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The relationship between hydrophobicity and surfactant properties of proteins Keshavarz, Elaheh 1977

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THE RELATIONSHIP BETWEEN HYDROPHOBICITY AND SURFACTANT PROPERTIES OF PROTEINS by ELAHEH KESHAVARZ B.Sc, University of Tehran, Iran, 1967 Pharm. D., University of Tehran, Iran, 1969 M.Sc, University of British Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Faculty of Graduate Studies Department of Food Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 1977 c^T) ELAHEH KESHAVARZ, 1977 In present ing th is thes is in p a r t i a l fu l f i lment of t h e r e q u i r e m e n t s f o r an advanced degree at the Un ivers i ty of B r i t i s h C o l u m b i a , I a g r e e t h a t the L ibrary sha l l make it f ree ly ava i l ab le for r e f e r e n c e and s t u d y . I fur ther agree that permission for extensive copying o f t h i s t h e s i s for scho la r ly purposes may be granted by the Head o f my D e p a r t m e n t o r by h is representat ives . It is understood that c o p y i n g o r p u b l i c a t i o n o f th is thes is fo r f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of T~QOV The Univers i ty of B r i t i s h Columbia 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D a t e \0, \ q 1 7 i ABSTRACT The c o m p o s i t i o n , s t r u c t u r e and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s a r e t h o u g h t t o be i n t e r r e l a t e d b u t t h e n a t u r e o f t h i s r e l a t i o n s h i p i s n o t c l e a r l y known. I n most s t u d i e s o f the s t r u c t u r e -f u n c t i o n r e l a t i o n s h i p s o f p r o t e i n s , more a t t e n t i o n has been f o c u s e d on the p o l a r r e s i d u e s w h i l e the i m p o r t a n c e of t h e n o n p o l a r i t y o f th e p r o t e i n m o l e c u l e has been o v e r l o o k e d . I t seems n e c e s s a r y t o s t u d y t h e r e l a t i o n s h i p s , i f any, between h y d r o p h o b i c i t y and f u n c t i o n a l p r o p e r t i e s w h i c h a r e i m p o r t a n t i n f o o d systems. The purpose o f t h i s s t u d y has been t w o - f o l d . F i r s t , e s t a b l i s h m e n t o f methods f o r d e t e r m i n a t i o n o f h y d r o p h o b i c i t y , and s e c o n d , t o c o r r e l a t e the h y d r o p h o b i c i t i e s w i t h t h e s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s . The h y d r o p h o b i c i t i e s c a l c u l a t e d so f a r ( B i g e l o w , 1967; T a n f o r d , 1962; Waugh, 1954) f r o m t h e amino a c i d c o m p o s i t i o n s do n o t appear t o c o r r e l a t e w i t h t h e p r o p e n s i t y o f p r o t e i n t o form h y d r o p h o b i c i n t e r a c t i o n s . I n an a t t e m p t t o d e t e r m i n e t h e e f f e c t i v e h y d r o p h o b i c i t y ( t h e c a p a c i t y t o p a r t i c i p a t e i n h y d r o p h o b i c i n t e r a c t i o n s ) , column chromatography on s u b s t i t u t e d Sepharose g e l s was employed. O l e i c a c i d , a l i p h a t i c and a r o m a t i c amines were c o u p l e d t o t h e CNBr a c t i v a t e d S e p h a r o s e 4B. However, t h e s e g e l s were n o t s u i t a b l e f o r d e t e r m i n a t i o n o f h y d r o p h o b i c i t y . T h i s may be due t o t h e p r e s e n c e o f u n d e s i r a b l e c h a r g e s o r t o t h e e x c e e d i n g l y s t r o n g h y d r o p h o b i c i n t e r a c t i o n s between t h e l i g a n d and t h e p r o t e i n s . Chromatography on Sephadex G-150 was a l s o employed i n t h e p