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UBC Theses and Dissertations

Studies on the structure and interaction of bovine k-casein, using fluorometric techniques Clarke, Reginald 1971

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STUDIES ON THE STRUCTURE AND INTERACTION OF BOVINE K -CASEIN, USING FLUOROMETRIC TECHNIQUES by REGINALD CLARKE B . S c # / S i r George W i l l i a m U n i v e r s i t y , 1961 M.Sc., U n i v e r s i t y o f Montreal, 1963 A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Food S c i e n c e We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d . THE UNIVERSITY OF June, BRITISH 1971 COLUMBIA In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f E r i t i s h Columbia, I agree 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 study. I f u r t h e r agree t h a t p e r m i s s i o n 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 gran 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 . I t i s understood t h a t copying 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 allowed without my w r i t t e n p e r m i s s i o n . REGINALD CLARKE Department o f Food Science The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The s t r u c t u r e and i n t e r a c t i o n s o f ;K - c a s e i n were s t u d i e d u s i n g s p e c t r o f l u o r o m e t r i c techniques i n c l u d i n g , f l u o r e s c e n t y i e l d , e m ission wavelength, energy t r a n s f e r , and f l u o r e s c e n c e d e p o l a r i s a t i o n . The e f f e c t o f denatu-r i n g f a c t o r s , temperature, sodium dodecyl s u l f a t e , i o n i c s t r e n g t h , urea and pH, on the f l u o r e s c e n t c h a r a c t e r i s t i c s o f a con j u g a t e o f K - c a s e i n and 8 - a n i l i n o n a p h t h a l e n e - l -s u l f o n a t e a r e i n d i c a t i v e o f s t r u c t u r a l changes i n the p r o t e i n . The importance o f hydrogen bonding and hydro-p h o b i c i n t e r a c t i o n s f o r the maintenance o f the s t r u c t u r a l i n t e g r i t y o f the molecule have been i n d i c a t e d . The s t r u c t u r a l changes a r e e f f e c t e d w i t h o u t the use o f d i s u l f i d e r e d u c i n g r e a g e n t s . The d i s s o c i a t i n g agents, urea and sodium d o d e c y l s u l f a t e , had v e r y s i g n i f i c a n t e f f e c t s upon the conformation o f the p r o t e i n . Temperature induced changes were r e l a t e d to the bonding which m a i n t a i n e d the conformation. Completely d i s s o c i a t e d o r random s t r u c t u r e s d i d not e x i s t except a t h i g h pH, i n 8M u r e a , o r i n 0.005M sodium dodecyl s u l f a t e . The i n t e r a c t i o n between a s i ~ a n a " < - c a s e i n was s t u d i e d by the f l u o r e s c e n c e p o l a r i s a t i o n t e c h n i q u e . The thermodynamic parameters A S and A H i n c r e a s e d w i t h temperature. A S and A H were p o s i t i v e . A F was n e g a t i v e and decreased f u r t h e r w i t h i n c r e a s i n g temperature. These data suggest t h a t the ease o r s p o n t a n e i t y o f i n t e r -a c t i o n i n c r e a s e s w i t h temperature, and t h a t the i n t e r a c t i o n i s h y drophobic i n n a t u r e . The r e s u l t s o f the i n t e r a c t i o n s t u d i e s a t 40 and 50°C demonstrated a 1:1 mole r a t i o f o r the i n t e r a c t i n g p r o t e i n s . B l o c k i n g o f the s i t e s f o 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 , the n e g a t i v e charges on the a s i - c a s e i n , by p o l y e t h y l i m i n e d i d not i n h i b i t the r e a c t i o n o r a f f e c t the i n t e r a c t i o n r a t i o . I t was t h e r e f o r e concluded t h a t a s i - a n d K c a s e i n s i n t e r a c t through hydrophobic i n t e r a c t i o n s i n a 1:1 mole r a t i o . Kappa-casein was m o d i f i e d i n a stepwise manner by (1) c a r b a m y l a t i o n o f the e -amino groups o f l y s i n e w i t h potassium i s o c y a n a t e (2) e s t e r i f i c a t i o n o f the f r e e c a r b o x y l i c groups u s i n g the c a r b o d i i m i d e method and g l y c i n e methyl e s t e r , s t r u c t u r a l changes i n the K - c a s e i n , - i v -thus m o d i f i e d , were f o l l o w e d by measuring changes i n f l u o r e s c e n t p r o p e r t i e s o f a conjugate o f the p r o t e i n s w i t h 8 ^ a h i l i n o - n a p h t h a l e n e - l - s u l f o n a t e . These s t r u c t u r a l changes were i n d i c a t i v e o f d e c r e a s i n g h y d r o p h o b i c i t y o f the c a s e i n s and l o s s o f s t a b i l i s i n g a b i l i t y o f a s 3. - c a s e i n a g a i n s t c a l c i u m i o n p r e c i p i t a t i o n . I t i s concluded t h a t the charged groups a r e r e s p o n s i b l e f o r the maintenance o f the p r o t e i n s t r u c t u r e , b ut may not be d i r e c t l y r e l a t e d t o s t a b i l i s i n g a b i l i t y o r i n t e r a c t i o n w i t h a s i - c a s e i n . -v-TABLE OF CONTENTS Page LIST OF TABLES v i i i LIST OF FIGURES i x LIST OF PLATES x i i i ACKNOWLEDGEMENT X i v INTRODUCTION 1 LITERATURE REVIEW 4 CHAPTER I . FLUORESCENT STUDIES OF K -CASEIN WITH  8-ANILINONAPHTHALENS-1-SUL FONAT S INTRODUCTION '14 MATERIALS AND METHODS 16 RESULTS 20 DISCUSSION 36 CHAPTER I I . KAPPA-g „ ,-CASEIN INTERACTION BY FLUORESCENCE  POLARISATION INTRODUCTION 43 MATERIALS AND METHODS 47 THEORY 49 RESULTS 54 DISCUSSION 6 5 CHAPTER I I I . EFFECT OF MODIFICATION OF CHARGED GROUPS  OF K -CASEIN ON ITS STRUCTURE AND  STABILISING ABILITY INTRODUCTION 71 - v i -MATERIALS AND METHODS 74 RESULTS 76 DISCUSSION 8 5 GENERAL DISCUSSION AND CONCLUSION 92 LITERATURE CITED 95 - v i i -LIST OF TABLES T a b l e Page I V a r i a t i o n i n P o l a r i s a t i o n o f a s 1 and a — K - c a s e i n Complex w i t h s 1 Temperature. 61 I I V a r i a t i o n i n A s s o c i a t i o n Constant o f the a s ^- K - c a s e i n I n t e r a c t i o n w i t h Temperature. 62 I I I Thermodynamic Parameters f o r the I n t e r -a c t i o n a s i ~ K - c a s e i n , and T h e i r Tem-p e r a t u r e Dependence. 64 IV Comparison o f the F l u o r e s c e n t Parameters o f a , , 3 . and. < - c a s e i n s . 84 s 1 - v i i i -LIST OF FIGURES Fi g u r e Page 1 E f f e c t o f K - c a s e i n c o n c e n t r a t i o n upon the number o f hydrophobic r e g i o n s and r e l a t i v e f l u o r e s c e n t i n t e n s i t y o f K -casein-ANS conjugate. 21 2 E f f e c t o f K - c a s e i n c o n c e n t r a t i o n on energy t r a n s f e r and p o l a r i s a t i o n o f K -casein-ANS conjugate. 22 3 E f f e c t o f s a l t c o n c e n t r a t i o n on r e l a t i v e f l u o r e s c e n t i n t e n s i t y and number o f hydro-phobic r e g i o n s f o r the < -casein-ANS conjugate. 24 . 4 E f f e c t o f s a l t c o n c e n t r a t i o n o f energy t r a n s f e r and p o l a r i s a t i o n o f K - c a s e i n -ANS conjugate. 25 5 E f f e c t o f urea on r e l a t i v e f l u o r e s c e n t i n -t e n s i t y and maximum emission wavelength of K -casein-ANS conjugate. 26 6 E f f e c t o f urea on energy t r a n s f e r and p o l a r i s a t i o n o f < -casein-ANS conjugate. 27 - i x -Page E f f e c t o f sodium d o d e c y l s u l f a t e on r e l a t i v e fluorescent., i n t e n s i t y and maximum emission wavelength o f K -casein-ANS conjugate. 29 E f f e c t o f sodium d o d e c y l s u l f a t e on energy t r a n s f e r and p o l a r i s a t i o n o f < - c a s e i n -ANS conjugate. 30 E f f e c t o f sodium d o d e c y l s u l f a t e on r e l a t i v e f l u o r e s c e n t i n t e n s i t y o f ANS. 31 E f f e c t o f pH on r e l a t i v e f l u o r e s c e n t i n -t e n s i t y and maximum emission wavelength o f < -casein-ANS conjugate. 32 E f f e c t o f temperature on r e l a t i v e f l u o r -escent i n t e n s i t y and number o f hydrophobic r e g i o n s o f < -casein-ANS conjugate. 34 E f f e c t o f temperature on energy t r a n s f e r o f K -casein-ANS conjugate., 35 P o l a r i s a t i o n o f d a n s y l a t e d a , - c a s e i n . s 1 _5 4 X 10 M t i t r a t e d w i t h < - c a s e i n a t v a r i o u s temperatures. 55 P o l a r i s a t i o n o f d a n s y l a t e d a - c a s e i n , s 1 F i g u r e Page 4 X 10" 5M w i t h and without added PEI t i t r a t e d w i t h K - c a s e i n a t v a r i o u s temperatures. 56 15 P o l a r i s a t i o n of d a n s y l a t e d a - c a s e i n , s 1 5 X 10~ 5M t i t r a t e d with K - c a s e i n a t 40°C and 4°C. 58 16 Log K f o r the a s s o c i a t i o n e q u i l i b r i u m p l o t -t e d a g a i n s t the e q u i l i b r i u m temperatures. 59 17 P e r r i n p l o t f o r a , - ic - c a s e i n complex. s 1 o Temperature range, 37 to 50 C. 18 E f f e c t o f m o d i f i c a t i o n o f c a r b o x y l i c a c i d f u n c t i o n s o f < - c a s e i n on r e l a t i v e f l u o r e s c e n t i n t e n s i t y , and wavelength o f maximum emission o f ANS conjugate. 77 19 E f f e c t o f m o d i f i c a t i o n o f c a r b o x y l i c a c i d f u n c t i o n s o f K - c a s e i n on , energy t r a n s f e r and p o l a r i s a t i o n o f ANS conjugate 78 - x i -F i g u r e Page 20 E f f e c t o f m o d i f i c a t i o n o f c a r b o x y l i c a c i d f u n c t i o n s o f < - c a s e i n on i t s a b i l i t y t o s t a b i l i s e a , - c a s e i n and on i t s own s 1 s o l u b i l i t y i n the presence o f 0.02M calcium i o n . ' 79 21 E f f e c t o f m o d i f i c a t i o n o f e -amino groups o f l y s i n e o f K - c a s e i n on r e l a t i v e f l u o r -escent i n t e n s i t y , and wavelength o f maximum emission o f ANS conjugate. 80 22 E f f e c t o f m o d i f i c a t i o n o f £ -amino group o f l y s i n e o f K - c a s e i n on .: energy t r a n s f e r and p o l a r i s a t i o n o f ANS conjugate. 81 23 E f f e c t o f m o d i f i c a t i o n o f £ -amino group o f l y s i n e o f K - c a s e i n on i t s a b i l i t y t o . s t a b i l i s e a s -^-casein and on i t s own s o l u b i l i t y i n the presence o f 0.02M cal c i u m i o n . 82 - x i i -LIST OF PLATES R e p l i c a t e o f E l e c t r o n m i c r o g r a p h o f Casein M i c e l l e i n Raw M i l k R e p l i c a t e o f E l e c t r o n m i c r o g r a p h o f Casein M i c e l l e i n Calcium Free M i l k - x i i i -ACKNOWLEDGEMENTS The author wishes t o express h i s deepest g r a t -i t u d e t o Dr. S. Nakai, A s s o c i a t e P r o f e s s o r , Department o f Food S c i e n c e , U n i v e r s i t y o f B r i t i s h Columbia, under whose s u p e r v i s i o n t h i s p r o j e c t was undertaken, and f o r h i s guidance i n the p r e p a r a t i o n o f t h i s t h e s i s . A p p r e c i a t i o n i s a l s o extended t o Dr. W.D. Powrie, Head o f the Department o f Food S c i e n c e ; Drs. J . F . Richards and M. Tung, Department o f Food S c i e n c e and Dr. R. Bose Canada Department o f F i s h e r i e s Research; f o r t h e i r d i s c u s s i o n and c r i t i c i s m d u r i n g the p e r i o d o f r e s e a r c h and i n t h e p r e p a r a t i o n o f t h i s t h e s i s . x i v -INTRODUCTION The c a s e i n s i n m i l k e x i s t p r e dominantly i n a m i c e l l a r s t r u c t u r e , which a l s o i n v o l v e s the i n o r g a n i c ions phosphate and c a l c i u m . G e l a t i o n o f m i l k p r o d u c t s o r p r e c i p i t a t i o n o f the c a s e i n s a r e a s s o c i a t e d w i t h d i s r u p t i o n o f t h i s s t r u c t u r e . Kappa-casein, which r e p r e s e n t s 10-15% o f the t o t a l c a s e i n i n m i l k i s r e s p o n s i b l e f o r the i n s e n -s i t i s a t i o n o f a and 8 - c a s e i n s t o c a l c i u m i o n p r e -c i p i t a t i o n . Because o f t h i s c h a r a c t e r i s t i c , < - c a s e i n i s a v e r y important c o n s t i t u e n t o f the m i l k , h i g h l y r e l a t e d to the t o t a l c a s e i n s t a b i l i t y ; s i n c e the n a t u r a l l e v e l s o f c a l c i u m i o n p r e s e n t c o u l d n o r m a l l y l e a d t o the p r e -c i p i t a t i o n o f a and 3 - c a s e i n s . Because o f t h i s importance to the s t a b i l i t y o f m i l k and m i l k p r o d u c t s , the s t r u c t u r e and mode o f i n t e r a c t i o n o f the c a s e i n s , p a r t i c -u l a r l y K - c a s e i n , have been areas o f i n t e n s e r e s e a r c h f o r some time. I n i t i a l l y the concept o f s t r u c t u r e l e s s c a s e i n s p r e v a i l e d . However, i n r e c e n t y e a r s , t h e r e have been s u g g e s t i o n s o f s t r u c t u r e a s s o c i a t e d w i t h the c a s e i n s , a l t h o u g h t h e r e i s s t i l l no una n i m i t y r e g a r d i n g t h i s o r the type o f i n t e r a c t i o n o r even the i n t e r a c t i o n r a t i o among the c a s e i n s . The r e a c t i o n s p e c i f i c i t y o f < - c a s e i n i n s e n s i t i v i t y t o c a l c i u m i o n p r e c i p i t a t i o n and i n s e n s i -t i s a t i o n o f a _ and 3 - c a s e i n t o c a l c i u m i o n would s 2. suggest t h a t t h e r e i s some s t r u c t u r e a s s o c i a t e d w i t h the < - c a s e i n . For these reasons i t was d e c i d e d t o i n v e s t i g a t e t h e s t r u c t u r e o f K - c a s e i n * i t s mode o f i n t e r a c t i o n w i t h a g ^ - c a s e i n and the i n t e r a c t i o n r a t i o * and f i n a l l y the e f f e c t o f charged groups o f the K - c a s e i n on i t s s t r u c t u r e and i n t e r a c t i o n w i t h a ^ - c a s e i n . The f l u o r e s c e n t t e c h n i q u e s , p o l a r i s a t i o n , f l u o r -e s c e n t y i e l d , wavelength o f maximum emission and energy t r a n s f e r a l l l e n d themselves r e a d i l y t o a study o f s t r u c t u r a l changes i n a macromolecule. P o l a r i s a t i o n i s a p r o p e r t y o f a macromolecule t h a t i s dependent upon the m o l e c u l a r volume, r i g i d i t y and shape o f the m o l e c u l e . F l u o r e s c e n t y i e l d and wavelength o f maximum emission o f a m a c r o m o l e c u l e - f l u o r e s c e n t dye conjugate a r e p r o p e r t i e s a s s o c i a t e d w i t h the p o l a r i t y o f the r e g i o n where the dye i s bound. Confo r m a t i o n a l changes w i l l u s u a l l y e f f e c t the l e v e l o f p e n e t r a t i o n o f the aequeous phase t o t h i s r e g i o n , o r the d i s t r i b u t i o n o f charged groups i n the v i c i n i t y ? a s -s o c i a t e d w i t h these would be changes i n the s o l v a t i o n c h a r a c t e r o f the r e g i o n , hence the p o l a r i t y and f l u o r e s c e n t p arameters. The energy t r a n s f e r i s a measure o f the e f f i c i e n c y o f the a b s o r b i n g chromophore ( i n the case o f p r o t e i n s , tryptophan o r t y r o s i n e ) t o g i v e up i t s energy, t o the e m i t t i n g c onjugated f l u o r e s c e n t dye. T h i s e f f i c i e n c y 3. depends not o n l y upon the d i s t a n c e o f s e p a r a t i o n o f the ab s o r b e r and e m i t t e r (more commonly r e f e r r e d to as the donor-acceptor) but a l s o the o r i e n t a t i o n o f the donor-a c c e p t o r . A s s o c i a t i o n , d i s s o c i a t i o n o r c o n f o r m a t i o n a l changes i n the p r o t e i n m o l e c u l e , s h o u l d r e s u l t i n changes o f some o r a l l the f l u o r e s c e n t parameters l i s t e d . The s t r u c t u r e and i n t e r a c t i o n o f K - c a s e i n were s t u d i e d u s i n g the above mentioned t e c h n i q u e s . 