UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on bovine casein. Kason, William Rudolph 1970

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STUDIES ON BOVINE CASEIN by WILLIAM R, KASON B.Sc. University of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of FOOD SCIENCE Faculty of A g r i c u l t u r a l Sciences We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1970 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or 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 ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of f^^A <>r_,e.^co The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada D a t e M i . . , « - 7 n ABSTRACT A model f o r e x p l a i n i n g t h e d i f f e r e n c e s i n p h y s i c a l c h e m i c a l p r o p e r t i e s o f t h e c a s e i n s was p r o p o s e d , and t h e r e s u l t s o f t h e e x p e r i m e n t s s u p p o r t i n g t h i s model a r e p r e s e n t e d . K, a , g and w h o l e - c a s e i n were shown t o e x h i b i t s i c i r c u l a r d i c h r o i c measurements t y p i c a l o f random c o i l e d o r d e n a t u r e d p r o t e i n s , a l l b e i n g s i m i l a r . M o d i f i c a t i o n o f t h e p r o t e i n s w i t h N a - t e t r a t h i o n a t e , S - m e r c a p t o - s u c c i n i c a n h y d r i d e , r e d u c t i o n and e s t e r i f i c a t i o n o r changes i n t h e s o l v e n t system w i t h u r e a o r p o l y e t h y l e n i m i n e e f f e c t e d t h e s p e c t r a o n l y s l i g h t l y . S i g n i f i c a n t d i c h r o i c changes were o b s e r v e d when pH a d j u s t m e n t s were made. The i n t e r a c t i o n o f p o l y e t h y l e n i m i n e o r i c - c a s e i n w i t h p r o d u c e d i n t e r a c t i o n p r o d u c t s w h i c h were d e t e c t a b l e w i t h a n u c l e a r m agnetic r e s o n a n c e s p e c t r o m e t e r . The i n t e r a c t i o n o f p o l y e t h y l e n i m i n e and a s ^ - c a s e i n were d e t e c t e d by e l e c t r o p h o r e s i s , u l t r a c e n t r i f l i g a t i o n , l i g h t s c a t t e r i n g , s o l u b i l i t y and n u c l e a r m a g n e t i c r e s o n a n c e . T h i s i n t e r a c t i o n was dependent upon pH, c o n c e n t r a t i o n and amino n i t r o g e n c o n t e n t . a , and K - c a s e i n s were o b s e r v e d t o - b e h e a t l a b i l e s i w h i l e t h e i r i n t e r a c t i o n p r o d u c t was more s t a b l e t h a n each i n d i v i d u a l c a s e i n . The r e s u l t s s u g g e s t t h a t t h e i n t e r a c t i o n o f c t ^ and K - c a s e i n s i s t h r o u g h c a r b p x y l and amino e l e c t r o s t a t i c bonds, and t h a t t h e i r a s s o c i a t i o n i s p u r e l y random due t o l a c k o f s t o i c h i o m e t r y i n t h e m o l e c u l e s . The r o l e o f u r e a i n t h e d i s s o c i a t i o n o f c a s e i n s i s t h e d i s r u p t i o n o f b o t h i o n i c and hydrogen bonds. - i v -TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 9 P r o t e i n P u r i f i c a t i o n 9 P r o t e i n M o d i f i c a t i o n 9 P o l y e t h y l e n i m i n e 10 P o l y e t h y l e n i m i n e M o d i f i c a t i o n 10 A c i d Base T i t r a t i o n 10 Amino A c i d A n a l y s i s 11 U l t r a c e n t r i f u g a t i o n 11 U l t r a v i o l e t Absorbance 11 N u c l e a r M a g n e t i c Resonance 12 C i r c u l a r D i c h r o i s m 12 T h e o r e t i c a l NMR S p e c t r a 13 T h e o r e t i c a l C a l c u l a t i o n s 13 E l e c t r o p h o r e s i s 13 RESULTS AND DISCUSSION 14 C i r c u l a r D i c h r o i s m 14 N u c l e a r M a g n e t i c Resonance 2 2 I n t e r a c t i o n 34 U l t r a v i o l e t Absorbance 41 T h e o r e t i c a l 48 Amino A c i d A n a l y s i s 51 U l t r a c e n t r i f u g a t i o n 5 3 E l e c t r o p h o r e s i s 56 CONCLUSION 5 8 BIBLIOGRAPHY 60 - V -T a b l e LIST OF TABLES C a l c u l a t i o n s f o r p l o t t i n g and p r e s e n t -a t i o n o f t h e o r e t i c a l s p e c t r a o f a ^ 8 and K - c a s e i n . Page 25 T a b l e I I T h e o r e t i c a l c a l c u l a t i o n s o f bond t y p e s and numbers i n a g i v e n m o l e c u l e from t h e a m i n o - a c i d a n a l y s i s o f a B and . _ . ^, „ * „ „ s i K-caseins. 5 2 T a b l e I I I A m i n o - a c i d a n a l y s i s o f m o d i f i e d ( e s t e r i f i e d and red u c e d ) K - c a s e i n and e f f e c t o f s t o r a g e a t room t e m p e r a t u r e and c o n d i t i o n s on t h e a n a l y s i s . 54 T a b l e IV S e d i m e n t a t i o n v a l u e s and e f f e c t o f pH on t h e s e v a l u e s f o r a s-^ and K - c a s e i n s as w e l l as p o l y e t h y l e n i m i n e and i t s i n t e r a c t i o n w i t h a , - c a s e i n . s i 55 - v i i -14. N u c l e a r m agnetic r e s o n a n c e s p e c t r a o f p o l y e t h y l e n i m i n e .25% i n D_0; pH 9.7 and 11.0; 100 Mc/sec 1 32 15. N u c l e a r m agnetic r e s o n a n c e s p e c t r a o f a , - c a s e i n 2.5% p o l y e t h y l e n i m i n e .25%; . pH 9.7, 10.6, 11.0; 100 Mc/sec - 33 16. N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f a , - c a s e i n 1.25% and K - c a s e i n 1.25% i n s l D 20; 40, 70 and 90°C; 100 Mc/sec 33 17. E f f e c t o f pH on c h e m i c a l s h i f t o f p o l y e t h y l e n i m i n e i n and o u t o f t h e p r e s e n c e o f a ^ c a s e i n ; p o l y e t h y l e n i m i n e / a ^ c a s e i n 1/1§; s ^ c a s e i n 2.5%; H^O room t e m p e r a t u r e s 35 36 .18. E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f t h e copolymer peaks o f a s ^ K c a s e i n 1/1 19. Changes i n peak h e i g h t o f s pectrum o f K and a c a s e i n 2.5% w/w D^O w i t h change i n t e m p e r a t u r e a s ^ - c a s e i n K - c a s e i n 37 20. E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f a , and K - c a s e i n 38 s l 21. E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f a , and K - c a s e i n 39 s l 22. E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f a , and K - c a s e i n 40 s l 23. Comparison o f u l t r a v i o l e t a bsorbance o f a s-^ and p o l y e t h y l e n i m i n e 42 24. Comparison o f t i t r a t i o n o f p o l y e t h y l e n i m i n e and a , c a s e i n w i t h a c i d and b a s e , .1 N NaOH and .S 1N HC1 PEI a , - c a s e i n 4 3 s± 25. . E f f e c t o f pH on t h e i n t e r a c t i o n o f p o l y e t h y l e n i m i n e and a , c a s e i n , a ,/PEI 44 10.0 S X S X 26. E f f e c t o f pH on t h e i n t e r a c t i o n o f p o l y e t h y l e n i m i n e and a c a s e i n a f t e r m o d i f i c a t i o n o f PEI A c i d H y d r o l y s i s 2 N HC1 i n b o i l i n g w a t e r 4 5 27. E f f e c t o f p o l y e t h y l e n i m i n e c o n c e n t r a t i o n on t h e f l o c c u l a t i o n o f a n c a s e i n 46 s l - v i i i -E f f e c t o f u r e a on t h e copolymer o f a ^ - c a s e i n p o l y e t h y l e n i m i n e E f f e c t o f pH on t h e u l t r a v i o l e t a bsorbance o f K - c a s e i n E f f e c t o f t e m p e r a t u r e and c o n c e n t r a t i o n on t h e a u t o p o l y m e r i z a t i o n o f K - c a s e i n ACKNOWLEDGMENTS I thank ray professor, Dr. S. Nakai for h i s patience and guidance Thanks to Dr. Rose and the F i s h e r i e s Research Board of Canada and to Dr. Hayward of the U.B.C. Chemistry Departement f o r the use of t h e i r equipement. Thanks to my many associates who helped to make these years an education. INTRODUCTION Casein was one of the f i r s t extensively studied proteins i n the f i e l d of b i o l o g i c a l chemistry. This, no doubt, was a r e s u l t of i t s ubiquitous supply, also i t s ease of separation from the other components in milk. The heterogenetiy of casein was f i r s t demonstrated by Lindstrom-Lang (1925, 1929) with observations of d i f f e r e n t s o l u b i l i t y i n a c i d i f i e d alcohol solutions and observations of varying amounts of phosphorous with these d i f f e r e n t i a l l y soluble f r a c t i o n s . Further d i f f e r e n t i a t i o n of casein f r a c t i o n s were demonstrated by Mellander (1939); by using e l e c t r o -phoresis, he showed that