r e s e n c e o f T r i t o n X-100 and t h e amount o f t h e n o n i o n i c d e t e r g e n t bound t o t h e p r o t e i n was d e t e r m i n e d . However, l a c k o f r e p e a t a b i l i t y i n t h e d e t e r m i n a t i o n o f t h e d e t e r g e n t bound t o t h e p r o t e i n p r o h i b i t e d the s a p p l i c a t i o n o f t h i s method f o r d e t e r m i n a t i o n o f t h e e f f e c t i v e h y d r o p h o b i c i t y . A l k y l e p o x y d e r i v a t i v e s o f Sepharose 4B (C4, C6 and C8) were s y n t h e s i z e d . H y d r o p h o b i c chromatography on the b u t y l and h e x y l d e r i v a t i v e s was s u c c e s s f u l i n d e t e r m i n a t i o n o f h y d r o p h o b i c i t y . However, s i n c e o c t y l e p o x y - S e p h a r o s e , because o f i t s h i g h h y d r o p h o b i c i t y , t i g h t l y i n t e r a c t e d w i t h t h e p r o t e i n s i t was found t o be i m p r a c t i c a l f o r t h e pu r p o s e o f m e a s u r i n g h y d r o p h o b i c i t y . I n an aqueous s o l v e n t phase, t h e s e a d s o r b e n t s d e m o n s t r a t e some o f t h e p r o p e r t i e s o f an o i l / w a t e r i n t e r f a c e , i n c l u d i n g t h e p o s s i b i l i t y o f d e n a t u r i n g some p r o t e i n s . T h e r e f o r e , i t i s assumed t h a t t h e p r o t e i n s were d e n a t u r e d on t h e o c t y l e p o x y - S e p h a r o s e g e l . The h y d r o p h o b i c p a r t i t i o n method was a l s o employed. Two phase polymer systems o f p o l y e t h y l e n e g l y c o l / d e x t r a n and p o l y e t h y l e n e g l y c o l p a l m i t a t e / d e x t r a n were used and t h e e x t e n t o f t h e h y d r o p h o b i c b i n d i n g o f t h e p r o t e i n s t o t h e p a l m i t a t e group was e x p r e s s e d as the " h y d r o p h o b i c c o e f f i c i e n t " o f t h e p r o t e i n s . The e f f e c t i v e h y d r o p h o b i c i t i e s o f p r o t e i n s d e t e r m i n e d by h y d r o p h o b i c chromatography w i t h b u t y l - and h e x y l e p o x y - S e p h a r o s e s and by h y d r o p h o b i c p a r t i t i o n s i g n i f i c a n t l y c o r r e l a t e d w i t h each o t h e r . A l l o f t h e s e t h r e e methods were found s u i t a b l e f o r d e t e r m i n a t i o n o f t h e e f f e c t i v e h y d r o p h o b i c i t y . No c o r r e l a t i o n was found between t h e e f f e c t i v e h y d r o p h o b i c i t y and t h e " a v e r a g e h y d r o p h o b i c i t y " ( B i g e l o w , 1967) n o r w i t h t h e m o l e c u l a r w e i g h t s o f t h e p r o t e i n s . The i n t e r f a c i a l t e n s i o n s o f t h e 0.2% p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e s were d e t e r m i n e d as a parameter o f t h e s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s . A n e g a t i v e c o r r e l a t i o n was found t o e x i s t between t h e e f f e c t i v e h y d r o p h o b i c i t i e s and t h e i n t e r f a c i a l t e n s i o n s o f t h e p r o t e i n s . T h i s r e s u l t s u g g e s t s t h a t h y d r o p h o b i c i t y i s i n v o l v e d i n t h e s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s . The more h y d r o p h o b i c t h e p r o t e i n , t h e b e t t e r t h e s u r f a c e a c t i v e p r o p e r t i e s , t h e r e b y f a c i l i t a t i n g t h e e m u l s i f y i n g p r o c e s s . i v TABLE OF CONTENTS Page INTRODUCTION . . 1 LITERATURE SURVEY 6 MATERIALS AND METHODS 31 1. A c t i v a t i o n o f Sepharose 4B w i t h cyanogen bromide . . . . 