8 - a n i l i n o - n a p h t h a l e n e - l - s u l f o n a t e and 1-dimethyl-amino n a p h t h a l e n e - 5 - s u l f o n y l c h l o r i d e were used as f l u o r e s c e n t c o n j u g a t e s . 4 LITERATURE REVIEW The p r i n c i p l e p r o t e i n s i n m i l k , the c a s e i n s , e x i s t p r e d o m i n a n t l y i n an aggregated s t a t e w i t h c a l c i u m and phosphate i n the form o f m i c e l l e s . Methods f o r s e p a r a t i o n and p u r i f i c a t i o n o f the i n d i v i d u a l c a s e i n s i n v o l v e the a c i d i n s o l u b i l i t y o f the c a s e i n f r a c t i o n s , the v a r i a t i o n o f s o l u -b i l i t y a t reduced temperatures, o r the e f f e c t o f c a l c i u m i o n s (36, 73, 85). I t i s t h i s v a r i a t i o n i n the e x t r a c t -a b i l i t y a t low temperatures, and the c a l c i u m s e n s i t i v i t y , which have been the most i n t r i g u i n g problems r e l a t i n g to the s t u d y and i n t e r a c t i o n o f the p r o t e i n s i n m i l k . Linderstrom-Lang (33) demonstrated the h e t e r o -g e n e i t y o f the c a s e i n s , and a l t h o u g h he d i d n o t s e p a r a t e the f r a c t i o n s a t t h a t time, he p o s t u l a t e d the e x i s t e n c e o f a f r a c t i o n which would be i n s e n s i t i v e t o c a l c i u m i o n , bu t s u s c e p t i b l e to the a c t i o n o f r e n n i n , a f t e r which the p r o t e i n i s p r e c i p i t a t e d by c a l c i u m i o n . H i s t h e s i s was, t h a t a l t h o u g h i n d e p e n d e n t l y one component would be s u s c e p t -i b l e to t h e c a l c i u m i o n c o n c e n t r a t i o n , the i n t e r a c t i o n between the components would l e a d t o i n s e n s i t i v i t y . In 19 56, Waugh and von H i p p e l (77) i d e n t i f i e d a c a s e i n f r a c t i o n which f i t t e d the d e f i n i t i o n o f the p r o t e i n d e s c r i b e d by L inderstrom-Lang. I t showed c a l c i u m i n s e n s i t i v i t y , was a t t a c k e d by r e n n i n , and i n t e r a c t e d w i t h a - and 3 - c a s e i n s , r e n d e r i n g these p r o t e i n s s o l u b l e i n the presence o f c a l c i u m i o n . D u r i n g the a c t i o n o f r e n n i n , a glycomacropeptide i s r e l e a s e d from K - c a s e i n . The remaining p r o t e i n p a r a -c a s e i n does n o t possess the a b i l i t y to s t a b i l i s e a - o r 3 - c a s e i n a g a i n s t c a l c i u m p r e c i p i t a t i o n , and furthermore, i s p r e c i p i t a t e d by c a l c i u m ions a l s o ( 7 7 ) . E x t e n s i v e s t u d i e s have been performed u s i n g s e d i m e n t a t i o n a n a l y s i s , g e l f i l t r a t i o n , d i f f e r e n c e s p e c t r a , e l e c t r o p h o r e s i s and l i g h t s c a t t e r i n g t e c h n i q u e s , w i t h a purpose o f u n d e r s t a n d i n g the s t r u c t u r e and t h e r e f o r e the i n t e r a c t i o n s o f the c a s e i n s . H e r s k o v i t s ( 1 7 ) and Noelken and R e i b s t e i n (49) used o p t i c a l r o t a r y d i s p e r s i o n d u r i n g t h e i r s t u d i e s on the s t r u c t u r e o f the c a s e i n s , and found a low l e v e l o f h e l i c a l c o n t e n t , which i s a u s u a l index o f o r d e r o r s t r u c t u r e i n a p r o t e i n . V i s c o s i t y and o p t i c a l r o t a t i o n measurement i n d i c a t e d t h a t the c a s e i n s had p r o p e r t i e s t y p i c a l o f denatured p r o t e i n s ( 2 2 ) . The above f a c t s a r e d i f f i c u l t to r e c o n c i l e w i t h the p r o p e r t i e s o f < - c a s e i n , namely c a l c i u m i n s e n s i t i v i t y 6. and r e n n i n s e n s i t i v i t y . R e c e n t l y , however, L e s l i e e t a l (32) have demonstrated by the use o f n u c l e a r magnetic resonance t h a t K - c a s e i n p o ssesses a r i g i d i t y which i s a p p a r e n t l y absent i n the o t h e r c a s e i n s . T h i s p o s s i b i l i t y o f s t r u c t u r e i n < - c a s e i n has been confirmed by o t h e r workers (5, 6) and a l s o has been suggested by. Z i t t l e (84). H i l l and Wake (21) have suggested a s p e c i a l r o l e f o r the carbohydrate moeity i n < - c a s e i n i n t h e i r d i s -c u s s i o n on the amphi p h i l e n a t u r e o f K - c a s e i n . However, M a c k i n l a y and. Wake (38) have demonstrated t h a t S-carboxy-methyl K - c a s e i n has f u l l s t a b i l i s i n g a b i l i t y f o r a s ^ -and 3 - c a s e i n s a g a i n s t c a l c i u m i o n p r e c i p i t a t i o n , a l t h o u g h the c a r bohydrate content i s v e r y low. These authors concluded t h a t the carbohydrate i s not n e c e s s a r y f o r t h e s t a b i l i s i n g a b i l i t y , n o r f o r t h e i n t e r a c t i o n between a -^o r 3 - c a s e i n and < - c a s e i n . The f a c t t h a t c a l c i u m ions and phosphate a r e i n c l u d e d i n the m i c e l l e s t r u c t u r e , has l e d many authors t o the c o n c l u s i o n t h a t the i n t e r a c t i o n s o f the c a s e i n s i n v o l v e p a r t i a l l y , o r e x c l u s i v e l y , c a l c i u m and/or, phosphate b r i d g e s . 7. Removal o f c o l l o i d a l c a l c i u m phosphate by-a d j u s t i n g the pH o f c h i l l e d m i l k to a pH o f 4.8 to 5.0 and d i a l y s i n g a g a i n s t a l a r g e excess o f m i l k (34) l e a d s to i n s t a b i l i t y and d i s r u p t i o n o f the m i c e l l e s t r u c t u r e . Schmidt and. Bucheim (59) found t h a t c a l c i u m was r e s p o n s i b l e f o r l a r g e m i c e l l e f o r m a t i o n by b r i d g i n g s m a l l e r c a s e i n m i c e l l e s t o g e t h e r ; t h i s i s i l l u s t r a t e d by r e p l i c a t e s o f t h e i r e l e c t r o n r a i c r o g r a p h s o f m i c e l l e s o f m i l k and c a l c i u m f r e e m i l k r e s p e c t i v e l y ( P l a t e s 1 and 2 ) . G a r n i e r and Ribadeau-Dumas (15) suggested, t h a t the m i c e l l e was composed, o f a K - c a s e i n core i n t e r a c t e d w i t h a s i - c a s e i n d i r e c t l y , which was i n t e r a c t e d f u r t h e r w i t h 3 - c a s e i n , a l l through hydrophobic bonds. These a u t h o r s suggested t h a t the c a l c i u m phosphate formed, b r i d g e s w i t h the h y d r o p h o b i c a l l y i n t e r a c t e d c a s e i n s l e a d i n g to the s t a b l e m i c e l l e s t r u c t u r e . P a r r y and. C a r r o l l (50) a l s o suggested t h a t the K - c a s e i n was i n the i n t e r i o r o f the m i c e l l e . Fox (11) found t h a t i o n i c c a l c i u m c o u l d e a s i l y r e p l a c e c o l l o i d a l phosphate l e a d i n g to a s t a b l e m i c e l l e s t r u c t u r e , which suggested t h e p o s s i b i l i t y o f n o n - s p e c i f i c e l e c t r o s t a t i c i n t e r a c t i o n . Nakai e t a l (42) found t h a t 8. PLATE I . REPLICATE OF ELECTRONMICROGRAPH OF CASEIN MICELLE IN RAW MILK PLATE I I . REPLICATE OF ELECTRONMICROGRAPH OF CASEIN MICELLE IN CALCIUM FREE MILK 9. calcium bridges alone could not be responsible for the maintenance of mi c e l l e structure. Rose and Colvin (56) suggested that the calcium and phosphate were instrumental in maintaining the structure of the mi c e l l e . These authors also suggested that the calcium and phosphate were probably l i n k e d with the e -amino group of l y s i n e from the a s -and 8 -caseins. Although the importance of calcium and phosphate i n the mi c e l l e s t a b i l i t y had been demonstrated, c e r t a i n temperature e f f e c t s were d i f f i c u l t to explain i f only s a l t bridges were involved. Fox (11) found the 8 -casein e a s i l y separates from the m i c e l l a r structure on cooling, and that the structure was not dependent on the 8 -casein. Downey and Murphy (8) found that as much as 50% of the 8-casein entered the serum on cooling, j u s t above f r e e z i n g . Rose and Colvin (57) found that 8 -casein which separated from the m i c e l l a r sturcture on cooling, d i d not re-enter the mi c e l l e on subsequent warming. Rose (55) suggested that the i n t e r a c t i o n between a - and 8 -casein was p r i n c i p a l l y hydrophobic in nature, s This i n t e r a c t i o n product was proposed to be in the core of the m i c e l l e , covered by a lay e r of « -casein. This hypothesis i s s i m i l a r to that of Talbot and Waugh (68), who envisaged a core of a s -casein surrounded by a layer of a _ K-casein. This model i s s i m i l a r to that of Payens (52) 10. who suggested t h a t a s \~ a n c ^ ^ - c a s e i n s were surrounded by < - c a s e i n , and t h a t the s t r u c t u r e was cemented by c a l c i u m phosphate b r i d g e s . Payens (52) suggested t h a t the i n t e r n a l s t r u c t u r e was m a i n t a i n e d by hydrophobic i n t e r a c t i o n , based on t h e temperature dependence o f a g g r e g a t i o n o f the i n d i v i d u a l c a s e i n s (60, 65, 70). T h i s h y p o t h e s i s i s sup-p o r t e d by Noelken (48) i n h i s study u s i n g hydrophobic d e t e r g e n t s . R e c e n t l y , the concept o f n o n - s p e c i f i c 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 between c a s e i n components has been suggested as the mechanism r e s p o n s i b l e f o r the s t a b i l i s i n g a b i l i t y o f < - c a s e i n . On p h o t o - o x i d a t i o n , < - c a s e i n l o s e s i t s s t a b i -l i s i n g a b i l i t y f o r a s i - a g a i n s t c a l c i u m i o n p r e c i p i t a -t i o n (20, 83). The p r i n c i p a l amino-acids d e s t r o y e d by t h i s p r o c e s s , as r e p o r t e d by Z i t t l e (83) and H i l l and L a i n g (20) were h i s t i d i n e and t r y p t o p h a n . These authors proposed a r o l e f o r h i s t i d i n e i n the s t a b i l i s i n g a b i l i t y o f K - c a s e i n , based on t h e i r r e s u l t s . Nakai e t a l (43), however, found t h a t a f t e r m o d i f i c a t i o n o f 1.5 r e s i d u e s o f h i s t i d i n e i n < - c a s e i n , t h e r e was no v a r i a t i o n i n i t s s t a b i l i s i n g a b i l i t y f o r a s i - c a s e i n . H i l l and C r a k e r (19), Woychik (81), Pepper e t a l (53), T a l b o t and Waugh (69) have 11. a l l suggested that l y s i n e residues i n K -casein are related to i t s a b i l i t y to s t a b i l i s e a j-casein against calcium ion p r e c i p i t a t i o n . However, there was some v a r i a t i o n i n the extent of l y s i n e modification necessary to produce a l o s s , or reduction, of s t a b i l i s i n g a b i l i t y and whether i t was due to s p e c i f i c charge e f f e c t s or conformational changes due to the increase i n net negative charge on the pro t e i n . Hoagland (23) found that 8 -caseins which were modified to give an increase i n net negative charge i n the protein were s t i l l s t a b i l i s e d by < -casein against calcium p e r c i p i t a t i o n , and secondly that the modified 8 -casein i t s e l f had become more soluble i n the presence of calcium i o n . H i l l (18) suggested that there i s a peptide region of high p o s i t i v e charge, containing l y s i n e , h i s t i d i n e and arginine, and that t h i s region was r e l a t e d to the s t a b i l i s i n g a b i l i t y o f K -casein f o r a -casein. Kason et a l (25) found that a s i -casein reacted with PEI, a polyethylenimine polymer with high primary amine content. These workers suggested that some p o s i t i v e region on the K -casein molecule could react s i m i l a r l y . 