casein contained three major fra c t i o n s a, 3 and y casein. Electrophoresis following these r e s u l t s was used quite extensively f o r the characterization of casein f r a c t i o n s but perhaps the most s i g n i f i c a n t treatment in the e l u c i d a t i o n of casein f r a c t i o n s was that used by Groh et a l . (1934). He used urea alcohol f r a c t i o n a t i o n which resulted i n the unknown electrophoretic patterns. This work was the f i r s t proof of a strongly i n t e r a c t i n g system; the components of which had not yet been f u l l y elucidated. With the development of f r a c t i o n a t i o n and p u r i f i c a t i o n procedures came an increase i n knowledge of the physical chemical properties of the caseins. Svedberg and Pedersen (1940) and Chanutin (1942) elucidated the combination of - 2 -c a l c i u m i o n s w i t h c a s e i n and t h e changes i n m o l e c u l a r w e i g h t w i t h changes i n c a l c i u m c o n t e n t . Nitschman and Gugginberg (1941) s t u d y i n g f l o w b i r e f r i n g e n c e and v i s c o m e t r y c o n c l u d e d t h a t c a s e i n was r o d shaped w i t h an a x i a l o r a t i o o f 8.7 and a l e n g t h o f 290 A. The e l u c i d a t i o n o f c h e m i c a l p r o p e r t i e s o f c a s e i n f r a c t i o n s s p e c i f i c a l l y p h o s p h o r u s , n i t r o g e n and amino a c i d c o n t e n t b r o u g h t about some o f t h e e a r l i e s t work r e l a t i n g s u b u n i t c o m p o s i t i o n t o p h y s i c a l p r o p e r t i e s o f t h e l a r g e m o l e c u l e , f o r example t h e c a l c u l a t i o n o f s p e c i f i c volumes by Gordon e_t a l . (1949) and McMeekin e t a l . ( 1 9 4 9 ) . The t e c h n i q u e o f u r e a f r a c t i o n -a t i o n has been v e r y e f f e c t i v e i n p u r i f y i n g c a s e i n f r a c t i o n s ; i t was t h o u g h t u r e a forms complexes w i t h t h e c a s e i n v e r y r e a d i l y t h u s b r e a k i n g down t h e v e r y s t r o n g i n t e r a c t i o n o f th e p r o t e i n s , t h i s a l l o w e d f o r t h e i r s e p a r a t i o n (McMeekin, 1954; N e u r a t h , 1944). E a r l y e x p e r i m e n t a l i s t s t h o u g h t u r e a t o be a v e r y d r a s t i c p r o c e d u r e w h i l e t h e use o f pH changes i n low t e m p e r a t u r e was t h o u g h t t o be q u i t e m i l d . However, b o t h t e c h n i q u e s produced i d e n t i c a l p r o d u c t s . Some e a r l y s t u d i e s on c a s e i n s u b u n i t s and t h e i r i n t e r a c t i o n s u g g e s t e d t h a t t h e i n t e r a c t i o n o f a and $ c a s e i n was i n such a way as t o mask i o n i c groups ( P e r l m a n n , 1952). These i n t e r a c t i o n s however, d i d not a f f e c t t h e t i t r a t i o n c u r v e s o f t h e s e components. The i n t e r a c t i o n s and t h e - 3 -r e s u l t i n g complex was t h o u g h t by F r a s e r and P o w e l l (1950) t o be such t h a t t h e complex was d i c t a t e d t o : t h u s ; a, 8 and y c a s e i n s were p r e s e n t i n a c o n s t a n t r a t i o . H ipp e t a l . (1952) e l u c i d a t i n g d i f f e r e n c e s i n p o t e n t i a l i n t e r a c t i n g groups on each f r a c t i o n showed t h a t a, 6 and y c a s e i n had d i f f e r e n t b u f f e r a b i l i t i e s f o r a c i d and b a s e , t h u s y c o u l d b u f f e r more a c i d t h a n a o r 8 and a c o u l d b u f f e r more base t h a n 8 o r y• He a l s o showed t h a t by s e l e c t i n g pH v a l u e s where i o n i c groups o f t h e p r o t e i n were e x p e c t e d t o d i s s o c i a t e , an e s t i m a t i o n o f t h e number o f i o n i z i n g groups p r e s e n t c o u l d be made. F u r t h e r a p p l i c a t i o n o f e x p e r i m e n t a l r e s u l t s i n t o p h y s i c a l p arameters was done by Hipp e t a l . (1952) by e x t r a p o l a t i o n o f r e d u c e d v i s c o s i t y t o z e r o c o n c e n t r a t i o n and a c q u i r i n g t h e i n t r i n s i c v i s c o s i t y t h e y c a l c u l a t e d a x i a l r a t i o s o f a, 8 , y a n d whole c a s e i n s as 10.2, 13.2, 9.3 and 12.0 r e s p e c t i v e l y . M e l l o n (1951) by d e t e r m i n i n g w a t e r a b s o r p t i o n o f s y n t h e t i c polymers d e t e r m i n e d t h e e f f e c t o f p o l a r groups on c a s e i n w a t e r a b s o r p t i o n . R e s u l t s i n d i c a t e d t h a t 25% o f w a t e r i n c a s e i n was f rom amino g r o u p s , 45% from p e p t i d e bonds and 10% from guanido g r o u p s . These, r e s u l t s d i d not d e t e r m i n e th e i n f l u e n c e o f h y d r o x y l o r c a r b o x y l groups on w a t e r a b s o r p t i o n . W i t h i n c r e a s e d s t u d y o f p h y s i c a l c h e m i c a l p r o p e r t i e s o f c a s e i n came i n c r e a s e d s t u d y i n f r a c t i o n a t i o n t e c h n i q u e s . I n 1956 Waugh and Von H i p p e l d e t e c t e d a new component o f - 4 -casein; t h i s component was K-casein, K-casein was i s o l a t e d and c h a r a c t e r i z e d by Wake (1959). T h i s new component now r e s u l t e d i n r e a p p r a i s a l o f t h e i n t e r a c t i o n s and s o l u t i o n phenomenon i n t h e c a s e i n s . The major c a s e i n s now o b s e r v e d by e l e c t r o p h o r e s i s a r e a , 6, K and y. S i n c e s t h e r e c o g n i t i o n o f t h e i m p o r t a n c e o f e l e c t r o p h o r e s i s i n t h e c h a r a c t e r i z a t i o n o f t h e p r o t e i n s , not o n l y g e n e t i c v a r i a t i o n s o f a g i v e n f r a c t i o n have been shown t o e x i s t but a l s o sub f r a c t i o n s o f a g i v e n v a r i a n t . S i n c e t h e improved i s o l a t i o n o f c a s e i n components by Wake and B a l d w i n (1961) and N e e l i n ( 1 9 6 4 ) , f u r t h e r p h y s i c a l studies on the c a s e i n s have been c a r r i e d o u t . The a c c u m u l a t i n g e v i d e n c e s u g g e s t s t h a t t h e c a s e i n s resemble d e n a t u r e d o r random c o i l p r o t e i n s ( K r e s c h e k , 1965). The work o f Noble and Waugh (1965) has shown t h a t a , and K - c a s e i n i n t e r a c t i o n s . ° s i ar e t e m p e r a t u r e dependent. The i n t e r a c t i o n o f 3 and K - c a s e i n o c c u r s s p o n t a n e o u s l y a t 30°C ( Z i t t l e and W a l t e r , 1967). B - C a s e i n i s h i g h l y heat s e n s i t i v e ( P a y e n s , Van M a r k w i j k , 1963). S i g n i f i c a n t work i n t h e a n a l y s i s o f t h e c o n f o r m a t i o n and e l a b o r a t i o n o f t h e i n t e r a c t i o n o f t h e p r o t e i n s were c a r r i e d out by H e r s k o v i t s (1966). U s i n g aqueous and a p o l a r o r g a n i c s o l v e n t s , d i s s o c i a t i n g a g e n t s , and pH and t e m p e r a t u r e p e r t u r b a t i o n , he has s u p p o r t e d t h e view t h a t t h e c a s e i n s a r e h i g h l y d i s o r g a n i z e d and as a r e s u l t would respond s i g n i f i c a n t l y t o s t r u c t u r a l f o r m i n g systems and t o t e m p e r a t u r e - 5 -changes, y e t v e r y l i t t l e t o systems c a u s i n g d i s o r i e n t a t i o n . These r e s u l t s s u p p o r t t h e h y p o t h e s i s t h a t t h e a s s o c i a t i o n o f a , 8 and K - c a s e i n i s random and not h i g h l y s t r u c t u r a l l y s o r i e n t e d . F u r t h e r e l u c i d a t i o n on t h e i n t e r a c t i o n o f a , , s l 8, and K - c a s e i n has been done by a p p l y i n g a m o d i f i e d method o f d e t e r m i n i n g i n o r g a n i c complexes, t h e i r c o m p o s i t i o n and t h e i r d i s s o c i a t i o n c o n s t a n t s ( J o b , 1928; F e l s e n f o l d and R i c h , 1957). G a r n i e r (1967) has shown t h a t t h e c o m p o s i t i o n o f t h e complex o f K, 3 and a - c a s e i n s have no r e l a t i o n s h i p s t o t h e c o n c e n t r a t i o n o f t h e p r o t e i n s i n m i l k o r t h e s t a b i l i z i n g c a p a c i t y o f K - c a s e i n ( Z i t t l e , 1 9 6 1), and t h a t t h e r e i s a u n i f o r m d i s t r i b u t i o n o f a , , 8 