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UBC Theses and Dissertations

Studies on the antigenic properties of reduced and carboxymethylated egg-white lysozyme Thompson, Karen E. 1969

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5 " i a o STUDIES ON THE ANTIGENIC PROPERTIES OF REDUCED AND CARBOXYMETHYLATED EGG-WHITE LYSOZYME by KAREN E. THOMPSON B.Sc. ( M i c r o b i o l o g y ) , The 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 , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f Mi c r o b i o l o g y 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 the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t , 1969 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the 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 , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree tha p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Microbiology The U n i v e r s i t y o f B r i t i s h Co lumbia V a n c o u v e r 8, Canada Date 28th September 1969 i i ABSTRACT T h i s t h e s i s i n v o l v e d a s t u d y o f t h e a n t i g e n i c p r o p e r t i e s o f reduced and S-carboxymethy1ated e g g - w h i t e lysozyme (CM-lysozyme). S i n c e work on the a n t i g e n i c p r o p e r t i e s o f n a t i v e lysozyme i s c u r r e n t l y i n p r o g r e s s i n two o t h e r l a b o r a t o r i e s , i t was th o u g h t t h a t a c o m p a r a t i v e s t u d y on CM-lysozyme might e l u c i d a t e t h e r o l e o f p r i m a r y and t e r t i a r y s t r u c t u r e i n d e t e r m i n i n g a n t i g e n i c i t y i n p r o t e i n s . T h i s m o l e c u l e was chosen m a i n l y because the complete amino a c i d sequence, and t h e X-ray c r y s t a l l o g r a p h y o f t h e m o l e c u l e have been c o m p l e t e d . T h i s makes i t p o s s i b l e t o c o r r e l a t e i n f o r m a t i o n on a n t i g e n i c d e t e r m i n a n t s w i t h t h e i r o r i e n t a t i o n on t h e c r y s t a l l i n e s t r u c t u r e . The n a t i v e m o l e c u l e i s r e l a t i v e l y r e s i s t a n t t o e n z y m a t i c d i g e s t i o n , but the reduced and S - c a r b o x y m e t h y l a t e d d e r i v a t i v e , l a c k i n g i t s d i s u l f i d e b r i d g e s , and c o n s e q u e n t l y i t s r i g i d t e r t i a r y s t r u c t u r e , i s r e a d i l y d i g e s t e d . The f i r s t e x p e r i m e n t s i n v o l v e d a t t e m p t s t o i s o l a t e fragments o f the CM-lysozyme m o l e c u l e e x h i b i t i n g h a p t e n i c a c t i v i t y . T r y p s i n was used, s i n c e t h i s i s a s p e c i f i c e n d o p e p t i d a s e , c l e a v i n g p r o t e i n s o r p e p t i d e s a t the c a r b o x y l o f l y s i n e and a r g i n i n e r e s i d u e s . Only one t r y p t i c p e p t i d e thus i s o l a t e d (T-11) e x h i b i t e d h a p t e n i c a c t i v i t y when v a r i o u s f r a c t i o n s were t e s t e d f o r t h e i r a b i l i t y t o i n h i b i t e i t h e r p r e c i p i t a t i o n o r complement f i x a t i o n between CM-lysozyme and i t s homologous a n t i s e r u m . There was no c r o s s -r e a c t i v i t y o b s e r v e d between CM-lysozyme and a n t i s e r u m d i r e c t e d a g a i n s t n a t i v e lysozyme. L i k e w i s e , the t r y p t i c d i g e s t o f CM-lysozyme o r any o f the p e p t i d e s i s o l a t e d from i t , d i d not i n h i b i t t h e immune r e a c t i o n o f n a t i v e lysozyme w i t h i t s homologous a n t i -serum. F u r t h e r e x p e r i m e n t s were c a r r i e d out i n a t t e m p t s t o p i n p o i n t t h e r e g i o n o f t h i s l a r g e 23 amino a c i d p e p t i d e , Tr 11,'.which was r e s p o n s i b l e f o r h a p t e n i c a c t i v i t y . From s e q u e n t i a l d e g r a d a t i o n o f th e p e p t i d e from both the C- and N - t e r m i n a l , u s i n g e n z y m a t i c and c h e m i c a l methods, t h e a n t i g e n i c r e g i o n was narrowed t o t h e N - t e r m i n a l p o r t i o n o f • T—11".. Two s y n t h e t i c p e p t i d e s , p r e p a r e d by t h e s o l i d phase method, c o m p r i s i n g t h e N - t e r m i n a l d e c a p e p t i d e o f T-11, and t h e d e c a p e p t i d e p l u s a r g i n i n e a t the N - t e r m i n a l , both showed h a p t e n i c a c t i v i t y by i n h i b i t i o n o f p r e c i p i t a t i o n between CM-lysozyme and i t s homologous a n t i s e r u m . The degree o f i n h i b i t i o n w i t h t h e two s y n t h e t i c p e p t i d e s were comparable but somewhat l e s s e f f i c i e n t than t h e whole T-11 p e p t i d e . i v TABLE OF CONTENTS Page INTRODUCTION AND LITERATURE REVIEW 1 I . The Importance o f M o l e c u l a r C o n f o r m a t i o n i n t he Immune R e a c t i o n 1 I I . C r o s s - R e a c t i n g A n t i g e n s 6 I I I . S y n t h e t i c A n t i g e n s and A n t i g e n i c D e t e r m i n a n t s 7 IV. D e t e r m i n a t i o n o f t h e S i z e o f A n t i g e n i c D e t e r m i n a n t s 8 V. A n t i g e n i c D e t e r m i n a n t s on P r o t e i n A n t i g e n s 12 1. Tobacco m o s a i c v i r u s p r o t e i n (TMVP) s u b u n i t . . . 12 2. B r a d y k i n i n 13 3. Human serum a l b u m i n . . 1A k. Sperm whale m y o g l o b i n 15 5. R i b o n u c l e a s e 17 6. B a c t e r i a l f e r r e d o x i n s 18 7. N a t i v e lysozyme 19 8. Reduced and c a r b o x y m e t h y l a t e d lysozyme (CM-lysozyme) 22 'MATERIALS AND METHODS 2k I. Immunization P r o c e d u r e s 2k V T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page II . Q u a n t i t a t i v e P r e c i p i t i n R e a c t i o n . . 25 I I I . Complement F i x a t i o n R e a c t i o n 26 IV. P r 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 3 0 V . P r o t e i n and P e p t i d e A n a l y s i s 34 V I . C a r b o x y p e p t i d a s e D i g e s t i o n s 36 V I I . Edman D e g r a d a t i o n o f T-11 42 V I I I . L e u c i n e Amino P e p t i d a s e E x p e r i m e n t s 46 1. E x p e r i m e n t a l s y s t e m 48 2. S u b s t r a t e c o n t r o l s 48 3. Enzyme c o n t r o l 48 IX. P u r i f i c a t i o n o f S p e c i f i c A n t i b o d i e s 50 X. S o l i d Phase P e p t i d e S y n t h e s i s 54 1. P e p t i d e s y n t h e s i s 54 2. R e d u c t i o n and a l k y l a t i o n o f s y n t h e s i z e d p e p t i d e s . . 57 3. P u r i f i c a t i o n o f reduced and c a r b o x y m e t h y l a t e d p e p t i d e 58 X I . ( l Z f C ) - A c e t y l a t ion o f P e p t i d e s 60 X I I . E q u i l i b r i u m D i a l y s i s 62 RESULTS AND DISCUSSION 64 I. H a p t e n i c A c t i v i t y in T r y p t i c D i g e s t s o f CM- lysozyme 64 v i T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page I I . C a r b o x y p e p t i d a s e D i g e s t i o n s 75 I I I . Edman D e g r a d a t i o n o f T-11 81 IV. L e u c i n e Amino P e p t i d a s e E x p e r i m e n t s 86 V. P u r i f i c a t i o n o f S p e c i f i c A n t i b o d i e s 90 V I . S o l i d Phase P e p t i d e S y n t h e s i s . 94 1. S y n t h e s i s 94 2. R e d u c t i o n and a l k y l a t i o n 96 3. P u r i f i c a t i o n o f reduced and carboxymethy 1ated p e p t i d e s 97 V I I . (1V)-Acetylation o f P e p t i d e s 104 V I I I . Equi 1 i b r i u m D i a l y s i s 107 GENERAL DISCUSSION 111 CONCLUDING COMMENTS 118 APPENDIX I 120 LITERATURE CITED 124 v i i L IST OF TABLES Page T a b l e I. F l ow c h a r t o f S o l i d Phase P e p t i d e S y n t h e s i s 56 T a b l e I I . Amino a c i d a n a l y s i s o f p e p t i d e T-11 67 T a b l e I I I . Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by c a r b o x y p e p t i d a s e B d i g e s t e d T-11 78 T a b l e IV. Amino a c i d a n a l y s e s d a t a o f s u b t r a c t i v e Edman's d e g r a d a t i o n o f T-11 83 T a b l e V. Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by Edman 's d e g r a d a t i o n o f T-11 84 T a b l e V I . Amino a c i d a n a l y s e s d a t a o f l e u c i n e amino p e p t i d a s e d i g e s t i o n o f T-11 p e p t i d e showing t h e amino a c i d s removed 87 T a b l e V I I . Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by l e u c i n e amino p e p t i d a s e d i g e s t e d T-11 88 T a b l e V I I I . Amino a c i d a n a l y s e s o f f ^ - A s n s y n t h e t i c d e c a p e p t i d e f rom the N - t e r m i n a l r e g i o n o f T-11 98 T a b l e IX. Amino a c i d a n a l y s e s o f NH^-Arg s y n t h e t i c p e p t i d e c o m p r i s i n g a r g i n i n e p l u s t he N - t e r m i n a l r e g i o n o f T-11 99 T a b l e X. Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by v a r y i n g c o n c e n t r a t i o n s o f t he f o l l o w i n g p e p t i d e s 102 v i i i LIST OF FIGURES Figure 1. Complement f i x a t i o n t i t r a t i o n curve of CM-lysozyme Figure 2 . Assay of enzyme a c t i v i t y of the trypsin preparation by hydrolysis of the substrate p - to luenesul fony l -L -arg in ine methyl ester Figure 3 . E lut ion p r o f i l e of t r y p t i c peptides of CM-lysozyme using the eight-chambered gradient system of Canf ield (1963a)j-Figure k. E lut ion p r o f i l e of t r y p t i c peptides of CM-lysozyme using the s i m p l i f i e d four -chambered gradient system Figure 5 . Typical scan of the T-11 peptide at a concentration of 0 . 1 0 mg/ml Figure 6 . Assay of enzyme a c t i v i t y of the carboxy-peptidase B preparation by the hydrolysis of the substrate h ippury l -L -arg in ine Figure 1. Assay of enzyme a c t i v i t y of the carboxy-peptidase A preparation by the hydrolysis of the substrate hippuryl -L -phenylalanine Figure 8 . Assay of enzyme a c t i v i t y of the activated leucine amino peptidase preparation by the hydrolysis of the substrate L - l e u c y l -p -n i t roani1 ide Figure 9 . E lut ion p r o f i l e of the p u r i f i c a t i o n of s p e c i f i c anti-CM-lysozyme antibodies on Sephadex G - 2 0 0 , Figure 1 0 . Inhib i t ion of immune p rec ip i ta t ion by peptide T-11 and the tota l t r y p t i c digest at varying concentrations Figure 11 . Inhibi t ion of complement f i xa t ion with CM-lysozyme and homologous antiserum by peptide T-11 at varying concentrations Figure 12. Inhibi t ion of complement f i xa t ion with CM-lysozyme and homologous antiserum by the tota l t r y p t i c digest at varying concentrations Page 29 31 33 35 37 39 M kl 51 68 70 71 i x L i s t o f F i g u r e s ( C o n t i n u e d ) Page F i g u r e 13. C o r r e l a t i o n o f i n h i b i t i o n o f complement f i x a t i o n a t a 1:62 d i l u t i o n o f complement and v a r y i n g c o n c e n t r a t i o n s o f T-11 and t r y p t i c d i g e s t 72 F i g u r e 14. E l u t i o n p r o f i l e o f s y n t h e t i c d e c a p e p t i d e and s y n t h e t i c d e c a p e p t i d e p l u s a r g i n i n e 101 F i g u r e 15. I n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n between CM-lysozyme and i t s homologous a n t i s e r u m by ( ^ C ) - T - 1 1 , Nh^-Arg s y n t h e t i c p e p t i d e , T-11 and NH^-Asn s y n t h e t i c p e p t i d e 103 F i g u r e 16. E l u t i o n p r o f i l e f o r the a c e t y l a t e d Nh^-Arg s y n t h e t i c p e p t i d e and a c e t y l a t e d T - 1 1 . 106 F i g u r e 17. C o r r e l a t i o n o f the v a r i o u s p e p t i d e e x h i b i t i n g h a p t e n i c a c t i v i t y sequences 113 ABBREVIATIONS = A n g s t r o m u n i t s = a-2,k-dinitropheny1 = B r o m o a c e t y l = R e d u c e d a n d c a r b o x y m e t h y l a t e d l y s o z y m e = c a r b o x y m e t h y l c y s t e i n e = C y a n o g e n b r o m i d e = D i c y c l o h e x y l c a r b o d i i m i d e = D i i s o p r o p y l f 1 u o r o p h o s p h a t e = Gamma-globu 1i n = 8-11S i m m u n o g l o b i n s , p r e s e n t i n s e c r e t i o n s s u c h a s c o l o s t r u m , s m a l l a m o u n t s i n s e r u m = 7S i m m u n o g l o b u l i n ; p r e s e n t i n s e r u m 19S i m m u n o g l o b u l i n ; p r e s e n t i n s e r u m = L e u c i n e a m i n o p e p t i d a s e = m i c r o c u r i e = m i c r o g r a m = m i c r o l i t e r = m i c r o m o l a r = mi 11i g r am = 0.02 M s o d i u m p h o s p h a t e b u f f e r , pH 6.0, 0.15 M N a C l x i TAME = p - t o l u e n e s u l f o n y l - L - a r g i n i n e methyl e s t e r t-BOC = t - b u t y l o x y c a r b o n y 1 TEA = t r i e t h y l a m i n e TMVP = Tobacco M o s a i c V i r u s P r o t e i n U-ApoMb = Apomyoglobin t r e a t e d w i t h 8 M urea 5% m e t h a n o l , pH 3 .0 . ACKNOWLEDGEMENTS F i r s t l y , I would l i k e t o e x p r e s s my s i n c e r e g r a t i t u d e t o Dr. J u l i a Levy f o r h e r encouragement, d i r e c t i o n and c o n s t r u c t i v e c r i t i c i s m o f both the r e s e a r c h and w r i t i n g o f t h i s t h e s i s . I would a l s o l i k e t o thank the members o f my committee, Dr. E. B e n j a m i n i , Dr. T.H. B l a c k b u r n , Dr. J.J.R. C a m p b e l l , Dr. D.M. McLean, Dr. G.M. Tener and Dr. J.H. Tremaine f o r t h e i r w i l l i n g a d v i c e and i n t e r e s t i n the e d i t i n g o f t h e t h e s i s . A l s o , I would l i k e t o thank Mrs. B a r b a r a M i t c h e l l , Mr. John C h r i s t e n s e n and Mr. Bob K e z i e r f o r t h e i r a s s i s t a n c e i n my r e s e a r c h e x p e r i m e n t s . L a s t l y , my thanks go t o Mrs. R i t a Rosbergen f o r h e r p a t i e n c e i n the t y p i n g o f t h i s f i n a l m a n u s c r i p t . 1 INTRODUCTION AND LITERATURE REVIEW The l i t e r a t u r e d i s c u s s e d here d e a l s w i t h the most r e c e n t o b s e r v a t i o n s made i n the g e n e r a l a r e a o f a n t i g e n i c c h a r a c t e r i s t i c s i n b o t h s y n t h e t i c and n a t u r a l s y stems, w i t h p a r t i c u l a r emphasis on the c h a r a c t e r i z a t i o n o f a n t i g e n i c d e t e r m i n a n t s i n p r o t e i n s . The r e s e a r c h on t h e h i s t o r i c a l development o f o u r p r e s e n t knowledge o f a n t i g e n i c i t y has been c o p i o u s and d i v e r g e n t . S e v e r a l good r e v i e w s have been p u b l i s h e d w i t h i n t h e p a s t f i v e y e a r s d e a l i n g w i t h t h e v a r i o u s s p e c i a l i z e d a s p e c t s ( K a b a t , 1966; 1968; S e l a , 1966; G i l l I I I , Kunz, Friedman and Doty, 1963; P r o c e e d i n g s o f The Royal S o c i e t y , S e r i e s B, 1966? Haber and R i c h a r d s , S e l a ) . I . The Importance o f M o l e c u l a r C o n f o r m a t i o n I ri t he Immune React i o n Beychok and Kabat (1965) and Kabat, L l o y d and Beychok (1969) have shown t h a t the c o n f o r m a t i o n o f o l i g o s a c c h a r i d e s can be r e a d i l y r e c o g n i z e d i n immune syste m s . T h e i r work has c o r r e l a t e d o p t i c a l r o t a r y d i s p e r s i o n and c i r c u l a r d i c h r o i s m measurements as an independent l i n e o f e v i d e n c e f o r c o n f o r m a t i o n , w i t h immunochemical s t u d i e s , thus e n a b l i n g them t o e x p l o r e the r o l e o f c o n f o r m a t i o n i n a n t i g e n - a n t i b o d y i n t e r a c t i o n s . O p t i c a l r o t a r y d i s p e r s i o n measurements 2 p r o v i d e d e s t i m a t e s o f t h e p r o p o r t i o n s o f a- and ( 3-1inked N - a c e t y l -D-glucosamine r e s i d u e s i n s t a p h y l o c o c c a l t e i c h o i c a c i d s i n a few h o u r s , whereas the immunochemical s t u d i e s were e x t e n s i v e and r e q u i r e d s e v e r a l weeks o f work t o a c h i e v e s i m i l a r d a t a . However, i t was o n l y by t h e more s e n s i t i v e immunochemical method t h a t T o r i i , Kabat and Bezer ( 1 9 6 A ) c o u l d d i s t i n g u i s h m i x t u r e s o f a- and g - t e i c h o i c a c i d s , from both l i n k a g e s on a s i n g l e p o l y r i b i t o l phosphate backbone. A l t h o u g h , t o d a t e , s t u d i e s on the c o r r e l a t i o n o f immunochemical p r o p e r t i e s w i t h o p t i c a l r o t a r y d i s p e r s i o n and c i r c u l a r d i c h r o i s m have been performed p r i n c i p a l l y w i t h o l i g o s a c c h a r i d e d e t e r m i n a n t s , the same t e c h n i q u e s s h o u l d prove a p p l i c a b l e t o the s t u d y o f a n t i g e n i c d e t e r m i n a n t s o f p o l y p e p t i d e s and p r o t e i n s . Crumpton and Small ( 1 9 6 7 ) demonstrated t h a t a t e t r a d e c a p e p t i d e from m y o g l o b i n , w h i c h i n the n a t i v e m o l e c u l e was p r e d o m i n a n t l y h e l i c a l , but w h i c h became n o n h e l i c a l i n aqueous s o l u t i o n , was a b l e t o combine w i t h a n t i b o d y t o metmyoglobin. The p e p t i d e , i n aqueous s o l u t i o n , may p o s s e s s a v a r i e t y o f c o n f o r m a t i o n s i n c o n t i n u o u s i n t e r c h a n g e , but i t was presumed t h a t the a n t i b o d i e s would r e a c t o n l y w i t h the f r a c t i o n o f m o l e c u l e s whose c o n f o r m a t i o n was the same as t h a t o f the c o r r e s p o n d i n g r e g i o n i n t h e n a t i v e a n t i g e n , namely h e l i c a l . T h i s p r e s e n t e d the p o s s i b i l i t y t h a t t h e p e p t i d e was s t a b i l i z e d i n t h e h e l i c a l form i n t h e a n t i b o d y c o m b i n i n g s i t e . S c h lossman, L e v i n e and Yaron ( 1 9 6 8 ) found t h a t the combining s i t e s o f a n t i b o d i e s produced a g a i n s t a - 2 , 4 - d i n i t r o p h e n y l - l y s i n e (a-DNP-(Lys)), were complementary t o t h e d e t e r m i n a n t a-DNP-(Lys)^, but t h o s e produced a g a i n s t a - D N P - ( L y s ) 1 2 Q 0 were complementary o n l y t o a-DNP-(Lys)^. Kabat s u g g e s t e d t h a t t h i s f i n d i n g might be r e l a t e d t o c o n f o r m a t i o n a l d i f f e r e n c e s between the two a n t i g e n s , and t h a t o p t i c a l r o t a r y d i s p e r s i o n and c i r c u l a r d i c h r o i s m s t u d i e s might be b e n e f i c i a l i n e l u c i d a t i n g t h i s o b s e r v a t i o n , t h e meaning o f wh i c h i s u n c l e a r a t t h i s t i m e . A n o t h e r s t u d y on the s t r u c t u r a l b a s i s f o r immune r e c o g n i t i o n was c a r r i e d o ut by B o n a v i d a , M i l l e r and S e r c a r z (1969) on n a t i v e hen eg g - w h i t e lysozyme. The two m e t h i o n i n e r e s i d u e s a t p o s i t i o n s 12 and 105 o f hen eg g - w h i t e lysozyme„were c l e a v e d by cyanogen bromide i n 10% f o r m i c a c i d . C o n v e r s i o n o f the n a t i v e lysozyme t o cyanogen bromide-t r e a t e d ( C N B r - t r e a t e d ) lysozyme caused a red s h i f t i n t h e a b s o r p t i o n s p e c t r u m . The d i f f e r e n c e s p e c t r u m showed a peak a t 2980 A^, wh i c h has been a s s o c i a t e d , i n some c a s e s , w i t h a s h i f t o f t r y p t o p h a n t o a more h y d r o p h o b i c e n v i r o n m e n t . These w o r k e r s were i n t e r e s t e d i n e s t a b l i s h i n g how much a l t e r a t i o n must t a k e p l a c e b e f o r e one e p i t o p e ( a n t i g e n i c d e t e r m i n a n t ) l o s e s i t s i d e n t i t y and may be r e c o g n i z e d as a n o t h e r , by the immune system. Immunochemical s t u d i e s w i t h t h e C N B r - t r e a t e d lysozyme showed t h a t t h i s d e r i v a t i v e caused 10% i n h i b i t i o n o f the b i n d i n g o f n a t i v e hen egg-white lysozyme by a n t i - h e n e g g - w h i t e lysozyme serum, w h i c h s u g g e s t s c o n s i d e r a b l e r e t e n t i o n o f n a t i v e c o n f o r m a t i o n . However, from g e l d i f f u s i o n s t u d i e s , u s i n g a n t i s e r u m t o C N B r - t r e a t e d lysozyme and t o n a t i v e lysozyme, i t was shown t h a t C N B r - t r e a t e d lysozyme had ^ a p p a r e n t l y l o s t some a n t i g e n i c d e t e r m i n a n t s , and g a i n e d new ones not 4 p r e s e n t on the n a t i v e (hen egg-white) lysozyme. The e x p l a n a t i o n proposed was t h a t , near t o the p o i n t s o f c l e a v a g e , t h e r e i s r e l a t i v e f l e x i b i l i t y i n the CNBr-lysozyme, p e r m i t t i n g r e a l i g n m e n t o f some o f the amino a c i d r e s i d u e s w h i c h , i n the p a r e n t m o l e c u l e , were i n an u n f a v o u r a b l e p o s i t i o n f o r r e a c t i n g w i t h a n t i b o d y . T h i s r e a l i g n m e n t c o u l d a c c o u n t f o r the a l t e r e d s p e c t r a l , e n z y m a t i c and immunological b e h a v i o u r o f C N B r - t r e a t e d lysozyme. A t a s s i and Habeeb (1969) demonstrated t h a t the n a t u r e o f a c h e m i c a l m o d i f i c a t i o n a t a g i v e n s i t e d e t e r m i n e s i t s e f f e c t on b i o l o g i c a l a c t i v i t y ( e n z y m a t i c o r a n t i g e n i c ) . N a t i v e hen egg - w h i t e lysozyme and the d e r i v a t i v e p r e p a r e d by n i t r a t i o n o f the t y r o s i n e s a t p o s i t i o n s 20 and 23 were employed. T h i s n i t r a t e d d e r i v a t i v e e x h i b i t e d c o n f o r m a t i o n a l changes as shown by i t s i n c r e a s e d s u s c e p t i b i l i t y t o t r y p t i c h y d r o l y s i s , and t h e a v a i l a b i l i t y o f one d i s u l f i d e group f o r r e d u c t i o n w h i c h was not r e a c t i v e i n the n a t i v e m o l e c u l e . The a n t i g e n i c r e a c t i v i t y was o n l y s l i g h t l y a f f e c t e d (namely, 7 7 - 9 0 % e f f e c t i v e n e s s r e l a t i v e t o t h e homologous r e a c t i o n ) , whereas the enzymic a c t i v i t y was d e c r e a s e d by 50%. When n i t r o -t y r o s i n e r e s i d u e s were reduced t o a m i n o t y r o s i n e most o f t h e a n t i g e n i c r e a c t i v i t y was r e c o v e r e d (namely, 95 "99% e f f e c t i v e n e s s r e l a t i v e t o the homologous r e a c t i o n ) , d e s p i t e the f a c t t h a t c o n f o r m a t i o n a l changes s t i l l e x i s t e d . T h i s was demonstrated by i t s s i m i l a r s u s c e p t i b i l i t y t o t r y p t i c h y d r o l y s i s , and a v a i l a b i l i t y o f t h e one d i s u l f i d e group f o r r e d u c t i o n , as w i t h the n i t r o t y r o s i n e d e r i v a t i v e . The l o s s i n enzymic ^ a c t i v i t y r e s u l t i n g from n i t r a t i o n was not r e c o v e r e d on r e d u c t i o n o f 5 t h e n i t r o t y r o s y l r e s i d u e s . T h i s s u g g e s t e d t h a t t h e l o s s o f e n z ymic a c t i v i t y was t h e r e s u l t o f t h e s e c o n f o r m a t i o n a l changes w h i c h appeared t o be s i m i l a r i n both the n i t r o t y r o s y l and a m i n o t y r o s y 1 d e r i v a t i v e s . I t a l s o s u g g e s t e d t h a t the amino a c i d sequences around t h e s e two t y r o s i n e s ( T y ^ Q and T y r ^ ^ ) were i m p o r t a n t f o r enzymic a c t i v i t y . The n i t r o g roup, i n the o r t h o p o s i t i o n t o t h e p h e n o l i c h y d r o x y l o f t y r o s i n e , through i t s e l e c t r o n w i t h d r a w i n g e f f e c t , i n c r e a s e d t h e a c i d i t y o f t h e p h e n o l i c h y d r o x y l (pK v a l u e s : t y r o s i n e , 1 0 . 1 ; 3 - n i t r o - t y r o s i n e , 7 . 2 ) . Because t h e r e d u c t i o n o f n i t r o t y r o s y l r e s i d u e s t o a m i n o t y r o s i n e ( t h e r e b y i n c r e a s i n g t h e pK n t o 1 0 . 0 ) r e s u l t e d i n a l m o s t complete r e c o v e r y o f a n t i g e n i c a c t i v i t y , w h i l e m a i n t a i n i n g the a l t e r e d c o n f o r m a t i o n , i t was s u g g e s t e d t h a t one o r b o t h o f t y r o s i n e s 20 and 23 were p r e s e n t i n an a n t i g e n i c r e g i o n o f lysozyme. I t s h o u l d be n o t e d t h a t t h i s r e c o v e r y i n a n t i g e n i c a c t i v i t y was o n l y between 5 t o 2 2 % , w h i c h d i d not p r o v i d e ' c o n c l u s i v e e v i d e n c e f o r t h e p r e s e n c e o f the two t y r o s i n e s i n an a n t i g e n i c r e g i o n . F u r t h e r , t h e s e w o r k e r s found 3 n i t r o t y r o s i n e , o r G l y - 3 - n i t r o t y r o s i i i e f a i l e d t o i n h i b i t t h e r e a c t i o n o f ( N 0 2 ) 2~ lysozyme w i t h i t s homologous p u r i f i e d a n t i b o d i e s . T h i s i n d i c a t e d t h a t the a n t i g e n i c s p e c i f i c i t y was not d i r e c t e d t o n i t r o t y r o s y l r e s i d u e s a l o n e , but more p r o b a b l y t o t h e n i t r o t y r o s y l r e s i d u e s i n c o n j u n c t i o n w i t h i t s n e i g h b o u r i n g r e s i d u e s . I I . C r o s s - R e a c t i r i g A n t i g e n s A comparison o f t h e a n t i g e n i c s t r u c t u r e o f the m u t u a l l y r e l a t e d enzymes, p a p a i n and chymopapain, was r e p o r t e d by Arnon and S h a p i r a (1968). By t h e method o f c r o s s - i m m u n o a d s o r p t i o n , t h e a n t i b o d i e s t o chymopapain were f r a c t i o n a t e d u s i n g s p e c i f i c p a p a i n c r o s s -immunoadsorbent, and the a n t i b o d i e s t o p a p a i n were s i m i l a r l y f r a c t i o n a t e d w i t h chymopapain cross-immunoadsorbent. These two a n t i b o d y s p e c i e s were d i r e c t e d t o a n t i g e n i c d e t e r m i n a n t s common t o bo t h enzymes, a l t h o u g h i t was u n c e r t a i n as t o whether t h e y were d i r e c t e d t o i d e n t i c a l amino a c i d sequences on t h e two p r o t e i n s . With r e s p e c t t o t h e q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n , t h e i r c a p a c i t y t o b i n d a n t i g e n s , and t h e i r i n h i b i t o r y c a p a c i t y toward each o f t h e two enzymes r e g a r d i n g t h e i r c a t a l y t i c a c t i v i t y on a l a r g e s u b s t r a t e , t h e two a n t i b o d y s p e c i e s were i d e n t i c a l . I t was a l s o found t h a t both a n t i b o d y s p e c i e s had a s i m i l a r i n h i b i t o r y e f f e c t on t h e c a t a l y t i c a c t i v i t y o f chymopapain on a low m o l e c u l a r w e i g h t s u b s t r a t e . However, a d i s s i m i l a r i t y between t h e two s p e c i e s was demonstrated when t h e i n h i b i t i o n o f p a p a i n on a low m o l e c u l a r w e i g h t s u b s t r a t e was t e s t e d . In t h i s c a s e , t h e a n t i b o d y s p e c i e s p r e p a r e d from a n t i - c h y m o p a p a i n serum by cross-immunoadsorbent was l e s s e f f i c i e n t as an i n h i b i t o r t han t h o s e ; f r o m a n t i - p a p a i n . T h i s phenomenon might be due t o a d i f f e r e n c e i n t h e a f f i n i t i e s o f t h e two s p e c i e s , r e s u l t i n g from t h e same a n t i g e n i c d e t e r m i n a n t s b e i n g p r e s e n t on d i f f e r e n t c a r r i e r s . 