32 2. C o u p l i n g amines to Sepharose 4B 32 3. C o u p l i n g o f 4-phenylbutylamine (PBA) to Sepharose . . . 33 4. C o u p l i n g o f o l e i c a c i d to Sepharose 33 5. Chromatography on a l k y l a m i n o - Sepharoses 34 6. S y n t h e s i s o f g l y c i d y l e t h e r s . 34 7. Treatment o f Sepharose 4B 35 8. C o u p l i n g g l y c i d y l e t h e r s t o Sepharose 35 9. D e t e r m i n a t i o n o f the dry weight o f Sepharose g e l su s p e n s i o n s 36 10. Hydrophobic column chromatography 36 11. G e l chromatography w i t h d e t e r g e n t s 37 12. Removal o f the T r i t o n X-100 37 13. Q u a n t i t a t i o n o f T r i t o n X-100 37 14. Hydrophobic p a r t i t i o n 38 15. S y n t h e s i s o f p o l y e t h y l e n e g l y c o l p a l m i t a t e 39 16. I n t e r f a c i a l t e n s i o n measurements 40 Con t i n u e d . TABLE OF CONTENTS ( C o n t i n u e d ) Page RESULTS AND DISCUSSION 41 1. Chromatography on Sepharoses s u b s t i t u t e d w i t h a l k y l amine 41 2. Chromatography on o l e i c Sepharose 4B 50. 3. Chromatography on a l k y l a m i n o - Sepharoses 51 4. Chromatography w i t h d e t e r g e n t s . 62 5. H y d r o p h o b i c p a r t i t i o n 69 6. I n t e r f a c i a l t e n s i o n measurements 78 SUMMARY AND CONCLUSION 84 BIBLIOGRAPHY 88 APPENDIX. • 93 The scheme o f t h e CNBr a c t i v a t i o n and c o u p l i n g o f Sepharose 4B 94 The scheme o f t h e r e a c t i o n s i n v o l v e d i n t h e p r e p a r a t i o n o f a l k y l epoxy Sepharoses 95 Table vx LIST OF TABLES Page I Retention c o e f f i c i e n t s of proteins determined by hydrophobic column chromatography (butylepoxy- Sepharose 4B) . 65 II Retention c o e f f i c i e n t s of proteins determined by hydrophobic column chromatography (hexylepoxy- Sepharose 4B) . 66 III Hydrophobicity of proteins determined by the hydrophobic p a r t i t i o n technique 70 IV I n t e r f a c i a l tension of 0.2% protein s o l u t i o n / corn o i l in t e r f a c e . 79 v i i LIST OF FIGURES F i g u r e Page 1 B e h a v i o u r o f p r o t e i n s on (15 x 1 cm) columns o f b u t y l - , h e x y l - , and o c t y l a m i n o Sepharose 4B 42 2 B e h a v i o u r o f b o v i n e serum a l b u m i n on Sepharose 4B and 4 - p h e n y l b u t y l a m i n o - S e p h a r o s e 4B . . . . 47 3 E l u t i o n o f a m i x t u r e of g - l a c t o g l o b u l i n and Y - g l o b u l i n f r o m p h e n y l b u t y l a m i n o - S e p h a r o s e 4B by subsequent a d d i t i o n o f 0.1 N a C l and 50% e t h y l e n e g l y c o l i n b u f f e r 49 4 R e t e n t i o n volume o f b o v i n e serum a l b u m i n on h e x y l e p o x y - S e p h a r o s e 4B a t d i f f e r e n t s a l t c o n c e n t r a t i o n s 56 5 R e t e n t i o n volume o f b o v i n e serum a l b u m i n and g l u c o s e pn h e x y l e p o x y - S e p h a r o s e 4B 58 6 C o r r e l a t i o n between h y d r o p h o b i c i t y d e t e r m i n e d on a h e x y l e p o x y - S e p h a r o s e 4B column and m o l e c u l a r w e i g h t o f p r o t e i n s 60 7 C o r r e l a t i o n between e f f e c t i v e h y d r o p h o b i c i t y measured on a h e x y l e p o x y - S e p h a r o s e 4B column and t h e "average h y d r o p h o b i c i t y " c a l c u l a t e d by B i g e l o w (1967) 61 8 R e t e n t i o n volume o f p r o t e i n s on b u t y l e p o x y -Sepharose 4B 63 9 R e t e n t i o n volume o f p r o t e i n s on h e x y l e p o x y -Sepharose 4B 64 10 C o r r e l a t i o n between r e t e n t i o n c o e f f i c i e n t s measured by two h y d r o p h o b i c chromatography t e c h n i q u e s . . . . . . 