and K - c a s e i n s s l ' w i t h i n m i c e l l e s o f v a r y i n g s i z e s ( G a r n i e r and Dumas, 1968). Recent t r e n d s i n p r o t e i n r e s e a r c h have been d i r e c t e d toward t h e e l u c i d a t i o n o f c o n f o r m a t i o n and p r o p e r t i e s from t h e c o m p o s i t i o n o f amino a c i d s . I t was t h e purpose o f t h i s s t u d y t o a r r i v e a t a model f o r t h e i n t e r a c t i o n o f t h e c a s e i n s as w e l l as an e x p l a n a t i o n f o r t h e i r d i f f e r i n g p r o p e r t i e s i n s o l u t i o n . The model proposed t h a t t h e i n t e r a c t i o n o f c a s e i n s was v i a amino c a r b o x y l i n t e r a c t i o n and t h e d i f f e r e n c e s i n a c t i v i t y i n s o l u t i o n was a r e s u l t o f d i f f e r i n g amounts o f hydrogen bonding p o t e n t i a l i n s o l u t i o n . The h y p o t h e s i s was t h a t a t a g i v e n pH the changes i n a g g r e g a t i o n o r a s s o c i a t i o n w h i c h r e s u l t e d w i t h c h a n g i n g t e m p e r a t u r e were - 6 -i n f a c t changes i n .phase from one o f aqueous o r hydrogen b o n d i n g i n s o l u t i o n t o one o f i o n i c o r e l e c t r o s t a t i c a s s o c i a t i o n o r p o l y m e r i z a t i o n . I f t h i s was t h e case t h e n t h e r e s u l t would show t h a t B - c a s e i n was t h e most t e m p e r a t u r e s e n s i t i v e due t o t h e f a c t t h a t i t c o n t a i n e d t h e most hydrogen bonds p e r m o l e c u l e f o l l o w e d by < and a ^ c a s e i n . The h y p o t h e s i s a l s o i m p l i e d t h a t d i f f e r e n c e s i n a s s o c i a t i o n s t r e n g t h s between t h e c a s e i n s would be p r e s e n t p u r e l y as a r e s u l t o f d i f f e r e n c e s i n t h e e l e c t r o s t a t i c i n t e r a c t i o n o f t h e p r o t e i n s . Thus, <* S 1 -K would be s t r o n g e r t h a n « s l - 3 o r 0-K i n t e r a c t i o n . A l s o a s . a r e s u l t o f c o m p a r i s o n o f c a r b o x y l g r o u p s , v a r y i n g c a l c i u m i o n s t a b i l i t y and s t a b i l i z i n g a b i l i t y o f one p r o t e i n o r a n o t h e r would be e x p l a i n e d by th e c h a r g e n e u t r a l i z a t i o n e f f e c t due t o t h e e l e c t r o s t a t i c i n t e r a c t i o n . A l l c a l c u l a t i o n s r e g a r d i n g hydrogen bonds, f r e e c a r b o x y l o r f r e e amino groups were made from amino a c i d a n a l y s i s . I n an at t e m p t t o pr o v e t h e h y p o t h e s i s a s y n t h e t i c p o l y m e r , p o l y e t h y l e n i m i n e , was used. T h i s polymer w h i c h has m o l e c u l a r w e i g h t 50,000-100,000 s i m u l a t e d a p r o t e i n c opolymer. The d i f f e r e n c e between t h e polymer and p r o t e i n i s t h a t t h e polymer c o n t a i n s o n l y c a r b o n , n i t r o g e n and hy d r o g e n , t h u s , i t s i n t e r a c t i o n mechanism would be r e a d i l y p o s t u l a t e d . T h i s p o l y m e r c o n t a i n e d 32.5% n i t r o g e n o f whi c h 60% was s e c o n d a r y amine, 20% p r i m a r y amine and 20% t e r t i a r y amine. - 7 -The amino a c i d polymer K c a s e i n c o n t a i n s a p p r o x i m a t e l y 15% n i t r o g e n , 70% s e c o n d a r y , 20% p r i m a r y and 10% t e r t i a r y . C l e a r l y t h e polymer would be a v e r y p o s i t i v e l y c h a r g e d random c o i l e d s t r u c t u r e w h i c h would be h i g h l y h y d r a t e d . The i n t e r a c t i o n o f t h i s polymer w i t h a n c a s e i n was i n v e s t -s l i g a t e d u s i n g s p e c t r o p h o t o m e t r y , n u c l e a r m agnetic r e s o n a n c e (NMR), u l t r a c e n t r i f u g a l , e l e c t r o p h o r e t i c and c i r c u l a r d i c h r o i c t e c h n i q u e s . C o n c u r r e n t l y , t h e s t u d y o f a .. and i c - c a s e i n was done t o i n v e s t i g a t e t h e e f f e c t o f pH and t e m p e r a t u r e on t h e i r i n d i v i d u a l and i n t e r a c t i o n c h a r a c t e r i s t i c s i n o r d e r t o e l a b o r a t e on t h e i r s o l u t i o n and i n t e r a c t i o n p r o p e r t i e s . The c a s e i n s were a l s o i n v e s t i g a t e d u s i n g c i r c u l a r d i c h r o i s m as a t o o l t o d e t e r m i n e and e l a b o r a t e t h e e f f e c t o f pH on t h e i r n a t i v e and m o d i f i e d s t a t e . The f u n c t i o n o f u r e a on t h e d i s r u p t i o n o f t h e c a s e i n s was a l s o s t u d i e d . I t was assumed t h a t u r e a d i d not f u n c t i o n on h y d r o p h o b i c r e g i o n s but r a t h e r on i o n i c and hydrogen bo n d i n g r e g i o n s o f t h e m o l e c u l e . The s e l f - p o l y m e r i z i n g p r o p e r t i e s and t h e e f f e c t s o f pH, t e m p e r a t u r e and c o n c e n t r a t i o n on t h e s e p r o p e r t i e s were a l s o o b s e r v e d u s i n g s p e c t r o p h o t o m e t r i c t e c h n i q u e s . The i d e a b e h i n d a l l o f t h e s e s t u d i e s was t h a t d i f f e r e n c e s i n p r o t e i n aqueous p r o p e r t i e s a t a g i v e n pH were a r e s u l t o f d i f f e r e n t amounts o f hydrogen bo n d i n g and t h a t d i f f e r e n c e s i n i n t e r -a c t i o n were a r e s u l t o f i o n i c i n t e r a c t i o n s . These two p r o p e r t i e s d i s c i p l i n e t h e a c t i v i t i e s o f t h e s e random c o i l e d p r o t e i n s . - 8 -Trends i n f o o d s c i e n c e r e s e a r c h have been t o produce s y n t h e t i c p r o d u c t s w h i c h have s i m i l a r p h y s i c a l and c h e m i c a l c h a r a c t e r i s t i c s o f n a t u r a l p r o d u c t s . The p r o d u c t i o n and m a n u f a c t u r i n g o f any f o o d r e q u i r e s an u n d e r s t a n d i n g o f t h e system c o n s i d e r e d , t h i s i s where a p p l i c a t i o n o f such r e s e a r c h as i s p r e s e n t e d i n t h i s t h e s i s may be. Our p e r s o n a l i n t e r e s t i n t h e ap p r o a c h t a k e n i n t h i s t h e s i s was t h a t m i l k b e i n g a 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 may be a s i m p l e r system f o r s t u d y i n g and e l a b o r a t i n g a c o n s i s t e n t phenomenon o f a l l 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 . - 9 -MATERIALS AND METHODS P r o t e i n P u r i f i c a t i o n Whole m i l k was c o l l e c t e d from a s i n g l e cow o f t h e U n i v e r s i t y o f B r i t i s h C o lumbia d a i r y h e r d . The cow used had p r e v i o u s l y been s e l e c t e d by e l e c t r o p h o r e t i c p h e n o t y p i n g f o r homozygous e o n d i t i o n o f the p r o t e i n d e s i r e d . The m i l k was skimmed by c e n t r i f u g a t i o n a t 3000Xg i n a S o r v a l l t y p e SS-34 c e n t r i f u g e f o r 25 minutes a t -2°C. The s o l i d f a t was removed and t h e skim m i l k h a r v e s t e d t h r o u g h cheese c l o t h . Whole c a s e i n was p r e p a r e d by slow d r o p w i s e a d d i t i o n o f 0.1 N HC1 a t room t e m p e r a t u r e u n t i l pH 4.5 was a c h i e v e d . The p r e c i p i t a t e was c o l l e c t e d by f i l t e r i n g t h r o u g h f o u r l a y e r s o f cheese c l o t h ; t h e p r e c i p i t a t e was t h e n washed t h r e e t i m e s i n 3 5°C w a t e r ; t h i s c a s e i n was t h e n r e d i s s o l v e d by s low a d d i t i o n o f 0.1 N NaOH u n t i l pH 7.5 was a c h i e v e d ; t h i s p r o c e d u r e was r e p e a t e d and t h e pure c a s e i n was f i n a l l y d i a l y z e d a g a i n s t 0.02 M N a C l , pH 7.0, p e r v a p o r a t e d and l y o p h i l i z e d . a , and K c a s e i n were p u r i f i e d a c c o r d i n g t o th e method o f Z i t t l e and C u s t e r ( 1 9 6 3 ) . \ \ 8 - c a s e i n was p u r i f i e d a c c o r d i n g t o t h e method o f A s c h a f f e n b u r g ( 1 9 6 3 ) . M o d i f i c a t i o n o f P r o t e i n s M o d i f i c a t i o n o f a , - c a s e i n by t h e i n t r o d u c t i o n o f s l s u l p h y d r y l groups 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 o f K l o t z