7 From t h i s method of s e l e c t i v e f r a c t i o n a t i o n o f the a n t i b o d y s p e c i e s by c r o s s - i m m u n o a d s o r b e n t s , chymopapain, which has a l a r g e r m o l e c u l a r w e i g h t than p a p a i n , was shown t o pos s e s s more a n t i g e n i c d e t e r m i n a n t s than p a p a i n . One of t h e s e d e t e r m i n a n t s e l i c i t e d t he f o r m a t i o n of a n t i b o d i e s which i n h i b i t p a p a i n a c t i v i t y on a m a c r o m o l e c u l a r s u b s t r a t e and a l s o on a s m a l l s u b s t r a t e . I I I . S y n t h e t i c A n t i g e n s and A n t i g e n i c D e t e r m i n a n t s From the s t u d y o f s y n t h e t i c p o l y p e p t i d e a n t i g e n s , s e v e r a l f a c t o r s i n f l u e n c i n g a n t i g e n i c i t y may be d e t e r m i n e d ( S e l a , 1966). G i l l , Kunz and P a p e r m a s t e r (1967) c a r r i e d out e x t e n s i v e s t u d i e s on the immuno-g e n i c p r o p e r t i e s o f s y n t h e t i c p o l y p e p t i d e a n t i g e n s i n o r d e r t o e l u c i d a t e s e v e r a l f a c t o r s i n f l u e n c i n g a n t i g e n i c i t y . The q u a n t i t a t i v e c o n t r o l o f a n t i b o d y p r o d u c t i o n was found t o depend upon ( i ) t h e ch e m i c a l c o m p o s i t i o n o f the a n t i g e n , where polymers c o n t a i n i n g a r o m a t i c amino a c i d s g e n e r a l l y e l i c i t e d more a n t i b o d y than t h o s e c o n t a i n i n g no a r o m a t i c res i d u e s ; ( i i ) an e x c e s s i v e l y h i g h charge on p o l y p e p t i d e s d e p r e s s e d t h e i r a n t i g e n i c p o t e n c y ; ( i i i ) t h e o p t i c a l c o n f i g u r a t i o n o f t he component amino a c i d s , where D-amino a c i d polymers e x h i b i t e d p o o r e r immunogenic!ty than t h o s e composed o f L-amino a c i d s . In o r d e r t o e l i c i t a n t i b o d y f o r m a t i o n , however, i t was e s s e n t i a l t o a d m i n i s t e r D-amino a c i d polymers i n s m a l l doses i n o r d e r t o p r e v e n t i m m u n o l o g i c a l p a r a l y s i s . J a t o n and S e l a (1968) used m u l t i c h a i n p o l y p r o l i n e as the c a r r i e r m a c r omolecule f o r attachment o f D- o r L-amino a c i d s t o t e s t t h e r o l e o f o p t i c a l c o n f i g u r a t i o n i n i m m u n o g e n i c i t y . These m u l t i -c h a i n p o l y p r o l i n e macromolecules o f e i t h e r o p t i c a l c o n f i g u r a t i o n d i d not provoke a n t i b o d y f o r m a t i o n i n r a b b i t s and t h e r e f o r e c o u l d be used as i n e r t o r n o n a n t i g e n i c c a r r i e r m a c r o m o l e c u l e s . Attachment o f as l i t t l e as 8.5% o f L-amino a c i d r e s i d u e s on t h e o u t s i d e o f a macromolecule composed e x c l u s i v e l y o f D-amino a c i d r e s i d u e s c o n v e r t e d i t t o a good immunogen. These w o r k e r s a l s o d e m o n s t r a t e d t h a t a macromolecule c o n t a i n i n g as much as 35.5% o f L-amino a c i d s , but h a v i n g a l l i t s p o l y m e r i c s i d e c h a i n s t e r m i n a t i n g i n D-amino a c i d s , was j u s t as poor an immunogen as a s i m i l a r m a c romolecule composed e x c l u s i v e l y o f D-amino a c i d s . These r e s u l t s o f J a t o n and S e l a ' s work s u b s t a n t i a t e d e x a c t l y t h e work o f G i l l e t a l . (1967). IV. D e t e r m i n a t i o n o f t h e S i z e o f A n t i g e n i c D e t e r m i n a n t s From work u s i n g s y n t h e t i c p e p t i d e s o f known sequence a t t a c h e d t o i n e r t c a r r i e r m o l e c u l e s , t h e s m a l l e s t a n t i g e n i c fragments have been shown t o be 6 t o 7 amino a c i d s i n l e n g t h . A r n o n , S e l a , Yaron and Sober (1965), who used p o l y l y s y l r a b b i t serum a l b u m i n w i t h an average c h a i n l e n g t h o f 5.5 l y s i n e s , showed t h a t the i n h i b i t i n g power o f t h e p e p t i d e s f o r t h e homologous a n t i g e n a t the e q u i v a l e n c e p o i n t o f t h e a n t i g e n - a n t i b o d y r e a c t i o n i n c r e a s e d s h a r p l y up t o p e n t a l y s i n e and then 9 o n l y s l i g h t l y more up t o noiia l y s i n e . The u l t i m a t e g o a l i n d e t e r m i n i n g t h e measurement o f the s i z e o f the a n t i b o d y c o m b i n i n g s i t e i s t o i s o l a t e a s i n g l e a n t i g e n i c d e t e r m i n a n t and know i t s s t r u c t u r e and c o n f o r m a t i o n i n s o l u t i o n . Immunochemical s t u d i e s on t h e polyTD-g l u t a m y l c a p s u l e o f Baci 1 1 us a n t h r a c i s by Goodman, N i t e c k i and S t o l t e n b e r g (1968) have shown t h a t the maximum s i z e f o r t h e r e g i o n on t h e p o l y p e p t i d e w h i c h combines w i t h the a n t i b o d y s i t e i s s i x r e s i d u e s o f D - g l u t a m i c a c i d . T h i s was t e s t e d by t h e q u a n t i t a t i v e p r e c i p i t i n i n h i b i t i o n r e a c t i o n . The d i m e n s i o n s o f h e x a g l u t a m i c a c i d a r e 36 x 10 x 6 . A i . f These d i m e n s i o n s a r e i n c l o s e agreement w i t h t h e d i m e n s i o n s found f o r t h e a n t i g e n i c d e t e r m i n a n t s on d e x t r a n w h i c h were 3^ x 12 x 7 Av! ( K a b a t , I 9 6 0 ) . I t i s i n t e r e s t i n g t o note t h e c l o s e agreement i n s i z e r e p o r t e d above, i n w h i c h i n s t a n c e s two d i f f e r e n t a n t i g e n s , a p o l y p e p t i d e and a c a r b o h y d r a t e , and two d i f f e r e n t s p e c i e s o f a n i m a l s , r a b b i t s and human b e i n g s , were used r e s p e c t i v e l y . The work on p e p t i d e s d e r i v e d from t h i s homologous c a p s u l a r p o l y p e p t i d e a l s o d e m o n s t r a t e d t h e h e t e r o g e n e i t y o f t h e c o m b i n i n g s i t e s o f a n t i -b o d i e s i n t h e s e r a t e s t e d from t h r e e r a b b i t s s i m i l a r l y immunized w i t h whole c a p s u l a r p o l y p e p t i d e . W i t h two o f t h e s e r a , i n c r e m e n t s o f p r e c i p i t i n - i n h i b i t i n g - e f f i c i e n c y were o b t a i n e d up t o t h e pentamer, w h i c h was equal i n i n h i b i t i n g c a p a c i t y t o t h e hexamer. However, t h e t h i r d a n t i s e r u m c o u l d d i s t i n g u i s h between t h e p e n t a p e p t i d e and hexa-p e p t i d e , by t h e g r e a t e r - i n h i b i t i n g - e f f i c i e n c y o f the h e x a p e p t i d e , i n d i c a t i n g t h a t t h e r e may be i n d i v i d u a l a n imal d i f f e r e n c e s i n the response t o a n t i g e n i c s t i m u l u s . 10 A number o f e x p e r i m e n t s demonstrated t h a t when a s i n g l e hapten i s a t t a c h e d t o a known r e s i d u e o f a pure p r o t e i n , o r t o a p o l y p e p t i d e backbone ( K a n t o r , Ojeda and B e n a c e r r a f , 1963; E i s e n and S i s k i n d , 1964) not a s i n g l e a n t i b o d y s p e c i e s , but whole f a m i l i e s o f a n t i b o d i e s were produced i n r e s p o n s e . T h i s h e t e r o -g e n e i t y o f most a n t i b o d i e s i s p a r t l y due t o the p r e s e n c e o f d i f f e r e n t a n t i b o d y c l a s s e s such as IgG, IgM and IgA and t h e i r r e s p e c t i v e s u b c l a s s e s , and t o g e n e t i c d i f f e r e n c e s , as w e l l as b e i n g dependent on t h e a n t i g e n i c c o m p l e x i t y o f t h e immunogen. Because the d i m e n s i o n s o f t h e a n t i b o d y combining s i t e a r e c o n s i d e r a b l y l a r g e r than most s m a l l o r g a n i c m o l e c u l e s used as haptens ( K a b a t , 1960; 1966; Sage, D e u t s c h , Fasman and L e v i n e , 1 964 ) , the s i t e s may encompass not o n l y the hapten but a l s o d i f f e r e n t s e c t i o n s o f the p r o t e i n t o w h i c h t h e hapten i s a t t a c h e d . Ri chards et_ aJL (1969) s t u d i e d the r e l a t i o n s h i p between a n t i g e n i c c o m p l e x i t y and h e t e r o g e n e i t y i n the a n t i b o d y r e s p o n s e . In t h i s r e s p e c t , R i c h a r d s and w o r k e r s t r i e d t o e l u c i d a t e the r o l e o f the immediate environment i n i t s i n t e r a c t i o n w i t h h a p t e n . They s y n t h e s i z e d amino a c i d copo1ymer w i t h a d e f i n e d sequence and an a p p r o x i m a t e m o l e c u l a r w e i g h t o f 10,000 w h i c h had t h e 2 , 4 - d i n i t r o p h e n y l - h a p t e n (2 ,4-DNP-hapten) a t r e g u l a r i n t e r v a l s o f about 30 A a l o n g the backbone, when the polymer was i n i t s e x t e n d e d form. The s t r u c t u r e was ( a - D N P - L - l y s - ( D - a l a - L - a l a ) , . ) n where n = 10.2 on the a v e r a g e . The m i c r o e n v i r o n m e n t around t h e hapten was r e l a t i v e l y homogeneous and n o n a n t i g e n i c , and i n i t s e x t e n d e d form t h e hapten groups were s e p a r a t e d by d i s t a n c e s g r e a t e r than the l a r g e s t d i m e n s i o n s e s t i m a t e d f o r t h e a n t i b o d y c o m b i n i n g s i t e , a p p r o x i m a t e l y 3^ AV-J as r e p o r t e d by Kabat (1960). In aqueous s o l u t i o n a t pH • 7.-0,. v t h e polymer i s a random c o i l w i t h no e v i d e n c e o f 3 _ c o n f o r m a t i o n o r a - h e l i x because o f the a l t e r n a t i n g D- and L-amino a c i d sequence. T h i s d e f i n e d sequence polymer w i t h t h e DNP-hapten was s t u d i e d a l o n g w i t h t h e DNP-hapten i n two o t h e r e n v i r o n m e n t s o f i n c r e a s i n g h e t e r o g e n e i t y . The second a n t i g e n was a s t a t i s t i c a l N - c a r b o x y a n h y d r i d e polymer w i t h a r e s i d u e r a t i o o f D L - a l a n i n e t o D N P - l y s i n e o f 1 0 . 6 : 1 , and h a v i n g the same amino a c i d c o m p o s i t i o n as the d e f i n e d sequence polymer The t h i r d , most complex a n t i g e n was randomly d i n i t r o p h e n y l a t e d b o v i n e y - g l o b u l i n . S t u d i e s o f t h e immune response produced i n r a b b i t s i n j e c t e d w i t h t h e s e t h r e e a n t i g e n s i n d i c a t e d t h a t as i n c r e a s i n g degrees o f " o r d e r " a r e imposed on the h a p t e n i c e n v i r o n m e n t , the a n t i b o d y response becomes more homogenous. The e x p e r i m e n t s i n v o l v e d d i s p e r s i o n o f m o l e c u l a r charge d i s t r i b u t i o n , and v a r i a t i o n s i n t h e magnitude o f n o n c o v a l e n t i n t e r a c t i o n s between t h e hapten and the a n t i b o d y p o p u l a t i o n s g i v e n as a s s o c i a t i o n c o n s t a n t s . These w o r k e r s a l s o produced e v i d e n c e t h a t a n t i b o d i e s t o the d e f i n e d - s e q u e n c e polymer had e a r l y m a t u r a t i o n p r o p e r t i e s showing no r i s e i n the aver a g e b i n d i n g e nergy l a t e r i n t h e immune r e s p o n s e , and were a l l o f t h e same c h a r g e . The N - c a r b o x y a n h y d r i d e polymer h a v i n g t h <same c o m p o s i t i o n , but a l e s s r i g i d l y o r d e r e d environment o f t h e dinitrophenyl-amino alkyl residue, gave rise to antibodies having intermediate maturation properties and some difference in charge distribution. Antibodies produced to randomly dinitrophenylated bovine ^f-globulin showed a marked rise in association constants with late response antibodies, and also showed a greater difference in charge distribution. V. Antigenic Determinants on Protein Antigens A major approach to the study of antigenic determinants and the dimensions of antibody combining sites must ultimately involve research on natural protein antigens. In instances where this approach has been undertaken, antigens have been degraded and the haptenic peptides isolated and characterized. It has been shown from these studies that proteins may contain many different antigenic determinants. 1. Tobacco mosaic virus protein subunit (TMVP) The most extensive of these studies so far have been those on TMVP by Benjamini and his associates (Benjamini, Young, Shimizu and Leung, 1964; Benjamini et_ al_., 1965). From the TMVP subunit, a single polypeptide chain of 158 amino acid residues, a twenty amino acid "peptide was found to be the only haptenic one present after trypsin digestion. Further investigation of this peptide showed that the * C - t e r m i n a l f i v e amino a c i d s (sequence L e u - A s p - A l a - T h r - A r g ) were the c r i t i c a l ones f o r a n t i g e n i c i t y (Young, B e n j a m i n i , S t e w a r t and Leung, I 9 6 7 ) . Subsequent work i n v o l v i n g s o l i d phase p e p t i d e s y n t h e s i s d e m o n s t r a t e d t h a t t h e h y d r o p h o b i c i t y o f t h e N - t e r m i n a l p o r t i o n o f t h i s p e n t a p e p t i d e enhanced t h e b i n d i n g o f a n t i b o d i e s ( B e n j a m i n i , S h i m i z u , Young and Leung, I 9 6 8 ) . The N - ( ^ C ) o c t a n o y l d e r i v a t i v e s o f t he C - t e r m i n a l t e t r a - , t r i - , and d i - p e p t i d e s o f the h a p t e n i c p e n t a p e p t i d e were s y n t h e s i z e d and t e s t e d f o r t h e i r c a p a c i t y t o b i n d s p e c i f i c a l l y w i t h anti-TMVP a n t i b o d i e s . The. r e s u l t s showed t h a t t h e o c t a n o y l t r i p e p t i d e and t e t r a p e p t i d e e x h i b i t e d s p e c i f i c b i n d i n g , but not the d i p e p t i d e ( B e n j a m i n i e t al... ,1968). By s o l i d phase p e p t i d e s y n t h e s i s , B e n j a m i n i ' s group showed t h a t t h e shape o f the a s p a r t i c a c i d r e s i d u e was more i m p o r t a n t than i t s n e g a t i v e c h a r g e , s i n c e a s p a r a g i n e c o u l d be s u b s t i t u t e d f o r a s p a r t i c a c i d , but g l u t a m i c a c i d c o u l d not (Young, B e n j a m i n i and Leung, I 9 6 8 ) . In a n o t h e r s e t o f e x p e r i m e n t s , the C - t e r m i n a l a r g i n i n e r e s i d u e was shown t o be more i m p o r t a n t than t h e N - t e r m i n a l l e u c i n e , i n t h a t removal o f the a r g i n i n e from t h e C - t e r m i n a l o f t h e p e p t i d e r e s u l t e d i n a pronounced d e c r e a s e i n h a p t e n i c a c t i v i t y , whereas removal o f t h e N - t e r m i n a l l e u c i n e o r both the l e u c i n e and a s p a r t i c a c i d d i d not (Young, B e n j a m i n i , S h i m i z u and Leung, I 9 6 6 ) . 2. B r a d y k i n i n , The work o f Spragg et_ a j . (1 968) on a n t i b o d y t o b r a d y k i n i n was a l s o an atte m p t t o e l u c i d a t e t h e s t r u c t u r a l and minimal r e q u i r e m e n t s f o r t h e b i n d i n g o f a n t i g e n t o a n t i b o d y . In t h i s s t u d y , a n t i b o d i e s were produced by r a b b i t s i n j e c t e d w i t h a b r a n c h - c h a i n coploymer o f b r a d y k i n i n , a n o n a p e p t i d e , and p o l y - L - l y s i n e . I n t r i n s i c a l l y l a b e l l e d ( C ) - b r a d y k i n i n and s i n g l y s u b s t i t u t e d a l a n i n e a n a l o g u e s o f b r a d y k i n i n and b r a d y k i n i n f r a g m e n t s were t e s t e d i n c o m p e t i t i o n e x p e r i m e n t s f o r b i n d i n g t o t h e a n t i b o d y . The amino a c i d sequence o f b r a d y k i n i n i s A r g - P r o - P r o - G l y - P h e - S e r - P r o - P h e - A r g . A l t e r a t i o n o f charge by r e p l a c i n g a r g i n i n e w i t h n i t r o a r g i n i n e o r t h e h y d r o p h o b i c c h a r a c t e r , by r e p l a c i n g p h e n y l a l a n i n e w i t h a l a n i n e , had r e l a t i v e l y l i t t l e e f f e c t on b i n d i n g t o a n t i b o d y . However, s u b s t i t u t i o n a t p r o l i n e , p o s i t i o n 3, o r g l y c i n e , p o s i t i o n k, which a r e o b l i g a t o r y f o r c o n f o r m a t i o n , produced t h e most p r o f o u n d change i n b i n d i n g . From the e x p e r i m e n t s u t i l i z i n g f r agments o f ' t h e sequence, i t was shown t h a t t he a n t i b o d y r e c o g n i z e d t h e p e p t i d e i n a p r e f e r r e d c o n f o r m a t i o n w h i c h r e q u i r e d t h e e n t i r e n o n a p e p t i d e sequence. 3. Human serum a l b u m i n L a p r e s l e and Webb (1965) , u s i n g p r o l o n g e d p e p t i c , c h y m o t r y p t i c and t r y p t i c d i g e s t i o n o f human serum a l b u m i n , showed t h a t one fragment w i t h a m o l e c u l a r w e i g h t o f 6 ,600 p o s s e s s e d o n l y one a n t i g e n i c d e t e r m i n a n t w h i c h p a r t i a l l y i n h i b i t e d t h e p r e c i p i t a t i o n r e a c t i o n between serum a l b u m i n and i t s homologous a n t i s e r u m . U s i n g t h e immunoadsorbent technique it antibody population to whole this determinant. was found that only about \% of the serum albumin was directed against k. Sperm whale myoglobin In sperm whale myoglobin, Crumpton and Wilkinson (1965) found eight chymotryptic peptides which had the capacity to inhibit precipitation using both antisera to metmyoglobin and apomyoglobin, and their respective antigens. However, the greatest degree of hapten inhibition obtained was 15% which indicates: that there are many more antigenic determinants present in other portions of the molecule; that these peptides could not assume the correct conformation to show maximal inhibition; or that these peptides did not represent the complete sequence of any single determinant. Atassi's work (1968) on nitrated apomyoglobin and metmyoglobin showed that one or both tyrosines 146 and 151 are present in a reactive region of the molecules, since the nitrated derivatives possessed lowered antigenic reactivities relative to apomyoglobin and metmyoglobin with their homologous antisera. A peptide fragment containing residues 56-131 and its derivative nitrated at tyrosine 103 (the third tyrosine in metmyoglobin) showed identical reactivity to the amount of antibody specifically adsorbed from antiserum to metmyoglobin, and therefore it was concluded that this tyrosine residue was not located in an antigenic region. By s i m i l a r methods, A t a s s i and Caruso (1968) showed t h a t a d e r i v a t i v e o f apomyoglobin m o d i f i e d a t t r y p t o p h a n 1 was i d e n t i c a l immunochemical 1y t o apomyoglobin p r e t r e a t e d w i t h 8.0 M ur e a - 5% methanol a t pH 3.0 (U-ApoMb) and w i t h metmyoglobin p r e p a r e d by complex f o r m a t i o n between f e r r i h e m e and U-ApoMb r e s p e c t i v e l y , and t h e r e f o r e i t was c o n c l u d e d t h a t t r y p t o p h a n 7 was not an e s s e n t i a l p a r t o f an a n t i g e n i c s i t e . I f c o n d i t i o n s were chosen so t h a t both t r y p t o p h a n s 7 and 14 were m o d i f i e d , t h e r e was a d r a s t i c d e c r e a s e i n a n t i g e n i c r e a c t i v i t y due p r o b a b l y t o changes i n c o n f o r m a t i o n o f the m o l e c u l e by u n f o l d i n g . U s i n g m i l d p e r i o d a t e o x i d a t i o n c o n d i t i o n s , so 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 a t p o s i t i o n s 55 and 131 were o x i d i z e d , A t a s s i (1967)showed t h a t t h e s e two r e s i d u e s were a l s o not e s s e n t i a l p a r t s o f the a n t i g e n i c d e t e r m i n a n t s o f metmyoglobin. An i n t e r e s t i n g o b s e r v a t i o n made from s t u d i e s on t r y p t i c d i g e s t s o f sperm whale myoglobin f o l l o w i n g i s o l a t i o n o f s o l u b l e p e p t i d e s by chromatography was t h a t the f i v e immunochemically r e a c t i v e t r y p t i c p e p t i d e s o c c u p i e d m a i n l y t h e f o u r c o r n e r s on the s u r f a c e i n t h e t h r e e -d i m e n s i o n a l s t r u c t u r e o f t h i s m o l e c u l e proposed by Kendrew e t a l . ( 1 9 6 1 ) . The p e p t i d e s o n l y showed i n h i b i t o r y a c t i v i t y by q u a n t i t a t i v e p r e c i p i t a t i o n , and d i d not p r e c i p i t a t e a n t i b o d y . T h e r e f o r e , each p e p t i d e was assumed t o c o n t a i n o n l y one a n t i g e n i c d e t e r m i n a n t . C l e a v a g e a t t h e two m e t h i o n i n e r e s i d u e s a t p o s i t i o n s 55 and 131 shown p r e v i o u s l y t o be n o n - e s s e n t i a l f o r a n t i g e n i c i t y , produced t h r e e f r a g m e n t s w h i c h a c c o u n t e d f o r a l m o s t a l l the immunochemical r e a c t i v i t y o f t h e i n t a c t m o l e c u l e . The two l a r g e s t f r a g m e n t s , 1 - 55 and 56 - 131 had the capacity to precipitate with antiserum against the whole protein, and therefore contain at least two antigenic determinants but fragment"' 132 - 153 acted as a true hapten possessing only the ab i l i t y to inhibit precipitation between the whole molecule and its antiserum. In the fragment containing the residues 1 - 55, the tryptophan at position 7 can be cleaved to yield a small peptide, 1 - 7, and the remaining 8 - 55. The small N-terminal peptide was found to possess no immunological reactivity. However, there was some evidence that this sequence might be important for the proper orientation of the reactive regions on fragment 1 - 55, since the precipitation obtained with fragment 8 - 55, for a given serum, was considerably less than that from the complete fragment 1 - 55. 5. Ribonuclease Oxidized ribonuclease was digested with trypsin, chymotrypsin, and pepsin, and various peptides were isolated. Brown (1962) found that peptides from two different regions of the protein specifically inhibited the immune reaction between rabbit antibody and oxidized ribonuclease. On a molar basis, the larger, 24-amino acid peptide caused 50% inhibition of the precipitin reaction between oxidized ribonuclease and its homologous antiserum. Further enzyme digestion of this peptide showed that the antigenic region lay within the f i r s t 15 amino acids of the sequence. The other peptide, 19 amino acids i n l e n g t h , and near t h e C - t e r m i n a l o f t h e a n t i g e n , caused 22% i n -h i b i t i o n o f t h e p r e c i p i t i n r e a c t i o n w i t h the same system. Both t h e s e h a p t e n i c p e p t i d e s a r e r i c h i n n o n - p o l a r amino a c i d s which may be i m p o r t a n t i n m a i n t a i n i n g t h e i r s t a b i l i t y and secondary s t r u c t u r e i n an aqueous e n v i r o n m e n t . I t i s i n t e r e s t i n g t o n o t e t h a t p e r f o r m i c a c i d o x i d i z e d , o r r e -duced and c a r b o x y m e t h y l a t e d r i b o n u c l e a s e , l a c k i n g d i s u l f i d e bonds and t h e r e f o r e much o f i t s t e r t i a r y s t r u c t u r e , does not p r e c i p i t a t e a n t i b o d y d i r e c t e d a g a i n s t n a t i v e r i b o n u c l e a s e . T h i s l a c k o f c r o s s -r e a c t i v i t y between a n t i b o d i e s d i r e c t e d t o t h e n a t i v e w e l 1 - c h a r a c t e r i z e d t h r e e - d i m e n s i o n a l form o f the p r o t e i n , and the reduced and c a r b o x y -m e t h y l a t e d form o f an a n t i g e n was a l s o demonstrated w i t h the lysozyme and CM-lysozyme system (Gerwing and Thompson, 1968). I t s h o u l d a l s o be noted here t h a t i n both t h e s e i n s t a n c e s , a l l h a p t e n i c p e p t i d e s so f a r d e m o n s t r a t e d , c o n t a i n one o r more c y s t e i n y l r e s i d u e s . 6. B a c t e r i a l f e r r e d o x i n s In p r o t e i n s , such as b a c t e r i a l f e r r e d o x i n s , i n w h i c h t h e t o t a l t e r t i a r y s t r u c t u r e has not been e s t a b l i s h e d , the immunological method may e l u c i d a t e i n p a r t the s i g n i f i c a n c e o f c o n f o r m a t i o n i n the a n t i b o d y c o m b i n i n g s i t e . Work on c l o s t r i d i a l f e r r e d o x i n s , u s i n g the p e r f o r m i c a c i d o x i d i z e d d e r i v a t i v e and r e a c t i n g i t w i t h a n t i b o d i e s t o n a t i v e f e r r e d o x i n , has shown t h a t a h i g h degree o f c r o s s - r e a c t i v i t y e x i s t s i n t h i s i n s t a n c e ( N i t z , M i t c h e l 1 , Gerwing and C h r i s t e n s e n , 1 9 6 9 ) . ' In f a c t , i t was found t h a t t h e immune r e a c t i o n appeared t o be g r e a t e r between o x i d i z e d f e r r e d o x i n and a n t i s e r u m a g a i n s t t h e n a t i v e m o l e c u l e than i t was w i t h t h e homologous system. T h i s might i n d i c a t e t h a t i n t h e animal body, t h e n a t i v e f e r r e d o x i n undergoes a l t e r a t i o n , p o s s i b l y r e s u l t i n g from t h e removal o f i r o n m o l e c u l e s w h i c h a r e bound t o the c y s t e i n e r e s i d u e s by n o n c o v a l e n t i n t e r m o l e c u l a r f o r c e s . The o b s e r v a t i o n t h a t a c o n s i d e r a b l e degree o f c r o s s - r e a c t i v i t y a l s o e x i s t e d between p e r f o r m i c a c i d o x i d i z e d and c a r b o x y m e t h y l a t e d f e r r e d o x i n and t h e i r r e s p e c t i v e a n t i s e r a a l s o i n d i c a t e d t h a t the c y s t e i n e r e s i d u e s i n t h i s s y stem, none o f w h i c h a r e i n v o l v e d i n d i s u l f i d e bonds, do not p l a y a d i r e c t r o l e i n t h e r e g i o n s r e s p o n s i b l e f o r a n t i g e n i c i t y . 7. N a t i v e lysozyme S h i n k a e t a_]_. (1967) r e c e n t l y p u b l i s h e d e v i d e n c e t h a t i n n a t i v e l y s o z y m e , f o u r p e p t i c p e p t i d e s e x h i b i t e d h a p t e n i c p r o p e r t i e s . A l l t h e s e p e p t i d e s c o n t a i n e d t h e sequence from G in 57 t o A l a 107 ( p e p t i d e 7 a ) , and d i f f e r e d o n l y i n t h e i r c l e a v a g e p o i n t s i n t h e lo o p between Cys 80 and Cys 3k. F u r t h e r work by t h i s group ( F u j i o , I m a n i s h i , N i s h i o k a and Amano, 1968a) showed t h a t a n o t h e r immuno-l o g i c a l l y a c t i v e p e p t i d e c o u l d be i s o l a t e d by l i m i t e d p e p s m / d i g e s t i o n , w h i c h c o n t a i n e d both the N- and C - t e r m i n a l p e p t i d e s l i n k e d by a s i n g l e d i s u l f i d e between Cys 6 and Cys 127. T h i s p e p t i d e c o n t a i n e d t h e sequence Lys 1 t o Asn 2 7 , and A l a 122 t o Leur=,129 and was d e s i g n a t e d p e p t i d e 17. E q u i l i b r i u m d i a l y s i s e x p e r i m e n t s showed t h a t K ] % o f the a n t i b o d y i n t h e 7 S a n t i - h e n e g g - w h i t e lysozyme was d i r e c t e d a g a i n s t t h i s p e p t i d e . These two a n t i g e n i c a l l y a c t i v e p e p t i d e s were independent o f each o t h e r , as shown by t h e o b s e r v a t i o n t h a t t h e r e was no i n h i b i t i o n o f b i n d i n g of one o f t h e s e p e p t i d e s t o a n t i s e r u m i n the p r e s e n c e o f the o t h e r ( F u j i o , I m a n i s h i , N i s h i o k a and Amano, 1968b) . Arnon and S e l a (1969) have a l s o s t u d i e d the a n t i g e n i c p r o p e r t i e s o f n a t i v e lysozyme. They p r e p a r e d a s y n t h e t i c c o n j u g a t e composed o f a fragment o f lysozyme (sequence Cys 64 t o Leu 83) w h i c h they d e s i g n a t e d as t h e " l o o p " p e p t i d e , and branched p o l y - D L - a l a n i n e . T h i s p e p t i d e c o m p r i s e s a p o r t i o n o f the l o n g e r h a p t e n i c p e p t i d e i s o l a t e d by S h i n k a e t _ a j _ . ( 1967) w h i c h was d e s i g n a t e d p e p t i d e 7 a . T h i s c o n j u g a t e was used t o immunize r a b b i t s t o produce a n t i b o d i e s s p e c i f i c a l l y a g a i n s t t h i s unique r e g i o n i n t h e n a t i v e lysozyme m o l e c u l e , and t h e p u r i f i c a t i o n o f t h e a n t i b o d i e s was c a r r i e d out u s i n g a l y s o z y m e - c e l 1 u l o s e immuno-a d s o r b e n t . By p r e p a r i n g an immunoadsorbent from t h i s " l o o p " p e p t i d e , Arnon and S e l a were a b l e t o i s o l a t e a n t i b o d i e s o f s i m i l a r s p e c i f i c i t y from a n t i - l y s o z y m e serum. These a n t i - " l o o p " a n t i b o d i e s were a b l e t o d i s t i n g u i s h between the " l o o p " p e p t i d e , c o n t a i n i n g a d i s u l f i d e bond, and the o p e n - c h a i n c a r b o x y m e t h y l a t e d p e p t i d e d e r i v e d from i t , s u g g e s t i n g t h a t they may be d i r e c t e d a g a i n s t a c o n f o r m a t i o n - d e p e n d e n t d e t e r m i n a n t . These w o r k e r s f e l t t h a t the a v a i l a b i l i t y o f t h e two p o p u l a t i o n s o f a n t i b o d i e s , d i r e c t e d most l i k e l y t o the same r e g i o n w i t h i n t h e n a t i v e p r o t e i n , w i l l p r o v i d e an o p p o r t u n i t y t o compare them and t o l e a r n t h e r o l e o f t h e a n t i g e n i c c a r r i e r i n t h e homogeneity o f a n t i -b o d i e s t o a s p e c i f i c d e t e r m i n a n t . T h i s p r o p o s e d work w i l l be i n t e r e s t i n g i n v i e w o f t h e p r e v i o u s l y mentioned work o f R i c h a r d e t aj_. (1969) on the r o l e o f t h e environment around the hapten i n the immune resp o n s e . The lysozyme m o l e c u l e was chosen f o r s t u d y f o r s e v e r a l r e a s o n s . F i r s t l y , the complete amino a c i d sequence i s known; s e c o n d l y , X-ray c r y s t a l l o g r a p h y has been c o m p l e t e d on the m o l e c u l e and a t h r e e -d i m e n s i o n a l s t r u c t u r e has been p r o p o s e d , s o t h a t the a r e a s found t o be h a p t e n i c can be c o r r e l a t e d w i t h t h e i r l o c a t i o n i n the c r y s t a l l i n e s t r u c t u r e ; t h i r d l y , t h e f o u r d i s u l f i d e bonds i n t h e s i n g l e p o l y p e p t i d e c h a i n g i v e t h e n a t i v e m o l e c u l e a f a i r l y r i g i d t e r t i a r y s t r u c t u r e ; f i n a l l y , lysozyme i s a h i g h l y b a s i c p r o t e i n w i t h a p i ( i s o e l e c t r i c p o i n t ) o f pH 11.05, and a s t u d y o f i t s a n t i g e n i c d e t e r m i n a n t s might be i n t e r e s t i n g i n voew o f the work o f S e l a and Mozes'. (1'9"66) d e m o n s t r a t i n g t h a t a n t i b o d i e s produced t o b a s i c p r o t e i n a n t i g e n s a r e found i n the a c i d i c f r a c t i o n o f immunoglobulins a f t e r chromatography on DEAE-Sephadex. 