 19,680 47,400 46,828 44,846 43,700 44,598 44,895 46,213 45,538 46,389 43,100 43,324 (1948') (22041) (2460' ) (2706') (39' ) (55 1) (71' ) (102') (134') (150') (1020') (1120') (1130') (1190') (1250') 19,143+338 1700 (17) 17,443 46,357+1340t 44,397+620+ 2600 (26) 42,582 44,794+1580+ *Number of succinyl groups i s i n parenthesis. tOv e r a l l mean (three sets of determinations), 45 182, Figure 18. Molecular weight determination of s u c c i n y l a 2 chain using interference optics method described by Van Holde. a) . C run. o b) . Sedimentation equilibrium run. The following equation was used for the c a l c u l a t i o n : 2 RT AJ M = w2 (1 - Vp) (b 2 - a 2) AJ . K sb i n which AJ , = C i n terms of fringes sb o ' ^ Jeq w a s °ktained ky p l o t t i n g no. of fringes i n sedimentation equilibrium run v.s. distance, For example 2 • 8.314 • IO 7 • 295.2 8.05 M = 0.2684 • 1.85 • 10 6 • 3.8363 10.3 = 19,153 The molecular weight of succinyl Hp 1-1 (prepared by Dr. G. F. Q. Chan) was also determined. These r e s u l t s are tabulated i n Table I I . As mentioned e a r l i e r i n the preparation of the 3 chain, the y i e l d of 8 chain was 4.93 gm, a 2 chain 0.917 gm, and a 1 chain 0.489 gm. Since the molecular weights of the a 1 and a 2 chains are known, and using the empirical composition 2 8 + a 1 + a 2 for haptoglobin 2-1, i t i s possible to estimate the molecular weight of the 6 chain. This was estimated to be 45,000, and gives a value for l y s i n e content of 26 residues/mole. By an i t e r a t i v e procedure, the observed molecular weight can be corrected and then recalculated. Thus the f i n a l molecular weight for 8 chain a f t e r correcting for the s u b s t i t u t i o n of 25 l y s y l and one N-terminal isoleucine i s 42,582, and for i t s polypeptide portion 34,431. (The carbohydrate content was determined as 19.4% i n the present studies). Gordon et a l . (104) have i s o l a t e d the 8 chain from reduced and alkylated haptoglobin by Sephadex G-200 chromato-graphy i n 5 M guanidinium chloride. Their values for residues of amino acid per a molecular weight of 40,000 for 8 chain ( e s t i -mated but not accurately measured) and correcting for a carbohydrat content of 15% are i n reasonably good agreement with those re-ported here. Since the amino acid sequence of a 2 chain i s known, and contains 16 l y s i n e residue, the subtraction of 17 suc c i n y l groups givej a value of 17 ,443 which i s i n excellent agreement with "/ /\ 17,300 + 3,000 reported by Connell et a l . (13). Similar c a l c u l a t -ions can be applied to succinyl haptoglobin 1-1, a f t e r subtracting 70 succinyl groups (2 X 25 l y s y l from $ chain + 2 X 8 l y s y l group of a 1 + 4 N-terminal) to give a value of 97,795. This value agrees well with other reported values (105, 106). Using the empirical composition 2 8 + a 1 + a 2 for haptoglobin 2-1, the "monomer" molecular weight would be equal to 2 X 42,58 2 + 9,057 + 16,685 = 110,906. Correcting for 19.4% carbohydrate i n the 6 chain, the molecular weight of the polypeptide portion of Hp 2-1 i s 94,546. These values were l a t e r used to c a l c u l a t e the number of residues of amino acid i n haptoglobin 2-1 and the separated chains. The same approach, using maleic anhydride to disaggregate protein subunits to determine eventually the accurate molecular weight of these maleylated subunits, has been reported recently 64 by Butler (107) on yeast alcohol dehydrogenase and Bruton and Hartley (108) on methionyl transfer RNA synthetase from E. c o l i . The Amino Acid Composition and Carbohydrate Composition of Haptoglobin 2-1 and Its Separated Chains Amino acid analyse were performed on the i n t a c t haptoglobin X 2-1 and on the separated 8/ a 2 and a 1 chains. These are shown i n Table I I I . Values for va l i n e , leucine, isoleucine and tyrosine used i n the c a l c u l a t i o n were taken at 73 hr, and those for threonine and serine were obtained by extrapolation to zero time. Cysteic acid determinations were made on performic acid oxidized samples and corrected for 6% loss during acid hydrolysis (99). The number of residues of each amino acid i n a 1 , a 2 , 6 chains and Hp 2-1 were calculated using molecular weights of 9,057, 16,685, 34,321 and 94,546 res p e c t i v e l y . The r e s u l t s of these c a l c u l a t i o n s i s shown i n Table IV. Also included i n Table IV i s the composition of haptoglobin 2-1 calculated on the basis of the empirical composition 2 8 + a 1 + a 2 . I t i s obvious that there i s good agreement between t h i s calculated value and the observed amino acid composition of haptoglobin 2-1 (l a s t column). A major discrepancy i s the 1/2 cystine analysis, 18 residues were recovered as carboxymethylcysteine (CMC) from i s o l a t e d carboxymethylamido chains, 5 residues i n each 8 chain, 5 i n a 2 and 3 i n a 1 chain. The values i n a 2 and a 1 chains are somewhat low. I t i s known that the CMC from acid hydrolysis i s frequently low and the loss appears to be approximately 20% i n the present case, so that the true value for a 2 chain i s 6.7 and a 1 chain 4 CMC residues. Sequence analysis of these two chains by Black 65 and D i x o n ( r e c e n t l y c o r r e c t e d by Malchy and Dixon) r e v e a l s t h e p r e s e n c e o f 7 CMC i n t h e a 2 c h a i n and 4 CMC i n t h e a 1 c h a i n . I n t h e c a s e of t h e 6 c h a i n , 5 d i s t i n c t c y s t e i c a c i d p e p t i d e s were i s o l a t e d by Kauffman and D i x o n ( 3 1 ) . When t h e s e c o r r e c t i o n s were made f o r a 2 and o ^ c h a i n , t h e sum o f 1/2 c y s t i n e from t h e i s o l a t e d c h a i n s was 21 r e s i d u e s . S i n c e 21 r e s i d u e s o f 1/2 c y s t i n e was^u^/-,/ r e c o v e r e d as c y s t e i c a c i d p e r mole o f p e r f o r m i c - a c i d - o x i d i z e d h a p t o g l o b i n 2-1, t h e d i s c r e p a n c y was r e s o l v e d . The 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 h a p t o g l o b i n and 3 c h a i n i s shown i n T a b l e I I I . These v a l u e s agree w i t h t h o s e p u b l i s h e d by Gerbeck e t a l . ( 6 4 ) . TABLE III Amino Acid Analyses of Whole Haptoglobin 2-1 and the Isolated Chains Hp 2-1 8 chain a 2 chain a 1 chain 16h 40h 73h 16h 40h 73h 16h 40h 73h 16h 40h 73h Lys 0. 293 0. 298 0. 298 0. 310 0. 323 0. 309 0. 369 0. 371 0. 363 0. 492 0. 494 0. 522 His 0.107 0. 118 0. 110 0. 129 0. 137 0. 132 0. 102 0. 098 0. 089 0. 122 0. 116 0. 124 Arg 0. 055 0. 071 0. 067 0. 066 0. 068 0. 075 0. 084 0. 087 0. 095 0. 116 0. 124 0. 116 Cysf 0. 079 - - 0. 058 - 0. 061 0. 113 0. 123 0. 121 0. 183 0. 192 0. 193 Asp 0-395 0. 385 0. 385 0. 412 0. 399 0. 392 0. 518 0. 518 0. 515 0. 895 0. 146 0. 853 Thr 0.185 0. 196 0. 162 0. 256 0. 253 0. 246 0. 114 0. 114 0. 112 0. 178 0. 176 0. 164 Ser 0.151 0. 147 0. 143 0. 220 0. 210 0. 196 0. 067 0. 068 0. 065 0. 116 0. 114 0. 101 Glu 0. 363 0. 362 0. 366 0. 416 0. 428 0. 420 0. 380 0. 377 0. 377 0. 582 0. 588 0. 569 Pro 0.178 0. 179 0. 178 0. 167 0. 173 0. 192 0. 263 0. 225 0. 241 0. 441 0. 439 0. 435 Gly 0.251 0. 248 0. 248 0. 280 0. 275 0. 285 0. 289 0. 286 0. 289 0. 447 0. 443 0 426 Ala 0. 236 0. 230 0. 243 0. 292 0. 283 0. 292 0. 189 0. 189 0. 195 0. 310 0. 308 0. 306 Val 0. 238 0. 285 0. 293 0. 286 0. 348 0. 362 0. 214 0. 254 0. 280 0. 391 0. 482 0. 483! Met 0. 039 0. 038 0. 038 0. 051 0. 052 0. 054 - - - - - -H e 0.116 0. 137 0. 144 0. 139 0. 173 0. 187 0. 105 0. 112 0. 116 0. 174 0. 184 0. 180 Leu 0. 230 0. 242 0. 244 0. 309 0. 328 0. 350 0. 140 0. 146 0. 151 0. 186 0. 197 0. 191 Tyr 0.147 0. 160 0. 166 0. 155 0. 166 0. 173 0. 188 0. 214 0. 227 0. 262 0. 308 0. 303 Phe 0. 068 0. 071 0. 077 0. 127 0. 125 0. 124 — — — — — — Hexose 7. 0% 9. 8% - - — - - -Glucosamine 4. 3% 6. 0% - - - - - -S i a l i c Acid 2. 4% 3. 6% - - - - - -tDetermined as carboxymethylcysteineexcept i n the case of the Hp 2-1 analyses where separate analyses of c y s t e i c acid i n hydrolysates of performic acid oxidized protein were carried out. Carbohydrate analyses were done on separate samples hydrolyzed i n 4 N HCl at 100°C for 8 h. TABLE IV Observed and Predicted Amino Acid Composition of Haptoglobin 2-1 0 a 2 a 1 (2 x 0 + a 2 + a 1 ) Hp 2-1 Lys 25 16 8 75 76 His 10 4 2 26 28 Arg 5 4 2 16 17 Asp 31 22 14 98 99 Thr 20 5 3 48 48 Ser 18 3 2 41 39 Glu 33 16 9 91 93 Pro 15 10 7 47 46 Gly 22 12 7 63 64 Ala 23 8 5 59 60 Val 28 12 8 76 75 l/2Cys* 5 (5) 5 (7) 3 (14) 18 (21) 21+ Met 4 0 0 8 10 H e 15 5 3 38 37 Leu 27 6 3 63 62 Tyr 14 10 5 43 42 Phe 10 0 0 20 18 Trp 5 2 1 13 14 Total 301 141 82 845 849 *l/2 Cystine determined as carboxymethylcysteine with no correction f o r hy d r o l y t i c losse. Value i n parenthesis indicates number of unique cy s t e i c acid sequences recovered i n cysteic acid peptides (Kauffman, D.L., and Dixon, G.H.: 31). t l / 2 Cystine determined as c y s t e i c acid and corrected for 6% loss on acid hydrolysis (99 ) . CHAPTER I I : THE FRACTIONATION OF CYANOGEN BROMIDE PEPTIDES OF HAPTOGLOBIN 3 CHAIN Introduction From the previous studies, the molecular weight of the 3 chain was shown to be 42,500, and i t contains 301 amino acid residues. To study the primary structure of a large polypeptide chain l i k e the haptoglobin 3 chain, the simple and l o g i c a l approach i s to obtain a l i m i t e d number of large fragments which can be studied and characterized i n d i v i d u a l l y . Various methods for s p e c i f i c , non-enzymatic cleavage of peptides at r e l a t i v e l y infrequent amino acid residues have been devised for the studies of the structure of proteins and polypeptide chains. Cyanogen bromide (CNBr), the most favourable reagent, reacts only with the methyl mercapto group of methionine and not with any of the common amino acids with the exception of cysteine with which ther i s a slow reaction. Since i t s f i r s t introduction by Gross and Witkop (109), t h i s reagent has the widest a p p l i c a b i l i t y i n se-quence determination due to i t s high s e l e c t i v i t y and quantitative cleavage. The primary structure of many proteins, ranging from ribonuclease (110), collagen (111), r a b b i t immunoglobulin (112), ferredoxin (113), human immunoglobulin (114), Staphylococcus % e x t r a c e l l u l a r nuclease (115), t h y r o - c a l c i t o n i n (116) have been p a r t i a l l y elucidated with the a p p l i c a t i o n of t h i s reagent. The mechanism of the cyanogen bromide cleavage reaction, o r i g i n a l l y postulated by Gross and Witkop i s shown as follows: CHR CHR Br The cyanosulfonium intermediate i s formed by i n t e r a c t i o n with CNBr. The displacement of the v o l a t i l e and stable methyl-thiocyanate, CH^SCN under the concerted attack of the iminolactone anion leads to the unstable imino ^-lactone which spontaneously hydrolyzes to homoserine lactone and a new amino terminal residue. Haptoglobin 6 chain contains four methionine residues accord-ing to the amino acid analysis. Ideally, f i v e fragments should be obtained a f t e r cleavage by cyanogen bromide. However, as can be seen i n t h i s chapter, d i f f i c u l t i e s were encountered due to the incomplete cleavage of a l l the methionine residues i n the 8 chain. T h i s p r e s e n t c h a p t e r r e p o r t s t h e f r a c t i o n a t i o n o f CNBr c l e a v e d p e p t i d e s and t h e c h a r a c t e r i z a t i o n o f t h e s e components. E x p e r i m e n t a l 1. C l e a v a g e o f 3_ c h a i n w i t h cyanogen bromide (94) H a p t o g l o b i n 3 c h a i n (600 mg, 14 ymoles) was d i s s o l v e d i n 110 ml o f 50% h e x a f l u o r o a c e t o n e s e s q u i h y d r a t e and 500 mg o f CNBr* (100 f o l d m o l a r e x c e s s per mole of m e t h i o n i n e r e s i d u e ) were added. The r e a c t i o n m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 24 hours and an e q u a l volume o f water was t h e n added. M e t h y l t h i o c y a n a t e and e x c e s s cyanogen bromide were f i r s t removed by w ater a s p i r a t i o n , and h e x a f l u o r o a c e t o n e s e s q u i h y d r a t e was l a t e r e v a p o r a t e d under vacuum a t room t e m p e r a t u r e - and r e c o v e r e d . The h o r n y , w h i t e r e s i d u e was d i s s o l v e d i n 0.2 N a c e t i c a c i d and l y o p h i l i z e d . The y i e l d o f t h e w h i t e f l u f f y m a t e r i a l was a p p r o x i -m a t e l y 58 0 mg. 2. S e p a r a t i o n o f h a p t o g l o b i n _6_ c h a i n f r a g m e n t s A p p r o x i m a t e l y 300 mg o f t h e p r o d u c t was d i s s o l v e d i n 8 ml o f 0.2 N a c e t i c a c i d and chromatographed on a Sephadex G-100 column (2.5 X 137 cm) a t room t e m p e r a t u r e u s i n g 0.2 N a c e t i c a c i d as e l u t i n g s o l v e n t ~a-nd 6.0 ml f r a c t i o n s were c o l l e c t e d . The e l u t i o n p r o f i l e i s shown i n F i g . 19. F u r t h e r p u r i f i c a t i o n o f i n d i v i d u a l components w i l l be d i s c u s s e d under t h e p r o p e r c o n t e x t . 3. M a l e y l a t i o n o f h a p t o g l o b i n 3 c h a i n fragments (107) The p r o c e d u r e f o r t h e m a l e y l a t i o n was e s s e n t i a l l y t h e same * Eastman O r g a n i c C h e m i c a l , R o c h e s t e r , New Y ork. as i n t h e s u c c i n y l a t i o n o f t h e h a p t o g l o b i n B c h a i n . Cyanogen bromide fragments were m a l e y l a t e d w i t h 100 f o l d e x c e s s o f m a l e i c a n h y d r i d e (Matheson, Coleman & B e l l , O h i o , Code No. 2837) i n 0.1 N phosphate b u f f e r f o r 1 h r , t h e pH b e i n g m a i n t a i n e d a t 8 - 9 w i t h NaOH. The r e a c t i o n m i x t u r e was l a t e r d e s a l t e d on a Sephadex G-25 column (2 X 30 cm) u s i n g 0.1 M NH 4HC0 3 as s o l v e n t . The m a l e y l a t e d p e p t i d e s were f i n a l l y l y o p h i l i z e d . R e s u l t s Chromatography o f Cyanogen Bromide f, C l e a v a g e fragments The e l u t i o n p r o f i l e o f t h e chromatography o f cyanogen bromide c l e a v a g e f r a g m e n t s on Sephadex G-100 column i s shown i n F i g . 19. A t l e a s t f i v e components can be d e m o n s t r a t e d . These components a r e d e s i g n a t e d as'8-CNBr-6-A, S-CNBr-6-B, B-CNBr-6-C and B-CNBr-, T 6-D r e s p e c t i v e l y . / t h e l a s t peak, 8~CNBr-6-D, c o n t a i n s two com- J r ponents t h a t a r e p a r t i a l l y r e s o l v e d . I n l a t e r chromatography - f on Sephadex G-100, b e t t e r r e s o l u t i o n was o b t a i n e d ^ t h i s i s shown i n F i g . 20 i n w h i c h t h e 8-CNBr-6-C r e g i o n r e s o l v e d i n t o two components, 8"CNBr-8-C and B-CNBr-8-D and s i m i l a r l y B~CNBr-6-D i n t o two components B-CNBr-8-E and B-CNBr-8-F. As can be seen a l s o i n F i g . 20, o n l y t h e f i r s t two components B-CNBr-8-A and B-CNBr-8-B c o n t a i n hexose. T h i s was s u b s e q u e n t l y c o n f i r m e d i n t h e c a r b o h y d r a t e a n a l y s i s . The use o f h e x a f l u o r o a c e t o n e s e s q u i h y d r a t e as t h e s o l v e n t f o r the cyanogen bromide r e a c t i o n r e q u i r e s some e x p l a n a t i o n . I n F r a c t i o n Number Figure 19. Chromatography of cyanogen bromide cleavage peptide of B chain (S-CNBr-8) on a Sephadex G-100 column (2.5 x 137 cm) using 0.2 N acetic acid as solvent. -J 0.2 -0. D. 280 0. D. 490 ? e o o - — ? ~ - o - -o o p 100 120 140 Fraction Number 220 Figure 20. Chromatography of cyanogen bromide cleavage peptides (6-CN-8) on a Sephadex G-100 column 15 x 150 cm) using 0.2 N acetic acid as solvent. CO 74 the o r i g i n a l experiment described by Gross and Witkop (109), 0.1 N HC1 was used. However, i t has been found by other workers (117) that the reaction was far from complete (approximately 8 0%) at least i n the case of g-galactosidase. This prompted these workers to s e l e c t instead 7 0% formic acid, a better denaturing agent. In studies of g a s t r i n by Gregory et a l . (118), t r i f l u o r o -a c e t i c acid was used. More recently, Burkhart and Wilcox (94) reported the use of hexafluoroacetone, an excellent solvent for protein and peptide f r a c t i o n a t i o n as well as for performing cyanogen bromide reactions. In the case of haptoglobin 3 chain, early experiments using 70% formic acid did not give a s a t i s f a c t o r y f r a c t i o n a t i o n pattern, since only two, or at the most three components could be v i s u a l i z e d i n the column. Hexafluoroacetone, on the other hand, was found to be a better solvent for disaggregating the 8 chain. However, as can be seen i n the analysis of sample 2 which i s an unfraction-ated mixture of cyanogen bromide cleavage products, the y i e l d of homoserine was only 75% even when t h i s solvent was used. Estimation of the Molecular Weight of the Cyanogen Bromide Peptides The size^of the cyanogen bromide fragments were estimated using two d i f f e r e n t methods, namely, the gel f i l t r a t i o n chromato-graphy method of Andrew (119) and the SDS d i s c gel electropho-r e s i s method recently reported by Weber and Osborn (101). E a r l i e r molecular weight estimations of these peptides were done on B-CNBr-5 (same e l u t i o n pattern as B-CNBr-6, except that the two components i n D region i n 8-CNBr-6 was not resolved) on a Sephadex G-100 column. The column was standardized against ovalbumin (M.W. = 45,000), chymotrypsinogen (25,000), cytochrome c (12,400), myoglobin (17,800) and b a c i t r a c i n (1,450). Since the f r a c t i o n a t i o n range of G-100 i s within 4,000 - 150,000, peptides which have molecular weights of 4,000 or less; with come o f f / t o -gether i n the hold-up volume. The apparent molecular weight of b a c i t r a c i n , which comes o f f i n the hold-up volume was taken as 4,000 instead. As can be seen i n F i g . 21a, a s t r a i g h t l i n e i s obtained when the log. M.W. of the standard proteins was plotted against the e l u t i o n volume of the proteins. The approximate molecular weight of the fragments A, B, C and D are 81,800, 17,000, 8,180 and 4,400 respe c t i v e l y . An obvious discrepancy i s noted. Since the molecular weight of the haptoglobin $ chain i s only 42,000, the molecular weight for fragment A (81,800) was too high. This indicated that fragment A i s either aggregated or represents the unreacted 3 chain which i s known to aggregate i n 0.2 N a c e t i c acid. The nature of the aggregate i n fragment A w i l l be discussed l a t e r . More recently, the molecular weights of these peptides were X reinvestigated using the sodium dodecyl s u l f a t e d i s c gel e l e c t r o -phoresis. This method has the advantage that a l l the peptides are disaggregated so that a more precise estimation of the mole-cular weight can be obtained for i n d i v i d u a l components. Fur-thermore, the homogeneity of the peptides can be examined at the same time. F i g . 22 shows the electrophoretic patterns of these peptides together with i n s u l i n , lysozyme, a 2 chain and 3 chain of haptoglobin. In order to estimate the molecular weight of the 76 5.0 4.0 3.0 o 4.0 3.0 -CNBr-5-A Ovalbumin sChy: motrypsinogen B-CNBr-5-B Myoglobin i-CNBr-5-C B-CNBr-5-D Ba c i t r a c i n Ve i n ml l 300 400 3 chain, CNBr-8-A, B-CNBr-8-B 500 a chain, B-CNBr-8-A, B-CNBr-8-fe-CNBr-8-c' 8-CNBr-8-A3, lyo 5-CNBr-8-o i n s u l i n B-CNBr-8-E B-CNBr-8-F i r yme D 3 4 Mob i l i t y i n cm Figure. 21 a. Molecular weight estimation of CNBr fragments (B-CNBr-5) on a Sephadex G-100 column. 21 b. Molecular weight estimation of CNBr fragments (B-CNBr-8) on SDS disc gel electrophoresis (10% gel) I 10 Figure 22. SDS disc gel electrophoresis of B-CNBr-8 fragments i n 10% g e l . From l e f t to r i g h t : 1. Insulin; 2. lysozyme; 3. a 2 chain; 4. 8 chain; 5. B-CNBr-8-A; 6. B-CNBr-8-B; 7. B"CNBr-8-C; 8. B-CNBr-8-D; 9. B-CNBr-8-E; 10. B-CNBr-8-F. 78 p e p t i d e s , t h e l o g . M.W. o f a 2 c h a i n , and lysozyme were p l o t t e d a g a i n s t t h e m o b i l i t y ( i n cm, from t h e o r i g i n ) , and a s t r a i g h t l i n e was o b t a i n e d . T h i s i s shown i n F i g . 21B. The m o l e c u l a r w e i g h t o f t h e 3 c h a i n was e s t i m a t e d as 45,710. S i n c e t h e p r e c i s e m o l e c u l a r w e i g h t o f t h e 3 c h a i n was d e t e r m i n e d as 42,000, t h e v a l u e e s t i m a t e d here i s s l i g h t l y h i g h e r . However, i t i s known t h a t 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 g e n e r a l l y d e v i a t e from normal g l o b u l a r p r o t e i n s so t h a t t h e m o l e c u l a r w e i g h t e s t i m a t i o n f o r t h e s e g l y c o p r o t e i n s i s u s u a l l y h i g h e r . T h i s i s p o s s i b l y due t o t h e h y d r a t i o n p r o p e r t i e s o f t h e c a r b o h y d r a t e s i d e c h a i n s . A s i m i l a r v a l u e (45,000) was o b t a i n e d when t h e r e s u l t s shown i n F i g . 12 of C h a p t e r I was used t o c a l c u l a t e t h e m o l e c u l a r w e i g h t o f t h e 3 c h a i n . Fragment 3~CNBr-8-A r e s o l v e s i n t o f o u r components h a v i n g the m o l e c u l a r w e i g h t s o f 45,710, 15,960, 14,300 and 10,000 r e s p e c t -i v e l y . The f i r s t component has t h e same m o b i l i t y as t h e h a p t o g l o -b i n 3 c h a i n , and p r o b a b l y r e p r e s e n t s t h e u n r e a c t e d m a t e r i a l . The f r a c t i o n a t i o n o f t h e s e components w i l l be d i s c u s s e d i n l a t e r s e c t i o n s . Fragment 3~CNBr-8-B has a m o l e c u l a r w e i g h t o f 25,000. S i n c e t h e g e l f i l t r a t i o n method gave 17,000, a d i s c r e p a n c y e x i s t s . The m o l e c u l a r w e i g h t c a l c u l a t e d from t h e amino a c i d c o m p o s i t i o n i s 18,034, w h i c h a g r e e s w i t h g e l f i l t r a t i o n s t u d i e s . S i n c e a 10% c r o s s - l i n k e d a c r y l a m i d e g e l was used, i t i s p o s s i b l e t h a t t h e g e l became l e s s d i s c r i m i n a t i n g f o r l a r g e r p e p t i d e s , and hence a l a r g e r m o l e c u l a r w e i g h t was e s t i m a t e d . Fragment 3-CNBr-8-C was e s t i m a t e d as 10,000, w h i c h a g r e e s w e l l w i t h t h e r e s u l t s from g e l f i l t r a t i o n method. Fragment 3-CNBr-8-D was e s t i m a t e d as 8,710. Fragment B-CNBr-8-E and fragment B-CNBr-8-F both have <j>/> V mobility s i m i l a r to the i n s u l i n marker. Since a l l the samples were reduced i n the presence of B-mercaptoethanol for 2 hr at 37°C, i n s u l i n which consists of A and B chains were s p l i t into /. i n d i v i d u a l A and B peptides. I t i s known that peptide A which consists of 21 amino acids does not s t a i n well, so that the one band observed i n the gel corresponds to the B chain which has 30 amino acids and a molecular weight of 3,39 6. Since the mobi-l i t y of the peptides B-CNBr-8-E and B-CNBr-8-F were s l i g h t l y slower than i n s u l i n B chain, the molecular weights of these two X components were approximately 4,000. This agrees with the r e s u l t s from gel f i l t r a t i o n studies. Studies on B-CNBr-8-A Cyanogen bromide cleavage fragment B-CNBr-8-A was the largest component according to the G-100 chromatography. The rechromato-graphy of t h i s component on a G-200 column, shown i n F i g . 23, indicates the presence of only one component. The molecular weight of t h i s fragment was estimated as 81,000 on gel f i l t r a t i o n chromatography, i n d i c a t i n g that t h i s fragment was either aggregated or represented the unreacted $ chain. Upon d i s c gel e l e c t r o -phoresis and SDS d i s c gel electrophoresis, four major components can be v i s u a l i z e d (Fig. 22). Since the four components have d i f f e r e n t m o b i l i t i e s and d i f f e r i n s i z e , namely 45,710, 15,960, 14,300 and 10,000, i t appeared that B-CNBr-8-A consisted of four components aggregated together under the experimental conditions. Dansylation studies shows the presence of two N-terminal amino acids, leucine and isoleucine, the l a t t e r being the minor component.. 2.0 h 120 Fraction Number Figure 23. Chromatography of B-CNBr-8-A on a Sephadex G-200 column (2 x 87 cm) using 0.2 N ace t i c acid as s o l vent. To c h a r a c t e r i z e t h e components f u r t h e r , B-CNBr-8-A was s u c c i n y l a t e d and t h e s u c c i n y l 8 -CNBr-8-A was th e n chromatographed on a G-200 column u s i n g 0.1 M NH^HCO^. As e x p e c t e d , f o u r d i s t i n c t components were r e s o l v e d . T h i s i s shown i n F i g . 