and Heiney (1961). - 10 -K - c a s e i n was m o d i f i e d as a r e s u l t o f d e t e r m i n a t i o n o f t h e number o f g l u t a m i n e and a s p a r a g i n e r e s i d u e s 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 W i l c o x (19630. The p r o c e d u r e was a l t e r e d by u s i n g a u t o m a t i c amino a c i d a n a l y z e r r a t h e r t h a n t h e manual c h r o m a t o g r a p h i c s e p a r a t i o n d e s c r i b e d . K - c a s e i n was a l s o m o d i f i e d by s u b s t i t u t i o n o f S - s u l p h e n y l s u l p h o n a t e . T h i s was c a r r i e d o u t a c c o r d i n g t o L i u ' s method ( 1 9 6 7 ) . P o l y e t h y l e n i m i n e P o l y e t h y l e n i m i n e , Separon C-12 0 i s a h i g h l y b ranched polymer 50-100,000 m o l e c u l a r w e i g h t . The polymer c o n t a i n e d 60% s e c o n d a r y amines, 20% p r i m a r y amines and 20% t e r t i a r y amines. I n f o r m a t i o n on polymer was o b t a i n e d from Dow C h e m i c a l Seminar N o t e s . M o d i f i c a t i o n o f P o l y e t h y l e n i m i n e P o l y e t h y l e n i m i n e was m o d i f i e d by t o t a l amide h y d r o l y s i s a c c o r d i n g t o Moore ( 1 9 6 3 ) . H y d r o l y s i s was f o l l o w e d by pH a d j u s t m e n t , l y o p h i l i z a t i o n and r e d i s s o l v i n g . A c i d and Base T i t r a t i o n T i t r a t i o n o f samples was c a r r i e d out a t room temp-e r a t u r e w i t h 50 ml o f 0.1% sample i n a 100 ml b e a k e r w i t h c o n s t a n t s i t r r i n g and i n c r e m e n t a l a d d i t i o n o f 0.1 N NaOH o r 0.1 N HC1. The pH change was r e c o r d e d on a P h o t o v o l t r e c o r d e r a t -t a c h e d t o a Beckman pH meter "Expandomatic" model. One minute was - 1 1 -a l l o w e d f o r e q u i l i b r a t i o n a f t e r a d d i t i o n o f a c i d o r base b e f o r e t h e r e c o r d i n g was t a k e n . Amino A c i d A n a l y s i s A n a l y s e s were performed on samples w h i c h had been t r e a t e d as f o l l o w s : A sample was d i s s o l v e d i n 6 N HC1 i n h y d r o l y s i s t u b e s and p l a c e d i n m e t h a n o l / d r y i c e b a t h and f r o z e n ; t h e f r o z e n sample was t h a n e v a c u a t e d t o 50 m i c r o n s Hg and f l u s h e d w i t h n i t r o g e n * , t h i s was r e p e a t e d t h r e e t i m e s . F i n a l l y , t h e t u b e s were e v a c u a t e d and s e a l e d . The sample was t h e n h y d r o l y z e d a t one hundred and f i v e d egrees C f o r 24 h o u r s . E x c e s s HC1 was removed by f l a s h e v a p o r a t i o n a t 35°C and t h e sample was r e d i s s o l v e d i n pH 2 . 2 1 c i t r a t e b u f f e r . An a l i q u o t was a n a l y z e d i n d u p l i c a t e on a P hoenix M i c r o Amino A c i d A n a l y z e r , model 6800, M o o r e - S t e i n system. U l t r a c e n t r i f u g a t i o n A l l s e d i m e n t a t i o n v e l o c i t i e s were d e t e r m i n e d i n a S p i n c o Model E Type u l t r a c e n t r i f u g e w i t h r o t o r r e g u l a t e d a t 25°C; samples were r u n i n 0.02 i o n i c s t r e n g t h b u f f e r s a t 44,770 rpm. P i c t u r e s were t a k e n a f t e r f u l l speed had been a t t a i n e d f o r 4 m i n u t e s . U l t r a v i o l e t Absorbance U l t r a v i o l e t absorbance was used f o r d e t e r m i n i n g 1% c o n c e n t r a t i o n by c o n v e r t i n g A 2 ° 0 t o c o n c e n t r a t i o n ; where 1% A ° n f o r K, a .,'and 8 - c a s e i n a r e 11.7, 10.9 ( N a k a i , 1965) l a u s l and 4.7 ( A s c h a f f e n b u r g , 1963). - 1 2 -A b s o r b a n c e a t 3 2 0 my ( l o n g u l t r a v i o l e t ) w a s u s e d t o m e a s u r e l i g h t s c a t t e r i n g a c c o r d i n g t o L a u f f e r ( 1 9 6 3 ) . A b s o r b a n c e o f p r o t e i n s o l u t i o n s f r o m 3 2 0 my t o 2 2 0 my w i t h c h a n g i n g p H w a s u s e d t o o b s e r v e s p e c t r a l s h i f t s a s T a n f o r d ( 1 9 5 2 ) d i d . A l l u l t r a v i o l e t a b s o r b a n c e s t u d i e s w e r e c a r r i e d o u t o n a B e c k m a n M o d e l D B S p e c t r o p h o t o m e t e r w i t h a P h o t o v o l t R e c o r d e r a t t a c h e d . N u c l e a r M a g n e t i c R e s o n a n c e N u c l e a r m a g n e t i c r e s o n a n c e s t u d i e s w e r e c a r r i e d o u t o n s a m p l e s w h i c h w e r e e x h a u s t i v e l y d i a l y z e d a g a i n s t 0 . 0 2 M N a C l . T h e s a m p l e s w e r e l y o p h i l i z e d . T h e l y o p h i l i z e d s a m p l e s w e r e e x c h a n g e d 3 t i m e s w i t h a n d t h e p H a d j u s t e d w i t h N a O D . S u c c e s s f u l r e s u l t s w e r e a c q u i r e d u s i n g s a m p l e s o l u t i o n s p r e p a r e d a p p r o x i m a t e l y 2 . 5 % W/W i n D 2 O . T h e i n s t r u m e n t u s e d w a s a V a r i a n H A 1 0 0 f i t t e d w i t h a t e m p e r a t u r e c o n t r o l l e d p r o b e . S a m p l e s w e r e r u n a n d r e s o n a n c e p o s i t i o n s r e c o r d e d r e l a t i v e t o a n e x t e r n a l l o c k s i g n a l o f t e t r a -m e t h y l s i l a n e . C i r c u l a r D i c h r o i s m T h e p r o t e i n s , m o d i f i e d p r o t e i n s a n d p r o t e i n - p o l y m e r s o l u t i o n s w e r e d i s s o l v e d a n d a l l o w e d t o e q u i l i b r a t e f o r a t l e a s t 4 h o u r s . T h e s e s t o c k s o l u t i o n s w e r e t h e n c h e c k e d f o r p r o t e i n c o n c e n t r a t i o n u s i n g u l t r a v i o l e t a b s o r b a n c e . C i r c u l a r d i c h r o i c m e a s u r e m e n t s w e r e m a d e o n a J a s c o M o d e l O R D / u v . T h e i n s t r u m e n t w a s c a l i b r a t e d b y u s i n g 5 a c h o l e s t a n - 3 - o n e . - 13 -i n 1,4 dioxane w h i c h had a d i f f e r e n c e *in m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t Ae,+1.13. Measurements were made i n 10 mm c e l l s a t v a r y i n g p r o t e i n c o n c e n t r a t i o n s . The r e s u l t s were r e p o r t e d i n terms o f mean r e s i d u e m o lar e l l i p t i c i t y ( 9 ) ; where (9) = K.Ae-]_c. 0 Where K i s mean r e s i d u e w e i g h t , 1 i s l i g h t p a t h l e n g t h and c i s c o n c e n t r a t i o n o f sample. T h e o r e t i c a l NMR S p e c t r a The t h e o r e t i c a l n u c l e a r m a g n e t i c r e s o n a n c e spectrum f o r 220 Mc/sec were c a l c u l a t e d as d e s c r i b e d by McDonald and P h i l i p s (196 7) f o r random c o i l c o n f i g u r a t i o n s . The amino a c i d a n a l y s i s f o r a ,, 8 , and K as g i v e n by McKenzie ( 1 9 6 8 ) . The n e c e s s a r y c o m p u t a t i o n s were done, t h i s f o l l o w e d by p l o t t i n g o f spectrum. T h e o r e t i c a l C a l c u l a t i o n s C a l c u l a t i o n s f o r t h e t o t a l number o f hydrogen and i o n i c bonds were made assuming c a r b o x y l and p r i m a r y amino groups were i o n i c and t h a t t h e number o f hydrogen bonds p e r r e s i d u e were as d e s c r i b e d i n T a b l e I I . The t o t a l numbers were c a l c u l a t e d from t h e amino a c i d a n a l y s i s . E l e c t r o p h o r e s i s Throughout t h i s r e s e a r c h b o t h p o l y a c r y l a m i d e and s t a r c h g e l e l e c t r o p h o r e s i s were used ( A s c h a f f e n b u r g , Thymann 1965; Thompson e t a l . , 1964). - 14 -RESULTS AND DISCUSSION C i r c u l a r D i c h r o i s m The purpose o f CD a n a l y s i s i n t h i s t h e s i s was t o d e t e r m i n e t h e CD s p e c t r a o f t h e major components o f c a s e i n : t o note changes i n t h e CD o f t h o s e components w i t h changes i n t h e e n v i r o n m e n t ; t o note t h e e f f e c t o f p r o t e i n m o d i f i c a t i o n on t h e CD s p e c t r a ; and t o o b s e r v e i f an i n t e r a c t i o n p r o d u c t c o u l d be d e t e c t e d between p r o t e i n and a s y n t h e t i c p olymer. The i m p o r t a n c e o f c i r c u l a r d i c h r o i s m as a t o o l f o r s t u d i e s on p r o t e i n s t r u c t u r e i s i t s extreme s e n s i t i v i t y t o m o l e c u l a r geometry. Most