8. Reduced and c a r b o x y m e t h y l a t e d lysozyme (CM-lysozyme) In t h i s s t u d y , reduced and c a r b o x y m e t h y l a t e d lysozyme (CM-lysozyme) was used as t h e a n t i g e n . R e s u l t s from i n i t i a l e x p e r i m e n t s i n d i c a t e d t h a t CM-lysozyme d i d not c r o s s - r e a c t a t t h e p r e c i p i t i n l e v e l w i t h n a t i v e lysozyme a n t i s e r u m , and none o f the t r y p t i c p e p t i d e s i s o l a t e d from i t showed any c a p a c i t y t o a c t as haptens w i t h t h e n a t i v e system. U n p u b l i s h e d work by Young and coworkers from B e n j a m i n i ' s group has s u b s t a n t i a t e d t h e s e o b s e r v a t i o n s showing t h e l a c k o f i n h i b i t i o n o f t h e l y s o z y m e - a n t i - l y s o z y m e r e a c t i o n by s m a l l p e p t i d e fragments i s o l a t e d from CM-lysozyme, when they t e s t e d them e i t h e r by complement f i x a t i o n o r radio-immunoassay t e c h n i q u e s . They found t h a t CM-lysozyme would r e a c t w i t h a n t i - l y s o z y m e serum a t a n t i b o d y c o n c e n t r a t i o n s h i g h e r than t h o s e r e q u i r e d f o r the b i n d i n g o f an e q u i v a l e n t amount o f lysozyme. T h i s C M - l y s o z y m e - a n t i - l y s o z y m e r e a c t i o n c o u l d not be i n h i b i t e d by c h y m o t r y p t i c d i g e s t s , but c o u l d be i n h i b i t e d by t r y p t i c d i g e s t s o f CM-lysozyme, o r by p e p t i d e s c o n t a i n i n g the N- and C - t e r m i n a l r e g i o n s , Lys 1 t o Met 12, and Asn 106 t o Leu 129, i s o l a t e d from cyanogen bromide (CNBr) c l e a v a g e o f CM-lysozyme. Because t h i s C M - l y s o z y m e - a n t i - l y s o z y m e r e a c t i o n c o u l d not be i n h i b i t e d by n a t i v e lysozyme u n l e s s the lysozyme was i n e x t r e m e l y h i g h c o n c e n t r a t i o n (1 ,000 f o l d e x c e s s ) , t h e s e w o r k e r s s u g g e s t e d t h a t the a n t i b o d i e s i n v o l v e d i n b i n d i n g the CM-lysozyme a r e , i n f a c t , more s p e c i f i c t o a d e n a t u r e d form o f lysozyme w h i c h may be p r e s e n t i n s m a l l amounts i n p r e p a r a t i o n s o f t h i s enzyme. The d e t e r m i n a n t a r e a s e x i s t i n g i n t h e t r y p t i c p e p t i d e s and t h e C - t e r m i n a l CNBr p e p t i d e may not have t h e same c o n f o r m a t i o n as i n t h e n a t i v e c r y s t a l l i n e lysozyme, and t h i s may e x p l a i n t h e i r i n h i b i t o r y a c t i v i t y . From t h e work r e p o r t e d i n t h i s t h e s i s on CM-lysozyme, i t was shown t h a t a s i n g l e t r y p t i c p e p t i d e encompassing the sequence Asn Ik -Lys 96 e x h i b i t e d s t r o n g h a p t e n i c a c t i v i t y . I t i s i n t e r e s t i n g .to note t h a t t h i s p e p t i d e f a l l s w i t h i n t h e fragment i s o l a t e d by S h i n k a e t a l . (I967) from p e p t i c d i g e s t s o f n a t i v e lysozyme w h i c h showed h a p t e n i c a c t i v i t y , and o v e r l a p s t h e " l o o p " p e p t i d e s t u d i e d by Arnon and S e l a . (1969). MATERIALS AND METHODS I. Immunization P r o c e d u r e s The hen e g g - w h i t e lysozyme used i n a l l e x p e r i m e n t s was purchased from 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 . R e d u c t i o n and a l k y l a t i o n o f th e f o u r d i s u l f i d e bonds o f t h e lysozyme m o l e c u l e w i t h i o d o a c e t i c a c i d was c a r r i e d o u t ^ a c c o r d i n g t o t h e method r e p o r t e d by C a n f i e l d and A n f i n s e n (1963)• The t r e a t e d l y s o z y m e , termed CM-lysozyme, was l y o p h i l i z e d and s t o r e d a t h C as a powder u n t i l r e q u i r e d . For i m m u n i z a t i o n , 30 mg o f CM-lysdzyme was suspended i n 1.5 ml s t e r i l e 0.9% s a l i n e and e m u l s i f i e d w i t h 1.5 ml complete Freund's a d j u v a n t ( B a c t o - D i f c o ) . Immunization o f t h e t h r e e r a b b i t s w i t h t h e m a t e r i a l was c a r r i e d o u t as f o l l o w s . R a b b i t s were a n a e s t h e t i z e d w i t h 1.0 ml o f nembutal (Abbott L a b o r a t o r i e s ) a d m i n i s t e r e d i n t r a v e n o u s l y . The e m u l s i o n (0.2 ml) was i n j e c t e d i n t o a f o o t pad o f each o f t h e f o u r e x t r e m i t i e s , and a f u r t h e r 0.2 ml was i n j e c t e d i n t r a m u s c u l a r l y i n the l e f t hincl l e g . On t h e f o l l o w i n g day, 1.0 ml o f a l u m - p r e c i p i t a t e d a n t i g e n c o n t a i n i n g 1.5 mg o f CM-lysozyme was i n j e c t e d i n t r a v e n o u s l y i n t o each a n i m a l . The a l u m - p r e c i p i t a t e d a n t i g e n was p r e p a r e d a c c o r d i n g t o t h e method d e s c r i b e d by Kabat and Mayer (1961 - p. 3 0 9 ) . The a n t i b o d y t i t e r was e l e v a t e d f o u r weeks a f t e r the l a s t i n j e c t i o n , and remained unchanged i n s u c c e s s i v e samples c o l l e c t e d w eekly f o r s i x \ months. The serum was s t o r e d i n 50 .0 ml ba t c h e s a t k C. A f t e r i t was d e t e r m i n e d t h a t t h e t i t e r o f t h e serum from two o f t h e r a b b i t s was i d e n t i c a l , t h e two s e r a (from r a b b i t s 1 and 2) were p o o l e d as th e y were c o l l e c t e d . M e r t h i o l a t e a t a f i n a l c o n c e n t r a t i o n o f 1:10,000 was added as a p r e s e r v a t i v e . A f t e r s i x months, t h e r a b b i t s were g i v e n b o o s t e r i n j e c t i o n s o f 0.5 ml Freund's a d j u v a n t e m u l s i o n c o n t a i n i n g 3.0 mg CM-lysozyme i n t r a -m u s c u l a r l y i n t h e l e f t h i n d l e g . A d d i t i o n a l serum samples were c o l l e c t e d a t we e k l y i n t e r v a l s s t a r t i n g two weeks a f t e r t h e b o o s t e r i n j e c t i o n , and s t o r e d as above. I I. Q u a n t i t a t i v e P r e c i p i t i n R e a c t i o n The immune p r e c i p i t a t i o n r e a c t i o n was used f o r i n i t i a l s t u d i e s on the a n t i g e n i c p r o p e r t i e s o f reduced and c a r b o x y m e t h y l a t e d lysozyme (CM - 1ysozyme). Optimal p r o p o r t i o n s were d e t e r m i n e d by i n c u b a t i n g v a r y i n g amounts o f a n t i g e n , i n a c o n s t a n t volume, w i t h c o n s t a n t amounts' o f a n t i s e r u m a t 37 C f o r 1 hr f o l l o w e d by 18 hr a t k C. The immune p r e c i p i t a t e s were c e n t r i f u g e d a t 2000 g, washed t w i c e w i t h 0.3% N a C l , and d i s s o l v e d i n 1.0 ml o f 0.1 N NaOH. Then the abso r b a n c e a t 2800 A1', was read on a Beckman DB-G s p e c t r o p h o t o m e t e r . A s t o c k s o l u t i o n o f CM-lysozyme c o n t a i n i n g 100 rug/ml i n 0.3% NaCl was used t o t i t r a t e s e r a from a l l t h r e e r a b b i t s . Sera 1 and 2ewh ich had i d e n t i c a l t i t e r s p r e c i p i t a t e d o p t i m a l l y w i t h 25.0. :;y;g o f CM-lysozyme/ml o f u n d i l u t e d serum. Serum 3 p r e c i p i t a t e d o p t i m a l l y w i t h 50 yg o f CM-lysozyme under t h e same c o n d i t i o n s . Because o f i t s h i g h e r t i t e r , serum 3 was s e l e c t e d f o r use i n a l 1 f u r t h e r e x t e n s i v e p r e c i p i t i n t e s t s . Hapten i n h i b i t i o n s t u d i e s w i t h t r y p t i c p e p t i d e s were performed as f o l l o w s . The p e p t i d e i n 0.1 ml o f 0.3% NaCl was mixed w i t h 0 . 4 ml of u n d i l u t e d serum and i n c u b a t e d a t 37 C f o r 1 h r f o l l o w e d by 18 h r a t 4 C. A n t i g e n was then added a t t h e c a l c u l a t e d amount t o y i e l d o p t i m a l p r e c i p i t a t i o n w i t h 0 . 4 ml of a n t i s e r u m , and t h e i n c u b a t i o n a t 37 C and 4 C was r e p e a t e d . C o n t r o l tubes u s i n g 0.1 ml o f s a l i n e i n s t e a d o f p e p t i d e were t r e a t e d i d e n t i c a l l y . The r e s u l t i n g p r e c i p i t a t e s were the n p r e p a r e d as p r e v i o u s l y d e s c r i b e d and read f o r 2800 A. The p e r c e n t i n h i b i t i o n was c a l c u l a t e d as t h e p e r c e n t a g e d e c r e a s e i n o p t i c a l d e n s i t i e s a t 2800 A t a k i n g t h e s a l i n e c o n t r o l as 100%. These s t u d i e s were always conducted i n t r i p l i c a t e u n l e s s o t h e r w i s e s t a t e d . I I I . Complement F i x a t i o n R e a c t i o n F r e e z e - d r i e d g u i n e a p i g serum (3.0 ml q u a n t i t i e s ) o b t a i n e d from H y l a n d Company was used as a s o u r c e o f complement. The serum was r e c o n s t i t u t e d f o r each t e s t i n the d i l u e n t p r o v i d e d a n d f u r t h e r d i l u t i o n s were made i n v e r o n a l b u f f e r e d s a l i n e , between 1:25 and 1:250, between 1:50 and 1:500, o r between 1:75 and 1:750 depending on the b a t c h o f g u i n e a p i g serum. The v e r o n a l b u f f e r e d s a l i n e used f o r a l l d i l u t i o n s o f a n t i g e n , h a p t e n s , complement and a n t i s e r u m was p r e p a r e d a c c o r d i n g t o the method o f Brooksby (1952). A p o o l e d sample o f a n t i s e r u m 1 and 2 , and a s t o c k s o l u t i o n o f CM-lysozyme c o n t a i n i n g 100 ug/ml were used t h r o u g h o u t . The complement f i x a t i o n t i t e r o f the a n t i s e r u m was de t e r m i n e d by u s i n g ten d i l u t i o n s o f complement between the v a l u e s g i v e n above, d o u b l i n g d i l u t i o n s o f the a n t i s e r u m s t a r t i n g a t 1:10 up t o 1:160, and one d i l u t i o n o f a n t i g e n , 0.4 ug/ml. The complement, a n t i g e n and a n t i s e r u m d i l u t i o n s were a l l used i n 1.0 ml q u a n t i t i e s p er tube. A s i m i l a r t e s t t o t e s t f o r a n t i c o m p l e m e n t a r y a c t i v i t y i n the a n t i s e r u m was s e t up s i m u l t a n e o u s l y u s i n g 1.0 ml o f v e r o n a l - b u f f e r e d s a l i n e i n s t e a d o:f the a n t i g e n . The tubes were mixed by s h a k i n g and i n c u b a t e d i o v e r n i g h t a t 4 C. A 2% s u s p e n s i o n o f twice-washed sheep e r y t h r o c y t e s i n v e r o n a l - b u f f e r e d s a l i n e was s e n s i t i z e d by m i x i n g i t w i t h an e q u a l volume o f a 1:50 d i l u t i o n o f the recommended s t a n d a r d s o l u t i o n o f h e m o l y s i n ( D i f c o - B a c t o A n t i s h e e p Hemolysin) f o r 10 min. T h i s p r e p a r a t i o n was added i n 1.0 ml a l i q u o t s t o the t e s t s e r i e s and i n c u b a t e d a t 37 C f o r 30 min i n a w a t e r b a t h . The tubes were shaken once at 15 min. F o l l o w i n g the 30 min i n c u b a t i o n , unhemolyzed red b l o o d c e l l s were sedimented by c e n t r i f u g a t i o n a t 1800 g_ and the s u p e r n a t a n t s were read on a K l e t t c o l o r i m e t e r u s i n g the green f i l t e r . End p o i n t s were c a l c u l a t e d as 50% h e m o l y s i s on a p r o b i t p l o t o f the a c t u a l p e r c e n t a g e s o f the K l e t t r e a d i n g s a c c o r d i n g t o the method d e s c r i b e d by W r i g h t ( 1963 ) . T i t e r s o f the p o o l e d t e s t a n t i s e r a 1 and 2 were 1:80 i n the p r e s e n c e o f the s t a n d a r d amount o f CM-lysozyme, 0.4 ug/ml. The f r e s h p o o l e d a n t i s e r u m was heated a t 55 C f o r 20 min t o i n a c t i v a t e any complement. No a n t i c o m p l e m e n t a r y a c t i v i t y was demonstrated by the p o o l e d a n t i s e r u m p r e p a r a t i o n (even a t a 1:20 d i l u t i o n ) a f t e r heat i n a c t i v a t i o n . The a n t i g e n was t i t r a t e d by r e p e a t i n g t h e t e s t s u s i n g 1 : 8 0 a n t i s e r u m t h r o u g h o u t w i t h d o u b l i n g d i l u t i o n s o f a n t i g e n r a n g i n g from 6.4 t o 0.0125 yg/ml. The d a t a showing t h e c a l c u l a t i o n o f the o p t i m a l a n t i g e n range a r e shown i n F i g u r e 1. Hapten i n h i b i t i o n s t u d i e s i n v o l v e d the use o f s t a n d a r d amounts o f a n t i g e n (0.8 yg/ml) and a n t i s e r u m ( 1 : 8 0 ) and v a r y i n g amounts o f complement and p e p t i d e . The t e s t p e p t i d e s i n 0.2 ml were a l l added i n i t i a l l y a t a c o n c e n t r a t i o n o f 50 yg i n 0.2 ml. P r e l i m i n a r y e x p e r i m e n t s w i t h a l l the t r y p t i c p e p t i d e s from CM-lysozyme i n d i c a t e d i n h i b i t i o n i n o n l y two p r e p a r a t i o n s , the po o l e d t r y p t i c d i g e s t and one p u r i f i e d p e p t i d e . The p o s i t i v e t e s t s were r e - a s s a y e d o v e r a range o f 50.0 t o 0.10 yg. The hapten i n h i b i t i o n t e s t s were c a r r i e d o u t as f o l l o w s . The p e p t i d e - a n t i s e r u m m i x t u r e s were i n c u b a t e d a t 37 C f o r 1 h r x a n d 18 h r a t 4 C. S u b s e q u e n t l y , 0.8 yg o f a n t i g e n i n 1.0 ml, and 1.0 ml o f complement c o n t a i n i n g d i l u t i o n s between 1:40 and 1:125 o f g u i n e a p i g serum, were added and t h e m i x t u r e s were l e f t o v e r n i g h t a t 4 C. On the t h i r d day, t h e i n d i c a t o r h e m o l y s i n system was added as p r e v i o u s l y d e s c r i b e d , and end p o i n t s were a g a i n taken as 50% h e m o l y s i s . 29 30 IV. P r 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 The c a r b o x y m e t h y l a t e d lysozyme was d i g e s t e d w i t h t r y p s i n ( W o r t h i n g t o n , l y o p h i l i z e d c o n t a i n i n g 125 units/mg) as d e s c r i b e d by C a n f i e l d ( 1963a ) . The t r y p s i n was as s a y e d u s i n g t h e h y d r o l y s i s o f p - t o l u e n e s u l f o n y 1 -L - a r g i n i n e methyl e s t e r (TAME) by t r y p s i n and measuring a n . i n c r e a s e i n absorbance a t 2^70 A (Hummel, 1959 ) . One u n i t o f t r y p s i n a c t i v i t y i s d e f i n e d as the h y d r o l y s i s o f 1 ymole o f TAME/min a t 25 C and pH 8.1 i n the p r e s e n c e o f 10 mM C a + + . The a s s a y f o r a c t i v i t y and the c a l c u l a t i o n o f the u n i t s o f a c t i v i t y i s shown i n F i g u r e 2. The d i g e s t i o n o f CM-lysozyme i n v o l v e d a 1% s o l u t i o n o f CM-lysozyme i n d i s t i l l e d w a t e r w i t h the pH m a i n t a i n e d a t 8.0 w i t h d i l u t e NH^OH, and t r y p s i n i n an amount e q u a l t o 2% o f t h e CM-lysozyme by w e i g h t . The s o l u t i o n was s t i r r e d a t 37 C f o r 2 hr f o r o p t i m a l t r y p s i n d i g e s t i o n and the pH was p e r i o d i c a l l y a d j u s t e d t o 8 . 0 . The i n s o l u b l e c o r e m a t e r i a l was removed by c e n t r i f u g a t i o n . D i g e s t s u s u a l l y l a c k e d the a b i l i t y t o p r e c i p i t a t e w i t h homologous a n t i s e r u m by the r i n g t e s t , but the p r e c a u t i o n was ta k e n o f p a s s i n g them through an u l t r a f i l t e r d e s i g n a t e d t o r e t a i n m a t e r i a l o f more th a n 10,000 m o l e c u l a r w e i g h t (Amicon u l t r a f i l t e r , UM-1 f i l t e r pad) b e f o r e p u r i f i c a t i o n o f t h e p e p t i d e s c h r o m a t o g r a p h i c a l l y . In the i n i t i a l s t a g e s o f t h e work, C a n f i e l d ' s method (1963a) f o r p e p t i d e s e p a r a t i o n , u s i n g a Dowex 50 x k (Bio-Rad) column o f d i m e n s i o n s (0 .7 x 150.0 cm) a t 35 C, w i t h an eight-chambered 31 F i g u r e 2. A s s a y o f enzyme a c t i v i t y o f t h e t r y p s i n p r e p a r a t i o n by h y d r o l y s i s o f t h e s u b s t r a t e p - t o l u e n e s u l f o n y 1 -L.-a r g i n i ne m e t h y l e s t e r . g r a d i e n t s y s t e m , was employed, w i t h each b u f f e r a t a volume o f 500.0 ml. In t h e f i r s t column s e p a r a t i o n , 100 mg o f t r y p t i c a l l y d i g e s t e d CM-lysozyme was a d j u s t e d t o pH 2.8 w i t h f o r m i c a c i d , a p p l i e d t o the column, and washed w i t h t h r e e 1.0 ml a l i q u o t s o f t h e s t a r t i n g b u f f e r . G r a d i e n t e l u t i o n w i t h an i n c r e a s i n g pH and s a l t c o n c e n t r a t i o n was used 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 s r a n g i n g from 0.10 M a c e t i c a c i d pH 3.8 up t o 2.0 M a c e t i c a c i d pH 5 .18. F r a c t i o n s o f 5.0 ml were c o l l e c t e d on an LKB f r a c t i o n c o l l e c t o r , and 0.3 ml a l i q u o t s o f e v e r y o t h e r tube were ta k e n and a n a l y z e d by the q u a n t i t a t i v e n i n h y d r i n r e a c t i o n a f t e r a l k a l i n e h y d r o l y s i s a c c o r d i n g t o t h e method of H i r s et_ aj_. (1956) . A r e p r e s e n t a t i v e e l u t i o n p r o f i l e w i t h the e i g h t - c h a m b e r e d g r a d i e n t system i s shown i n F i g u r e 3. Each peak was p o o l e d and d r i e d by f l a s h e v a p o r a t i o n a t 45 C, washed w i t h d i s t i 1 l e d w a t e r , f r e e z e d r i e d , and d i s s o l v e d o r resuspended (some p e p t i d e s were o n l y s p a r i n g l y s o l u b l e ) i n 0.3% N a C l . From i n i t i a l i m m u n o l o g i c a l s t u d i e s , s t r o n g h a p t e n i c a c t i v i t y was o b s e r v e d i n t h e peak c o n t a i n i n g the T-11 p e p t i d e ( C a n f i e l d ' s (1963b) n o m e n c l a t u r e ) , and a c o n c o m i t a n t l a c k o f h a p t e n i c b e h a v i o u r on t h e p a r t o f any o t h e r p e p t i d e f r a c t i o n was n o t e d . T h e r e f o r e , i n f u r t h e r p e p t i d e s e p a r a t i o n s , the main purpose o f w h i c h was t o c o l l e c t T-11 m a t e r i a l , a simp 1 i f i e d four-chambered g r a d i e n t system was s e t up u s i n g the f i r s t f o u r p y r i d i n e - a c e t a t e b u f f e r s recommended by C a n f i e l d , r a n g i n g from 0.1 M a c e t i c a c i d pH 3.8 t o 0 .4 M a c e t i c a c i d pH 4 . 4 7 . In t h i s m o d i f i e d s ystem, 500 ml o f each b u f f e r were a g a i n used and t h e p e p t i d e s F i g u r e 3. E l u t i o n p r o f i l e o f t r y p t i c p e p t i d e s o f CM-lysozyme u s i n g t h e e i g h t - c h a m b e r e d g r a d i e n t s y s t e m o f C a n f i e l d (1963a). were e l u t e d t h r o u g h t h e Dowex 50 x k column a t 35 C u n t i l 1.0 1 had been c o l l e c t e d . Then t h e r e m a i n i n g adsorbed p e p t i d e s were e l u t e d by r u n n i n g 2.0 M b u f f e r a t pH 5.18 t h r o u g h the column. F r a c t i o n s o f 10.0 ml were c o l l e c t e d from w h i c h 0.3 ml a l i q u o t s were ta k e n and a n a l y z e d by t h e q u a n t i t a t i v e n i n h y d r i n r e a c t i o n as d e s c r i b e d above. F i g u r e k shows a r e p r e s e n t a t i v e e l u t i o n p r o f i l e o f t h e m o d i f i e d system. The T-11 p e p t i d e was e l u t e d as t h e f i r s t s h a r p l y d e l i n e a t e d peak, and was f r e e from o t h e r p e p t i d e s e l u t e d l a t e r . Amino a c i d a n a l y s i s o f t h i s m a t e r i a l e s t a b l i s h e d i t s p u r i t y and i d e n t i t y w i t h C a n f i e l d ' s T-11 p e p t i d e . T a b l e M p r e s e n t s the r e s u l t s o f t h e amino a c i d a n a l y s i s o f t h i s m a t e r i a l as w e l l as t h e r e s u l t s r e p o r t e d by C a n f i e l d . The n i n h y d r i n p o s i t i v e m a t e r i a l e l u t i n g w i t h t h e 2.0 M p y r i d i n e -a c e t a t e b u f f e r a t pH 5.18 from the column was p o o l e d and t e s t e d f o r h a p t e n i c a c t i v i t y a t a c o n c e n t r a t i o n o f 200 yg j t o c o n f i r m t h a t no h a p t e n i c p e p t i d e s were p r e s e n t i n t h i s m i x t u r e o f t r y p t i c p e p t i d e s . V. P r o t e i n and P e p t i d e A n a l y s i s Q u a n t i t a t i v e e s t i m a t i o n o f c a r b o x y m e t h y l a t e d lysozyme was c a l c u l a t e d i n i t i a l l y by t h e Lowry method (Lowry e t a l ,,, 1951) f o r p r o t e i n d e t e r m i n a t i o n , and equated on a w e i g h t b a s i s w i t h i t s e x t i n c t i o n a t 2800 A'L.:. S u b s e q u e n t l y , e s t i m a t i o n s were made on a s p e c t r o p h o t o m e t r i c b a s i s . The T-11 and o t h e r p e p t i d e s were q u a n t i t a t e d i n i t i a l l y on a d r y w e i g h t b a s i s . C l o s e r e s t i m a t i o n s o f o o ID IO o 0.5. 400 800 1200 ML ELUENT 1600 F i g u r e h. E l u t i o n p r o f i l e o f t r y p t i c p e p t i d e s o f CM-lysozyme u s i n g the s i m p l i f i e d f o u r - c h a m b e r e d g r a d i e n t system. Arrow d e s i g n a t e s change t o 2.0 M - b u f f e r a t pH 5.18. the T-11 p e p t i d e were o b t a i n e d from t h e c a l c u l a t i o n o f mic r o m o l e s o f s i n g l y o c c u r r i n g amino a c i d r e s i d u e s p r e s e n t a f t e r q u a n t i t a t i v e amino a c i d a n a l y s i s . Amino a c i d a n a l y s e s were c a r r i e d o ut a c c o r d i n g t o t h e method o f Spackman e t a l . (1-958) on a Beckman model 120 Amino A c i d A n a l y s e r . The T-11 p e p t i d e c o u l d not be q u a n t i t a t e d on t h e b a s i s o f i t s e x t i n c t i o n s p e c t r o p h o t o m e t r i c a l l y s i n c e i t c o n t a i n s no a r o m a t i c amino a c i d s . However, i t s a b s o r p t i o n spectrum a t l e a s t i n d i c a t e d no c o n t a m i n a t i o n w i t h t r y p t o p h a n - c o n t a i n i n g p e p t i d e s ( t h i s would not show up on amino a c i d a n a l y s i s ) . A t y p i c a l scan o f t h i s p e p t i d e from 3000 > 2200 Af) i s shown i n F i g u r e 5. The main i n d i c a t i o n o f t h e p u r i t y o f t h e T-11 p e p t i d e was t h a t f i n g e r p r i n t a n a l y s i s showed t h e p r e s e n c e o f o n l y one n i n h y d r i n p o s i t i v e s p o t . High v o l t a g e e l e c t r o p h o r e s i s was c a r r i e d o ut f o r 45 min a t 3000 V on a G i l s o n High V o l t a g e E l e c t r o p h o r e t o r i n p y r i d i n e - a c e t a t e b u f f e r (10%; 0.k%) pH 6.5 on Whatman #3 f i l t e r paper w i t h 0.1 mg o f t h e T-11 p r e p a r a t i o n . For t h e second d i m e n s i o n , t h e e l e c t r o p h o r e s e d s t r i p was sewn o n t o a n o t h e r p i e c e o f Whatman #3 f i l t e r paper and d e s c e n d i n g chromatography was c a r r i e d o u t i n b u t a n o l - p y r i d i n e - a c e t i c a c i d - w a t e r s o l v e n t (10:15 : 3:12, v:v:v:v) a t 25 C f o r 18 h r . V I . C a r b o x y p e p t i d a s e D i g e s t i o n s C a r b o x y p e p t i d a s e B ( W o r t h i n g t o n C0,B, not D F P - t r e a t e d ) was as s a y e d f o r enzyme a c t i v i t y a c c o r d i n g t o t h e r a t e o f h y d r o l y s i s o f 37 F i g u r e 5. T y p i c a l s c a n o f t h e T-11 p e p t i d e a t a c o n c e n t r a t i o n o f 0.10 mg/ml. h i p p u r y l - L - a r g i n i n e (Fo 1 k et_ a]_., 1960). The r a t e o f h y d r o l y s i s o f the s u b s t r a t e was de t e r m i n e d by r e c o r d i n g t h e i n c r e a s e i n absorban c y a t 2540 A o v e r one minute i n t e r v a l s . One u n i t o f enzyme a c t i v i t y i s d e f i n e d as the amount o f enzyme r e q u i r e d t o h y d r o l y z e 1.0 umole o f s u b s t r a t e per min under t h e c o n d i t i o n s s p e c i f i e d . Once th e a c t i v i t y o f the c a r b o x y p e p t i d a s e B (COB) had been d e t e r m i n e d (shown i n F i g u r e 6), an e x p e r i m e n t was s e t up u s i n g 1.0 ml o f T-11 i n s a l i n e ( c o n t a i n i n g 1.875 mg o r 0.75 ymole) and 0.4 ml o f COB i n 0.025 M T r i s b u f f e r ( c o n t a i n i n g 120 yg which p o s s e s s e d 0.25 u n i t s o f enzyme a c t i v i t y ) a t pH 8.5 f o r 4 hr a t 37 C. A s u b s t r a t e c o n t r o l c o n t a i n i n g 0.5 ml o f T-11 and 0.2 ml o f 0.25 M T r i s b u f f e r a t pH 8.5, and an enzyme c o n t r o l c o n t a i n i n g 1.0 ml s a l i n e and 0.4 ml o f COB i n T r i s b u f f e r a t pH 8.5 were t r e a t e d s i m i l a r l y . A f t e r 4 h r a t 37 C, d u p l i c a t e samples o f 0.1 ml were t a k e n from the T-11 d i g e s t and the c o n t r o l t u b e s , and the pH was a d j u s t e d t o below 4.0 w i t h g l a c i a l a c e t i c a c i d t o t e r m i n a t e the r e a c t i o n and d e n a t u r e the enzyme. D u p l i c a t e hapten i n h i b i t i o n t e s t s u s t n g 0.4 ml o f a n t i s e r u m 3 were s e t up. D i g e s t i o n o f the re m a i n i n g m a t e r i a l i n the tubes was a l l o w e d t o c o n t i n u e o v e r n i g h t a t 37 C and then d u p l i c a t e samples o f 0.1 ml from a l l tubes were a g a i n a d j u s t e d t o below pH 4.0 and t e s t e d f o r hapten i n h i b i t i o n . High 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 1.9 i n f o r m a t e - a c e t a t e b u f f e r (2.5% f o r m i c a c i d ; 7-8% a c e t i c a c i d ) was done on samples from a l l t h r e e tubes a f t e r o v e r n i g h t d i g e s t i o n . 