24a. S i m i l a r l y , m a l e y l a t i o n o f B-CNBr-8-A produced a s i m i l a r e l u t i o n p r o f i l e ( F i g . 24b). The m a l e y l a t e d components, namely M-B-CNBr-8-A I , M-B-CNBr-8-A I I , M-B-CNBr-8-A I I I and M-B-CNBr-8-A IV were f u r t h e r p u r i f i e d by g e l f i l t r a t i o n chromatography on Sephadex G-200 and G-100 r e s p e c t i v e l y , as can be seen i n F i g . 25 and F i g . 26. The homogeneity o f t h e s e components was d e t e r m i n e d by d i s c g e l e l e c t r o p h o r e s i s , w h i c h i s shown i n F i g . 27. Component M-8-CNBr-8-A I d i d n o t s t a i n w e l l w i t h Amido B l a c k and c o n t a i n s a l a r g e amount o f c a r b o h y d r a t e . Component M-B-CNBr-8-A I I I s t i l l c o n t a i n s two components w h i l e M-6-CNBr-8-A I I and M-g-CNBr-8-A IV were p u r e I t i s l i k e l y t h a t t h e f o u r components o b s e r v e d i n SDS d i s c g e l e l e c t r o p h o r e s i s c o r r e s p o n d t o components I I , I I I and IV t o g e t h e r . A t t e m p t s t o s e p a r a t e t h e two components o f M-B-CNBr-8-A I I I u s i n g DEAE Sephadex o r DE52 were n o t s u c c e s s f u l . The amino a c i d c o m p o s i t i o n 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 t h e s e components a r e shown i n T a b l e V and w i l l be d i s c u s s e d i n more d e t a i l under t h e amino a c i d a n a l y s i s s e c t i o n . S t u d i e s on B-CNBr-8-B Fragment B -CNBr-8-B i s t h e major c a r b o h y d r a t e - c o n t a i n i n g p e p t i d e . The hexosamine and hexose c o n t e n t i n t h e peptide___w.as-" 10% and 6% r e s p e c t i v e l y . S t u d i e s o f t h i s component were c o m p l i c a t e d by t h e p r e s e n c e o f a t t a c h e d c a r b o h y d r a t e . The e l u t i o n p r o f i l e o f t h i s component 20 40 60 80 100 120 F r a c t i o n Number Chromatography o f s u c c i n y l B-CNBr-8 -A on a Sephadex G-200 column (2 x 87 cm) w i t h 0.005 N phosphate b u f f e r pH 7.0 as s o l v e n t . Chromatography o f m a l e y l B-CNBr-8-A on a Sephadex G-200 column (2 x 172 cm) u s i n g 0.1 M NH4HCC>3 as s o l v e n t . F i g u r e 24 a. 24 b. 83 40 60 80 100 Fraction Number 120 Figure 25 a. Chromatography of M-6-CNBr-8-A I on a Sephadex G-200 column (2 x 172 cm) using 0.1 M NH"4HC03 as solvent. 2S b. Chromatography of M-6-CNBr-8-A II on a Sephadex G-200 column (2 x 172 cm) using 0.1 M NH4HCC>3 as solvent. 84 1 0 Fiqure 26 a 26 b 30 40 50 Fraction Number 60 Chromatography of M-B-CNBr-8-A III on a Sephadex G-100 column (2 x 89 cm) using 0.1 M NH^ HCO-j as solvent. Chromatography of M-B-CNBr-8-A IV on a Sephadex G-100 column (2 x 89 cm) using 0.1 M NH^HCO^ as solvent. 85 F i g u r e 27. D i s c g e l e l e c t r o p h o r e s i s o f m a l e y l a t e d components of 3~CNBr-8. 1. M-3-CNBr-8-A I; 2. M-B-CNBr-8-A II; 3. M-B-CNBr-8-A III; 4. M-B-CNBr-8-A IV. 86 from Sephadex G-100 and Sephadex G-200 chromatography ( F i g . 28a) was a s y m m e t r i c a l i n d i c a t i n g some h e t e r o g e n e i t y i n t h e component. Upon d i s c g e l e l e c t r o p h o r e s i s , a p p r o x i m a t e l y e l e v e n components can be d e t e c t e d ( F i g . 2 9 ) . On t h e o t h e r hand, d a n s y l a t i o n s t u d i e s r e v e a l e d v a l i n e as t h e s o l e N - t e r m i n a l amino a c i d , s u g g e s t i n g t h a t t h e s e components have t h e same N - t e r m i n a l amino a c i d . V a r i o u s methods were employed t o i n v e s t i g a t e t h e h e t e r o -g e n e i t y o f t h i s fragment. The f i r s t a p proach i n v o l v e d t h e m o d i f i -c a t i o n o f t h i s component by s u c c i n y l a t i o n . The r e a s o n f o r t h i s was t h a t t h i s method had been s u c c e s s f u l i n e l u c i d a t i n g t h e a g g r e g a t e d n a t u r e o f 8 c h a i n and B-CNBr-8-A. The s u c c i n y l a t e d 6-CNBr-8-B was chromatographed on a Sephadex G-200 column (2 X 87 cm). A s i n g l e peak h a v i n g an e l u t i o n p r o f i l e s i m i l a r t o t h e u n t r e a t e d p e p t i d e ( F i g . 28b) was seen. U n l i k e t h e s u c c i n y l a t i o n o f p-CNBr-8-A, no d i s t i n c t s e p a r a t i o n o f t h e components r-we-r-e—-o b t a i n e d . These r e s u l t s i n d i c a t e d t h a t t h e components were s i m i l a r i n s i z e , i f n o t i d e n t i c a l . T h i s argument was s u p p o r t e d by t h e chromatography o f B-CNBr-8-B on G-7 5 i n t h e p r e s e n c e o f 0.2% SDS, a c o n d i t i o n used t o d i s a g g r e g a t e p r o t e i n and p e p t i d e a g g r e g a t e s . As can be seen i n F i g . 30, o n l y one component was d e m o n s t r a t e d . I t i s t h e r e f o r e l i k e l y t h a t t h e h e t e r o g e n e i t y o f t h e s e components o b s e r v e d on d i s c g e l e l e c t r o p h o r e s i s may a r i s e from t h e p r e s e n c e of c a r b o h y d r a t e . There a r e a p p r o x i m a t e l y 8 r e s i d u e s o f n e g a t i v e l y c h a r g e d s i a l i c a c i d s i n t h e h a p t o g l o b i n 8 c h a i n . These s i a l i c a c i d r e s i d u e s w h i c h o c c u r a t t h e end o f t h e c a r b o -h y d r a t e s i d e c h a i n a r e a c i d - l a b i l e . Some of t h e s i a l i c a c i d r e s i d u e s may be removed d u r i n g 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 of t h e 8 c h a i n and t h e cyanogen bromide p e p t i d e s , t h u s p r o d u c i n g 87 Figure 28 a. 28 b. 60 80 Fraction Number Chromatography of B-CNBr-8-B on a Sephadex G-200 column (2.2 x 186 cm) with 0.2 N acetic acid as solvent. Chromatography of succinyl B-CNBr-8-B on a Sephadex G-200 column (2 x 87 cm) with 0.005 N phosphate buffer, pH 7.0, as solvent. 88 1 2 Figure 29. 8 M urea disc gel electrophoresis of 8-CNBr-8-B. 1. Before mild acid treatment. 2. After mild acid treatment. 89 Fraction Number Figure 30. Chromatography of B-CNBr-8-B on a Sephadex G-75 column (1 x 94 cm) using 0.05 phosphate buffer, pH 7.0/ containing 0.2% SDS as solvent. the electrophoretic v a r i a t i o n observed. In order to check t h i s idea, the fragment was subjected to mild acid hydrolysis with 0.01 N HC1 at 100°C for one hour to remove the s i a l i c acid residues The acid-treated sample was then analyzed by disc gel electropho-r e s i s . As expected, the pattern (Fig. 29) i s much simpler. There are four components migrating more slowly ( i . e . with a lower anodal mobility) a f t e r acid hydrolysis compared to at l e a s t 10 i n 3~CNBr-8-A. Furthermore, as can be seen i n F i g . 22, only one component was detected a f t e r SDS d i s c gel electrophoresis i n contrast with the r e s u l t s from urea disc g e l . I t would be expect-ed that the v a r i a t i o n i n s i a l i c acid content of the i n d i v i d u a l components w i l l have l i t t l e e f f e c t on the mobility of the peptide i n the presence of SDS. This type of heterogeneity has also been observed i n other glycoproteins. Rabbit serum t r a n s f e r r i n gives r i s e to two bands on starch gel electrophoresis (120). These bands contain one and two s i a l i c acid residues r e s p e c t i v e l y . A f t e r removal of the s i a l i c acid by neuraminidase, only one component (containing no s i a l i c acid) was obtained. S i m i l a r l y , porcine RNase produced seven bands on d i s c gel electrophoresis, and contained only two major bands a f t e r extensive neuraminidase treatment (121). The electrophoretic behaviour of these proteins i s obviously deter-mined by the v a r i a t i o n i n the content of terminal s i a l i c acid residues. I t i s l i k e l y that only one carbohydrate u n i t occurs i n t h i s fragment despite the heterogeneity observed. One i n d i r e c t piece of evidence was the i s o l a t i o n of a t r y p t i c glycopeptide (T-G II) from the haptoglobin 6 chain. This glycopeptide, when chro-matographed on a Dowex 1-X4 ion exchange column also gave r i s e to a heterogeneous pattern reminiscent of those obtained by disc gel electrophoresis of B-CNBr-8-B. As with the electrophoresis of B-CNBr-8-B, the heterogeneity of the e l u t i o n p r o f i l e of t h i s t r y p t i c glycopeptide i s probably due to the v a r i a t i o n s i n the s i a l i c acid content. Dansylation studies, high voltage e l e c t r o -phoresis (after removal of s i a l i c acid) and amino acid analysis show that t h i s peptide contains only a si n g l e , short, amino acid sequence. Direct evidence to support the above argument was the actual i s o l a t i o n of only one pure thermolysin glycopeptide from B-CNBr-8-B. These glycopeptides w i l l be discussed l a t e r i n Chapter V. However, i t i s pertinent to summarize the p a r t i a l amino acid sequence of the carbohydrate attachment region as CHO I Val-(Leu,Gly,lieu)Leu-His-Gln-Asx-Asn-Ser-Thr-Ala-Lys-Asx in which the carbohydrate uni t i s attached to asparagine linkage by an aspartiamido-glycosyl linkage. The C-terminal sequence of t h i s fragment overlaps with an arginine peptide, T-Arg 4 which was i s o l a t e d from the t r y p t i c digest of haptoglobin B chain and has the sequence; Val-Ser-Val-Asn-Glu-Arg. Another arginine peptide S-Argj_ was i s o l a t e d from a s u b t i l i s i n digest of B-CNBr-8-B and has the sequence; Glu-Arg-Val-Hsr. Since the peptide contains homoserine as the C-terminal residue, the combined sequence, Val-Ser-Val-Asn-Glu-Arg-Val-Hsr i s 92 therefore the C-terminal of B-CNBr-8-B. Studies on 3-CNBr-8-C and $-CNBr-8-D Fragments 8-CNBr-8-C and 8-CNBr-8-D were rechromatographed on G-100 columns. The homogeneity of these two peptides was examined by SDS disc gel electrophoresis as shown i n F i g . 22. Both components are r e l a t i v e l y pure. Peptide g-CNBr-8-C contains one major component having a molecular weight of 10,000 together with two slower running components. Since peptide g-CNBr-8-C has a s i m i l a r molecular weight to the f a s t e s t component i n 3-CNBr-8-A, i t was thought that 8~CNBr-8-C and M-6-CNBr-8-A IV were i d e n t i c a l . However, amino acid analys/is of these two / components were d i f f e r e n t . Peptide 8-CNBr-8-C contains homoserine while M-8-CNBr-8-A IV does not and the l a t t e r may be the C-terminal peptide of haptoglobin 8 chain. This C-terminal peptide would be the only fragment predicted to be without a C-terminal homo-se r y l lactone residue after cyanogen bromide cleavage. Dansylation studies showed that 8-CNBr-8-C and 8~CNBr-8-D have valine and leucine as the N-terminal amino acids res p e c t i v e l y . The l a t t e r fragment was obtained only i n low y i e l d and i t s amino acid composition i s shown i n Table V. Studies on 8~CNBr-8-E and 3-CNBr-8-F In e a r l i e r fractionations of cyanogen bromide cleavage peptides of 8 chain, as i n B-CNBr-6, B-CNBr-6-D contained 93 two components t h a t a r e o n l y p a r t i a l l y r e s o l v e d . D a n s y l a t i o n s t u d i e s d e m o n s t r a t e d t h e p r e s e n c e o f N - t e r m i n a l i s o l e u c i n e and p r o l i n e . To s e p a r a t e t h e s e two components, c a r b o x y m e t h y l - c e l l u l o s e chromatography was used. As can be seen i n F i g . 31, 8-CNBr-6-D was r e s o l v e d i n t o two major and two minor components u s i n g a g r a d i e n t o f 200 ml o f 0.05 M NH^Ac b u f f e r , pH 5.0 and 200 ml o f 0.5 M NH^Ac b u f f e r , pH 5.0. Components B-CNBr-6-D 3 (which c o n t a i n s i s o l e u c i n e as t h e N - t e r m i n a l ) and B-CNBr-6-D^ (which c o n t a i n s p r o l i n e as t h e N - t e r m i n a l ) were found t o be homogeneous as judged by d a n s y l a t i o n , 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 d i s c g e l e l e c t r o -p h o r e s i s . However, i n l a t e r s t u d i e s , such as i n t h e f r a c t i o n a t i o n p a t t e r n shown i n 8-CNBr-8, t h e two components were w e l l s e -p a r a t e d so t h a t a p r o c e d u r e u s i n g C M - c e l l u l o s e chromatography was found t o be u n n e c e s s a r y . Fragment 8-CNBr-8-E ( e q u a l t o B-CNBr-6-D 4) and B-CNBr-8-F ( e q u a l t o 8-CNBr-8-D 3) can be p u r i -f i e d , s i m p l y by r e c h r o m a t o g r a p h y on G-100 t h u s making t h e p r e p a r a t i o n much e a s i e r . B o t h of t h e s e components a r e s i m i l a r i n s i z e as e s t i m a t e d by SDS d i s c g e l e l e c t r o p h o r e s i s , h a v i n g a m o l e c u l a r w e i g h t s i m i l a r t o t h e B c h a i n o f i n s u l i n . Thus, one would not have e x p e c t e d t h e s e p a r a t i o n o f t h e s e two components on g e l f i l t r a t i o n c hromatography. However, as was shown l a t e r i n t h e a n a l y s i s , B-CNBr-8-E c o n t a i n s h a l f c y s t i n e i n s t e a d o f c a r b o x y m e t h y l c y s t e i n e (CMC), so t h a t t h i s p e p t i d e c o u l d have e x i s t e d as a dimer d u r i n g f r a c t i o n a t i o n and t h u s would have been s e p a r a t e d from B-CNBr-8-F. In t h e p a r t i c u l a r b a t c h o f 8 c h a i n used f o r CNBr c l e a v a g e , t h e a l k y l a t i o n o f t h e 3 c h a i n was not c o m p l e t e , and t h e p r e s e n c e of 94 10 20 30 40 50 60 Fraction Number Figure 31. Chromatography of 8_CNBr-6-D on carboxymethyl-cellulose column (1.0 x 26.5 cm). The gradient consisted of 200 ml of 0.05 M NH^Ac, pH 5.0 and 200 ml of 0.5 M NH.Ac, pH 5.0. h a l f c y s t i n e c o u l d be d e t e c t e d i n s i g n i f i c a n t amounts t o g e t h e r w i t h CMC a f t e r a c i d h y d r o l y s i s . P e p t i d e B-CNBr-8-F i s t h e o n l y p e p t i d e t h a t c o n t a i n s i s o -l e u c i n e as t h e N - t e r m i n a l , t h u s s u g g e s t i n g t h a t i t must r e p r e s e n t s " t h e N - t e r m i n a l p i e c e o f t h e 3 c h a i n ( F i g . 3 2 ) . P e p t i d e 3-CNBr-8-E has p r o l i n e as t h e N - t e r m i n a l ( F i g . 3 2 ) . T h i s i m p l i e s t h a t a Met-Pro bond was c l e a v e d by cyanogen bromide. U n l i k e o t h e r amino a c i d s , p r o l i n e p o s s e s s e s an i m i d o group i n s t e a d o f a-amino group. The c l e a v a g e o f Met-Pro l i n k a g e by cyanogen bromide has p r e v i o u s l y been o b s e r v e d by Ambler (122) i n cytochrome 551 from Pseudomonas f l u o r e s c e n s . Amino A c i d A n a l y s i s The amino a c i d analyses o f t h e cyanogen bromide c l e a v a g e p e p t i d e s a r e shown i n T a b l e V. These v a l u e s r e p r e s e n t an a v e r a g e o f d u p l i c a t e a n a l y s e . The v a l u e s f o r homoserine have been c o r r e c t -A ed f o r t h e p r e s e n c e of homoserine a-amide and t h e d e s t r u c t i o n o f homoserine d u r i n g a c i d h y d r o l y s i s . None of t h e o t h e r amino a c i d s were c o r r e c t e d , and t r y t o p h a n was n o t d e t e r m i n e d . The p r e s e n c e o f homoserine a-amide, w h i c h was u n e x p e c t e d , w i l l be d i s c u s s e d i n some d e t a i l i n t h e l a s t s e c t i o n . Sample 1 i s t h e c o n t r o l h a p t o g l o b i n 3 c h a i n w h i c h was not s u b j e c t e d t o cyanogen bromide r e a c t i o n . As can be seen i n t h e a n a l y s i s , t h e r e a r e f o u r m e t h i o n i n e r e s i d u e s i n t h e m o l e c u l e . No m e t h i o n i n e s u l f o n e nor s u l f o x i d e was p r e s e n t . F u r t h e r m o r e , a p p r o x i m a t e l y one r e s i d u e o f h a l f c y s t i n e was p r e s e n t , w h i c h s u g g e s t s t h a t t h e 3 c h a i n was i n c o m p l e t e l y a l k y l a t e d . 96 4 14 F i g u r e 3 2 . D a n s y l a t i o n s t u d i e s o f cyanogen bromide c l e a v a g e p e p t i d e s o f 3 c h a i n . 1. L e u c i n e ; 2, 3. |3-CNBr-6-A; 4. L e u c i n e ; 5. g-CNBr-6-B; 6. I s o l e u c i n e , v a l i n e (not r e s o l v e d ) ; 7. D i - t y r o s i n e ; 8. B-CNBr-6-C; 9. P r o l i n e ; 10. 6-CNBr-6-D; 11. D i - t y r o s i n e ; 12. I s o l e u c i n e ; 13. NH_; 14. P r o l i n e . Sample 2 i s the t o t a l cyanogen bromide reaction mixture before any f r a c t i o n a t i o n , and except for the values of methionine and homoserine, a l l the other amino acids are unaltered a f t e r * cyanogen bromide treatment, thus confirming the s p e c i f i c i t y of the reagent. However, there were only three homoserine residues detected instead of the four expected from complete reaction, so that only 7 5% reaction occurred. One methionine was recovered i n the analysis. The incomplete cleavage of the 8 chain by ^ cyanogen bromide was distressing'. As can be seen i n the e a r l i e r discussion, more than f i v e components were produced, making the complete f r a c t i o n a t i o n of each component d i f f i c u l t . I t i s there-fore not possible at the present stage to piece a l l the fragments together. Since a hundred f o l d excess of reagent over methionine residues was used, the f a i l u r e of complete reaction to occur was somewhat unexpected. Cyanogen bromide, when applied to other proteins, generally produces s a t i s f a c t o r y r e s u l t s . I t seems most l i k e l y that the aggregation of 3 chain may constitute the main b a r r i e r to complete reaction. As shown i n Chapter I, the 8 chain was s t i l l highly aggregated i n a c i d i c aqueous conditions such as 0.2 N acetic acid. Though hexafluoroacetone sesquihydrate i s a better solvent than a c e t i c acid, disaggregation may not be complete. A s i m i l a r s i t u a t i o n has also been encountered by Putnam and co-workers during the studies of immunoglobulin u chain with cyanogen bromide (personal communication to Dr. Dixon). I t has also been reported by Gregory et. a l . (118) that the normal condition for the cyanogen bromide reaction (0.1 N HCl) was not suitable for g a s t r i n since t h i s peptide hormone was not 98 s o l u b l e under t h e s e c o n d i t i o n s . T r i f l u o r o a c e t i c a c i d was used and the p e p t i d e s were i s o l a t e d i n low y i e l d . I n T a b l e V, sample 3 t o 6 a r e t h e m a l e y l a t e d components i s o l a t e d a f t e r m a l e y l a t i o n o f B-CNBr-8-A. Sample 3 (M-8-CNBr-8-A I) c o n t a i n s a l a r g e amount o f c a r b o h y d r a t e m a t e r i a l and d i d n ot s t a i n w e l l w i t h Amido B l a c k a f t e r d i s c g e l e l e c t r o p h o r e s i s . The c o n t e n t o f amino a c i d s i n t h i s component i s low compared t o o t h e r f r a g m e n t s . A s i m i l a r p i c t u r e has been o b t a i n e d d u r i n g t h e i s o l a t i o n o f t r y p t i c g l y c o p e p t i d e s from h a p t o g l o b i n 8 c h a i n . A g l y c o p e p t i d e , d e s i g n a t e d T-G I was i s o l a t e d . T h i s component c o n t a i n s a l a r g e amount o f hexose. I t i s v e r y l i k e l y t h a t t h e g l y c o p e p t i d e T-G I d e r i v e s from M-B-CNBr-6-A I . Component M-B-CNBr-6-A I I has a m o b i l i t y i n SDS a c r y l a m i d e g e l s s i m i l a r t o t h e i n t a c t h a p t o g l o b i n 8 c h a i n , and might be t h e u n r e a c t e d m a t e r i a l . On t h e o t h e r hand, s i n c e t h i s component c o n t a i n s some homoserine, i t might p o s s i b l e r e p r e s e n t a l a r g e , X p a r t i a l l y c l e a v e d component w h i c h has a m o l e c u l a r w e i g h t c l o s e t o t h a t o f 8 c h a i n . Sample 5 r e p r e s e n t s t h e a n a l y s i s of a m i x t u r e o f t h e two components of M-B-CNBr-8-A I I I t h a t i t was n o t p o s s i b l e t o r e -s o l v e . Sample 6 (M-B-CNBr-8-A IV) shows t h e a n a l y s i s o f t h e f a s t e s t m i g r a t i n g component i n 8 -CNBr-8-A, vand w h i c h c o n t a i n s 82 amino / a c i d s from th e a n a l y s i s . F u r t h e r m o r e , t h i s component i s t h e o n l y cyanogen bromide fragment t h a t d i d not have any homoserine i n t h e a n a l y s i s , and i s most l i k e l y t o be the C - t e r m i n a l o f t h e 8 c h a i n . Fragment 8-CNBr-8-B, as d i s c u s s e d e a r l i e r , i s t h e major 99 c a r b o h y d r a t e c o n t a i n i n g p e p t i d e and c o n t a i n s a p p r o x i m a t e l y 12 6 amino a c i d s i n t h e a n a l y s i s , and gave a m o l e c u l a r w e i g h t o f 14,428 f o r t h e p o l y p e p t i d e p o r t i o n . I f t h e c a r b o h y d r a t e c o n t e n t ( a p p r o x i m a t e l y 6% o f hexose and 10% o f hexosamine) was t a k e n i n t o c o n s i d e r a t i o n , a m o l e c u l a r w e i g h t of 18,000 was o b t a i n e d w h i c h i s c o n s i s t e n t w i t h t h e r e s u l t s from g e l f i l t r a t i o n s t u d i e s . Fragment g-CNBr-8-C (sample 8) and 3-CNBr-8-D (sample 9) have v e r y s i m i l a r amino a c i d c o m p o s i t i o n s . Fragment 3 -CNBr-8-C c o n t a i n e d 8 0 amino a c i d s and 3-CNBr-S-D,59 amino a c i d s . The / m o l e c u l a r w e i g h t c a l c u l a t e d on t h e b a s i s o f t h e amino a c i d com-p o s i t i o n gave t h e s e two components v a l u e s o f 8,851 and 6,600 wh i c h a r e i n r e a s o n a b l y good agreement w i t h p r e v i o u s e s t i m a t i o n s . As mentioned e a r l i e r , b o t h components p o s s e s s s i m i l a r amino a c i d c o m p o s i t i o n s , n o t a b l y t h e same r e s i d u e s o f h i s t i d i n e , c a r b o x y -m e t h y l c y s t e i n e , g l u t a m i c a c i d , p r o l i n e and i s o l e u c i n e . S i n c e 3-CNBr-8-C c o n t a i n s m e t h i o n i n e and homoserine, w h i l e 3 _CNBr-8-D c o n t a i n s o n l y homoserine, i t i s v e r y l i k e l y t h a t 3~CNBr-8-C i s a p a r t i a l c l e a v a g e p r o d u c t w h i c h would y i e l d 3 -CNBr-8-D upon complete r e a c t i o n . The two s m a l l e s t fragments 3~CNBr-8-E and 3~CNBr-8-F b o t h c o n t a i n a p p r o x i m a t e l y 35 - 37 amino a c i d s . T h i s i s c o n s i s t e n t w i t h t h e m o l e c u l a r w e i g h t e s t i m a t e d from g e l f i l t r a t i o n and SDS d i s c g e l e l e c t r o p h o r e s i s (4,000 - 4,400). L a t e r sequence a n a l y s i s o f 3 -CNBr-8-E gave t h i s p e p t i d e a p r e c i s e m o l e c u l a r w e i g h t of 4,146. Fragment 3 _CNBr-8-E r e q u i r e s f u r t h e r e x p l a n a t i o n . I t c o n t a i n s two a r g i n i n e r e s i d u e s and h a l f c y s t i n e i n t h e a n a l y s i s i n s t e a d o f c a r b o x y m e t h y l c y s t e i n e . The p r e s e n c e of h a l f c y s t i n e s u g g e s t s 100 t h a t t h e p e p t i d e i s e i t h e r l i n k e d t o i t s e l f • f o r m i n g a dimer o r t o some o t h e r p e p t i d e s . However, i n t h e SDS d i s c g e l 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 sample was p r e v i o u s l y r e d u c e d w i t h 3 - m e r c a p t o e t h a n o l , o n l y one band was o b s e r v e d , s u g g e s t i n g t h a t B-CNBr-8-E i s a dimer. T h i s i s f u r t h e r s u p p o r t e d by t h e p r e s e n c e o f p r o l i n e as t h e o n l y N - t e r m i n a l amino a c i d , and t h e i n t e g r a l amino a c i d com-p o s i t i o n w h i c h c o r r e s p o n d s t o t h e e s t i m a t e d m o l e c u l a r w e i g h t . F u r t h e r m o r e , Malchy and D i x o n (28) i s o l a t e d a cyanogen bromide p e p t i d e , P C I I I , from whole h a p t o g l o b i n 1-1 w h i c h upon r e d u c t i o n and a l k y l a t i o n , gave r i s e t o i n t a c t a c h a i n and p e p t i d e E. P e p t i d e E was t h e n i s o l a t e d i n a p u r e s t a t e and c h a r a c t e r i z e d . B o t h p e p t i d e E and 8~CNBr-8-E were i d e n t i c a l i n m o l e c u l a r w e i g h t , amino a c i d c o m p o s i t i o n , t r y p t i c f i n g e r p r i n t , and N and C - t e r m i n a l r e s i d u e s . Sequence a n a l y s i s o f 8-CNBr-8-E c o n f i r m e d t h e above argument. Malchy and D i x o n (28) have o b s e r v e d t h a t w h i l e P C I I I was s o l u b l e i n aqueous s o l u t i o n , p e p t i d e E ( B-CNBr-8-E) a g g r e g a t e d r e a d i l y upon r e d u c t i o n and a l k y l a t i o n . T h i s may p o s s i b l y e x p l a i n t h e i n c o m p l e t e a l k y l a t i o n o f t h i s component. F i g u r e 33 i l l u s t r a t e s t h e p r e s e n t i n f o r m a t i o n o b t a i n e d from t h e s t u d i e s o f t h e cyanogen bromide p e p t i d e . P e p t i d e B-CNBr-8-F w h i c h c o n t a i n s i s o l e u c i n e as t h e N - t e r m i n a l was a s s i g n e d as t h e N - t e r m i n a l r e g i o n o f t h e B c h a i n . On t h e o t h e r hand, M-8-CNBr-8-A IV, w h i c h does n o t c o n t a i n any homoserine i n t h e a n a l y s i s , o c c u p i e s t h e C - t e r m i n a l p o s i t i o n o f t h e 8 c h a i n . The o t h e r p e p t i d e s such as B-CNBr-8-B, B-CNBr-8-C, B~CNBr-8-D and B-CNBr-8-E a r e a l l d e r i v e d from t h e i n t e r n a l r e g i o n of t h e 8 c h a i n . The e x a c t p o s i t i o n c a nnot be d e t e r m i n e d a t p r e s e n t . The a c h a i n o f h a p t o g l o b i n i s j o i n e d t o t h e 8 c h a i n by anaB i n t e r c h a i n d i s u l f i d e TABLE V Amino Acid Composition of Cyanogen Bromide Cleavage Fragments Sample 1 Sample 2 Sample 3 Sample 4 B Chain B-CNBr-8 M-8-CNBr-A I M-B-CNBr-8-A II umole Lysine 0.085 25.01 (25) Hist i d i n e 0.033 9.73 (10) Arginine 0.02 5.8 ( 6 ) Carboxymethyl-cysteine 0.015 3.9 ( 4) Aspartic Acid 0.106 31.2 (31) Threonine 0.069 20.3 (20) Serine 0.055 16.2 (16) Homoserine n i l Glutamic Acid 0.120 35.1 (35) Proline 0.042 12.4 (12) Glycine 0.063 18.6 (19) Alanine 0.078 23.0 (23) Half Cystine 0.003 0.91 ( 1) Valine 0.103 30.4 (30) Methionine 0.013 3.8 ( 4 ) Isoleucine 0.052 15.3 (15) Leucine 0.095 28.01 (28) Tyrosine 0.048 14.0 (14) Phenyalanine 0*036 10.6 (11) Total , 304 Hexose 8.7% Glucosamine 3.4% Calculated M.W. from Amino Acid Composition 42,700 Observed M.W. from SDS disc Gel Electrophoresis 45,000 umole ymole 0. 