12. Kenkare and Hansen (27) suggested t h a t the i n t e r -a c t i o n between a s - and K - c a s e i n i s h y d r o p h i l l c i n n a t u r e , based on the temperature dependence o f a s s o c i a t i o n and d i s s o c i a t i o n o f thes e p r o t e i n s . Waugh (74) suggested t h a t t h i s temperature e f f e c t c o u l d be due t o a d i f f e r e n t type o f c a l c i u m b i n d i n g among the p r o t e i n s . The problems o f the type o f bonding w i t h i n the p r o t e i n become even more complex when i t i s r e a l i s e d t h a t 3 - c a s e i n can l e a v e the m i c e l l e s t r u c t u r e and not r e - e n t e r i t , and a l s o by the f a c t t h a t a l t h o u g h the r a t i o o f a : s B : K - c a s e i n i n m i l k i s 3 : 2 : 1, Z i t t l e (82) demon-s t r a t e d t h a t r a t i o s o f a : K o f 10 : 1 can be s t a b i l i s e d s i n t he presence o f c a l c i u m i o n . Many authors have attempted t o a s c e r t a i n the i n t e r a c t i o n r a t i o o f a „ , - t o K - c a s e i n s 1 i n model systems. Waugh and von H i p p e l (77) suggested t h a t the i n t e r a c t i o n r a t i o o f a - : K - c a s e i n was 4 : 1 . However, s Noble and Waugh (47) l a t e r suggested t h a t the i n t e r a c t i o n r a t i o was c l o s e t o u n i t y . G a r n i e r (13) suggested t h a t the i n t e r a c t i o n r a t i o i s u n i t y , b ut l a t e r t h i s a uthor (12) suggested t h a t the K - c a s e i n s p e c i e s i n v o l v e d i n the i n t e r -a c t i o n was a t r i m e r , and t h a t t h i s combined w i t h a monomer o f a _ - c a s e i n , hence the i n t e r a c t i o n r a t i o o f a - : < -s • ' s i c a s e i n was 1 : 3 on a monomer b a s i s . P a r r y e t a l (51) u s i n g g e l f i l t r a t i o n t e c h n i q u e s , showed a s i n g l e i n t e r -a c t i o n p r o d u c t w i t h a weight r a t i o o f u n i t y , whereas Kenkare and Hansen (27) suggested a v a r i a b l e i n t e r a c t i o n r a t i o but pre d o m i n a n t l y a s 1 ~ • K - c a s e i n o f 4 : 1, I t i s obvious from the l i t e r a t u r e t h a t the type o f i n t e r a c t i o n s i n v o l v i n g the c a s e i n s and the i n t e r -a c t i o n r a t i o s among the d i f f e r e n t s p e c i e s a r e not c l e a r l y e s t a b l i s h e d . 14. CHAPTER I . FLUORESCENT STUDIES OF K -CASEIN WITH 8- ANILIN ONAPHTHALEN E -1- SULFONATE. INTRODUCTION Recent s t u d i e s by H e r s k o v i t s (17) and Noelken and R e i b s t e i n (49), u s i n g o p t i c a l r o t a t o r y d i s p e r s i o n have demonstrated t h a t c a s e i n s a r e unordered w i t h l i t t l e . h e l i c a l s t r u c t u r e . A c c o r d i n g to the c l a s s i c a l d e f i n i t i o n o f p r o t e i n s t r u c t u r e c a s e i n s e x i s t as random c o i l s . How-ever, such a s t r u c t u r e i s r a t h e r d i f f i c u l t t o r e c o n c i l e w i t h the p h y s i c a l and chemical p r o p e r t i e s o f c a s e i n s , p a r t i c u l a r l y o f < c a s e i n : T a l b o t and Waugh (69) sug-g e s t e d a S-S-bonded open chain polymer as a p o s s i b l e form f o r K - c a s e i n . Swaisgood and Erunner (66), McKenzie and Wake (36), McKenzie (35) and Mackinlay and Wake (37) demons-t r a t e d the e x i s t e n c e o f d i s u l f i d e b r i d g e s i n K - c a s e i n . Payens (52) p o s t u l a t e d the p o s s i b i l i t y o f hydrophobic i n t e r a c t i o n s as a p r i n c i p a l cause o f a g g r e g a t i o n and c o n s i d e r e d the v a r i a t i o n i n the sedimentation c o e f f i c i e n t w i t h c o n c e n t r a t i o n to be due to entanglement o f the random c o i l f i b r e s of K - c a s e i n . Recently, P a r r y and C a r r o l l (50), and Chesseman and Knight (6) r e p o r t e d t h a t 1 the K. - c a s e i n i n m i l k i s s t r u c t u r e d l i k e - a n aggregated, g l o b u l a r p r o t e i n . I t has been domonstrated by Weber and Laurence (80) and S t r y e r (62, 63) t h a t 8 - a n i l i n o n a p h t h a l e n e - l -s u l f o n a t e (ANS)^ can be used as a probe, f o r hydrophobic r e g i o n s and the quantum y i e l d o f f l u o r e s c e n c e and the wavelength o f maximum -emission o f the probe bound to the p r o t e i n v a r i e s depending upon the p o l a r i t y o f the r e g i o n o f attachment. R e s u l t s o f s p e c t r o f l u o r o m e t r i c s t u d i e s on K -c a s e i n a r e p r e s e n t e d to demonstrate the e x i s t e n c e o f hydrophobic r e g i o n s i n the molecule and changes i n molecula conformation, under v a r y i n g environmental c o n d i t i o n s , measured by the v a r i a t i o n s i n energy t r a n s f e r (10, 63, 64) and the p o l a r i s a t i o n o f f l u o r e s c e n c e (78, 79). The f o l l o w i n g a b b r e v i a t i o n was used; ANS, 8 - a n i l i n o n a p h t h a l e n e - l - s u l f o n a t e MATERIALS AND METHODS 16. Kappa-casein was prepared by the method o f Z i t t l e and C u s t e r (85) and was e l e c t r o p h o r e t i c a l l y pure on p o l y -a c r y l a m i d e g e l . The magnesium s a l t o f ANS was pr e p a r e d from the t e c h n i c a l grade sodium s a l t (Eastman Kodak Company, Rochester, N.Y.), by p r e c i p i t a t i o n w i t h s a t u r a t e d mag-nesium a c e t a t e . The crude p r o d u c t was r e c r y s t a l l i s e d from h o t water and f i l t e r e d through a bed o f a c t i v a t e d c h a r c o a l and H y f l o Super C e l . T h i s was rep e a t e d u n t i l a c o n s t a n t molar absorbance, 5700, a t 350 nm i n 0.1M phosphate b u f f e r pH 6.5, was o b t a i n e d . F l u o r e s c e n c e s p e c t r a were r e c o r d e d on a Moseley, Model 7004A X-Y Recorder, manufactured by Hewlett-Packard, connected through a F h o t o m u l t i p l i e r Microphotometer t o an Aminco-Bowman S p e c t r o p h o t o f l u o r o m e t e r , No. 4-8202. Temperatures above and below ambient were mai n t a i n e d by a Sargent Thermoniter and by a Blue M C o n t r o l Flow C o o l i n g U n i t , r e s p e c t i v e l y . The number o f hydrophobic r e g i o n s were c a l c u l a t e d _P 1 (1 + K ) u s i n g the K l o t z (29) e q u a t i o n , Dx = n ( l - x ) D (1) 17. where P = the p r o t e i n concentrat ion D = the ANS concentrat ion x = the f r a c t i o n o f the dye bound to the pro te in K = the d i s s o c i a t i o n constant of the dye-prote in complex n = the number of b inding s i t e s . _P 1 A p l o t o f Dx against ( l -x )D gives a s t r a i g h t l i n e with 1 in tercept n . For t h i s c a l c u l a t i o n , a constant concentrat ion of 4.0 X 10~^M was used for K - case in throughout the studies except for the r e s u l t s appearing i n Figures 1 and 2. The concentrat ion of ANS was v a r i e d between 2.6 and 6.6 X 10" 5 M. Fluorescence data i s reported as r e l a t i v e f luorescent i n t e n s i t y , which i s the r a t i o of the f luorescent i n t e n s i t y of the sample to that o f a so lu t ion of < - case in 4.0 X 10" 5 M, when both are conjugated with ANS a t a concent-r a t i o n o f 2 X 10" 6 M. The p o l a r i s a t i o n , a t room temperature, was c a l -cu la ted us ing the equation of Azumi and KcGlynn (1) , I I E E " I E B P = BB (2) I *EE + I E B - S l BB where P = p o l a r i s a t i o n I = f luorescent i n t e n s i t y 1 8 . T h e p o l a r i s e r w a s t w o d i s k s o f p o l a c o a t f o r m u l a 1 0 5 U V f i l m s e t a t m u t u a l r i g h t a n g l e s . T h e a n a l y s e r w a s t w o d i s k s o f p o l a r o i d H N 3 8 f i l m s e t a t m u t u a l r i g h t a n g l e s . A n e x c i t a t i o n w a v e l e n g t h o f 3 5 0 n m , a n d a n e m i s s i o n w a v e l e n g t h o f 4 8 0 nm o r t h e w a v e l e n g t h . o f m a x i m u m e m i s s i o n o f s a m p l e s , w h e r e a p p l i c a b l e , w e r e u s e d . E n e r g y t r a n s f e r w a s c a l c u l a t e d b y t h e d e c r e a s e i n t h e e m i s s i o n a t 3 5 0 n m , e x c i t a t i o n 2 9 0 n m , o f t h e K - c a s e i n , w h e n c o n j u g a t e d w i t h A N S . T h e m o l a r r a t i o o f p r o t e i n t o A N S v/as c o n s t a n t a t 1 0 : 1 . S l i t p o s i t i o n s o n t h e s p e c t r o f l u o r o r n e t e r w e r e a s f o l l o w s : f o r f l u o r e s c e n t y i e l d a n d e m i s s i o n w a v e l e n g t h d e t e r m i n a t i o n s - # 2 , # 5 , 4mm; # 3 , #4 , # 7 , 3mm; f o r e n e r g y t r a n s f e r - # 2 , # 5 , n o n e ; # 3 , # 4 , # 7 , 1mm; f o r p o l a r i s a t i o n - # 2 , 4mm; # 3 , # 4 , 3mm; # 5 , n o n e ; # 7 , 2mm. T h e m o l e c u l a r w e i g h t o f 2 0 , 0 0 0 w a s a s s u m e d f o r K - c a s e i n m o n o m e r . P r o t e i n c o n c e n t r a t i o n s w e r e d e t e r m i n e d f r o m t h e a b s o r b a n c e a t 2 8 0 n m . S o d i u m d o d e c y l s u l f a t e s o l u t i o n s w e r e p r e p a r e d i n 0 . 0 5 M T r i s - H C l b u f f e r p H 8 . 0 . U r e a s o l u t i o n s w e r e a d j u s t e d t o p H 7 . 0 . P h o s p h a t e b u f f e r s , 0 . 0 5 M , w e r e u s e d f o r p H v a l u e s b e t w e e n 5 . 0 a n d 7 . 0 ; 0 . 0 5 T r i s - H C l f o r p H 8 . 0 a n d 9 . 0 ; 0 . 0 5 b i c a r b o n a t e f o r p H 1 0 . 0 ; p H 1 1 . 0 a n d 12.0 were o b t a i n e d by adding NaOH to the sample. The r e s u l t s p r e s e n t e d are averages of f o u r (4) r e a d i n g s * two d u p l i c a t e s a t d i f f e r e n t times. 20. RESULTS E f f e c t o f K - C a s e i n C o n c e n t r a t i o n F i g u r e 1 shows the r e l a t i o n s h i p between r e l a t i v e f l u o r e s c e n t i n t e n s i t y , hydrophobic r e g i o n s and c o n c e n t r a t i o n o f K - c a s e i n . The r e l a t i o n s h i p between r e l a t i v e f l u o r -e s c e n t i n t e n s i t y and hydrophobic r e g i o n s i s i n v e r s e . The number o f hydrophobic r e g i o n s tended to approach 0.5 per molecule as the c o n c e n t r a t i o n o f K - c a s e i n i n c r e a s e d , and ap p r o x i m a t e l y f o u r r e g i o n s p e r molecule were o b t a i n e d by d i l u t i n g t o 5.0 X 10"" M. A t low c o n c e n t r a t i o n s o f < - c a s e i n , the number o f hydrophobic r e g i o n s was l a r g e and. the r e l a t i v e f l u o r e s c e n t i n t e n s i t y low. However, the r e l a t i v e f l u o r -e scent i n t e n s i t y i n c r e a s e d and the number of hydrophobic r e g i o n s decreased w i t h i n c r e a s i n g c o n c e n t r a t i o n s . Concen-t r a t i n g d i l u t e s o l u t i o n s o r d i l u t i n g c o n c e n t r a t e d s o l u t i o n s y i e l d e d the same v a l u e s f o r the number o f hydrophobic r e g i o n s and r e l a t i v e f l u o r e s c e n t i n t e n s i t y as those i n f i g u r e 1 a t the same c o n c e n t r a t i o n o f K - c a s e i n . A l s o as the concen-t r a t i o n o f < - c a s e i n was i n c r e a s e d , the energy t r a n s f e r i n c r e a s e d w i t h a s l i g h t d e crease i n p o l a r i s a t i o n ( F i g . 2 ) . 21. to Z o 0 2 4 6 8 10 5 k-CASEIN CONCENTRATION X10 MOLAR F i g u r e 1. E f f e c t o f < - c a s e i n c o n c e n t r a t i o n upon the number o f hydrophobic r e g i o n s ( © - © ) and r e l a t i v e f l u o r e s c e n t i n t e n s i t y ( & ) o f K -casein-ANS conjugate. pK 6.5, 0.05M phosphate b u f f e r . Figure 2. E f f e c t o f K - case in concentrat ion on energy t r a n s f e r ( • - • ) and p o l a r i s a t i o n (A-A ). 23 E f f e c t o f NaCl A t low c o n c e n t r a t i o n s o f s a l t from zero t o 0.01M, t h e r e was a marked i n c r e a s e i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y and a decrease i n the number o f hydrophobic r e g i o n s ( F i g . 3 ) . These phenomena were accompanied by a sharp r i s e i n energy t r a n s f e r , and a decrease i n p o l a r i s a t i o n ( F i g . 4 ) . A t NaCl c o n c e n t r a t i o n s h i g h e r than 0.01M these changes c o n t i n u e d , however, a t a reduced r a t e . E f f e c t o f Urea As t h e c o n c e n t r a t i o n o f urea was i n c r e a s e d t h e r e l a t i v e f l u o r e s c e n t i n t e n s i t y r a p i d l y dropped, then l e v e l l e d o f f a f t e r 4M; a l s o , t h e r e was a s i g n i f i c a n t r e d s h i f t i n the wavelength o f maximum emission from 480 to 518 nm ( F i g . 5 ) . F i g u r e 6 i n d i c a t e s t h a t t h e r e was a v e r y low l e v e l o f energy t r a n s f e r and the p o l a r i s a t i o n de-c r e a s e d w i t h i n c r e a s i n g urea c o n c e n t r a t i o n . E f f e c t o f SDS 2 As the c o n c e n t r a t i o n o f SDS was i n c r e a s e d up t o The f o l l o w i n g a b b r e v i a t i o n was used: SDS, sodium d o d e c y l s u l f a t e . 24. NaCl CONCENTRATION MOLAR Figure 3. E f f e c t o f s a l t concentrat ion of r e l a t i v e f luorescent i n t e n s i t y ( A - A ) and number o f hydrophobic regions (•— •) for the < - c a s e i n -ANS conjugate. pH adjusted to 7.0 25> Figure 4. E f f ec t o f s a l t concentrat ion on energy t rans fer ( • - © ) and p o l a r i s a t i o n ( A — A ) o f K - c a s e i n . 26. UREA CONCENTRAT ION, MOLAR Figure 5. E f f e c t o f urea on r e l a t i v e f luorescent i n t e n s i t y (•—•) and maximum emission wavelength (A—A) f o f K -casein-ANS conjugate. pH 7.0 27 0 2 4 6 a UREA C O N C E N T R A T I O N , MOLAR Figure 6. E f f e c t of urea on energy transfer (•—•) and p o l a r i s a t i o n ( A — A ) of K-casein. 28. 0.005M t h e r e was a r a p i d decrease i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y f o l l o w e d by a s l i g h t i n c r e a s e as SDS c o n c e n t r a t i o n was f u r t h e r i n c r e a s e d t o 0.1M. Concomitant w i t h t h i s v a r i a t i o n i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y w i t h SDS con-c e n t r a t i o n t h e r e was a marked r e d s h i f t i n the wavelength o f maximum e m i s s i o n . A t h i g h e r SDS c o n c e n t r a t i o n s t h e r e was a g a i n a s l i g h t b l u e s h i f t ( F i g . 7 ) . As SDS c o n c e n t r a t i o n i n c r e a s e d the energy t r a n s f e r and the p o l a r i s a t i o n decreased r e v e a l i n g the minimum a t an SDS c o n c e n t r a t i o n o f 0.01M. A t h i g h e r c o n c e n t r a t i o n o f SDS t h e r e was ag a i n a s l i g h t i n c r e a s e i n the energy t r a n s f e r and the p o l a r i s a t i o n ( F i g . 8 ) . F i g u r e 9 shows the e f f e c t o f SDS c o n c e n t r a t i o n on ANS a l o n e . D o d e c y l s u l f a t e i t s e l f i n c r e a s e d the r e l a t i v e f l u o r e s c e n t i n t e n s i t y but to a l e s s e r extent than the K -casein-SDS m i x t u r e . E f f e c t o f pH As the pH was i n c r e a s e d from 5.0 the r e l a t i v e f l u o r e s c e n t i n t e n s i t y passed through a minimum a t pH 5.5 and a maximum a t pH 6.0. As the pH was f u r t h e r i n c r e a s e d t h e r e was a continuous d e c r e a s e i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y up t o pH 12.0. There was no apparent change i n wavelength o f maximum emi s s i o n w i t h pH u n t i l i t exceeded 11.0 ( F i g . 