67 11 C o r r e l a t i o n between h y d r o p h o b i c i t i e s measured by h y d r o p h o b i c chromatography on b u t y l e p o x y -Sepharose 4B and h y d r o p h o b i c p a r t i t i o n 74 12 C o r r e l a t i o n between h y d r o p h o b i c i t i e s measured by h y d r o p h o b i c chromatography on h e x y l e p o x y -Sepharose 4B and h y d r o p h o b i c p a r t i t i o n 75 C o n t i n u e d . . . . v i l i L I S T OF F I G U R E S ( C o n t i n u e d ) F i g u r e P a g e 13 C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y d e t e r m i n e d o n b u t y l e p o x y - S e p h a r o s e 4B a n d i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e 80 14 C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y d e t e r m i n e d o n h e x y l e p o x y - S e p h a r o s e 4B a n d i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e 81 15 C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y o f p r o t e i n s d e t e r m i n e d b y t h e h y d r o p h o b i c p a r t i t i o n m e t h o d a n d i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e 82 ACKNOWLEDGEMENT I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o D r . S . N a k a i , P r o f e s s o r , D e p a r t m e n t o f F o o d S c i e n c e , f o r h i s s u p e r v i s i o n a n d i n v a l u a b l e a d v i c e t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . My a p p r e c i a t i o n i s a l s o e x t e n d e d t o D r . W.D. P o w r i e a n d D r . P . M . T o w n s l e y o f t h e D e p a r t m e n t o f F o o d S c i e n c e , a n d t o D r . B. R o u f o g a l i s o f t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s f o r t h e i r i n v a l u a b l e s u g g e s t i o n s a n d g u i d a n c e . I am g r e a t l y i n d e b t e d t o D r . B. S k u r a o f t h e D e p a r t m e n t o f F o o d S c i e n c e f o r h i s i n v a l u a b l e a d v i c e d u r i n g t h e p r e p a r a t i o n o f t h e m a n u s c r i p t . My g r a t i t u d e a l s o e x t e n d s t o D r . F . J a m a l i f o r h i s e n c o u r a g e m e n t a n d a d v i c e d u r i n g t h i s w o r k . 1 INTRODUCTION T h e s t r u c t u r e a n d f u n c t i o n o f p r o t e i n s h o u l d b e i n t e r r e l a t e d . I n f a c t , t h i s t y p e o f r e l a t i o n s h i p h a s b e e n c o n f i r m e d i n many c a s e s , f o r e x a m p l e , i n e n z y m e s ( B a k e r , 1967; B e r n h a r d , 1968). I n v e s t i g a t i o n o f t h e s t r u c t u r e o f p r o t e i n s p r o v i d e s i n f o r m a t i o n w h i c h c a n l e a d t o a b e t t e r u n d e r s t a n d i n g o f t h e t y p e o f r e l a t i o n s h i p b e t w e e n s t r u c t u r e a n d f u n c t i o n . T h e c o n f o r m a t i o n o f p r o t e i n s m u s t b e c r i t i c a l i n many m e c h a n i s m s g o v e r n i n g t h e i r f u n c t i o n a l i t y i n f o o d s y s t e m s . T h e f l e x i b i l i t y o f p r o t e i n , w h i c h i s t h e a b i l i t y t o c h a n g e c o n f o r m a t i o n u n d e r c e r t a i n c o n d i t i o n s , i s i m p o r t a n t i n f u n c t i o n a l a p p l i c a t i o n s ( F r a n k s , 1975). T h e p r i m a r y s t r u c t u r e i s t h o u g h t t o d e t e r m i n e t h e c o n f o r m a t i o n o f t h e p r o t e i n s ( S c o t t e t a l . , 1967). I t h a s b e e n s u g g e s t e d t h a t t h e t e r t i a r y a n d q u a t e r n a r y s t r u c t u r e may b e r e l a t e d t o t h e b e h a v i o u r o f s e v e r a l f o o d p r o t e i n s ( K i n s e l l a , 1976). A l t h o u g h t h e a m i n o a c i d c o m p o s