085 24. 8 (25) 0.039 0.034 9. 9 ( 9) 0.018 0.02 5. 8 ( 6) 0.018 0. 014 4. 2 ( 4) 0.105 30. 6 (31) 0. 063 0. 067 19. 51 (20) 0.019 0. 055 16. 0 (16) 0. 032 0.01 3. 01 ( 3) 0.118 34. 4 (34) 0. 062 0. 045 13. 1 (13) 0. 028 0.064 18. 6 (19) 0.0175 0. 079 23. 0 (23) 0. 042 0. 004 1. 00 ( 1) . 0.106 30. 9 (31) 0.057 0. 003 0. 9 ( 1) 0. 004 0.048 13. 9 (14) 0. 024 0. 092 26. 8 (27) 0. 05 0.047 13. 68 (14) 0. 02 0.031 9. 02 (10) 0. 02 301 4.6% 3.2% ymole 1. 95 (2) 0. 047 15. 6 (16) 0. 9 (1) 0. 019 6. 3 ( 6) 0. 9 (1) 0. 014 4. 6 ( 5) 0. 005 1. 6 ( 2) 3. 1 (3) 0. 069 23. 0 (23) 1. 45 (2) 0. 048 16 (16) 1. 6 (2) 0. 035 11. 0 (11) 0. 002 0. 6 ( 1) 3. 1 (3) 0. 077 25. 6 (26) 1. 4 (1) 0. 024 9. 0 ( 9) 0. 875 (1) 0. 043 14 (14) 2. 1 (2) 0. 049 16 (16) 0. 004 1. 3 ( 1) 2. 85 (3) 0. 067 22 (22) 0. 2 0. 003 1 ( 1) 1. 2 (1) 0. 031 10 (10) 2. 5 (3) 0. 064 21 (21) 1 (1) 0. 026 8. 6 ( 9) 1 (1) 0. 024 8. 0 ( 8) 24 206 19.5% 5.2% trace 10% 31,000 45,000 TABLE V- Continued Sample 5 Sample 6 Sample 7 Sample 8 M-B- CNBr-8-A III M-S--CNBr-8-A IV B-CNBr-•8-B 8- CNBr-•8-C ymole ymole ymole ymole Lysine 0. 082 8. 2 ( 8) 0. 055 5. 5 ( 6) 0. 099 11 (11) 0. 208 7 5 (8) Hi s t i d i n e 0. 024 2. 4 ( 2) 0. 016 1. 6 ( 2) 0. 043 4. 7 ( 5) 0. 076 2 7 (3) Arginine 0. 014 1. 4 ( 1) 0. 012 1. 4 ( 1) 0. 064 2 4 (2) CMC 0. 022 2. 2 ( 2) 0. 01 1 ( 1) 0. 002 0. 27 0. 054 1 9 (2) Aspartic Acid 0. 128 12. 8 (13) 0. 10 10. 0 (10) 0. 126 14 (14) 0. 109 7 4 (7) Threonine 0. 094 9. 4 ( 9) 0. 071 7 ( 7) 0. 084 9 ( 9) 0. 110 3 9 (4) Serine 0. 077 7. 7 ( 8) 0. 065 6. 5 ( 7) 0. 051 5. 6 ( 6) 0. 124 4. 4 (5) Homoserine 0. 001 0. 2 0. 009 1. 00 ( 1) 0. 028 1 0 (1) Glutamic Acid 0. 178 17. 8 (18) 0. 13 13 (13) 0. 123 13. 6 (14) 0. 21 7 5 (8) Proline 0. 055 5. 5 ( 6) 0. 013 1. 3 ( 1) 0. 047 5. 2 ( 5) 0. 164 5. 6 (6) Glycine 0. 095 9. 5 (10) 0. 066 6. 6 ( 7) 0. 061 6. 7 ( 7) 0. 172 6 1 (6) Alanine 0. 115 11. 5 (12) 0. 100 10 (10) 0. 078 8. 6 ( 9) 0. 121 4 3 (4) Half Cystine 0. 015 1. 5 ( 2) n i l trace n i l Valine 0. 140 14. 0 (14) 0. 105 10. 5 (11) 0. 12 13. 3 (13) 0. 24 8 6 (9) Methionine 0. 01 1. 0 ( 1) 0. 008 0. 8 ( 1) 0. 001 0. 1 0. 008 0 3 (1) Isoleucine 0. 062 6. 2 ( 6) 0. 049 5. 0 ( 5) 0. 065 7. 2 ( 7) 0. 088 3 2 (3) Leucine 0. 069 6. 9 ( 7) 0. 043 4 3 ( 4) 0. 157 17. 4 (17) 0. 126 4 5 (5) Tyrosine 0. 077 7. 7 ( 8) 0. 066 6. 6 ( 7) 0. 04 4. 4 ( 4) 0. 10 3. 6 (4) Phenyalanine 0. 045 4.' ( 5) 0. 034 3. 4 ( 3) . 0. 026 2. 8 ( 3) 0. 08 2 9 (3) Total 132 82 126 81 Hexose n i l n i l 5.4% n i l Glucosamine n i l n i l 9. 67% n i l Calculated M.W. from Amino Acid Composition Observed M.W. from SDS disc Gel Electrophoresis 13,859 14,300 10,284 10,000 18,034 25,000 8,851 10,000 TABLE V- Continued Sample 9 8-CNBr-8-D Sample 10 B-CNBr-8-E Sample 11 B-CNBr-8-F ymole ymole ymole Lysine 0. 136 3. . 8 (4) 0. 063 2. , 4 (3) 0. 075 3. .6 (4) H i s t i d i n e 0. 087 2. . 5 (3) 0. 02 0. .78 (1) 0. 031 1. . 5 (2) Arginine 0. 042 1, .2 (1) 0. 04 1. ,6 (2) 0. 011 0. , 52 (1) CMC 0. 079 2. .1 (2) 0. 002 0. , 07 ' n i l Aspartic Acid 0. 17 4. .8 (5) 0. 089 3. , 5 (4) 0. 058 2. , 76 (3) Threonine 0. 114 4. , 1 (4) 0. 022 0. , 85 (1) 0. 008 0. . 38 Serine 0. 100 2. , 85 (3) 0. 043 1, . 62 (2) 0. 042 2. , 0 (2) Homoserine 0. 035 1. ,00 (1) 0. 026 1. , 00 (1) 0. 021 1. . 00 (1) Glutamic Acid 0. 296 8. ,4 (8) 0. 025 •o. , 96 (1) 0. 038 1. , 8 (2) Proline 0. 21 6. , 00 (6) 0. 046 1. , 77 (2) 0. 038 1. , 8 (2) Glycine 0. 145 4. , 2 (4) 0. 100 3. . 82 (4) 0. 11 4. . 76 (5) Alanine 0. 097 2. , 8 (3) 0. 048 1. , 85 (2) 0. 074 3. .5 (4) Half Cystine n i l 0. 017 0. . 65 (1) Valine 0. 186 5. ,3 (5) 0. 098 3. .78 (4) 0. 034 1. . 6 (2) Methionine 0. 002 0. ,07 n i l Isoleucine 0. 09 2. , 57 (3) 0. 022 0. . 85 (1) 0. 035 1. .7 (2) Leucine 0. 098 2. , 8 (3) 0. 051 1. , 96 (2) 0. 075 3. . 6 (4) Tyrosine 0. 051 1. , 5 (2) 0. 064 2, , 6 (3) 0. 022 0. , 95 (1) Phenyalanine 0. 051 1. ,5 (2) 0. 046 1. . 84 (2) 0. 047 2. , 2 (2) Total 59 36 37 Hexose n i l n i l n i l Glucosame n i l n i l n i l Calculated M.W. from Amino Acid Composition Observed M.W. from SDS Disc Gel Electrophoresis 6574 8710 4146 4000 3905 4000 a 1 chain CHO l i e u Hsr (Val 1 Hsr, Pro- -Hsr, Leu Hsr, Val Hsr) Leu- COOH B-CNBr-8-F B-CNBr-8-B B-CNBr-8-E B"CNBr-8-D B-CNBr-8-C M-B~CNBr-8-A IV Figure 33. The alignment of the cyanogen bromide cleavage peptides, o t h r o u g h t h e h a l f c y s t i n e r e s i d u e i n p e p t i d e 8-CNBr-8-E. The E q u i l i b r i u m Between Homoserine - Homoserine L a c t o n e and P r e s e n c e o f Homoserine Amide As can be seen i n t h e mechanism o u t l i n e d p r e v i o u s l y (p. 6 9 ) , when m e t h i o n y l r e s i d u e s r e a c t s ^ w i t h cyanogen bromide, a C - t e r m i n a l / h o m o s e r y l l a c t o n e i s formed. The e q u i l i b r i u m between homoserine and homoserine l a c t o n e has been s t u d i e d p r e v i o u s l y by A r m s t r o n g (123) . I t was f o u n d t h a t i n n e u t r a l and b a s i c aqueous s o l u t i o n , homoserine i t s e l f i s s t a b l e and does n o t t r a n s f o r m t o i t s l a c t o n e . However, i n a c i d i c s o l u t i o n , homoserine i s i n e q u i l i b r i u m w i t h i t s l a c t o n e , t h e c o n c e n t r a t i o n o f t h e l a t t e r i n c r e a s i n g w i t h a c i d c o n c e n t r a t i o n . On t h e o t h e r hand, i n b a s i c s o l u t i o n , t h e l a c t o n e r i n g o f homoserine l a c t o n e i s opened t o form homoserine. Upon a c i d h y d r o l y s i s o f cyanogen bromide t r e a t e d p r o t e i n s , t h e y i e l d o f r e a c t e d m e t h i o n i n e w i l l be t h e sum o f homoserine and homoserine l a c t o n e . I n o r d e r t o s i m p l i f y t h e p r o c e d u r e , t h e h y d r o l y s a t e i s u s u a l l y c o n v e r t e d e i t h e r t o l a c t o n e by h e a t i n g i n a c i d s o l u t i o n o r opened t o homoserine by t r e a t m e n t w i t h a l k a l i a t room t e m p e r a t u r e . Tang and H a r t l e y (75) t r e a t e d t h e l a c t o n e form w i t h 2 N NH^OH a t 37°C f o r 2 hours b e f o r e amino a c i d a n a l y s i s . T h i s method was adopted i n t h e p r e s e n t s t u d i e s . Though t h e homoserine l a c t o n e c o m p l e t e l y d i s a p p e a r e d a f t e r t h i s t r e a t m e n t , a m i n or component, d i s t i n c t from homoserine and homoserine l a c t o n e was d e t e c t e d on t h e B i o - R a d A-5 column f o r t h e a n a l y s i s o f b a s i c amino a c i d s . T h i s component was b a s i c , and emerged s l i g h t l y l a t e r 106 t h a n h i s t i d i n e . The c o n c e n t r a t i o n o f t h i s component i n c r e a s e s w i t h t h e ammonium h y d r o x i d e c o n c e n t r a t i o n used. The i d e n t i t y o f t h i s component was i n v e s t i g a t e d b e f o r e any c o r r e c t i o n f o r homo-s e r i n e d u r i n g a c i d h y d r o l y s i s was made. / T h i s component was i s o l a t e d i n pure form from a homoserine s t a n d a r d s o l u t i o n . The homoserine s t a n d a r d was f i r s t c o n v e r t e d t o t h e l a c t o n e by h e a t i n g i n 6 N H C l a t 110°C f o r 6 h o u r s . The d r i e d sample was t h e n t r e a t e d w i t h c o n c e n t r a t e d ammonium h y d r o x i d e . T h i s component was s e p a r a t e d f rom homoserine by pH 1.9 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 . The s e p a r a t i o n i s shown i n F i g . 34. S i n c e t h i s component appeared a f t e r t h e ammonium h y d r o x i d e t r e a t -ment o f homoserine l a c t o n e , homoserine a-amide CH o0H I 2 CH 2 NH„ C—CONH„ Z | 2. H was s u g g e s t e d f o r t h i s component. T h i s i s f u r t h e r i n d i c a t e d by t h e r e v e r s i b l e c o n v e r s i o n o f homoserine amide t o homoserine upon a c i d h y d r o l y s i s . The sum o f homoserine and homoserine amide, a f t e r c o r r e c t i o n f o r t h e d e s t r u c t i o n o f homoserine d u r i n g a c i d h y d r o l y s i s was t h e r e f o r e used f o r t h e c a l c u l a t i o n o f t h e number o f r e s i d u e s o f t o t a l homoserine i n cyanogen bromide p e p t i d e s . 107 The following equations i l l u s t r a t e the r e l a t i o n s h i p s between homoserine, homoserine lactone and homoserine a-amide, CM, CH„ <^ CH 2OH 'O + NH. CH"2OH H H Ammonolysis^ CH 2 / W 0 N H „ — C -C0NHo I H I 2 / V NH C -C 2 I H + NH, Homoserine lactone + OH Hydrolysis HjOH NR^C—C I" \ H 0 Homoserine a-amide Homoserine Homoserine Figure 34. The i s o l a t i o n of homoserine amide from the acid hydrolysate of homoserine afte r NH.OH treatment. 109 CHAPTER I I I : STUDIES ON CYANOGEN BROMIDE CLEAVAGE FRAGMENTS B-CNBr-8-E AND B-CNBr-8-F Introduction In the previous chapter, the p u r i f i c a t i o n of cyanogen bromide cleavage peptides was discussed. I t was found that the two small-est fragments, B-CNBr-8-E and 3-CNBr-8-F were homogeneous so that amino acid sequence studies could be undertaken. Peptide B-CNBr-8-F has is o l e u c i n e as the N-terminal, suggesting that i t i s the o r i g i n a l N-terminal of the 8 chain. Peptide g-CNBr-8-E was found to l i n k to i n t a c t a chain of haptoglobin molecule (28) i n d i c a t i n g that t h i s peptide i s involved i n the aB interchain d i s u l f i d e linkage of the haptoglobin molecule. Malchy and Dixon i s o l a t e d a component PCIII from the cyanogen bromide cleavage products of whole haptoglobin on urea-phosphocellulose columns. Upon re-duction and a l k y l a t i o n , fragment PCIII gives r i s e to a 1 chain and B-CNBr-8-E. Preliminary r e s u l t s indicate that cysteine 73 of a 1 chain i s involved i n the a,8 interchain linkage with B-CNBr-8-E. The structure^of these two components were therefore ^ investigated. Experimental 1. T r y p t i c digest of B-CNBr-8-E Peptide B-CNBr-8-E (8.0 mg) was suspended i n 2 ml of 0.1 M NH.HCO.,, pH 8.0, and 10 X of porcine t r y p s i n ( c r y s t a l l i n e porcine t r y p s i n Novo, Novo I n d u s t r i A/S) (1 mg i n 100 A) was added. The d i g e s t was i n c u b a t e d a t 37°C f o r 3 h o u r s , and l y o p h i l i z e d t w i c e w i t h w a t e r . The p e p t i d e m i x t u r e , a f t e r b e i n g d i s s o l v e d i n 0.2 ml o f pH 6.5 h i g h v o l t a g e b u f f e r , was a p p l i e d t o 14 cm Whatman 3. MM paper. 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 pe r f o r m e d a t pH 6.5 3 KV f o r 1 hour. C o n t r o l p e p t i d e s s t r i p s were f i r s t s t a i n e d w i t h p henanthrenequinone f o r t h e d e t e c t i o n o f a r g i n i n e p e p t i d e s . A f t e r washing w i t h e t h a n o l and 5% a c e t i c a c i d i n a c e t o n e , t h e s t r i p s were d i p p e d w i t h c a d m i u m - n i n h y d r i n r e a g e n t . I n l a t e r p u r i f i c a t i o n s t a g e s , 0.5% n i n h y d r i n and E h r l i c h r e a g e n t were used s u c c e s s i v e l y f o r t h e d e t e c t i o n o f t r y p t o p h a n p e p t i d e s . P e p t i d e s were xd f i n a l l y e l u t e d w i t h 30% a c e t i c a c i d (124) and d r i e d . ^ 2. C h y m o t r y p t i c d i g e s t o f B-CNBr-8-E P e p t i d e B-CNBr-8-E (4.5 mg) was suspended i n 1.0 ml o f 0.1 M NH^HCO-j i pH 8.0, 0.1 mg o f c h y m o t r y p s i n (3 X c r y s t a l l i n e , W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n ) was added. Soybean t r y p s i n i n h i b i t o r (3 X c r y s t a l l i z e d , W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n ) 0.02 mg, was added t o i n h i b i t any c o n t a m i n a t i n g t r y p t i c a c t i v i t y . The d i g e s t was k e p t a t 37°C f o r 1 1/2 hours and l y o p h i l i z e d . P e p t i d e s were p u r i f i 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 a t pH 6.5 and pH 1.9 s u c c e s s i v e l y . R e s u l t s S t u d i e s on B-CNBr-8-F P e p t i d e B~CNBr-8-F c o n t a i n s a p p r o x i m a t e l y 37 amino a c i d s and has i s o l e u c i n e as i t s N - t e r m i n a l . S i n c e t h i s i s t h e o n l y CNBr fragment to possess an N-terminal isoleucine, i t i s the N-terminal fragment of the haptoglobin B chain. The Dansyl-Edman procedure and leucine aminopeptidase d i -gestion were used to study the amino acid sequence i n the N-terminal region of t h i s peptide. The Edman procedure was only p a r t i a l l y successful with t h i s peptide^ leucine and glycine were / detected a f t e r the f i r s t and second Edman cycles, but further degradation did not release any more amino acids. This suggests that the fourth residue a f t e r Ileu-Leu-Gly may be a glutamine which, when i t occurs at the N-terminal, i s found to c y c l i z e r e a d i l y to pyrrolidone carboxylic acid which i s then r e s i s t a n t to further coupling with phenylisothiocyanate (12 5). From the r e s u l t s of the leucine aminopeptidase digestion, shown i n F i g . 35, i t was possible to delineate the sequence at the N-terminal, as Ileu-Leu-Gly-Gln-Ala-Lys-Glu-Val. The f i r s t three residues are, therefore, consistent with the r e s u l t s from Dansyl-Edman degradation and the cause of the i n a b i l i t y to detect the fourth residue i s shown to be the presence of glutamine at t h i s p o s i t i o n . To investigate further the i d e n t i t y of t h i s peptide, haptoglo bin 2-1 was digested with leucine aminopeptidase. Since both a 1 and a 2 have Val-Asn-Asp at the N-terminal region, only v a l i n e and asparagine would come from the a chains. The f i r s t leucine i s at p o s i t i o n 43 i n a chains so that the early release of leucine from haptoglobin would suggest that the N-terminal se-quence of B chain i s Val-Leu-. Valine, i s o l e u c i n e , and leucine were released i n approximately 5% y i e l d . The early release of leucine, therefore, indicated that i t was located near the N-l . o h 8 10 Time i n Hours 12 14 Figure 35. Leucine aminopeptidase digest on 6-CNBr-8-F, t e r m i n u s o f t h e 8 c h a i n , so t h a t g-CNBr-8-E v e r y l i k e l y c o m p r i s e s t h e N - t e r m i n a l o f t h e 6 c h a i n . The sequence a t t h e C - t e r m i n a l o f B-CNBr-8-F was i n v e s t i g a t e d u s i n g c a r b o x y p e p t i d a s e A. The r e s u l t s ^ shown i n T a b l e V I . t TABLE VI C a r b o x y p e p t i d a s e A D i g e s t o f 8-CNBr-8-F Time ( i n Hours) Amino A c i d 8 10 ymole ymole S e r i n e 0. 010 (0. 41) 0. 013 (0. 54) A l a n i n e 0. 016 (0. 66) 0.018 (0. 75) Homoserine 0.024 (1. 00) 0. 025 (1. 04) Asn The r e s u l t s s u g g e s t Ser - A l a - H s r t o be t h e C - t e r m i n a l amino G i n a c i d sequence o f B -CNBr-8-F. Asn The n a t u r e o f t h e t h i r d r e s i d u e Ser cannot be e s t a b l i s h e d G i n s i n c e a l l o f them emerge a t t h e same p o s i t i o n on t h e a n a l y t i c a l system used on t h e Beckman 120C. An a t t e m p t was made t o deamidate t h e d i g e s t u s i n g 2 N HCl a t 100°C f o r 3 h o u r s . However, because o f t h e s m a l l s i z e o f t h e p e p t i d e , some o f t h e p e p t i d e was a b s o r b -ed t o g e t h e r w i t h t h e amino a c i d s on t h e Dowex 50-X8 r e s i n , making t h e i n t e r p r e t a t i o n d i f f i c u l t . S t u d i e s on 6-CNBr-8-E The t r y p t i c p e p t i d e s o f B-CNBr-8-E 1 1 4 The amino acid compositions of the t r y p t i c peptides from r B-CNBr-8-E are shown i n Table VIlQ -t:he N-terminal amino acid of each peptide was also included. The porcine t r y p s i n used i n t h i s experiment had s i g n i f i c a n t chymotryptic a c t i v i t y , as can be demonstrated by the actual i s o -l a t i o n of chymotryptic peptides T^, T,., T^ and T g. These pep-t i d e s , however, assisted the e l u c i d a t i o n of the primary structure. Peptide T^ (Pro-Ileu-Cys-Pro-Leu-Ser-Lys) had p r o l i n e as the N-terminal and contained half cystine i n the analysis. Kauffman and Dixon (31) had previously i s o l a t e d and characterized a c y s t e i c acid peptide from the thermolysin digest of B chain. The amino acid sequence of t h i s c y s t e i c acid peptide was shown to be Ileu-Cys-Pro-Leu-Ser-Lys-Asp. This suggests that peptide T^ i s Pro-Ileu-Cys-Pro-Leu-Ser-Lys. Dansyl isoleucine, dansyl h a l f cystine and dansyl p r o l i n e were the N-terminal amino acids a f t e r f i r s t , second and t h i r d Edman degradations respectively on peptide T^. A subtractive amino acid analysis a f t e r the t h i r d Edman degradation cycle i s shown i n Table VIII. The structure of peptide T^ i s therefore elucidated. Peptide (Asp-Tyr) was a minor component. The y i e l d of tyrosine i n the peptide was low. However, dansyl tyrosine was revealed a f t e r the f i r s t Edman degradation cycle. Studies of the mobility of the peptide at pH 6.5 confirmed the above assign-ment. Peptide T^ (Asp-Tyr-Ala-Glu-Val-Gly-Arg) was i s o l a t e d and characterized from the t r y p t i c digest of B chain during the studies TABLE VII Amino Acid Composition of Tryptic Peptides of B-CNBr-8-E Lysine H i s t i d i n e Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Half Cystine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Homoserine Total Color Reaction* Mo b i l i t y 6.5** Mobility 1.9*** N-terminal amino acid T 1 T 2 T3 T4 ymole ymole ymole ymole 0.053 (0. 82) 1 0. 01 (0. 15) 0.09 (0. 93) 1 0.046 (0. 92) 1 0.04 (1. 00) 1 0.103 (1. 07) 1 0.067 (1. 0) 1 0.008 (0. 2) 0.007 (0. 2) 0. 086 (0. 89) 1 0. 054 (1. 08) 1 0.137 (2. 1) 2 0. 036 (0. 6) 1 0. 023 (0. 3) 0. 01 (0. 25) 0.109 (1. 13) 1 0. 054 (1. 08) 1 0.098 (1. 02) 1 0.05 (1. 00) 1 0.096 (1. 00) 1 0.045 (0. 92) 1 0.052 (0. 81) 1 0.064 (1. 00) 1 0. 01 (0. 25) 1 0.086 (0. 89) 1 7 2 7 5 P.Q. P.Q. 0.43 -0.62 -0.30 0. 00 Proline Aspartic Acid 0. 87 Aspartic Acid 1.12 Alanine *P.Q. = Phenathrone quinone, E = E h r l i c h **Mobility r e l a t i v e to as p a r t i c acid, negative sign means same d i r e c t i o n as aspartic acid. ***Mobility r e l a t i v e to serine. TABLE VII- Continued T T T T 5 6 7 8 ymole ymole ymole ymole Lysine H i s t i d i n e Arginine 0.08 (0.8) 1 0.02 (0.8) 1 Aspartic Acid Threonine Serine 0.1 (1.00) 1 0.027 (0.81) 1 Glutamic Acid Proline Half Cystine Glycine 0.025 (1.00) 1 0.29 (2.9) 3 0.039 (1.2) 1 0.025 (1.00) 1 Alanine Valine 0.025 (1.00) 1 0.16 (1.6) 2 0.033 (1.00) 1 Methionine Isoleucine Leucine Tyrosine 0.21 (0.8) 1 0.06 (0.6) 1 0.011 (0.33) Phenylalanine Homoserine Total 3 + + 9 E + 4 P O + 2 Color Reaction* P.Q. , E v ' Mobility 6.5** 0.00 0.30 0.00 0.79 Mobility 1.9*** 0.72 0.51 1.93 N-terminal Valine Valine Valine Glycine amino acid *P.Q. = Phenathrone quinone, E = E h r l i c h **Mobility r e l a t i v e to as p a r t i c acid, negative sign means same d i r e c t i o n as aspartic acid. ***Mobility r e l a t i v e to serine. TABLE VII - Continued T, 12 13 14 Lysine H i s t i d i n e Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Half Cystine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Homoserine Total Color Raction* Mobi l i t y 6.5** Mobili t y 1.9*** N-terminal amino acid ymole 0.076 (0.86) 0.16 (1.81) 0.088 (1.00) 1 0.07 (0.8) 0.43 Aspartic Acid ymole 0. 08 0. 089 0. 084 0. 089 ymole ymole (0.9) (1.00) (0.95) (1.00) 0.30 Phenylalanine 1 1 1 1 0.089 (1.00) 1 0.079 (0.89) 1 0.007 (0.3) 0.005 (0.2) 0.005 (0.2) 0.008 (0.3) 0.006 (0.26) 0.023 (1.00) 1 0.023- (1.00) 1 0.02 (0.86) 0.018 (0.8) 0.00 1.1 Tyrosine 1 1 3 0.024 (1.04) 0.026 (1.13) 0.55 Tyrosine 1 1 3 *P.Q. = Phenathrone quinone, E = E h r l i c h **Mobility r e l a t i v e to aspartic acid, negative sign means same d i r e c t i o n as aspartic a c i d . ***Mobility r e l a t i v e to serine. TABLE VIII Amino Acid Analysis of Major Tryptic Peptides of $-CNBr-8-E Aft e r Edman Degradation Procedure x l x3 T6 T 9 T12 (3rd Cycle) (2nd Cycle) (2nd Cycle) (2nd Cycle) (2nd Cycle) Tl2 (4th Cycle) Lysine H i s t i d i n e Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Half Cystine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Homoserine 1st E d m a n 2nd Edman 3rd Edman 4th Edman 0.004 (1) 0.002 0.006 0.005 (1) 0.0071 (1) Dansyl l i e u Dansyl Half Cys Dansyl Pro 0.03 (1) 0.003 0. 03 0.03 (1) 0.027 (1) 0.023 (1) 0.0024 Dansyl Tyr Dansyl Ala Dansyl Glu 0.01 (1) 0.01 (1) 0.02 (2) 0.008 (1) 0.007 (1) Not Clear Not Clear 0. 