1 0 ) . 00001 0001 001 1-0 S D S C O N C E N T R A T I O N , MOLAR F i g u r e 7. E f f e c t o f sodium d o d e c y l s u l f a t e on r e l a t i v e f l u o r e s c e n t i n t e n s i t y ( © — ©) and maximum emission wavelength ( •& ) o f < - c a s e i n -ANS conjugate. pH 8.0. F i g u r e 8 . E f f e c t o f sodium d o d e c y l s u l f a t e on energy t r a n s f e r ( ©—•©) and p o l a r i s a t i o n ( &) of. K -casein-ANS conjugate. 31. F i g u r e 9. E f f e c t o f sodium d o d e c y l s u l f a t e on r e l a t i v e f l u o r e s c e n t i n t e n s i t y o f ANS. pH 8.0. F i g u r e 10. E f f e c t o f pH on r e l a t i v e f l u o r e s c e n t i n t e n s i t y (A — A ) a n d maximum e m i s s i o n w a v e l e n g t h (•— #) o f K - c a s e i n - A N S c o n j u g a t e . 33 E f f e c t o f Temperature As the temperature i n c r e a s e d from 4 t o 60°C t h e r e was a decrease i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y , but no change i n the number o f hydrophobic r e g i o n s ( F i g . 11). T h i s d e crease was accompanied by a decrease i n the energy t r a n s f e r ( F i g . 12). F i g u r e 11. E f f e c t o f temperature on r e l a t i v e f l u o r e s c e n t i n t e n s i t y (•'—•) and number o f hydrophobic r e g i o n s ( A — A ) o f < - c a s e i n . 3 5 . Figure 12. E f f e c t o f temperature on energy t rans fer of K - c a s e i n . .36. DISCUSSION The r e l a t i o n s h i p between the p o l a r i t y o f the r e g i o n o f bound ANS, f l u o r e s c e n t y i e l d and emission wavelength, has been demonstrated by S t r y e r ( 6 3 ) . The more non-polar the environment o f the probe, the g r e a t e r i s the f l u o r e s c e n t y i e l d and the s h o r t e r i s the wavelength o f maximum e m i s s i o n . S t r y e r (62) and S t r y e r and Haugland (64) f u r t h e r demonstrated the r e l a t i o n s h i p between energy t r a n s f e r and d i s t a n c e o f s e p a r a t i o n o f donor and a c c e p t o r based on the F o r s t e r ' s equation (10). The v a r i a t i o n s i n f l u o r e s c e n t y i e l d , emission wavelength and energy t r a n s f e r when c o n s i d e r e d along with p o l a r i s a t i o n (78, 79) should, g i v e a good i n d i c a t i o n o f c o n f o r m a t i o n a l changes, a g g r e g a t i o n o r d i s s a g g r e g a t i o n , s i z e and r i g i d i t y i n a macromolecule. From f i g u r e 1 i t i s apparent t h a t t h e r e was some co n f o r m a t i o n a l change as t h e r e were changes i n the p o l a r i t y o f the environment o f the ANS, j u d g i n g from v a r i a t i o n i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y , and a l s o a change i n the number o f hydrophobic r e g i o n s w i t h c o n c e n t r a t i o n . These changes i n p o l a r i t y and number o f hydrophobic r e g i o n s w i t h c o n c e n t r a t i o n are s u g g e s t i v e o f c o n c e n t r a t i o n dependence o f a g g r e g a t i o n through hydrophobic i n t e r a c t i o n s as p o s t u l a t e d by Payens (52) and. o f a s i m i l a r c o n c e n t r a t i o n dependence o f a g g r e g a t i o n f o r a s i~ (67), and 6 - c a s e i n s ( 6 0 ) . How-ever, the data on energy t r a n s f e r and p o l a r i s a t i o n ( F i g . 2 ) d i d not support t h i s concept. Although i t i s p o s s i b l e t o have i n c r e a s e d energy t r a n s f e r due to a g g r e g a t i o n , i t sh o u l d r e s u l t i n a concomitant i n c r e a s e i n p o l a r i s a t i o n because o f a l a r g e r m o l e c u l a r volume and an i n c r e a s e d r e l a x a t i o n time, f o r a r i g i d m o l e c u l e . I f the molecule i s n o t r i g i d i t i s u n l i k e l y to l e a d t o i n c r e a s e d energy t r a n s f e r . In f a c t t h e r e was a v e r y s l i g h t change i n p o l a r -i s a t i o n which i s c o n t r a r y to the e x p e c t a t i o n . T h i s de-cre a s e i n p o l a r i s a t i o n may be due t o a g r e a t e r degree o f i n t e r n a l r o t a t i o n which decreases m o l e c u l a r r i g i d i t y , a c l o s e r approach to a sphere o r a decreased m o l e c u l a r volume (2, 78, 79). A decreased m o l e c u l a r volume c o u l d e a s i l y account f o r i n c r e a s e d energy t r a n s f e r due to decreased donor-a c c e p t o r d i s t a n c e o f s e p a r a t i o n , decreased number o f b i n d i n g r e g i o n s due to s t e r i c l i m i t a t i o n s r e s u l t i n g from th e d e c r e a s e d m o l e c u l a r volume, and i n c r e a s e d r e l a t i v e f l u o r e s c e n t i n t e n s i t y due to more c l o s e l y packed non-polar s i d e c h a i n s . T h i s phenomenon i s s i m i l a r t o the c o n c e n t r a -t i o n dependence o f se d i m e n t a t i o n c o e f f i c i e n t ( 5 8 ) . I f K - c a s e i n a t a n e u t r a l pH forms e l o n g a t e d macromolecules as Payens (52) suggested i t i s p o s s i b l e t h a t the s t e r i c e x c l u s i o n which c o n t r i b u t e s t o the g e o m e t r i c a l lower v i s c o s i t y o f s u r r o u n d i n g r e g i o n s (3, 58) w i l l d e crease as the c o n c e n t r a t i o n o f < - c a s e i n i n c r e a s e s because of entanglement o f molecules (52). T h i s i n c r e a s e s the a c t u a l v i s c o s i t y i n the s u r r o u n d i n g r e g i o n s u b s t a n t i a l l y and decreased m o l e c u l a r volume and S t o k e 1 s r a d i u s o f m o l e c u l e s . I n c r e a s i n g the i o n i c s t r e n g t h by the a d d i t i o n o f NaCl l e a d s to a more compact s t r u c t u r e as judged from a dec r e a s e i n p o l a r i s a t i o n , an i n c r e a s e i n energy t r a n s f e r and r e l a t i v e f l u o r e s c e n t i n t e n s i t y , and a decrease i n the number o f hydrophobic r e g i o n s . Due to the h i g h content o f a p o l a r s i d e c h a i n s i n < - c a s e i n ( 2 1 ) , i t i s expected t h a t i n c r e a s i n g the concen-t r a t i o n o f NaCl i n the s o l v e n t would r e s u l t i n a more i n t e n s e h ydrophobic i n t e r a c t i o n (71) as was observed f o r ° s 1" a n d ^ - c a s e i n s by Waugh, e t a l . ( 7 5 ) . Furthermore i n t e n s i f i c a t i o n o f hydrogen bonds may not be n e g l e c t e d ( 9 ) . Such i n t e r a c t i o n s might l e a d t o a more compact s t r u c t u r e . I n c r e a s i n g the i o n i c s t r e n g t h would have an e f f e c t o f r e d u c i n g 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 , l e a d i n g to i n c r e a s e d d i s o r g a n i s a t i o n i f the molecule e x i s t e d as a monomer, o r t o d i s s o c i a t i o n i f a polymer. In the former case t h i s change would l e a d t o decreased p o l a r i s a t i o n , r e l a t i v e f l u o r e s c e n t i n t e n s i t y and energy t r a n s f e r ? i n the l a t t e r case i t would a l s o l e a d to decreased p o l a r i s a t i o n and p r o b a b l y decreased r e l a t i v e f l u o r e s c e n t i n t e n s i t y and energy t r a n s f e r . The experimental r e s u l t s d i d not support t h i s , thus i t appears t h a t 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 do not p l a y a major r o l e i n the s t r u c t u r e . Treatment w i t h urea and SDS, which r u p t u r e s hydrogen bonds and hydrophobic i n t e r a c t i o n s (26, 39, 40), amply demonstrate t h a t t h e r e i s some o r d e r a s s o c i a t e d w i t h the K - c a s e i n m o l e c u l e . C o n s i d e r a b l e r e d u c t i o n i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y , energy t r a n s f e r , r e d s p e c t r a l s h i f t and d e c r e a s e d p o l a r i s a t i o n ( F i g . 5, 6, 7 and 8) a r e a l l s u g g e s t i v e o f l o s s o f o r g a n i s a t i o n i n m o l e c u l e s . A s i g -n i f i c a n t decrease i n p o l a r i s a t i o n i s i n d i c a t i v e o f a h i g h degree o f i n t r a m o l e c u l a r f r e e r o t a t i o n due t o a decrease i n m o l e c u l a r r i g i d i t y . T h i s i s i n agreement w i t h the work o f L e s l i e , e t a l . (32) who demonstrated the r i g i d i t y o f the * - c a s e i n aggregate. From NMR s t u d i e s the ag-greg a t e possessed some l e v e l o f r i g i d i t y as compared w i t h the o t h e r c a s e i n s . The a r o m a t i c region, was not r e s o l v e d w i t h K - c a s e i n t r e a t e d w i t h 7M urea which then l e a d s to some f l e x i b i l i t y o f the amino a c i d r e s i d u e s . Cheeseman and J e f f c o a t (5) and Cheeseman and Knight (6) a l s o demon-40. s t r a t e d the d i s r u p t i v e e f f e c t o f SDS on < - c a s e i n by a marked decrease i n the s e d i m e n t a t i o n c o e f f i c i e n t and v a r i a t i o n s i n the s p e c t r a l c h a r a c t e r i s t i c s . A t h i g h e r c o n c e n t r a t i o n s o f SDS t h e r e was an apparent o r d e r generated as judged by a b l u e s p e c t r a l s h i f t and i n c r e a s e s i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y , energy t r a n s f e r and p o l a r i s a t i o n . T h i s c o u l d be due p a r t l y t o d e c r e a s e d s o l v e n t p o l a r i t y as i s suggested from f i g u r e 9 where the r e l a t i v e f l u o r e s c e n t i n t e n s i t y o f ANS a l o n e was i n c r e a s e d w i t h i n c r e a s i n g SDS c o n c e n t r a t i o n s and s i m u l -t a n e o u s l y due t o the development o f some h e l i x - l i k e s t r u c t u r e as suggested by J i r g e n s o n s and C a p e t i l l o ( 2 4 ) . The e f f e c t o f pH upon the < - c a s e i n s t r u c t u r e was r e f l e c t e d by the r e l a t i v e f l u o r e s c e n t i n t e n s i t y and t h e number o f hydrophobic r e g i o n s ( F i g . 10). A p a r t from a minimum a t pH 5.5 t h e r e l a t i v e f l u o r e s c e n t i n t e n s i t y g r a d u a l l y decreased up to pH 12.0. T h i s d ecrease i s r e a d i l y e x p l a i n e d by an i n c r e a s e i n m o l e c u l a r volume due t o e l e c t r o s t a t i c r e p u l s i o n . The l a r g e s p e c t r a l s h i f t above pH 11.0 i s i n d i c a t i v e of a h i g h l y s i g n i f i c a n t s t r u c t u r a l change. S i n c e hydrophobic i n t e r a c t i o n s a r e endothermic 41. i n c h a r a c t e r (45), i n c r e a s i n g temperatures s h o u l d i n t r o d u c e a h i g h e r l e v e l o f a g g r e g a t i o n through hydrophobic i n t e r a c t i o n s as suggested by Payens (52) o r a t l e a s t to a more compact s t r u c t u r e due to i n c r e a s e d i n t r a - m o l e c u l a r hydrophobic i n t e r a c t i o n s . C o n v e r s e l y d e c r e a s i n g temperatures s h o u l d i n v i t e d i s s o c i a t i o n . The r e s u l t s p r e s e n t e d i n f i g u r e s 11 and 12 d i d not support t h i s assumption. Low temperatures i n c r e a s e d the r e l a t i v e f l u o r e s c e n t i n t e n s i t y and the energy t r a n s f e r compared w i t h e l e v a t e d temperatures i n d i c a t i n g a r o l e f o r hydrogen bonding i n m a i n t a i n i n g the m o l e c u l a r s t r u c t u r e o f < - c a s e i n . However, even a t 60°C t h e r e was s t i l l c o n s i d e r a b l e s t r u c t u r e remaining as r e l a t i v e f l u o r -e s c e n t i n t e n s i t y and energy t r a n s f e r ( F i g . 11 and 12) were h i g h e r than those f o r K - c a s e i n s d i s s o c i a t e d w i t h u r e a and SDS ( F i g . 5, 6, 7 and 8 ) . A t those e l e v a t e d temperatures the c o n t r i b u t i o n o f hydrogen bonds t o the maintenance o f s t r u c t u r e i s not expected t o be v e r y s i g n i f i c a n t and the s t r u c t u r a l i n t e g r i t y would be due predominantly to hydro-p h o b i c i n t e r a c t i o n s . These r e s u l t s a r e i n a c c o r d w i t h the f i n d i n g s o f Z i t t l e (84), P a r r y and C a r r o l l (50), L e s l i e , e t a l . (32) and T a l b o t and Waugh (69) t h a t the K - c a s e i n e x i s t s i n a p o l y m e r i c form w i t h a s t r u c t u r e but not n e c e s s a r i l y a - h e l i x c o n f i g u r a t i o n . Kappa-casein, 4.0 X 10" M i n 0.05M phosphate b u f f e r pH 7.0, had a r e l a x a t i o n time o f 480 nanoseconds. T h i s would compare wi t h a sphere o f m o l e c u l a r weight 600,000 o r an e l l i p s o i d o f a x i a l r a t i o 3:1 i f the m o l e c u l a r weight o f 200,000 (69) was used. As p o l a r i s a t i o n i n urea and SDS without d i s u l f i d e r e d u c i n g agents was remarkably low, i t i s p o s s i b l e t h a t d i s u l f i d e b r i d g e s a r e not important f o r the conformation o f the K - c a s e i n molecule s e p a r a t e d by the method o f Z i t t l e and C u s t e r (85). T h i s s t r u c t u r e seems to depend, m a i n l y upon hydrogen bonds and hydrophobic i n t e r a c t i o n s . F i n a l l y , from these d a t a , i t i s p o s s i b l e t h a t t h e r e i s no c o n c e n t r a t i o n dependent a g g r e g a t i o n but a c o n c e n t r a t i o n dependent c o n f o r m a t i o n a l or shape change i n K - c a s e i n m o l e c u l e s . 43. CHAPTER I I . INTERACTION OF K - WITH a s 1-CASEIN BY  FLUORESCENCE POLARISATION. INTRODUCTION Si n c e the f r a c t i o n a t i o n o f a - c a s e i n i n t o f r a c t i o n s <* and < , c a l c i u m s e n s i t i v e and i n s e n s i t i v e r e s p e c t i v e l y (77), t h e r e has been a g r e a t i n t e r e s t i n the < - c a s e i n f r a c t i o n because o f i t s unique s t a b i l i s i n g a b i l i t y towards t h e o t h e r c a s e i n s . S t u d i e s have been done on the i n t e r a c t i o n between K - and a ^ - c a s e i n s , which s t a b i l i s e s ct g ^ - c a s e i n a g a i n s t c a l c i u m p r e c i p i t a t i o n , and c o u l d a c t as p r e c u r s o r to s t a b l e c a s e i n m i c e l l e s (47, 65). The techniques used and the r e s u l t s o b t a i n e d were v a r i e d r e g a r d i n g the i n t e r -a c t i n g r a t i o o f a s -j^- t o K - c a s e i n and the type o f i n t e r -a c t i o n , i . e . h y d r o p h i l i c , h ydrophobic o r e l e c t r o s t a t i c . Waugh and von H i p p e l (77) s t u d i e d the i n t e r a c t i o n between a s 1~ a n d K - c a s e i n and found a v a r i a b l e i n t e r a c t i o n r a t i o , but found t h a t t h e 0 1 s 1 s K r a t i o was predomin-a n t l y 4. Noble and Waugh (47) found t h a t the a , : < S JL r a t i o was low and c l o s e to u n i t y . G a m i e r (12) found an 44. i n t e r a c t i o n r a t i o o f 1 mole a to 3 mole < - c a s e i n . 