s p e c t r a l p r o p e r t i e s o f m o l e c u l e s a r e more o r l e s s a d d i t i v e and depend upon th e c o n s t i t u e n t c h e m i c a l g r o u p s . The r e l a t i v e g e o m e t r i e s o f t h e c o n s t i t u e n t groups i n t h e m o l e c u l e a l t e r p r o p e r t i e s and depending on t h e s e n s i t i v i t y o f t h e method a s e c o n d a r y e f f e c t on t h e m o l e c u l e s as a r e s u l t o f t h e s e groups w i l l be o b s e r v e d . C i r c u l a r d i c h r o i s m i s bound t o t h e i n t e r a c t i o n s among the groups and t h e r e f o r e t o m o l e c u l a r geometry. W i t h th e e x c e p t i o n o f a few r a r e m o l e c u l e s w h i c h have i n t r i n s i c a l l y a symmetric chromophores, CD o r i g i n a t e s d i r e c t l y from o r i e n t -a t i o n o f t h e m o l e c u l e i n a g i v e n e n v i r o n m e n t . T h i s o r i e n t a t i o n o f t r a n s i t i o n moments i n c o n s t i t u e n t groups i s d e t e c t e d b u t n o t th e o r i e n t a t i o n o f atoms o r bonds. - 15 -The u t i l i t y o f CD i n s t u d y i n g p r o t e i n c o n f o r m a t i o n a l changes depends upon t h e p o s s i b i l i t y o f c o r r e l a t i o n o f s p e c i f i c s t r u c t u r e s w i t h c h a r a c t e r i s t i c CD s p e c t r a . The CD o f a - h e l i x has been w e l l c h a r a c t e r i z e d by Beychok, T i m a s h e f f e t a l . (1967) and Yang (1967) i n terms o f wave l e n g t h s and e l l i p t i c i t i e s o f i t s t h r e e bonds. C o n t r a s t i n g i s t h e d e f i n i t i o n o f CD f o r random c o i l e d g l o b u l a r t y p e s . These have been s t u d i e d by C a r v e r , S c h e c h t e r and B l o u t (1966) b u t a r e not w e l l u n d e r s t o o d . The d e f i n i t i o n s p r o p osed have a l l been on the a s s u m p t i o n t h a t a p o l y p e p t i d e c h a i n w i t h c h a r g e d s i d e c h a i n groups i s a s a t i s f a c t o r y model f o r a random c o i l o r u n o r d e r e d s t r u c t u r e . L o i s , T i f f a n y , K r i m and Mark (19 64) have shown t h a t even random c o i l e d m o l e c u l e s have l o c a l extended h e l i x s t r u c t u r e . Thus t h e r e s u l t s o f CD s p e c t r a on u n o r d e r e d o r random c o i l cannot be i n t e r p r e t e d s t r i c t l y . C o n s i d e r a b l e changes i n o p t i c a l a c t i v i t y have been noted when r e m o v a l o f a s u p p o r t i n g e l e c t r o l y t e w h i c h causes t h e s t r u c t u r e t o expand and s t r e t c h out (Yang, 1965) was done. A l s o i n . p r o t e i n e s p e c i a l l y g l o b u l a r o r random c o i l , t h e s e a r e C o t t o n e f f e c t s a r i s i n g from a r o m a t i c chromophores. From our r e s u l t s comparing t h e c a s e i n f r a c t i o n s 8, K and whole c a s e i n we may c o n c l u d e t h a t t h e r e i s l i t t l e d i f f e r e n c e s t r u c t u r a l l y a t pH 7.0 between - 16 -any o f t h e s e f r a c t i o n s and whole c a s e i n ( f i g u r e 1 ) . I n f i g u r e 2 t h e o n l y c o n c l u s i o n s we may draw i s t h a t a t pH 10.5 £ c a s e i n appears t o m a i n t a i n i t s s t r u c t u r e s l i g h t l y more t h a n e i t h e r a , o r K c a s e i n . The e f f e c t o f pH on K • s i . r c a s e i n was t h a t extremes i n pH a f f e c t t h e s e c o n d a r y s t r u c t u r e ( f i g u r e 3 ) . Our r e s u l t s on K c a s e i n may be i n t e r p r e t e d as b e i n g (a) an i n c r e a s e d d i s s o c i a t i o n o f p r o t e i n s a t b o t h low and h i g h pH, r e s u l t i n g from d e c r e a s e d i n t r a m o l e c u l a r e l e c t r o s t a t i c i n t e r a c t i o n s and (b) development o f a p o s i t i v e l y a b s o r b i n g peak b e i n g a t t r i b u t e d t o t y r o s i n e (Beychok and Fasman, 1964). S i n c e t h e n a t u r e o f t h e m o d i f i c a t i o n s o f K c a s e i n a r e known t h e r e s u l t s t e n d t o c o n f i r m t h a t i n f i g u r e 4 and 5 t h a t t h e p r o t e i n i s more o r d e r e d i n t h e n a t i v e s t a t e a l s o t h a t t h e e f f e c t o f pH on t h e d i f f e r e n t forms o f K c a s e i n show t h a t t h e 240-270 nuj p o s i t i v e c o n t o u r e f f e c t i s t h e r e s u l t o f a r o m a t i c chromophore p r o b a b l y t y r o s i n e . T h i s i s s u p p o r t e d by t h e f a c t t h a t m e t h y l a t e d and r e d u c e d K c a s e i n i n f i g u r e 4 has p r o b a b l y some a l t e r a t i o n t o t h e OH o f t y r o s i n e . The o b s e r v e d e f f e c t o f 3 M u r e a on K c a s e i n , pH 7.0, f i g u r e 6, may c o n f i r m t h e h y p o t h e s i s t h a t c a s e i n s a r e \ i n a p a r t i a l l y d e n a t u r e d c o n d i t i o n . F i g u r e 7 f u r t h e r c o n f i r m s t h a t the c a s e i n s , s p e c i f i c a l l y <*s^ > a r e random c o i l e d i n t h e n a t i v e s t a t e because t h e r e i s e f f e c t i v e l y no - 17 -220 240 260 WAVE LENGTHrrijj 1. C i r c u l a r d i c h r o i c s p e c t r u m : whole c a s e i n ; a ^ - c a s e i n ; B - c a s e i n ; K - c a s e i n ; pH 7 . 0 , H^O-HCl 220 240 260 WAVE LENGTH JTILI 2. C i r c u l a r d i c h r o i c spectrum: a ^-casein; 3-casein; ic-casein; pH 10.5, s H^O-NaOH - 19 -220 240 260 WAVE LENGTH mp 3. C i r c u l a r d i c h r o i c s p e c t r u m : K - c a s e i n ; pH 7 . pH 3.6 and pH 10.5; H o0 ' 220 240 260 WAVE LENGTHrnp C i r c u l a r d i c h r o i c s p e c t r u m : K - c a s e i n ; sodium t e t r a t h i o n a t e ; m e t h y l a t e d and r e d u c e d ; n a t i v e pH 10.5, H 20-Na0H - 21 -t a ff J 220 240 260 WAVE LENGTH mp C i r c u l a r d i c h r o i c s p e c t r u m : K - c a s e i n ; sodium t e t r a t h i o n a t e ; m e t h y l a t e d and r e d u c e d ; n a t i v e ; pH 7..0, H 20 - 2 2 -c h a n g e i n t h e s t a t u s o f t h e C D u p o n m o d i f i c a t i o n w i t h S - a c e t y l m e r c a p t o s u c c i n i c a n h y d r i d e . I n t h e p r e s e n c e o f p o l y e t h y l e n i m i n e n o c h a n g e i n t h e C D s p e c t r a w a s o b s e r v e d a t p H 9 . 5 o r p H 1 1 . 0 . O u r r e s u l t s , i n b r i e f , h a v e s h o w n t h a t t h e c a s e i n s a -, , 8 a n d K h a v e a s y m m e t r i c r a n d o m c o i l s t r u c t u r e s w h i c h a s s o c i a t e i n a r a n d o m l y a s y m m e t r i c f a s h i o n , a n d f i n a l l y f o r m w h o l e c a s e i n w h e n t h e i r r e l a t i v e c o m p o s i t i o n s a l l o w . N u c l e a r M a g n e t i c R e s o n a n c e N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y h a s b e e n u s e d a s a t o o l t o i d e n t i f y t h e r e s o n a n c e s o f i n d i v i d u a l r e s i d u e s i n p r o t e i n s a n d t h e e f f e c t ' o f e n v i r o n m e n t a l c h a n g e s o n t h e s e g r o u p s . S i n c e l o w m o l e c u l a r w e i g h t c o m p o u n d s o f s i n g l e s t r u c t u r e h a v e b e e n s h o w n t o g i v e a d i s t i n c t s i g n a l , i t h a s b e e n s h o w n t h a t i t i s p o s s i b l e t o f i n d i n t e r a c t i o n s i t e s . T h e w i d t h s a n d s h a p e s o f r e s o n a n c e p e a k s h a v e b e e n u s e d t o o b t a i n d a t a o n t h e m o b i l i t i e s o f c h e m i c a l g r o u p i n g s i n s o l u t i o n . O u r o b j e c t i v e s w e r e i n a p p l y i n g t h i s m e t h o d t o s t u d y c o n f o r m a t i o n a l c h a n g e i n c a s e i n a n d , i f p o s s i b l e , t o d e t e r m i n e i n t e r a c t i o n p r o d u c t s . F i g u r e 8 , 9 a n d 1 0 a r e t h e o r e t i c a l l y c a l c u l a t e d 2 2 0 M c / s e c n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f c a s e i n s . T h e y w e r e c a l c u l a t e d f r o m t h e d a t a o n T a b l e I . T h e s e c a l c u l a t e d p a t t e r n s a r e i n g o o d s u b j e c t i v e a g r e e m e n t w i t h t h e 2 0 0 M c / s e c s p e c t r a w h i c h h a v e b e e n d o n e b y L e s l i e e j t a l . ( 1 9 6 9 ) a n d a l s o w i t h o u r r e s u l t s f o r a g l a n d < c a s e i n s ( f i g . 