39 10 8 6 4 MINUTES F i g u r e 6. A s s a y o f enzyme a c t i v i t y o f t h e c a r b o x y p e p t i d a s e B p r e p a r a t i o n by t h e h y d r o l y s i s o f t h e s u b s t r a t e h i p p u r y l - L - a r g i n i n e . A second c a r b o x y p e p t i d a s e e x p e r i m e n t was t r i e d a f t e r t he i n i t i a l COB e x p e r i m e n t i n d i c a t e d t h a t t he removal o f l y s i n e (and p o s s i b l y a l a n i n e ) from t h e C - t e r m i n a l o f the T-11 p e p t i d e had no e f f e c t on i t s h a p t e n i c a c t i v i t y . In the second e x p e r i m e n t , both COA and COB were used t o t r y t o degrade the p e p t i d e from t h e C-t e r m i n a l s e q u e n t i a l l y as f a r as p o s s i b l e , t o t e s t the l i m i t o f d e g r a d a t i o n b e f o r e l o s s o f h a p t e n i c a c t i v i t y o c c u r r e d . The COA ( W o r t h i n g t o n , D F P - t r e a t e d ) was ass a y e d f o r e n z y m a t i c a c t i v i t y and i t s r a t e o f h y d r o l y s i s o f h i p p u r y l - L - p h e n y l a l a n i n e measured by the i n c r e a s e i n absorb a n c y a t 2540 A a t 25 C a c c o r d i n g t o the method o f F o l k and S c h i r m e r (19&3)• 0 n e u n i t o f enzyme a c t i v i t y i s d e f i n e d as t h e amount o f enzyme r e q u i r e d t o h y d r o l y z e 1.0 umole o f s u b s t r a t e p e r min under t h e c o n d i t i o n s s p e c i f i e d . F i g u r e 1 shows the r e s u l t s o f t h e a s s a y o f t h i s enzyme. The methods used f o r the second e x p e r i m e n t were b a s i c a l l y t h o s e d e s c r i b e d by Young — (1966) i n t h e i r work on an i m m u n o l o g i c a l l y a c t i v e t r y p t i c p e p t i d e from t o b a c c o mosaic v i r u s p r o t e i n . For t h i s e x p e r i m e n t 3.0 ml o f T-11 i n s a l i n e c o n t a i n i n g 0 .75 umole, and 3.0 ml o f s a l i n e as a c o n t r o l were used. The pH o f t h e t e s t and the c o n t r o l were a d j u s t e d t o 8.0 w i t h 2% NaHCO^, and 0 . 0 1 m l o f COB (100 ug c o n t a i n i n g 0 .4 u n i t s o f a c t i v i t y ) was added t o each tube and the r e a c t i o n was a l l o w e d t o pro c e e d f o r 60 min a t 37 C. Then 0.01 ml o f COA (250 ug c o n t a i n i n g 23 u n i t s o f enzyme a c t i v i t y ) and 0.1 ml o f 10% L i C l were added t o both tubes and the i n c u b a t i o n a t 37 C was c o n t i n u e d . Because COA i s known t o aut o d e g r a d e on l e n g t h y d i g e s t i o n , an 41. Figure 7. Assay of enzyme a c t i v i t y of the carboxypeptidase A preparation by the hydrolysis of the substrate hippuryl-L-phenylalanine. a d d i t i o n a l 0.01 ml o f t h e enzyme was added a t 2 h r , 15 hr and 2k hr t o b oth t u b e s . Samples o f 0.3 ml were t a k e n from both t e s t and c o n t r o l tubes a t 2, 6, 15, 2k and kO h r . From t h e a l i q u o t s t a k e n , 0.1 ml was d r i e d and ta k e n up i n the s t a r t i n g b u f f e r f o r amino a c i d a n a l y s i s , and d u p l i c a t e hapten i n h i b i t i o n t e s t s were c a r r i e d o ut on the remainder. V I I . Edman D e g r a d a t i o n o f T-11 . Because t h e r e s u l t s from C - t e r m i n a l d e g r a d a t i o n i n d i c a t e d t h a t you c o u l d remove s e v e r a l amino a c i d s from the C - t e r m i n a l end o f th e T-11 p e p t i d e w i t h o u t l o s s o f h a p t e n i c a c t i v i t y , t h e next e x p e r i m e n t s i n v o l v e d d e g r a d i n g the p e p t i d e from the N - t e r m i n a l . Edman's d e g r a d a t i o n was t r i e d f i r s t . A Sephadex G-25 (Pharmacia F i n e C h e m i c a l s ) column (2.5 x kO.O cm) was e q u i l i b r a t e d w i t h d i s t i l l e d w a t e r and 2.0 ml o f T-11 c o n t a i n i n g 1.5 umoles i n s a l i n e was d e s a l t e d by r u n n i n g i t th r o u g h t h e column. The p e p t i d e , w h i c h came out near the v o i d volume was f l a s h e v a p o r a t e d a t k5 C t o d r y n e s s and used as the s t a r t i n g m a t e r i a l f o r s e r i a l Edman's d e g r a d a t i o n . The method f o l l o w e d was d e s c r i b e d by K o n i g s b e r g and H i l l (1962) w i t h minor m o d i f i c a t i o n s . The f i r s t s t a g e i n the Edman's r e a c t i o n i n v o l v e s the c o u p l i n g o f t he u n p r o t o n a t e d amino group ( t h e r e f o r e t h e r e a c t i o n i s c a r r i e d out a t an a l k a l i n e pH) o f t h e p e p t i d e w i t h t h e t h i o c a r b o n y l group o f phenyl i s o t h i o c y a n a t e (Eastman O r g a n i c ) . A s u i t a b l e s o l v e n t g i v i n g b oth t h e p e p t i d e and t h e reagent a p p r e c i a b l e s o l u b i l i t y was found t o be 50% aqueous p y r i d i n e c o n t a i n i n g 2% t r i m e t h y l a m i n e . In t h i s f i r s t c o u p l i n g r e a c t i o n , i t i s a l s o i m p o r t a n t t o e x c l u d e oxygen by f l u s h i n g the r e a c t i o n v e s s e l w e l l w i t h n i t r o g e n and s t o p p e r i n g the t u b e , s i n c e oxygen w i l l r e p l a c e s u l f u r i n the t h i o c a r b o n y l group o f t h e p h e n y l t h i o c a r b a m y l p e p t i d e and w i l l p r e v e n t t h e l a t e r c y c l i z a t i o n and e l i m i n a t i o n o f t h e p h e n y l t h i o h y d a n t o i i o f the t e r m i n a l amino a c i d . The c o u p l i n g s t a g e was c a r r i e d out f o r 2 hr a t k5 C u s i n g a 1:50 molar r a t i o o f p e p t i d e t o phenyl i s o t h i o -c y a n a t e i n a volume o f 0 .215 ml. The second s t a g e , a f t e r removal o f e x c e s s r e a g e n t s w i t h two benzene e x t r a c t i o n s o f 1.0 - 2 .0 m l , was the c y c l i z a t i o n o f t h e p h e n y l t h i o c a r b a m y l group on t h e amino group w i t h the c a r b o n y l c a r b o n o f the amino t e r m i n a l amino a c i d , r e s u l t i n g i n the f o r m a t i o n o f a t h i a z o l i n o n e d e r i v a t i v e . I t i s n e c e s s a r y t o p e r f o r m t h e c y c l i z a t i o n under anhydrous c o n d i t i o n s w i t h s t r o n g a c i d i n o r d e r t o p r e v e n t h y d r o l y t i c c l e a v a g e o f a c i d - s e n s i t i v e bonds. The r e l a t i v e l y m i l d c o n d i t i o n s p e r m i t t e d by anhydrous t r i f 1 u o r o a c e t i c a c i d a t k5 C f o r 30 min i n a volume o f 0 .3 ml under n i t r o g e n were used on the T-11 p e p t i d e . F o l l o w i n g c y c l i z a t i o n , the t r i f l u o r o a c e t i c a c i d was removed by vacuum e v a p o r a t i o n a t 25 C o v e r P2®5' a n c ' t n e r e s ' d u e w a s d i s s o l v e d i n 0 .2 ml o f 0 .2 M a c e t i c a c i d and e x t r a c t e d t w i c e w i t h 1.0 -2.0 ml amounts o f benzene t o remove t h e p h e n y 1 t h i o c a r b a m y l amino a c i d s and p h e n y 1 t h i o h y d a n t o i n s o f most a c i d i c and n e u t r a l ami no a c i d s . P e p t i d e d e v o i d o f t h e N - t e r m i n a l amino a c i d remained i n the aqueous phase. The d r i e d r e s i d u e was resuspended i n 0 . 1 ml o f 0 . 2 N a c e t i c a c i d and adsorbed o n t o a s m a l l Dowex 5 0 column (k x 5 0 mm) i n the p y r i d i n e form e q u i l i b r a t e d w i t h 0 . 2 N a c e t i c a c i d . The column was washed by r u n n i n g a p p r o x i m a t e l y 1 0 ml o f 0 . 2 N a c e t i c a c i d t hrough i t , f o l l o w e d by 5 - 1 0 ml o f 2 . 0 M p y r i d i n e - a c e t a t e b u f f e r a t pH 5 . 0 . The a c e t i c a c i d washings c o n t a i n e d n i n h y d r i n n e g a t i v e m a t e r i a l w h i c h , a f t e r a c i d h y d r o l y s i s was shown by K o n i g s b e r g and H i l l ( 1 9 6 2 ) t o c o n t a i n a l l the amino a c i d s p r e s e n t i n the p a r e n t p e p t i d e , but t h i s p e p t i d e d e r i v a t i v e i s d e v o i d o f f r e e amino groups and t h e r e f o r e i s not adsor b e d under the s t a t e d c h r o m a t o g r a p h i c c o n d i t i o n s . The t r u e , r e m a i n i n g p e p t i d e w i t h f r e e amino groups e l u t e s 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 . In t h e p r e s e n t i n s t a n c e , t h i s m a t e r i a l was c o l l e c t e d and d r i e d down and resuspended i n 50% aqueous p y r i d i n e w h i c h c o m p l e t e d one c y c l e . T h i s p u r i f i c a t i o n s t e p cannot be used i f t h e p e p t i d e t o be degraded c o n t a i n s c y s t e i c a c i d s i n c e i t w i l l not be a d s o r b e d , o r i f i t c o n t a i n s a h i g h c o n c e n t r a t i o n o f b a s i c o r a r o m a t i c r e s i d u e s , s i n c e i t w i l l be e l u t e d o n l y w i t h d i f f i c u l t y w i t h t h e 2 . 0 M p y r i d i n e - a c e t a t e b u f f e r . However, t h e T - 1 1 p e p t i d e c o n t a i n s o n l y one l y s i n e r e s i d u e , no a r o m a t i c r e s i d u e s , and the c y s t e i n e r e s i d u e s a r e a l l c a r b o x y m e t h y l a t e d , so no problems o f t h i s type were e n c o u n t e r e d . At t h e end o f one c y c l e , which removed one amino a c i d , t h r e e samples o f 10.0 y l ( a p p r o x i m a t e l y 0.05 ymoles) were t a k e n , one f o r amino a c i d a n a l y s i s , and two f o r s e t t i n g up d u p l i c a t e hapten i n h i b i t i o n t e s t s . Three c y c l e s were r e p e a t e d , the o n l y d i f f e r e n c e b e i n g t h a t the volumes o f the a l i q u o t s taken a f t e r the second and t h i r d c y c l e were 20 and 30 y l r e s p e c t i v e l y t o compensate f o r l o s s e s i n p e p t i d e r e s u l t i n g from h a n d l i n g and column chromatography. The c o n c e n t r a t i o n o f p e p t i d e s was d e t e r m i n e d by amino a c i d a n a l y s i s and then hapten i n h i b i t i o n t e s t s were performed on the t h r e e samples d e s i g n a t e d T-11-1, T-11-2 and T-11-3. A l i q u o t s used f o r hapten i n h i b i t i o n t e s t s were d r i e d and r e d i s s o l v e d i n 0.9% N a C l . Each time a c y c l e was c o m p l e t e d , the r e m a i n i n g p e p t i d e was taken up i n the amount o f 50% p y r i d i n e t o g i v e t h e same m i c r o m o l a r c o n c e n t r a t i o n p r e s e n t i n the s t a r t i n g m a t e r i a l . C o n t r o l t e s t s f o r the hapten i n h i b i t i o n were s e t up i n d u p l i c a t e u s i n g t h r e e c o n c e n t r a t i o n s o f T-11, 0.04, 0.02 and 0.01 y m o l e s. N e g a t i v e c o n t r o l s c o n t a i n e d 0.9% s a l i n e i n e q u i v a l e n t volumes, i n p l a c e o f p e p t i d e . S i n c e t h e s e e x p e r i m e n t s i n d i c a t e d the importance o f t h e N-t e r m i n a l p o r t i o n o f the T-11 p e p t i d e as an a n t i g e n i c d e t e r m i n a n t , the c o m plete e x p e r i m e n t was r e p e a t e d , w h i c h c o n f i r m e d the o b s e r v a t i t h a t removal o f t h r e e amino a c i d s from the N - t e r m i n a l caused l o s s o f h a p t e n i c a c t i v i t y . Because Edman's d e g r a d a t i o n i n v o l v e s somewhat r i g o r o u s t r e a t m e n t o f b i o l o g i c a l l y a c t i v e m a t e r i a l s , the p o s s i b i l i t y was c o n s i d e r e d t h a t the l o s s o f h a p t e n i c a c t i v i t y c o u l d be due t o m a n i p u l a t i o n r a t h e r than t o t h e importance o f the t h r e e N - t e r m i n a l r e s i d u e s . In o r d e r t o t e s t t h i s , a n o t h e r s e r i e s o f e x p e r i m e n t s were s e t up u s i n g e n z y m a t i c c l e a v a g e a t t h e N - t e r m i n a l t o a s c e r t a i n whether o r not the o b s e r v a t i o n s made here were v a l i d . V I M . L e u c i n e Amino P e p t i d a s e E x p e r i m e n t s The l e u c i n e amino p e p t i d a s e ( L A P ) , d i i s o p r o p y l phosphoro-f l u o r i d a t e - t r e a t e d , was o b t a i n e d from W o r t h i n g t o n B i o c h e m i c a l Company. B e f o r e use i n any e x p e r i m e n t s , t h e enzyme p r e p a r a t i o n was assayed f o r a c t i v i t y by n o t i n g i t s r a t e o f h y d r o l y s i s o f t h e s u b s t r a t e L - l e u c i n e p - n i t r o a n i 1 i d e . B e f o r e t h i s enzyme can be a s s a y e d , i t i s n e c e s s a r y t o a c t i v a t e i t i n t h e p r e s e n c e o f 2 mM M g C ^ i n 0.14 M t r i e t h y l a m i n e a d j u s t e d t o pH 8.5 w i t h a c e t a t e a t 40 C f o r 2.5 h r . I t has been found t h a t magnesium i o n i s b e t t e r than manganese i o n f o r a c t i v a t i o n o f t h i s m e t a l - r e q u i r i n g enzyme, as manganese i o n s l o w l y forms manganese d i o x i d e under s l i g h t l y a l k a l i n e c o n d i t i o n s , and t h i s w i l l a l l o w a r e v e r s i b l e o x i d a t i o n - r e d u c t i o n system t o be formed c a p a b l e o f d e s t r o y i n g t r y p t o p h a n and s u l f u r - c o n t a i n i n g amino a c i d s . The as s a y o f the enzyme a c t i v i t y showing the d e t e r m i n a t i o n o f t h e u n i t s o f a c t i v i t y p e r mg i s shown i n F i g u r e 8. When the a c t i v i t y o f t h e a c t i v a t e d LAP was d e t e r m i n e d , an e x p e r i m e n t f o l l o w i n g t h e p r o c e d u r e o f L i g h t (1967) and u s i n g t h e f o l l o w i n g s o l u t i o n s was c a r r i e d o u t . F i g u r e 8. A s s a y o f enzyme amino p e p t i d a s e t h e s u b s t r a t e L-a c t i v i t y o f t h e a c t i v a t e d l e u c i n e p r e p a r a t i o n by t h e h y d r o l y s i s o f l e u c y l - p - n i t r o a n i 1 i d e . 1. E x p e r i m e n t a l system 8.0 mg (3.2 ymoles ) o f T-11 p e p t i d e was d i s s o l v e d i n 1.0 ml o f 0.14 M t r i e t h y l a m i n e a c e t a t e a t pH 8.5 c o n t a i n i n g 0.002.M M g C l 2 . At z e r o t i m e , 0.25 mg (0.3 ml o f the a c t i v a t e d enzyme p r e p a r a t i o n c o n t a i n i n g 25.0 u n i t s o f enzyme a c t i v i t y ) o f LAP was added. 2. S u b s t r a t e c o n t r o l s a. 1.0 mg (0.4 ymoles) o f T-11 p e p t i d e d i s s o l v e d i n 0.1 ml o f t r i e t h y l a m i n e a c e t a t e b u f f e r c o n t a i n i n g M g C l 2 a t pH 8.5. b. 4.0 mg (1.6 ymoles) o f T-11 p e p t i d e d i s s o l v e d i n 0.5 ml o f t r i e t h y l a m i n e a c e t a t e b u f f e r c o n t a i n i n g M g C l 2 a t pH 8.5. At z e r o t i m e , 0.125 mg (0.15 ml o f t h e a c t i v a t e d enzyme p r e p a r a t i o n c o n t a i n i n g 12.5 u n i t s o f a c t i v i t y ) o f h e a t - i n a c t i v a t e d ( b o i l e d ) LAP was added. 3. Enzyme c o n t r o l 0.125 mg (12.5 u n i t s o f a c t i v a t e d enzyme p r e p a r a t i o n ) o f LAP was d i s s o l v e d i n 0.5 ml o f t r i e t h y l a m i n e a c e t a t e b u f f e r c o n t a i n i n g M g C l 2 . A l l t he tubes were i n c u b a t e d a t 37 C, and samples o f 0.15 ml were t a k e n from the t e s t , t h e enzyme c o n t r o l and the s u b s t r a t e c o n t r o l s a t 8, 16, 24, 40 and 48 h r . A sample o f 0.15 ml was t a k e n from the t e s t s o l u t i o n a t 32 hr a l s o j u s t b e f o r e f r e s h enzyme i n the same amount as a t the s t a r t was added. H e a t - i n a c t i v a t e d enzyme, a t an e q u i v a l e n t amount t o t h a t added a t the s t a r t , was added t o t h e a p p r o p r i a t e c o n t r o l , as w e l l as 0.125 mg o f enzyme t o the enzyme c o n t r o l a t 32 h r . As a l i q u o t s were t a k e n , the pH was lowered i n each one t o below 3.5 w i t h a c e t i c a c i d (0.1 N) i n o r d e r t o t e r m i n a t e t h e a c t i o n o f the LAP. From th e samples removed a t the t i m e s g i v e n above, a l i q u o t s o f 0.03 ml were taken from the t e s t s e r i e s , d r i e d down and t a k e n up i n 0.5 ml o f s t a r t i n g b u f f e r (pH 2 . 2 ) , and an amino a c i d a n a l y s i s was c a r r i e d o u t on each one t o d e t e r m i n e t h e e x t e n t o f d i g e s t i o n . A 0.03 ml a l i q u o t o f t h e sample t a k e n a t 48 hr from t h e enzyme c o n t r o l was a l s o a n a l y s e d f o r f r e e amino a c i d c o n t e n t , t o check f o r any a u t o d e g r a d a t i o n o f the LAP. Under th e above c o n d i t i o n s , 0.004 ymoles o f l e u c i n e o n l y was d e t e c t e d i n the enzyme c o n t r o l , a v a l u e w h i c h was about 10% o f the amount o f l e u c i n e r e l e a s e d i n t h e t e s t s e r i e s . C o r r e c t i o n was made f o r t h i s i n c a l c u l a t i n g t h e amount o f d i g e s t i o n i n the e x p e r i m e n t a l samples. When q u a n t i t a t i v e amino a n a l y s e s had been c a r r i e d o u t on a l l the d i g e s t s a m p l e s , and t h e e x t e n t o f enzyme c l e a v a g e had been d e t e r m i n e d , hapten i n h i b i t i o n t e s t s were done i n t r i p l i c a t e on 0.01 ml (60 ug o f t h e o r i g i n a l T-11) a l i q u o t s o f the t e s t samples and c o n t r o l s , With t h e T-11 d i g e s t , a l l t i m e s were t e s t e d , but w i t h t h e b o i l e d LAP c o n t r o l , o n l y 8, 16 and 48 h r samples were t e s t e d , and w i t h t h e enzyme c o n t r o l t e s t s were run on the 8, 24 and 48 hr samples. The T-11 c o n t r o l was t e s t e d o n l y a t 48 hr t o see \\f t h e p r o l o n g e d i n c u b a t i o n a t 37 C had had any e f f e c t on i t s h a p t e n i c a c t i v i t y , s i n c e s h o r t e r times would have l e s s e f f e c t . A c o n t r o l s e t i n t r i p l i c a t e u s i n g an e q u i v a l e n t amount o f t r i e t h y l a m i n e a c e t a t e b u f f e r c o n t a i n i n g M g C l 2 r a t h e r than d i g e s t was done t o o b t a i n the maximum p r e c i p i t a t i o n l e v e l . IX. P u r i f i c a t i o n o f Sp e c i f i c Ant i b o d i e s R a b b i t a n t i - C M - l y s o z y m e a n t i b o d y was s p e c i f i c a l l y p u r i f i e d from p o o l e d s e r a from r a b b i t s 1 and 2 a c c o r d i n g t o t h e method o f F u j i o e t ^ a l _ . (1968a) . One hundred ml o f p o o l e d s e r a was p r e c i p i t a t e d a t the e q u i v a l e n c e p o i n t w i t h a c a l c u l a t e d amount o f t h e s t o c k s o l u t i o n o f CM-lysozyme, by i n c u b a t i n g t h e m i x t u r e f o r 1 h r a t 37 C and then f o r 2 days a t k C. The s p e c i f i c immune p r e c i p i t a t e was c e n t r i f u g e d a t 20,000 £ f o r 20 min and washed t h r e e t i m e s w i t h 0 .02 M sodium p h o s p h a t e , 0.15 M N a C l , pH 6.0 b u f f e r (PBS). The washed p r e c i p i t a t e , c o n t a i n i n g about 25 mg o f a n t i b o d y , was d i s s o l v e d i n 5.0 ml o f 0 .2 N a c e t i c a c i d and i n c u b a t e d a t 37 C f o r 2 h r w i t h s t i r r i n g t o d i s s o c i a t e t h e a n t i g e n -a n t i b o d y complex. The d i s s o c i a t e d m i x t u r e was passed t h r o u g h an a s c e n d i n g Sephadex G-200 column (2 .5 x 90 .0 cm) e q u i l i b r a t e d w i t h 0.2 N a c e t i c a c i d . The g e l f i l t r a t i o n was c a r r i e d out a t room t e m p e r a t u r e and 5.0 ml f r a c t i o n s were c o l l e c t e d . The f l o w r a t e was a d j u s t e d t o 12.0 m l / h r . The absorbance o f each f r a c t i o n was read a t 2800 A, and t h e e l u t i o n p r o f i l e i s shown i n F i g u r e 9 . B l u e d e x t r a n was run t h r o u g h f i r s t t o d e t e r m i n e v o i d volume. There was no 19S 51 ML E L U E N T i g u r e 9. E l u t i o n p r o f i l e o f t h e p u r i f i c a t i o n o f s p e c i f i c a n t i - C M - l y s o z y m e a n t i b o d i e s on Sephadex G-200. The d o t t e d l i n e r e p r e s e n t s b l u e d e x t r a n and t h e s h a r p , m a j o r s o l i d l i n e peak r e p r e s e n t s t h e IgG (7S) s p e c i f i c a n t i b o d i e s . gamma g l o b u l i n peak w h i c h would have come out i n t h e v o i d volume so t h a t o n l y t h e r e t a r d e d IgG peak i s d e m o n s t r a t e d . The f r a c t i o n s c o n t a i n i n g t he IgG were p o o l e d and d i a l y s e d a g a i n s t 100 volumes o f 0.02 M PBS a t pH 6.0 a t k C f o r 3 days w i t h s t i r r i n g and changes i n b u f f e r t w i c e d a i l y . The s t o c k - s o l u t i o n o f p u r i f i e d a n t i b o d y was c o n c e n t r a t e d by u l t r a f i l t r a t i o n t h r ough an Amicon u l t r a f i l t e r a p p a r a t u s u s i n g a UM-1 f i l t e r pad, so t h a t t h e f i n a l c o n c e n t r a t i o n was 1.10 mg p r o t e i n / m l . T h i s m a t e r i a l was s t o r e d a t - 15 C u n t i l r e q u i r e d . N o n s p e c i f i c r a b b i t gamma g l o b u l i n w h i c h had been c o n c e n t r a t e d by p r e c i p i t a t i o n w i t h 50% (NH^^SO^ and p u r i f i e d by DEAE-cel l u l o s e (Calbiochem) column chromatography i n 0.05 M phosphate b u f f e r a t pH 7 - 5 , a c c o r d i n g t o the method used by F u j i o et^ a_J_. (1968a) , was used f o r t h e gamma g l o b u l i n c o n t r o l i n e q u i l i b r i u m d i a l y s i s e x p e r i m e n t s . The gamma g l o b u l i n p r e p a r e d i n t h i s way was s t o r e d as a l y o p h i l i z e d powder, and was d i s s o l v e d i n s m a l l amounts o f 0.02 M PBS a t pH 6.0 and d i a l y s e d a g a i n s t t h i s b u f f e r f o r 3 days a t k C as r e q u i r e d . The d i a l y s e d p r e p a r a t i o n c o n t a i n e d 17 mg p r o t e i n / m l . A n o t h e r method f o r t h e p u r i f i c a t i o n o f s p e c i f i c a n t i b o d y was a l s o employed u s i n g a s p e c i f i c immunoadsorbent. The method used f o r t h e p r e p a r a t i o n o f the immunoadsorbent and t h e p u r i f i c a t i o n o f a n t i b o d y was b a s i c a l l y t h a t o f Robbins e_t a_J_. (1967). The a n t i g e n , CM-lysozyme was r e a c t e d w i t h t he bro m o a c e t y l c e l l u l o s e ( o b t a i n e d by r e a c t i o n o f b r o m o a c e t i c a c i d and bro m o a c e t y l bromide w i t h powdered Whatman c e l l u l o s e ) i n a manner p e r m i t t i n g c o v a l e n t bonding t o o c c u r between t h e a n t i g e n and t h e a d s o r b e n t . The c o n j u g a t e o f a n t i g e n and bromoacetyl c e l l u l o s e was then used t o a d s o r b s p e c i f i c a n t i b o d y from p o o l e d s e r a from r a b b i t s 1 and 2 i n 0.15 M phosphate b u f f e r pH 7.k a t k C. Immunoadsorbent c o n j u g a t e s p r e p a r e d i n t h i s way have a h i g h c a p a c i t y f o r e x t r a c t i n g IgM and IgG c l a s s e s o f s p e c i f i c a n t i b o d i e s from a n t i s e r a , and t h e y a r e s t a b l e f o r months and may be used r e p e a t e d l y w i t h o u t s i g n i f i c a n t l o s s o f a n t i b o d y b i n d i n g c a p a c i t y . W i t h l a r g e p r o t e i n a n t i g e n - c e l l u l o s e c o n j u g a t e s , most o f the d i f f e r e n t t y p e s o f a n t i g e n i c d e t e r m i n a n t s a r e s t i l l a v a i l a b l e f o r i n t e r a c t i o n w i t h a n t i b o d y . The serum t o be adsorbed was c l a r i f i e d f i r s t by c e n t r i f u g a t i o n a t 20,000 g_ f o r 1 hr a t k C, and t h e f l o a t i n g l i p i d m a t e r i a l and any sedimented m a t e r i a l was removed. Then the immunoadsorbent was d i s p e r s e d i n t h e serum, and the s u s p e n s i o n was s t i r r e d a t k C f o r 2 h r . At t h e end o f t h i s t i m e , the c e l l u l o s e c o n j u g a t e was c e n t r i f u g e d a t 20,000 g f o r 20 m i n , and a l l t h e s u p e r n a t a n t serum d r a i n e d o f f ( t e s t s on t h i s m a t e r i a l i n d i c a t e d t h a t most o f t h e s p e c i f i c a n t i b o d y had been removed i n t h i s s t e p ) . A f t e r washing the a d s o r b e n t w i t h 0.15 M NaCl arid r e c e n t r i f u g i n g u n t i l t he a b s o r b a n c e o f t h e washes was l e s s than 0.08 a t 2800 A-, > the a n t i b o d y was e l u t e d from t h e immunoadsorbent by s t i r r i n g t he complex i n 0.1 M a c e t i c a c i d (pH 2.8) f o r 1 hr a t 37 C. The s u s p e n s i o n was c e n t r i f u g e d f o r 30 min a t 20,000 g_ t o remove t h e c e l l u l o s e - a n t i g e n c o n j u g a t e , and t h e s u p e r n a t a n t f l u i d c o n t a i n i n g the a n t i b o d y , was d i a l y s e d a g a i n s t 350 - 700 volumes o f 0.1 M NaCl - 0.01 M T r i s HC1 a t pH 7 - 0 . T h i s p u r i f i e d a n t i b o d y s t o c k s o l u t i o n was s t o r e d a t - 1 5 C u n t i l r e q u i r e d a f t e r m i x i n g i t w i t h an equal volume o f g l y c e r i n e . X. S o l i d Phase P e p t i d e S y n t h e s i s 1. P e p t i d e s y n t h e s i s The M e r r i f i e l d (1964) method o f s o l id phase p e p t i d e s y n t h e s i s was f o l l o w e d w i t h t h e m o d i f i c a t i o n s as g i v e n i n t h e r e c e n t book S o l i d Phase P e p t i d e S y n t h e s i s ( S t e w a r t and Young, 1 969 ) . The method b a s i c a l l y i n v o l v e s the growth o f p e p t i d e s by s t e p w i s e a d d i t i o n s o f the r e q u i r e d amino a c i d s , w h i l e the p e p t i d e i s c o v a l e n t l y bonded t o an i n s o l u b l e s u p p o r t . The i n s o l u b l e s u p p o r t i s c h l o r o m e t h y l a t e d c o p o l y s t y r e n e * " d i v i n y l b e n z e n e r e s i n ( B i o - R a d ) . The r e a c t i v e g r o u p s , t h e c h l o r o m e t h y l g r o u p s , r e a c t w i t h the s a l t o f a t - b u t y l o x y c a r b o n y l ( t - B o c ) amino a c i d ( o b t a i n e d from Mann Research B i o c h e m i c a l s and Sigma C h e m i c a l s ) t o form an e s t e r bond between t h e C 0 0 H group o f the amino a c i d and t h e r e s i n . Thus the p e p t i d e i s le n g t h e n e d from the C - t e r m i n a l amino a c i d toward the N - t e r m i n a l . The t-Boc group b l o c k s t h e f r e e amino group o f each incoming amino a c i d so t h a t o n l y t h e c a r b o x y l group i s f r e e t o r e a c t . The t-Boc group o f the a t t a c h e d amino a c i d i s removed by t r e a t m e n t w i t h anhydrous HCl t o -allow t h i s f r e e amino a c i d group t o r e a c t w i t h t h e incoming p r o t e c t e d amino a c i d . The c o u p l i n g r e a c t i o n i n v o l v i n g t h e f o r m a t i o n o f a p e p t i d e bond r e q u i r e s t h e p r e s e n c e o f d i c y c l o h e x y l c a r b o d i imide and n e u t r a l pH i n most i n s t a n c e s , but a c t i v e e s t e r s o f incoming t - B o c amino a c i d s may be used w i t h no c o u p l i n g r e a g e n t . The e x c e s s amino a c i d s and o t h e r r e a g e n t s a r e e a s i l y washed out o f the r e s i n by a p p r o p r i a t e s o l v e n t s , thus e l i m i n a t i n g l a b o r i o u s p u r i f i c a t i o n p r o c e d u r e s at i n t e r m e d i a t e s t e p s d u r i n g t h e s y n t h e s i s . A f t e r t h e s y n t h e t i c c y c l e s a r e c o m p l e t e d , t h e p e p t i d e i s c l e a v e d from the s o l i d r e s i n s u p p o r t by p a s s i n g HBr gas ( B a k e r , Matheson Go.) t h r o u g h a s u s p e n s i o n o f t h e r e s i n and p e p t i d e i n anhydrous t r i f 1 u o r o a c e t i c a c i d . The t e r m i n a l - t - B o c group sti11 p r e s e n t a t t h i s t i m e i s removed by an e l i m i n a t i o n r e a c t i o n , and the p e p t i d e i s c l e a v e d from t h e r e s i n by a n u c j e o p h i 1 i c d i s p l a c e m e n t r e a c t i o n . C e r t a i n o t h e r s i d e c h a i n p r o t e c t i n g groups such as b e n z y l groups on the h y d r o x y l s o f s e r i n e and t h r e o n i n e a r e a l s o removed by t h i s p r o c e d u r e . However, t h e s i d e c h a i n p r o t e c t i n g groups such as the b e n z y l groups on the s u l f h y d r y l o f c y s t e i n e must be removed by r e d u c t i o n w i t h sodium i n l i q u i d ammonia. A f t e r a l l t h e s i d e c h a i n p r o t e c t i n g groups a r e removed, the c l e a v e d p e p t i d e i s p u r i f i e d by a s u i t a b l e c h r o m a t o g r a p h i c t e c h n i q u e . A f l o w c h a r t f o r a s y n t h e t i c c y c l e i s shown i n T a b l e l». In t h i s i n s t a n c e , the p e p t i d e s y n t h e s i z e d , w h i c h c o n s t i t u t e d a p o r t i o n o f t h e T-11 p e p t i d e , was c l e a v e d from the r e s i n and t r e a t e d w i t h sodium i n 1iquid'ammonia ( B a k e r , Matheson Co.) t o remove t h e S-benzyl groups on the c y s t e i n e r e s i d u e s . T a b l e I, F low c h a r t o f S o l i d Phase P e p t i d e S y n t h e s i s . C H 3 O H R, O I I! ' I I !! C H 3 — C — O — C — N — C — C — O - + C l — C H 2 — C H 3 H Boc amino acid P O L Y M E R chloromethyl polymer C H 3 O H Ri O C H 3 H Boc amino acyl polymer HCl—dioxane; Et 3 N P O L Y M E R DEPROTECT; NEUTRALIZE C H , i! C H 3 — C I C H 3 , isobutylene H Ri O I I II / = \ + C O , + H — N — C — C — O — C H , — < ^ h— H amino acyl polymer Boc amino acid I diimide P O L Y M E R COUPLE C H 3 O H R 2 O H Ri O I II I I II I I II C H 3 — C — O — C — N — C — C — N — C — C — O — C H 2 — • I I I C H , H H Boc peptide polymer P O L Y M E R H B r — F 3 C C O O H CLEAVE C H , H Rj O H R, O C H 3 — C + C O , + H N — C — C — N — C — C — O H + Br—CH 2 —( x >— P O L Y M E R C H 3 isobutylene H H peptide Reproduced f r om S o l i d Phase P e p t i d e S y n t h e s i s by John S t e w a r t and J a n i s Young, p. 3-2. R e d u c t i o n and a l k y l a t i o n o f s y n t h e s i z e d p e p t i d e s The method f o l l o w e d f o r r e d u c t i o n and a l k y l a t i o n o f the s y n t h e s i z e d p e p t i d e was a m o d i f i c a t i o n o f the c a r b o x y m e t h y l a t i o n p r o c e d u r e used f o r n a t i v e lysozyme as r e p o r t e d by C a n f i e l d and A n f i n s e n (1963a). The p e p t i d e was f l a s h e v a p o r a t e d a t 45 C f o l l o w i n g r e d u c t i o n i n l i q u i d ammonia, washed w i t h d i l u t e a c e t i c a c i d (0.1 N) and suspended i n 5-0 ml o f d i l u t e a c e t i c a c i d . A 5 0 - f o l d e x c e s s o f 2-mercaptoethanol was added, and the pH o f the s o l u t i o n was a d j u s t e d t o 8.6 w i t h d i l u t e NH^OH. The f l a s k was f l u s h e d w i t h n i t r o g e n , s t o p p e r e d , and i n c u b a t e d a t 37 C f o r 4 hr to e n s u r e the r e d u c t i o n o f the c y s t e i n e r e s i d u e s i n the p e p t i d e . F o l l o w i n g the r e d u c t i o n s t e p , the pH was lowered t o 2.0 w i t h d i l u t e HC1 t o p r e v e n t r e f o r m a t i o n o f any d i s u l f i d e bonds, and t h e p r e p a r a t i o n was f l a s h e v a p o r a t e d a t 45 C t o d r y n e s s and washed once w i t h a c i d i f i e d d i s t i l l e d w a t e r . The reduced p e p t i d e was d i s s o l v e d i n 5-0 ml o f d i s t i l l e d w a t e r , and 2.5 u C i o f ( ^ C ) - i o d o a c e t i c a c i d (New England N u c l e a r Co. c o n t a i n i n g 0.19 ymole) was added. The pH was r a i s e d t o 8.6 and a l k y l a t i o n w i t h t h e l a b e l l e d i o d o a c e t a t e was a l l o w e d t o t a k e p l a c e f o r 20 min b e f o r e c o l d i o d o a c e t i c a c i d ( p u r i f i e d by one c r y s t a l l i z a t i o n from p e t r o l e u m e t h e r ) was added i n a 1:1 r a t i o on a w e i g h t b a s i s w i t h the amount o f p e p t i d e p r e s e n t . T h i s c o n s t i t u t e d a l a r g e molar e x c e s s , and the r e a c t i o n was a l l o w e d t o c o n t i n u e f o r a f u r t h e r 5 min a t pH 8.6 t o e n s u r e t h a t a l 1 the c y s t e i n e r e s i d u e s had been a l k y l a t e d . A 1 2 5 - f o l d m o l a r e x c e s s ( t o the moles o f c y s t e i n e r e s i d u e s p r e s e n t i n t h e p e p t i d e ) o f 2 - m e r c a p t o e t h a n o l was the n added t o the r e a c t i o n m i x t u r e t o reduce the e x c e s s i o d o a c e t a t e . The pH was m a i n t a i n e d a t 8.6 f o r 1 min a f t e r t he a d d i t i o n o f 2-me r c a p t o e t h a n o l and then lowered t o pH 2.0 w i t h d i l u t e HG1. The a l k y l a t e d p e p t i d e was d r i e d by f l a s h e v a p o r a t i o n a t kS C, washed once w i t h d i s t i l l e d w a t e r , d r i e d a g a i n , and suspended i n 10.0 ml o f d i s t i l l e d w a t e r w i t h the pH a d j u s t e d t o 2.8 w i t h f o r m i c a c i d . 