5 X However, i n an e a r l i e r paper, G a m i e r e t a l . (13) r e p o r t e d an i n t e r a c t i o n r a t i o o f u n i t y . P a r r y et a l . (51) found the i n t e r a c t i o n r a t i o to be u n i t y , u s i n g g e l f i l t r a t i o n . Kenkare and Hansen (27) found t h a t the i n t e r -a c t i o n p r o d u c t was o f v a r i a b l e composition, and suggested t h a t the i n t e r a c t i o n between the a s i ~ and < - c a s e i n was h y d r o p h i l i c i n n a t u r e . T h i s i s i n c o n t r a s t w i t h the work of G a m i e r e t a l . (14) who found a p o s i t i v e e ntropy f o r the r e a c t i o n , s u g g e s t i v e o f hydrophobic i n t e r a c t i o n . T h i s p o s t u l a t i o n o f hydrophobic i n t e r a c t i o n seems t o be f e a s i b l e , i n the l i g h t o f t h e i n h i b i t i o n o f the i n t e r a c t i o n a t temperatures 2 t o 6°C and s p o n t a n e i t y o f the same r e a c t i o n a t 37°C ( 4 7 ) . Payens (52) suggested t h a t the main i n t e r a c t i n g f o r c e between the c a s e i n s , i n m a i n t a i n i n g the m i c e l l e s t r u c t u r e , i s hyd r o p h o b i c i n n a t u r e , d i r e c t e d w i t h i n the m i c e l l e . T h i s concept was based on the amino-acid compo-s i t i o n o f t h e c a s e i n s , the temperature dependence o f a g g r e g a t i o n o f the c a s e i n s and the e s t a b l i s h e d hydrophobic n a t u r e o f a g g r e g a t i o n o f a s ^- and 8 - c a s e i n s . H i l l and wake (21) d i s c u s s e d the a m p h i p h i l e n a t u r e o f K - c a s e i n and suggested t h a t the a s 1~ < ~ c a s e i n i n t e r a c t i o n i s s i g n i f i c a n t l y hydrophobic i n n a t u r e . However, i n a d d i t i o n t o the s u g g e s t i o n o f hydro-p h i l i c and h ydrophobic i n t e r a c t i o n s , t h e r e have r e c e n t l y been s u g g e s t i o n s o f n o n - s p e c i f i c 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 . Woychik (81), Pepper e t a l . (53), T a l b o t and Waugh (69) have m o d i f i e d l y s i n e r e s i d u e s i n K - c a s e i n u s i n g v a r i o u s t e c h n i q u e s and found t h a t the m o d i f i c a t i o n o f i n c r e a s i n g numbers o f l y s i n e r e s i d u e s reduced the s t a b i l i s i n g a b i l i t y towards a g ^ - c a s e i n u n t i l i t was l o s t c o m p l e t e l y a f t e r 5 o r more r e s i d u e s were m o d i f i e d . H i l l and L a i n g (20) found t h a t the m o d i f i c a t i o n by p h o t o - o x i d a t i o n o f h i s t i d i n e r e s i d u e s i n < - c a s e i n reduced i t s s t a b i l i s i n g a b i l i t y f o r a s i - c a s e i n . Nakai, e t a l . (43) noted t h a t the m o d i f i c a t i o n o f h i s t i d i n e i n K - c a s e i n caused d e c r e a s e d s t a b i l i s i n g a b i l i t y when t h e r e was a g g r e g a t i o n . I t i s obvious t h a t the i n t e r a c t i o n s between a - and K - c a s e i n a r e f a r from c l e a r . V a r y i n g i n t e r -a c t i o n r a t i o s have been r e p o r t e d , and mechanisms i n v o l v i n g h y d r o p h i l i c , h ydrophobic and n o n - s p e c i f i c e l e c t r o s t a t i c 46 i n t e r a c t i o n s have heen p o s t u l a t e d . In t h i s c h a p t e r a r e r e p o r t e d the r e s u l t s o f a stud y on the i n t e r a c t i o n o f a s i ~ and K - c a s e i n s , u s i n g f l u o r e s c e n c e p o l a r i s a t i o n . 47. MATERIALS AND METHODS Kappa and a g ^ - c a s e i n s were pr e p a r e d a c c o r d i n g to the method o f Z i t t l e and C u s t e r (85). Dan s y l a t e d a ^  ^ - c a s e i n was pre p a r e d by r e a c t i n g a s ^ - c a s e i n , 280 mg/40 ml, i n carbonate b u f f e r (NaHCOg, 0.4M - Na 2C0 3, 0.1M, pH 9.2) w i t h a suspension o f 6 mg dimethylamino-naphthalene s u l f o n y c h o l r i d e , i n 1 ml o f the same b u f f e r . The r e a c t i o n was c a r r i e d out i n an i c e bath f o r 20 minutes w i t h c o n s t a n t s t i r r i n g . The r e a c t i o n m i x t u r e was then c e n t r i f u g e d a t 4°C, the supernatant d i a l y s e d a g a i n s t d i s t i l l e d water a t 4°C f o r 48 hours and l y o p h i l i s e d . One d a n s y l r e s i d u e was i n t r o d u c e d per mole o f a s ^ - c a s e i n (mol.wt. 27,000). Imidazole b u f f e r , 0.081, pH 6.8, was pre p a r e d by mixi n g 0.01M i m i d a z o l e and 0.01M HCl i n a r a t i o : o f 6:4, w i t h 0.07M NaCl added t h e r e a f t e r . Samples o f < - and d a n s y l a t e d a g ^ - c a s e i n s were p r e p a r e d i n 0.08M i m i d a z o l e b u f f e r , pH 6.8 Temperatures above and below ambient were m a i n t a i n e d by a Sargent Thermonitor and a Blue M co n s t a n t f l o w c o o l i n g u n i t , r e s p e c t i v e l y . P o l a r i s a t i o n was measured 48. u s i n g an Aminco-Bowman Spec t r o p h o t o f l u o r o m e t e r #4-8202 w i t h Zenon lamp and Bl a n k - S u b t r a c t P h o t o m u l t i p l i e r . S l i t s were as f o l l o w s : #2, 5? 4mm* #3, 4? 2mm* #7, 1mm. P o l a c o a t UV 105 and P o l a r o i d HN38 were used f o r the p o l a r i s e r and a n a l y s e r , r e s p e c t i v e l y . E x c i t a t i o n and emission wavelengths were 350 nm and 508 nm, r e s p e c t i v e l y . P o l y e t h y l e n i m i n e PEI 1000 from Dow Chemicals i n 0.08M i m i d a z o l e b u f f e r , pH 6.8 was added t o the a s ^-_ 5 c a s e i n s o l u t i o n , 4.0 X 10 M, to g i v e a f i n a l c o n c e n t r a t i o n o f PEI o f 0.04 mg/ml. 49 THEORY Fl u o r e s c e n c e p o l a r i s a t i o n o f a macromolecule i s m a i n l y dependent on the geometry and r i g i d i t y o f the m o l e c u l a r s t r u c t u r e . Changes i n m o l e c u l a r conformation due to a g g r e g a t i o n and i n t e r a c t i o n w i l l , t h e r e f o r e , r e s u l t i n changes i n the p o l a r i s a t i o n , because these changes modify m o l e c u l a r geometry and volume i n the macromolecule (16, 28, 61, 78, 79). Thus the p o l a r i s a t i o n t e chnique w i l l be a u s e f u l t o o l f o r the stud y o f the i n t e r a c t i o n between a s 1"" a n d K _ c a s e i n « B Y l a b e l l i n g one o f the c a s e i n s w i t h d a n s y l c h l o r i d e , i n our case a g ^ - c a s e i n , i t i s p o s s i b l e t o f o l l o w the i n t e r a c t i o n between t h e two c a s e i n s by measuring changes i n p o l a r i s a t i o n . Weber (78) demonstrated t h a t i f more than one o s c i l l a t o r , c o r r e s p o n d i n g t o more than one macromolecule i s s o l u t i o n , a r e s i m u l t a n e o u s l y e x c i t e d , the observed p o l a r i s a t i o n , P, and t h e f l u o r e s c e n t i n t e n s i t y , F, emit t e d by the components, a r e r e p r e s e n t e d by the e q u a t i o n : p = £Ti F i F J P i ( 3 ) i F i The observed p o l a r i s a t i o n o f the i n t e r a c t i n g system a ^. and K - c a s e i n , would t h e r e f o r e be: 50. P = F l P l + F 2 P 2 + F 3 P 3 (4) F l + F 2 + F 3 where the subscr ipts 1, 2, 3 r e f e r to a s K - case in and t h e i r i n t e r a c t i o n product . However, s ince only « , - c a s e i n was f luorescent s i by dansy la t ion , the concentrat ion of the dansylated a s 1" casein was kept constant and there was no change i n f luorescent i n t e n s i t y with i n t e r a c t i o n , equation (4) can be s i m p l i f i e d t o : P = ( F 0 - F 3 ) P X + F 3 P 3 ^ F 0 where F Q i s the f luorescent i n t e n s i t y of o r i g i n a l dansylated a . - c a s e i n , as the unreacted < -case in would not con-t r i b u t e to the f luorescence and. p o l a r i s a t i o n of the mixture. Rearranging equation (5) we o b t a i n : P - P = F 3 1 _ (6 p 3 " P l F 0 This equation i s eventual ly the same as the equation used by Dandl iker et a l . (7) . Since the i n t e r a c t i o n between a s i ~ a n d K casein i s known to be n e g l i g i b l e a t 2 to 6 ° C and to be spontaneous above 37 C ( 4 7 ) i t i s p o s s i b l e , u s i n g equation ( 6 ) , t o p r e d i c t the p o l a r i s a t i o n changes o f d a n s y l a t e d a g -^-casein d u r i n g t i t r a t i o n w i t h n o n - f l u o r e s c e n t < -c a s e i n a t these and i n t e r m e d i a t e temperatures. S i n c e t h e r e i s no r e a c t i o n , i . e . the e q u i l i b r i u m c o n s t a n t i s i n d e t e r m i n a t e a t 2 to 6°C, F_ and p a r e equal 3 3 to 0, equation (6) s i m p l i f i e s t o : P - P1 = 0, t h e r e f o r e P = P 1 (7.) Thus t h e r e s h o u l d be no change i n the p o l a r i s a t i o n o f a g ^ - c a s e i n t i t r a t e d w i t h < - c a s e i n i n t h i s temperature range. A t 37°C, the a s s o c i a t i o n constant i s i n f i n i t e and t h e r e i s spontaneous c o n v e r s i o n o f a g ^ - c a s e i n to a s 2_- K - c a s e i n complex. I f enough < - c a s e i n i s added t o the a , - c a s e i n , such t h a t a l l the a , - c a s e i n i s s ± s j. r e a c t e d , the o n l y f l u o r e s c e n t s p e c i e s p r e s e n t i n the mix-t u r e would be a s \~ K - c a s e i n complex. In equation (6) F 3 = F Q and the equation s i m p l i f i e s to P = P 3 , i . e . the observed p o l a r i s a t i o n i s due o n l y to the a s 1~ K - c a s e i n complex. F u r t h e r a d d i t i o n o f * - c a s e i n to the r e a c t i o n m i x t u r e , a f t e r a l l the f l u o r e s c e n t l a b e l l e d a - c a s e i n s 1 has r e a c t e d , should not a f f e c t the observed f l u o r e s c e n t y i e l d as the u n r e a c t e d K - c a s e i n i s n o n - f l u o r e s c e n t . 52. Equation (6) can be used to c a l c u l a t e F3 from the observed p o l a r i s a t i o n P s i n c e l?-^ and P 3 are constant f o r a given temperature and v i s c o s i t y , and F Q i s c o n s t a n t i f the c o n c e n t r a t i o n o f a s ^ - c a s e i n i s kept constant d u r i n g t h e experiment, and t h e r e i s no change i n f l u o r e s c e n t i n -t e n s i t y w i t h i n t e r a c t i o n . The observed p o l a r i s a t i o n should, t h e r e f o r e , v a r y d i r e c t l y w i t h the amount of K - c a s e i n added to the r e a c t i o n m i x t u r e . For c o n d i t i o n s o f complete i n t e r a c t i o n , i . e . 37°C o r above, P should i n c r e a s e l i n e a r l y w i t h added K - c a s e i n as a l l would be c o n v e r t e d to the a - K - c a s e i n complex. S i As s t a t e d p r e v i o u s l y , a f t e r a l l o f the a g ^ - c a s e i n has r e a c t e d , t h e r e i s no f u r t h e r i n c r e a s e i n observed p o l a r i s a t i o n . For c o n d i t i o n s o f incomplete i n t e r a c t i o n , between 6 and 37°C, because o f the e q u i l i b r i u m c o n d i t i o n s e x i s t i n g between r e a c t a n t s and p r o d u c t s , a l l the K - c a s e i n added i s not converted t o a g _ K - c a s e i n complex; t h e r e f o r e the i n c r e a s e i n p o l a r i s a t i o n w i t h added K - c a s e i n would be n o n - l i n e a r and l e s s than t h a t f o r the complete i n t e r a c t i o n . As the e q u i l i b r i u m c o n s t a n t decreases a t lower temperatures ( 4 7 ) a f i x e d amount o f K - c a s e i n added to a , - c a s e i n s 1 would r e s u l t i n l e s s i n t e r a c t i o n a t reduced temperatures, and, as a r e s u l t , a lower l e v e l o f i n c r e a s e i n the observed p o l a r i s a t i o n . 53. From e q u a t i o n (6) i t i s p o s s i b l e t o c a l c u l a t e t h e e q u i l i b r i u m c o n s t a n t a t a n y t e m p e r a t u r e a n d c o n c e n t r a t i o n F o f a d d e d K - c a s e i n , s i n c e i s e q u i l i v a l e n t t o t h e f r a c t i o n o f a s ^ - c a s e i n r e a c t e d , a n d t h e r e a c t i n g m o l e r a t i o i s 0.93:1 ( F i g . 13 a n d 1 5 ) . 54. RESULTS _ 5 The changes i n p o l a r i s a t i o n o f a 4.0 X 10 M s o l u t i o n o f d a n s y l a t e d a s ^ - c a s e i n due to t i t r a t i o n w i t h K - c a s e i n a t 4, 17, 24, 30, 35 and 40°C a r e p r e s e n t e d i n f i g u r e 13. A t 4°C t h e r e i s no change i n p o l a r i s a t i o n o f a s ^ - c a s e i n when t i t r a t e d w i t h < - c a s e i n . A t 40°C t h e r e i s a l i n e a r i n c r e a s e i n the p o l a r i s a t i o n o f a ^ ^ - c a s e i n when t i t r a t e d w i t h < - c a s e i n up to a c o n c e n t r a t i o n o f _ 5 4.3 X 10 M. F u r t h e r a d d i t i o n o f K - c a s e i n does not e f f e c t the p o l a r i s a t i o n o f the a ^ - c a s e i n . A t the i n t e r m e d i a t e temperatures, 17, 24, 30 and 35°C the p o l a r i s a t i o n o f the a ^ - c a s e i n i n c r e a s e s c u r v i -l i n e a r l y when t i t r a t e d w i t h K - c a s e i n . The s l o p e s o f these l i n e s decreases w i t h d e c r e a s i n g temperature. The i n t e r a c t i o n r a t i o o f a K - c a s e i n i s 0.93:1 based on t h e s t u d i e s s l o c a r r i e d out a t 4o C, The e f f e c t o f PEI and temperature on the p o l a r -i s a t i o n o f d a n s y l a t e d a , - c a s e i n when t i t r a t e d w i t h s 1 K - c a s e i n a r e p r e s e n t e d i n f i g u r e 14. The PEI r e s u l t s i n a h i g h e r v a l u e f o r the p o l a r i s a t i o n o f d a n s y l a t e d a , -J 1 I ' ' 2 4 6 8 10 12 5 k-CASEIN CONCENTRATION X10 MOLAR Figure 13. P o l a r i s a t i o n of dansylated <* -casein, S X 4.0 X 10"" ^  t i t r a t e d with < -casein at various temperatures. O - G 40°C : 35°C : A-A 30°C A - A 2 4 ° C i » - « 1 7 0 C : D-D4 ° C 018L 0 56 k- CASEIN CONCENTRATION X10 MOLAR Figure 14. P o l a r i s a t i o n of dansylated a - c a s e i n , s l * 4.0 X IO"*5** with and without added PEI t i t r a t e d with < - c a s e i n at var ious temperatures. • - • 50°C No PEI* 6 - D 4 0 ° C No PEI • - • 4 0 ° C PEI added; A - A 4 ° C No PEI A - A 4 ° C PEI added. 