007 0.011 (1) 0.007 (1) Dansyl Ala Dansyl Asp Dansyl Val Dansyl Phe 0.011 (1) 0.014 (1) 0.019 (1) 0.016 (1) Dansyl Thr Dansyl Asp Dansyl Leu 0.003 (1) 0.002 0.057 (1) 119 o f a r g i n i n e p e p t i d e s . The a c t u a l sequence d e t e r m i n a t i o n o f t h i s p e p t i d e w i l l be d i s c u s s e d under Chapter V I . I n t h e p r e s e n t s t u d i e s , t h r e e Edman d e g r a d a t i o n s were p e r f o r m e d , and d a n s y l t y r o s i n e , d a n s y l a l a n i n e and d a n s y l g l u t a m i c a c i d were found t o be t h e N - t e r m i n a l amino a c i d i r e s p e c t i v e l y . A s u b t r a c t i v e amino c a c i d a n a l y s i s a f t e r second c y c l e i s a l s o shown i n T a b l e V I I I . P e p t i d e T^ ( A l a - G l u - V a l - G l y - A r g ) had a amino a c i d c o m p o s i t i o n i d e n t i c a l t o p e p t i d e T^ a f t e r a s p a r t i c a c i d and t y r o s i n e were removed. S i n c e a l a n i n e was found t o be t h e N - t e r m i n a l o f t h i s p e p t i d e , t h e i d e n t i t y o f t h i s p e p t i d e was e s t a b l i s h e d as a p a r t -i a l c l e a v a g e p e p t i d e o f T^ by t h e c h y m o t r y p t i c a c t i v i t y a s s o c i a t e d w i t h t h e enzyme. The r e l e a s e o f g l u t a m i c a c i d by l e u c i n e amino-p e p t i d a s e e s t a b l i s h e d t h a t the g l u t a m i c a c i d was i n t h e f r e e a c i d i n s t e a d o f t h e amide form. T h i s was c o n f i r m e d by t h e mobi-l i t y o f t h e p e p t i d e a t pH 6.5. P e p t i d e T,. ( V a l - G l y - T y r ) had v a l i n e as N - t e r m i n a l . C a r -b o x y p e p t i d a s e A d i g e s t i o n o f t h e p e p t i d e r e l e a s e d o n l y t y r o s i n e a f t e r 30 m i n u t e s i n c u b a t i o n . No s i g n i f i c a n t r e l e a s e o f g l y c i n e and v a l i n e were o b s e r v e d under t h e s e c o n d i t i o n s . I t i s known t h a t g l y c i n e i s o n l y s l o w l y r e l e a s e d by c a r b o x y p e p t i d a s e A (92) and d i p e p t i d e s a r e v e r y r e s i s t a n t t o the enzyme. P e p t i d e T g ( V a l - G l y - ( V a l , T y r , S e r , T r y , G l y , A r g ) ) was p a r t i a l -l y c h a r a c t e r i z e d . A s u b t r a c t i v e amino a c i d a n a l y s i s a f t e r two Edman d e g r a d a t i o n c y c l e s i s shown i n T a b l e V I I I . P e p t i d e T 7 ( V a l - ( T r y , G l y , S e r ) ) had v a l i n e as i t s t e r m i n a l . P e p t i d e Tg (Gly-Arg) had g l y c i n e as i t s N - t e r m i n a l . P e p t i d e Tg (Asn-Ala-Asp-Phe-Lys) was n e u t r a l a t pH 6.5, t h e r e f o r e one o f t h e a s p a r t i c a c i d r e s i d u e was i n i t s amide form. / 120 The sequence o f t h i s p e p t i d e was e s t a b l i s h e d by t h r e e Edman d e g r a d a t i o n c y c l e s and a l s o by l e u c i n e a m i n o p e p t i d a s e d i g e s t i o n . The r e s u l t o f l e u c i n e a m i n o p e p t i d a s e d i g e s t i o n i s shown i n T a b l e IX. P e p t i d e T ^ (Lys) and T ^ (Lys-Phe) b o t h g i v e d i - d a n s y l l y s i n e as t h e N - t e r m i n a l . P e p t i d e T^g had t h e same m o b i l i t y as l y s i n e , s u g g e s t i n g t h a t i t was a f r e e amino a c i d . P e p t i d e T ^ had t h e m o b i l i t y o f l y s y l - p h e n y l a l a n i n e . The p r e s e n c e o f l y s y l -p h e n y l a l a n i n e was l a t e r c o n f i r m e d by t h e s t u d i e s o f t h e chymo-t r y p t i c p e p t i d e s . P e p t i d e T ^ (Phe-Thr-Asp-His-Leu-Lys) was e s t a b l i s h e d u s i n g t h e d a n s y l Edman d e g r a d a t i o n p r o c e d u r e and l e u c i n e a m i n o p e p t i d a s e . The r e s u l t s a r e shown i n T a b l e V I I I and T a b l e IX. However, t h e p e p t i d e d e s e r v e s some comments due t o t h e p r e s e n c e o f h i s t i d i n e . D a n s y l h i s t i d i n e was d e s t r o y e d d u r i n g a c i d h y d r o l y s i s (85) wh i c h would 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 any d a n s y l amino a c i d s a f t e r t h e t h i r d Edman c y c l e . S i g n i f i c a n t amounts o f h i s t i d i n e were p r e s e n t i n t h e amino a c i d a n a l y s i s a f t e r t h e f o u r t h c y c l e , p r o b a b l y b e c a u s e ^ P T H - h i s t i d i n e r e m a i n s p a r t i a l l y i n t h e aqueous phase d u r i n g e x t r a c t i o n , and was c o n v e r t e d back t o h i s t i d i n e d u r i n g a c i d h y d r o l y s i s (126). P e p t i d e T ^ a n& T ^ ( T y r - V a l - H s r ) b o t h c o n t a i n e d homoserine and showed t h e same amino a c i d c o m p o s i t i o n s u s i n g t h e homoserine system f o r a n a l y s i s . However, t h e s e two p e p t i d e s d i f f e r e d i n t h e m o b i l i t y d u r i n g pH 6.5 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 , w h i l e T ^ was n e u t r a l under t h e s e c o n d i t i o n s : ? T ^ was i n t h e homoserine / l a c t o n e form and m i g r a t e d l i k e a b a s i c p e p t i d e . D a n s y l a t i o n r e -v e a l e d t y r o s i n e as t h e N - t e r m i n a l , and v a l i n e was t h e n e x t r e s i d u e TABLE I X L e u c i n e A m i n o p e p t i d a s e D i g e s t s o f t h e T r y p t i c P e p t i d e s o f B - C N B r - 8 - E Time ( i n H o u r s ) A m i n o A c i d -> 0 i -> y m o l e y m o l e y m o l e y m o l e y m o l e P e p t i d e T4 G l u t a m i c A c i d .026 (1.0) G l y c i n e .026 (1.0) A l a n i n e .026 (1.0) V a l i n e .022 (.84) A r g i n i n e .018 (.69) P e p t i d e T9 A s p a r t i c A c i d 0. 001 (0. 25) .004 (0.1) A s p a r a g i n e 0. 015 (0. 37) . 02 (0.5) ( s e r i n e p o s i t i o n o n a n a l y z e r ) A l a n i n e 0. 006 (0. 15) . 016 (0.4) P e p t i d e T h r e o n i n e .001 (0. 05) .007 (.35) .013 (.65) P h e n y l a l a n i n e .21 (1. 05) .02 (1.0) . 019 (.95) 122 a f t e r one Edman c y c l e . C a r b o x y p e p t i d a s e A s t u d i e s c o n f i r m e d t h e above a s s i g n m e n t . F i g . 36 i l l u s t r a t e s t h e o v e r a l l p i c t u r e o f t h e p r i m a r y s t r u c t u r e o f B-CNBr-8-E. The sum o f n o n o v e r l a p p i n g p e p t i d e s added (T^+T^+Tg+Tg+T^"4" T^^) i s c o n s i s t e n t w i t h t h e o v e r a l l amino a c i d c o m p o s i t i o n o f t h e u n d i g e s t e d p e p t i d e . T h i s c o m p a r i s o n i s shown i n T a b l e X. E x c e p t f o r t h e t r y p t o p h a n r e s i d u e w h i c h was n o t d e t e r m i n e d i n fragment B-CNBr-8-E, a l l t h e o t h e r amino a c i d s i n t h i s f ragment have been ac c o u n t e d f o r . S i n c e t h e C - t e r m i n a l d i p e p t i d e v a l y l - h o m o s e r i n e was n o t a c t u a l l y i s o l a t e d from t h e c h y m o t r y p t i c d i g e s t , t h e sum of amino a c i d s from c h y m o t r y p t i c d i g e s t was n o t t a b u l a t e d . How-e v e r , i t gave t h e same r e s u l t i f t h i s p e p t i d e v a l y l - h o m o s e r i n e was i n c l u d e d i n t h e c a l c u l a t i o n . The c h y m o t r y p t i c p e p t i d e s o f B-CNBr-8-E The c h y m o t r y p t i c d i g e s t gave a much s i m p l e r p i c t u r e t h a n d i d t h e t r y p t i c d i g e s t . The amino a c i d c o m p o s i t i o n s o f t h e c h y m o t r y p t i c p e p t i d e s a r e shown i n T a b l e X I . I t can be seen t h a t t h e y i e l d o f t y r o s i n e was low i n t h e a n a l y s i s . T h i s phenomenon has a l s o been e n c o u n t e r e d by o t h e r w o r k e r s . I t may p o s s i b l y be due t o t h e s e l e c t i v e d e s t r u c t i o n o f t y r o s i n e r e s i d u e s d u r i n g a c i d h y d r o l y s i s when t h e amino a c i d o c c u p i e s t h e C - t e r m i n a l o f p e p t i d e s . However, t h e use o f c a r b o x y p e p t i d a s e A d e m o n s t r a t e d u n e q u i v o c a l l y t h e p r e s e n c e o f t y r o s i n e i n t h e s e p e p t i d e s and t h e d i s c r e p a n c y was r e s o l v e d . P e p t i d e C^ ( P r o - I l e u - C y s - P r o - L e u - S e r - L y s - A s p - T y r ) c o n t a i n e d p r o l i n e as t h e N - t e r m i n a l ; i t i s , t h e r e f o r e t h e o r i g i n a l N - t e r m i n a l Pro-Ileu-Cys-Pro-Leu-Ser-Lys-Asp-Tyr-Ala-Glu-Val-Gly-Arg-Val-Gly-Tyr--¥ 4 r «--> 4-Val-Ser-Gly-Try-Gly-Arg-Asn-Ala-Asp-Phe-Lys-Phe-Thr-Asp-His-Leu-Lys-Tyr-Val-Hsr -» * > «-12 13 & 14 11 < »• T 10 Figure 36. Proposed amino acid sequence of g-CNBr-8-E. to Co TABLE X A Comparison Between the Amino Aci d Composition of 3~CNBr-8-E and the Sum of I t s T r y p t i c Peptides Amino Acid B-CNBr-8-E Sum of Tryptic Peptides Lysine 2.5 3 His t i d i n e 0.8 1 Arginine 1.6 2 Aspartic A c i d 3.5 4 Threonine 0. 85 1 Serine 1.62 2 Glutamic A c i d 0. 96 1 Proline 1.8 2 Half Cystine 0.6 1 Glycine 3.8 4 Alanine 1.8 2 Valine 3.8 4 Methionine Isoleucine 0.85 1 Leucine 1. 96 2 Tyrosine 2.6 3 Phenylalanine 1.8 2 Homoserine 1.00 1 Tryptophan not calculated 1 TABLE XI Amino Acid Composition of Chymotryptic Peptides of 8-CNBr-8-E C l C2 C3 C 4 C5 Lysine 0.15 (0.7) 1 0. 004 0.14 (1. 4) 2 His t i d i n e 0.09 (0. 9) 1 Arginine 0. 02 0. 046 (0.86) 1 0. 088 (0. 88) 1 Aspartic Acid 0.28 (1.2) 1 0. 01 0.21 (2. 1) 2 0.12 (1. 2) 1 Threonine 0. 007 0.10 (1. 0) 1 Serine 0. 23 (1.2) 1 0. 009 0.036 (1.00) 1 0. 01 0. 008 (0. 1) Glutamic Acid 0. 04 0. 053 (1.00) 1 0. 008 Proline 0. 45 (2.00) 2 0. 02 Half Cystine 0.11 (0.5) 1 Glycine 0.07 0.10 (1.88) 2 0.042 (1.12) 1 0.063 (0. 63) 1 0. 01 Alanine 0. 03 0. 043 (0.81) 1 0.13 (1. 3) 1 Valine 0. 03 0.095 (1.8) 2 0.032 (0.88) 1 0.003 Methionine Isoleucine 0.16 (0.71) 1 0. 0065 Leucine 0.18 (0.8) 1 0. 006 0.009 0.10 (1. 0) 1 Tyrosine 0. 03 1 0. 005 (0.1) 1 Phenylalanine 0. 01 0.099 (1. 00) 1 0.10 (1. 0) 1 Homoserine Total 9 1. 8 A 4 4- 6 8 Color Reaction P.Q. E + P.Q. Mob i l i t y 6.5 0. 35 0. 35 0.23 0.55 0.75 Mobility 1.9 0.92 0. 82 0.71 1.05 1.7 N-terminal Proline Alanine Valine Glycine Lysine amino acid 126 o f 3-CNBr-8-E. S i n c e t h e sequence o f t h e t r y p t i c p e p t i d e P r o - I l e u - C y s - P r o - L e u - S e r - L y s was d e t e r m i n e d , and f u r t h e r m o r e , t h e sequence o f t h e c y s t e i c a c i d p e p t i d e i s o l a t e d by Kauffman and Di x o n i s I l e u - C y s - P r o - L e u - S e r - L y s - A s p , The above sequence i s sug g e s t e d f o r C^. The amino a c i d c o m p o s i t i o n showed s l i g h t con-t a m i n a t i o n by p e p t i d e and was low i n t y r o s i n e . However, t h e use o f c a r b o x y p e p t i d a s e r e l e a s e d t y r o s i n e q u a n t i t a t i v e l y as t h e o n l y amino a c i d , t h u s c o n f i r m i n g t h e p r e s e n c e o f t y r o s i n e as the C - t e r m i n a l o f t h e p e p t i d e . The r e s u l t o f t h e c a r b o x y p e p t i d a s e A d i g e s t i s shown i n T a b l e X I I . P e p t i d e C 2 ( A l a - G l u - V a l - G l y - A r g - V a l - G l y - T y r ) was a l s o i s o -l a t e d from t h e c h y m o t r y p t i c d i g e s t o f 6 c h a i n d u r i n g t h e s t u d i e s o f a r g i n i t t e p e p t i d e s . The s t u d i e s o f t h i s p e p t i d e w i l l be d i s -c u s s e d i n more d e t a i l under C h a p t e r V I . P e p t i d e C 2 was c o n t a -m i n a t e d by p e p t i d e C.^ . T h i s was because b o t h p e p t i d e s C 1 and had s i m i l a r m o b i l i t i e s f ' a l i 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 . However, i n t h e s t u d i e s o f a r g i n i n e p e p t i d e , p e p t i d e C 2 (C-Arg^) was f r e e o f o t h e r c o n t a m i n a n t s (p. 188) . P e p t i d e C^ ( V a l - S e r - G l y - T r y ) c o n t a i n e d t r y p t o p h a n and had t h e same amino a c i d c o m p o s i t i o n as . Edman d e g r a d a t i o n s u g g e s t e d t h e above as s i g n m e n t . P e p t i d e C^ (Gly-Arg-Asn-Ala-Asp-Phe) w i l l be d i s c u s s e d under C h a p t e r V I . P e p t i d e C,. ( L y s - P h e - T h r - A s p - H i s - L e u - L y s - T y r ) gave l y s i n e as th e N - t e r m i n a l , and p h e n y l a l a n i n e a f t e r t h e f i r s t Edman d e g r a d a t i o n . I t i s l i k e l y t h a t t h e p r e s e n c e o f N - t e r m i n a l l y s i n e a d j a c e n t t o p h e n y l a l a n i n e r e n d e r s t h e p h e n y l a l a n i n e r e s i d u e r e f r a c t o r y t o c h y m o t r y p t i c c l e a v a g e . The p r e s e n c e o f t y r o s i n e a t t h e C - t e r m i n a l 127 TABLE XII Carboxypeptidase A Digests of the T r y p t i c and Chymotryptic Peptides of e-CNBr-8-E Time Peptide Amino Acid 30 min 6 hr ymole ymole T 5 Tyrosine .021 (1.00) T 1 4 Homoserine .008 (1.00) Valine .006 (0.75 Tryosine .005 (0.62) C1 Glycine .006 (0.12) Valine .005 (0.1) Tyrosine .042 (0.84) C2 Leucine Lysine Tyrosine . 006 .007 . 007 (0.85) (1.00) (1.00) was demonstrated with the use of carboxypeptidase A. The r e s u l t s are shown i n Table XII. The alignment of the t^yjrptic and chymotryptic peptides i s shown i n F i g . 36. Peptide T^ and are derived from the N-terminal region of the fragment 8-CNBr-8-E. Peptide contains two more amino acids Asp-Tyr than peptide T^ and thus provided the bridge f o r l i n k i n g peptides T^ and T^. The i s o l a t i o n of peptide by the chymotryptic a c t i v i t y of the tr y p s i n provided further evidence for the connection. S i m i l a r l y , peptide T^ ^ 4 A^ Tg are connected by peptide C^. Peptide T^, T^ and Tg are a l l derived from the chymotryptic a c t i v i t y of the porcine t r y p s i n . As mention e a r l i e r , peptide T^ and peptide are the same peptide. Peptide joined peptide Tg and T^ together, and f i n a l l y peptide C,. linked the three peptides, T^, T 1 2 and together, and thus completed the o v e r a l l sequence of B-CNBr-8-E. CHAPTER IV: THE ISOLATION OF METHIONINE PEPTIDES Introduction In order to a l i g n the cyanogen bromide cleavage peptides i n the r i g h t order, i t was necessary to i s o l a t e a set of over-lapping methionine peptides, from enzymic digests, or from other chemical cleavage reaction 5. Since i t i s generally pre- % ferable that these overlapping peptides be small enough for d i r e c t amino acid sequence studies, enzymic digestion i s usually the method of choice. However, enzymic digests of large proteins give r i s e to large numbers of peptides which may be d i f f i c u l t to f r a c t i o n a t e using conventional methods. Thus, i t i s extremely h e l p f u l i f the peptides of p a r t i c u l a r i n t e r e s t can be p u r i f i e d s e l e c t i v e l y from the t o t a l enzymic digest. Methods involving diagonal paper electrophoresis have been devised f o r the s e l e c t i v e p u r i f i c a t i o n of c e r t a i n peptides. The e s s e n t i a l features of such a method must be: (1) that a X s p e c i f i c chemical modification of a residue can be c a r r i e d out on a paper s t r i p ; (2) that the modification should change the net charge of the peptide; (3) that the modified peptides should be stable and f i n a l l y , (4) that the residue modified should be r e l a t i v e l y rare i n the protein; otherwise too many peptides would emerge from the diagonal. The diagonal technique most often used was devised by Brown and Hartley (127, 128) for the i s o l a t i o n of c y s t e i c acid 130 peptides from the d i s u l f i d e bridges of proteins. Cystine peptides are separated by paper electrophoresis and oxidized on paper by performic acid vapour. Electrophoresis at r i g h t angles to the f i r s t d i r e c t i o n produces groups of cy s t e i c acid peptides l y i n g o f f the diagonal. Pairs of c y s t e i c a c i d peptides from any given cystine containing peptide l i n e up i n the axis of the second electrophoresis.and can therefore be rela t e d to each other. Recently, Tang and Hartley (75) developed a diagonal technique for the s e l e c t i v e p u r i f i c a t i o n of methionine peptides by u t i l i z i n g a l k y l a t i o n of methionine by iodoacetamide. This reaction was discussed previously by Lawsonet. a l . (129) as being very s p e c i f i c at acid pH i n y i e l d i n g the sulfonium s a l t , the carboxymethylamide d e r i v a t i v e , CMA-methionine, which has an extra p o s i t i v e charge. CH 3 CH 3 S +S-CH2-CO-NH2 c, CH 2 + I-CH 2CO-NH 2—Y H2 +l" CH 2 CH 2 -NH-CH-CO- -NH-CH-CO-Using the above technique, these workers i s o l a t e d two methionine peptides from S-aminoethyl-Chymotrypsinogen A, which have the expected sequences Asp-Ala-Met-Ileu-AECys, and Met-Gly-Asp-Ser-Gly-Pro-Leu-Val-AEcys-. This technique was therefore applied to a t r y p t i c digest of reduced, alkylated B chain. Three methionine peptides were s e l e c t i v e l y p u r i f i e d , and p a r t i a l l y characterized. E x p e r i m e n t a l 1. A n a l y t i c a l d i a g o n a l paper e l e c t r o p h o r e s i s H a p t o g l o b i n 8 c h a i n (16 mg) was suspended i n 4 ml o f 0.1 M NH.HCO.,, pH 8.0 and p o r c i n e t r y p s i n ( — — molar r a t i o ) was 4 J 20 added. The d i g e s t was k e p t a t 37°C f o r 4 h o u r s , and l y o p h i l i z e d t w i c e w i t h w a t e r . The sample was d i s s o l v e d i n 0.2 ml o f p y r i d i n e a c e t a t e b u f f e r , pH 3.6 and c e n t r i f u g e d . D u p l i c a t e samples o f t h e su p e r -n a t a n t (25 A each) were a p p l i e d on two s e p a r a t e s h e e t s o f Whatman 3 MM paper. 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 performed on pH 4.0, a t 2.5 KV and pH 6.5 a t 3 KV r e s p e c t i v e l y . A f t e r d r y i n g , one s t r i p from each r u n was c u t , w e t t e d c a r e f u l l y w i t h f r e s h l y ^ p r e p a r e d i o d o a c e t a m i d e s o l u t i o n (0.1 M i n p y r i d i n e a c e t a t e b u f f e r , pH 3.6). The s t r i p s were t h e n a r r a n g e d on a g l a s s r a c k i n a d e s s i c a t o r s a t u r a t e d w i t h t h e p y r i d i n e a c e t a t e b u f f e r , c a r e b e i n g t a k e n t o a v o i d c o n t a c t between d i f f e r e n t p a r t s o f t h e paper. A l k y l a t i o n was a l l o w e d t o c o n t i n u e a t room t e m p e r a t u r e f o r 16 h o u r s . The s t r i p s were t h e n d r i e d , washed s e v e r a l t i m e s w i t h a c e t o n e , s t i t c h e d t o a f u l l s h e e t o f Whatman 3 MM paper and sub-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 r i g h t angles t o t h e o r i g i n a l d i r e c t i o n a t t h e same pH. A c o n t r o l s t r i p w i t h o u t a l k y l a t i o n was t r e a t e d s i m i l a r l y . The pap e r s were d r i e d and dipped^w.i-th^cadmium-nin- * h y d r i n , r e a g e n t . 2. P r e p a r a t i v e d i a g o n a l paper e l e c t r o p h o r e s i s F o r p r e p a r a t i v e paper d i a g o n a l e l e c t r o p h o r e s i s , a p p r o x i m a t e -l y 60 mg o f t h e t r y p t i c d i g e s t o f 3 c h a i n was a p p l i e d t o a 40 cm 132 Whatman 3 MM paper. F l u o r e s c e n t markers c o n t a i n i n g DNS-Arg and D N S - s u l f o n i c a c i d were a l s o a p p l i e d a l o n g w i t h t h e sample on t h e o r i g i n . These markers a r e u s e f u l i n c a l i b r a t i n g t h e m o b i l i t y o f t h e p e p t i d e s and a c t as g u i d e s f o r e l u t i n g bands i n p r e p a r a t i v e r u n s . 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 pe r f o r m e d a t pH 4.0, 1.5 KV f o r 3 h o u r s . A c o n t r o l s t r i p was c u t , a l k y l a t e d and 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 g a i n . T h i s d i a g o n a l " f i n g e r p r i n t " was d e v e l o p e d w i t h c a d m i u m - n i n h y d r i n t o r e v e a l t h e p o s i t i o n s o f m e t h i o n i n e p e p t i d e s i n t h e p r e p a r a t i v e e l e c t r o p h o r e t o g r a m s . The bands o f m e t h i o n i n e p e p t i d e were c u t o u t and a l k y l a t e d f o r 16 h o u r s . A f t e r washing w i t h a c e t o n e and d r y i n g , t h e bands were s t i t c h e d t o Whatman 3 MM paper and e l e c t r o p h o r e s i s was pe r f o r m e d p e r p e n d i c u l a r t o t h e band. A g u i d e s t r i p was c u t o f f and s t a i n e d w i t h c a d m i u m - n i n h y d r i n . The CMA-methionine p e p t i d e s , due t o t h e i r e x t r a p o s i t i v e c h a r g e r u n ahead o f t h e u n s u b s t i t u t e d p e p t i d e s , t o ward t h e c a t h o d e . The CMA-methionine p e p t i d e s , were t h e n c u t o u t , e l u t e d 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 6.5 ( p r e p a r e d from r e d i s t i l l e d p y r i d i n e ) , and d r i e d . R e s u l t s The M e t h i o n i n e Paper D i a g o n a l The r e s u l t s o f t h e pH 4.0 a n a l y t i c a l d i a g o n a l e l e c t r o p h o r e s i s a r e shown i n F i g . 37. As can be seen i n F i g . 37a, t h e c o n t r o l e l e c t r o p h o r e s i s was c l e a n . A l l t h e p e p t i d e s i n t h e c o n t r o l l i e i n a s t r a i g h t l i n e a t a p p r o x i m a t e l y a 45 degree a n g l e w i t h r e s -p e c t t o 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 . However, i n t h e paper t r e a t e d w i t h i o d o a c e t o m i d e , f i v e p e p t i d e s (3 major and 2 minor) can be seen t o l i e o f f t h e d i a g o n a l . The l i m i t e d number o f p e p t i d e s w h i c h a r e o f f t h e d i a g o n a l i n d i c a t e d t h e s e l e c t i v i t y a b Figure 37. A n a l y t i c a l methionine paper diagonal of t r y p t i c digest of 8 chain at pH 4.0. a) Control. b) After a l k y l a t i o n with iodoacetamide for 16 hours at room temperature. LO o f t h e r e a g e n t . As would be e x p e c t e d , t h e m e t h i o n i n e p e p t i d e s o f t h e 8 c h a i n , a f t e r a c q u i r i n g one e x t r a p o s i t i v e c h a r ge m i g r a t e d f u r t h e r towards t h e c a t h o d e , so t h a t t h e y were s e p a r a t e d from o t h e r p e p t i d e s h a v i n g t h e same m o b i l i t y b e f o r e t h e a l k y l a t i o n . The pH 6.5 a n a l y t i c a l d i a g o n a l e l e c t r o p h o r e s i s , w h i c h was no t shown h e r e , gave l e s s s a t i s f a c t o r y s e p a r a t i o n s . The m e t h i o n i n e p e p t i d e s , though m i g r a t e d o f f t h e d i a g o n a l a f t e r a l k y l a t i o n , were t o o c l o s e d t o each o t h e r so t h a t t h e s e p a r a t i o n s were not as good Y-as t h e pH 4.0 e l e c t r o p h o r e s i s . B e f o r e t h e pH 4.0 d i a g o n a l was s e l e c t e d f o r p r e p a r a t i v e p u r p o s e s , t h e i d e n t i t y o f t h e s e p e p t i d e s jwe-r-e- f u r t h e r examined. V A p e p t i d e f i n g e r p r i n t t e c h n i q u e was pe r f o r m e d a t pH 4.0 u s i n g i d e n t i c a l c o n d i t i o n s , and l a t e r chromatographed on BAWP (81) system. The p e p t i d e map was t h e n s t a i n e d w i t h p l a t i n i c i o d i d e reagent.. Two m e t h i o n i n e p o s i t i v e s p o t s were l o c a t e d . T h e i r p o s i t i o n s c o r r e s p o n d t o p e p t i d e M^ and i n t h e e l e c t r o p h o r e t i c r u n . However, M,- was n o t d e t e c t e d by t h i s r e a g e n t p r o b a b l y due t o i t s low s e n s i t i v i t y . The y i e l d o f p e p t i d e was low as compared t o and . These r e s u l t s , t h e r e f o r e , c o n f i r m e d t h e s e l e c t i v i t y o f t h e m e t h i o n i n e d i a g o n a l t e c h n i q u e , such t h a t o n l y m e t h i o n i n e r e s i d u e s were a l k y l a t e d under t h e s e c o n d i t i o n s . S i n c e t h e s e p e p t i d e s i s o l a t e d from t h e d i a g o n a l a r e i n t h e form o f t h e s u l f o n i u m s a l t , t h e y were f i r s t h e a t e d i n d i s t i l l e d w a t er a t 100°C f o r 2 hours t o c l e a v e a t m e t h i o n i n e w i t h t h e con-c o m i t a n t p r o d u c t i o n o f two p e p t i d e s , t h e f i r s t c o m p r i s i n g t h e N-t e r m i n a l r e g i o n of t h e CMA-methionine p e p t i d e w i t h homoserine l a c t o n e C - t e r m i n a l and the second, t h e p o r t i o n of t h e o r i g i n a l CMA-methionine p e p t i d e C - t e r m i n a l t o t h e m e t h i o n y l r e s i d u e . 135 A mechanism, s i m i l a r to CNBr cleavage reaction was postu-lated by Lawson et a l . (129) to be as follow: CH 0 . + l 3 I S-CH*-C0NH„ CH„ 0 CH 2 COOH R'-C-NH-C-C-N -CH-R • I I- I H O H COOH H2N-C-R + H 0 CH„ 0 COOH II i 2 I + I -r R'_ C-NH-C- -ON-C-R H 0 CH: H H H 20 R'—C-NH-C-I H \ 0 The peptide mixture aft e r heating i n water was hydrolyzed i n 6 N HC1 as i n the usual procedure for amino acid analysis. The amino acid compositions of these methionine peptides are Y. tabulated i n Table XIII. The mobility- of these peptides before y and a f t e r a l k y l a t i o n at pH 4.0 are also shown. As can be seen i n the analysis, a l l three of the methionine peptides contained homoserine instead of methionine. This suggests that the conversion of CMA-methionine to homoserine was quanti-t a t i v e under these conditions. Peptide M^ , being the largest peptide, contained arginine and i s r i c h i n aspartic acid, glutamic acid and va l i n e . Peptide M^ , on the other hand i s r i c h i n proline and contains carboxymethylcysteine. The y i e l d of peptide M^  was low, and the analysis was not very good. 136 TABLE XIII Amino Acid Composition of Methionine Peptides M5 Lysine - 0. , 05 1. ,1 0. 0015 1. ,9 H i s t i d i n e - -Arginine 0. , 013 1. , 1 -Cysteic - 0. 005 Carboxymethyl - 0. 036 0. 8 Cysteine Aspartic Acid 0. , 034 2. ,93 0. , 008 0. , 008 1 Threonine 0. . 0015 0. ,006 Serine 0. . 003 0. , 061 1. , 3 0. , 0017 2. , 0 Homoserine 0. , 12 1. , 0 0. , 045 1. , 0 0. , 005 0. , 6 Glutamic A c i d 0. , 035 3. ,0 0. , 013 0. , 008 1 Proline - 0. , 088 1. , 95 -Glycine 0. . 007 0. , 56 0. , 016 0. , 0012 1. ,4 Alanine 0. . 018 1. , 6 0. , 005 0. , 001 1. , 2 Valine 0. . 034 ; 2. , 95 0. , 055 1. , 2 0. , 007 1. , 0 Methionine - - - -Isoleucine 0. . 015 1. , 3 0. , 0471 1. , 0 -Leucine 0. , 015 1. , 3 0. , 053 1. ,2 -Tyrosine 0. . 013 1. , 1 - -Phenyalanine - - -u* (before) 0. , 42 0. . 73 0. 91 u (after) 0. ,63 1. , 06 1. , 31 E h r l i c h E" E" E" * - Mo b i l i t y corresponds to DNP Agmatine. 