3. P u r i f i c a t i o n o f reduced and c a r b o x y m e t h y l a t e d p e p t i d e The reduced and S - c a r b o x y m e t h y l a t e d p e p t i d e i n d i l u t e f o r n i i c a c i d a t pH 2.8 was a p p l i e d t o a Dowex 50 x k, Bio-Rad column (2.0 x 25.0 cm) wh i c h had been e q u i l i b r a t e d w i t h 0.1 M p y r i d i n e -a c e t i i c a c i d b u f f e r a t pH 3.8. The p e p t i d e p r e p a r a t i o n c o n t a i n e d some i n s o l u b l e b l a c k m a t t e r , but t h i s g r a d u a l l y d i s s o l v e d on t h e column. The four-chambered g r a d i e n t m o d i f i e d from t h e o r i g i n a l C a n f i e l d p r o c e d u r e (1963a) was used as t h e i n i t i a l p u r i f i c a t i o n s t e p f o r the s y n t h e t i c p e p t i d e . F r a c t i o n s o f 3-0 ml were c o l l e c t e d , and t h e p e p t i d e was d e t e c t e d by c o u n t i n g 0.1 ml a l i q u o t s from e v e r y o t h e r tube i n 8.0 ml o f s c i n t i 1 l a t i o n f l u i d . The l i q u i d ' s c i n t i l l a t i o n f l u i d was p r e p a r e d wi t h kl ml o f l i q u i f l u o r (New England N u c l e a r ) i n 1 1 o f t o l u e n e - m e t h a n o l .(60 : k 0 , v : v ) . A l l 59 c o u n t i n g was c a r r i e d o u t on a N u c l e a r C h i c a g o l i q u i d s c i n t i l l a t i o n c o u n t e r (Model 725). U s i n g t h e s e c h r o m a t o g r a p h i c p r o c e d u r e s , t h e u n r e a c t e d i o d o -a c e t i c a c i d e l u t e d a t the f r o n t . The second major peak c o n t a i n e d p e p t i d e m a t e r i a l w h i c h , a f t e r amino a c i d a n a l y s i s was found t o c o n s i s t o f the d e s i r e d s y n t h e t i c p e p t i d e . The p o o l e d peak m a t e r i a l was c a r e f u l l y f l a s h e v a p o r a t e d a t 45 C u s i n g NaOH p e l l e t s in a t r a p in c a s e any u n r e a c t e d ( C ) - i o d o a c e t i c a c i d was p r e s e n t . The m a t e r i a l was taken up in 4.0 ml o f d i s t i l l e d wa te r and 0.02 ml was a n a l y z e d a f t e r a c i d h y d r o l y s i s f o r i t s amino a c i d c o n t e n t . In o r d e r to p u r i f y the p e p t i d e f u r t h e r and f r e e i t f rom low m o l e c u l a r w e i g h t c o n t a m i n a n t s wh ich were sometimes p r e s e n t , the m a t e r i a l f rom the Dowex column was run t h r o u g h a Sephadex G-15 column (2.5 x 35.0 cm) e q u i l i b r a t e d w i t h 0.1 N a c e t i c a c i d . The column was run a t 12 m l / h r and 4.5 ml f r a c t i o n s were c o l l e c t e d . The f r a c t i o n s were read f o r 2300 A' a b s o r b a n c e on a DB-G s p e c t r o p h o t o m e t e r . The p e p t i d e m a t e r i a l thus d e t e c t e d was c o n c e n t r a t e d by f l a s h e v a p o r a t i o n a t 45 C and r e d i s s o l v e d in 4.0 ml o f d i s t i l l e d w a t e r . A sample o f 0.01 ml was h y d r o l y z e d and a n o t h e r amino a c i d a n a l y s i s was per formed on t h i s . Some improvement in the amino a c i d r a t i o s was u s u a l l y o b s e r v e d a f t e r t h i s s t e p . H igh 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 h e p e p t i d e m a t e r i a l was c a r r i e d o u t u s i n g a G i l s o n High V o l t a g e E l e c t r o p h o r e t o r . The s a m p l e , a t 0.1 mg q u a n t i t y was r u n a t pH 6 . 5 i n p y r i d i n e - a c e t i c a c i d b u f f e r (10% p y r i d i n e - 0 . 4 % a c e t i c a c i d ) a t 3000 v o l t s / c m f o r 30 m i n . O n l y o n e n i n h y d r i n p o s i t i v e s p o t was p r e s e n t i n t h e s e s a m p l e s . T h e h a p t e n i c a c t i v i t y o f t h e s y n t h e s i z e d p e p t i d e s was t e s t e d by t h e p r e c i p i t a t i o n i n h i b i t i o n t e c h n i q u e p r e v i o u s l y d e s c r i b e d a t c o n c e n t r a t i o n s o f 5 0 , 25 a n d 1 2 . 5 , 6 . 2 5 , 3 . 0 , 1.5 a n d 0 . 4 y g a n d a l s o by e q u i l i b r i u m d i a l y s i s s t u d i e s d e s c r i b e d l a t e r . 1 k X I . ( C ) - A c e t y l a t i o n o f P e p t i d e s T h e p r e p a r a t i o n o f (l-^V)-acetyl p e p t i d e s was c a r r i e d o u t a c c o r d i n g t o t h e m e t h o d d e s c r i b e d by F u j i o e t a l . (1968a) w i t h s l i g h t m o d i f i c a t i o n s . T h e . (1V)-acetic a n h y d r i d e (5 mCi/mM) was o b t a i n e d i n 0 .1 C l b r e a k s e a l t u b e s f r o m New E n g l a n d N u c l e a r Co. T h e b r e a k s e a l t u b e was f r o z e n i n a d r y i c e - a c e t o n e b a t h u n t i l t h e t u b e a n d t h e vacuum s e a l was b r o k e n . Once t h e vacuum was b r o k e n , t h e b e n z e n e was d r a w n down i n t o t h e t u b e . A f t e r w a r m i n g t h e b e n z e n e s o l u t i o n w h i c h now c o n t a i n e d t h e l a b e l l e d a c e t i c a n h y d r i d e , i t was w i t h d r a w n w i t h a s y r i n g e a n d t r a n s f e r r e d t o a s m a l l t u b e . F o r t h e a c e t y l a t i o n r e a c t i o n , t h e p e p t i d e t o be s o t r e a t e d was d i s s o l v e d i n 1 .0 M s o d i u m a c e t a t e a t pH 8 . 0 , a n d t h e b e n z e n e s o l u t i o n o f ( l - ^ C ) - a c e t i c a n h y d r i d e was l a y e r e d o n t o p o f t h e p e p t i d e s o l u t i o n w h i c h had b e e n c o o l e d t o 0 C. T h e m o l a r r a t i o o f p e p t i d e t o a c e t i c a n h y d r i d e was 1:20. The r e a c t i o n was a l l o w e d t o c o n t i n u e f o r a t l e a s t 2k hr a t k C. The aqueous phase was passed t h r o u g h a Sephadex G-15 column (2.5 x 40.0 cm) e q u i l i b r a t e d w i t h 50% a c e t i c a c i d a t 25 C and e l u t e d w i t h the same s o l v e n t . P e p t i d e s t r e a t e d i n t h i s way had m o l e c u l a r w e i g h t s of a p p r o x i m a t e l y 1200, so t h a t they were o n l y s l i g h t l y r e t a r d e d on Sephadex G-15• However, the u n r e a c t e d l a b e l l e d a c e t i c a n h y d r i d e which under t h e s e c o n d i t i o n s would be c o m p l e t e l y h y d r o l y z e d t o a c e t i c a c i d , d i d not e l u t e . u n t i l t o t a l exchange i n the column w i t h c o l d a c e t i c a c i d had o c c u r r e d a t a p p r o x i m a t e l y one column volume. F r a c t i o n s of k.5 ml were c o l l e c t e d and 0.1 ml o f e v e r y o t h e r f r a c t i o n was mixed w i t h 8.0 ml o f s c i n t i l l a t i o n f l u i d and the r a d i o a c t i v i t y was counted on a N u c l e a r C h i c a g o s c i n t i l l a t i o n c o u n t e r (Model 7 2 5 ) . The p e p t i d e peak was p o o l e d and the a c e t i c a c i d was removed by f l a s h e v a p o r a t i o n through a NaOH t r a p a t k$ C. The p e p t i d e was washed w i t h d i s t i l l e d w a t e r and l y o p h i l i z e d . A f t e r t a k i n g the p e p t i d e up i n a known volume of 0.02 M PBS a t pH 6.0, c o n t a i n i n g a s m a l l amount o f sodium a z i d e ( 0 . 0 2 % ) , a s m a l l sample was taken t o be h y d r o l y z e d f o r amino a c i d a n a l y s i s . A n o t h e r sample o f 0.01 ml was put i n t o 8.0 ml o f s c i n t i l l a t i o n f l u i d and the r a d i o a c t i v i t y was c o u n t e d , so t h a t q u a n t i t a t i v e e v a l u a t i o n o f c o u n t s per m icromole o f p e p t i d e c o u l d be e s t i m a t e d . 62 XII. Equ ? .1i br ium D ialys ? s The dialysis apparatus used in these studies was a small sample vial with a screw cap. In the outside compartment 4.0 ml of the peptide solution was placed (all peptides had previously been quantitated with respect to specific counts per micromole). The various antibody preparations (purified as described previously), anti-CM-lysozyme, non-specific gamma globulin, and PBS as a buffer control, a l l in 1.0 ml quantities, were placed in the inside (dialysis sac) compartment. The PBS, in which the peptides and antibody were dissolved was at pH 6.0 and 0.02 M and contained 0.15% NaCl and 0.02% sodium azide. The antibody concentrations were calculated from their extinctions at 2800 A"., The semipermeable membrane separating the outside and inside compartments consisted of Visking dialysis tubing (Union Carbide). The dialysis apparatus was rocked gently during incubation on the apparatus designed for this purpose. Dialysis was continued at 4 C for a number of days, the control well containing only PBS being checked at 48 hr intervals until equilibriumrwas established. This was tested by removing aliquots of 0.05 ml at these times and counting the radioactivity in the outside and inside compartments. When the counts were comparable, i t was assumed that equilibrium had been reac h e d . T h i s u s u a l l y took between k and 6 days. At t h i s t i m e , 0.05 ml a l i q u o t s were t a k e n from a l l compartments, and c o u n t s were made i n 10 ml o f s c i n t i 1 l a t i o n f l u i d t o t e s t f o r t h e p r e s e n c e o f s p e c i f i c b i n d i n g . RESULTS AND DISCUSSION I. H a p t e n i c A c t i v i t y i n T r y p t i c D i g e s t s o f CM-lysozyme The a n t i g e n i c p r o p e r t i e s o f reduced and S - c a r b o x y m e t h y l a t e d lysozyme were s t u d i e d i n o r d e r t o e l u c i d a t e the r e g i o n s o f t h i s m o l e c u l e r e s p o n s i b l e f o r a n t i g e n i c s p e c i f i c i t y . The lysozyme m o l e c u l e was chosen as t h e a n t i g e n , s i n c e the a n a l y s i s o f t h e e n t i r e amino a c i d sequence ( C a n f i e l d , 1963b) , and X-ray c r y s t a l l o g r a p h y o f t h e m o l e c u l e showing i t s t e r t i a r y s t r u c t u r e had been completed ( B l a k e e t _ a _ l ^ ,1965). I n f o r m a t i o n r e g a r d i n g a n t i g e n i c i t y c o u l d be more m e a n i n g f u l i n t h i s l i g h t . To approach the problem o f i s o l a t i n g and c h a r a c t e r i z i n g the a n t i g e n i c d e t e r m i n a n t s on the CM-lysozyme m o l e c u l e s , e n z y m a t i c d e g r a d a t i o n was chosen. T r y p s i n was used t o o b t a i n l a r g e p e p t i d e f r a c t i o n s , s i n c e t h i s i s a s p e c i f i c e n d o p e p t i d a s e c l e a v i n g p r o t e i n m o l e c u l e s o n l y a t the C - t e r m i n a l o f l y s i n e and a r g i n i n e r e s i d u e s . The lysozyme m o l e c u l e i s a h i g h l y b a s i c p r o t e i n c o n t a i n i n g 6 l y s i n e and 11 a r g i n i n e r e s i d u e s w h i c h a l l o w s t h e c a r b o x y m e t h y l a t e d d e r i v a t i v e t o be c l e a v e d i n t o as many as f o u r t e e n s m a l l e r p e p t i d e f r a g m e n t s and some s i n g l e amino a c i d s . The n a t i v e lysozyme w i t h i t s d i s u l f i d e bonds i n t a c t i s not s u s c e p t i b l e t o p r o t e o l y t i c c l e a v a g e by t r y p s i n . T h i s i s c h a r a c t e r i s t i c of many n a t i v e m o l e c u l e s which have a r i g i d f o l d e d t e r t i a r y s t r u c t u r e h e l d t o g e t h e r by d i s u l f i d e bonds. A f t e r d e n a t u r a t i o n by r e d u c t i o n and a l k y l a t i o n o f d i s u l f i d e bonds t h e s e p r o t e i n m o l e c u l e s a r e s u s c e p t i b l e t o c o n s i d e r a b l e enzyme c l e a v a g e . A s t u d y on the a n t i g e n i c p r o p e r t i e s o f n a t i v e lysozyme had been c a r r i e d out by a n o t h e r group o f w o r k e r s ( S h i n k a e t a l . , f l 9 & 7 ) , and i t was f e l t t h a t a c o m p a r a t i v e s t u d y on the reduced and c a r b o x y m e t h y l a t e d d e r i v a t i v e would be i n t e r e s t i n g and might throw some l i g h t on the i n t e r r e l a t e d r o l e o f p r i m a r y and t e r t i a r y s t r u c t u r e i n p r o t e i n s w i t h r e s p e c t t o t h e i r a n t i g e n i c p r o p e r t i e s . The d i g e s t i o n o f CM-lysozyme w i t h t r y p s i n , and t h e s e p a r a t i o n o f the t r y p t i c p e p t i d e s have been d e s c r i b e d i n the p r e v i o u s s e c t i o n on methods. I n i t i a l s c r e e n i n g f o r h a p t e n i c a c t i v i t y by p r e c i p i t i n i n h i b i t i o n i n v o l v e d m i x i n g o f t r i p l i c a t e samples o f 0.4 ml o f a n t i -serum o f known t i t e r wi.th.0.1 ml q u a n t i t i e s o f the v a r i o u s p e p t i d e f r a c t i o n s ( c o n t a i n i n g between 200 and 1000 ;" Ug/ml depending on t h e i r s o l u b i l i t i e s ) d i s s o l v e d i n 0.3% N a C l , and i n c u b a t i n g t h e m i x t u r e s a t 37 C f o r 1 hr f o l l o w e d by 18 hr a t 4 C. A t the same t i m e , s a l i n e c o n t r o l s were s e t up i n t r i p l i c a t e . On the f o l l o w i n g day, a n t i g e n was added t o a c o n c e n t r a t i o n e q u i v a l e n t t o o p t i m a l p r o p o r t i o n s ( t h i s v a r i e d w i t h the s e r a b e i n g u s e d ) , and t h e i n c u b a t i o n was r e p e a t e d . The immune p r e c i p i t a t e s formed were c e n t r i f u g e d , washed i n 0.3% N a C l , and then d i s s o l v e d i n 1.0 ml o f 0.1 N NaOH t o be read f o r 2800 A;.' a b s o r b a n c e . These e x p e r i m e n t s were r e p e a t e d on t h e s e r a from t h r e e i n d i v i d u a l a n i m a l s . In each i n s t a n c e the o n l y p e p t i d e f r a c t i o n w h i c h showed e v i d e n c e o f h a p t e n i c a c t i v i t y was t h a t which c o r r e l a t e d w i t h the T-11 p e p t i d e i s o l a t e d from the Dowex column run as recommended by C a n f i e l d (1963a). An amino a c i d a n a l y s i s on t h i s p e p t i d e f r a c t i o n e s t a b l i s h e d i t s i d e n t i t y w i t h t h e T-11 p e p t i d e i s o l a t e d by C a n f i e l d . The c l o s e agreement between our r e s u l t s and C a n f i e l d ' s d a t a i s shown i n T a b l e I I . The p r e p a r a t i o n was v i r t u a l l y f r e e o f a l l o t h e r c o n t a m i n a t i n g amino a c i d s as e v i d e n c e d by t h e t r a c e amounts o f G l u and G l y p r e s e n t ( t h e s e a r e not p r e s e n t i n the p e p t i d e ) i n a n a l y s e s a t c o n c e n t r a t i o n s o f l e s s than 2.0% o f o t h e r r e s i d u e amino a c i d s i n the p e p t i d e . Ring t e s t s on t h i s f r a c t i o n were n e g a t i v e , i n d i c a t i n g t h a t no p r e c i p i t a b l e and u n d i g e s t e d m a t e r i a l was p r e s e n t . For most o f the t e s t s r e p o r t e d h e r e , a s t o c k s o l u t i o n o f the p e p t i d e was used w h i c h was a t a c o n c e n t r a t i o n o f 500 ug/ml. A q u a n t i t a t i v e hapten i n h i b i t i o n t e s t was c a r r i e d o ut on T-11 u s i n g between 50 and 0.1 ug o f t h e p e p t i d e , and compared on a w e i g h t b a s i s w i t h a s i m i l a r t e s t on t h e t o t a l t r y p t i c d i g e s t . Because serum from r a b b i t 3 had a h i g h e r p r e c i p i t a t i n g t i t e r than d i d the o t h e r two s e r a , i t was used f o r t h e s e t e s t s . F i g u r e 10 shows t h e r e s u l t s o f t h e s e t e s t s which were run i n t r i p l i c a t e . These f i g u r e s r e p r e s e n t mean v a l u e s f o r each c o n c e n t r a t i o n and were found t o be c l o s e l y r e p r o d u c i b l e , s i n c e on r e t e s t i n g t h e v a r i a t i o n was found t o be between 1.5 and 2.0%. Maximum i n h i b i t i o n o f 60% was o b s e r v e d T a b l e I I . Amino a c i d a n a l y s i s o f p e p t i d e T - 1 1 . The m i c r o m o l a r r a t i o s a r e i n agreement w i t h t h o s e d e t e r m i n e d by C a n f i e l d (1963a) and shown i n column 1. Amino a c i d M i c r o m o l e s 1 2 R e s i d u e s Lys i ne 0.27 0.0211 1 S-CM-cystei ne 0.73 0.0597 3 A s p a r t i c a c i d 1.11 0.0722 4 Threon i ne 0.27 0.0195 1 Ser i ne 1.03 0.0618 4 P r o l i ne 0.28 0.0154 1 A l a n i n e 0.78 O.O5I6 3 V a 1 i ne 0.25 0.0173 1 1 s o l e u c i n e 0.55 0.0341 2 Leuc i ne 0.82 0.0559 3 68 10.0 1.0 01 M HAPTEN F i g u r e 1 0 . I n h i b i t i o n o f immune p r e c i p i t a t i o n by p e p t i d e T - 1 1 (o-o) and the t o t a l t r y p t i c d i g e s t (o -o) at v a r y i n g c o n c e n t r a t i o n s . C a l c u l a t i o n of i n h i b i t i o n was based o n l y on 50% hemolys is end p o i n t . w i t h 25 yg o n v l a r g e r q u a n t i t i e s o f T - 1 1 . In o r d e r t o e s t a b l i s h t h a t the i n h i b i t i o n o b s e r v e d h e r e was not unique f o r p r e c i p i t a t i o n , i n h i b i t i o n t e s t s u s i n g complement f i x a t i o n w i t h p o o l e d s e r a from r a b b i t s 1 and 2 were c a r r i e d o u t . With serum t i t e r s a t 1 :80, the o p t i m a l a n t i g e n l e v e l s f e l l between 0.2 and 0 .8 yg ( F i g . 1 ) . For hapten i n h i b i t i o n t e s t i n g , 0 .8 yg o f CM-lysozyme was used s t a n d a r d l y i n o r d e r t o work i n a r e g i o n o f s l i g h t a n t i g e n e x c e s s . In t h e i n i t i a l t e s t on both T-11 and the t o t a l t r y p t i c d i g e s t , o n l y two d i l u t i o n s o f complement ( 1 :hi and 1:50). were used, as t h e s e had p r e v i o u s l y been c a l c u l a t e d t o produce 50% h e m o l y s i s i n t h e r e a c t i o n o f a n t i g e n and a n t i b o d y . Under t h e s e c o n d i t i o n s , t h e T-11 p e p t i d e caused t o t a l i n h i b i t i o n a t c o n c e n t r a t i o n s between 50 and 10 y g , but t h e t r y p t i c d i g e s t a c t e d i n t h e same way o n l y a t 50 and 25 y g . To d e t e r m i n e the a c t u a l degree o f i n h i b i t i o n , a n o t h e r t e s t was s e t up e x p a n d i n g t h e range o f complement and hapten l e v e l s . T e s t s were.run c o n c u r r e n t l y on o t h e r t r y p t i c p e p t i d e s which had f a i l e d t o cause i n h i b i t i o n o f the p r e c i p i t a t i o n r e a c t i o n . These o t h e r t r y p t i c p e p t i d e s were t e s t e d a t o n l y one l e v e l v a r y i n g between 50 and 200 yg depending on t h e maximum s o l u b i l i t y o f the p a r t i c u l a r p e p t i d e . As i n the p r e v i o u s t e s t s , t h e o n l y f r a c t i o n s showing i n h i b i t i o n were t h e t o t a l d i g e s t and T - 1 1 . The r e s u l t s f o r t h e s e two p r e p a r a t i o n s a r e documented i n F i g u r e s 11 and 1 2 , and are c o r r e l a t e d i n F i g u r e 13 , ' n w h i c h , on a w e i g h t b a s i s , p e p t i d e T-11 showed c o n s i d e r a b l y more i n h i b i t o r y c a p a c i t y than the t r y p t i c d i g e s t . The T-11 demonstrated d e t e c t a b l e |:60 l :80 |:|00 1-120 COMPLEMENT DILUTION gure 11. Inh ib i t ion of complement f i x a t i o n with CM-lysozyme and homologous antiserum by peptide T-11 at varying concentrat ions ; ©-©, 50 yg; &-A, 25 yg; a - ^ 10 • yg; o - o , 1.0 : Ug; A - A , 0.1 yg ; andQj-Q no hapten c o n t r o l . C a l c u l a t i o n of i n h i b i t i o n was based only on 50% hemolysis end po in t . 71 M50 1^ 60 V70 VQO 1^ 90 COMPLEMENT DILUTION F i g u r e 1 2 . I n h i b i t i o n o f complement f i x a t i o n w i t h C M - l y s o z y m e and homologous a n t i s e r u m by the t o t a l t r y p t i c d i g e s t a t v a r y i n g c o n c e n t r a t i o n s ; o-o, 50 y g ; 2.5 y g ; 10 y g ; o - o , 1 .0 y g ; A - A , 0 .1 .. y g ; a n d O - Q , no h a p t e n c o n t r o l . C a l c u l a t i o n o f i n h i b i t i o n was b a s e d o n l y on 50% h e m o l y s i s end p o i n t . 72 0 I 10 100 jurg HAPTEN F i g u r e 13. C o r r e l a t i o n o f i n h i b i t i o n o f c o m p l e m e n t f i x a t i o n a t a 1 :62 d i l u t i o n o f c o m p l e m e n t a n d v a r y i n g c o n c e n t r a t i o n s o f T-11 ( o - o ) a n d t r y p t i c d i g e s t ( © - © ) . C a l c u l a t i o n o f i n h i b i t i o n was b a s e d o n l y o n 50% h e m o l y s i s e n d p o i n t . i n h i b i t i o n i n c o n c e n t r a t i o n s as low as 1.0 y g , whereas the t o t a l d i g e s t showed o n l y s l i g h t i n h i b i t i o n a t 10 y g . The t e s t i n g o f the s p e c i f i c i t y o f the i n h i b i t i o n o b s e r v e d w i t h the T-11 p e p t i d e was the f i n a l c o n t r o l measure taken i n t h e s e e x p e r i m e n t s . For t h i s p u r p o s e , a system o f n a t i v e lysozyme and i t s homologous a n t i s e r u m was used, s i n c e i t had p r e v i o u s l y been found t h a t a n t i s e r u m a g a i n s t n a t i v e lysozyme d i d not c r o s s - r e a c t a t the p r e c i p i t a t i o n l e v e l w i t h CM-lysozyme. The a n t i s e r u m had been p r e p a r e d and t i t e r e d i n an i d e n t i c a l manner to t h a t d e s c r i b e d here f o r CM-lysozyme a n t i s e r a . No d e t e c t a b l e i n h i b i t i o n was produced by the T-11 p e p t i d e , i n d i c a t i n g s p e c i f i c i t y f o r the CM-lysozyme system, and no a f f i n i t y f o r the a n t i s e r u m a g a i n s t the n a t i v e a n t i g e n . From t h e s e e x p e r i m e n t s , i t would appear t h a t the T-11 r e g i o n o f t h e CM-lysozyme m o l e c u l e p l a y s a s i g n i f i c a n t r o l e as an a n t i g e n i c d e t e r m i n a n t , by i t s a b i l i t y t o i n h i b i t the homologous imm u n o l o g i c a l r e a c t i o n , u s i n g both p r e c i p i t a t i o n and complement f i x a t i o n as t e s t s ystems. The p o s s i b i l i t y t h a t our i n h i b i t o r y p r e p a r a t i o n s o f T-11, c o n t a i n e d u n d i g e s t e d p r o t e i n , o r a c o n t a m i n a t i n g p e p t i d e which c o u l d be r e s p o n s i b l e f o r the o b s e r v e d r e a c t i o n s was d i s c o u n t e d on s e v e r a l grounds: ( ' l) the i n a b i l i t y o f p e p t i d e p r e p a r a t i o n s t o p r e c i p i t a t e w i t h a n t i b o d y when t e s t e d e i t h e r by r i n g t e s t o r by t h e t e s t i n g o f v a r y i n g p e p t i d e c o n c e n t r a t i o n s w i t h a n t i s e r u m ; ( i i ) the amino a c i d a n a l y s i s w h i c h a g r e e s c l o s e l y w i t h C a n f i e l d 1 s . (1963a) a n a l y s i s o f the p e p t i d e ; ( i i i ) the f i n g e r p r i n t a n a l y s i s which demonstrated o n l y one n i n h y d r i n - p o s i t i v e s p o t ; ( i v ) the a b s o r p t i o n scan on T-11 w h i c h showed no c o n t a m i n a t i o n w i t h t r y p t o p h a n - c o n t a i n i n g p e p t i d e s ; (v) th e v e r y low l e v e l s a t w h i c h the p e p t i d e d e m o n s t r a t e d h a p t e n i c a c t i v i t y ; and ( v i ) the l a c k o f complement f i x i n g c a p a c i t y on the p a r t o f t h e p e p t i d e f r a c t i o n . A n o t h e r i m p o r t a n t c o n t r o l was t h e p r o o f t h a t t h e i n h i b i t i o n by t h e T-11 p e p t i d e was s p e c i f i c f o r the homologous CM-lysozyme - a n t i - C M - l y s o z y m e system. T h i s e f f e c t was shown by the i n a b i l i t y o f T-11 t o i n h i b i t p r e c i p i t a t i o n between n a t i v e lysozyme and i t s homologous a n t i s e r u m . E a r l y e x p e r i m e n t s had shown t h a t t h e r e was no c r o s s - r e a c t i v i t y between CM-lysozyme and n a t i v e lysozyme a n t i s e r u m a t t h e p r e c i p i t i n l e v e l s t e s t e d , and f u r t h e r , t h a t none o f t h e t r y p t i c p e p t i d e s o r whole d i g e s t showed any c a p a c i t y t o . a c t as haptens w i t h the n a t i v e system. On a w e i g h t b a s i s , i t was found t h a t the T-11 p e p t i d e was a p p r o x i m a t e l y two t o t h r e e t i m e s as e f f i c i e n t as t h e t r y p t i c d i g e s t as an i n h i b i t o r when t e s t e d e i t h e r by p r e c i p i t a t i o n o r complement f i x a t i o n . I f t h i s p e p t i d e r e p r e s e n t e d the o n l y d e t e r m i n a n t p r e s e n t i n t h e t r y p t i c d i g e s t , on a w e i g h t b a s i s , one would e x p e c t about f i v e t i m e s t h e s p e c i f i c a c t i v i t y f o r t h e p e p t i d e , s i n c e i t c o m p r i s e s a p p r o x i m a t e l y 20% (23 amino a c i d r e s i d u e s t o 129 r e s i d u e s i n t h e t o t a l m o l e c u l e ) o f the t o t a l