57. c a s e i n a t 4 and 40°C, although the s l o p e o f the curves a t the r e s p e c t i v e temperatures a r e the same when t i t r a t e d w i t h K - c a s e i n . -5 The changes i n p o l a r i s a t i o n o f a 6.0 X 10 M s o l u t i o n o f d a n s y l a t e d <*s ^ - c a s e i n due t o t i t r a t i o n w i t h K - c a s e i n a t 4 and 40°C a r e p r e s e n t e d i n f i g u r e 15 and show the same c h a r a c t e r i s t i c s as the lower c o n c e n t r a t i o n ( F i g . 13). 1 F i g u r e 16 shows a p l o t o f l o g K a g a i n s t ^ from which A H was c a l c u l a t e d . F i g u r e 17 shows the P e r r i n p l o t o f the a - K s 1 c a s e i n complex. Temperature ranges 37°C t o 50°C. From t h i s f i g u r e i t i s p o s s i b l e t o c a l c u l a t e the p o l a r i s a t i o n o f the a - K - c a s e i n complex a t lower temperatures, s 1 T a b l e I shows the v a r i a t i o n i n p o l a r i s a t i o n o f d a n s y l a t e d a ^ - c a s e i n and p o l a r i s a t i o n o f d a n s y l a t e d a s i ~ K - c a s e i n complex w i t h temperature. Ta b l e I I shows the v a r i a t i o n i n a s s o c i a t i o n con-s t a n t as c a l c u l a t e d w i t h the a i d o f equation (6) and. data p r e s e n t e d i n T a b l e I and f i g u r e 13, w i t h temperature. Con-_5 c e n t r a t i o n o f d a n s y l a t e d a - c a s e i n was 4.0 X 10 M. 58. i F i g u r e 1 6 . Log K f o r t h e a s s o c i a t i o n e q u i l i b r i u m p l o t t e d a g a i n s t t h e e q u i l i b r i u m t e m p e r a t u r e s . 60 TABLE I 61. VARIATION IN POLARISATION OF  a s i - AND a s 1 - K CASEIN COMPLEX WITH TEMPERATURE. P o l a r i s a t i o n o f Caseins Temperature °C a , a ,- K -complex s 1 s 1 4 0.283 17 0.238 0.322 24 0.232 0.305 30 0.216 0.292 35 0.200 0.288 40 0.185 0.268 50 0.170 0.246 TABLE II 62. VARIATION IN ASSOCIATION CONSTANT OF THE a ,- K -CASEIN s 1 INTERACTION WITH TEMPERATURE. C o n c e n t r a t i o n o f A s s o c i a t i o n Constant X 10~ 4 K - c a s e i n X 10 5M 17°C 24°C 30 UC 35 UC 1.0 0.99 1.71 2.81 6.80 2.0 0.96 1.63 2.88 6.81 3.0 1.02 1.68 2.93 7.61 4.0 1.00 1.61 3.00 7.51 6.0 1.02 1.60 3.05 7.40 8.0 1.06 1.74 3.05 7,27 10.0 0.97 1.63 3.09 6.82 12.0 0.97 1.70 3.00 6.87 Mean 1.00 1.66 2.97 7.16 A F and A S were c a l c u l a t e d from the f o l l o w i n g equations and a r e l i s t e d i n T a b l e I I I . AF = -RT In K A S = AH - ^ F ( 8 ) ( 9 ) TABLE I I I 64. THERMODYNAMIC PARAMETERS FOR THE INTERACTION o - K -s 1 CASEIN AND THEIR TEMPERATURE DEPENDENCE. s 1 Temperature E q u i l i b r i u m A F k c a l / A H k c a l / As EU °C Constant X 1 0 mole mole 1 7 1 . 0 0 - 5 . 2 9 2 4 1 . 6 6 - 5 . 7 1 3 . 3 3 3 0 . 4 3 0 2 . 9 7 - 6 . 1 9 4 . 6 7 3 5 . 8 3 5 7 . 1 6 - 6 . 8 2 DISCUSSION 65. D a n d l i k e r e t a l . (7) Kierzenbaum e t a l . (28) r e -p o r t e d a method f o r e s t i m a t i o n o f the a s s o c i a t i o n constant between a n t i g e n and a n t i b o d y by f l u o r e s c e n c e p o l a r i s a t i o n The b a s i c equation i n those papers and ours a r e the same i f t h e r e i s no change i n the f l u o r e s c e n t i n t e n s i t y a f t e r i n t e r -a c t i o n . F b Q b = P - P F D a n d l i k e r " s (10) F f Q f P b " P F b Q b + E f Q f = 1 + p b " p = p b - P f F b Q b P - P f P - P f = f o F 3 (11) The molecules i n a s o l u t i o n f l u o r e s c e i n d e p e n d e n t l y and the observed i n t e n s i t y i s s i m p l y the sum o f the i n t e n s i t i e s from a l l the i n d i v i d u a l m o l e c u l e s . There was no change i n f l u o r e s c e n t i n t e n s i t y upon i n t e r a c t i o n which was i n d i c a t e d by c o n s t a n t i n t e n s i t y f o r d a n s y l a t e d a -^-casein w i t h i n c r e a s i n g K - c a s e i n c o n c e n t r a t i o n a t 40°C. In t h i s case D a n d l i k e r { s Q^, and Q become equal and t h e i r equation (10) can be s i m p l i f i e d . In the case o f 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 the h e t e r o g e n e i t y o f b i n d i n g s i t e s f o r a n t i b o d y was r e p o r t e d ( 4 6 ) . However, i n our case the b i n d i n g s i t e s were assumed to be homogeneous (a = 1 ) w i t h i n samples used o f a s 1~ and K - c a s e i n s , and t h i s i s supported by the i n t e r a c t i n g mole r a t i o o f ap p r o x i m a t e l y 1 and by the f a c t t h a t t h e r e i s one hydrophobic s i t e p e r monomer u n i t . The e q u i l i b r i u m c o n s t a n t s d e s c r i b e d i n t h i s paper a r e average a s s o c i a t i o n c o n s t a n t s f o r the p o l y - d i s p e r s e system. These c o n s t a n t s a r e f o r the a s s o c i a t i o n between d a n s y l a t e d a - c a s e i n and u n l a b e l l e d K - c a s e i n which s 1 s h o u l d not be s i g n i f i c a n t l y d i f f e r e n t from those between u n l a b e l l e d a _ and < - c a s e i n , as demonstrated f o r s 1 bovine serum albumin and i t s a n t i b o d y by Kierzenbaum e t a l . (28), s i n c e the l e v e l o f d a n s y l a t i o n was one molecule p e r monomer u n i t o f a g ^ - c a s e i n . Such a low l e v e l o f modi-f i c a t i o n would p e r f e r a b l y be a t the t e r m i n a l amino- group, and as such s h o u l d not s i g n i f i c a n t l y i n t e r f e r e w i t h the i n t e r a c t i o n . In the c a l c u l a t i o n o f the a s s o c i a t i o n c o n s t a n t , c o n c e n t r a t i o n s o f a -and K - c a s e i n were determined, as s 1 monomer u n i t s . I f the i n t e r a c t i n g s p e c i e s i s a polymer, f o r example N-mer,with the i n t e r a c t i o n r a t i o b e i n g 1 : 1 , the e q u i l i b r i u m c o n s t a n t would be decreased by the f a c t o r N, as the molar c o n c e n t r a t i o n s o f a l l the s p e c i e s would be decreased, by t h i s f a c t o r . Values o f N f o r a - c a s e i n s 1 range from 3 t o 5 (52). Such a v a l u e would have a v e r y s l i g h t e f f e c t upon the v a l u e s of the thermodynamic p a r a -meters and would, not change the d i r e c t i o n o f v a r i a t i o n o f the parameters w i t h temperature. The s t a b i l i t y o f c a s e i n m i c e l l e s i s p r o b a b l y a r e f l e c t i o n o f many parameters, one of which i s undoubtedly the i n t e r a c t i o n between the c a s e i n s . The endothermic n a t u r e o f i n t e r a c t i o n o f the c a s e i n s has been known f o r sometime. T h i s endothermic n a t u r e o f a s s o c i a t i o n s o f the c a s e i n s has been suggested as b e i n g due to c o n f o r m a t i o n a l change i n the p r o t e i n and subsequent changes i n the c a l c i u m i o n b i n d i n g (74). However, ISIemethy and Scheraga (45) have p o s t u l a t e d t h a t the endothermic n a t u r e of i n t e r a c t i o n s c o u l d be due t o hydrophobic i n t e r a c t i o n s i n the p r o t e i n s , w i t h a subsequent i n c r e a s e i n both the enthalpy and en t r o p y o f the system. The r e s u l t s p r e s e n t e d i n T a b l e I I I c l e a r l y demonstrated the endothermic n a t u r e o f the i n t e r a c t i o n between a - and K - c a s e i n . The . a s s o c i a t i o n c o n s t a n t s 1 i n c r e a s e d w i t h temperature, and a t 40°C the i n t e r a c t i o n was complete. The i n c r e a s i n g ease o f r e a c t i o n was a l s o r e f l e c t e d i n the lower l e v e l o f f r e e energy w i t h temperature. 68. In agreement w i t h the t h e o r y o f hydrophobic i n t e r a c t i o n s (45) t h e r e was a p o s i t i v e e n t h a l p y and entropy. These parameters both i n c r e a s e d w i t h temperature, i n d i c a t i v e o f the hydrophobic nature o f the i n t e r a c t i o n . These r e s u l t s c o n f i r m the s u g g e s t i o n s o f G a r n i e r e t a l . ( 1 4 ) who found a p o s i t i v e e ntropy f o r the a s 1~ K * " c a s e i n i n t e r a c t i o n , which i n d i c a t e d the hydrophobic n a t u r e of t h e i n t e r a c t i o n , and a l s o Payens (52) who suggested t h a t the i n t e r a c t i o n would be hydrophobic i n n a t u r e . From the 40 o r 50°C curves i n f i g u r e s 13, 14 and 15 the i n t e r a c t i o n r a t i o a ^ - : K was 0.93:1 on a molar b a s i s , as evidenced by the l i n e a r i n c r e a s e i n p o l a r i s a t i o n o f a s i ~ w i t h added < - c a s e i n , u n t i l the above r a t i o was reached; then t h e r e was no f u r t h e r i n c r e a s e i n p o l a r i s a t i o n , t h a t i s no f u r t h e r i n t e r a c t i o n . The l i n e a r r e l a t i o n s h i p between p o l a r i s a t i o n and added K - c a s e i n , i s i n d i c a t i v e o f o n l y one r e a c t i o n p r o d u c t . I f t h e r e were an i n t e r a c t i o n p r o d u c t o r p r o d u c t s w i t h a h i g h e r r a t i o , such a l i n e a r r e l a t i o n s h i p would not e x i s t . The f i r s t a d d i t i o n o f K - c a s e i n would l e a d t o a maximum i n c r e a s e i n p o l a r i s a t i o n , c o r r e s p o n d i n g to the h i g h e s t p o s s i b l e r a t i o o f a g ^ s K under the experimental con-d i t i o n s ; as t h i s would r e p r e s e n t the l a r g e s t p o s s i b l e molecule. 6 9 . F u r t h e r a d d i t i o n o f < - c a s e i n would decrease the g K r a t i o ? t h e r e f o r e the m o l e c u l a r weight o f the i n t e r a c t i o n p r o d u c t , and hence the p o l a r i s a t i o n would a l s o decrease. S i n c e a c u r v i l i n e a r r e l a t i o n s h i p was not o b t a i n e d the p o s s i b i l i t y o f m i l t i p l e i n t e r a c t i o n products i s p r e c l u d e d . T h i s r e a c t i o n r a t i o was independent o f c o n c e n t r a t i o n as shown i n f i g u r e s 13, 14 and 15. These r e s u l t s are i n agreement w i t h those o f Noble and Waugh (47), who found t h a t a s -j^ - and K - c a s e i n r e a c t e d i n a low weight r a t i o and a l s o o f P a r r y et a l . (51) who found, t h a t the i n t e r a c t i n g weight r a t i o o f a K - c a s e i n i s u n i t y . Kason e t a l . (25) have shown t h a t t h e r e i s an i n t e r a c t i o n between a s ^ - c a s e i n and p o l y e t h e l e n i r a i n e which i s e l e c t r o s t a t i c i n n a t u r e . That such an i n t e r a c t i o n does o c c u r i s e v i d e n t from f i g u r e 14, where the i n t e r a c t i o n be-tween p o l y e t h y l e n i m i n e - t r e a t e d a ^ - c a s e i n and K - c a s e i n was compared w i t h the i n t e r a c t i o n between a s ^- and < -c a s e i n a t 4 and 40°C. In both cases the s l o p e s a t the two temperatures were i d e n t i c a l , however, the p o l a r i s a t i o n v a l u e s f o r the p o l y e t h y l e n i m i n e - t r e a t e d a s -^-casein was h i g h e r than f o r the u n t r e a t e d a s -^-casein. Two con-c l u s i o n s can be drawn from these r e s u l t s . F i r s t l y , the p o l y e t h y l e n i m i n e - t r e a t e d a g ^ - c a s e i n i s a l a r g e r molecule, i n d i c a t i v e o f an i n t e r a c t i o n p r o d u c t as demonstrated by 7 0 . Kason e t a l . (25). Secondly, the n o n - s p e c i f i c e l e c t r o -s t a t i c n a ture o f the i n t e r a c t i o n p r o d u c t 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 n e i t h e r i n t e r f e r e w i t h the a , - K - c a s e i n s 1 s x i n t e r a c t i o n , nor a f f e c t the i n t e r a c t i o n r a t i o . T h i s i n d i c a t e s t h a t the i n t e r a c t i o n i s q u i t e s p e c i f i c and not e l e c t r o s t a t i c i n n a t u r e . In the p r e v i o u s chapter < - c a s e i n was shown to have one hydrophobic r e g i o n . T h i s i s i n t o t a l agreement w i t h the r e s u l t s p r e s e n t e d above, namely t h a t the i n t e r -a c t i o n r a t i o o f a K i s u n i t y and a l s o t h a t the s l r e a c t i o n i s hydrophobic i n n a t u r e . 71. CHAPTER I I I . EFFECT OF MODIFICATION OF CHARGED GROUPS OF  K -CASEIN ON ITS STRUCTURE AND STABILISING  ABILITY. INTRODUCTION The c a l c i u m i n s e n s i t i v e f r a c t i o n o f whole c a s e i n , < - c a s e i n , i s unique among the c a s e i n s i n i t s a b i l 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 o f o t h e r c a s e i n s i n t h e presence o f ca l c i u m i o n s ( 7 7 ) . However, i n t e r a c t i o n o f the c a s e i n s i n the presence o f c a l c i u m i o n s l e a d t o the v e r y s t a b l e c a s e i n m i c e l l e s which e x i s t i n m i l k . I t i s g e n e r a l l y accepted, t h a t the unusual s t a b i l -i t y o f the c a s e i n m i c e l l e i s due to s t r u c t u r e m a i n t a i n e d by hydrophobic i n t e r a c t i o n s , n o n - s p e c i f i c e l e c t r o s t a t i c a t t r a c t i o n s and c a l c i u m phosphate s a l t b r i d g e s . Payens (52) and H i l l and Wake (21) suggested t h a t the main i n t e r a c t i n g f o r c e i s hydrophobic i n nat u r e , d i r e c t e d w i t h i n the m i c e l l e . T a l b o t and Waugh (69) Pepper et a l . (53), Woychik (81) and H i l l and L a i n g (20), suggested t h a t non-s p e c i f i c e l e c t r o s t a t i c a t t r a c t i o n p l a y s a r o l e i n the K -c a s e i n s t a b i l i s i n g a b i l i t y , p r o b a b l y i n v o l v i n g the e -amino groups o f l y s i n e i n < - c a s e i n . However, i t was not c l e a r whether the l y s i n e r e s i d u e s i n the K - c a s e i n p l a y e d a d i r e c t r o l e i n i n t e r a c t i o n , o r whether t h i s was due to s t r u c t u r a l changes induced i n the p r o t e i n as a r e s u l t o f charge d i f f e r e n c e s . T a l b o t and Waugh ( 6 9 ) found a decrease i n s t a b i l i s i n g a b i l i t y a f t e r one l y s i n e r e s i d u e was m o d i f i e d , whereas Pepper e t a_l. (53) found i t nec e s s a r y t o modify f i v e r e s i d u e s i n < - c a s e i n b e f o r e t h e r e was any l o s s i n s t a b i l -i s i n g a b i l i t y . Rose and. C o l v i n (57) on the other hand, suggested the p o s s i b i l i t y o f a c a l c i u m phosphate b r i d g e i n v o l v i n g the e -amino group o f a _ ,- and B - c a s e i n s . Hoagland (23) S JL however, found t h a t the e -amino group of 8 - c a s e i n was not n e c e s s a r y f o r s t a b i l i s a t i o n with K - c a s e i n . H i l l (18) suggested the presence-of a p o s i t i v e r e g i o n i n K - c a s e i n i n v o l v i n g a p e p t i d e sequence w i t h l y s i n e , a r g i n i n e and h i s t i d i n e , which was ne c e s s a r y f o r i n t e r a c t i o n . H i l l and L a i n g (20) had. p r e v i o u s l y demon-s t r a t e d t h a t m o d i f i c a t i o n o f h i s t i d i n e by p h o t o - o x i d a t i o n l e a d s t o l o s s o f s t a b i l i s i n g a b i l i t y . Nakai e t a_l. (43) m o d i f i e d the h i s t i d i n e o f < - c a s e i n r e s i d u e s w i t h d i -i s o p r o p y l f l uorophosphate and found no change i n i t s s t a b i l i s i n g a b i l i t y . 