137 Amino A c i d Sequence S t u d i e s From t h e amino a c i d a n a l y s i s , m e t h i o n i n e p e p t i d e c o n t a i n s a r g i n i n e . The amino a c i d sequence o f t h i s p e p t i d e , t h e r e f o r e o v e r l a p s w i t h t h e l a t e r s t u d i e s on a r g i n i n e p e p t i d e s . B e s i d e s t h e t r y p t i c p e p t i d e i s o l a t e d i n t h e p r e s e n t c a s e , two more a r g -i n i n e - c o n t a i n i n g p e p t i d e s w h i c h a l s o c o n t a i n m e t h i o n i n e have been i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d from c h y m o t r y p s i n and thermo-l y s i n d i g e s t s o f h a p t o g l o b i n 3 c h a i n . The sequences o f t h e s e v p e p t i d e s a r e shown a s : (a) T r y p t i c d i g e s t V a l - ( A s p 3 , G l u ^ , G l y , A l a 2 , V a l ^ , I l e u 1 , L e u 1 , T y r 1 , M e t ) A r g . (b) T h e r m o l y s i n d i g e s t I l e u - A r g - H i s . (c) C h y m o t r y p s i n d i g e s t V a l - A s p - ( A l a , A s p , G l u 2 , P r o , G l y , V a l , M e t , l i e u , L e u ) A r g - H i s - T y r . A f t e r c o m b i n i n g t h e t r y p t i c , c h y m o t r y p t i c and t h e r m o l y t i c p e p t i d e s , t h e sequence around t h e p e p t i d e M^ i s V a l - ( G l u , A s p , A l a ) T y r - V a l - A s p - ( A l a , A s p , P r o , M e t / G l u 2 , G l y , V a l , L e u ) l i e u - A r g - H i s - T y r . An minor c h y m o t r y p t i c p e p t i d e w h i c h has been i s o l a t e d has t h e amino a c i d c o m p o s i t i o n o f V a l - ( A s p 2 , A l a , P r o , G l u , G l y , V a l , M e t , L e u ) . T h i s f i t s t h e above sequence a s s i g n m e n t . F u r t h e r m o r e , t h i s s u g g e s t s t h e c l e a v a g e a t a L e u - I l e u l i n k a g e o r V a l - I l e u l i n k a g e . T h i s i m p l i e s a sequence o f V a l - I l e u - A r g - H i s o r L e u - I l e u - A r g - H i s . S i n c e t h e N - t e r m i n a l amino a c i d s o f t h e cyanogen bromide fragments were l i e u ( N - t e r m i n a l o f 8 c h a i n ) , p r o l i n e (found t o be i n p e p t i d e M.), l e u c i n e and v a l i n e , t h i s would s u g g e s t a sequence o f M, as 138 Leu Met- -Ileu-Arg-His. Val The suggested p a r t i a l sequence of M^ i s shown as follows: Val-(Glu,Asp,Ala)Tyr-Val-Asp-(Ala,Asp,Pro, Val Glu 2,Gly)Met- -Ileu-Arg-His-Tyr. Leu Peptide M^  contained carboxymethylcysteine; i t was therefore compared with the c y s t e i c acid peptides i s o l a t e d from the 8 chain by Kauffman and Dixon (31). One of the c y s t e i c acid peptides i s o l a t e d from the thermolysin digest has the following sequence: Ileu-Cys-Pro-Leu-Ser-Lys-Asp. Furthermore, sequence analysis of B-CNBr-8-E established the N-terminal region as Pro-Ileu-Cys-Pro-Leu-Ser-Lys-Asp, and implies a sequence of -Met-Pro-Ileu-Cys-Pro-Leu-Ser-Lys-Asp-which was cleaved at Met-Pro by the cyanogen bromide to y i e l d 8-CNBr-8-E. The sequence of M^  i s therefore established to be Val-Met-Pro-Ileu-Cys-Pro-Leu-Ser-Lys. The detection of valine as the N-terminal amino acid of the peptide M^  confirmed the above sequence assignment. Due to the poor y i e l d of M,_ from the diagonal, the peptide was not further characterized. However, during the studies of arginine peptides from thermolysin digests, a methionine peptide was i s o l a t e d together with an arginine peptide, Val-Gly-Arg. Edman degradation of the mixture indicated the sequence of t h i s methionine peptide as Ala-Met-Asn-Lys. It i s l i k e l y that t h i s peptide was derived from peptide M-. Since there are four methionine residues i n the 8 chain, the fourth methionine has not been i s o l a t e d . D i f f i c u l t i e s i n i s o l a t i o n may ar i s e either because of ready oxidation of t h i s residue, or more l i k e l y as a r e s u l t of i t s occurrence i n a large peptide that i s hard to resolve from the diagonal. CHAPTER V: STUDIES ON THE GLYCOPEPTIDES OF HAPTOGLOBIN 3 CHAIN I n t r o d u c t i o n The c a r b o h y d r a t e a n a l y s i s o f h a p t o g l o b i n and i t s s e p a r a t e d c h a i n s showed t h a t t h e 8 c h a i n c o n t a i n s a l l t h e c a r b o h y d r a t e s i d e 1 2 chaxns whxle a and a c h a i n s a r e f r e e o f c a r b o h y d r a t e m a t e r i a l (14 ) . The c a r b o h y d r a t e m a t e r i a l c o n s t i t u t e s 20% o f the 8 c h a i n by w e i g h t . There a r e a p p r o x i m a t e l y 22 r e s i d u e s o f hexose, 12 r e s i d u e s o f g l u c o s a m i n e , 8 r e s i d u e s o f s i a l i c a c i d and 0.5 r e s i -due o f f u c o s e p e r mole o f 8 c h a i n (130). The f u n c t i o n o f c a r b o h y d r a t e i n g l y c o p r o t e i n s i s s t i l l m o s t l y unknown. R i b o n u c l e a s e B (131) , c h o l i n e e s t e r a s e (132), p r o t h r o m b i n (133), t r a n s f e r r i n , f i b r i n o g e n , i m m u n o g l o b u l i n s (134) and some o t h e r s a l l have b i o l o g i c a l a c t i v i t i e s w h i c h appear t o be i n d e p e n d e n t o f the bound c a r b o h y d r a t e . The case o f r i b o n u c l e a s A and B i s c l e a r c u t . Both enzymes have i d e n t i c a l s u b s t r a t e s p e c i f i c i t y and e n z y m a t i c a c t i v i t y . The c a r b o h y d r a t e u n i t does n o t a f f e c t o r c o n t r i b u t e t o t h e b i o l o g i c a l a c t i v i t y . On t h e o t h e r hand, n e u r a m i n i d a s e t r e a t m e n t , w h i c h removes t h e s i a l i c a c i d from the c a r b o h y d r a t e s i d e c h a i n i n t h e M and T a n t i g e n s o f human e r y t h r o c y t e s , i n d i c a t e d t h a t t h e c a r b o h y d r a t e m i g h t be i n v o l v e d i n f u n c t i o n . F u r t h e r m o r e , the b i o l o g i c a l a c t i v i t y o f human c h o r i o n i c g o n a d o t r o p i n and o v i n e f o l l i c l e - s t i m u l a t i n g hormones were l o s t when the s i a l i c a c i d s were removed from t h e s e hormones. T h i s was f i r s t o b s e r v e d by W h i t t e n (135), and l a t e r c o n f i r m e d by Brossmer and W a l t e r (136) and G o t t s c h a l k , W h i t t e n and Graham (137) 141 I n t he case o f h a p t o g l o b i n , the s i a l i c a c i d can be removed w i t h o u t the l o s s o f H b - b i n d i n g a b i l i t y (35, 36). S i n c e many s e c r e t e d p r o t e i n s a re g l y c o p r o t e i n i n n a t u r e , E y l a r (134) s u g g e s t e d t h a t the c a r b o h y d r a t e u n i t might s e r v e as a p a s s p o r t w h i c h would promote t h e e x i t o f the p r o t e i n s from t h e c e l l s . Even though the 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 h a p t o g l o b i n has been d e t e r m i n e d , the f u n c t i o n o f the c a r b o h y d r a t e u n i t , t h e s t r u c -t u r e o f the c a r b o h y d r a t e s i d e c h a i n s , t h e n a t u r e o f t h e l i n k a g e as w e l l as the amino a c i d sequence o f t h e p e p t i d e c h a i n around the a t t a c h m e n t s i t e s remain t o be s t u d i e d . T h i s c h a p t e r c o n c e r n s m a i n l y 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 t r y p t i c g l y c o p e p t i d e s from t h e 3 c h a i n , the e s t a b l i s h -ment o f the n a t u r e o f t h e l i n k a g e between the c a r b o h y d r a t e and p r o t e i n m o i e t y , and an a t t e m p t t o d e t e r m i n e t h e amino a c i d sequence around the c a r b o h y d r a t e - a t t a c h m e n t s i t e . E x p e r i m e n t a l 1. T r y p t i c d i g e s t o f h a p t o g l o b i n 3--chain H a p t o g l o b i n 8 c h a i n (250 mg) i n 65 ml o f 0.1 M NH 4HC0 3 pH 8.0 was d i g e s t e d w i t h 7.0 mg o f p o r c i n e t r y p s i n a t 37°C f o r 4 h o u r s . The d i g e s t was l y o p h i l i z e d t w i c e a f t e r r e d i s s o l v i n g i n d i s t i l l e d w a t e r . 2. Ion exchange chromatography o f t r y p t i c d i g e s t o f h a p t o -g l o b i n 8_ c h a i n 142 (a) Dowex 50-X2 chromatography The t r y p t i c d i g e s t was d i s s o l v e d i n 2.0 ml o f 0.2 N p y r i -d i n e - a c e t a t e b u f f e r pH 3.15, and a p p l i e d t o a Dowex 50-X2 (AG-50W-X2, 200-400 mesh,Bio-Rad L a b o r a t o r y , Richmond, C a l i f o r n i a ) column (2.2 x 92 cm) a c c o r d i n g t o the p r o c e d u r e d e s c r i b e d by S c h r o e d e r (138). The e l u t i o n g r a d i e n t c o n s i s t e d o f t h r e e b u f f e r chambers. The f i r s t two chambers had 750 ml o f 0.2 N p y r i d i n e -a c e t a t e b u f f e r pH 3.1 and t h e l a s t chamber c o n t a i n e d 750 ml o f 2.0 N p y r i d i n e - a c e t a t e b u f f e r pH 5.0. The column was o p e r a t e d a t 50°C a t a f l o w r a t e o f 70 ml p e r hour. Nine ml f r a c t i o n s were c o l l e c t e d o f w h i c h 0.3 ml was used f o r n i n h y d r i n a s s a y (139) and 0.2 ml f o r c a r b o h y d r a t e a n a l y s i s ( 7 8 ) . The e l u t i o n p r o f i l e i s shown i n F i g . 38. The g l y c o p e p t i d e s were n o t r e t a r d e d , and were t h e r e f o r e r e c h r o m a t o g r a p h e d on a Dowex 1-X4 column. (b) Dowex 1-X4 chromatography The g l y c o p e p t i d e s were d i s s o l v e d i n 2.0 ml o f pH 9.4 N - e t h y l - m o r p h o l i n e b u f f e r and t h e pH was r a i s e d t o about 10.5 w i t h NaOH. The sample was t h e n a p p l i e d t o a Dowex 1-X4 (AG-1W-X4, 200-400 mesh, Bio-Rad) column (1 x 100 cm) a t room tempera-t u r e . The e l u t i o n g r a d i e n t was s e t up a c c o r d i n g t o S c h r o e d e r (140) and c o n s i s t e d o f a c o n s t a n t volume m i x e r and a s e r i e s o f v o l a t i l e b u f f e r s (140) s u c c e s s i v e l y i n t r o d u c e d i n t o the c h a n g i n g s o l u t i o n i n t h e m i x e r . I n t h i s e x p e r i m e n t , a c l o s e d 250 ml f l a s k c o n t a i n i n g 135 ml o f pH 9.4 b u f f e r was use d , and 40 ml o f pH 9.4 b u f f e r , 120 ml o f pH 8.4 b u f f e r , 160 ml o f pH 6.5 b u f f e r , 240 ml o f 0.5 N a c e t i c a c i d , 400 ml o f 2 N a c e t i c a c i d and 200 ml of 4 N a c e t i c a c i d were r u n i n s u c c e s s i v e l y . F i v e ml f r a c t i o n s Figure 38. Separation of t r y p t i c peptides of haptoglobin chain on Dowex 50-x 2 ion exchang chromatography. 0. D. at 570 nm ©—-—o , .0. D. at 490 nm © -© were c o l l e c t e d , and the f r a c t i o n s were a n a l y z e d as i n S e c t i o n 2 ( a ) . The e l u t i o n p r o f i l e i s shown i n F i g . 39. 3. Repeated pronase d i g e s t o f B-CNBr-8-B Ten m i l l i g r a m s o f t h e c a r b o h y d r a t e - c o n t a i n i n g cyanogen bromide fragment o f 8 c h a i n , 8~CNBr-8-B, were d i s s o l v e d i n 3.0 ml o f 0.1 M NR^HCO^, pH 8.0, and d i g e s t e d w i t h p o r c i n e t r y p s i n a t an e n z y m e / s u b s t r a t e r a t i o o f 1/100 f o r 2 1/2 hours a t 37°C. The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n o f a few drops o f g l a c i a l a c e t i c a c i d , and t h e s o l u t i o n was l y o p h i l i z e d . The t r y p t i c d i g e s t o f t h i s p e p t i d e was t h e n s u b j e c t e d t o p r o n a s e d i g e s t i o n i n 1.0 ml o f 0.2 M N - e t h y l - m o r p h o l i n e b u f f e r , pH 8.5, c o n t a i n i n g 0.01 M C a C ^ a t an e n z y m e / s u b s t r a t e r a t i o o f 1/50. The d i g e s t was c o v e r e d w i t h t o l u e n e and i n c u b a t e d a t 37°C f o r 48 h o u r s . The pronase d i g e s t was t h e n l y o p h i l i z e d , d i s s o l v e d i n 0.5 ml o f 0.2 N a c e t i c a c i d and chromatographed on a B i o - G e l P-4 column (0.8 x 94 cm) u s i n g 0.2 N a c e t i c a c i d as s o l v e n t . The g l y c o p e p t i d e s were w e l l s e p a r a t e d from o t h e r p e p t i d e s as can be seen i n F i g . 44a. The g l y c o p e p t i d e s were th e n d r i e d and d i g e s t e d w i t h pronase a g a i n w i t h an e n z y m e / s u b s t r a t e r a t i o o f 1/20 f o r 4 8 hours and f i n a l l y chromatographed on t h e same P-4 column ( F i g . 44b). 4. T h e r m o l y s i n d i g e s t o f p e p t i d e g-CNBr-8-B G l y c o p e p t i d e B~CNBr-8-B from cyanogen bromide c l e a v a g e was f i r s t d i g e s t e d w i t h p e p s i n i n 5% f o r m i c a c i d f o r 4 hours a t 37°C u s i n g an e n z y m e / s u b s t r a t e r a t i o o f 1/50. The p e p t i c g l y c o -p e p t i d e s , due t o t h e i r a c i d i c n a t u r e , were n o t r e t a i n e d i n Dowex Fraction Number Figure 39. Chromatography of t r y p t i c glycopeptide on.Dowex 1-x 4 ion exchange chromatography. 50-X2 (H ) column, and were s e p a r a t e d from o t h e r p e p t i d e s . A f t e r d r y i n g , t h e g l y c o p e p t i d e s were d i s s o l v e d i n 1 ml o f N - e t h y l -m o r p h o l i n e b u f f e r and were th e n d i g e s t e d w i t h t h e r m o l y s i n f o r 4 hours a t 37°C, and f i n a l l y l y o p h i l i z e d . The t h e r m o l y t i c d i g e s t was chromatographed on a B i o - G e l P-10 column (0.8 x 94 cm) u s i n g 0.2 N a c e t i c a c i d as s o l v e n t . The s e p a r a t i o n o f t h e t h e r m o l y t i c p e p t i d e s was shown i n F i g . 4 3b. R e s u l t s The T r y p t i c G l y c o p e p t i d e s o f H a p t o g l o b i n 8 C h a i n I n t h e s t u d i e s o f g l y c o p e p t i d e s , most i n v e s t i g a t o r s g e n e r a l l y a t t e m p t t o p r e p a r e g l y c o p e p t i d e s by t h e d e g r a d a t i o n o f g l y c o p r o -t e i n s w i t h p r o t e o l y t i c enzymes o f b r o a d s p e c i f i c i t y . A l t h o u g h t h e s e p r o t e a s e s cause e x t e n s i v e d e g r a d a t i o n o f t h e p r o t e i n , t h e y y i e l d a complex m i x t u r e o f g l y c o p e p t i d e s w h i c h i s d i f f i c u l t t o f r a c t i o n a t e (141, 142). On the o t h e r hand, th e use o f h i g h l y s p e c i f i c p r o t e a s e s , such as t r y p s i n and c h y m o t r y p s i n , has e n a b l e d B a h l (143) and M e l c h e r s (144) t o i s o l a t e homogenous g l y c o p e p t i d e s f rom human c h o r i o n i c g o n a d o t r o p i n and mouse i m m u n o g l o b u l i n l i g h t c h a i n r e s p e c t i v e l y , and H o w e l l , Hood and Sanders (145) t o p e r f o r m t h e c o m p a r a t i v e s t u d i e s o f IgG heavy c h a i n s from v a r i o u s s o u r c e s . T r y p s i n was s e l e c t e d i n t h i s i n v e s t i g a t i o n . The s e p a r a t i o n o f t r y p t i c p e p t i d e s on Dowx 50-X2 i o n exchange chromatography 'v i s shown i n F i g . 38. Because o f t h e a c i d i c n a t u r e o f t h e 147 g l y c o p e p t i d e s , t h e y were n o t r e t a i n e d by t h e column and hence s e p a r a t e d from most of t h e p e p t i d e s . Upon u s i n g chromatography on Dowex 1-X4 i o n exchange column f o r t h e s e p a r a t i o n o f a c i d i c p e p t i d e s , t h r e e major components can be v i s u a l i z e d ( F i g . 39). The f i r s t component T-G I c o n t a i n e d a l a r g e amount o f hexose and was n o t r e t a i n e d by the column. T h i s s u g g e s t e d t h a t component T-G I might be n e u t r a l and c o n t a i n s a l a r g e n e u t r a l c a r b o h y d r a t e s i d e c h a i n . Component T-G I I and T - G - I I I were, however r e t a i n e d by t h e column. The n i n h y d r i n p r o -f i l e $ o f t h e s e two components were somewhat h e t e r o g e n o u s . The X ^ > y h e t e r o g e n i t y o f t h e g l y c o p e p t i d e s may p o s s i b l y be due t o t h e ' d i f f e r e n c e i n s i a l i c a c i d c o n t e n t as was seen b e f o r e i n g l y c o -9 X p e p t i d e s , B-CNBr-8-B. The a p p a r e n t h e t e r o g e n i t y o f g l y c o p e p - ' (\ t i d e s d u r i n g f r a c t i o n a t i o n has a l s o been e n c o u n t e r e d by v a r i o u s w o r k e r s . F o r example, M e l c h e r s (144) i s o l a t e d t h r e e t r y p t i c g l y c o p e p t i d e s from mouse i m m u n o g l o b u l i n l i g h t c h a i n s . These p e p t i d e s have the same amino a c i d c o m p o s i t i o n and sequence b u t d i f f e r e d o n l y i n t h e amount o f s i a l i c a c i d r e s i d u e . These pep-t i d e s c o n t a i n e d z e r o , one and two r e s i d u e s o f s i a l i c a c i d . I f t h e h e t e r o g e n i t y was i n d e e d d e r i v e d from th e f r a c t i o n a -t i o n o f t h e s i a l i c a c i d c o n t e n t , t h e s e g l y c o p e p t i d e s s h o u l d be v e r y s i m i l a r i n s i z e and most i m p o r t a n t , o f t h e same amino a c i d c o m p o s i t i o n . Components T-G I I and T-G I I I were s u b j e c t e d t o m i l d a c i d t r e a t m e n t t o remove the s i a l i c a c i d , and l a t e r r e c h romatographed on a B i o - G e l P-10 column. The e l u t i o n p r o f i l e i s shown i n F i g . 40, The c a r b o h y d r a t e - c o n t a i n i n g p e p t i d e s appeared homogenous and ){ V 148 F i g u r e 40. 20 40 60 F r a c t i o n Number Chromatography o f d e s i a l i z e d t r y p t i c g l y c o p e p t i d e s on a BioRad P-10 column (1 x 100 cm) u s i n g 0.2 N a c e t i c a c i d as s o l v e n t . 0. D. a t 230 nm c o 0. D. a t 490 nm 149 eluted at the same p o s i t i o n i n d i c a t i n g these components were si m i l a r , i f not i d e n t i c a l , i n s i z e . Further studies on the N-terminal amino acid of T-G II and T-G III revealed that both components had dansyl-valine as the only N-terminal amino acid. Amino acid analysis established that these two components are indeed i d e n t i c a l as can be seen from F i g . 41 and Table XIV, respectively. The homogenity of T-G II \ was further investigated by high voltage electrophoresis at pH 1.9, X 4 KV for 45 min. Only one band with some streaking was observed (Fig. 42). The appearance of the ninhydrin color was slow which i s consistent with the assignment of valine at the N-terminal. The peptide was also Pauly-positive with the same streaking. The carbohydrate compositions of these glycopeptides are also i n - j{ eluded i n Table XIV. The values of glucosamine and hexose of T-G I ( i . e . , 6 residues of hexose, and 4 for glucosamine per residue of lysine) are the same t'p those reported e a r l i e r by A Gerbeck et a l . (64) and Cheftel et a l . (146). However, T-G I gave an extremely high value for hexose, which indicates that o t h i s component probably does not contain stoichimetric quantities X of amino acids and thus does not represent the carbohydrate moiety covalently bound to a peptide. Component T-G I was d i f f i c u l t to characterize. I t con-tained mainly carbohydrate material. The amino acid analysis of t h i s component was low. Dansylation of thi s component did not give any p o s i t i v e i d e n t i f i c a t i o n of amino acid. It i s pos-s i b l e that the y i e l d of the amino acids were low when hydrolyzed i n the presence of large amounts of hexose. Another p o s s i b i l i t y 150 ~ •Mtttf t* - ' 1 6 11 Figure 41. Dansylation studies of t r y p t i c glycopeptides. I. Pro, His; 2. T-G I; 3. Leu, Lys; 4. Val, Asp; 5. T-GII; 6. Val, Asp; 7. T-G II; 8. Val, Asp; 9. Leu, Lys; II . Pro, His. 151 TABLE XIV Amino Acid Analysis of Tryptic Glycopeptides T-G I T -G II T-•G III ymole ymole ymole Lysine 0. 008 (1) 1 0. 043 (0. 78) 1 0. 06 (0. 66) 1 His t i d i n e 0. 0075 (0. 93) 1 0. 06 (1. 09) 1 0. 09 (1. 00) 1 Arginine 0. 003 (0. 37) Aspartic A c i d 0. 017 (2. 1) 2 0. 106 (1. 92) 2 0. 163 (1. 8) 2 Threonine 0. 01 (1. 25) 1 0. 056 (1. 01) 1 0. 08 (0. 9) 1 Serine 0. 011 (1. 37) 1 0. 054 (0. 98) 1 0. 07 (0. 78) 1 Glutamic A c i d 0. 014 (1. 75) 2 0. 046 (0. 83) 1 0. 08 (0. 88) 1 Proline 0. 009 (1. 12) 1 0. 02 (0. 36) 0. 039 (0. 43) Glycine 0. 0094 (1. 17) 1 0. 04 (0. 72) 1 0. 055 (0. 61) 1 Alanine 0. 013 (1. 62) 1 0. 054 (0. 98) 1 0. 074 (0. 82) 1 Valine 0. 012 (1. 5) 2 0. 055 (1. 00) 1 0. 09 (1. 00) 1 Methionine 0. 007 (0. 12) 0. 009 (0. 1) Isoleucine 0. 003 (0. 37) 0. 04 (0. 72) 1 0. 07 (0. 78) 1 Leucine 0. 012 (1. 5) 2 0. 11 (2. 00) 2 0. 176 (1. 95) 2 Tyrosine 0. 003 (0. 37) 0. 018 (0. 32) 0. 03 (0. 33) Phenyalanine 0. 005 (0. 62) 1 0. 022 (0. 40) 0. 03 (0. 33) Glucosamine* 0. 0159 (1. 98) 0. 2 (3. 6) 0. 284 (3. 15) Total 16 13 13 Hexose 218 6 6 Glucosamine 2.3 4 4 * Determined! a f t e r 21 hours hydrolysis. 152 Figure 42. High voltage electrophoresis of T-G II at pH 1.9, 4 KV for 45 minutes. would be that an 0-ester linkage e x i s t s between the carbohydrate and the peptide moiety so that T-G I represents the l a b i l e carbo-hydrate side chain. The o r i g i n and the nature of th i s component remain to be elucidated. Dobryszycka and Lisowska (147) have e a r l i e r reported the i s o l a t i o n of t r y p t i c glycopeptides from whole haptoglobin. I t i s , however, d i f f i c u l t to compare those reports with the present i n v e s t i g a t i o n . F i r s t of a l l , no amino acid composition and homogeneity of the glycopeptides were reported. Secondly, d i f -ferent p u r i f i c a t i o n methods were used. These workers reported the f r a c t i o n a t i o n of three components (I, II and III) from a Sephadex G-100 column and four components (I', I I " , I I I " , IV") from DEAE Sephadex. However, component I and II had i d e n t i c a l carbohydrate compositions (both have 5.1% s i a l i c acid, and 10% ^ hexose). S i m i l a r l y , component I 1 and I I ' , component I I I ' and IV' are also i d e n t i c a l i n carbohydrate composition. I t i s l i k e l y that only two major components which might correspond to T-G I and T-G II were present i n 6 chain. Two d i f f e r e n t kinds of t r y p t i c glycopeptides have also been observed by Cheftel et a l . (148). The f i r s t type was non-dialyzable and contained 74% of the t o t a l s i a l i c acid residues which can be removed by treatment with neuraminidase. The other type, was however, dialyzable, and the s i a l i c acid (26%) residues were r e s i s t a n t to neuraminidase treatment. Since Gerbeck et_ a l . (149) have i s o l a t e d a glycopeptide from the pronase digest of haptoglobin which has the amino acid composition; aspartic acid-, serine, threonine and alanine, i t 154 would be i n t e r e s t i n g i f t h i s pronase glycopeptide could be derived from the larger peptide, T-G I I . Therefore, peptide T-G II was digested with pronase for 54 hours at an enzyme/substrate r a t i o of 1/50 and the glycopeptides chromatographed on Bio-Gel P-4 (Fig. 43a) and Dowex 50-X2 column successively. The amino acid analysis at each stage i s shown i n Table XV. Upon high voltage electrophoresis at pH 1.9, 3.5 KV for 40 min., the glycopeptides a f t e r the Bio-Gel column and Dowex 50-X2 ion exchange chromatography contain one major and one minor component, the l a t t e r component being Pauly-positive. Since only 0.5 residue of h i s t i d i n e and leucine were detected by the amino acid analysis, t h i s suggested that the minor component, containing h i s t i d i n e and leucine, i s present i n 50% y i e l d of the major com-ponent. Thus the conclusion from these data i s that the glyco-peptide at t h i s stage consists of two d i f f e r e n t carbohydrate-containing peptides. The major component contains threonine, aspartic acid2, serine, alanine and glutamic acid while the minor component contains h i s t i d i n e , leucine and possibly aspartic acid. Similar r e s u l t s have been observed by Gerbeck et al_. (64) e a r l i e r on the pronase digest of haptoglobin. These workers i s o l a t e d two types of pronase glycopeptide from haptoglobin having d i f f e r e n t amino acid composition but i d e n t i c a l carbo-hydrate composition. The f i r s t type contained threonine and a l a -nine, but no h i s t i d i n e , the other one was r i c h i n h i s t i d i n e but had no serine. These workers suggested that these two types of glycopeptides might be produced as a r e s u l t of d i f f e r e n t s i t e s of cleavage by pronase of the same carbohydrate attachment region. • 0.1 10 20 30 40 50 60 F r a c t i o n Number b 10 20 30 40 50 60 F r a c t i o n Number F i g u r e 43 a. Chromatography o f pr o n a s e d i g e s t o f T-G I I on a B i o Rad P-4 column (0.8 x 94 cm) u s i n g 0.2 N a c e t i c a c i d as s o l v e n t , b. Chromatography o f t h e r m o l y s i n d i g e s t o f 8-CNBr-8-B on a BioRad P-10 column (0.8 x 94 cm). o — o o.D. a t 230 nm O—-o 0. D. a t 490 nm 156 TABLE XV Amino Acid Composition of Pronase Peptide from T-G II P-4-A Dowex 50 ymole ymole Lysine H i s t i d i n e 0. 034 (1.2) 0.01 (0.5) Arginine Aspartic Acid 0.064 (2.2) 0. 046 (2.3) Threonine 0. 027 (0.94) 0. 022 ( 1 ) Serine 0.030 (1.05) 0.020 ( 1 ) Glutamic Acid 0. 023 (0.8) 0.019 ( 1 ) Proline Glycine 0. 005 (0.2) 0.020 ( 1 ) Alanine 0. 