m o l e c u l e . However, ?n t h e s e e x p e r i m e n t s , t h i s 5 - f o l d a c t i v i t y was n e v e r o b s e r v e d , and a t most a 3 _ f o l d g r e a t e r s p e c i f i c a c t i v i t y o f the p e p t i d e t o t h e d i g e s t was n o t e d . T h i s r e s u l t does not d i s c o u n t t h e p r o b a b i l i t y t h a t o t h e r r e g i o n s o f t h e CM-lysozyme m o l e c u l e p o s s e s s a n t i g e n i c p r o p e r t i e s , but r a t h e r t h a t the T-11 r e g i o n i s the o n l y one . " r e t a i n i n g c o n s i d e r a b l e a c t i v i t y a f t e r 75 t r y p s i n d i g e s t i o n . Because a l l t h e o t h e r p e p t i d e s i s o l a t e d from t r y p t i c d i g e s t s o f CM-lysozyme on the e i g h t - c h a m b e r e d g r a d i e n t system recommended by C a n f i e l d (1963a) showed no h a p t e n i c a c t i v i t y , a s i m p l i f i e d f o u r -chambered system was s e t up. T h i s used the f i r s t f o u r b u f f e r s used i n t h e o r i g i n a l s y stem, and p e r m i t t e d t h e ' e l u t i o n o f T-11 as t h e f i r s t s h a r p l y d e l i n e a t e d peak. The purpose o f t h e s e f u r t h e r p e p t i d e s e p a r a t i o n s was m a i n l y t o c o l l e c t l a r g e amounts o f T-11 f o r f u r t h e r s t u d y . The f a c t t h a t t h e s e r a from t h r e e d i f f e r e n t r a b b i t s a l l r e a c t e d i d e n t i c a l l y i n t h e systems t e s t e d , e x c e p t w i t h r e s p e c t t o a n t i b o d y t i t e r , i n d i c a t e d t h a t the s p e c i f i c i t y o f the system i s not r e l a t e d t o i n d i v i d u a l a n i m a l s . I t i s i n t e r e s t i n g t o n o t e t h a t t h i s T-11 p e p t i d e i s t h e l a r g e s t t r y p t i c p e p t i d e found i n CM-lysozyme, c o n t a i n i n g 23 amino a c i d r e s i d u e s o c c u p y i n g between t h e 7 ^ t h and 9 6 th p o s i t i o n i n the m o l e c u l e . The sequence o f the p e p t i d e i s as f o l l o w s : NH^-Asn-Leu,- CMC - Asn - M e - Pro - CMC - Ser - A l a - Leu - Leu - Ser - Ser -Asp - l i e - Thr - A l a - Ser - V a l - Asn - CMC - A l a - Lys COOH ( C a n f i e l d , 1963b ) . I I. C a r b o x y p e p t i d a s e . D i g e s t i o n s . S i n c e T-11 i s such a l a r g e p e p t i d e , t h e n e x t s e r i e s o f e x p e r i m e n t s i n v o l v e d s e q u e n t i a l d e g r a d a t i o n o f t h e m o l e c u l e i n a t t e m p t s t o d e t e r m i n e w h i c h p o r t i o n o f the whole p e p t i d e was r e s p o n s i b l e f o r i t s h a p t e n i c a c t i v i t y . The f i r s t e x p e r i m e n t s employed t h e c a r b o x y p e p t i d a s e enzymes w h i c h , as t h e name i n d i c a t e s , s e q u e n t i a l l y c l e a v e amino a c i d s from t h e c a r b o x y ' t e r m i n a l o f p e p t i d e s and p r o t e i n s . I n i t i a l l y , c a r b o x y p e p t i d a s e B was used t o t e s t whether o r not the C - t e r m i n a l r e s i d u e ( l y s i n e ) o f T-11 was i m p o r t a n t t o i t s h a p t e n i c a c t i v i t y . C a r b o x y p e p t i d a s e B i s an e x o p e p t i d a s e o b t a i n e d from p o r c i n e pancreas w h i c h s p e c i f i c a l l y c l e a v e s b a s i c amino a c i d s ( l y s i n e , a r g i n i n e o r o r n i t h i n e ) from the C - t e r m i n a l o f p e p t i d e s o n j p r o t e i n s . The commercial p r e p a r a t i o n s c o n t a i n h i g h l y a c t i v e homogeneous enzyme, e s s e n t i a l l y d e v o i d o f c a r b o x y p e p t i d a s e A a c t i v i t y w hich p r e f e r e n t i a l l y c l e a v e s n e u t r a l and a c i d i c ami no a c i d s s e q u e n t i a l l y from the C - t e r m i n a l . A f t e r t h e enzyme a c t i v i t y ,-of'the p r e p a r a t i o n had been d e t e r m i n e d , an e x p e r i m e n t was s e t up f o r d e g r a d a t i o n o f t h e T-11 p e p t i d e . S i m u l t a n e o u s l y , a s u b s t r a t e c o n t r o l , u s i n g o n l y T-11 and no enzyme, and an enzyme c o n t r o l c o n t a i n i n g enzyme but no p e p t i d e , were s e t up and t r e a t e d i n a manner i d e n t i c a l t o t h e t e s t d i g e s t s o l u t i o n as r e p o r t e d i n the Methods. The s u b s t r a t e c o n t r o l t e s t e d whether the i n c u b a t i o n c o n d i t i o n s had any n o n s p e c i f i c e f f e c t on t h e h a p t e n i c a c t i v i t y o f T-11. The enzyme c o n t r o l t e s t e d f o r any n o n s p e c i f i c e f f e c t t h e enzyme might have on t h e subsequent i m m u n o l o g i c a l t e s t i n g o f t h e d i g e s t , as w e l l as f o r a u t o d e g r a d a t i v e r e l e a s e o f amino a c i d s from t h e enzyme i t s e l f r a t h e r than from the p e p t i d e . Samples were removed f o r h a p t e n i c t e s t i n g a t k h r and a t 18 h r , and the a d d i t i o n o f g l a c i a l a c e t i c a c i d t e r m i n a t e d t h e r e a c t i o n by d e n a t u r i n g and p r e c i p i t a t i n g t h e enzyme. The s u b s t r a t e c o n t r o l was used as the p o s i t i v e c o n t r o l i n t e s t i n g f o r h a p t e n i c a c t i v i t y . The enzyme c o n t r o l a c t e d as the n e g a t i v e c o n t r o l and showed no n o n s p e c i f i c i n h i b i t i o n o f t h e p r e c i p i t a t i o n between th e homologous CM-lysozyme - a n t i - C M - l y s o z y m e system. A n o t h e r n e g a t i v e c o n t r o l was a l s o s e t up u s i n g an e q u i v a l e n t amount o f s a l i n e - T r i s b u f f e r as used f o r t h e t e s t s o l u t i o n t o e n s u r e t h a t no n o n s p e c i f i c i n h i b i t i o n c o u l d be a t t r i b u t e d t o the enzyme c o n t r o l , due p o s s i b l y t o p r o d u c t s o f a u t o d i g e s t i o n . A l l the hapten t e s t s were done i n d u p l i c a t e . The amount o f T-11 p r e s e n t i n a l i q u o t s t aken f o r t e s t i n g were e q u i v a l e n t t o 65 ug, w hich from p r e v i o u s t e s t i n g was known t o y i e l d maximum i n h i b i t i o n . The r e s u l t s a r e p r e s e n t e d i n T a b l e M l . There was e s s e n t i a l l y no l o s s i n the h a p t e n i c a c t i v i t y of the T-11 p e p t i d e a f t e r COB d i g e s t i o n f o r 18 h r . The enzyme c o n t r o l gave v i r t u a l l y no i n h i b i t i o n o f p r e c i p i t a t i o n and t h e r e f o r e the enzyme was assumed t o have no n o n s p e c i f i c e f f e c t . 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 e s t and c o n t r o l samples run w i t h a l y s i n e s t a n d a r d showed t h a t l y s i n e had been c l e a v e d from the C - t e r m i n a l o f the p e p t i d e . There was a l s o a n o t h e r n i n h y d r i n -p o s i t i v e s p o t i n a d d i t i o n t o t h a t f o r the remainder o f t h e p e p t i d e . The s u b s t r a t e c o n t r o l c o n t a i n i n g o n l y T-11 gave o n l y one n i n h y d r i n -p o s i t i v e s p o t , and the enzyme c o n t r o l s i m i l a r l y gave one major s p o t and a minor one which c o r r e s p o n d e d t o t h e unknown one o b s e r v e d i n the t e s t sample. Thus, th e second n i n h y d r i n - p o s i t i v e s p o t i n t h e d i g e s t and the enzyme c o n t r o l was thought t o r e s u l t from a u t o d i g e s t i o n T a b l e I I I . Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by c a r b o x y p e p t i d a s e B d i g e s t e d T-11. 0 D * P e r c e n t D i g e s t i o n Time ' ' 2 8 0 0 A I n h i b i t i o n k hr C a r b o x y p e p t i d a s e B d i g e s t e d T-11 (65 yg = 0.026 ymoles) 0.0805 24.8 S u b s t r a t e (T-11) c o n t r o l (65 yg = 0.026 ymoles) 0.0835 22.0 Enzyme c o n t r o l 0.107 0.0 (100% p r e c i p i t a t i o n l e v e l ) S a l i n e c o n t r o l 0.107 0.0 18 hr C a r b o x y p e p t i d a s e B d i g e s t e d T-11. (65 yg = 0.026 ymoles) 0.078 22.0 S u b s t r a t e (T-11) c o n t r o l (65 yg = 0.026 ymoles) 0.079 .21.0 Enzyme c o n t r o l 0.107 0.0 (100% p r e c i p i t a t i o n l e v e l ) S a l i n e c o n t r o l 0.106 0.0 R e p r e s e n t s the mean v a l u e s o f the d u p l i c a t e samples. o f t h e enzyme. In the second c a r b o x y p e p t i d a s e e x p e r i m e n t , both COB and COA were used t o degrade the p e p t i d e s e q u e n t i a l l y from t h e C - t e r m i n a l i n as f a r as p o s s i b l e , t o t e s t t h e l i m i t o f d e g r a d a t i o n b e f o r e l o s s o f h a p t e n i c a c t i v i t y o c c u r r e d . COA i s stopped i n i t s c l e a v a g e by p r o l i n e as the C - t e r m i n a l amino a c i d . Commercial c a r b o x y p e p t i d a s e A i s t h e b o v i n e enzyme and s t i l l c o n t a i n s c o n s i d e r a b l e a c t i v i t y a g a i n s t b a s i c amino a c i d s a l t h o u g h i t has been h i g h l y p u r i f i e d . The COA used here had been t r e a t e d w i t h d i i s o p r o p y l f l u o r o p h o s p h a t e (DFP) t o i n h i b i t any t r y p t i c or c h y m o t r y p t i c e n d o p e p t i d a s e a c t i v i t y . The COB was added f i r s t f o r 1 hr t o e n s u r e the removal of l y s i n e from the C - t e r m i n a l o f T-11 b e f o r e the COA was added. A d d i t i o n a l a l i q u o t s of COA were added a f t e r 2 , 15 and 2k hr s i n c e t h i s enzyme i s known to undergo a u t o d e g r a d a t i o n upon p r o l o n g e d i n c u b a t i o n . C o n t r o l t u b e s , both a s u b s t r a t e c o n t r o l and an enzyme c o n t r o l as d e s c r i b e d above, were s e t up and samples of 0.3 ml were removed from both the t e s t and c o n t r o l s a t 2 , 6, 15 , 2k and kO h r . The r e a c t i o n i n the samples taken was t e r m i n a t e d by d e n a t u r a t i o n o f t h e enzyme by the a d d i t i o n o f 20 y l o f g l a c i a l a c e t i c a c i d . A l i q u o t s o f 0.1 m l , c o n t a i n i n g 0.070 ymole o f p e p t i d e i n the c a s e of the t e s t samples, were removed from the 0.3 ml samples t a k e n a t v a r i o u s t i m e s , and d r i e d and taken up i n s t a r t i n g b u f f e r f o r amino a c i d a n a l y s i s . A n a l y s i s on t h e s h o r t column o f the Beckman A n a l y z e r showed t h a t between 80 and 30% o f the C - t e r m i n a l l y s i n e had been removed from T - 1 1 . However, t h e r e s u l t s o f t o t a l amino a c i d a n a l y s e s were not c l e a r c u t s i n c e an i s o l e u c i n e peak and a c a r b o x y m e t h y l -80 c y s t e i n e were p r e s e n t , but none o f the o t h e r e x p e c t e d r e s i d u e s (Asp, T h r , S e r , A l a and V a l r e s p e c t i v e l y ) were d e t e c t e d i n e q u i v a l e n t amounts. I s o l e u c i n e i s n i n e amino a c i d s i n from t h e C - t e r m i n a l , and a l l the above mentioned amino a c i d s would have t o be c l e a v e d b e f o r e i t would be p r e s e n t i n d i g e s t s o f T-11. I t i s a l s o known t h a t S - c a r b o x y m e t h y l c y s t e i n e i s o n l y r e l e a s e d e x t r e m e l y s l o w l y by COA, and s i n c e t h i s i s the t h i r d amino a c i d i n from t h e C - t e r m i n a l , a l l f u r t h e r d e g r a d a t i o n would be g r e a t l y r e d u c e d , so t h a t one would e x p e c t o n l y t r a c e amounts o f t h e o t h e r amino a c i d s . Because o f the h i g h l e v e l s o f enzyme used i n t h i s second e x p e r i m e n t , and the f a c t t h a t some f r e e amino a c i d s were d e t e c t e d i n t h e enzyme c o n t r o l on amino a c i d a n a l y s i s , i t c o u l d not be c o n c l u s i v e l y d e t e r m i n e d how f a r i n from the C - t e r m i n a l the COA had degraded t h e p e p t i d e . The d i g e s t s , upon a c i d i f i c a t i o n t o t e r m i n a t e the r e a c t i o n , gave r i s e t o c o n s i d e r a b l e p r e c i p i t a t e s which i n t e r f e r e d w i t h the hapten i n h i b i t i o n t e s t s . In f a c t , t h e r e appeared t o be more p r e c i p i t a t i o n o b s e r v e d between t h e homologous a n t i g e n - a n t i b o d y s ystem, w h i c h was used t o y i e l d the 100% p r e c i p i t i n l e v e l . T h e r e f o r e , t h e r e s u l t s from t h e COA and COB e x p e r i m e n t were not v a l i d and d i d not g i v e the s o u g h t - f o r answer. However, the o r i g i n a l e x p e r i m e n t i n d i c a t e d t h a t removal o f the C - t e r m i n a l l y s i n e had no e f f e c t on h a p t e n i c a c t i v i t y . Because o f the t e c h n i c a l d i f f i c u l t i e s e n c o u n t e r e d u s i n g COA i n t h e s e e x p e r i m e n t s , f u r t h e r work a t t h i s time i n v o l v e d i n v e s t i g a t i o n o f the N - t e r m i n a l o f T-11. I t was d e c i d e d t h a t i f the N - t e r m i n a l r e g i o n o f T-11 was found t o be u n i n v o l v e d i n a n t i g e n i c i t y , then the c a r b o x y p e p t i d a s e e x p e r i m e n t s would be r e p e a t e d , u s i n g more i n t r i c a t e t e c h n i q u e s f o r p u r i f y i n g each d i g e s t a l i q u o t b e f o r e t e s t i n g f o r hapten i n h i b i t i o n . I I I . Edmari D e g r a d a t i o n o f T-11 D e g r a d a t i o n of the T-11 p e p t i d e s e q u e n t i a l l y from t h e N-t e r m i n a l was c a r r i e d out u s i n g t h e Edman's method. The sub-t r a c t i v e Edman t e c h n i q u e was used i n which the N - t e r m i n a l amino a c i d i s removed and an a l i q u o t of t h e r e m a i n i n g p e p t i d e i s s u b j e c t e d t o h y d r o l y s i s and amino a c i d a n a l y s i s . Comparison o f th e r e m a i n i n g amino a c i d s w i t h the known a n a l y s i s o f t h e t o t a l p e p t i d e e s t a b l i s h e s the removal o f t h e N - t e r m i n u s . a The f i r s t s t a g e i n the r e a c t i o n i n v o l v e s the a t t a c h m e n t o f the t h i o c a r b a m y l group o f phenyl i s o t h i o c y a n a t e t o the u n p r o t o n a t e d ami no group o f t h e N - t e r m i n a l amino a c i d . A f t e r the c o u p l i n g r e a c t i o n has o c c u r r e d and e x c e s s r e a g e n t s have been removed, t h e p h e n y l t h i o c a r b a m y l group on t h e amino group i s c o u p l e d w i t h t h e carbamyl c a r b o n o f t h e amino t e r m i n a l amino a c i d , r e s u l t i n g i n the f o r m a t i o n o f a t h i a z o l i n o n e d e r i v a t i v e . The c y c l i z a t i o n r e a c t i o n and c l e a v a g e o f t h e N - t e r m i n a l amino a c i d from t h e p e p t i d e must be performed under anhydrous c o n d i t i o n s t o p r e v e n t h y d r o l y t i c c l e a v a g e o f a c i d - s e n s i t i v e bonds. A f t e r benzene e x t r a c t i o n , t o remove t h e p h e n y 1 t h i o c a r b a m y l amino a c i d s and p h e n y l h y d a n t o i n s o f most a c i d i c and n e u t r a l amino a c i d s , the r e m a i n i n g p e p t i d e was p u r i f i e d on a s m a l l Dowex 50 column e q u i l i b r a t e d w i t h 0.2 N a c e t i c a c i d . A c e t i c a c i d was used t o e l u t e n i n h y d r i n - n e g a t i v e m a t e r i a l , and t h e p e p t i d e was then e l u t e d w i t h 2.0 N p y r i d i n e - a c e t i c a c i d b u f f e r a t pH 5-0. The T-11 p e p t i d e c o n t a i n s o n l y one b a s i c r e s i d u e l y s i n e , no a r o m a t i c r e s i d u e s , and the t h r e e c y s t e i n e r e s i d u e s a r e a l l c a r b o x y m e t h y l a t e d , and t h e r e f o r e not s t r o n g l y a c i d i c . Thus, no d i f f i c u l t i e s were e n c o u n t e r e d when u s i n g t h i s p u r i f i c a t i o n t e c h n i q u e . The e l u t e d , p u r i f i e d r e m a i n i n g p e p t i d e was d r i e d and resuspended i n 50% aqueous p y r i d i n e from w h i c h 3 samples were t a k e n . One sample was used f o r amino a c i d a n a l y s i s a f t e r i t had been d r i e d and h y d r o l y z e d f o r 18 h r . The o t h e r two samples were d r i e d and r e d i s s o l v e d i n 0.3% NaCl f o r hapten i n h i b i t i o n t e s t s . The amino a c i d a n a l y s i s y i e l d e d q u a n t i t a t i o n o f the amounts o f p e p t i d e p r e s e n t i n the samples used f o r hapten i n h i b i t i o n t e s t i n g . Each t i m e a c y c l e was r e p e a t e d , t h e r e . w e r e l o s s e s i n m a t e r i a l r e s u l t i n g from h a n d l i n g and the column p u r i f i c a t i o n s t e p , and i n o r d e r t o compensate f o r t h i s , l a r g e r samples were removed each t i m e as s t a t e d i n t h e methods. D u p l i c a t e samples of T-11 were s e t up as c o n t r o l s a t t h r e e c o n c e n t r a t i o n s o f 0.0k, 0.02 and 0.01 y m o l e s , w h i c h were w i t h i n t h e range o f amounts o f the T-11-1 , T-11-2 and T-11-3 samples t e s t e d . N e g a t i v e c o n t r o l s u s i n g e q u i v a l e n t volumes o f 0.3% NaCl i n p l a c e o f p e p t i d e were a l s o s e t up. The r e s u l t s o f amino a c i d a n a l y s e s and the hapten i n h i b i t i o n t e s t s a r e shown i n T a b l e s IV and V r e s p e c t i v e l y , and demo n s t r a t e T a b l e IV. Amino a c i d a n a l y s e s d a t a o f s u b t r a c t i v e Edman's d e g r a d a t i o n o f T - 1 1 . Amino a c i d M i c r o m o l e s T - 1 1 - 1 Res i dues M i c r o m o l e s T - 1 1 - 2 Res i d u e s M i c r o m o l e s T - 1 1 - 3 Res i dues R e s i d u e s o f T - 1 1 p e p t i d e S - C M - c y s t e i n e 0 . 2 1 2 3 . 0 0 . 0 3 6 3 . 3 0 . 0 2 0 1 . 8 " 3 A s p a r t i c a c i d 0 .217 3 . 1 * 0 . 0 3 3 3 . 0 " 0 . 0 3 1 2 . 8 " 4 T h r e o n i ne 0 . 0 6 8 0 .97 0 .013 1 . 2 0 . 0 1 2 1 .1 1 Se r i ne 0 . 2 6 1 3 . 7 0 . 0 4 2 3 . 8 0 . 0 4 0 3 . 6 4 P r o l i ne 0 . 0 9 7 1 . 4 0 . 0 1 6 1 . 5 0 . 0 1 5 1 . 4 1 A l a n i n e 0 . 2 4 2 3 . 5 0 . 0 3 5 3 . 2 0 . 0 3 7 3 . 4 3 V a l i n e 0 . 0 7 7 1.1 0 . 0 1 2 1.1 0 . 0 1 4 1 . 3 1 1 s o l e u c i ne 0 . 1 5 1 2 . 2 0 . 0 2 2 2 . 0 0 . 0 2 2 2 . 0 2 L e u c i ne 0 . 2 2 2 3 . 2 0 . 0 2 5 2 . 3 " 0 . 0 2 4 2 . 2 " 3 mcM i n s a m p l e a n a l y z e d 0.070 0 . 0 1 1 0 . 0 1 1 Amino a c i d s removed . Sequence o f N - t e r m i n a l r e g i o n o f T - 1 1 s h o w i n g amino a c i d s removed a t each Edman 1 s d e g r a d a t i o n c y c l e : N H 2 - A s n - Leu -/ CMC - Asn - 1 l e - P r o T - 1 1 - 1 > \ T - 1 1 - 2 ? s / T - 1 1 - 3 ? •\ \ / T a b l e V. Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by Edman's d e g r a d a t i o n o f T-11. T e s t °' D ,2800 A P e r c e n t Inh i b i t ion T-11-1 (0.07 ymoles) 0.118 18.6 T-11-2 (0.011 ymoles) 0.143 0.0 T-11-3 (0.011 ymoles) 0.167 0.0 C o n t r o l s A. P o s i t i v e hapten i n h i b i t i o n T-11 (0.04 ymoles) 0.063 57.3 T-11 (0.02 ymoles) 0.067 53.8 T-11 (0.01 ymoles) 0.073 49.7 B. N e g a t i v e hapten i n h i b i t i o n (maximum p r e c i p i t a t i o n l e v e l ) 0.145 0.0 R e p r e s e n t s the mean v a l u e s o f d u p l i c a t e samples. t h a t t h e removal o f t h e a s p a r a g i n e from the N - t e r m i n a l o f T-11 d e c r e a s e d t h e h a p t e n i c a c t i v i t y by67.5%, and removal o f t h e next amino a c i d , l e u c i n e , caused e s s e n t i a l l y 100% l o s s o f h a p t e n i c a c t i v i t y . The T-11 -3, when mixed w i t h a n t i s e r u m , became i n s o l u b l e i m m e d i a t e l y , which a c c o u n t e d f o r t h e h i g h e r r e a d i n g s o f p r e c i p i t a t e a f t e r c o m p l e t i o n of. t h e t e s t . T h i s was p o s s i b l y caused by l o s s o f s o l u b i l i t y o f t h e p e p t i d e a f t e r t h e removal o f the 3 N - t e r m i n a l amino a c i d s , o r by the r i g o r o u s m a n i p u l a t i o n s i n v o l v e d i n the t e c h n i q u e . Because t h e s e r e s u l t s i n d i c a t e d t h e importance o f t h e N-t e r m i n a l r e g i o n -of T-11'. .'.irv i t s h a p t e n i c a c t i v i t y , the complete e x p e r i m e n t was r e p e a t e d and the r e s u l t s were c o m p l e t e l y c o m p a r a b l e , even t o the l o s s o f s o l u b i 1 i t y o f the T-11-3. However, because Edman's d e g r a d a t i o n i n v o l v e s f a i r l y r i g o r o u s t r e a t m e n t o f b i o l o g i c a l l y a c t i v e m a t e r i a l s , the p o s s i b i 1 i t y was c o n s i d e r e d t h a t t h e l o s s o f h a p t e n i c a c t i v i t y was due t o m a n i p u l a t i o n o f the p e p t i d e r a t h e r than t o the removal o f t h e N - t e r m i n a l . a m i no a c i d s . To t e s t t h i s p o s s i b i 1 i t y , a n o t h e r s e r i e s o f e x p e r i m e n t s was c a r r i e d o ut t o a s c e r t a i n whether o r not t h e s e o b s e r v a t i o n s were v a l i d . These i n v o l v e d a second method o f s e q u e n t i a l d e g r a d a t i o n o f p e p t i d e s from t h e N - t e r m i n a l , namely e n z y m a t i c c l e a v a g e w i t h l e u c i n e amino p e p t i d a s e . IV. L e u c i n e Amino P e p t i d a s e Experimerits L e u c i n e amino p e p t i d a s e i s an e x o p e p t i d a s e o b t a i n e d from swine k i d n e y , which removes ami no a c i d s s e q u e n t i a l l y from t h e N-terminus o f a p r o t e i n o r p e p t i d e . T h i s e x o p e p t i d a s e i s stopp e d i n i t s c l e a v a g e r e a c t i o n when p r o l i n e i s a t t h e N - t e r m i n u s , and i s s l o w when a s p a r t i c a c i d i s i n t h i s p o s i t i o n . L e u c i n e i s removed f a s t e r than a l l o t h e r amino a c i d s as t h e name i m p l i e s . The commercial p r e p a r a t i o n o f t h i s enzyme has been t r e a t e d w i t h DFP t o i n h i b i t s e r i n e p r o t e a s e a c t i v i t y such as t r y p s i n and c h y m o t r y p s i n . The enzyme p r e p a r a t i o n must be a c t i v a t e d i n the p r e s e n c e o f 0.002 M M g C l 2 a t pH 8.5 a t 40 C f o r 2 t o 3 h r b e f o r e use. A f t e r a c t i v a t i o n and a s s a y o f the enzyme p r e p a r a t i o n , the e x p e r i m e n t was s e t up as d e s c r i b e d i n t h e methods. Amino a c i d a n a l y s i s d a t a on t h e samples t a k e n a t t h e v a r i o u s t i m e s from t h e LAP d i g e s t a re p r e s e n t e d i n T a b l e V I . C a l c u l a t i o n o f the p e r c e n t a g e c l e a v a g e a t t h e v a r i o u s t i m e s was d e t e r m i n e d by comparing t h e m i c r o m o l a r amounts o f a s i n g l e f r e e amino a c i d r e l e a s e d w i t h the m i c r o m o l a r amount o f t h e p e p t i d e p r e s e n t i n t h e sample, which would r e p r e s e n t the m i c r o m o l a r v a l u e f o r 100% c l e a v a g e . The r e s u l t s o f t h e hapten i n h i b i t i o n t e s t s by p r e c i p i t a t i o n a r e g i v e n i n T a b l e V I I . In o r d e r t o c o n s e r v e a n t i s e r u m , o n l y t h r e e t i m e s ( 8 , 16 and 48 h r ) were t e s t e d w i t h the b o i l e d LAP -T-11 c o n t r o l , and ( 8 , 24 and 48 h r ) w i t h the enzyme c o n t r o l . T a b l e V I . Amino a c i d a n a l y s e s d a t a o f l e u c i n e amino p e p t i d a s e d i g e s t i o n o f T-11 p e p t i d e showing t h e amino a c i d s removed. Amino a c i d 48 hr % % % % % % b o i l e d LAP + 8 hr c l e a v a g e 16 hr c l e a v a g e 24 hr c l e a v a g e 32 hr c l e a v a g e 40 hr c l e a v a g e 48 hr c l e a v a g e T-11 c o n t r o l S -CM-cysteine 0.0124 16 .8 0.028 37. 8 0.047 63 .5 0.052 70 .3 0.094 75 . 8 0.302 202.7 0.0 A s p a r a g i ne 0.016 22.0 0.028 37. 8 0.043 58 .1 0.054 73 .0 0.165 133. 0 0 .360 241 . 6 0.0 Threon i ne 0.0 0.0 0.0 0. 0 0 .0 0 .0 0 .0 0 .0 0 .0 0. 0 0 .0 0.0 0.0 P r o l i ne 0.0 0.0 0.0 0. 0 0.0 0 .0 0 .0 0 .0 0.0 0. 0 0 .032 21 .5 0.0 1 s o l e u c i ne 0.0 0.0 0.0 0. 0 0 .0 0 .0 0 .0 0 .0 0.0 0. 0 0.051 34.2 0.0 L e u c i n e 0.022 24 . 3 0.037 44. 6 0.057 71 .6 0.058 73 .0 0.102 79. 0 0.163 109.4 0.004 P e r c e n t c l e a v a g e ( 0.016 ( 0.074 x 100 = 22J Sequence o f N - t e r m i n a l r e g i o n o f T-11 showing amino a c i d s removed by the enzyme: Nhy-Asn - Leu - CMC • j 8-32 hr • Asn «. - M e - Pro - CMC -' — t r a c e s \ / 40 hr V , 48 hr • Lys-COOH 00 T a b l e V I I . Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by l e u c i n e amino p e p t i d a s e d i g e s t e d T-11. Sampling times (hr) A. L e u c i n e amino p e p t i d a s e d i g e s t e d T-11 (60 yg = 0.024 ymoles) 8 °- D'2800 A 0 .175 P e r c e n t i n h i b i t i o n 37.7 16 0.165 41.3 24 0.247 12.1 32 0.275 2.0 40 0.271 3.8 48 0.280 0.0 B. B o i l e d l e u c i n e amino p e p t i d a s e + T-11 ( p o s i t i v e hapten c o n t r o l , 60 yg = 0.024 ymoles) 8 16 0.147 0.162 47.7 42.2 48 0.150 46.5 C. Enzyme (LAP) c o n t r o l (no i n h i b i t i o n , maximum p r e c i p i t a t i o n ) 8 0.276 1.7 24 0.278 1.0 48 0.281 0.0 D. P o s i t i v e hapten (T-11) c o n t r o l (60 yg = 0.024 ymoles) 0 0.146 48.0 E. N e g a t i v e c o n t r o l (no i n h i b i t i o n , maximum p r e c i p i t a t i o n ) 0 0.281 0.0 R e p r e s e n t s the mean v a l u e s o f the t r i p l i c a t e samples. These i n h i b i t i o n t e s t s c o n f i r m e d t h e r e s u l t s o f Edman d e g r a d a t i o n , i n d e m o n s t r a t i n g the importance o f the N - t e r m i n a l r e g i o n o f T-11 i n i t s h a p t e n i c a c t i v i t y . The a l i q u o t s t e s t e d f o r hapten a c t i v i t y i n t r i p l i c a t e were e s t i m a t e d t o c o n t a i n 60 ug o f the o r i g i n a l T-11 p r e p a r a t i o n , w h i c h s h o u l d y i e l d maximum i n h i b i t i o n i f enzyme c l e a v a g e a t the N - t e r m i n a l had no e f f e c t . The b o i l e d LAP - T-11 c o n t r o l a c t e d as a p o s i t i v e hapten c o n t r o l , but t o r u l e out t h e p o s s i b i l i t y o f a n o n s p e c i f i c e f f e c t on p r e c i p i t a t i o n by b o i l e d enzyme, a T - 1 1 . c o n t r o l a t the same m i c r o m o l a r c o n c e n t r a t i o n was s e t up. The n e g a t i v e c o n t r o l s were r e p r e s e n t e d by the enzyme c o n t r o l s a mples, and t o d i s c o u n t n o n s p e c i f i c enzyme e f f e c t , a n e g a t i v e c o n t r o l was s e t up u s i n g the same b u f f e r as i n the d i g e s t . T h i s l a t t e r n e g a t i v e c o n t r o l gave the maximum p r e c i p i t a t i o n l e v e l w h i c h was used as t h e 100% f i g u r e . The r e s u l t s show t h a t a t 8 and 16 h r , v i r t u a l l y 100% o f the h a p t e n i c a c t i v i t y remained. T h i s c o r r e l a t e d w i t h the amino a c i d a n a l y s i s d a t a , which i n d i c a t e d t h a t even a t 16 h r , about 60% o f t h e i n t a c t p e p t i d e was s t i l l p r e s e n t . T h i s lowered the amount o f hapten i n t h e s e samples t o 36 ug w h i c h s t i l l f e l l w i t h i n the range o f maximum h a p t e n i c a c t i v i t y (25 ug o r g r e a t e r ) . The b o i l e d LAP - T-11 c o n t r o l s a t ^ c o m p a r a b l e times showed e s s e n t i a l l y maximum i n h i b i t i o n . A t 2k h r , t h e r e was o n l y s l i g h t i n h i b i t i o n , and none was o b s e r v e d from 32 hr on. At 2h h r , t h e r e was 58% c l e a v a g e o f the N - t e r m i n u s , and the amount i n c r e a s e d u n t i l a t hQ h r , e s s e n t i a l l y 100% o f the p e p t i d e had been degraded w i t h t h e t o t a l r e l e a s e o f a t l e a s t the f i r s t t h r e e N - t e r m i n a l amino a c i d s . A t 48 h r , t h e b o i l e d LAP - T-11 c o n t r o l s t i l l e x h i b i t e d maximum i n h i b i t i o n , as d i d the p o s i t i v e c o n t r o l o f T-11 a l o n e . V. P u r i f i c a t i o n o f S p e c i f i c A n t i b o d i e s In o r d e r t o o b t a i n h i g h l y s p e c i f i c a n t i b o d y f o r the CM-lysozyme m o l e c u l e , the p o o l e d a n t i s e r u m 1 and 2 was p u r i f i e d by two d i f f e r e n t t e c h n i q u e s . Once p u r i f i e d s p e c i f i c a n t i b o d i e s have been o b t a i n e d , the d e t e c t i o n o f the s p e c i f i c immune r e a c t i o n i s made more s e n s i t i v e . T h i s was e s p e c i a l l y i m p o r t a n t i n the CM-lysozyme system, s i n c e t h e a n t i b o d y t i t e r s o f the whole serum were low. A s i d e from t h i s , i m m u n o s p e c i f i c p u r i f i c a t i o n o f a n t i -b o d i e s i s an e s s e n t i a l s t e p i n u n d e r s t a n d i n g immunological phenomena a t t h e m o l e c u l a r l e v e l . In t h e f i r s t method (Fuj i o e_t a j _ . , 1 968a ) , t h e s p e c i f i c a n t i -b o d i e s i n t h e p o o l e d a n t i s e r u m were p r e c i p i t a t e d by the homologous a n t i g e n a t o p t i m a l p r o p o r t i o n s . The p r e c i p i t a t e thus formed was c e n t r i f u g e d and washed w e l l w i t h PBS b u f f e r . The a n t i g e n - a n t i b o d y complex was d i s s o c i a t e d under a c i d c o n d i t i o n s , and the d i s s o c i a t e d m i x t u r e was passed through an a s c e n d i n g Sephadex G-200 column. The major a n t i b o d y component has a M.W. o f about 150 ,000 , and thus i s s l i g h t l y r e t a r d e d under t h e s e c o n d i t i o n s . However, any 19 S (igM) immunog1obins would come out i n the v o i d volume. In our p r e p a r a t i o n s , no 19 S a n t i b o d y was d e t e c t e d . T h i s was e x p e c t e d , s i n c e the i m m u n i z a t i o n s c h e d u l e used here s e l e c t e d f o r l a t e r e s p o n s e or IgG (7S) a n t i b o d y . IgM a n t i b o d y may be o b t a i n e d w i t h v e r y low a n t i g e n doses and t h e serum s h o u l d be c o l l e c t e d w i t h i n two weeks o f a n t i g e n i c s t i m u l u s . In t h e p r e s e n t i n s t a n c e , c o l l e c t i o n o f a n t i s e r a was not s t a r t e d u n t i l f o u r weeks a f t e r i m m u n i z a t i o n , and a l a r g e a n t i g e n i c s t i m u l u s was g i v e n . The CM-lysozyme a n t i g e n , r e p r e s e n t e d by t h e t h i r d peak i n f i g u r e 9 , was e x t r e m e l y r e t a r d e d on the Sephadex G-200 column. In f a c t i t was more r e t a r d e d than e x p e c t e d , p o s s i b l y due t o the h i g h number o f a r o m a t i c and b a s i c amino a c i d r e s i d u e s i t c o n t a i n s , which a r e known t o adsorb w e a k l y t o Sephadex under some c o n d i t i o n s . The IgG thus c o l l e c t e d was d i a l y z e d e x h a u s t i v e l y a g a i n s t PBS and then c o n c e n t r a t e d t o a f i n a l c o n c e n t r a t i o n o f 1.10 mg p r o t e i n / m l . N o n s p e c i f i c r a b b i t y - g l o b u l i n was a l s o p u r i f i e d from n o n s p e c i f serum by p r e c i p i t a t i o n w i t h (NH^^SO^ and subsequent chromatography on D E A E - c e l 1 u l o s e (Humphrey and P o r t e r , 1957 ) - T h i s was used as c o n t r o l n o n s p e c i f i c y - g l o b u l i n i n e q u i l i b r i u m d i a l y s i s e x p e r i m e n t s . The y - g ' o b u l i n p r e p a r e d i n t h i s way was l y o p h i l i z e d and s t o r e d as a d r y powder u n t i l i t was r e q u i r e d . I t was then d i s s o l v e d i n PBS and d i a l y z e d e x h a u s t i v e l y a g a i n s t t h i s b u f f e r a t k C i m m e d i a t e l y b e f o r e use. The second method used f o r p u r i f i c a t i o n o f a n t i b o d i e s i n v o l v e d the use o f a s p e c i f i c immunoabsorbent. Ther e a r e s e v e r a l c r i t e r i a f o r an i d e a l immunoadsorbent: ( i ) i t s h o u l d be h i g h l y s o l u b l e ; ( i i ) i t s h o u l d b i n d a l l the s p e c i f i c a n t i b o d i e s under c o n d i t i o n s a t w hich i t w i l l not r e t a i n o t h e r p r o t e i n s ; ( i i i ) i t s h o u l d r e l e a s e the a d s o r b e d a n t i b o d i e s q u a n t i t a t i v e l y w i t h o u t s i g n i f i c a n t change i n t h e i r a c t i v i t y ; ( i v ) i t s h o u l d be s t a b l e w i t h r e s p e c t t o t i m e . The s y n t h e s i s o f b r o m o a c e t y l c e l l u l o s e (BAC) met t h e above r e q u i r e m e n t s and p e r m i t t e d t h e p r e p a r a t i o n o f h i g h l y s u b s t i t u t e d p r o t e i n - c e l l u l o s e c o n j u g a t e s w h i c h r e t a i n e d much o f the b i o l o g i c a l a c t i v i t y o f t h e p r o t e i n . T h i s method can be used t o p u r i f y a n t i -h a p ten a n t i b o d i e s which cannot be o b t a i n e d by the f i r s t method g i v e n . A l s o , t h e r e a r e s e v e r a l examples o f a n t i b o d i e s t h a t do not form i n s o l u b l e complexes a f t e r they i n t e r a c t w i t h a n t i g e n , and t h e r e f o r e c o u l d not be p u r i f i e d by t h e immune p r e c i p i t a t i o n method. However, i n t h e case o f the an t i - C M - l y s o z y m e serum, whole a n t i g e n was c o n j u g a t e d w i t h t h e b r o m o a c e t y l c e l l u l o s e immunoadsorbent. The p r e p a r a t i o n o f b r o m o a c e t y l c e l l u l o s e f o l l o w e d t h e m o d i f i e d method g i v e n by Robb i ns e t _ a j _ . (1967). The r e a c t i o n i n v o l v e d m i x i n g b r o m o a c e t i c a c i d and powdered Whatman c e l l u l o s e i n a b s o l u t e d i o x a n e f o r 20 h r , and the n a d d i n g b r o m o a c e t y l bromide, ' a n d - a l l o w i n g ' t h e r e a c t i o n t o c o n t i n u e f o r 10 t o 12 h r . The Whatman c e l l u l o s e had been e x h a u s t i v e l y washed w i t h acetone-and d i o x a n e , and d r i e d t o a c o n s t a n t w e i g h t b e f o r e b e i n g used. The b r o m o a c e t y l c e l l u l o s e (BACr c e l l u l o s e ) was then p r e c i p i t a t e d by a d d i n g the r e a c t i o n m i x t u r e t o a l a r g e e x c e s s o f d e i o n i z e d w a t e r which had p r e v i o u s l y been c o o l e d t o A C . The B A C - c e l l u l o s e was then washed w i t h d e i o n i z e d w a t e r and 0.1 M NaHCO^ o v e r a s i n t e r e d g l a s s f u n n e l t o g e t r i d o f e x c e s s u n r e a c t e d r e a g e n t s . I t was found t h a t the B A C - c e l l u l o s e changed i n p h y s i c a l p r o p e r t i e s i f i t was d r i e d out c o m p l e t e l y , and the b i n d i n g c a p a c i t y w i t h a n t i g e n s was redu c e d , so t h e p r e p a r e d immunoadsorbent was s t o r e d i n a m o i s t s t a t e a t 4 C. The average degree o f s u b s t i t u t i o n o b t a i n e d by t h i s p r o c e d u r e was 1.3 meq Br/g c e l l u l o s e ( t h e range was 1.0 - 1.4) and t h e B A C - c e l 1 u l o s e was s t a b l e f o r up t o t h r e e months. The amount o f a n t i b o d y bound t o BAC-prote i n c o n j u g a t e s was found t o be low when compared w i t h r e p o r t s by Robbins e t a l . (1967) on s e v e r a l d i f f e r e n t a n t i g e n s t e s t e d i n wh i c h r e c o v e r y o f , s p e c i f i c a n t i b o d y ranged between 20 and 40% as opposed t o 80 - 90% r e p o r t e d by Robbins e t a l . The a n t i b o d y adsorbed t o the immunoadsorbent was e l u t e d w i t h 0.1 N a c e t i c a c i d (pH 2 .8 ) f o r 1 h r a t 37 C, and then t h e c e l l u l o s e -a n t i g e n c o n j u g a t e was c e n t r i f u g e d o u t , and t h e s u p e r n a t a n t f l u i d c o n t a i n i n g t h e a n t i b o d y was d i a l y z e d e x h a u s t i v e l y a g a i n s t 0.1 M NaCl - 0.01 M T r i s HCl a t pH 7 . 0 . The p u r i f i e d s t o c k a n t i b o d y 1 p r e p a r a t i o n was s t o r e d a t 4 C a f t e r m e r t h i o l a t e : t o a f i n a l c o n c e n t r a t i o n o f 1 :10,000 was added. The complement f i x a t i o n t i t e r o f t h e p u r i f i e d a n t i b o d y p r e p a r a t i o n was 1:300 when the p r o t e i n c o n c e n t r a t i o n was 0 .8 mg/ml whi c h r e p r e s e n t e d o n l y 40% r e c o v e r y but a marked i n c r e a s e i n s p e c i f i c a c t i v i t y when compared w i t h whole serum which had a t i t e r o f 1 : 8 0 and c o n t a i n e d about 30 mg p r o t e i n / m l . V I . S o l i d Phase P e p t i d e S y n t h e s i s 1. S y n t h e s i s S i n c e the r e s u l t s from s e q u e n t i a l d e g r a d a t i o n of the T-11 p e p t i d e i n d i c a t e d the i m p o r t a n c e o f the N - t e r m i n a l p o r t i o n i n i t s h a p t e n i c a c t i v i t y , s o l i d phase p e p t i d e s y n t h e s i s was chosen t o c o n f i r m t h e s e r e s u l t s and p o s s i b l y p i n p o i n t t h e a n t i g e n i c r e g i o n . The u l t i m a t e t e s t f o r i d e n t i f i c a t i o n o f an a n t i g e n i c d e t e r m i n a n t i s t h e s y n t h e s i s o f t h e amino a c i d sequence thought t o be h a p t e n i c and show t h e d e m o n s t r a t i o n o f i t s a b i l i t y t o r e a c t w i t h a n t i b o d i e s d i r e c t e d a g a i n s t the n a t u r a l a n t i g e n . The M e r r i f i e l d method f o r p e p t i d e s y n t h e s i s was a s i g n i f i c a n t advance i n p e p t i d e s y n t h e s i s , because i t a l l o w e d the p e p t i d e b e i n g s y n t h e s i z e d t o be a t t a c h e d t o an i n s o l u b l e s u p p o r t , the c h l o r o -m e t h y l a t e d c o p o l y s t y r e n e - d i v i n y l b e n z e n e r e s i n , and t h e r e f o r e e x c e s s r e a g e n t s and amino a c i d s o f t h e s o l u b l e phase c o u l d be washed out o f t h e r e s i n . T h i s e l i m i n a t e s the n e c e s s i t y f o r e x t e n s i v e p u r i f i c a t i o n p r o c e d u r e s a t i n t e r m e d i a t e s t e p s i n t h e s y n t h e s i s . The r e a c t i v e c h l o r o m e t h y l groups on t h e r e s i n r e a c t w i t h t h e s a l t o f a t - b u t y l o x y c a r b o n y l (t-BOC) amino a c i d t o form an e s t e r l i n k between the c a r b o x y l group o f the amino a c i d and the r e s i n . The growth o f the p e p t i d e o c c u r s from the C - t e r m i n a l amino a c i d toward t h e N - t e r m i n a l . The f r e e amino group o f each incoming amino a c i d i s b l o c k e d by t h e t-BOC l e a v i n g o n l y the c a r b o x y l group f r e e t o r e a c t . Anhydrous HCl i s used t o remove the t-BOC group from the a t t a c h e d amino a c i d so t h a t t h e amino group i s f r e e t o r e a c t w i t h the incoming p r o t e c t e d amino a c i d . For t h e c o u p l i n g r e a c t i o n , which i n v o l v e s the f o r m a t i o n of a p e p t i d e bond, d i c y c l o h e x y l c a r b o d i i m i d e (DCC) and a n e u t r a l pH a r e r e q u i r e d i n most i n s t a n c e s , but a c t i v e e s t e r s o f incoming amino a c i d s may be used w i t h no c o u p l i n g r e a g e n t . The t-BOC r e s i d u e s o f Asn and G i n must be added as a c t i v e p - n i t r o -phenyl e s t e r s s i n c e the DCC may d e h y d r a t e t h e w-amide f u n c t i o n s o f Asn and G i n t o n i t r i l e s d u r i n g the c o u p l i n g r e a c t i o n , r e s u l t i n g i n t h e permanent i n c o r p o r a t i o n of a number o f t h e s e w-cyano d e r i v a t i v e s i n t o the p e p t i d e c h a i n . A f t e r c o m p l e t i o n o f the s y n t h e t i c c y c l e s , HBr gas i s used t o c l e a v e t h e p e p t i d e from t h e s o l i d r e s i n s u p p o r t . Anhydrous t r i -f l u o r o a c e t i c a c i d i s used f o r s u s p e n s i o n o f the p e p t i d e - r e s i n f o r c l e a v a g e . The t e r m i n a l t-BOC group s t i l l p r e s e n t a t t h i s t i m e i s removed by an e l i m i n a t i o n r e a c t i o n , as w e l l as the c l e a v a g e o f the p e p t i d e from the r e s i n by a n u c l e o p h i l i c d i s p l a c e m e n t r e a c t i o n . The s i d e c h a i n h y d r o x y l s o f s e r i n e and t h r e o n i n e and the s u l f h y d r y l o f c y s t e i n e must be p r o t e c t e d w i t h b e n z y l groups d u r i n g the s y n t h e t i c c y c l e s . These must be removed from the p e p t i d e a f t e r c l e a v a g e from t h e r e s i n b e f o r e i t can be t e s t e d f o r b i o l o g i c a l a c t i v i t y . The b e n z y l group on s e r i n e and t h r e o n i n e h y d r o x y l s a r e removed by the HBr c l e a v a g e s t e p , but the b e n z y l group on the s u l f h y d r y l o f c y s t e i n e r e q u i r e s a more r i g o r o u s r e d u c t i o n i n v o l v i n g Na i n l i q u i d ammonia. When c y s t e i n e , methionine or tyros ine are present in s y n t h e t i c p e p t i d e s , e thyl methyl s u l f i d e must be used to protect these reduced side chains from oxidat ion and d e s t r u c t i o n during t h i s s tep . A f t e r a l l the s ide chain protec t ing groups have been removed, the cleaved peptide is p u r i f i e d by a s u i t a b l e chromatographic technique. Table if is a flow chart for a s y n t h e t i c c y c l e . The peptides synthesized in t h i s study, which cons t i tu ted the N-terminal decapeptide of T - 1 1 , and the decapeptide plus Arg at the N-terminal ( this is the next amino a c i d along in the sequence of lysozyme), required the sodium in l i q u i d ammonia r e d u c t i o n , s ince they contained 2 cysteine res idues . These synthet peptides required the ac t ive es ter coupling react ion for the 2 Asn r e s i d u e s . The sequence of the two s o l i d phase peptides was: NH 2 - (Arg) - Asn - Leu - Cys - Asn - Ile - Pro - Cys - Ser -A l a - Leu - COOH. 2. Reduction and a l k y l a t i o n Once the benzyl groups had been removed from the cysteine residues of the cleaved pept ide , the s y n t h e t i c peptides were immediately reduced and carboxymethylated to prevent d i s u l f i d e bond formation between the two cyste ine residues . The reduction step with sodium in l i q u i d ammonia a l s o converts the n i t r o - a r g i n i n e residue to a r g i n i n e . Although the cysteine residues should be in the reduced s u l f h y d r y l f o r m , 2-mercaptoethanol was added p r i o r t o the a l k y l a t i o n t o e n s u r e t h a t they were r e d u c e d . For the a l k y l a t i o n , ( ^ C ) - i o d o a c e t i c a c i d was used, so t h a t l a t e r t e s t i n g f o r the b i n d i n g o f p e p t i d e s w i t h s p e c i f i c a n t i b o d i e s c o u l d be d e t e c t e d by the r a d i o a c t i v e l a b e l l i n g . However, i t was l a t e r found t h a t the degree o f l a b e l l i n g i n cpm/ymole was f a r t o o low f o r b i n d i n g s t u d i e s . The l a r g e e x c e s s o f i o d o a c e t i c a c i d r e q u i r e d f o r a l k y l a t i o n made i t i m p r a c t i c a l t o use o n l y r a d i o a c t i v e i o d o a c e t i c a c i d . T h e r e f o r e , a s m a l l amount o f ( ^ C ) - i o d o a c e t i c a c i d was added t o r e a c t w i t h the c y s t e i n e r e s i d u e s i n the p e p t i d e s f o r 20 min, f o l l o w e d by a l a r g e amount of c o l d i o d o a c e t i c a c i d , and t h e r e a c t i o n was c o n t i n u e d f o r a n o t h e r 5 min. A f t e r t h e e x c e s s i o d o a c e t i c a c i d had been reduced by a d d i t i o n of 2 - m e r c a p t o e t h a n o l , the reduced and a l k y l a t e d p e p t i d e s were d r i e d by f l a s h e v a p o r a t i o n a t k5 C, washed w i t h d i s t i l l e d w a t e r , and suspended i n 10.0 ml w a t e r . The pH o f the p e p t i d e m i x t u r e s was a d j u s t e d t o 2.8 w i t h f o r m i c a c i d . An amino a c i d a n a l y s i s was done a t t h i s t i m e on an a l i q u o t o f the p e p t i d e p r e p a r a t i o n s ( T a b l e s VII I and I X ) . 3. P u r i f i c a t i o n o f reduced and c a r b o x y m e t h y l a t e d p e p t i d e s The p u r i f i c a t i o n p r o c e d u r e used f o r t h e reduced and a l k y l a t e d p e p t i d e s was s i m i l a r t o t h a t used f o r whole T-11 p e p t i d e , namely Dowex 50 c a t i o n exchange r e s i n w i t h the m o d i f i e d four-chambered g r a d i e n t system ( d e s c r i b e d i n the methods). Because o f the p r e s e n c e T a b l e V I M . Amino a c i d a n a l y s e s o f NH -Asn s y n t h e t i c d e c a p e p t i d e from t h e N - t e r m i n a l r e g i o n o f T-11. A. A f t e r c l e a v a g e B. A f t e r Dowex 50 C. A f t e r Sephadex G-15 from r e s i n p u r i f i c a t i o n p u r i f i c a t i o n D. Expected Amino a c i d ymoles R e s i d u e s ymoles R e s i d u e s ymoles R e s i d u e s r e s i d u e s S -CM-cysteine 0.075 0.45 0.160 1.7 0.140 2.2 2 A s p a r t i c a c i d 0 .512 3-0 0.250 2.7 0.146 2.2 2 S e r i n e 0 .194 1.2 0.084 0.90 0.081 1.2 1 P r o l i n e 0.167 1.0 0.093 1.0 O.O65 1.0 1 A l a n i n e 0.376 2.3 0.155 1.7 O.O83 1.3 1 I s o l e u c i n e 0.161 O.96 O.O87 0 .94 0.071 1.1. 1 L e u c i n e 0.335 2.0 0.187 2.0 0.143 2.2 2 Expected sequence o f c o r r e c t s y n t h e t i c d e c a p e p t i d e : . NH- - Asn - Leu - CMC - Asn - M e - Pro - CMC - Ser - A l a - Leu p o s i t i o n in 2 CM-lysozyme: 74 — - - 83 T a b l e IX. Amino a c i d a n a l y s e s o f NH^-Arg s y n t h e t i c p e p t i d e c o m p r i s i n g a r g i n i n e p l u s t h e N - t e r m i n a l r e g i o n o f T-11. Amino a c i d A. A f t e r from ymoles c l e a v a g e res i n Res idues B. A f t e r Dowex 50 p u r i f i c a t i o n ymoles R e s i d u e s C. A f t e r Sephadex G-15 p u r i f i c a t i o n ymoles R e s i d u e s D. Expected res i dues S-CM-cystei ne 0.051 2.4 0.037 2.5 0.064 2.3 2 A s p a r t i c a c i d 0.048 2.3 0.036 2.4 0.054 1 .9 2 Ser i ne 0.019 0.90 0.012 oi;8o 0.026 0.93 1 P r o l i ne 0.030 1.4 0.021 1.4 0.034 1.2 1 A l a n i n e 0.021 1.0 0.013 0.87 0.030 1.1 1 1 s o l e u c i ne 0.022 1.0 0.016 1.1 0.031 1.1 1 Leuc i ne 0.048 2.3 0.033 2.2 0.051 1.8 2 A r g i n i ne 0.027 1 .3 0.018 1 .2 0.032 1.2 1 Expected sequence o f above c o r r e c t 11 amino a c i d s y n t h e t i c p e p t i d e r e p r e s e n t i n g N - t e r m i n a l d e c a p e p t i d e o f T-11 p l u s a r g i n i n e on N- t e r m i n u s : p o s i t i o n i n CM-lysozyme: NH 2 - Ar g - Asn - Leu - CMC - Asn - M e - Pro - CMC - Ser - A l a - Leu 73 8 3 o f t h e C}-carboxymethy 1 group on some o f t h e c y s t e i n e r e s i d u e s , t h e s y n t h e t i c p e p t i d e s were d e t e c t e d by s c i n t i l l a t i o n c o u n t i n g r a t h e r than by a l k a l i n e h y d r o l y s i s and q u a n t i t a t i v e n i n h y d r i n r e a c t i o n s on a l i q u o t s o f f r a c t i o n s . The u n r e a c t e d i o d o a c e t i c a c i d e l u t e d i n t h e v o i d volume o f t h e column, and t h e second major r a d i o a c t i v e peak, coming out w e l l s e p a r a t e d from t h e u n r e a c t e d i o d o a c e t i c a c i d , was shown t o c o n t a i n p e p t i d e m a t e r i a l w h i c h , a f t e r amino a c i d a n a l y s i s ( T a b l e s V I I I and IX) was shown t o c o n t a i n t h e d e s i r e d s y n t h e t i c p e p t i d e . F i g u r e 14 r e p r e s e n t s the e l u t i o n p r o f i l e s o f t h e two s y n t h e t i c p e p t i d e s . The s y n t h e t i c p e p t i d e s were e l u t e d from two s e p a r a t e Dowex columns but t h e r e s u l t s a r e graphed t o g e t h e r . The m a t e r i a l from t h i s peak was p o o l e d and d r i e d o v e r a NaOH t r a p i n case any u n r e a c t e d i o d o a c e t i c a c i d was p r e s e n t . The e v a p o r a t e d and washed p e p t i d e m a t e r i a l was the n t a k e n up i n a known volume o f d i s t i l l e d w a t e r , and a sample was t a k e n f o r h y d r o l y s i s and amino a c i d a n a l y s i s . In o r d e r t o p u r i f y f u r t h e r t h e p e p t i d e s , and f r e e them from low m o l e c u l a r w e i g h t c o n t a m i n a n t s which were sometimes p r e s e n t , the m a t e r i a l from t h e Dowex column s e p a r a t i o n was pa s s e d through a Sephadex G-^ 15 column e q u i l i b r a t e d w i t h 0.1 N a c e t i c a c i d . A f t e r r e a d i n g t h e f r a c t i o n s f o r 2300 AfV a b s o r b i n g m a t e r i a l ( t h e p e p t i d e s i n t h e s e i n s t a n c e s have m o l e c u l a r w e i g h t s o f about 1300 and o n l y s l i g h t l y r e t a r d e d on G-15), the e l u t e d p e p t i d e was c o n c e n t r a t e d by f l a s h e v a p o r a t i o n , washed and r e d i s s o l v e d . i n d i s t i l l e d w a t e r . Then a sample was t a k e n , h y d r o l y z e d , and a n o t h e r amino a c i d a n a l y s i s was 101 180 240 ML ELUENT 300 F i g u r e ^k. E l u t i o n p r o f i l e o f s y n t h e t i c d e c a p e p t i d e ( o o ) a n d s y n t h e t i c d e c a p e p t i d e p l u s a r g i n i n e ( © - © ) . T h e f i r s t l a r g e p e a k r e p r e s e n t s t h e u n r e a c t e d C \ ) - i o d o a c e t i c a c i d. / T a b l e X. Hapten i n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n by v a r y i n g c o n c e n t r a t i o n s o f the f o l l o w i n g p e p t i d e s . P e r c e n t I n h i b i t i o n 1 Hapten ( 1 V T - 1 1 A r g N - t e r m i n a l s y n t h e t i c p e p t i d e Asn N - t e r m i n a l s y n t h e t i c p e p t i d e 5 0 . 0 51 . 9 4 0 . 6 42.1 60.1 25-0 4 6 . 5 34 .5 31 .4 59-4 12 .5 37 .2 28 .6 26 .4 56.1 6 .25 3 0 . 7 3 0 . 7 19 . 3 5 8 . 6 3 .0 27 .5 12.1 19 . 3 47.1 1.5 23 . 6 5 .0 10.7 22 .0 0 .4 15-0 0 0 3 .6 103 1 0 . 0 1 . 0 0 . 1 M g HAPTEN F i g u r e 15. I n h i b i t i o n o f s p e c i f i c immune p r e c i p i t a t i o n between CM-lysozyme and i t s homologous a n t i s e r u m by: ( 1 4C>T-11; , NH 2-Arg s y n t h e t i c p e p t i d e ; o-o, T-11; A-A , N r L - A s n s y n t h e t i c p e p t i d e . c a r r i e d out ( T a b l e V I I I and I X ) . There was i n v a r i a b l y some improvement i n t h e amino a c i d r a t i o s . High 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 c o n f i r m e d t h a t i n both c a s e s , o n l y one n i n h y d r i n p o s i t i v e component was p r e s e n t a f t e r p u r i f i c a t i o n o f t h e p e p t i d e s . The h a p t e n i c a c t i v i t y o f the s y n t h e s i z e d p e p t i d e s was t e s t e d by t h e p r e c i p i t a t i o n i n h i b i t i o n t e c h n i q u e , u s i n g serum 3 as p r e v i o u s l y d e s c r i b e d . The p e p t i d e s were t e s t e d o v e r a range between 50 and 0.4 „tyg s i m u l t a n e o u s l y w i t h T-11 and (N-< C ) - a c e t y l a t e d T-11 (see the f o l l o w i n g s e c t i o n ) . The r e s u l t s a r e p r e s e n t e d i n T a b l e X and i n F i g u r e 15. T e s t s were run s i m u l t a n e o u s l y w i t h t h e v a r i o u s p e p t i d e s u s i n g n a t i v e lysozyme and i t s homologous a n t i s e r u m . No n o n - s p e c i f i c p r e c i p i t i n i n h i b i t i o n was n o t e d i n t h e s e i n s t a n c e s , so i t was assumed t h a t t h e i n h i b i t i o n o b s e r v e d w i t h the CM-lysozyme system was t h e r e s u l t o f s p e c i f i c h a p t e n i c a c t i v i t y . I t can be seen from t h e s e d a t a t h a t both s y n t h e t i c p e p t i d e s cause i n h i b i t i o n o f p r e c i p i t a t i o n , a l t h o u g h on a q u a n t i t a t i v e b a s i s , they do not a c t as e f f i c i e n t l y as the T-11 p e p t i d e . V I I . ^1 ^ Q-Acety 1 a t i on o f Pept i des Because t h e l a b e l l i n g o f t h e s y n t h e t i c p e p t i d e s w i t h r a d i o -a c t i v e i o d o a c e t i c a c i d d i d not g i v e h i g h enough s p e c i f i c a c t i v i t y f o r u s i n g t h e s e p r e p a r a t i o n s i n e q u i l i b r i u m d i a l y s i s s t u d i e s , (1 4 A a n o t h e r r a d i o a c t i v e l a b e l , Q - a c e t i c a n h y d r i d e , was used. T h i s 105 l a b e l adds an a d d i t i o n a l C ) a c e t y l group o n t o t h e N - t e r m i n a l r e s i d u e o f t h e p e p t i d e , and a l s o some secondary groups such as the e-amino o f l y s i n e . However, under the c o n d i t i o n s chosen, and c o n s i d e r i n g t h e amino a c i d sequence o f t h e p e p t i d e s a c e t y l a t e d , (l4 the N - t e r m i n a l a c e t y l a t i o n r e a c t i o n i s h i g h l y f a v o u r e d . The Q-a c e t i c a n h y d r i d e was d i s s o l v e d i n benzene as s t a t e d i n t h e methods, and then l a y e r e d o n t o t h e p a r t i c u l a r p e p t i d e m a t e r i a l i n 1.0 M sodium a c e t a t e a d j u s t e d t o pH 8.0 which i s t h e optimum pH f o r N - t e r m i n a l a c e t y l a t i o n . F o l l o w i n g a c e t y l a t i o n , t h e p e p t i d e s were s e p a r a t e d from unbound (14 Q - a c e t a t e by p a s s i n g the aqueous phase t h r o u g h a Sephadex G-15 column e q u i 1 i b r a t e d w i t h 50% a c e t i c a c i d . The e l u t i o n was a l s o c a r r i e d o ut w i t h 50% a c e t i c a c i d u s i n g a v e r y s l o w f l o w r a t e . F i g u r e 16 shows the e l u t i o n p r o f i l e f o r the a c e t y l a t e d s y n t h e t i c p e p t i d e ( t h e d e c a p e p t i d e p l u s Arg) and a l s o f o r a c e t y l a t e d T - 1 1 . The s y n t h e t i c p e p t i d e had a m o l e c u l a r w e i g h t o f about 1300. and was s l i g h t l y r e t a r d e d on Sephadex G-15'. The l a r g e r T-11 p e p t i d e , c o n t a i n i n g 23 amino a c i d r e s i d u e s and h a v i n g a m o l e c u l a r w e i g h t o f about 2700 came out w i t h t h e v o i d and e l u t e d j u s t ahead o f t h e s y n t h e t i c p e p t i d e . The u n r e a c t e d l a b e l l e d a c e t i c a n h y d r i d e , w h i c h under t h e s e c o n d i t i o n s would be c o m p l e t e l y h y d r o l y z e d t o a c e t i c a c i d , came out much l a t e r a f t e r t o t a l exchange i n t h e column w i t h c o l d a c e t i c a c i d had o c c u r r e d a t one column volume. The p e p t i d e peaks were d e t e c t e d by s c i n t i l l a t i o n c o u n t i n g on 0.01 ml samples taken from e v e r y o t h e r f r a c t i o n c o l l e c t e d . The p e p t i d e m a t e r i a l was p o o l e d and f l a s h e v a p o r a t e d t o remove — • \ 105,000 -90,000 75,000 _j 2 • — Z 2 60,000 A JNTS • ° 45,000 K ) 30,000 i i i i 15,000 < * 0 t it . A • <> i 0 k 1 ' 1 1 LJkBL \ > ( 60 120 180 240 M L E L U E N T F i g u r e 16. E l u t i o n p r o f i l e f o r the a c e t y l a t e d N H 2 - A r g s y n t h e t i c p e p t i d e ( © - - - © ) , and a c e t y l a t e d T - 1 1 f o - o ) . The l a s t l a rge peak represen ts the unreac ted 0 Vacet i c a c i d . a c e t i c a c i d , washed w i t h d i s t i l l e d w a t e r and l y o p h i l i z e d . A sample o f whole T-11 p e p t i d e was a c e t y l a t e d t o t e s t i t s h a p t e n i c a c t i v i t y by e q u i l i b r i u m d i a l y s i s and hapten i n h i b i t i o n s i m u l t a n e o u s l y w i t h the s y n t h e t i c a c e t y l a t e d p e p t i d e . The l y o p h i l i z e d p e p t i d e s were t a k e n up i n a known volume o f PBS a t pH 6 .0 ( c o n t a i n i n g 0.02% sodium a z i d e ) and two s m a l l samples from each were t a k e n , one f o r h y d r o l y s i s and amino a c i d a n a l y s i s , and one f o r r a d i o -a c t i v i t y c o u n t i n g , so t h a t q u a n t i t a t i v e c o u n t s per micromole o f p e p t i d e c o u l d be c a l c u l a t e d . These d a t a showed t h a t the s y n t h e t i c p e p t i d e had a s p e c i f i c a c t i v i t y o f 362,426 cpm/ umole, and T-11 had 808,691 cpm/umole. Hapten i n h i b i t i o n t e s t s by t h e p r e c i p i t i n method were s e t up on a c e t y l a t e d T-11 p e p t i d e . The r e s u l t s a r e r e p o r t e d i n T a b l e X and F i g u r e 15> and show t h a t a c e t y l a t i o n does not i n h i b i t the h a p t e n i c a c t i v i t y o f T - 1 1 . . V I I I . Equ i 1ibr?um D i a l y s i s The e q u i l i b r i u m d i a l y s i s method f o r p r e c i s e measurement of h a p t e n - a n t i b o d y i n t e r a c t i o n i s the b e s t method a v a i l a b l e i n immunochemistry because i t y i e l d s a d i r e c t measurement o f the b i n d i n g o f a n t i b o d y w i t h the hapten. The o t h e r methods o f hapten t e s t i n g by p r e c i p i t i n i n h i b i t i o n and complement f i x a t i o n i n h i b i t i o n both measure a n e g a t i v e r e a c t i o n i n w h i c h t h e r e i s more room f o r n o n s p e c i f i c e f f e c t s . The e q u i l i b r i u m d i a l y s i s method i s a l s o a thermodynamical ly sound way to obtain measurements of the association constants for the interaction of hapten with an antibody combining site, since the measurements are made at equilibrium. The following equation expresses the interaction of a low molecular weight hapten with an antibody combining site, and the equilibrium or association constant. H + B . -*• • HB (HB) _ THTW " K The molar quantities of antibody and of bound and free hapten must be determined in order to evaluate K. In setting up the equilibrium dialysis experiments, known amounts of antibody and of hapten are used, and therefore the equilibrium dialysis essentially determines the amount of bound and free hapten. The antibody preparation is placed on one side of a dialysis membrane and hapten on the other, and the apparatus is rocked at constant temperature (4 C) for establishment of equilibrium. The dialysis membrane is such that hapten can pass through but antibody molecules cannot. When equi1ibrium has been established, the free hapten concentration is measured by counting the radioactivity in samples from the hapten side of the dialysis system. At infinite hapten concentration, if the volumes on both sides of the membrane are equal, and also the free hapten concentration on both sides of the membrane are equal at e q u i l i b r i u m , then the amount o f bound hapten e q u a l s t h e t o t a l hapten added minus t w i c e t he f r e e hapten. E x p r e s s e d on a molar b a s i s , i t i s p o s s i b l e t o c a l c u l a t e t he r a t i o o f t h e number o f moles o f hapten bound per mole o f a n t i b o d y a t the c o n c e n t r a t i o n o f f r e e hapten measured. C o n t r o l s u s i n g normal ( n o n s p e c i f i c ) y - g l o b u l i n and b u f f e r must be s e t up a t t h e same time as t h e t e s t p r e p a r a t i o n s c o n t a i n i n g s p e c i f i c a n t i b o d y . When n o n s p e c i f i c Y~9l°bulin i s used, the hapten s h o u l d e q u i l i b r a t e and d i s t r i b u t e e q u a l l y between the two s i d e s . I f any hapten i s bound i t i s due t o a n o n s p e c i f i c r e a c t i o n . The b u f f e r c o n t r o l i s i m p o r t a n t a l s o , s i n c e a s m a l l amount of a d s o r p t i o n o f t h e hapten t o the membrane o c c u r s w h i c h may be measured by the d i f f e r e n c e between the amounts o f hapten added and t h e amount r e c o v e r e d . A g a i n , the hapten s h o u l d d i s t r i b u t e e q u a l l y between th e two s i d e s i n the b u f f e r c o n t r o l . T h i s method o f e q u i l i b r i u m d i a l y s i s was used t o t e s t h a p t e n i c a c t i v i t y o f t h e a c e t y l a t e d 11 r e s i d u e s y n t h e t i c p e p t i d e and the a c e t y l a t e d T-11 p e p t i d e . U n f o r t u n a t e l y , t he l e v e l s o f s p e c i f i c l a b e l l i n g i n the a c e t y l a t e d p e p t i d e s were not p a r t i c u l a r l y good. T h i s , c o u p l e d w i t h t he f a c t t h a t t h e s p e c i f i c a n t i b o d y c o n c e n t r a t i o n i n t he t e s t was low (0.005 ymole/ml o f s p e c i f i c a n t i b o d y t o CM-lysozyme) made t h i s a poor e x p e r i m e n t . However, the f o l l o w i n g p r e p a r a t i o n s were s e t up f o r e q u i l i b r i u m d i a l y s i s . Both the a c e t y l a t e d 11 r e s i d u e s y n t h e t i c p e p t i d e and t h e a c e t y l a t e d T-11 p e p t i d e were t e s t e d a t 0.0075 umoles/ml and 0.0038 ymoles/ml. The a c e t y l a t e d p e p t i d e s were used i n k ml q u a n t i t i e s o f the above c o n c e n t r a t i o n s , and 1 ml o f the a n t i b o d y p r e p a r a t i o n a t 0.005 pmo.les was p l a c e d i n d i a l y s i s t u b i n g and suspended i n the p e p t i d e s o l u t i o n s . C o n t r o l s were s e t up u s i n g the same two c o n c e n t r a t i o n s o f p e p t i d e w i t h n o n s p e c i f i c y - g l o b u l i n and a l s o w i t h PBS. A f t e r e q u i l i b r i u m had been e s t a b l i s h e d i n k d a y s , as shown by e s s e n t i a l l y equal c o u n t s i n s i d e and o u t s i d e d i a l y s i s sac i n PBS c o n t r o l t u b e s , the p e p t i d e s o l u t i o n s , s p e c i f i c y - g l o b u l i n and n o n s p e c i f i c g l o b u l i n tubes were c o u n t e d . The r e s u l t s were i n c o n c l u s i v e , w i t h o n l y 1-3% s p e c i f i c b i n d i n g w i t h a n t i - C M - l y s o z y m e serum which i s w i t h i n the l i m i t s o f v a r i a b i l i t y . Because the amount o f s p e c i f i c r a d i o s a c t i v e l a b e l l i n g was low i n t h e two p e p t i d e p r e p a r a t i o n s , i t was f e l t t h a t p o s s i b l y o n l y 10% o f the p e p t i d e m a t e r i a l was r a d i o a c t i v e l y l a b e l l e d . T h i s would mean t h a t 90% c o l d , u n l a b e l l e d p e p t i d e would compete w i t h the 10% l a b e l l e d p e p t i d e f o r the s p e c i f i c a n t i b o d y m o l e c u l e s . A t the low c o n c e n t r a t i o n o f p e p t i d e used h e r e , the c o u n t s would not be s i g n i f i c a n t t o d e t e c t s p e c i f i c b i n d i n g t o a n t i - C M -lysozyme a n t i b o d i e s . 111 GENERAL DISCUSSION In t h i s t h e s i s , t h e a n t i g e n i c p r o p e r t i e s o f reduced and S - c a r b o x y m e t h y l a t e d lysozyme were s t u d i e d , i n o r d e r t o d e t e r m i n e t h e r e g i o n s i n t h e d e n a t u r e d m o l e c u l e r e s p o n s i b l e f o r a n t i g e n i c s p e c i f i c i t y . The r e s u l t s o b t a i n e d from h a p t e n i c t e s t i n g o f t r y p t i c p e p t i d e s o f CM-lysozyme demonstrated the p a r t i c i p a t i o n o f t h e T-11 p e p t i d e , on t h e b a s i s o f i t s a b i 1 i t y t o i n h i b i t the homologous i m m u n o l o g i c a l r e a c t i o n by p r e c i p i t i n and complement f i x a t i o n i^h-i13l^rqnrV~ As d i s c u s s e d i n t h e p r e v i o u s s e c t i o n , , t h e p o s s i b i 1 i t y t h a t the T-11 p e p t i d e p r e p a r a t i o n s c o n t a i n e d u n d i g e s t e d p r o t e i n o r a c o n t a m i n a t i n g p e p t i d e , was r u l e d o u t . T h i s h a p t e n i c e f f e c t was a l s o shown t o be s p e c i f i c f o r t h e homologous CM-lysozyme - a n t i - C M - l y s o z y m e system s i n c e the p e p t i d e caused no i n h i b i t i o n o f t h e immune r e a c t i o n o f n a t i v e lysozyme and i t s homologous a n t i s e r u m . A l l o t h e r t r y p t i c p e p t i d e s , even when t e s t e d a t h i g h c o n c e n t r a t i o n s l a c k e d h a p t e n i c a c t i v i t y . T h i s does not mean t h a t o t h e r r e g i o n s o f the CM-lysozyme m o l e c u l e a r e not i n v o l v e d i n a n t i g e n i c i t y , but r a t h e r t h a t o t h e r a n t i g e n i c s i t e s may be s p l i t by t r y p s i n . The s p e c i f i c i t y o f t h i s system does not appear t o be r e l a t e d t o . i n d i v i d u a l a n i m a l s , s i n c e the s e r a from tihree d i f f e r e n t r a b b i t s a l l r e a c t e d i dent i c a l l y , . e x c e p t w i t h r e s p e c t t o a n t i b o d y t i t e r . T h i s h a p t e n i c p e p t i d e , T - 1 1 , i s t h e l a r g e s t t r y p t i c p e p t i d e found i n CM-lysozyme. I t c o n t a i n s 23 r e s i d u e s and encompasses t h e sequence from A s n ^ t o L y s " ^ . The amino a c i d sequence i s NH 2-Asn-Leu-CMC-Asn-Ile-Pro-CMC-Ser-Ala-Leu-Leu-Ser-Ser-Asp-Ile-Thr-Ala-Ser-Val-Asn-CMC-Ala-Lys-COOH ( C a n f i e l d , 1963b ) . T h i s p e p t i d e shows s i m i l a r i t i e s t o the p e p t i d e T -8 from t o b a c c o mosaic v i r u s p r o t e i n demonstrated t o have h a p t e n i c a c t i v i t y by B e n j a m i n i e_t a_l_. ( 1 964 ) , i n t h a t i t c o n t a i n s no a r o m a t i c amino a c i d s , i t i s r i c h i n n o n p o l a r r e s i d u e s and i s thus s p a r i n g l y s o l u b l e i n aqueous s o l u t i o n s . The most i n t e r e s t i n g a s p e c t o f t h e s e o b s e r v a t i o n s i s t h a t , a l t h o u g h T-11 does not i n h i b i t t he p r e c i p i t a t i o n r e a c t i o n between n a t i v e lysozyme and i t s homologous a n t i s e r u m under the c o n d i t i o n s 74 96 used h e r e , the sequence Asn t o Lys f a l l s w i t h i n the l o n g e r p e p t i d e G l n " ^ t o A l a ^ ^ demonstrated by S h i n k a et_ aj_. (1967) t o e x h i b i t h a p t e n i c a c t i v i t y i n t h e n a t i v e system ( F i g u r e 1 7 ) . These w o r k e r s used p e p s i n d i g e s t i o n o f t h e n a t i v e lysozyme m o l e c u l e s i n c e i t i s r e l a t i v e l y r e s i s t a n t t o e n z y m a t i c c l e a v a g e by more s p e c i f i c enzymes such as t r y p s i n . They were a b l e t o i s o l a t e f o u r p e p t i c p e p t i d e s from t h e r e g i o n G l n * ^ t o A l a ^ ' ' w h i c h a l l p o s s e s s e d t h e c a p a c i t y t o i n h i b i t p r e c i p i t a t i o n by 25%, and a l l had c l o s e l 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 . In f a c t , they d i f f e r e d o n l y i n t h e i r p o i n t s o f 80 94 c l e a v a g e w i t h i n t h e loop between Cys and Cys , wh i c h o c c u r r e d a t Pept ic _o cleavages i | ^ ^ ^ a) NH, G i n 5 7 Cys 6** . C y s 7 6 C y s 8 ° 83 84 85 Cys9** A l a 1 0 7 COOH l J r  b) " N H 2 T r p 6 2 C y s 6 / < A r g 6 8 COOH NH^Asn 7 ^ Cys76 C y s 8 ° C y s 9 4 L y s 9 6 COOH 1 J r _ _ c) N H 2 C y s 6 < 4 C y s 8 0 L e u 8 3 COOH - - -d) N H ^ s n 7 * * C M C 7 6 CMC 80 Cys9** L y s 9 6 COOH NH 2Arg 7 3 C M C 7 6 C M C 8 0 L e u 8 3 COOH N H 2 A s n 7 / < C M C 7 6 CMC8° L e u 8 3 COOH Figure 17. C o r r e l a t i o n of the various peptide sequences e x h i b i t i n g haptenic a c t i v i t y . a) Haptenic p e p t i c peptide i s o l a t e d by Shinka et a_l_.(1967) from nat ive lysozyme; b) haptenic t r y p t i c peptide i s o l a t e d from pept ic peptide (a) ; Shinka et a l . ( 1 9 6 7 ) ; c) haptenic " l o o p " peptide of Arnon and Sela (1969); d) haptenic T-11 peptide i s o l a t e d from CM-lysozyme in this t h e s i s ; e) two haptenic s o l i d phase peptides synthesized in th is t h e s i s . 114 Leu 83 Leu 84 and Ser 85 I t s h o u l d be mentioned t h a t t h i s 80 arid between Cys and Cys , whi c h would g i v e t h i s a r e a c o n s i d e r a b l e t e r t i a r y s t r u c t u r e -When S h i n k a ' s group d i g e s t e d t h e i r l a r g e r p e p t i d e w i t h t r y p s i n , they found one fragment c a p a b l e o f d e l a y i n g p r e c i p i t a t i o n i n t h e homologous system, but no o v e r a l l i n h i b i t i o n was o b s e r v e d . From t h e i r amino a c i d a n a l y s i s d a t a , i t appeared t h a t t h i s fragment r e p r e s e n t e d t h e sequences A s n ^ t o A l a " ^ , and T y r ^ 2 t o A r g ^ ( F i g . 17) , j o i n e d a t t h e C y s ^ and C y s ^ p o s i t i o n s . T h e i r c o n c l u s i o n s a r e i n agreement w i t h o ur f i n d i n g s w i t h CM-lysozyme a l t h o u g h , w i t h o ur sy s t e m , i n h i b i t i o n o f i m m u n o l o g i c a l r e a c t i v i t y by T-11 was f a r more pronounced. T h e r e f o r e i t i s p o s s i b l e t h a t t he d e t e r m i n a n t s i n t h i s a r e a may be the same f o r n a t i v e lysozyme and i t s reduced and a l k y l a t e d d e r i v a t i v e , even though the two a n t i s e r a do not appear t o c r o s s -r e a c t . I t i s p o s s i b l e t h a t r a d i c a l d i f f e r e n c e s i n c o n f o r m a t i o n between the two a n t i g e n i c p r e p a r a t i o n s may be r e s p o n s i b l e f o r t h i s . However, f u r t h e r work on th e s e systems i s n e c e s s a r y b e f o r e t h i s can be e s t a b l i s h e d . In v i e w o f t h i s l a t t e r p r o p o s a l , i t i s i n t e r e s t i n g t o note the : work o f Arnon and S e l a (1969) on the n a t i v e lysozyme m o l e c u l e . They p r e p a r e d a s y n t h e t i c c o n j u g a t e by c o v a l e n t bonding o f a lysozyme fragment (sequence C y s ^ t o L e u ^ , denoted " l o o p " p e p t i d e ) t o a branched p o l y - D L - a l a n i n e p e p t i d e , and used t h i s t o e l i c i t i n r a b b i t s , t he f o r m a t i o n o f a n t i b o d i e s s p e c i f i c a l l y d i r e c t e d 115 a g a i n s t t h i s r e g i o n o f t h e lysozyme m o l e c u l e . T h i s loop p e p t i d e i s p a r t o f the l o n g e r p e p t i d e shown by S h i n k a ' s group t o be h a p t e n i c ( F i g . 1 7 ) . These a n t i b o d i e s were i s o l a t e d i m m u n o s p e c i f i c a l l y on a l y s o z y m e - c e l l u i o s e immunoadsorbent, so no a n t i b o d i e s t o t h e D L - a l a n i n e p o l y p e p t i d e p o r t i o n were p r e s e n t . In a d d i t i o n , they o b t a i n e d a n t i b o d i e s w i t h a s i m i l a r s p e c i f i c i t y from a n t i - n a t i v e lysozyme serum, w i t h an immunoadsorbent p r e p a r e d from t h e same loop p e p t i d e . T h i s loop p e p t i d e c o n t a i n s a d i s u l f i d e b r i d g e between C y s ^ and C y s ^ , g i v i n g i t a r i g i d c o n f o r m a t i o n . These w o r k e r s d e m o n s t r a t e d t h a t t h e a n t i - l o o p a n t i b o d i e s were a b l e t o d i s t i n g u i s h between t h e loop p e p t i d e c o n t a i n i n g a d i s u l f i d e bond, and the open c h a i n p e p t i d e d e r i v e d from i t , s u g g e s t i n g t h a t t h e a n t i b o d i e s may be d i r e c t e d t o a c o n f o r m a t i o n - d e p e n d e n t d e t e r m i n a n t . However, t h e a n t i - l o o p a n t i b o d i e s o b t a i n e d from i m m u n i z a t i o n w i t h t h e s y n t h e t i c c o n j u g a t e c o n t a i n i n g t h e loop p e p t i d e , were a b l e t o b i n d h a p t e n i c a l l y w i t h both t h e loop p e p t i d e and i t s reduced and c a r b o x y m e t h y l a t e d d e r i v a t i v e , a l t h o u g h t o a somewhat l e s s e r degree i n t h e l a t t e r c a s e . In c o n t r a s t , the a n t i - l o o p a n t i b o d i e s d e r i v e d from a n t i s e r u m p r e p a r e d a g a i n s t n a t i v e lysozyme, were a b l e t o b i n d h a p t e n i c a l l y o n l y w i t h loop p e p t i d e c o n t a i n i n g t h e d i s u l f i d e b r i d g e , thus d i s c r i m i n a t i n g t o a g r e a t e r e x t e n t between t h e '''loop" and "open-c h a i n " form o f t h e p e p t i d e , than a n t i b o d i e s o b t a i n e d from the s y n t h e t i c p o l y p e p t i d e c o n j u g a t e . T h e r e f o r e , t h e s e two p o p u l a t i o n s o f a n t i b o d i e s , d i r e c t e d most l i k e l y t o t h e same r e g i o n o f t h e n a t i v e p r o t e i n , w i l l p r o v i d e an o p p o r t u n i t y t o compare them i n o r d e r t o l e a r n more on t h e r o l e o f t h e a n t i g e n i c c a r r i e r i n the homogeneity o f a n t i b o d i e s t o a s p e c i f i c d e t e r m i n a n t . F o l l o w i n g the 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 the h a p t e n i c T-11 p e p t i d e , the next s e r i e s o f e x p e r i m e n t s were aimed a t p i n p o i n t i n g t h e c r i t i c a l a n t i g e n i c r e g i o n o f t h i s l a r g e 23 amino a e i d p e p t i d e . As r e p o r t e d i n the l i t e r a t u r e r e v i e w , w i t h both s y n t h e t i c and n a t u r a l p r o t e i n a n t i g e n s , such as the TMVP, a sequence o f f i v e amino a c i d s i s f r e q u e n t l y a b l e t o a c t as an a n t i g e n i c d e t e r m i n a n t . From t h e r e s u l t s o b t a i n e d from s e q u e n t i a l d e g r a d a t i o n o f T-11 from t h e C- and N - t e r m i n u s , i t appeared t h a t t h e N - t e r m i n a l r e g i o n was i m p o r t a n t i n h a p t e n i c a c t i v i t y . T h i s i s i n t e r e s t i n g , s i n c e t h e loop p e p t i d e p r e p a r e d by A m on and S e l a (1969) over 1aps t h e N - t e r m i n a l r e g i o n o f T-11 by 10 ami no a c i d r e s i d u e s ( F i g . 1 7 ) . T h i s i m p l i e d t h e impo r t a n c e o f t h e N - t e r m i n a l r e g i o n , i f t h e s t a t e d h y p o t h e s i s o f a common a n t i g e n i c d e t e r m i n a n t i n n a t i v e and CM-lysozyme i s c o r r e c t . F i g u r e 17 i l l u s t r a t e s t he v a r i o u s p e p t i d e sequences found t h a t e x h i b i t h a p t e n i c a c t i v i t y , and shows t h a t the o n l y sequence common t o a l l o f t h e s e i s t h e d e c a p e p t i d e 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 o f T - 1 1 . Because o f t h e s e i m p l i c a t i o n s , t h e N - t e r m i n a l r e g i o n o f T-11 was s y n t h e s i z e d by t h e s o l i d phase method, i n o r d e r t o d e t e r m i n e i f t h i s s m a l l e r p e p t i d e would e x h i b i t h a p t e n i c a c t i v i t y . The p e p t i d e c o m p r i s e d t h e sequence A s n ^ t o L e u ^ , and a n o t h e r p e p t i d e c o m p r i s i n g t h i s p e p t i d e p l u s A r g , were s y n t h e s i z e d . The l a t t e r was p r e p a r e d ' t o d e t e r m i n e whether o r not t h i s Arg r e s i d u e would improve h a p t e n i c a c t i v i t y . It was s y n t h e s i z e d b e c a u s e S e l a and Mozes (1966) had shown t h a t a h i g h p e r c e n t a g e o f the a n t i b o d i e s d i r e c t e d a g a i n s t n a t i v e l y s o z y m e , a h i g h l y b a s i c p r o t e i n , .were p r e s e n t in the a c i d i c y~ g l o b u l i n f r a c t i o n i s o l a t e d by DEAE-Sephadex c h r o m a t o g r a p h y , and an o v e r a l l p o s i t i v e c h a r g e might enhance the h a p t e n i c a c t i v i t y o f a n t i g e n i c d e t e r m i n a n t s . However, f rom the r e s u l t s o f p r e c i p i t i n i n h i b i t i o n t e s t i n g o b t a i n e d w i t h the two s y n t h e t i c p e p t i d e s , i t a p p e a r e d t h a t they were both e q u a l l y h a p t e n i c w i t h i n the l i m i t s o f v a r i a b i l i t y o f the t e s t . When t h e s e two p e p t i d e s were t e s t e d s i m u l t a n e o u s l y w i t h n a t i v e lysozyme and i t s homologous a n t i s e r u m , no i n h i b i t i o n o f p r e c i p i t a t i o n was d e t e c t e d . T h e r e f o r e , the i n h i b i t i o n o f the C M - l y s o z y m e system was assumed to be the r e s u l t o f s p e c i f i c h a p t e n i c a c t i v i t y . On a q u a n t i t a t i v e b a s i s , n e i t h e r o f the s y n t h e t i c p e p t i d e s were as e f f i c i e n t as the T -11 p e p t i d e in c a u s i n g s p e c i f i c i n h i b i t i o n o f p r e c i p i t a t i o n . However , the c a l c u l a t i o n o f the m i c r o m o l a r q u a n t i t i e s o f the s y n t h e t i c p e p t i d e s f rom amino a c i d a n a l y s i s d a t a may not be as a c c u r a t e as w i t h t h e T -11 p e p t i d e . Smal l amounts o f b i o l o g i c a l l y i n a c t i v e p e p t i d e m a t e r i a l may be p r e s e n t . A n o t h e r p o s s i b l e e x p l a n a t i o n f o r the g r e a t e r h a p t e n i c e f f i c i e n c y o f T -11 might i n v o l v e the g r e a t e r d e g r e e o f h y d r o p h o b i c ! t y in the t o t a l s e q u e n c e . In t h i s r e s p e c t , Ben jamin i ( B e n j a m i n i , et_ a]_.., 1968a; 1968b) has shown t h a t , w i t h TMVP, i n c r e a s i n g h y d r o p h o b i c i t y by a d d i n g t he C^C)-acetyl group t o the N-terminus of t h e h a p t e n i c p e n t a p e p t i d e , s i g n i f i c a n t l y i n c r e a s e d i t s h a p t e n i c b i n d i n g t o s p e c i f i c a n t i b o d i e s compared t o the n o n - a c e t y l a t e d p e n t a p e p t i d e . In f a c t , i f t h e N- o c t a n o y l d e r i v a t i v e s o f the C-t e r m i n a l t e t r a - and t r i p e p t i d e s were used, s p e c i f i c b i n d i n g w i t h anti-TMVP serum was e x h i b i t e d , whereas the n o n - o c t a n o y l a t e d C-t e r m i n a l t e t r a - and t r i p e p t i d e s d i d n o t . Thus, by i n c r e a s i n g h y d r o p h o b i c i t y on the end o f t h e p e p t i d e s c o n t a i n i n g the amino a c i d s not e s s e n t i a l f o r a n t i g e n i c i t y , i n c r e a s e d b i n d i n g w i t h s p e c i f i c a n t i s e r u m was o b s e r v e d . From the work o f Met z g e r et_ aj_. (1963) on h a p t e n - a n t i b o d y i n t e r a c t i o n , i t i s known t h a t i n the b i n d i n g o f an a n t i g e n i c a r e a w i t h s p e c i f i c a n t i b o d y , a h y d r o p h o b i c i n t e r a c t i o n i s i m p o r t a n t . The enhancement o f h a p t e n i c b i n d i n g may be due t o the o r i e n t a t i o n o f t he p e p t i d e away from w a t e r and i n t o t h e a n t i b o d y s i t e , o r due to a s t a b i l i z i n g e f f e c t o f t h e f a t t y a c i d ( o c t a n o i c a c i d i n the c a s e o f the work mentioned above) on a g i v e n c o n f o r m a t i o n e x h i b i t i n g t h e b e s t f i t t o th e co m b i n i n g s i t e o f the a n t i b o d y . CONCLUDING COMMENTS The f o l l o w i n g i s a summary o f the f i n d i n g s r e p o r t e d here on the a n t i g e n i c p r o p e r t i e s o f CM-lysozyme: 1) A t r y p t i c p e p t i d e (T-11) was i s o l a t e d w h i c h e x h i b i t e d h a p t e n i c a c t i v i t y by c a u s i n g i n h i b i t i o n o f the immune r e a c t i o n between CM-lysozyme and i t s homologous a n t i s e r u m when t e s t e d by p r e c i p i t a t i o n and complement f i x a t i o n . 2) The N - t e r m i n a l r e g i o n o f T-11 was shown t o be i m p o r t a n t f o r t he h a p t e n i c a c t i v i t y o f t h e p e p t i d e . T h i s was demonstrated by s t u d i e s on s e q u e n t i a l l y degraded samples o f T-11, u s i n g both e n z y m a t i c and c h e m i c a l methods. 3) U l t i m a t e p r o o f o f t h e importance o f the N - t e r m i n a l d e c a p e p t i d e o f T-11 f o r h a p t e n i c a c t i v i t y was e s t a b l i s h e d by i t s s y n t h e s i s u s i n g the s o l i d phase method. The s y n t h e t i c p e p t i d e demonstrated h a p t e n i c a c t i v i t y by i n h i b i t i o n o f p r e c i p i t a t i o n between CM-lysozyme and i t s homologous a n t i s e r u m . k) The o b s e r v a t i o n was made t h a t CM-lysozyme d i d not c r o s s - r e a c t w i t h a n t i s e r u m p r e p a r e d a g a i n s t n a t i v e lysozyme. I t was f u r t h e r noted t h a t t r y p t i c p e p t i d e s i s o l a t e d from d i g e s t s o f CM-lysozyme d i d not i n h i b i t p r e c i p i t a t i o n between n a t i v e lysozyme and i t s homologous a n t i s e r u m . APPENDIX I? Cheriii c a l s arid M a t e r i a l s A c e t i c a c i d A cetone ^ ^ C ) - A c e t i c a n h y d r i d e Aluminum p o t a s s i u m s u l f a t e (alum) Ammonium c h l o r i d e Ammon i um s u1 f a t e A n t i s h e e p Hemolysin B a r b i t u r i c a c i d Benzene t-BOC-amino a c i d s B r o m o a c e t i c a c i d B r o m o a c e t y l bromide B u t a n o l Cadmium a c e t a t e C a l c i u m c h l o r i d e C a r b o x y p e p t i d a s e A, D F P - t r e a t e d C a r b o x y p e p t i d a s e B, not D F P - t r e a t e d C h l o r o m e t h y l a t e d c o p o l y s t y r e n e d i v i n y l b e n z e n e r e s i n C i t r i c a c i d P l a c e P u rchased F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c New England N u c l e a r F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c Di f c o - B a c t o B r i t i s h Drug House F i s h e r S c i e n t i f i c Mann Research B i o c h e m i c a l s and Sigma C h e m i c a l s Eastman O r g a n i c Chemicals Eastman O r g a n i c C h e m i c a l s F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c W o r t h i n g t o n B i o c h e m i c a l s W o r t h i n g t o n B i o c h e m i c a l s Bio-Rad F i s h e r S c i e n t i f i c Complete Freund's A d j u v a n t B a c t o - D i f c o D E A E - c e l l u l o s e C a l b i o c h e m D i c y c l o h e x y c a r b o d i imide Eastman O r g a n i c C h e m i c a l s N,N-D i m e t h y l f o r m a m i d e F i s h e r S c i e n t i f i c Dioxane F i s h e r S c i e n t i f i c Dowex 50 x k c a t i o n exchange r e s i n Bio-Rad E t h y l M e t h y l S u l f i d e Eastman O r g a n i c Chemicals F i 1 t e r paper Whatman Co. Formi c a c i d F i s h e r S c i e n t i f i c G l y c e r i ne F i s h e r S c i e n t i f i c G u inea p i g serum (complement) Hyland Co. H i p p u r y l - L - a r g i n i n e Mann Research B i o c h e m i c a l H i p p u r y l - L - p h e n y l a l a n i n e Mann Research B i o c h e m i c a l H y d r o c h l o r i c a c i d F i s h e r S c i e n t i f i c Hydrogen Bromide B a k e r , Matheson l o d o a c e t i c a c i d Eastman O r g a n i c C h e m i c a l s ^ V ) - i o d o a c e t i c a c i d New England N u c l e a r L - l e u c i n e - p - n i t r o a n ? 1 i d e Sigma C h e m i c a l s L e u c i n e amino p e p t i d a s e W o r t h i n g t o n B i o c h e m i c a l s L i q u i d ammonia B a k e r , Matheson L i q u i f l u o r New En g l a n d N u c l e a r L i t h i u m c h l o r i d e F i s h e r S c i e n t i f i c Lysozyme W o r t h i n g t o n B i o c h e m i c a l s Magnesium c h l o r i d e F i s h e r S c i e n t i f i c M e r t h i o l a t e 2 - M e r c a p t o e t h a n o l Methanol M e t h y l c e l l u l o s e M e t h y l e n e c h l o r i d e Nembutal N i n h y d r i n N i t r o g e n P e t r o l e u m Phosphorous p e n t o x i d e Phenyl i s o t h i o c y a n a t e P y r i d i n e Sephadex Sodi um a c e t a t e Sodium a z i d e Sodium b a r b i t o n e Sodium b i c a r b o n a t e Sodium c h l o r i d e Sodium h y d r o x i d e Sodium metal Sodium ph o s p h a t e , d i b a s i c Sodium ph o s p h a t e , monobasic Stannous c h l o r i d e T o l u e n e B r i t i s h Drug House Eastman O r g a n i c Chemical F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c A b b o t t L a b o r a t o r i e s P i e r c e C h e m i c a l s Canadian L i q u i d A i r F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c Eastman O r g a n i c Chemical F i s h e r S c i e n t i f i c P harmacia F i n e C h e m i c a l s F i s h e r S c i e n t i f i c FTsher S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c p - t o l u e n e s u l f o n y l - L - a r g i n i n e methyl e s t e r T r i f 1 u o r o a c e t i c a c i d T r i m e t h y l a m i n e T r i s ( h y d r o x y m e t h y 1 ) a m i no methane T r y p s i n l y o p h i l i z e d V i s k i n g d i a l y s i s t u b i n g C e l l u l o s e powder Sigma C h e m i c a l s F i s h e r S c i e n t i f i c F i s h e r S c i e n t i f i c Sigma C h e m i c a l s W o r t h i n g t o n B i o c h e m i c a l s Union C a r b i d e Whatman Co. 124 LITERATURE .CITED 1. 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