73. K o l a r and Brunner (30) were a b l e to p a r t i a l l y d i s r u p t the m i c e l l a r s t r u c t u r e by c o o l i n g , a n d Rose (55) suggested t h a t the a s s o c i a t i o n o f a - and 3 - c a s e i n s i s through hydrophobic bonds. In chapters I and I I i t has been demonstrated t h a t < - c a s e i n possesses -some l e v e l o f s t r u c t u r e which can be d i s r u p t e d by the normal d i s s o c i a t i n g agents, and a l s o t h a t the i n t e r a c t i o n between a - and < - c a s e i n s 1 was accompanied by thermodynamic changes s u g g e s t i v e o f h ydrophobic i n t e r a c t i o n . I t was d e c i d e d t o t e s t whether these two p o s i t i o n s were r e c o n c i l a b l e . The l y s i n e r e s i d u e s o f < - c a s e i n were modified, step-wise by c a r b a m y l a t i o n (53), to g i v e a s e r i e s o f K - c a s e i n s w i t h i n c r e a s e d net n e g a t i v e charge. The f r e e c a r b o x y l i c a c i d f u n c t i o n s o f g l u t a m i c and a s p a r t i c a c i d were e s t e r i f i e d i n a step-wise manner to g i v e a s e r i e s o f K - c a s e i n samples w i t h i n c r e a s e d p o s i t i v e c h a r a c t e r . The e f f e c t s o f these m o d i f i c a t i o n s on s t r u c t u r e and s t a b i l -i s i n g a b i l i t y o f K - c a s e i n were then i n v e s t i g a t e d . MATERIALS AND METHOD 74 Kappa and ^ ^ caseins- were prepared by the method o f Z i t t l e and Cu s t e r (8 5) arid were e l e c t r o p h o r e t i c a l l y pure on p o l y a c r y l a m i d e g e l . B - c a s e i n was perpared by the method o f Nakai e t a l . (44). Carbamylated K - c a s e i n s were pr e p a r e d a c c o r d i n g t o the procedure o f Pepper e t a l . (53), u s i n g r e a c t i o n times o f 0, 15, 30, 45, 60, 90, 120 minutes, and 20 h o u r s . The f r e e c a r b o x y l i c a c i d groups were c o n v e r t e d t o g l y c i n e methyl e s t e r s , a c c o r d i n g t o the method o u t l i n e d by L i n and Koshland (31). Rea c t i o n times used were 5, 10, 15, 27, 40 and 60 minutes. G l y c i n e methyl e s t e r c o n c e n t r a t i o n used was 0.25M. Exten t o f l y s i n e m o d i f i c a t i o n was c a l c u l a t e d from the percentage decrease i n the l y s i n e peak a f t e r amino a c i d a n a l y s i s , and assuming the l y s i n e content t o be n i n e r e s i d u e s p e r m o l e c u l a r weight o f 20,000. C a r b o x y l i c a c i d groups m o d i f i e d were determined from the i n c r e a s e i n the g l y c i n e peak, c a l c u l a t e d as above, assuming t h r e e g l y c i n e r e s i d u e s p e r m o l e c u l a r weight o f 20,000. Changes i n e l e c t r o p h o r e t i c m o b i l i t y o f the m o d i f i e d samples were determined u s i n g a 10% p o l y a c r y l a m i d e g e l , T r i s - g l y c i n e b u f f e r pH 8.7 w i t h 4.5 M urea and 2- mer-c a p t o e t h a n o l . The s t a b i l i s i n g a b i l i t y was determined a c -c o r d i n g t o the method o f Pepper e t a l . (53) w i t h the f o l l o w i n g m o d i f i c a t i o n s : (a) the r a t i o a s K - c a s e i n was 5, and (b) the f i n a l c o n c e n t r a t i o n o f c a l c i u m c h l o r i d e was 0.02M. R e l a t i v e f l u o r e s c e n t i n t e n s i t y i s the r a t i o o f the f l u o r e s c e n t i n t e n s i t i e s o f the sample t o t h a t o f < _ 5 c a s e i n a t a p r o t e i n c o n c e n t r a t i o n o f 4.0 X 10 M and an ANS c o n c e n t r a t i o n o f 2.0 X 10 M. These c o n c e n t r a t i o n s o f p r o t e i n and ANS were used throughout the experiment. P o l a r -i s a t i o n and energy t r a n s f e r were determined u s i n g the t e c h n i q u e and i n s t r u m e n t a t i o n p r e v i o u s l y d e s c r i b e d . ANS from Eastman Chemicals was p u r i f i e d as p r e v i o u s l y d e s c r i b e d . S o l -u t i o n s were p r e p a r e d i n 0.01M Imidazole b u f f e r . 76. RESULTS The changes i n p o l a r i s a t i o n , r e l a t i v e f l u o r e s -cent i n t e n s i t y , p ercentage energy t r a n s f e r , wavelength o f maximum emission and s t a b i l i s i n g a b i l i t y w i t h d i f f e r e n t l e v e l s o f e s t e r i f i c a t i o n o f c a r b o x y l i c f u n c t i o n s o f K c a s e i n a r e p r e s e n t e d i n f i g u r e s 18, 19 and 20. As the l e v e l o f m o d i f i c a t i o n i n c r e a s e s , t h e r e i s a decrease i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y and an i n c r e a s e i n wavelength i n maximum emi s s i o n ( F i g . 18). Both o f these changes a r e i n d i c a t i v e o f a decrease i n h y d r o p h o b i c i t y . I n c r e a s i n g the l e v e l o f m o d i f i c a t i o n decreases the energy t r a n s f e r and p o l a r i s a t i o n o f K - c a s e i n ( F i g . 19), i n d i c a t i v e o f s t r u c t u r a l change. Concomintant w i t h t h i s s t r u c t u r a l change i s a l o s s i n s t a b i l i s i n g a b i l i t y and c a l c i u m i n s e n s i t i v i t y ( F i g . 2 0 ) . The changes i n p o l a r i s a t i o n , r e l a t i v e f l u o r e s c e n t i n t e n s i t y , p e r c e n t a g e energy t r a n s f e r , wavelength o f maximum emi s s i o n and s t a b i l i s i n g a b i l i t y , w i t h d i f f e r e n t l e v e l s o f ca r b a m y l a t i o n o f E - amino group o f the l y s i n e r e s i d u e s i n < - c a s e i n a r e p r e s e n t e d i n f i g u r e s 21, 22.and 23. As the l e v e l o f l y s i n e m o d i f i c a t i o n i n c r e a s e s , t h e r e i s i n i t i a l l y an i n c r e a s e i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y . A f t e r one r e s i d u e i s m o d i f i e d , the r e l a t i v e f l u o r e s c e n t i n t e n s i t y d e c r e a s e s p r o g r e s s i v e l y w i t h f u r t h e r m o d i f i c a t i o n . Concom-7 7 . CARBOXYLIC ACID RESIDUES MODIFIED F i g u r e 18. E f f e c t o f m o d i f i c a t i o n o f c a r b o x y l i c a c i d f u n c t i o n s o f K - c a s e i n on r e l a t i v e f l u o r e s c e n t i n t e n s i t y 9—0 , and wavelength o f maximum emis s i o n o f ANS conjugate A—A . F i g u r e 1 9 . E f f e c t o f m o d i f i c a t i o n o f c a r b o x y l i c a c i d f u n c t i o n s o f K - c a s e i n o n % e n e r g y t r a n s f e r , a n d p o l a r i s a t i o n A —A . 01 — J 1 I I I L 0 2 4 6 8 10 12 CARBOXYLIC A C I D RESIDUES MODIFIED F i g u r e 20. E f f e c t o f m o d i f i c a t i o n o f c a r b o x y l i c a c i d f u n c t i o n s o f K - c a s e i n on i t s a b i l i t y t o s t a b i l i s e a - c a s e i n O —©and o n i t s own s l s o l u b i l i t y i n t h e p r e s e n c e o f 0.02M c a l c i u m i o n A— A . 8 0 . LYSINE RESIDUES MODIFIED E f f e c t 2 1 . E f f e c t o f m o d i f i c a t i o n o f E - a m i n o g r o u p s o f l y s i n e o f < - c a s e i n o n r e l a t i v e f l u o r e s c e n t i n t e n s i t y V — V , a n d w a v e l e n g t h o f maximum e m i s s i o n o f ANS c o n j u g a t e O—O . 81. DC UJ l i _ CO Z < o F i g u r e 22. E f f e c t o f m o d i f i c a t i o n o f e - a m i n o g r o u p o f l y s i n e o f < - c a s e i n o n % e n e r g y t r a n s f e r C^O a n d p o l a r i s a t i o n V^-v . 82 o« 1 1 I i i _ 0 2 4 6 8 10 LYSINE RESIDUES M O D I F I E D F i g u r e 2 3 , E f f e c t o f m o d i f i c a t i o n o f e - a m i n o g r o u p o f l y s i n e o f * - c a s e i n o n i t s a b i l i t y t o s t a b i l i s e a s j ^ - c a s e i n O - 0 a n d on i t s own s o l u b i l i t y i n t h e p r e s e n c e o f 0.02M c a l c i u m i o n v — v . 83. t a n t w i t h these changes i n r e l a t i v e f l u o r e s c e n t i n t e n s i t y , t h e r e i s i n i t i a l l y a decrease i n the wavelength o f maximum emis s i o n f o l l o w e d by a r a p i d i n c r e a s e ( F i g . 21). There a r e v e r y s m a l l changes i n the energy t r a n s f e r and p o l a r -i s a t i o n o f K - c a s e i n w i t h m o d i f i c a t i o n o f up to f i v e l y s i n e r e s i d u e s . F u r t h e r m o d i f i c a t i o n l e a d s t o v e r y r a p i d decreases i n energy t r a n s f e r , p o l a r i s a t i o n and s t a b i l i s i n g a b i l i t y f o r a g .^-casein ( F i g s . 22, 23). T a b l e IV shows the p o l a r i s a t i o n , percentage energy t r a n s f e r , r e l a t i v e f l u o r e s c e n t i n t e n s i t y and wave-l e n g t h o f maximum emission o f a .-, B and < - c a s e i n s . The e l e c t r o p h o r e t i c p a t t e r n s o f the m o d i f i e d samples were as expected f o r p r o t e i n s w i t h changes i n the charge c h a r a c t e r i s t i c s . With i n c r e a s i n g l e v e l s o f l y s i n e m o d i f i c a t i o n the a n i o n i c c h a r a c t e r , and hence t h e e l e c t r o -p h o r e t i c m o b i l i t y , i n c r e a s e d . With the m o d i f i c a t i o n o f c a r b o x y l i c f u n c t i o n s , however, the a n i o n i c c h a r a c t e r decreased and m o b i l i t y decreased, and e v e n t u a l l y the m i g r a t i o n was towards the cathode a t h i g h l e v e l s o f m o d i f i c a t i o n . TABLE IV 84. COMPARISON OF THE FLUORESCENT PARAMETERS OF a - j - , AND K -CASEINS. P r o t e i n R e l a t i v e P o l a r i s a t i o n %Energy Xmax nm F l u o r e s c e n t I n t e n s i t y T r a n s f e r K - c a s e i n 1.00 0.246 12.0 477.5 a , - c a s e i n 0.37 0.198 7.0 482.4 s 1 8 - c a s e i n 0.31 0.173 12.0 482.4 DISCUSSION 85 The m o d i f i c a t i o n o f charged groups i n < - c a s e i n s has been shown t o l e a d t o a l o s s i n s t a b i l i s i n g a b i l i t y (18, 19, 20, 53, 69, 81, 84), and suggests the p o s s i b i l i t y o f 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 between the c a s e i n s , p a r t -i c u l a r l y i n v o l v i n g the e -amino group of l y s i n e . The r e s u l t s p r e s e n t e d here support the f i n d i n g t h a t m o d i f i c a t i o n o f charged groups induces a l o s s o f s t a b i l i s i n g a b i l i t y ( F i g s . 20, 23). However, t h i s l o s s o f a b i l i t y t o s t a b i l i s e a s ^ - c a s e i n a g a i n s t p r e c i p i t a t i o n was a c h i e v e d by modify-i n g e i t h e r the e -animo group o f l y s i n e r e s i d u e s as the above authors have done, o r by m o d i f i c a t i o n o f c a r b o x y l i c groups. Furthermore, a t h i g h l e v e l s o f m o d i f i c a t i o n o f c a r b o x y l i c groups, the K - c a s e i n r e a c t e d l i k e a - and s 1 8 - c a s e i n s and i t was p r e c i p i t a t e d by c a l c i u m i o n s . F i n a l l y , a l t h o u g h low l e v e l s o f m o d i f i c a t i o n o f l y s i n e r e s i d u e s changed the s t a b i l i s i n g a b i l i t y o n l y s l i g h t l y m o d i f i c a t i o n o f c a r b o x y l i c f u n c t i o n s even a t low l e v e l s had a more pronounced e f f e c t upon the s t a b i l i s i n g a b i l i t y . Changes i n f l u o r e s c e n t parameters o f a macromole-c u l e a r e a good i n d i c a t i o n o f s t r u c t u r a l changes (63 ) . F i g u r e s 18, 19, 21, 22 show v e r y s i g n i f i c a n t changes i n the f l u o r e s c e n t parameters measured, i n d i c a t i v e o f s t r u c t u r a l 86. changes i n the macromolecule and f u r t h e r t h a t these changes i n c r e a s e w i t h i n c r e a s i n g l e v e l s o f m o d i f i c a t i o n . A c c o r d i n g t o S t r y e r (63), i f the 8 - a n i l i n o n a p h -t h a l e n e - l - s u l f o n a t e i s bound t o a r e g i o n o f a macromole-c u l e , s t r u c t u r a l changes which r e s u l t i n an i n c r e a s e i n p o l a r i t y o f the r e g i o n o f the molecule, l e a d to a decrease i n f l u o r e s c e n t i n t e n s i t y , and i n c r e a s e i n wavelength o f maximum e m i s s i o n . These s p e c t r a l changes a r e due t o s o l v e n t r e o r i e n t a t i o n around the e x c i t e d s t a t e d u r i n g i t s l i f e t i m e . The s h i f t s i n wavelength o f maximum emi s s i o n and the decrease i n f l u o r e s c e n t i n t e n s i t y , as measured r e l a t i v e t o unmodified < - c a s e i n , ( F i g s . 