i t i o n o f m o s t common f o o d p r o t e i n s i s k n o w n , t h e p r i m a r y s e q u e n c e , h a s b e e n d e t e r m i n e d f o r o n l y a f e w , s u c h a s m i l k a n d e g g p r o t e i n s . F u n c t i o n a l p r o p e r t i e s o f p r o t e i n s t h a t a r e i m p o r t a n t i n f o o d s y s t e m s , s u c h a s e m u l s i f i c a t i o n a n d f o a m i n g , c a n b e c a t e g o r i z e d a s s u r f a c t a n t p r o p e r t i e s a n d r e l a t e d t o t h e s u r f a c e a n d i n t e r f a c i a l t e n s i o n s o f p r o t e i n s o l u t i o n s . T h e r e i s e v i d e n c e t o s u g g e s t t h a t s u r f a c e a c t i v i t y may b e r e l a t e d t o t h e h y d r o p h o b i c i t y o f a p r o t e i n ( D a v i s e_t a l . , 1973) . F o r e x a m p l e , a p o l i p o p r o t e i n s a r e h y d r o p h o b i c a n d s u r f a c e a c t i v e , g - c a s e i n i s h y d r o p h o b i c a n d a l s o s u r f a c e a c t i v e ( D a v i s e_t a l . , 1973). 2 A l m o s t a l l p r o t e i n s c o n t a i n a r e l a t i v e l y h i g h c o n c e n t r a t i o n o f a m i n o a c i d s w i t h n o n p o l a r s i d e c h a i n s s u c h a s t h e i s o p r o p y l o f v a l i n e a n d t h e b e n z y l o f p h e n y l a l a n i n e . S i n c e t h e n o n p o l a r s i d e c h a i n s h a v e a l o w a f f i n i t y f o r w a t e r , t h e y h a v e t h e t e n d e n c y t o a d h e r e t o o n e a n o t h e r , i n a n a q u e o u s e n v i r o n m e n t , a n d f o r m w h a t i s p r o b a b l y o n e o f t h e m o s t i m p o r t a n t f a c t o r s i n v o l v e d i n s t a b i l i z a t i o n o f t h e f o l d e d c o n f i g u r a t i o n i n many n a t i v e p r o t e i n s ( K a u z m a n n , 1959). T h i s c o n f i g u r a t i o n c a n a l s o h a v e a n i m p o r t a n t r e l a t i o n t o t h e f u n c t i o n a l p r o p e r t i e s . F i s h e r (1964) s u g g e s t e d t h e e x i s t e n c e o f a c o r r e l a t i o n b e t w e e n some p h y s i c a l p r o p e r t i e s o f p r o t e i n a n d t h e c o n t e n t o f n o n p o l a r a m i n o a c i d r e s i d u e s i n t h e m o l e c u l e . K a u z m a n n (1959) p o i n t e d o u t t h a t n o n p o l a r a m i n o a c i d s a r e e x p e c t e d t o o c c u r i n t h e p r o t e i n i n t e r i o r b e c a u s e t h e y a r e h y d r o p h o b i c . P o l a r r e s i d u e s , w h e t h e r i n t e r n a l l y h y d r o g e n b o n d e d o r n o t , w e r e t h o u g h t t o b e h y d r o p h i l i c a n d t h e r e f o r e t o o c c u r o n t h e p r o t e i n s u r f a c e . C h o t h i a (1974) s u g g e s t e d t h a t t h i s m o d e l s h o u l d b e m o d i f i e d . T h e s u r f a c e s o f a c o n s i d e r a b l e n u m b e r o f p r o t e i n s c o n t a i n e x p o s e d h y d r o -p h o b i c " p o c k e t s " s u c h a s b i n d i n g s i t e s o r s u b s i t e s o r t h e c r e v i c e s a t s u b u n i t j u n c t i o n s . T h e d i s t r i b u t i o n p a t t e r n o f h y d r o p h o b i c s i t e s s h o u l d h a v e a p r o n o u n c e d e f f e c t o n t h e c h a r a c t e r a n d b e h a v i o u r o f t h e s e m a c r o -m o l e c u l e s ( T a n f o r d , 1973). H y d r o p h o b i c i n t e r a c t i o n s a p p e a r t o b e t h e m a j o r f a c t o r c o n t r i b u t i n g t o l i p i d - p r o t e i n b i n d i n g ( G u r r a n d J a m e s , 1 9 7 1 ) . A s s o c i a t i o n o r i n t e r -a c t i o n b e t w e e n l i p i d s a n d p r o t e i n s i s t a k e n t o mean t h e f o r m a t i o n o f b