1 1 a n d 1 2 ) w i t h t h e 1 0 0 M c / s e c s p e c t r o m e t e r . T h e s e 10 I4L 220 240 260 WAVE LENGTHrnu 6. CD s p e c t r u m : A - c a s e i n ( ), pH 7.0 H 2 0 ; A - c a s e i n . (• ), pH 7-0 3M urea,. - 24 -220" 240 260 WAVE LENGTH JTIM 7. CD spectrumrcCSI - c a s e i n ( -), pH 7 .0 and pH I I . 0 ; m o d i f i e d & S l - c a s e i n (• ), pH 7.0 and pH 1 1 . 0 . - 2 5 -Table I. Calculations For Theoretical Spectra P r o t o n t y p e L e u c i n e E q u i v a l e n t R e s o n a n c e p r o t o n s / t r i a n g l e r e s i d u e p o s i t i o n T r i a n g l e b a s e H , H Z T r i a n g l e A l t i t u d e 3 K a s i C H 3 6 1 9 5 3 0 4 9 4 . 8 7 3 6 . 0 4 0 8 . 0 B - C H 2 + Y C H 3 3 3 6 0 4 0 1 8 5 . 5 2 7 6 . 0 1 5 3 . 0 I s o l e u c i n e C H 3 6 1 8 3 4 0 2 9 1 . 0 2 4 4 . 8 4 1 5 . 2 C H 2 1 2 0 0 6 0 3 2 . 0 2 6 . 9 4 5 . 7 C H 2 1 3 1 0 • 6 0 3 2 . 0 2 6 . 9 4 5 . 7 B - C H 1 4 2 5 5 0 3 8 . 8 3 2 . 6 5 5 . 4 V a l i n e C H 3 6 2 0 5 3 4 2 9 9 . 9 5 8 0 . 1 4 3 6 . 5 B - C H 1 4 9 5 5 0 3 4 . 1 6 5 . 9 4 9 . 6 A l a n i n e C H 3 3 3 1 0 T h r e o n i n e C H 3 3 2 7 0 L y s i n e . - C H 2 2 3 1 5 6 - C H 2 + 0 - C H 2 4 3 7 0 e - C H 2 2 6 6 5 A r g i n i n e - C H 2 2 3 6 5 B - C H 2 2 4 0 5 S - C H 2 2 7 0 4 P r o l i n e - C H 2 2 4 4 5 B - C H 2 2 4 6 5 S - C H 2 2 7 2 5 G l u t a m i c A c i d B - C H 2 2 4 3 5 - C H 2 2 5 0 0 3 6 1 1 7 . 6 7 2 . 3 2 7 9 . 9 3 2 8 9 . 9 1 3 4 . 8 2 7 2 . 7 6 0 8 6 . 3 6 2 . 8 7 8 . 9 6 0 1 7 1 . 6 1 2 4 . 6 1 5 6 . 7 4 4 1 1 7 . 2 8 5 . 3 1 0 7 . 1 5 6 3 1 . 9 2 3 . 6 5 2 . 7 4 8 3 7 . 3 2 7 . 6 6 1 . 5 2 8 6 3 . 5 4 6 . 9 1 0 4 . 7 1 4 0 . 0 2 6 2 . 2 2 3 5 . 4 5 0 1 1 7 . 9 2 2 0 . 8 1 9 8 . 2 6 0 9 8 . 8 1 8 4 . 9 1 6 6 . 0 4 0 3 3 2 . 6 3 2 6 . 4 3 2 0 . 6 4 0 3 3 2 . 6 3 2 6 . 4 3 2 0 . 6 - 26 -P r o t o n t y p e A s p a r t i c A c i d Resonance E q u i v a l e n t t r i a n g l e p r o t o n s / p o s i t i o n T r i a n g l e r e s i d u e H_z base HZ T r i a n g l e A l t i t u d e 8 K a s l B-CH 2 2 590 110 43.2 27.1 55.8 M e t h i o n i n e CHo 3 454 20 126.6 153.6 69.6 B-CH 2 2 454 44 38.4 46.6 21.1 -CH 2 2 565 32 52.8 64.0 29.0 C y s t e i n e B-CH 2 2 665 24 - - 11. 3 H i s t i d i n e B-CH 2 2 700 56 31. 8 334.6 31. 9 i m i d a z o l e C-H 1 1555 • 20 44.4 48 . 8 44.6 i m i d a z o l e C-Z 1 1740 20 44.4 38. 8 44.6 T y r o s i n e B-CH 2 2 685 60 58.1 21.4 73.2 a r o m a t i c o r t h o t o OH 2 1500 34 102.1 37 . 8 128.9 a r o m a t i c meta t o OH 2 1560 34 102 .1 37.8 128.9 P h e n y l a l a n i n e B-CH 2 1 650 60 17.6 24.6 17.9 B-CH 2 1 700 60 17.6 24.6 17 . 9 a r o m a t i c 5 1598 60 89.0 124.4 90.9 T r y p t o p h a n B-CH 2 2 745 84 14.7 7.7 7.4 I n d o l e C-2 1 1584 20 19. 9 10.4 10.0 I n d o l e C.-S 1 1549 30 13.3 6.97 6.7 I n d o l e C-6 1 1566 30. 13.3 6.97 6.7 I n d o l e C-4 1 1638 36 11.1 5.82 5.6 I n d o l e C-7 1 1658 36 11.1 5.82 5.6 Gl u t a m i n e B-CH 2 2 455 40 - - -Y - C H 2 2 510 40 - - -A s p a r a g i n e B-CH2 1 613 60 Y-CH2 1 637 60 - - -THEORETICAL N.MR SPECTRUM CCSI CASEIN 8 . T h e o r e t i c a l l y c a l c u l a t e d n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r u m o f a - c a s e i n , 2 2 0 M c / s e c PPM 9. T h e o r e t i c a l l y c a l c u l a t e d n u c l e a r m a g n e t i c r e s o n a n c e spectrum o f 8 - c a s e i n , 220 Mc/sec THEORETICAL NMR SPECTRUM K CASEIN 10. T h e o r e t i c a l l y c a l c u l a t e d n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r u m o f K - c a s e i n , 220 Mc/sec - 30 6.0 5.0 N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f a -, c a s e i n 2.5% i n D o0; 40°C, 70°C and 90°cf 100 Mc/sec N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f K c a s e i n 2.5% i n D 20; 40, 70 and 90°C; 100 Mc/sec - 31 -s p e c t r a were c o n s t r u c t e d from t h e r e s o n a n c e s o f i n d i v i d u a l amino a c i d r e s i d u e s c o n f i r m i n g t h e a d d i t i v i t y o f n u c l e a r -m agnetic r e s o n a n c e . These r e s u l t s i n d i c a t e d t h a t i n c r e a s e d t e m p e r a t u r e d e c r e a s e s hydrogen b o n d i n g . Hydrogen bonds, b o t h i n t r a m o l e c u l a r and i n t e r m o l e c u l a r , w i l l s h i e l d p r o t o n s as a r e s u l t o f t h e i r r e s t r i c t e d m o t i o n which i s an e f f e c t o f t h e bon d i n g on the 100 Mc/sec compared t o the t h e o r e t i c a l 220 Mc/sec NMR. An e f f e c t o f h e a t i s d e s h i e l d i n g w h i c h • r e s u l t s i n peak h e i g h t e n i n g , i n c r e a s e d r e s o l u t i o n and s l i g h t d o w n f i e l d s h i f t i n g . E f f e c t s o f pH on t h e re s o n a n c e o f polymers a r e peak h e i g h t e n i n g p r o b a b l y as a r e s u l t o f i n c r e a s e d h y d r a t i o n and s h i f t t o h i g h f i e l d r e s o n a n c e w i t h i n c r e a s e d pH, i n d i c a t i n g a s h i e l d i n g e f f e c t . T h i s c o u l d be i n t e r p r e t e d as t h a t t h e p r o t e i n has t h e combined e f f e c t o f d e s h i e l d i n g and s h i e l d i n g a t a g i v e n pH ( f i g u r e 1 3 ) . T h i s was s i m i l a r l y n o t i c e d i n a s y n t h e t i c o r g a n i c p o l y m e r , p o l y e t h y l e n i m i n e ( f i g u r e 1 4 ) . F u r t h e r s t u d i e s r e v e a l e d t h a t an i n t e r a c t i o n p r o d u c t between a , and PEI was pH dependent ( f i g u r e 1 5 ) . A l s o t h e i n t e r a c t i o n was o b s e r v e d between a ., and K c a s e i n , s l as i n f i g u r e 16. F i g u r e 16 a l s o i n d i c a t e d t h e s t a b i l i z i n g e f f e c t o f t h e c o p o l y m e r i z a t i o n when a h e a t t r e a t m e n t i s a p p l i e d . - 32 -13. N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f a c a s e i n 2.5% i n D O ; pH 9 . 7 'and 11.0; 100 Mc/sec 3.5 3.0 25 14. N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f p o l y e t h y l e n i m i n e .25% i n D O ; pH 9.7 and 11.0; 100 Mc/sec 1 3.0 2.0 1.0 15. N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f a , - c a s e i n 2.5% p o l y e t h y l e n i m i n e .25%; pH 9.7, 10.6, 11.0; 100 Mc/sec 16. N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f a , - c a s e i n 1.25% and K - c a s e i n 1.25% i n s l D 20; 40, 70 and. 90°C; 100 Mc/sec - 34 -F o l l o w i n g t h e a c q u i r i n g o f t h e s e r e s u l t s , a s e r i e s o f t r e a t m e n t s were pursued i n o r d e r t o a n a l y z e t h e changes o f peak w i d t h , h e i g h t s and p o s i t i o n s r e s u l t i n g f r om t e m p e r a t u r e , pH and i n t e r a c t i o n . F i g u r e s 17 and 18 show the e f f e c t o f c o p o l y m e r i z a t i o n on t h e peak p o s i t i o n s . F i g u r e 17 d e m o n s t r a t e s t h e pH dependence o f c o p o l y m e r i z a t i o n o f a ^.and'PEI. F i g u r e 18 shows t h e e f f e c t o f h e a t i n g on a . and K c a s e i n c o p o l y m e r i z a t i o n and s t a b i l i z a t i o n . F i g u r e 19 d e m o n s t r a t e s t h e e f f e c t o f t e m p e r a t u r e on t h e peak, h e i g h t s o f a ^ and K c a s e i n . T h i s was done i n o r d e r t o compare h e a t s t a b i l i t y o f t h e p r o t e i n s . F i g u r e s 20, 21 and 22 f u r t h e r e l a b o r a t e d t h e h e a t s t a b i l i t y .of a , r e l a t i v e t o < c a s e i n by comparing the degree o f c h e m i c a l s h i f t i n g as a r e s u l t o f h e a t t r e a t m e n t . F i g u r e 23 compares the s h i f t i n K / C X^ copolymer t o a ^ and K c a s e i n as i n t h e f i g u r e s 20, 21 and 22 showing by NMR^the copolymer e f f e c t . F u r t h e r e m p i r i c a l d a t a c o u l d be d e r i v e d from t h e s e e x p e r i m e n t s , however, i t was deemed u n r e l i a b l e s i n c e i t was o n l y q u a l i t a t i v e . I n t e r a c t i o n The s t u d y o f i n t e r a c t i o n and c o p o l y m e r i z a t i o n o f a ., c a s e i n and PEI was s t u d i e d t o e l a b o r a t e on t h e i n t e r a c t i o n s i o f t h e c a s e i n s . F i g u r e 24 compares the uv absorbance o f a , and P E I . PEI shows l i t t l e o r no 