018 (0.6) Valine 0.009 (0.3) Methionine Isoleucine Leucine 0.015 (0.52) 0. 010 (0.5) Tyrosine Phenyalanine Glucosamine 0.168 (5.6) 0.11 (5.9) Later studies on the thermolysin glycopeptide v e r i f i e d the above argument. The amino acid composition of the thermolysin peptides consisted of leucine, h i s t i d i n e , a s p a r t i c a c i d 2 , threo-nine, serine, alanine, and glutamic acid. Since only N-terminal leucine was found i n t h i s peptide, leucine and h i s t i d i n e must be associated with other amino acids i n the same carbohydrate attach ment region. The sequence studies around the carbohydrate attachment s i t e w i l l be discussed i n the l a s t section of t h i s chapter. The Nature of the Linkage During the past few years, the chemical properties of g l y -coproteins from a v a r i e t y of sources have been examined. The glycosylamine linkage of the carbohydrate to the amide group of asparagine has been demonstrated or postulated to occur i n oval-bumin (150), immunoglobulin (151), a^-acid glycoprotein (152), soybean hemagglutinin (153), ovomucoid (154) ribonuclease B (155) and more recently pancreatic DNase (156) and yeast inver-tase (157). A second type of linkage involving 0-glycosidic ether bonds to serine or threonine residues has been suggested for mucins (158, 159) as well as for an enzyme, taka-amylase A (160). The presence of both types of carbohydrate-amino acid linkage has been demonstrated recently by Dawson and Clamp (161) i n an A myeloma globulin, type K, and by Bahl (14 3) i n human chorionic gonadotropin. In the case of haptoglobin, the f i r s t type of linkage appears to be the most l i k e l y for reasons discus-sed below. This has i n fact been suggested but not proved by 158 Gerbeck et a l . (149). Two d i f f e r e n t approaches were taken i n the present studies to e s t a b l i s h the linkage between the carbohydrate and the protein moiety. I t i s known that an 0-glycosidic linkage between carbohydrate and serine or threonine i s l a b i l e to mild a l k a l i treatment. These two amino acids w i l l be converted to dehydroalanine and a-amino crotonic a c i d respectively by a 8~elimination reaction (162) under these conditions. Glycopeptide T-G I and T-G II were dissolved i n 1.0 ml of 0.5 N NaOH, flushed with N 2 and kept at 4°C for 19 hours. Con-centrated HCl, 1.0 ml, was added, and the samples were hydrolyzed for 17 hours. Control samples were hydrolyzed d i r e c t l y with 6 N HCl. The amino acid analysis of these samples are shown i n / Table XVI. As can be seen i n the table, the serine and threonine values were i d e n t i c a l both i n control and a l k a l i - t r e a t e d samples. The resistance of both amino acids to a l k a l i treatment makes i t very u n l i k e l y that an O-glycosidic bond involving these amino acids i s present i n haptoglobin. The a l k a l i - r e s i s t a n c e of these two amino acids has also been demonstrated by Neumann and Lampen (157) i n yeast invertase and by Wagh, Bornstein and Winz-l e r (163) i n human orosomucoid and these authors came to the same conclusion. Unequivocal evidence f o r the aspartamido-hexose linkage would be the actual demonstration of a s p a r t i c acid-carbohydrate complex. Di f f e r e n t combinations of enzymes have been used to digest glycoproteins. In general, i t has proved d i f f i c u l t to TABLE XVI Amino Acid Composition of Tryptic Glycopeptides on A l k a l i Treatment T-G-II T-G I NaOH T-G II NaOH Control Treatment Control Treatment Lysine 0.75. 0. 75 His t i d i n e 1 1 Arginine Aspartic Acid 1.18 1.17 2.1 2.1 Threonine 1 1 1 1 Serine 1 1 1 1 Glutamic Acid 1 1 1 0.93 Proline Glycine 0.6 1 0. 67 0. 64 Alanine 1 1 1.1 1.1 Valine 0.6 0.6 0. 88 0. 88 Isoleucine 0.46 0.48 0.7 0. 67 Leucine 0. 94 1 2 1.9 Tyrosine Phehyalanine 0. 35 0.28 0.5 0.92 160 prepare a glycopeptide containing aspartic acid as the only amino acid. One i s tempted to suggest that the carbohydrate moiety causes s t e r i c hindrance to the attack of enzymes. A method for the i s o l a t i o n of aspartic acid-carbohydrate complex was described by Fletcher et a l . (150). The glycoprotein was f i r s t digested with pronase, fractionated on G-25 column and the a-NH^ group of the glycopeptide was blocked with benzyloxy-carbonyl chloride before treatment with carboxypeptidase A. However, glycopeptides prepared i n t h i s manner s t i l l contain s i g n i f i c a n t amounts of serine and threonine. More recently, Conchie et a_l. (164) i s o l a t e d an aspartic-carbohydrate complex free of other amino acids from ovalbumin using repeated pronase digestion followed by chromatography on Sephadex G-25. This method was applied i n an attempt to i s o l a t e an aspartic-carbohydrate complex from haptoglobin. Glycopeptide, B-CNBr-8-B, from cyanogen bromide cleavage was used and was digested with tr y p s i n before the f i r s t pronase digest. The pronase digest was chromatographed on a Bio-Gel P-4 column as shown i n F i g . 44a. However, repeated pronase digestion of the glycopeptide from the P-4 column did not release any further amino acids (Fig. 44b and Table XVII) and the glycopeptides s t i l l contain other amino acids i n addition to aspartic acid. The ease of i s o l a t i o n of an aspartic-carbohydrate complex from ovalbumin i s most l i k e l y due to the simple carbohydrate moiety of t h i s protein. Ovalbumin contains only N-acetylglucosamine and mannose, while haptoglobin has a much bul k i e r carbohydrate side chain. Another approach was sought therefore. From the analysis of pronase digestion, two aspartic acid residues were found i n 161 0.6 0.4 0.2 ° — o O. D. at 230 nm O—-O o. D. at 490 nm o-OnrSrc-p-0* 10 0.3 0.2 0.1 20 30 40 Fraction Number g- Q-g-0 -o o—O—O »,*<>• -o-o- •p*o* "O- • 10 20 30 40 Fraction Number 50 0.1 -04-10 20 30 40 Fraction Number 50 Figure 44 a. b. Pronase digest of t r y p t i c 8~CNBr-8-B. _ Second pronase digest on A. c. P a r t i a l acid hydrolysis on B. A l l samples were chromatographed on the same P-4 column using 0.2 N acetic acid as solvent. 162 TABLE XVII Amino Acid Composition of Glycopeptide a f t e r Pronase and P a r t i a l Acid Hydrolysis Treatment F i r s t Second P a r t i a l Acid Pronase Pronase Hydrolysis (Peak A) (Peak B) (Peak C) Lysine H i s t i d i n e 0. 04 (0. 57) 0. 054 (0. 54) 0. 04 (0. 36) Arginine Aspartic Acid 0. 07 (1. 00) 0. 100 (1. 0) 0. 11 (1. 00) Threonine 0. 017 (0. 24) 0. 024 (0. 24) 0. 004 (0. 03) Serine 0. 022 (0. 32) 0. 028 (0. 28) 0. 01 (0. 1) Glutamic Acid 0. 02 (0. 28) 0. 027 (0. 27) 0. 01 (0. 1) Proline 0. 018 (0. 25 0. 028 (0. 28) Glycine 0. 0069 (0. 1) Alanine 0. 018 (0. 25) 0. 026 (0. 26) 0. 018 (0. 16) Valine Methionine Isoleucine Leucine 0. 0042 (0. 06) -Tyrosine Phenyalanine Glucosamine 0. 11 (1. 6) 0. 21 (2. 1) 0. 34 (3. 00) the glycopeptide. Since most l i k e l y only one aspartic acid r e s i -due i s involved i n the linkage, p a r t i a l acid hydrolysis was per-formed on the second pronase-digested sample, and l a t e r chro-matographed on Bio-Gel P-4 column (Fig. 44c). The front peak (c) was found to contain mainly aspartic acid and glucosamine upon amino acid analysis (Table XVII). The values for threonine and serine were too low to be of any s i g n i f i c a n c e . Furthermore, there are three glucosamine residues i n t h i s analysis compared to only 1.6 residue i n the f i r s t pronase digestion. Since there are 4 glucosamines i n the glycopeptide as determined e a r l i e t h i s suggests that a l l the glucosamine could be accounted for i n t h i s complex i f the correction of glucosamine during acid hydroly s i s i s considered. The nature of the linkage i s therefore estab-l i s h e d as an aspartylglycosylamine linkage by the i s o l a t i o n of t h i s pure aspartic-carbohydrate complex. As expected, t h i s linkage i s stable under d i l u t e a l k a l i treatment, which was i n d i -cated i n the e a r l i e r experiment. The Amino Acid Sequence of Glycopeptides The amino acid sequence^ i n the v i c i n i t y of the carbohy-drate attachment s i t e of several glycoproteins have been charac-t e r i z e d by various workers. The sequence^of these glycopeptides are shown i n Table XVIII (165). They have been grouped on the basis of the nature of the attached polysaccharide. Class I glycopeptides are associated with a complex polysaccharide moiety that may contain fucose, galatose, galactosamine, glucosamine, mannose, and s i a l i c acid. Class II glycopeptides contain only glucosamine and mannose. TABLE XVIII Amino Acid Sequence of Glycopeptides 164 Source Amino Acid Sequence Porcine RNase Porcine RNase IgG Heavy Chains Stem Bromelain T r a n s f e r r i n Fibrinogen Fibrinogen a^-Acid Glycoprotein a^-Acid Glycoprotein a^-Acid Glycoprotein Ovomucoid Class I Glycopeptides CHO I Ser-Ser-Ser-Asn-Ser-Ser-Asn Tyr-Gln-Ser-Asn-Ser-Thr-Met Gln-Gln-Phe-Asn-Ser-Thr-Ile Gln-Ser-Asn-Asn-Glu-Ser-Ser Leu-Ile-His-Asn-Arg-Thr-Gly Val-Gly-Glu-Asn-Arg--Asn-Lys-Thr -Pro-Asn-Lys--Thr-Asn-Lys--Glu-Ser-Asn-Thr-Gly -Asn-Thr-Porcine RNase Egg White Avidin Ovalbumin Bovine RNase B Thyroglobulin Bovine DNase • Ovotransferrin Class II. Glycopeptides CHO I Ser-Arg-Arg-Asn-Met-Thr-Gln Leu-Gly-Ser-Asn-Met-Glu-Lys-Tyr-Asn-Leu-Thr-Ser Lys-Ser-Arg-Asn-Leu-Thr-Lys Ala-Leu-Gly-Asn-Ala-Thr-Arg -Ser-Asn-Ala-Thr-Leu-Ile-His-Asn-Arg-Thr-Gly 165 W i t h th e e x c e p t i o n o f t h e r e p o r t e d sequence i n a ^ - a c i d g l y c o p r o t e i n , t h e s e sequences a r e a l l o f t h e t y p e : CHO -A-Asn-B-(Ser o r T h r ) - C I n c l a s s I g l y c o p e p t i d e s , res±dfu§^h-4s?-always p o l a r , w h i l e i n C l a s s I I , w i t h a s i n g l e e x c e p t i o n , i t i s always a p o l a r . E y l a r (134) has p o i n t e d o u t e a r l i e r t h a t a hydroxyamino a c i d c h a i n c l o s e t o t h e a t t a c h m e n t s i t e appears t o be a d e t e r m i n -a n t f o r t h e s p e c i f i c i t y o f t h e enzyme w h i c h c a t a l y z e s t h e r e a c t i o n o f p e p t i d e - b o u n d a s p a r a g i n e w i t h U D P - N - a c e t y l g l u c o s a m i n e . The hydroxyamino a c i d was l o c a t e d on t h e c a r b o n y l s i d e o f t h e a s p a r a -g i n e r e s i d u e s w h i c h form t h e a t t a c h m e n t s i t e , w i t h an i n t e r v e n i n g r e s i d u e B a c t i n g i n the c a p a c i t y o f a s p a c e r . I t i s p o s s i b l e t h a t t h e p o l a r n a t u r e o f r e s i d u e B i n f l u e n c e s t h e t y p e o f p o l y -s a c c h a r i d e s i d e c h a i n t o be a t t a c h e d d u r i n g b i o s y n t h e s i s . I t w a s ' i n t e r e s t i n g t h e r e f o r e t o f i n d o ut whether t h e h a p t o -g l o b i n c a r b o h y d r a t e - b i n d i n g sequence had t h e same c h a r a c t e r i s t i c s and t o w h i c h c a t e g o r y i t b e l o n g e d . The amino a c i d c o m p o s i t i o n o f t h e g l y c o p e p t i d e from t h e p r onase d i g e s t i o n o f t y p t i c 6 c h a i n g l y c o p e p t i d e shows a s p a r t i c a c i d 2 , t h r e o n i n e , s e r i n e , g l u t a m i c a c i d and a l a n i n e . Thus h a p t o -g l o b i n by v i r t u r e o f t h e complex n a t u r e o f t h e c a r b o h y d r a t e m o i e t y (mannose, g l y c o s a m i n e , f u c o s e , and s i a l i c a c i d ) , and t h e • p r e s e n c e of t h r e o n i n e and s e r i n e appears t o b e l o n g t o c a t e g o r y I g l y c o p e p t i d e s . A t h e r m o l y s i n g l y c o p e p t i d e (T-P-8-CNBr-8-B) from the p e p t i c d i g e s t o f g-CNBr-8-B was i s o l a t e d . The c o m p o s i t i o n o f t h i s p e p t i d e i s shown i n T a b l e XIX. The N - t e r m i n a l amino 166 TABLE XIX Amino Acid Composition of Thermolysin Glycopeptide Th-P-6-CNBr-8-B Th-P-B -CNBr-8-B (2nd Edman) Lysine 0. 004 His t i d i n e 0. 05 ( 1 ) 1 0. 015 (0.75) Arginine Aspartic A c i d 0.107 (2.1) 2 0. 037 (1.85) 2 Threonine 0. 033 (0.67) 1 0. 016 (0.9) 1 Serine 0. 04 (0.8) 1 0. 021 (1.0) 1 Glutamic Acid 0.043 (0.86) 1 0. 021 (1.0) 1 Proline 0. 007 Glycine Alanine 0. 047 (0.94) 1 0. 021 (1.0) 1 Valine 0. 002 Methionine Isoleucine Leucine 0. 058 (1.1) 1 0. 0034 Tyrosine Phenyalanine 0. 01 (0.2) Total 8 6 167 a c i d was d e t e r m i n e d as l e u c i n e and was s l i g h t l y c o n t a m i n a t e d by p h e n y a l a n i n e . A s u b t r a c t i v e Edman d e g r a d a t i o n was p e r f o r m e d on t h i s p e p t i d e and the r e s u l t i s a l s o i n c l u d e d i n T a b l e XIX. The r e s u l t s s u g g e s t e d the sequence a t t h e N - t e r m i n a l as L e u - H i s - . ( G i n , A s n , Asp, Thr, S e r , A l a ) . However, d a n s y l a t i o n a f t e r t h e second Edman d i d n o t r e v e a l any amino a c i d . S e v e r a l p o s s i b i l i -t i e s m i g h t a c c o u n t f o r t h e s e o b s e r v a t i o n s ; i f g l u t a m i n e was p r e s e n t i n p o s i t i o n 3, the n the f a c i l e c y c l i z a t i o n t o p y r r o l i -done d i c a r b o x y l i c a c i d c o u l d be o c c u r r i n g . A l t e r n a t i v e l y , i f r e s i d u e t h r e e were the a s p a r t a m i d o - c a r b o h y d r a t e complex l i n k a g e , t h e a s p a r t y l amino group would be s t e r i c a l l y h i n d e r e d from r e a c t i n g w i t h d a n s y l c h l o r i d e . No d i f f i c u l t i e s have been r e p o r t e d between the c o u p l i n g o f p h e n y l i s o t h i o c y a n a t e w i t h t h e a s p a r t i a m i d o -c a r b o h y d r a t e complex; thus the c a r b o h y d r a t e does n o t i n t e r f e r e w i t h the Edman d e g r a d a t i o n (144). T h e r e f o r e the l a t t e r p o s s i -b i l i t y i s u n l i k e l y , and t h e p r e s e n c e o f g l u t a m i n e a t p o s i t i o n 3 was t e n t a t i v e l y a s s i g n e d . L e u c i n e a m i n o p e p t i d a s e d i g e s t d i d n o t r e l e a s e amino a c i d s t o any s i g n i f i c a n t e x t e n t . S t e r i c h i n d r a n c e by the c a r b o h y d r a t e s i d e c h a i n s t o enzymic d i g e s t i o n o f g l y c o -p e p t i d e s has been e n c o u n t e r e d by o t h e r w o r k e r s (14 4 ) . A p a r t i a l amino a c i d sequence o f t h i s t h e r m o l y s i n g l y c o -p e p t i d e i s su g g e s t e d as f o l l o w s : CHO I L e u - H i s - G l n - ( A s n , A s x, Thr, S e r , . A l a ) . By c o m b i n i n g the t r y p t i c g l y c o p e p t i d e (13 amino a c i d s ) and the above t h e r m o l y s i n g l y c o p e p t i d e , the p a r t i a l sequence o f t h e t r y p t i c p e p t i d e i s 168 CHO V a l L e u - H i s - G l n - ( A s n , Asp, T h r , S e r , A l a ) A l a i n w h i c h t h r e e amino a c i d s ( g l y c i n e , l e u c i n e and i s o l e u c i n e ) from the t r y p t i c p e p t i d e cannot be d e f i n i t e l y a s s i g n e d . I n o r d e r t o c h a r a c t e r i z e the amino a c i d sequence around the a ttachment s i t e f u r t h e r , a t h e r m o l y s i n p e p t i d e was a g a i n i s o l a t e d from p e p t i d e g-CNBr-8-B. The g l y c o p e p t i d e was f i r s t i s o l a t e d from B i o - G e l P-10 column from t h e t h e r m o l y s i n d i g e s t , p a s s i n g t h r o u g h Dowex 50-X2 r e s i n t o remove any l a r g e n o n g l y c o -p e p t i d e s . The s i a l i c a c i d was t h e n removed by m i l d a c i d t r e a t -ment and f i n a l l y r echromatography was c a r r i e d o u t on t h e same B i o - G e l P-10 column. The amino a c i d c o m p o s i t i o n o f t h i s g l y c o -p e p t i d e , d e s i g n a t e d Th-g-CNBr-8-B i s shown i n T a b l e XX. A com-p a r i s o n between Th-P-g-CNBr-8-B and Th-g-CNBr-8-B r e v e a l e d t h e p r e s e n c e o f one more a s p a r t i c a c i d and one l y s i n e i n Th-g-CNBr-8-B. S i n c e Th-P-8-CNBr-8-B had been s u b j e c t e d t o p e p s i n d i g e s t b e f o r e t h e r m o l y s i n c l e a v a g e , i t c o n t a i n e d fewer amino a c i d ^ t h a n Th-8- X CNBr-8-B. D a n s y l a t i o n s t u d i e s showed t h a t , l i k e Th-P-6-CNBr-8-B, p e p t i d e Th-8-CNBr-8-B a l s o c o n t a i n s l e u c i n e as t h e N - t e r m i n a l ( F i g . 4 5 ) , so t h a t the e x t r a a s p a r t i c a c i d and l y s i n e occupy the C - t e r m i n a l p o s i t i o n s . S i n c e t h e t r y p t i c g l y c o p e p t i d e T-G I I c o n t a i n s one l y s i n e and two a s p a r t i c a c i d i n s t e a d o f t h r e e , t h i s X would s u g g e s t t h a t i n p e p t i d e Th-B-CNBr-8-B, a s p a r t i c a c i d i n s t e a d o f l y s i n e , occupy' the C - t e r m i n a l p o s i t i o n . The amino a c i d o f ^ ^ Th-g-CNBr-8-B i s t h e r e f o r e CHO L e u - H i s - G l n - ( A s n , Asx, T h r , S e r , A l a ) L y s - A s x . TABLE XX Amino Acid Composition of Th-B-CNBr-8-B and Its S u b t i l i s i n Peptides Th-6- CNBr-8-B S l S2 S3 S4 S5 Lys 0. 03.7 (0.74) 1 0.0097 (0. 74) 1 0. 011 (1. 00) 1 0.021 (0.84) 1 0. 022 (1. 00) 1 His 0.05 (1.00) 1 Asp 0.153 (3.00) 3 0.11 (1.00) 1 0.012 (0. 92) 1 0. 011 (1. 00) 1 Thr 0. 046 (0.92) 1 0.0089 (0. 68) 1 0.018 (0.72) 1 Ser 0.046 (0.92) 1 0.006 (0. 46) Glu 0.043 (0.86) 1 0.003 (0. 23) Ala 0. 063 (1.26) 1 0.013 (1. 00) 1 0. 012 (1. 00) 1 0.025 (1.00) 1 0. 021 (1. 00) 1 Leu 0. 055 (1.1) 1 Total 10 1 4 3 3 2 Mobil-i t y 1.9 1.34 1. 5 1.62 1. 91 N-term-i n a l Leucine Threonine Threonine 170 Figure 45. Dansylation studies of Th-3-CNBr-8-B. a) 1,9. DNS-isoleucine; 2,8. DNS alanine; 3,7. DNS l y s i n e ; 4,6. DNS leucine; 5. Th-6-CNBr-8-B. b) Same plate, rechromatographed i n solvent I I . 5. Contains leucine and e DNS l y s i n e . The sequence o,f, T-G. II i s CHO Val-(Gly, Leu, lieu) Leu-His-Gln-(Asn, Asp, Thr, Ser, Ala) Lys in which glycine, leucine and isoleucine are assigned at the N-terminal region. S u b t i l i s i n digestion of peptide Th-B-CNBr-8-B released f i v e peptides when analyzed on high voltage electrophoresis, pH 1.9, 4 KV f o r 1 hour. The separation of these peptides i s shown i n F i g . 46, and the amino acid compositions of these peptides, S^, S 2, S 3, S 4 and S 5 are included i n Table XX. Peptide S^, which i s the major component, contains only aspartic acid. Peptide S 2 i s a minor component, and contains threonine, alanine, aspartic acid and l y s i n e i n the analysis. This peptide i s contaminated by serine and glycine. However, the mobility of th i s peptide at pH 1.9 high voltage electrophoresis i s consistent with the above composition. Peptide S^ contains aspartic acid, alanine and l y s i n e . Peptide S^ contains threonine, alanine and l y s i n e . Peptide S,. contains only alanine and l y s i n e . I t i s apparent that these peptides are the p a r t i a l cleavage products of peptide Th-B~CNBr-8-B. Due to the s t e r i c hindrance of the carbohydrate unit, the enzyme released these peptides slowly (12 hours incubation, 1/50 enzyme substrate ratio) and i n poor y i e l d . Furthermore, those amino acids such as leucine, h i s t i d i n e and glutamine i n the N-terminal region remained i n t a c t , so that peptides S, , S„, S-,, S. and S^ are a l l derived from the 172 F i g u r e 46. S e p a r a t i o n o f s u b t i l i s i n p e p t i d e s o f Th-£-CNBr-8-B on pH 1.9 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 4 KV f o r 1 hour. 173 C - t e r m i n a l r e g i o n o f t h e p e p t i d e . The C - t e r m i n a l t e t r a p e p t i d e amino a c i d sequence was d e t e r m i n e d as CHO L e u - H i s - G l n - ( A s n , Asp, S e r ) T h r - A l a - L y s - A s x . The amino a c i d sequence o f Th-B-CNBr-8-B i s t h e r e f o r e s u g g e s t e d as CHO I L e u - H i s - G l n - A s x - A s n - S e r - T h r - A l a - L y s - A s x i n w h i c h Asx i s t e n t a t i v e l y a s s i g n e d as p o s i t i o n 4, so t h a t a g e n e r a l p a t t e r n A s p - S e r - T h r can be o b t a i n e d . I t i s s t i l l pos-s i b l e t h a t s e r i n e may occupy p o s i t i o n 4 i n s t e a d o f Asx. F i g . 47 summarizes t h e amino a c i d sequence o f t h e c a r b o -h y d r a t e a t t a c h m e n t s i t e o f t h e 3 c h a i n , w i t h a l l t h e o v e r l a p p i n g p e p t i d e s i n d i c a t e d . I t i s i n t e r e s t i n g t h a t the IgG heavy c h a i n has an amino a c i d sequence CHO G l n - P h e - A s n - S e r - T h r - I l e u , i n t h i s c a s e , p h e n y a l a n i n e r e p l a c e s Asx and i s o l e u c i n e r e p l a c e s a l a n i n e . T h i s a g a i n , d e m o n s t r a t e s a s t r u c t u r a l homology between t h e s e two t y p e s o f m o l e c u l e s w h i c h has been d i s c u s s e d i n t h e I n t r o d u c t i o n . CHO Val-(Gly, Leu, lieu) Leu-His-Gln-Asx-Asn-Ser-Thr'-Aia-Lys i iAsx 4 — T-G-II < . ) Th-B-CNBr-8-B * ; : — t Th-P-B-CNBr-8-B 4 » Pronase peptide « — Figure 4 7 . The amino acid sequence of the carbohydrate attach ment s i t e of haptoglobin 8 chain. CHAPTER VI: THE FRACTIONATION OF TRYPTIC MALEYLATED PEPTIDES AND THE ISOLATION, CHARACTERIZATION OF ARGININE PEPTIDES Introduction As can be seen from the studies of the CNBr cleavage of haptoglobin 6 chain, i t i s d i f f i c u l t to a l i g n the methionine peptides. This d i f f i c u l t y arises from the incomplete cleavage of a l l the methionine residues. This i s further complicated by the f a i l u r e to characterize a l l the t r y p t i c methionine peptides for overlapping purposes. Another approach i n determining the primary structure was therefore investigated. I t i s known that the ly s i n e residue can Mr-he. modified-with-various ways such as carbamylation (166), t r i -fl u o r o a c e t y l a t i o n (167) and maleylation (168). The modified l y s y l residue becomes immune to the action of t r y p s i n , and cleavage occurs only at arginine residues. The larger fragments obtained by t r y p t i c digestion can be cleaved again at the l y s y l p o s i t i o n a f t e r the blocking group i s removed. Maleylation of the l y s y l group was selected for various reasons. Blocking by the mal'eyl group i s r e v e r s i b l e and i t can be removed under mild conditions. Furthermore, the maleylated fragments are soluble at neutral pH, making the f r a c t i o n a t i o n procedure simple. This approach, using maleylation, has enabled De Lange et a l . (1969) to elucidate the amino acid sequence of c a l f thymus histone IV. T h i s c h a p t e r c o n s i s t s o f two s e c t i o n s . The f i r s t s e c t i o n d i s c u s s e s t h e c h a r a c t e r i z a t i o n o f the a r g i n i n e p e p t i d e s f o r the o v e r l a p p i n g p r o c e d u r e . The l a t e r s e c t i o n d e a l s w i t h t h e 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 o f t h e m a l e y l a t e d fragments a f t e r the t r y p t i c d i g e s t on t h e m o d i f i e d h a p t o g l o b i n 3 c h a i n . S i n c e t h e r e a r e o n l y f i v e a r g i n i n e r e s i d u e s i n the 8 c h a i n , t h e r e s h o u l d be s i x fragments from the m a l e y l a t e d 6 c h a i n upon t r y p t i c d i g e s t i o n . E x p e r i m e n t a l 1. T r y p t i c d i g e s t o f h a p t o g l o b i n B_ c h a i n The same p r e p a r a t i o n o f t h e Dowex 5 0-X2 chromatography used f o r t h e i s o l a t i o n o f t r y p t i c g l y c o p e p t i d e s w^e-re- a l s o used f o r t h e i s o l a t i o n o f a r g i n i n e p e p t i d e s . The p e p t i d e s were l o c a t e d u s i n g the phenanthrone quinone s t a i n . F r a c t i o n 70-76 ( T - A r g ^ ) , 149-159 ( T - A r g 2 3 ) , 175-194 ( T - A r g 4 ) and 232-242 ( T - A r g 5 ) were p o o l e d and t h e a r g i n i n e p e p t i d e s were l a t e r p u r i f i 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 . 2. C h y m o t r y p t i c d i g e s t o f h a p t o g l o b i n 6_ c h a i n H a p t o g l o b i n 6 c h a i n (150 mg) was d i g e s t e d w i t h 0.79 mg c h y m o t r y p s i n (3 x c r y s t a l l i n e , W o r t h i n g t o n B i o c h e m i c a l C o r p o r -a t i o n ) i n 50 ml o f 0.1 M NH 4HC0 3 pH 8.0. Soybean t r y p s i n i n h i b -i t o r (3 X c r y s t a l l i n e , W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n ) , 0.2 mg was added. The d i g e s t was k e p t a t 37°C f o r 2 h o u r s , and f i n a l l y l y o p h i l i z e d t w i c e w i t h w a t e r . The p e p t i d e m i x t u r e , was chromatographed on Dowex 50-X2 (H +) i o n exchange columns (2.2 x 86 cm) at 40°C. The gradient used was the same as i n the i s o l a -t i o n of t r y p t i c digest peptides. Phenanthrone quinone p o s i t i v e fractions were pooled, and the arginine peptides were further p u r i f i e d by paper high voltage electrophoresis. 