18, 21) a r e i n d i c a t i o n s t h a t t h e r e i s i n c r e a s e d exposure o f the ANS b i n d i n g r e g i o n t o the s o l v e n t water m o l e c u l e s , o r a t l e a s t the molecule a c q u i r e s a new conformation, whereby charged groups have caused an i n c r e a s e i n p o l a r i t y o f the r e g i o n . T h i s s i t u a t i o n c o u l d be brought about by a s w e l l i n g o f the macromolecule, o r by d i s r u p t i o n o f bonds m a i n t a i n i n g the m o l e c u l a r r i g i d i t y thus a l l o w i n g f r e e r r o t a t i o n and a reduced c l u s t e r i n g o f the a p o l a r g r o u p i n g s . The f a c t t h a t t h e r e i s a decrease i n percentage energy t r a n s f e r (10) from the tryphophan t o ANS, ( F i g s . 19, 22) c o u l d support e i t h e r o f the concepts, as both would l e a d t o an i n c r e a s e d d i s t a n c e o f s e p a r a t i o n o f 87. the a b s o r b i n g typtophan group and the e m i t t i n g ANS mole-c u l e . However, the v a r i a t i o n i n the p o l a r i s a t i o n o f the m o l e c u l e cannot be accounted f o r by s w e l l i n g i n the mole-c u l e . Any s w e l l i n g would l e a d to an i n c r e a s e d m o l e c u l a r volume v/ith concomitant increased, r e l a x a t i o n time and p o l a r i s a t i o n , assuming t h a t t h e r e was no change i n m o l e c u l a r r i g i d i t y (16, 61, 78, 79). The observed decrease i n p o l a r i s a t i o n can o n l y be the r e s u l t o f two e f f e c t s , a decreased m o l e c u l a r volume, and/or a d e c r e a s e i n the m o l e c u l a r r i g i d i t y . I f t h e r e were a decrease i n m o l e c u l a r volume, w h i l e m a i n t a i n i n g r i g i d i g y , the p e r c e n t a g e energy t r a n s f e r would i n c r e a s e as t h i s would p r o b a b l y l e a d t o a s h o r t e r donor-acceptor d i s t a n c e o f s e p a r a t i o n . Secondly, a decreased m o l e c u l a r volume c o u l d l e a d t o h i g h e r f l u o r e s c e n t y i e l d and s h o r t e r wavelength o f maximum e m i s s i o n , as the a p o l a r groups would be f o r c e d i n t o c l o s e r c o n t a c t w i t h each o t h e r , away from the s o l v e n t environment. Hence a s m a l l e r l o s s o f energy would o c c u r d u r i n g the l i f e t i m e o f the e x c i t e d s t a t e , due t o s o l v e n t r e o r i e n t a t i o n . Thus the decrease i n p o l a r i s a t i o n cannot be due t o a c o n t r a c t i o n i n m o l e c u l a r volume. Such a c o n t r a c t i o n would be d i f f i c u l t t o envisage when the m o d i f i c a t i o n s l e a d to c o n d i t i o n s o f i n c r e a s e d r e p u l s i v e f o r c e s . 88. The o n l y e x p l a n a t i o n f o r the decrease i n p o l a r -i s a t i o n , which i s compatible w i t h the data p r e s e n t e d i n f i g u r e s 18, 19, 21, 22 i s t h a t t h e r e i s some d i s r u p t i o n i n the m o l e c u l e which l e a d s t o a decrease i n r i g i d i t y . Such a decrease i n r i g i d i t y would a l l o w a c e r t a i n degree o f f r e e r o t a t i o n , and c o u l d q u i t e e a s i l y decrease the non-p o l a r i t y o f the hydrophobic r e g i o n , e i t h e r by d i s r u p t i n g the s t r u c t u r e which maintained the conformation o f the hydro-p h o b i c group, o r by i n c r e a s e i n g the aqueous nature o f the environment o f the hydrophobic r e g i o n . T h i s would r e s u l t i n d e creased f l u o r e s c e n c e and i n c r e a s e d wavelength o f maximum e m i s s i o n . The f r e e r o t a t i o n a l s o would e f f e c t the percentage energy t r a n s f e r by a l l o w i n g v a r i a t i o n s i n the donor-acceptor o r i e n t a t i o n f a c t o r (10). I t would appear, t h e r e f o r e , t h a t by m o d i f i c a t i o n o f K - c a s e i n , some o f the r i g i d i t y i n h e r e n t i n the molecule i s l o s t . In t h e p r e v i o u s c h a p t e r s , evidence has been p r e s e n t e d i n support o f a hydrophobic i n t e r a c t i o n between K -and d ^.-casein, which p r e v e n t e d the l a t t e r from b e i n g p r e c i p i t a t e d by c a l c i u m . The f a c t t h a t i n c r e a s i n g l e v e l s o f m o d i f i c a t i o n o f < - c a s e i n ( F i g s . 20, 23), l e a d s t o de-c r e a s i n g s t a b i l i s i n g a b i l i t y , c o n c o m i t a n t l y w i t h d e c r e a s i n g h y d r o p h o b i c i t y (as measured by d e c r e a s i n g r e l a t i v e f l u o r -escence, and i n c r e a s e d wavelength o f maximum e m i s s i o n ) , 89. lends support to the concept o f hydrophobic i n t e r a c t i o n between a - and K - c a s e i n , s 1 I f the data f o r a „ and B - c a s e i n a r e s l compared w i t h t h a t f o r K - c a s e i n , (Table I V ) , the low v a l u e s o f r e l a t i v e f l u o r e s c e n t i n t e n s i t y and p o l a r i s a t i o n , and the h i g h wavelength o f maximum emission o f the two former c a s e i n s a r e q u i t e r e a d i l y apparent. I t would seem t h a t a ^- and $ - 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 s e d , random c o i l s and have a h i g h l e v e l of f r e e r o t a t i o n com-pared t o < - c a s e i n . However, when 12 c a r b o x y l i c a c i d s r -f u n c t i o n s o f < - c a s e i n were m o d i f i e d , t h i s p r o t e i n more c l o s e l y resembled a s i ~ a n d & - c a s e i n , i n i t s hydro-p h o b i c c h a r a c t e r i s t i c s and s u s c e p t i b i l i t y t o c a l c i u m i o n p r e c i p i t a t i o n ( F i g s . 18 - 20; Table I V ) . T h i s lends f u r t h e r support to the concept o f hydrophobic i n t e r a c t i o n among the c a s e i n s . A l though the e l e c t r o s t a t i c f o r c e s a r e not d i r e c t -l y r e l a t e d t o the a b i l i t y o f < - c a s e i n to s t a b i l i s e a s -and B - c a s e i n a g a i n s t c a l c i u m i o n p r e c i p i t a t i o n , these f o r c e s a r e important i n m a i n t a i n i n g the s t r u c t u r a l i n t e g r i t y o f the m o l e c u l e . C o n v e r s e l y , the m o l e c u l a r changes c o u l d be due s o l e l y t o an i n c r e a s e i n r e p u l s i v e f o r c e s i n the m o l e c u l e w i t h i n c r e a s i n g l e v e l s o f m o d i f i c a t i o n . F i n a l l y , 90. as can be seen from f i g u r e s 21, 22, 23, low l e v e l s o f m o d i f i c a t i o n o f l y s i n e r e s i d u e s i n K - c a s e i n do not have as pronounced an e f f e c t on the K - c a s e i n as low l e v e l s o f c a r b o x y l i c r e s i d u e m o d i f i c a t i o n . Whereas the c a r b o x y l i c a c i d m o d i f i c a t i o n l e a d s t o an immediate e f f e c t upon s t r u c t u r e and s t a b i l i s i n g a b i l i t y , ( F i g s . 18, 19, 20) the f l u o r e s c e n t parameters f o r s i m i l a r l e v e l s o f l y s i n e m o d i f i c a t i o n suggest i n c r e a s e d h y d r o p h o b i c i t y ( F i g . 21). These data might sug-g e s t t h a t some l y s i n e r e s i d u e s are on the s u r f a c e , and not d i r e c t l y r e l a t e d t o maintenance o f the s t r u c t u r e . Mod-i f i c a t i o n would t h e r e f o r e o n l y have the e f f e c t o f re d u c i n g the p o l a r i t y w i t h a concomitant apparent i n c r e a s e i n h y d r o p h o b i c i t y . The c a r b o x y l i c a c i d f u n c t i o n s seem t o be more r e l a t e d t o s t r u c t u r a l i n t e g r i t y , p r o b a b l y i n t e r n a l l y . I f t h i s s i t u a t i o n e x i s t s i n < - c a s e i n , i t o f f e r s an e x p l a n a t i o n f o r i t s s o l u b i l i t y i n the presence o f c a l c i u m ions due to p r e f e r e n t i a l i n t r a m o l e c u l a r c a l c i u m i o n b i n d i n g . T h i s i s due to the l o c a t i o n o f the c a r b o x y l i c f u n c t i o n s i n the i n t e r i o r o f the m o l e c u l e . Secondly, the l y s i n e r e s i d u e s on the s u r f a c e would r e p e l the approaching c a l c i u m i o n s . T h i s concept o f s t r u c t u r a l changes i n < - c a s e i n w i t h i n c r e a s i n g l e v e l s o f m o d i f i c a t i o n i s supported by the work o f Pepper e t a l . (53) and Woychik (81) who found a marked decrease i n the sed i m e n t a t i o n c o n s t a n t s o f < - c a s e i n 91. w i t h h i g h l e v e l s o f m o d i f i c a t i o n o f the l y s i n e e -amino groups. The c a l c i u m b i n d i n g c a p a c i t y o f a - and < - c a -s s e i n s i s s i m i l a r (41). The reason f o r h i g h e r s o l u b i l i t y o f K - c a s e i n than a - and 3 - c a s e i n s i n the presence s 1 o f c a l c i u m i o n , c o u l d be r e l a t e d to the f a c t t h a t t h e r e i s s t r u c t u r e i n K - c a s e i n which i s p r e d o m i n a n t l y l a c k i n g i n a - , 3 - c a s e i n , and h i g h c h a r g e - m o d i f i e d < - c a s e i n . Because o f the more open s t r u c t u r e i n « . 3 , and h i g h l y c h a r g e - m o d i f i e d « - c a s e i n , i n t e r m o l e c u l a r c a l c i u m i o n b i n d i n g might o c c u r , l e a d i n g to p o l y m e r i s a t i o n whereas, i n t h e more r i g i d < - c a s e i n s t r u c t u r e , i n t r a m o l e c u l a r c a l c i u m i o n b i n d i n g may predominate. The p o s s i b i l i t y o f hydro-p h o b i c i n t e r a c t i o n l e a d i n g t o a g g r e g a t i o n because o f a d e crease i n e l e c t r o s t a t i c r e p u l s i o n due to c a l c i u m b i n d i n g cannot be o v e r l o o k e d . However, the weak hydrophobic c h a r a c t e r o f a g ^ - c a s e i n as judged from T a b l e IV would seem to negate t h i s , u n l e s s t h e r e were a c o n f o r m a t i o n a l change a f t e r c a l c i u m i o n b i n d i n g , r e s u l t i n g i n i n c r e a s e d hydrophob-i c i t y . GENERAL DISCUSSION AND CONCLUSIONS The l o n g h e l d b e l i e f t h a t K - c a s e i n was s t r u c t u r e -l e s s seems to be unfounded. Kappa-casein a l t h o u g h h i g h l y aggregated, assumes a s t r u c t u r e s i m i l a r to t h a t of a g l o b u l a r p r o t e i n . T h i s s t r u c t u r e i s r e l a t e d t o i t s a b i l i t y t o s t a b i l i s e a - c a s e i n a g a i n s t c a l c i u m p r e c i p i t a t i o n . s 1 Kappa-casein has a w e l l d e f i n e d hydrophobic r e g i o n , changes i n which a l o n g w i t h o t h e r f l u o r e s c e n t c h a r a c t e r -i s t i c s , have been used as an index o f s t r u c t u r a l change i n the m o l e c u l e . The i n t e r a c t i o n w i t h a . - c a s e i n i n v o l v e s S X t h i s h y drophobic r e g i o n and the thermodynamic parameters AS and AH a r e i n d i c a t i v e o f an i n t e r a c t i o n which i s h i g h l y h y d r o p h o b i c i n c h a r a c t e r . Chemical m o d i f i c a t i o n o f < - c a s e i n l e a d i n g to changes i n p o s i t i v e o r n e g a t i v e charges on the p r o t e i n e f f e c t the h y d r o p h o b i c c h a r a c t e r o f K - c a s e i n , and sub-s e q u e n t l y t o a decrease i n the s t a b i l i s i n g a b i l i t y o f < c a s e i n f o r a ^ - c a s e i n a g a i n s t c a l c i u m i o n p r e c i p i t a t i o n . I t i s t h e r e f o r e concluded t h a t the charged groups o f K - c a s e i n a r e important i n m a i n t a i n i n g the s t r u c t u r a l i n t e g r i t y o f the m o l e c u l e , but a r e not d i r e c t l y r e l a t e d t o the i n t e r a c t i o n w i t h the o t h e r c a s e i n s . I t i s a l s o suggested t h a t i n t e r a c t i o n w i t h c a l c i u m i o n might be p r e d o m i n a n t l y i n t r a m o l e c u l a r i n K - c a s e i n and i n t e r -m o l e c u l a r i n a s - and. 6 - c a s e i n , hence the p o s s i b i l i t y o f h i g h l e v e l s o f a g g r e g a t i o n o f the l a t t e r p r o t e i n s i n the p r e s e n c e o f c a l c i u m i o n s . Questions n a t u r a l l y a r i s e as t o the r e l a t i o n -s h i p o f these data to m i c e l l e s t r u c t u r e . S i n c e the i n t e r -a c t i o n between a - and < - c a s e i n i s on a 1:1 mole s 1 b a s i s , a l l the a s i ~ a n d & - c a s e i n cannot be d i r e c t l y i n t e r a c t e d w i t h K - c a s e i n . A l s o 8 - c a s e i n r e a d i l y s e p a r a t e s from the m i c e l l e on c o o l i n g , thus the i n t e r a c t i o n i n v o l v i n g 8 - c a s e i n i s hydrophobic and not e l e c t r o s t a t i c i n n a t u r e . S i n c e the h y d r o p h o b i c i t y o f < - c a s e i n i s much h i g h e r than <* s ^ - o r 8 - c a s e i n , t h i s would suggest t h a t the weakest hydrophobic bond would i n v o l v e a ^- and 8 -c a s e i n . Hence i t i s suggested t h a t the 8 - c a s e i n i s i n t e r -a c t e d w i t h a - c a s e i n through hydrophobic bonding. The s 1 polymer o f a , - 8 - c a s e i n i s then bound t o the < - c a s e i n s 1 through hydrophobic i n t e r a c t i o n , to form a s m a l l c a l c i u m f r e e m i c e l l e . Such an i n t e r a c t i o n must have th e e f f e c t o f e i t h e r l i m i t i n g the l e v e l o f a g g r e g a t i o n , i n the presence o f c a l c i u m i o n , o r o f o b s c u r i n g p a r t i a l l y the n e g a t i v e charges on the <* _ and 6 - c a s e i n s . I t i s concluded t h a t the l a t t e r p o s s i b i l i t y i s more l i k e l y , as the more random c o i l a „ , - and 8 - c a s e i n s would have t o assume s l some geometric r i g i d i t y a f t e r the i n t e r a c t i o n w i t h the more s t r u c t u r e d < - c a s e i n . The r o l e o f c a l c i u m i o n i n the m i c e l l e would t h e r e f o r e be i n v o l v e d i n i n c r e a s i n g the m i c e l l e s i z e by b r i d g i n g s m a l l e r c a s e i n m i c e l l e s t o g e t h e r . Such b r i d g e s c o u l d p r o b a b l y i n v o l v e c a r b o x y l i c and p h o s p h o r i c a c i d f u n c t i o n s . The i n c r e a s e d r i g i d i t y produced by such b r i d g i n g would f u r t h e r l i m i t the random c h a r a c t e r o f the p r o t e i n s , and l e n d f u r t h e r s t a b i l i t y t o the m i c e l l a r s t r u c t u r e . 95. LITERATURE CITED 1. Azumi, T. and McGlynn, S.P. (1962) J . Chem. Phys. 37, 2413. 2. 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