280 my a b s o r b a n c e , a l l 17. E f f e c t o f pH on c h e m i c a l s h i f t o f p o l y e t h y l e n i m i n e i n and o u t o f t h e p r e s e n c e o f a c a s e i n ; p o l y e t h y l e n i m i n e / a ^ c a s e i n 1/10; ^ c a s e i n 2.5%; H»0 room t e m p e r a t u r e - 36 -2.701-40 ~~50 ©o " 7 0 80 90 TEMPERATURE °C 18. E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f t h e co p o l y m e r peaks o f U^^/K c a s e i n 1/1 • o 20 40 60 80 TEMPERATURE °C 3. Changes i n peak h e i g h t o f s p e c t r u m o f K and a , c a s e i n 2.5% w/w D 20 w i t h change i n t e m p e r a t u r e a , - c a s e i n K - c a s e i n S X TEMPERATURE °C 2 0 . E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f o . and K - c a s e i n TEMPERATURES 2 1 . E f f e c t o f t e m p e r a t u r e on t h e c h e m i c a l s h i f t o f a and K - c a s e i n FEMPERATURE °C 22. E f f e c t of temperature on the chemical s h i f t of a , and K-casein s i - 4 1 -absorbance below 2 7 0 my r e s u l t s from t h e a z i r i d i n e r i n g . F u r t h e r c o n t r a s t i n g PEI t o a ,, as w e l l showing i t s m o n o t e r i c s t a t u s , was t h e t i t r a t i o n o f a , and PEI w i t h s i a c i d and base as shown i n f i g u r e 2 4 . The i n t e r a c t i o n o f PEI and a .. was o b s e r v e d t o s i be pH dependent and a l s o i t was shown t h a t t h e i n t e r a c t i o n p r o d u c t o r copolymer had an i s o l e c t r i c o r f l o c c u l a t i o n p o i n t w h i c h was o b s e r v e d a p p r o x i m a t e l y a t pH 9 . 5 ( f i g u r e 2 5 ) . H y d r o l y s i s o f t h e polymer l i b e r a t i n g a l l p r i m a r y amine r e s u l t e d i n a s h i f t o f f l o c c u l a t i o n p o i n t w h i c h was a p p r o x i m a t e l y t h e i s o e l e c t r i c p o i n t o f whole c a s e i n , pH 4 . 6 ( f i g u r e 2 6 ) . The i n t e r a c t i o n o f PEI and a . c a s e i n was s i m o n i t o r e d by measuring. OD 3 2 0 my. The p o l y m e r i z a t i o n o f a - c a s e i n w i t h a d d i t i o n o f i n c r e m e n t a l amounts o f PEI s showed t h a t t h e p o l y m e r i z a t i o n was c o n c e n t r a t i o n dependent and a l s o t h a t r e m o v a l o f p r i m a r y amino groups r e a d i l y a f f e c t e d t h e c o p o l y m e r i z a t i o n w h i c h was c o n c e n t r a t i o n dependent ( f i g u r e 2 7 ) . U r e a , a w e l l known d i s s o c i a t i n g a g e n t , was used i n an a ttempt t o d i s s o c i a t e t h e c o p o l y m e r s . F i g u r e 2 8 c o n f i r m s t h e a b i l i t y o f u r e a t o d i s s o c i a t e t h i s i n t e r a c t i o n t y p e . U l t r a v i o l e t Absorbance P r o t e i n s o l u t i o n s commonly have shown maximum a b s o r p t i o n a t w a v e l e n g t h s w i t h i n 2 7 0 - 2 9 0 my. W i t h changes - 42 -240 280 320 'AVE LENGTH mu 23. Comparison o f u l t r a v i o l e t a b s o r b a n c e o f a g l and p o l y e t h y l e n i m i n e > pH 26. E f f e c t o f pH on t h e i n t e r a c t i o n o f p o l y e t h y l e n i m i n e and a , c a s e i n . a f t e r m o d i f i c a t i o n o f PEI s l A c i d H y d r o l y s i s : 2 N HC1 i n b o i l i n g w a t e r ^—A-Ar HYDR0LY2ED PEI 0 08 ro O 0.0 0.002 0.006 PEI ttSI 27. E f f e c t o f p o l y e t h y l e n i m i n e c o n c e n t r a t i o n on t h e f l o c c u l a t i o n o f a n c a s e i n s l 0 1 2 3 4 5 M UREA 28. E f f e c t of urea on the copolymer of a ^-casein polyethylenimine - 48 -i n t h e environment changes i n t h i s a bsorbance have been o b s e r v e d . I n p a r t i c u l a r , l a r g e s h i f t s i n p r o t e i n absorbance i n t h e r e g i o n 240-300 my have been a t t r i b u t e d t o changes i n t h e s t a t u s o f t r y p t o p h a n , t y r o s i n e and p h e n y l a l a n i n e (Beaven, 1961; W e t l a u f e r , 1962). The f a r - u l t r a v i o l e t r e g i o n 240-185 my has been shown t o c o n t a i n absorbance o f t h e p e p t i d e o r amide chromophore. I t has been shown t h a t p e r t u r b a t i o n s o f t h e s e s p e c t r a a r e i n f l u e n c e d by th e c o n f o r m a t i o n o f p o l y p e p t i d e c h a i n s ( G r o t z e , 1967). The r e s u l t s o f K-casein ( f i g u r e 29) show, w i t h an i n c r e a s i n g pH, t h e r e was a marked i n c r e a s e i n t h e absorbance maximum a t 280 my and a s l i g h t d e c r e a s e i n t h e 320 my absorbance ( G l a z e r , McKenzie and Wake, 1957) have shown t h a t t h e s e s p e c t r a l changes a r e i n f l u e n c e d and c o n t i n u o u s w i t h t h e m o l e c u l a r e x p a n s i o n o f t h e p r o t e i n m o l e c u l e . F i g u r e 3 0 was/attempted t o show t h e e f f e c t o f t e m p e r a t u r e and c o n c e n t r a t i o n on t h e a u t o p o l y m e r i z a t i o n o f K-casein. The r e s u l t s s u p p o r t t h e h y p o t h e s i s t h a t i n c r e a s e d t e m p e r a t u r e a f f e c t s t h e hydrogen b o n d i n g o f t h e m o l e c u l e i n t o s o l u t i o n and ,thus i n c r e a s e d t e m p e r a t u r e a t a s p e c i f i e d c o n d i t i o n enhances phase t r a n s i t i o n from aqueous t o i o n i c c o n d i t i o n . T h e o r e t i c a l The r e s u l t s o f T a b l e I I show i n o r d e r o f i n c r e a s i n g number o f hydrogen bonds p e r m o l e c u l e K, a , and 8 c a s e i n ; 29. E f f e c t o f pH on t h e u l t r a v i o l e t a b s o r b a n c e o f K-casein - 51 -i n c r e a s i n g number o f c a r b o x y l groups p e r m o l e c u l e K , 8 and a , c a s e i n ; i n c r e a s i n g number o f amide groups p e r S -L m o l e c u l e 8, a and K c a s e i n ; i n c r e a s i n g number o f charg e d S JL i o n i c groups p e r hydrogen bond p e r m o l e c u l e 8, K and a ,. c a s e i n . The i n t e r p r e t a t i o n o f t h i s d a t a was t h a t was t h e most e l e c t r o n e g a t i v e w h i l e K was t h e most e l e c t r o -p o s i t i v e . Assuming t h a t t h e m o l e c u l a r i n t e r a c t i o n s were p u r e l y e l e c t r o s t a t i c t h e i n t e r a c t i o n o f a g and < c a s e i n s w ould be t h e s t r o n g e s t , r a t h e r t h a n a -,-8, o r 8-a -, i n t e r a c t i o n s . I n t e r p r e t i n g t h e s i g n i f i c a n c e o f hydrogen bond numbers p e r m o l e c u l e would r e s u l t i n c o n c l u s i o n t h a t 8 c a s e i n was t h e most h e a t s e n s i t i v e - s i n c e . i t i s w e l l documented t h a t hydrogen bonds a r e v e r y h e a t l a b i l e . The r e s u l t s may a l s o be i n t e r p r e t e d t h a t K c a s e i n i s t h e most a c i d s o l u b l e w h i l e a , i s t h e most base s o l u b l e o f t h e s e s l . t h r e e f r a c t i o n s . C o n s i d e r i n g s o l u b i l i t y i n aqueous i o n i c s o l u t i o n s t h e r e s u l t s show t h a t i n b a s i c c o n d i t i o n s K c a s e i n was most h e a t l a b i l e w h i l e i n a c i d c o n d i t i o n a ., s l was t h e most h e a t l a b i l e . Amino A c i d A n a l y s i s The r e s u l t s o f our a m i n o - a c i d a n a l y s i s o f m o d i f i e d ( e s t e r i f i e d and red u c e d ) K - c a s e i n and m o d i f i e d and s t o r e d a t room t e m p e r a t u r e and a t m o s p h e r i c c o n d i t i o n s f o r s i x months. T a b l e I I I showed t h a t most o f t h e a s p a r t i c and g l u t a m i c a c i d r e s i d u e s were i n t h e amidated form. The r e s u l t s showed t h a t - 52 -Ta b l e I I . T h e o r e t i c a l C a l c u l a t i o n o f Bonds and I o n i z a b l e Groups Number of \ hydrogen bonds p e r r e s i d u e K & °Sl G l y c i n e 2 6.8 10.4 17.0 A l a n i n e 2 24. 8 11.2 19. 2 S e r i n e 3 32.4 48.3 44.7 T h r e o n i n e 2 21. 3 32.1 19. 8 P r o l i n e 2 35.6 65.6 36.4 V a l i n e 2 17.6 43.4 24.6 I s o l e u c i n e 2 19.6 . 21.0 21.2 L e u c i n e 2 19.6 44.2 35.6 P h e n y l a l a n i n e 2 9.6 17.4 16.3 T y r o s i n e •3 32.4 13.2 31. 8 Try p t o p h a n 3 5.4 3.0 8.4 Cys/2 3 3.3 0.0 0.0 M e t h i o n i n e 2 3.0 11.4 8.8 A s p a r t i c A c i d 4 4 5.6 36. 8 61.6 G l u t a m i c A c i d 4 98.0 15 7.6 153.2 A r g i n i n e 5 26.0 24. 