3. Thermolysin (170) digest of haptoglobin 6_ chain Haptoglobin B chain (20 mg) i n 5 ml of 0.2 M N-ethyl-morpholine buffer containing 0.002 M Ca C l 2 was digested with 0.1 mg of thermolysin ( g i f t of Dr. T. Ando) at 37°C f o r 5 hours. A f t e r l y o p h i l i z a t i o n , the peptide mixture was applied on Whatman 3 MM paper, and high voltage electrophoresis at pH 4.0 was per-formed at 2 KV for 2 1/2 hours. Three arginine peptides Th-Arg 1 Th-Arg 2 and Th-Arg 3 were l a t e r p u r i f i e d on pH 6.5 and pH 1.9 hig] voltage electrophoresis and eluted with 30% acetic acid. 4. S u b t i l i s i n digest of B~CNBr-8-B Fragment B~CNBr-8-B (20 mg) i n 2 ml of 0.1 M NH4HC03 was digested with 0.05 mg of s u b t i l i s i n ( S u b t i l i s i n (B. S u b t i l i s ) Mann. Research Laboratory Incorporation, New York) at 37°C f o r 6 hours. The peptide mixture was l y o p h i l i z e d and fractionated on high voltage electrophoresis. One arginine peptide S-Arg^ was i s o l a t e d and characterized. 5. Preparation of mixed d i s u l f i d e B_ chain One gram of haptoglobin 2-1 was reduced at room tempera-ture i n 50 ml of sodium borate buffer, pH 8.6, (0.1 M boric acid 0.05 M sodium hydroxide) containing 0.05 M B -mercaptoethanol. After 1 hr, 10-fold excess of 2-hydroxyethyl d i s u l f i d e HEDS A (Aldrich Chemical Co., Milwaukee, Wisconsin) ^ -were" added and l e f t at room temperature for 3 hrs to allow d i s u l f i d e exchange to occur. The reaction mixture was then desalted i n a G-25 column (hold up volume 228 ml) e q u i l i b r a t e d with 0.2 N acetic acid, 0.01 M HEDS. The peptides-containing f r a c t i o n s were pooled, l y o p h i l i z e d , and l a t e r chromatographed i n a G-75 column (hold up volume 900 ml) e q u i l i b r a t e d with the same solvent. The mixed d i s u l f i d e B chain was well resolved from the mixed d i s u l f i d e s 2 1 a and a chains. Fractions containing the mixed d i s u l f i d e B chain were pooled and l y o p h i l i z e d . 6. Preparation. of maleylated mixed d i s u l f i d e B_ chain Mixed d i s u l f i d e Bchain (100 mg) i n 15 ml of 0.2 N phos-phate buffer (pH 8.0) was added with 100-fold molar excess of maleic anhydride (in terms of lysin e content) and the pH was maintained at 8.9 with NaOH at 4°C for 1 hr. The sample was then desalted on a Sephadex G-25 column (2 x 25 cm) using 0.1% NH4HC03 as solvent and the sample l a t e r l y o p h i l i z e d . The extent of maleylation of the B chain was measured by the trinitrobenzene-s u l f o n i c a c i d procedure (174) using L-leucine as standard amino acid. The r e s u l t s showed that 94% of the free NH2 groups were blocked. 7. T r y p t i c digest of maleylated B_ chain and chromatography of  maleylated peptides by gel f i l t r a t i o n The maleylated B chain (100 mg) was digested with 1 mg of TPCK-treated t r y p s i n (Calbiochem. B grade, l o t . 52441) i n 10 ml of 0.1 M NH.HCO., at 37°C for 2 hrs, and l y o p h i l i z e d d i r e c t l y . 179 The t r y p t i c d i g e s t o f t h e m a l e y l a t e d 8 c h a i n (80 mg) was t h e n a p p l i e d on a Sephadex G-75 column (2 x 150 cm) u s i n g 0.1 M NH^HCO^ as s o l v e n t . The e l u t i o n p r o f i l e i s shown i n F i g . 52. R e s u l t s Amino A c i d Sequence S t u d i e s on t h e A r g i n i n e P e p t i d e s from T r y p t i c D i g e s t The amino a c i d c o m p o s i t i o n o f t h e a r g i n i n e p e p t i d e s from t r y p t i c d i g e s t i s t a b u l a t e d i n T a b l e XXI. The N - t e r m i n a l amino a c i d s o f t h e s e p e p t i d e s a r e a l s o shown i n T a b l e XXI. The a c t u a l i d e n t i f i c a t i o n o f t h e s e d a n s y l amino a c i d s on t h i n l a y e r chromatography i s shown i n F i g . 48. P e p t i d e T - A r g 2 w h i c h c o n t a i n s m e t h i o n i n e w i l l n o t be d i s c u s s e d i n t h i s c h a p t e r , t h e r e a s o n b e i n g t h a t T - A r g 2 , when p o o l e d f r o m t h e Dowex column, was a l k y l a t e d i n s o l u t i o n w i t h X i o s o a c e t a m i d e and re-chromatographed on t h e Dowex column u s i n g t h e same c o n d i t i o n s . The a l k y l a t e d p e p t i d e , a f t e r a c h i e v i n g X one e x t r a p o s i t i v e c h a r g e , was r e t a r d e d and e l u t e d l a t e r t h a n o t h e r p e p t i d e s . However, t h e a l k y l a t e d p e p t i d e s were found t o decompose d u r i n g s t o r a g e and t h i s p r e v e n t e d f u r t h e r s t u d i e s on t h i s p e p t i d e . P e p t i d e T-Arg^ ( A s p - T y r - A l a - G l u - V a l - G l y - A r g ) i s a c i d i c a t pH 6.5 (e = - 1 ) ; t h e r e f o r e , b o t h a s p a r t i c a c i d and g l u t a m i c a c i d a r e i n t h e a c i d form. The sequence o f t h i s p e p t i d e was e s t a b l i s h e d u s i n g d a n s y l Edman and s u b t r a c t i v e Edman p r o c e d u r e s . The i d e n t i -f i c a t i o n o f t h e d a n s y l amino a c i d s i s shown i n F i g . 49 and F i g . 50. TABLE XXI Amino Acid Composition of Tr y p t i c Arginine Peptides T--Arg 1 T-Arg 2 T--Arg 3 T-•Arg 4 T-Arg 5 Lysine 0. 022 (0.072) Hist i d i n e Arginine 0.2 (1 .00) 1 0.013 (1.1) 1. 0.02 (0 . 6 4 ) 1 0.23 (0.82) 1 0.25 (1.2) 1 Aspartic Acid 0.2 (1.00) I 0.0 34 (2.9 3) 2 0.0 OS (0.14) 0.28 (1.00) 1 Threonine 0.0015 0.025 (0.83) 1 Serine 0.0029 0.03 (1.0) 1 0.258 (0.92) 1 Glutamic Acid 0.23 (1.16) 1 0.035 (2.99) 3 0.316 (1.11) 1 Proline 0.052 (1.65) 2 Glycine 0.22 (1-10 1 0'. 065 CO. 56 ) I 0.009 (0.29) 0.02:8 (0.1) 0.204 (1.0) 1 Alanine 0. 262 (1.32) 1 0.018 (1.55) 2 0.067 (2 . 0 8 ) 2 0.028 (0.1) Valine 0.218 (1.08) 1 0.034 (2.95) 3 0.032 (1.00) 1 0.595 (2.1) 2 Methionine 0.011 (1.0) 1 Isoleucine 0.015 (1.26) 1 Leucine 0.015 (1.28) I 0.031 (1.00) I 0. 024 (0.1) Tyrosine 0.194 (0.94) 1 0.013 (1.1) 1 Phenyalanine Total residues Mobility 6.5 -0.25 Mobility 1.9 0.88 N-terminal A s p a r t i c amino acid acid 16 Tyrosine 0.25 Valine 0.00 1.02 Valine 0.76 1.78 Glycine 181 0 l i t 11 22 Figure 48. N-terminal amino acid of arginine peptides. I, 16. Ileu, Met, Glu; 2, 17. Val, Ala, Asp; 3, 18. Pro, His; 4, 19. Leu, Gly; 5, 20. Phe, Thr; 6, 21, Lys, Ser; 7, 22. Tyr, Arg; 8. T-Arg, (aspartic acid); 9. T-Arg_ (valine); I I , 13. T-Arg 5(glycine); 12. T-Arg 4 (valine). 182 t I 18 Fiqure 49. Dansylation studies of arginine peptides a f t e r 1st and 2nd Edman degradations. I, 12. Ileu, Met, Glu; 2, 13. Val, Ala, Asp; 3, 14. Pro, His; 4, 15. Leu, Gly; 5, 16. Phe, Thr; 6, 17. Lys, Ser; 7, 18. Tyr, Arg; 8. T-Arg^ 1st cycle (tyrosine); 9. T-Arg 1 2nd cycle (alanine); 10. T-Arg^ 1st cycle (serine); I I . T-Arg. 2nd cycle (valine). 183 1 9 18 Figure 50. Dansylation studies of arginine peptides a f t e r 3rd and 4th Edman degradations. I, 12. Ileu, Met, Glu; 2, 13. Val, Ala, Asp; 3, 14. Pro, His; 4, 15. Leu, Gly; 5, 16. Phe, Thr; 6, 17. Lys, Ser; 7, 18. Thr, Arg; 8. T-Arg^ 3rd cycle (glutamic acid); 9. T-Arg^ 4th cycle (valine); 10. T-Arg. 3rd cycle (aspartic a c i d ) ; I I . T-Arg. 4th cycle (glutamic a c i d ) . The amino acid analysis, a f t e r second Edman and fourth Edman f degradations, are tabulated i n Table XXII. This peptide has also been i s o l a t e d from the t r y p t i c digest of 8-CNBr-8-E. T-Arg 3 (Val-Ala-(Thr, Ser)(Pro 2, Ala, Leu)Arg)was only p a r t i a l l y characterized due to the poor y i e l d of t h i s peptide. The dansyl-Edman method revealed alanine a f t e r one degradation. Valine, alanine, threonine and serine are the four amino acids released by leucine aminopeptidase. I t was expected that the presence of proline i n the peptide would retard the further degradation of the peptide by the enzyme. The r e s u l t of leucine aminopeptidase digestion i s shown i n Table XXIII. Peptide T-Arg 4(Val-Ser-Val-Asp-Glu-Arg) i s neutral at pH 6.5, i n d i c a t i n g that one of the d i c a r b o x y l i c acids i s i n i t s amide form. The sequence of t h i s peptide was established using the same method as with peptide T-Arg^, and the r e s u l t s are shown i n F i g . 49 and 50 and Table XXII for dansyl amino acids and subtractive amino acid analysis r e s p e c t i v e l y . The nature of the amino acid bearing the amide group was established using leucine aminopeptidase (91). I t can be seen i n Table XXIII that aspartic acid was completely absent i n the analysis, and an amide (appearing i n serine p o s i t i o n on the Analyzer) was quanti-t a t i v e l y recovered, making a t o t a l of two "serine" residues (one serine and one asparagine). However, glutamic acid was recovered as such. This suggests that only aspartic acid i s i n the form of asparagine. Peptide T-Arg,- (Gly-Arg) l i k e peptide T-Arg^, i s i n peptide B-CNBr-8-E. Since i t was found that the tryptophan residue was located at the N-terminal side of glycine, the production of 185 TABLE XXIII Leucine Aminopeptidase Digest of Arginine Peptides Time (in Hours) 8 14 20 ymole ymole ymole T--Arg 3 Arginine* 0.0057 (0. 38) Threonine - 0. 007 (0. 46) Serine 0.0094 (0. 62) Alanine 0.0087 (0. 58) Valine 0. 015 (1. 00) Leucine 0. 004 (0. 26) T--Arg 4 Argine 0.0495 (0. 99) Serine 0.1038 (2. 07) Glutamic Acid 0.059 (1. 1) Valine • 0.100 (2. 0) c-" A r9*4 Aspartic Acid 0. 007 (0. 46) • Serine 0. 003 (0. 2) Glutamic Acid 0.0026 (0. 17) Glycine 0.0016 (0. 11) Alanine 0.0047 (0. 3) Valine 0.01 (0. 66) * The arginine peak might represent the peptide instead of free arginine. I 186 TABLE XXII Amino Acid Analysis of T-Arg^ and T-Arg^ a f t e r Edman Degradation T-Arg 1 T-Arg x T-Arg 4 T-Arg 4 (2nd Cycle) (4th Cycle) (2nd Cycle) (4th Cycle) Lysine H i s t i d i n e Arginine 0. 016 (1) 0.012 (1) 0.0184 (0.91) 0.018 (0.9) Aspartic Acid 0.001 0.001 0.02 (1) 0. 009 Threonine Serine 0.066 0. 002 Glutamic Acid 0. 017 (1) 0.006 0.023 (1.15) 0.021 (1.0) Proline Glycine 0.016 (1) 0.014 (1) Alanine 0. 016 (1) 0. 005 Valine 0.016 (1) 0.012 (1) 0.018 (0.9) 0. 006 Methionine Isoleucine Leucine Tyrosine Phenyalanine 1st Edman cycle 2nd Edman cycle 3rd Edman cycle 4th Edman cycle Dansyl tyrosine Dansyl alanine Dansyl glutamic acid Dansyl va l i n e Dansyl serine Dansyl va l i n e Dansyl aspartic a c i d Dansyl glutamic acid t h i s peptide i s due to the chymotryptic-type cleavage of the porcine t r y p s i n . Studies on the Chymotryptic Arginine Peptides The chromatography of chymotryptic peptides on Dowex 50-X2 column was less s a t i s f a c t o r y . The arginine peptides were eluted late i n the gradient and were not very well resolved. However, four d i s t i n c t arginine peptides were p u r i f i e d by paper high voltage electrophoresis. The amino acid compositions of these peptides are shown i n Table XXIV. Peptides C-Arg^ and C-Arg,- contained approximately 25 amino acids each. The y i e l d of these two peptides from paper was low, hence no further studies were ca r r i e d out on these peptides. Peptide C-Arg 2(Gly-Arg-Asn-Ala-Asp-Phe) has been charac-t e r i z e d during the studies of 8-CNBr-8-E (Chapter I I I , p. 126). Peptide C-Arg 3(Ala-Glu-Val-Gly-Arg-Val-Gly-Tyr) overlaps with peptide T-Arg^. The structure of t h i s peptide was estab-l i s h e d using carboxypeptidase A. These r e s u l t s are shown i n Table XXV where i t may be seen that the sequence around the C-terminal of t h i s peptide i s Val-Gly-Tyr. This i s confirmed by the presence of dansyl valine and dansyl alanine from the t r y p t i c digest of th i s peptide. Since alanine i s the o r i g i n a l N-terminal of the peptide, the appearance of valine a f t e r the digest established the above sequence. ; Peptide C-Arg 4 (Val-Asp-(Ala, Asp, Glu2/ Pro, Gly, Val Met, l i e u , Leu, Arg)His-Tyr) was p a r t i a l l y characterized. The N-terminal region was studied using leucine aminopeptidase and TABLE XXIV Amino Acid Composition of Chymotryptic Arginine Peptides C-Arg-j^ C-Arg 2 C-Arg 3 C-Arg 4 C-Arg 5 Lysine 0. 064 (2. 00) 2 Hi s t i d i n e 0. 011 (0. 85) 1 0. 04 (1. 02) 1 0. 017 (0. 53) 1 Arginine 0. 013 (1. 00) 1 0.014 (1. 00) 1 0. 035 (1. 02) 1 0.042 (1. 13) 1 0. 036 (1. 1) 1 Aspartic Acid 0. 04 (3. 07) 3 0.029 (2. 07) 2 0. 072 (1. 97) 2 0. 123 (3. 84) 4 Threonine 0. 003 (0. 23) 0. 044 (1. 37) 1 Serine 0. 011 (0. 85) 1 0.004 (0. 28) 0.012 (0. 32) 0. 065 (2. 03) 2 Glutamic Acid 0. 037 (2. 84) 3 0.004 (0. 28) 0. 033 (0. 96) 1 0.088 (2. 3) 2 0. 137 (4. 2) 4 Proline 0. 025 (1. 9) 2 0.042 (1. 13) 1 0. 032 (1. 00) 1 Glycine 0. 025 (1. 9) 2 0.014 (1. 00) 1 0. 062 (1. 8) 2 0. 042 (1. 13) 1 0. 06 (1. 87) 2 Alanine 0. 076 (5. 84) 6 0.02 (1. 4) 1 0. 03 (0. 9) 1 0.045 (1. 12) 1 0. 058 (1. 81) 2 Valine 0. 026 (2. 00) 2 0. 061 (1. 8) 2 0.08 (2. 16) 2 0. 046 (1. 43) 1 Methionine 0. 007 (0. 54) 1 0. 027 (0. 72) 1 Isoleucine 0. 014 (1. 07) 1 0.026 (0. 72) 1 0. 022 (0. 7) 1 Leucine 0. 018 (1. 38) 1 0.027 (0. 72) 1 0. 059 (1. 84) 2 Tyrosine 0. 015 (1. 1) 1 0. 035 (1. 02) 1 0.037 (1. 00) 1 0. 025 (0. 8) 1 Phenyalanine 0.013 (1. 00) 1 0. 027 (0. 84) 1 Total 25 6 8 15 26 Mobili t y 6.5 0. 00 0.00 0. 00 0.0 -0. 025 Mobility 1.9 0. 79 0. 96 0. 94 0. 87 N-terminal Glycine Alanine Valine TABLE XXV Carboxypeptidase A Digest of Arginine Peptides 3 Time (in Hours) 15 20 7 5 C-Arg 3 Valine ymole ymole .0056 ymole (.22) ymole ymole Glycine . 0024 (. 096) . 008 (.32) Tyrosine .024 (. 96) .027 (1.08) C-Arg 4 H i s t i d i n e Tyrosine .023 (0.52) .045 (1.02) Th-Arg 1 H i s t i d i n e .016 (.8) S-Arg 1 Homoserine Valine .06 .073 (1.15) (1.4) CO VO 190 t h e C - t e r m i n a l r e g i o n by c a r b o x y p e p t i d a s e A. T h e s e r e s u l t s a r e shown i n T a b l e s X X I I a nd XXV, r e s p e c t i v e l y . S i n c e o n l y h i s t i d i n e a n d t y r o s i n e w e r e r e l e a s e d b y c a r b o x y p e p t i d a s e A, t h i s i n d i c a t e d t h a t t h e r e s i d u e a d j a c e n t t o h i s t i d i n e i s r e f r a c t o r y t o d i g e s t i o n , w h i c h may be p r o l i n e o r a r g i n i n e . L a t e r s t u d i e s o n t h e t h e r m o l y -s i n p e p t i d e T h - A r g 3 ( I l e u - A r g - H i s ) v e r i f i e d t h e a b o v e o b s e r v a t i o n . S t u d i e s on t h e T h e r m o l y s i n A r g i n i n e P e p t i d e s S i n c e t h e c h y m o t r y p s i n p e p t i d e s w e r e d i f f i c u l t t o c h a r a c t e r -i z e , a t h e r m o l y s i n d i g e s t was p e r f o r m e d o n h a p t o g l o b i n $ c h a i n , h o p i n g t h a t s m a l l e r p e p t i d e s w o u l d r e s u l t w h i c h c o u l d t h e n be c h a r a c t e r i z e d more r e a d i l y . U n l i k e c h y m o t r y p s i n , t h e r m o l y s i n h a s a much b r o a d e r s p e c i f i c i t y ( 1 7 1 , 1 7 2 ) . T h e r m o l y s i n u s u a l l y h y d r o l y z e s t h e p e p t i d e b o n d s i n v o l v i n g t h e a-NH^ g r o u p o f h y d r o -p h o b i c amino a c i d s w i t h b u l k y s i d e c h a i n s . T h i s s p e c i f i c i t y i s b o r n o u t by t h e i s o l a t i o n o f t h r e e a r g i n i n e p e p t i d e s h a v i n g v a l i n e , a l a n i n e a n d i s o l e u c i n e a s N - t e r m i n a l r e s i d u e s . The amino a c i d c o m p o s i t i o n o f t h e s e p e p t i d e s i s shown i n T a b l e X X V I . P e p t i d e T h - A r g 1 ( V a l - A s n - G l u - A r g ) d e r i v e s f r o m p e p t i d e T - A r g ^ ( V a l - S e r - V a l - A s n - G l u - A r g ) . The s e q u e n c e was c o n f i r m e d b y two d a n s y l Edman d e g r a d a t i o n s . P e p t i d e T h - A r g 2 ( V a l - G l y - A r g + A l a - M e t - A s n - L y s ) c o n t a i n s two p e p t i d e s w h i c h w e r e d i f f i c u l t t o s e p a r a t e due t o t h e i r i d e n t i c a l m o b i l i t y a t d i f f e r e n t p H's. D a n s y l a t i o n o f t h e m i x t u r e r e v e a l e d v a l i n e and a l a n i n e as t h e N - t e r m i n a l r e s i d u e s . G l y c i n e a n d s m a l l amounts o f m e t h i o n i n e " a p p e a r e d a f t e r t h e f i r s t Edman c y c l e . S i m i l a r l y , a r g i n i n e and a s p a r t i c a c i d w e r e r e v e a l e d TABLE XXVI Amino Acid Composition of Thermolysin Arginine Peptides and S u b t i l i s i n Peptides Th-Arg 1 Th-Arg 2 Th-Arg. S - A ^ Lysine H i s t i d i n e Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenyalanine Homoserine Lactone Total Residues Mobility 6.5 0.00 Mobility 1.9 1.21 N-terminal Valine 0.05 (1.00) 1 0.051 (1.0) 1 0.054 (1.08) 1 0.045 (0.9) 1 0.053 (1.03) 0.051 (1.0) 0.039 (0.76) 0.056 (1.09) 0.048 (0.94) 0.037 (0.72) 0.013 (0.3) 0.6 Valine, Alanine 0.063 (1.00) 1 0.063 (1.00) 1 0.0068 (1.07) 1 0. 81 Isoleucine 0.054 (1.00) 1 0.016 (0.29) 0.072 (1.3) 1 0.013 (0.24) 0.08 (1.4) 1 0.021 (0.38) 1 0. 00 1. 22 Glutamic Acid a f t e r the second Edman c y c l e . S i n c e V a l - G l y - A r g has been d e t e r -mined p r e v i o u s l y w i t h p e p t i d e C - A r g 3 ( A l a - G l u - V a l - G l y - A r g - V a l -G l y - T y r ) , t h e sequence o f A l a - M e t - A s n - L y s was t h e r e f o r e deduced. I t can be seen t h a t t h e r e l e a s e o f p e p t i d e V a l - G l y - A r g from p e p t i d e C - A r g 3 a g r e e s w i t h t h e s p e c i f i c i t y o f t h i s enzyme. P e p t i d e T h - A r g 3 ( I l e u - A r g - H i s ) was d e t e r m i n e d u s i n g t h e d a n s y l Edman p r o c e d u r e . T h i s sequence i s c o n s i s t e n t w i t h t h e c a r b o x y p e p t i d a s e A s t u d i e s o f p e p t i d e C-Arg^. As e x p e c t e d , c a r -b o x y p e p t i d a s e A d i g e s t i o n r e l e a s e d o n l y h i s t i d i n e from p e p t i d e T h - A r g 3 . S t u d i e s on t h e S u b t i l i s i n - A r g i n i n e P e p t i d e o f B-CNBr-8-B From t h e s t u d i e s o f t r y p t i c , c h y m o t r y p t i c and t h e r m o l y t i c a r g i n i n e p e p t i d e s , t h e N - t e r m i n a l and C - t e r m i n a l r e g i o n s around the a r g i n i n e r e s i d u e were e s t a b l i s h e d f o r t h r e e o f t h e a r g i n i n e p e p t i d e s . The C - t e r m i n a l r e g i o n o f t h e o t h e r two p e p t i d e s how-ev e r remained t o be e l u c i d a t e d b e f o r e a complete o v e r l a p p i n g o f the m a l e y l a t e d fragments was p o s s i b l e . D u r i n g t h e s t u d i e s o f t h e g l y c o p e p t i d e s o f B-CNBr-8-B, an a r g i n i n e p e p t i d e c o n t a i n i n g o n l y g l u t a m i c a c i d and a r g i n i n e was i s o l a t e d from a pronase d i g e s t . T h i s s u g g e s t e d t h e p r e s e n c e o f p e p t i d e T - A r g 4 i n fragment B-CNBr-8-B. S u b t i l i s i n (173) was used, due t o i t s d i f f e r e n t s p e c i f i c i t y f rom t r y p s i n and t h e r m o l y s i n . The use o f t h e s e l a t t e r enzymes would most l i k e l y produce t h e same p e p t i d e s T-Arg^ and Th-Arg.^. One s u b t i l i s i n p e p t i d e S-Arg-j^ ( G l u - A r g - V a l - H s r ) was i s o -l a t e d from t h i s d i g e s t . The amino a c i d c o m p o s i t i o n o f t h i s 193 p e p t i d e i s shown i n T a b l e XXVI. The y i e l d o f homoserine l a c t o n e was somewhat low, because o f t h e e q u i l i b r i u m between homoserine and homoserine l a c t o n e . The t r e a t m e n t w i t h 2 N NH^OH was n o t perf o r m e d on t h e a c i d h y d r o l y s a t e because t h e p r e s e n c e o f homo-s e r i n e was n o t o r i g i n a l l y s u s p e c t e d . S i n c e g l u t a m i c a c i d i s t h e N - t e r m i n a l o f t h i s p e p t i d e , t h e above sequence i s s u g g e s t e d . T h i s i s c o n f i r m e d from t h e c a r b o x y p e p t i d a s e A d i g e s t s t u d i e s . The p e p t i d e was f i r s t t r e a t e d w i t h 2 N NH^OH t o c o n v e r t a l l t h e homo-s e r i n e l a c t o n e i n t o homoserine w h i c h was r e l e a s e d r a p i d l y by t h e enzyme. Homoserine (which, i n t h i s case emerged i n t h e same p o s i t i o n as g l u t a m i c a c i d i n t h e s i n g l e column system) and v a l i n e were r e c o v e r e d i n q u a n t i t a t i v e y i e l d s . The r e s u l t o f t h i s s t u d y i s shown i n T a b l e XXV. The Amino A c i d Sequence o f t h e A r g i n i n e P e p t i d e s The o v e r a l l amino a c i d sequence o f t h e a r g i n i n e p e p t i d e s i s o l a t e d from d i f f e r e n t enzyme d i g e s t s i s shown i n F i g . 51. A r g i n i n e group I p e p t i d e s c o n s i s t o f T - A r g , T h - A r g 2 and C-Arg^. T-Arg^ and C-Arg^ a r e i d e n t i c a l w i t h p e p t i d e T^ and C 2, r e s p e c t i v e l y , from fragment g-CNBr-8-E. These group I p e p t i d e s o f t e n amino a c i d s a r e t h e r e f o r e d e r i v e d from fragment 6-CNBr-8-E. A r g i n i n e group I I p e p t i d e s c o n s i s t o f T - A r g 2 , C-Arg^ and Th-A r g ^ and were o n l y p a r t i a l l y c h a r a c t e r i z e d . S i n c e t h i s group c o n t a i n s m e t h i o n i n e , i t o v e r l a p s w i t h t h e s t u d i e s o f m e t h i o n i n e p e p t i d e . A r g i n i n e group I I I p e p t i d e c o n s i s t s o f o n l y one t r y p t i c a r g i n i n e p e p t i d e T-Arg... T h i s p e p t i d e c o n t a i n s two p r o l i n e Group I: T _ A r g i Asp-Tyr-Ala-Glu-Val-Gly-Arg-Val-Gly-Tyr < * Th-Arg 2 C-Arg 3 Group I I : T-Arg„ ^ *> (Glu,Val,Ala)-Tyr-Val-Asp-Ala-(Asp,Glu 2,Gly 2,Val,Pro,Met,Leu)Ileu-Arg-His-Tyr Th-Arg 3 Group III C-Arg 4 T-Arg 3 Group IV: Val-Ala-Ser-Thr-(Pro 2,Ala,Leu)Arg T-Arg r-($ly-Arg-Asn-Ala-Asp-Phe < > C-Arg 0 Group V: T-Arg 4 Val-Ser-Val-Asn-Glu-Arg-Val-Hsr Th-Arg^j^ S-Arg-^ Figure 51. The amino acid sequence of arginine peptides of haptoglobin 6 chain. vo residues located on the N-terminal side of the arginine residue. Arginine group IV (T-Argj. + C-Arg2) l i k e arginine group I peptides were derived from fragment B-CNBr-8-E. Id e n t i c a l pep-tides T^g and have been i s o l a t e d from the above fragment B-CNBr-8-E. Arginine group V peptides (T-Arg^, Th-Arg^, S-Arg^) contain homoserine as the C-terminal amino acid, and therefore are located at the C-terminal of fragment 8~CNBr-8-B. Another i n t e r e s t i n g feature of t h i s group of peptides i s that t h i s over-laps with the N-terminal region of B-CNBr-8-E (Pro-Ileu-Cys-Pro-Leu-Ser-Lys) with the use of methionine peptide (Val-Met-Pro-Ileu-Cys-Pro-Leu-Ser-Lys). These two fragments,B -CNBr-8-B and B-CNBr-8-E, are therefore linked. Dansylation Studies of the T r y p t i c Malaylated Peptides As can be seen i n F i g . 52, 7 major components can be f r a c -tionated under these conditions. These are designated as T-M-B I, T-M-B I I , T-M-B I I I , T-M-B IV, T-M-B V, T-M-B VI and T-M-B VII, respectively. Dansylation studies of the unfractionated mixture are shown in F i g . 53. The control maleylated 8 chain which had not been subjected to enzymic digestion did not reveal any DNS-amino acid. This suggests that the N-terminal (isoleucine) amino acid and the e-NB^ groups of l y s i n e were completely blocked as had already been indicated by the measurement of the free NB^ groups using the trinitrobenzene s u l f o n i c acid procedure. On the other hand, i n the sample treated with t r y p s i n , dansyl-valine, glycine, 0.6 o oo 0, (N -P (0 Q 0.2 o-Q-^-o 50 75 100 125 -o-o Figure 52. Fraction Number Chromatography of t r y p t i c maleylated peptides of haptoglobin 6 chain on a Sephadex G-75 column (2 x 150 cm) using 0.1 M NH^HCC^ as solvent. U5 16 Figure 53. Dansylation of t r y p t i c digest of maleylated 6 chain. 1, 10. Ileu, Met, Glu; 2, 11. Val, Ala, Asp; 3, 12. Pro, His; 4, 13. Leu, Gly; 5, 14. Phe, Thr; 6. Maleylated 8 chain control; 7. Tryptic digest of maleylated 8 chain; 8, 15. Lys, Ser; 9, 16. Tyr, Arg. 198 aspartic acid, alanine and glutamic acid were present. Since the o r i g i n a l N-terminal of the 6 chain was blocked, the absence of dansyl isoleucine was expected. Furthermore, as only f i v e N-terminal amino acids were detected a f t e r the enzyme digestion, t h i s indicated that the cleavage was r e l a t i v e l y s p e c i f i c . 6 From the studies of the arginine peptides, one would pre-d i c t the N-terminal amino acids for the maleylated peptides of 8 chain as va l i n e (arginine group I and group V), h i s t i d i n e (group I I ) , and aspartic acid (arginine group IV) assuming that tr y p s i n only cleaved at the arginine residues without attacking the modified l y s i n e residue or other amino acids. The N-terminal amino acid from group III remained unknown. Since dansyl h i s t i -dine i s unstable upon acid hydrolysis, the presence of t h i s amino acid was d i f f i c u l t to demonstrate. In our present TLC system, dansyl glycine and 0-dansyl-tyrosine were not separated, so that alanine and glutamic acids were the two amino acids that appeared and these were inconsistent with cleavage at arginine. This would imply that at l e a s t one of the two amino acids was derived from a non-specific cleavage by t r y p s i n . This has, i n f a c t , been encountered previously during the studies of 8 -CNBr-8-E using porcine t r y p s i n . Although the TPCK-treated enzyme should be more s p e c i f i c than porcine t r y p s i n , the chymotryptic cleavages must s t i l l have occurred as w i l l be seen l a t e r by dansylation of the i n d i v i d u a l components. The present section therefore serves as a preliminary report of the f r a c t i o n a t i o n of these maleylated peptides and the p a r t i a l characterization of these peptides. 199 C h a r a c t e r i z a t i o n o f M a l e y l a t e d P e p t i d e s Fragment T-M-3 V I I was t h e s m a l l e s t component among t h e m a l e y l a t e d t r y p t i c p e p t i d e s . D a n s y l a t i o n r e v e a l e d t h e p r e s e n c e o f b o t h D N S - v a l i n e and D N S - a s p a r t i c a c i d , w i t h D N S - v a l i n e b e i n g p r e s e n t i n l a r g e r amounts. F i g . 54 t h u s i n d i c a t e s t h e p r e s e n c e o f more th a n one component. To c h a r a c t e r i z e these' p e p t i d e s , t h e y were a n a l y 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 a t pH 6.5. Three major p e p t i d e s can be s e p a r a t e d under t h e s e c o n d i t i o n s as can be seen i n F i g . 55. T-M-3 V I I (3) T-M-B V I I (2) T-M-B V I I (1) F i g u r e 55. High v o l t a g e e l e c t r o p h o r e s i s o f T-M-3 V I I on pH 6.5. Both p e p t i d e s T-M-3 V I I (2) and T-M-B V I I (3) a r e p h e n a n t h r e n -equinone and E h r l i c h p o s i t i v e , i n d i c a t i n g t h e p r e s e n c e o f b o t h a r g i n i n e and t r y t o p h a n r e s i d u e s . F u r t h e r m o r e , s i n c e b o t h p e p t i d e s are b a s i c under t h e s e c o n d i t i o n s , a s p a r t i c a c i d , g l u t a m i c a c i d and e - m a l e y l - l y s i n e were u n l i k e l y t o be p r e s e n t . C a r e f u l i n s p e c t i o n o f t h e amino a c i d sequence o f B-CNBr-8-E would s u g g e s t t h e sequence as V a l - S e r - G l y - T r y - G l y - A r g and V a l - G l y -T y r - V a l - S e r - G l y - T r y - G l y - A r g f o r V I I (3) and V I I ( 2 ) , r e s p e c t i v e l y . T h i s was i n f a c t c o n f i r m e d by the amino a c i d c o m p o s i t i o n o f t h e s e p e p t i d e s a f t e r f u r t h e r p u r i f i c a t i o n by pH 1.9 h i g h v o l t a g e e l e c t r o -DNP l y s i n e 200 1 11 Figure 54. Dansylation of t r y p t i c maleylated fragments of 8 chain. 1, 15. Ileu, Met, Glu; 2, 16. Val, Ala, Asp; 3, 17. Pro, His; 4, 18. Leu, Gly; 5, 19. Phe, Thr; * 6, 20. Lys, Ser; 7, 21. Tyr, Arg; 8. T-M-B I; 9. T-M-B II; 10. T-M-B I I I ; 11. T-M-B IV; 12. T-M-B V; 13. T-M-B VI; 14. T-M-B VII. phoresis. The amino acid composition^of these peptides are Y shown i n Table XXVII. Since these two peptides have valine as the N-terminal, t h i s agrees with the dansylation studies of these maleylated fragments. Peptide T-M-B VII (1), which i s neutral at pH 6.5, resolved into two components. One of the components gave an analysis of v a l i n e , glycine and tyrosine consistent with the sequence Val-Gly-Tyr, which has i n f a c t been established i n B-CNBr-8-E so that t h i s peptide must be the chymotryptic cleavage product. Maleylated fragment T-M-B VI, i s the second smallest i n size and also contains valin e as the major N-terminal amino acid. Alanine, glycine (or O-dansyl-tyrosine) and aspartic acid were present i n minor quantities.' The amino acid compos-i t i o n of the unfractionated mixture i s shown i n Table XXVII. From the studies of arginine peptides, both group I and group V peptides contain valine at the C-terminal side of a r g i -nine. Since T-M-6 VII was linked to group I, peptide T-M-6 VI was compared with arginine group V peptides. I t was known previously from the sequence studies of B-CNBr-8-E that the N-terminal region of B-CNBr-8-E was connected to C-terminal of B-CNBr-8-B. The amino acid sequence of t h i s enlarged fragment of 6-CNBr-8-E i s shown i n F i g . 56. As can be seen i n the fi g u r e , arginine group V peptide and arginine I peptides are close to each other. The amino acid sequence between these two arginine residues i s Arg-Val-Met-Pro-Ileu-Cys-Pro-Leu-Ser-Lys-Asp-Tyr-Ala-Glu-Val-Gly-Arg. The amino acid composition of T-M-B VI did i n f a c t correspond TABLE XXVII Amino Acid Composition of Maleylated Peptides T-M-B III T-M-B V T-M-B VI T-M-B VII (3) T-M-B VII (2) Lysine 0.087 (6.2) 0.2 (1. 64) Hi s t i d i n e 0. 025 (1.8) 0. 083 (1. 7) n i l (0. 0 Arginine 0.014 (1.00) 0. 04 (0. 8) 0. 077 (0. 65) 0.04 (0. 51) 1 0. 008 (0. 5) 1 Cysteic Acid 0. 093 (1. 9) 0.108 (0. 9) Aspartic Acid 0.095 (6.8) 0.25 (5. 1) 0.27 (3. 08) Threonine 0.061 (4.35) 0.17 (3. 06) 0.078 (0. 65) Serine 0.046 (5.28) 0.21 (4. 28) 0.199 (1. 62) 0. 077 (1. 00) 1 0. 016 (1. 00) 1 Glutamic Acid 0.14 (2. 87) 0.30 (2. 5) Proline 0. 082 (1. 67) 0.155 (1. 4) Glycine 0.094 (6.7) 0.23 (4. 7) 0.163 (1. 35) 0.229 (2. 89) 3 0.03 (1. 87) 2 Alanine 0.053 (3.8) 0.19 (3. 87) 0.258 (2. 1) Valine 0. 078 (5.57) 0.13 (2. 7) 0. 355 (2. 91) 0.140 (1. 77) 2 0. 011 (0. 68) 1 Met-Sulfone 0. 05 (1. 00) 0.12 (1. 00) Isoleucine 0. 054 (3.84) 0.059 (1. 2) 0.194 (1. 61) Leucine 0.121 (8.64) 0.12 (2. 4) 0.153 (1. 27) Tyrosine 0. 018 (1.28) 0.043 (0. 88) 0. 04 (0. 03) 0.03 (0. 35) 1 Phenyalanine 0.0134 (0.95) 0. 051 (1. 03) n i l N-terminal Amino Acid . Blocked (Isoleucine) Aspartic Acid Valine Valine Valine Val-Ser-Val-Asn-Glu-Arg-Val-Met-Pro-Ileu-Cys-Pro-Leu-Ser-Lys-Asp-Tyr-Ala-« y T-Arg, S-Ar^g1 M, Glu-Val-Gly-Arg-Val-Gly-Tyr-Val-Ser-Gly-Try-Gly-Arg-Asn-Ala-Asp-f V ,T-'t 6-CNBr-8-E Phe-Lys-Phe-Thr-Asp-His-Leu-Lys-Tyr-Val-Hsr Figure 56. Enlarged sequence of 3_CNBr-8-E. 204 clo s e l y to t h i s sequence of 16 amino acids notably by the presence and the expected number of methionine sulfone (1), c y s t e i c acid (1), p r o l i n e (2), leucine (1), tyrosine (1), isoleucine (1) and arginine (1) i n the analysis. Some of the other amino acids e s p e c i a l l y the a c i d i c amino acids were somewhat higher due probably to the presence of the other minor components revealed by dansylation. Peptide T-M-B V contains mainly aspartic acid as the N-terminal. The amino acid composition i s shown i n Table XXVII. Since t h i s peptide contains aspartic acid as the N-terminal, i t i s l i k e l y that t h i s peptide i s rela t e d to the arginine group IV peptide which i s i n B-CNBr~8-E. Furthermore, the amino acid eomposition of t h i s peptide revealed the presence of two CMC residues and one arginine which i s cl o s e l y s i m i l a r to B-CNBr-8-C and B"CNBr^8-iD. This would suggest that peptide T-M-B V i s composed of the l a t e r portion of B-CNBr-8-E and some major part of B-CNBr-8-D. Peptide T-M-B III did not show any amino acid upon dansy-l a t i o n . This suggests that e i t h e r pepetide T-M-B III contains h i s t i d i n e as the N-terminal, and di-DNS-histidine was destroyed during acid hydrolysis or a l t e r n a t i v e l y , peptide T-M-B III represents the blocked N-terminal of the 8 chain. A leucine aminopeptidase digest of t h i s peptide d i d not reveal any amino acid, i n d i c a t i n g that N-terminal i s , i n f a c t , blocked, thus T-M'-B H I must be the N-terminal of the B chain. Peptide T-M-B 1/ contained aspartic acid as i t s N-terminal, but, the amino acid analysis was not s a t i s f a c t o r y . Since T-M-B I contains a large amount of hexose, i t i s rela t e d to M-8-CNBr-8-A I from t h e cyanogen e s t u d i e s . P e p tidesT-M-g I I and T-M-g IV were s t i l l h e t erogeneous so -/ t h a t amino a c i d c o m p o s i t i o n s t u d i e s were n o t p e r f o r m e d . How-e v e r , T-M-g I I c o n t a i n s c a r b o h y d r a t e and i s t h u s r e l a t e d t o CNBr-8-B. The i n f o r m a t i o n a c h i e v e d from t h e s t u d i e s o f a r g i n i n e pep-t i d e s and t r y p t i c m a l e y l a t e d p e p t i d e s i s i l l u s t r a t e d i n F i g . 57 o f C h a p t e r V I I t o g e t h e r w i t h t h e s t u d i e s o f cyanogen bromide p e p t i d e s . P e p t i d e T-M-g I I I w h i c h c o n t a i n s the b l o c k e d N - t e r m i n a l c o n s t i t u t e s t h e N - t e r m i n a l r e g i o n o f t h e h a p t o g l o b i n g c h a i n . A r g i n i n e g r o u p I I p e p t i d e o c c u p i e s t h e C - t e r m i n a l p o s i t i o n o f p e p t i d e T-M-g I I I and v e r y l i k e l y t h e N - t e r m i n a l o f T-M-g I I . The e v i d e n c e o f t h i s l i n k a g e w i l l be d i s c u s s e d l a t e r i n t h e l a s t c h a p t e r . B o t h p e p t i d e s VI and V I I a r e d e r i v e d from cyanogen bromide c l e a v a g e p e p t i d e g-CNBr-8-E. The sequence*, o f t h e s e two p e p t i d e s have t h e r e f o r e been e s t a b l i s h e d . A r g i n i n e group I and IV pep-t i d e s hence o v e r l a p w i t h t h e s e p e p t i d e s and t h e i r p o s i t i o n s i n t h e whole c h a i n i s e s t a b l i s h e d . F u r t h e r m o r e , s i n c e p e p t i d e T-M-g V c o n t a i n s a s p a r t i c a c i d as t h e N - t e r m i n a l , t h i s p e p t i d e i s t h e r e f o r e l i n k e d t o p e p t i d e T-M-g V I I t h r o u g h a r g i n i n e group IV p e p t i d e . I n t h e s t u d i e s o f cyanogen bromide and m e t h i o n i n e p e p t i d e s , i t has e s t a b l i s h e d t h a t g-CNBr-8-B and g-CNBr-8-E a r e a d j a c e n t t o each o t h e r . Though p e p t i d e T-M-g I I was not pure enough f o r s ubsequent s t u d i e s , s i n c e i t c o n t a i n s c a r b o h y d r a t e s i t i s r e a s o n a b l e t o assume t h a t p e p t i d e I I and VI a r e j o i n e d by a r g i n i n e group V p e p t i d e s . The p r e s e n t i n f o r m a t i o n c o u l d t h e r e f o r e a c c o u n t f o r f o u r o f t h e a r g i n i n e p e p t i d e s and f i v e o f t h e t r y p t i c m a l e y l a t e d f r a g -ments, l e a v i n g a r g i n i n e p e p t i d e group I I I , T-M-B I , T-M-B IV u n a s s i g n e d . However, s i n c e p e p t i d e T-M-B V c o n t a i n s a r g i n i n e i n the a n a l y s i s , t h i s i m p l i e s t h a t a r g i n i n e o c c u p i e s the C - t e r m i n a l p o s i t i o n i n t h i s p e p t i d e . F u r t h e r m o r e , s i n c e a r g i n i n e group I I I p e p t i d e i s t h e o n l y group o f a r g i n i n e p e p t i d e s u n l o c a t e d , i t t h e r e f o r e must occupy t h i s p o s i t i o n . The amino a c i d c o m p o s i t i o n s o f p e p t i d e T-M-B V i s c o n s i s t e n t w i t h t h e c o m p o s i t i o n o f a r g i n -i n e group I I I , n o t a b l y t h e p r e s e n c e o f two. p r o l i n e r e s i d u e s . As t h e n a t u r e o f T-M-B I i s not d e t e r m i n e d , p e p t i d e T-M-B IV i s s u g g e s t e d t o be t h e C - t e r m i n a l p e p t i d e o f t h e B c h a i n , and p e p t i d e T-M-B I might p o s s i b l y be d e r i v e d from t h i s p a r -t i c u l a r r e g i o n o f t h e B c h a i n . 207 CHAPTER VII: THE PRIMARY STRUCTURE OF THE HAPTOGLOBIN 8 CHAIN The primary structure of the haptoglobin 8 chain has been studied i n the present i n v e s t i g a t i o n . Two d i f f e r e n t methods were employed to characterize t h i s polypeptide chain. Though the studies of cyanogen bromide cleavage peptides and the t r y p t i c maleylated peptides are not yet completed, i t i s possible to construct an o v e r a l l structure for 8 chain. Figure 57 i l l u s t r a t e s the structure deduced from the present studies. As discussed i n Chapter I I , the cyanogen bromide reaction, though very s p e c i f i c for methionine residues, was not complete. The f a i l u r e to achieve quantitative cleavage might be r e l a t e d to the highly aggregated nature of the 8 chain. Though only f i v e fragments were expected, nine fragments were a c t u a l l y i s o l a t e d and p a r t i a l l y characterized. Peptide 8~CNBr-8-B, 8~CNBr-8-C, B-CNBr-8-D, 8~CNBr-8-E and 8~CNBr-8-F contained homoserine and no i n t a c t methionine i n the analysis and must therefore, repre-sent complete cleavage products of the cyanogen bromide reaction. The sum of the amino acid compositions of these peptides plus the C-terminal peptide, 8~CNBr-8-A IV, gives a t o t a l of 339 amino acid residues. Since the 8 chain, from i t s molecular weight and amino acid analysis, contains only 301 amino acids, t h i s value implies some redundancy. However, peptide B-CNBr-8-A IV, though i t did not contain any homoserine, s t i l l has one methionine by analysis, so that i t was assumed to be an incomplete cleavage product. The difference between the number of residues 208 comprising the sum of the cyanogen bromide peptides and the t o t a l 8 chain i s 38. Fragments B-CNBr-8-D, 8-E and 8-F are a l l approxi-mately t h i s size and any one could be a part of B-CNBr-8-A IV. This being the case, the p a r t i c u l a r 38 residue fragment would be included twice i n the summation and would, therefore, account for the extra 38 residues. Since B-CNBr-8-F derives from the N-terminal region of the B chain, the presence of t h i s fragment i n M-B-CNBr-8 IV i s u n l i k e l y . I t i s therefore possible that M-B-CNBr-8 IV might produce a f t e r CNBr cleavage, 8-E, or 8-D together with a unique C-terminal peptide i f complete cleavage had taken place. However, no C-terminal fragment containing no methionine or homoserine was i s o l a t e d . Peptide B-CNBr-8-C also contained methionine and homoserine by analysis. Prelim-inary experiments indicated that B~CNBr-8-C might consist of B-CNBr-8-E and B~CNBr-8-D. Tryptic d i g e s t i f B-CNBr-8-C gave two arginine peptides having the same mobility i n pH 6.5 high voltage electrophoresis as Gly-Arg and Asp-Tyr-Ala-Glu-Val-Gly-Arg which are present i n B~CNBr-8-E. Furthermore, the sum of A B-CNBr-8-E and B-CNBr-8-D i s close to that V C N B r-8-C. Though <. B-CNBr-8-C was thought to contain valin e as the N-terminal, i t i s possible that the N-terminus could be pr o l i n e instead because valine and proline have very s i m i l a r m o b i l i t i e s i n our TLC system. However, i t should be possible to e s t a b l i s h unequivocally whether 8-D and 8-E are derived from 8-C by preparing peptide maps to see i f the map of 8-C contains the sum of the peptide i n 8-D and 8-E. Peptide B~CNBr-8-F i s the only peptide which contains isoleucine as the N-terminal amino acid and therefore must r e p r e s e n t t h e N - t e r m i n a l p o r t i o n o f t h e 8 c h a i n . The N - t e r m i n a l amino a c i d sequence o f 8-F and t h e r e f o r e t h a t o f the h a p t o g l o b i n 6 c h a i n i s e s t a b l i s h e d as I l e u - L e u - G l y - G l n - A l a - L y s - G l u - V a l - . The Ser C - t e r m i n a l r e g i o n o f 8-CNBr-8-F i s Asn - A l a - M e t . The C - t e r m i n a l G i n p o r t i o n s o f 8-B, 8-E, and 8-F a r e known and o n l y 8-F c o r r e s p o n d s c l o s e l y t o t h e amino a c i d c o m p o s i t i o n s o f m e t h i o n i n e p e p t i d e M^. The C - t e r m i n a l o f 8-B i s l i n k e d t o the N - t e r m i n a l p o r t i o n o f 8-E by m e t h i o n i n e p e p t i d e M^, so t h a t i t cannot o v e r l a p w i t h M^. On the o t h e r hand, t h e C - t e r m i n a l amino a c i d sequence o f 8 -CNBr-8-E i s d i f f e r e n t from t h e amino a c i d c o m p o s i t i o n o f M^, n o t a b l y w i t h r e s p e c t t o t h e absence o f l y s i n e , h i s t i d i n e , and p h e n y l a l a n i n e i n t h e l a t t e r . The amino a c i d sequence o f 8 _CNBr-8-D i s unknown a t t h e moment; however, i n v i e w o f t h e p o o r y i e l d o f t h i s pep-t i d e , i t i s u n l i k e l y t h a t p e p t i d e M^ w h i c h was i s o l a t e d i n major q u a n t i t y , d e r i v e s from t h i s r e g i o n . The above argument, t h e r e -f o r e , i n d i c a t e s t h e l i n k a g e between M^ and B-CNBr-8-F. The g l y c o p e p t i d e c o n t a i n s v a l i n e as t h e N - t e r m i n a l and i t i s sug-g e s t e d t h a t i t c o n n e c t s w i t h t h e C - t e r m i n a l o f 8-F t h r o u g h M^. A l t h o u g h a l i g n m e n t i s i n d i r e c t , the s t u d i e s o f a r g i n i n e pep-t i d e s s u p p o r t t h e above a s s i g n m e n t s as d i s c u s s e d below. I n t h e s t u d i e s o f a r g i n i n e p e p t i d e s and t r y p t i c m a l e y l a t e d p e p t i d e s , f i v e groups o f a r g i n i n e p e p t i d e s were i s o l a t e d . Group I , IV and V p e p t i d e s were e s t a b l i s h e d as b e i n g l o c a t e d i n 8 -CNBr-8-B and B-CNBr-8-E, l e a v i n g a r g i n i n e group I I and I I I p e p t i d e s u n d e c i d e d . A r g i n i n e p e p t i d e I I c o n t a i n s m e t h i o n i n e p e p t i d e M^, so t h a t i t must be a s s o c i a t e d w i t h t h e r e g i o n l i n k i n g B-CNBr-8-F t o 8-CNBr-8-B, t h e g l y c o p e p t i d e . A r g i n i n e p e p t i d e I I I c o n t a i n s a h i g h c o n t e n t o f p r o l i n e and has p r e v i o u s l y been n o t e d 210 t h a t p e p t i d e 3-CNBr-8-D and T-M-3 V c o n t a i n a l a r g e amount o f p r o l i n e so t h a t t h e a r g i n i n e group I I I p e p t i d e p r o b a b l y d e r i v e s from B-CNBr-8-D r a t h e r t h a n from B-CNBr-8-F. F u r t h e r m o r e , pep-t i d e T-M-B V, w h i c h has an amino a c i d c o m p o s i t i o n s i m i l a r t o 3-CNBr-8-D, o v e r l a p s w i t h t h e C - t e r m i n a l r e g i o n o f B-CNBr-8-E. As can be seen i n F i g . 57, t h i s i m p l i e s t h a t T-M-3 V c o n t a i n s the m e t h i o n i n e r e s i d u e o f 8 -CNBr-8-E. I f T-M-B V were a l s o t o c o n t a i n a r g i n i n e group I I p e p t i d e s , i t would have t o c o n t a i n two r e s i d u e s o f m e t h i o n i n e i n s t e a d o f one. The amino a c i d a n a l y s i s f a i l s t o show more t h a n one m e t h i o n i n e so t h a t T-M-B V cannot c o n t a i n a r g i n i n e group I I p e p t i d e s . T h i s i s c o n s i s t e n t w i t h t h e above s u g g e s t i o n t h a t p e p t i d e 3~CNBr-8-D and T-M-B V c o n t a i n s t h e a r g i n i n e group I I I p e p t i d e . A r g i n i n e group I I p e p t i d e , t h e r e f o r e , i s l o c a t e d n e a r the N - t e r m i n a l p o r t i o n o f t h e 8 c h a i n . The m a l e y l a t e d t r y p t i c f r agment, p e p t i d e T-M-3 I I / w h i c h c o n t a i n s t h e c a r b o h y d r a t e , i s a s s i g n e d t o t h e N - t e r m i n a l r e g i o n o f 3 -CNBr-8-E, and hence would be c l o s e t o t h e N - t e r m i n a l o f t h e 3 c h a i n . S i n c e T-M-3 I I c o r r e s p o n d s t o B-CNBr-8-B, the l i n k a g e between B-CNBr-8-F and 3~CNBr-8-B i s t h e r e f o r e i n d i c a t e d . The c a r b o h y d r a t e a t t a c h m e n t s i t e , i n w h i c h t h e amino a c i d sequence has been p a r t i a l l y e l u c i d a t e d , i s l o c a t e d i n t h e i n t e r n a l r e g i o n o f B~CNBr-8-B. P e p t i d e 3~CNBr-8-B and p e p t i d e B~CNBr-8-E are j o i n e d by m e t h i o n i n e p e p t i d e M^. F u r t h e r m o r e , p e p t i d e o v e r l a p s w i t h a r g i n i n e p e p t i d e group V and a t o t a l amino a c i d sequence o f 45 amino a c i d s has been d e t e r m i n e d i n t h i s p a r t i c u l a r r e g i o n . P e p t i d e B~CNBr-8-E c o n t a i n s two a r g i n i n e r e s i d u e s and t h e s e c o r r e s p o n d t o a r g i n i n e group I and group IV p e p t i d e s , r e s p e c t i v e l y . The a c t u a l i s o l a t i o n o f a r g i n i n e group I and IV 211 p e p t i d e s and t h e t r y p t i c m a l e y l a t e d fragments .T-M-B V I I and T-M-B VI p r o v i d e d f u r t h e r e v i d e n c e f o r t h e amino a c i d sequence o f B-CNBr-8-E. As can be seen i n the f i g u r e , t h e s e two m a l e y l a t e d fragments T-M-B V I I and T-M-B VI a r e p o r t i o n s o f t h e B-CNBr-8-E, w h i c h can be o b t a i n e d upon t r y p t i c c l e a v a g e o f t h e m a l e y l a t e d B c h a i n . The l i n k a g e between B-CNBr-8-E and B-CNBr-8-D i s p r o v i d e d by the t r y p t i c a r g i n i n e p e p t i d e T-M-B V. T h i s p e p t i d e c o n t a i n s a s p a r t i c a c i d as t h e N - t e r m i n a l and has an amino a c i d c o m p o s i t i o n s i m i l a r t o B -CNBr-8-D. Though i t s h o u l d be p o s s i b l e t o o v e r l a p t h e s e p e p t i d e s by a m e t h i o n i n e p e p t i d e as i n B-CNBr-8-B and B-CNBr-8-E by m e t h i o n i n e p e p t i d e M^, i t has been found d i f f i c u l t t o i s o l a t e the m e t h i o n i n e p e p t i d e s r e s p o n s i b l e f o r t h i s l i n k a g e and t h a t between B~CNBr-8-D and M-B-CNBr-8-A IV. However, i f p e p t i d e B-CNBr-8-C does i n f a c t c o n s i s t s o f B~CNBr-8-E and B-CNBr-8-D, t h i s would p r o v i d e the a d d i t i o n a l c o n f i r m a t i o n o f t h e above l i n k a g e . I t i s i n t e r e s t i n g t o n o t i c e t h a t p e p t i d e B-CNBr-8-D c o n t a i n s 2 c a r b o x y m e t h y l c y s t e i n e r e s i d u e s i n t h e a n a l y s i s . I n the case o f T-M-B V, t h e two h a l f c y s t i n e s are i n Y. the form o f a mixed d i s u l f i d e w i t h B ~ m e t c a p t o e t h a n o l . So f a r i t \ i s n o t p o s s i b l e t o a s s i g n the c y s t e i c a c i d p e p t i d e s i s o l a t e d and sequenced by Kauffman and D i x o n . Two d i f f e r e n t methods c o u l d be used t o d e t e r m i n e w h i c h two o f t h e c y s t e i c a c i d p e p t i d e s c o r -r e s p o n d t o t h e two c a r b o x y m e t h y l c y s t e i n e r e s i d u e s i n t h i s r e g i o n . One s i m p l e approach would be the d i r e c t i s o l a t i o n o f the CMC p e p t i d e s from B -CNBr-8-D o r a l t e r n a t i v e l y , t o c o n v e r t t h e two mixed d i s u l f i d e o f T-M-B V i n t o S - a m i n o - e t h y l c y s t e i n e by e t h y l e n e -i m i n e . T h i s would produce two s i t e s w h i c h c o u l d be c l e a v e d by 212 t r y p s i n . Upon d a n s y l a t i o n , t h e two N - t e r m i n a l a m i n o a c i d s s h o u l d c o r r e s p o n d t o t h e a m i n o a c i d s a d j a c e n t t o h a l f c y s t i n e s i n t h e c y s t e i c a c i d p e p t i d e s . P e p t i d e M-B-CNBr-8-A I V c o r r e s p o n d s t o t h e C - t e r m i n a l o f t h e 8 c h a i n . The i n f o r m a t i o n r e g a r d i n g t h i s r e g i o n i s l e s s s a t i s f a c t o r y , c o m p a r e d t o t h e o t h e r s . F u r t h e r c h a r a c t e r i z a t i o n o f M-B-CNBr-8-A I V a n d T-M-B I V w i l l u n d o u b t e d l y c l a r i f y t h e a m b i g u i t y , a n d t h e f i g u r e shown i n F i g . 57 c a n be u s e d f o r f u r t h e r e x p e r i m e n t a t i o n a n d t h e u l t i m a t e d e t e r m i n a t i o n o f t h e a m i n o a c i d s e q u e n c e o f t h e B c h a i n . n.u-L.u-oiy-ci„-»i.-i.y.-oi«-v.l( >v«l (aw. »u> -Tyr-v.l-»*p- (oi«,, L . u . M p , ely,. Pro, B •• •• «...-M„ u . - - , oo Cll> * 1 )Lau-aW-Gln-»a»»an-a. r -Tt ir L y . » . « ( ) « C N 8 F » < C N 8 B « Mi 1 < Arg II » « T M 6 III 1 « T M 6 II Val-Sar-Val-Aan-Glu-Arg-Val-Net-Pro-ilau-Cya-Pro-Lau -aar-Lya-Aap-Tyr-Ma-OW-val-Gly-Ara-Val-Gly-Tyr- Va l- lar-Gly-Try-Gly-te«-*an-Ma-kap-Pha-Lya-Pha T t i r - M p - H i . - L a u - l * . - T y r - v a l » , CN 8 E — I M 4 1 «— Arg V • ' - A r g 1 • — A r g IV » » » T M & VI i— —• i T M I VII • I ™ M L . u -( )-val-IUa-aar-Thr<Pro , A l a . 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