5 29.0 L y s i n e 3 27.9 33.3 81.0 Ammonia 1 24.7 2 8.6 27.0 H i s t i d i n e 4 7.6 20.0 18.4 461. 8 621.9 654. 0 Number of h hydrogen bonds p e r m o l e c u l a r w e i g h t 20,000 25,000 27,000 h Hydrogen bonds/20,000 M.W. 461.8 497.9 484.3 h Hydrogen bonds/20,000 M.W. i f NH^ Ammonia Number i o n i z a b l e groups coo"/NH 3 Number charged groups p e r hydrogen bonds Number o f coo- groups Number o f NH~ + groups 437.1 0.2523 0.1272 .056 . .222 474.6 0.4040 0.1171 .080 .198 464.6 0.49 0 8 0.1297 .099 .201 - 53 -when t h e m o d i f i e d p r o t e i n was s t o r e d a t room t e m p e r a t u r e and a t m o s p h e r i c c o n d i t i o n s t h e r e s u l t s became a l t e r e d t h i s p r o b a b l y was a r e s u l t o f o x i d a t i o n r e a c t i o n . I f t h e mode o f K-a , s t a b i l i z a t i o n i s v i a c a r b o x y l -s l J amide i n t e r a c t i o n t h e n r e m o v a l o f amide would d e c r e a s e s t a b i l i z i n g a b i l i t y . T h i s l o s s o f s t a b i l i z i n g a b i l i t y as a r e s u l t o f amide h y d r o l y s i s has been s u g g e s t e d by N a k a i and T a t t o ( 1 9 6 8 ) . I t was t h e purpose o f t h i s m o d i f i c a t i o n p r o c e d u r e t o d e t e r m i n e amide from g l u t a m i c and a s p a r t i c r e s i d u e s and a l s o t o form a m o d i f i e d p r o t e i n . U l t r a c e n t r i f u g a t i o n U l t r a c e n t r i f u g a t i o n was c a r r i e d out i n o r d e r t o d e t e r m i n e t h e e f f e c t o f pH on S v a l u e s o f t h e p r o t e i n s a -, s l and t c - c a s e i n as w e l l as t h e e f f e c t o f pH on t h e a s s o c i a t i o n o f p o l y e t h y l e n i m i n e and a 1 c a s e i n , t a b l e IV. Our r e s u l t s showed t h a t i n c r e a s e d pH r e s u l t s i n d i s s o c i a t i o n o f a , and < c a s e i n . T h i s i s c o n s i s t e n t s l w i t h t h e r e s u l t s o f McKenzie and Wake (1 9 5 9 ) . Our sediment-a t i o n v a l u e f o r c a s e i n was s l i g h t l y l o w e r t h a n t h e i r v a l u e , p r o b a b l y t h i s i s e x p e r i m e n t a l e r r o r . The l a r g e polymer s i m u l a t e d a c o m p l e t e l y d i s s o c i a t e d p r o t e i n such as K c a s e i n a t pH 11.0 p o s s e s s i n g a s e d i m e n t a t i o n v a l u e o f 1.3. I n t e r a c t i o n o b s e r v e d d u r i n g t h e r u n o f a , - c a s e i n s l and p o l y e t h y l e n i m i n e was p r o b a b l y due t o amino c a r b o x y l - 54 -T a b l e I I I . Amino A c i d A n a l y s i s o f < C a s e i n Amino A c i d s C o n t r o l M o d i f i e d S t o r e d M o d i f i e d A s p a r a g i n e 5.74 5.94 T h r e o n i n e 7.29 8.08 7.70 S e r i n e 8.64 5.22 6.16 G l u t a m i n e 10.32 16.70 P r o l i n e 12.39 11.1 1.69 G l y c i n e 1.49 1.08 2 .48 A l a n i n e 8.38 6.36 9.46 H a l f c y s t i n e V a l i n e 6.20 6.42 6.83 M e t h i o n i n e 1.16 . 84 1.52 I s o l e u c i n e 6.92 7.41 7.00 L e u c i n e 5.10 5.10 5.1 T y r o s i n e 5.46 4.94 2.49 P h e n y l a l a n i n e 2.77 2.43 2.51 A s p a r t i c a c i d 7.65 1.91 •71 G l u t a m i c a c i d 16.03 5.71 - 55 -T a b l e IV. R e s u l t s o f U l t r a c e n t r i f u g a t i o n S t u d i e s S e d i m e n t a t i o n V a l u e s M a t e r i a l C o n d i t i o n S V a l u e a , - c a s e i n pH 11.0 1.1 s i r a T - c a s e i n pH 7.0 3.0 s i P o l y e t h y l e n i m i n e pH 11.0 1.3 P o l y e t h y l e n i m i n e / a s ^ pH 10.8 1.5 K-casein pH 7.0 5.9. K-casein pH 11.0 - 56 -i n t e r a c t i o n s . Very s l i g h t i n t e r a c t i o n would be a n t i c i p a t e d s i n c e n o t a l l o f c a r b o x y l groups would be d i s s o c i a t e d a l s o l e s s t h a n 10% o f t h e amino group would be p r o t o n a t e d . These r e s u l t s appeared t o s u p p o r t o u r h y p o t h e s i s o f a s s o c i a t i o n a l s o o u r NMR r e s u l t s . E l e c t r o p h o r e s i s R e s u l t s o f e l e c t r o p h o r e s i s were used t o phenotype f r a c t i o n s o f a , , B and K c a s e i n . We s h o u l d m e n t i o n t h a t t h i s s l ' t e c h n i q u e would o n l y d e t e c t d i f f e r e n c e s i n a changed amino a c i d on d i f f e r e n c e s i n e x t e n t s o f a m i d a t i o n assuming t h a t phosphate and s u g a r c o n t e n t s were c o n s t a n t . D i f f e r e n c e s i n amino a c i d s u b s t i t u t i o n such as g l y c i n e f o r a l a n i n e o r g l u t a m i c f o r a s p a r t i c would not be d e t e c t e d . A l s o s i n c e e x t e n t s o f o x i d a t i o n a r e d i f f e r e n t one might s u s p e c t t h a t s u b f r a c t i o n s c o u l d show v a r i a t i o n i n t h e i r amide n i t r o g e n c o n t e n t . However, s i n c e e l e c t r o p h o r e s i s i s me a s u r i n g t h e mean change we might s u s p e c t t h a t w i t h i n a g i v e n e l e c t r o p h o r e t i c band o r p o p u l a t i o n o f m o l e c u l e s s e v e r a l v a r i a t i o n s would be p r e s e n t , t h e mean change from band t o band b e i n g o b s e r v e d . B e s i d e s u s i n g e l e c t r o p h o r e s i s as a c r i t e r i a o f p u r i t y i n t h e s e i n v e s t i g a t i o n s , i t was a l s o used t o o b s e r v e t h e i n t e r a c t i o n o f a ^ c a s e i n and p o l y e t h y l e n i m i n e . I t was found t h a t p o l y e t h y l e n i m i n e PEI m i g r a t e d t o t h e o p p o s i t e p o l e t h a t t h e c a s e i n s d i d and i t showed s l i g h t amido b l a c k s t a i n i n g . - 5 7 -In the presence of urea at pH 9.1 no i n t e r a c t i o n product was observed however i n the absence of urea absolutely no migration i n the gel was shown upon st a i n i n g . - 58 -CONCLUSION The r e s u l t s o f t h i s t h e s i s d i d not l e n d t h e m s e l v e s t o a s p e c i f i c c o n c l u s i o n r e g a r d i n g t h e m o l e c u l a r s t r u c t u r e o r i e n t a t i o n o r i n t e r a c t i o n o f K , and 8 - c a s e i n , however, t h e y d i d a l l o w f o r a g e n e r a l c o m p a r i s o n and c o n c l u s i o n . The c i r c u l a r d i c h r o i s m o f a ., , 6, and K - c a s e i n were s l n o t s i g n i f i c a n t l y d i f f e r e n t a t e i t h e r pH 7.0 o r pH 10.5. M o d i f i c a t i o n o f i c - c a s e i n w i t h s o d i u m - t e t r a t h i o n a t e o r . r e d u c t i o n and m e t h y l a t i o n d i d not r e s u l t i n a . p r o t e i n d e r i v a t i v e w h i c h had o p t i c a l p r o p e r t i e s d i f f e r e n t f r om n a t i v e K - c a s e i n a t pH 7.0. The t h e o r e t i c a l l y c a l c u l a t e d n u c l e a r m a g n e t i c s p e c t r a o f o t g ^ , 8 and K - c a s e i n were s i g n i f i c a n t l y d i f f e r e n t i n t h e i r r e l a t i v e d i s t r i b u t i o n o f r e s o n a t i n g p r o t o n s . The a c t u a l n u c l e a r m a g n e t i c s p e c t r u m o f and K - c a s e i h were d i f f e r e n t and r e a d i l y d i s t i n g u i s h a b l e . They were e a s i l y r e l a t e d t o t h e t h e o r e t i c a l s p e c t r a . I n t e r a c t i o n p r o d u c t s o f c c ^ - c a s e i n w i t h p o l y e t h y l -e n i m i n e and K - c a s e i n were d e t e c t e d and o b s e r v e d . The i n t e r a c t i o n o f and p o l y e t h y l e n i m i n e was found t o be not o n l y pH and c o n c e n t r a t i o n dependent b u t a l s o dependent upon t h e p r i m a r y amide groups i n t h e po l y m e r . The i n t e r a c t i o n p r o d u c t o f a g l and p o l y e t h y l e n i m i n e was d i s p e r s e d by u r e a . - 59 -K-casein c o n t a i n s a p p r o x i m a t e l y 75% o f i t s a s p a r t i c a c i d as a s p a r a g i n e and a p p r o x i m a t e l y 63% o f i t s g l u t a m i c a c i d as g l u t a m i n e . - 60 -BIBLIOGRAPHY A s c h a f f e n b u r g , R. 1963. P r e p a r a t i o n o f 3 - c a s e i n by m o d i f i e d u r e a f r a c t i o n a t i o n method. J . D a i r y Res. 30_: 2 59. A s c h a f f e n b u r g , R. and Thymann, M. 1965. 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