o n d s ( h o w e v e r w e a k ) b e t w e e n t h e s e t w o c o m p o n e n t s s o t h a t c o n t a c t i s made a t t h e m o l e c u l a r l e v e l . T h e s t a b i l i z a t i o n o f a n y s u c h e m u l s i o n o r s t r u c t u r e m u s t 3 be c r i t i c a l l y dependent upon th e above-mentioned i n t e r a c t i o n s . A l t h o u g h f o r m a t i o n o f a d i e l e c t r i c d o u b l e l a y e r and r e p u l s i o n between th e o i l d r o p l e t s i s s u g g e s t e d t o be r e s p o n s i b l e f o r t h e s t a b i l i t y o f t h e o i l / w a t e r e m u l s i o n s , t h i s mechanism i s o f l i t t l e i m p o r t a n c e t o t h e s t a b i l i z a t i o n o f w a t e r / o i l e m u l s i o n s ( F r i b e r g > 1976). M o r e o v e r , n o n i o n i c s u r f a c t a n t s do n o t owe t h e i r s t a b i l i z a t i o n c a p a c i t i e s t o d o u b l e l a y e r e f f e c t s . The c o a l e s c e n c e o f t h e o i l d r o p l e t s r a t h e r depends on t h e s t a b i l i t i e s o f t h e i r s u r r o u n d i n g f i l m (Overbeek, 1952). P r o t e i n s f o r m an i m p o r t a n t c l a s s o f e m u l s i f i e r s w h i c h have e a r l i e r been t r e a t e d m a i n l y as t w o - d i m e n s i o n a l m o l e c u l e s a t t h e o i l / w a t e r i n t e r f a c e . Weak i n t e r a c t i o n s between t h e e m u l s i f i e r m o l e c u l e and b o t h t h e o i l and w a t e r m o l e c u l e s a r e i m p o r t a n t i n t h e f o r m a t i o n o f p r o t e c t i v e m u l t i l a y e r s around t h e o i l d r o p l e t s (Krog and L a u r i d s e n , 1976). I n an o i l / w a t e r i n t e r f a c e , a more h y d r o p h o b i c p r o t e i n was t h o u g h t t o have a g r e a t e r tendency t o u n f o l d and o r i e n t on the s u r f a c e s o f t h e o i l d r o p l e t s p r o v i d i n g a t h i c k l a y e r and t h e r e b y p r e v e n t i n g c o a l e s c e n c e and adherence o f t h e o i l g l o b u l e s . L onger h y d r o c a r b o n c h a i n s i n t h e e m u l s i f i e r m o l e c u l e have been r e p o r t e d t o be more a c t i v e and p r o v i d e b e t t e r a s s o c i a t i o n between o i l and w a t e r ( B a r e t , 1969). I t seemed n e c e s s a r y t o s t u d y t h e r e l a t i o n s h i p , i f any, between h y d r o p h o b i c i t y and s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s , so t h a t i t w o u l d be p o s s i b l e t o p r e d i c t t h e l a t t e r f r o m t h e i n f o r m a t i o n w h i c h i s p r o v i d e d on the f o r m e r . A l t h o u g h t h e r e have been many s p e c u l a t i o n s on t h e r e l a t i o n s h i p between h y d r o p h o b i c i t y o f a p r o t e i n and i t s f u n c t i o n a l p r o p e r t i e s ( K i n s e l l a , 1976), r e s e a r c h i n t h i s f i e l d i s s t i l l i n i t s i n f a n c y and no c l e a r c u t c o n c l u s i o n has been made on t h e s u b j e c t . T h e r e f o r e , f u r t h e r r e s e a r c h i s r e q u i r e d t o e l u c i d a t e t h e p h y s i c o c h e m i c a l mechanisms r e s p o n s i b l e f o r s p e c i f i c f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s . Many i n v e s t i g a t o r s ( T a n f o r d , 1962; F i s h e r , 1964; B i g e l o w , 1967) a t t e m p t e d t o measure the h y d r o p h o b i c i t y o f p r o t e i n s m a i n l y t h r o u g h t h e i r c o n s t i t u e n t a p o l a r amino a c i d s o r s o l u b i l i t y p r o p e r t i e s . However, c o n s i d e r i n g t h a t i n most c a s e s even the s i d e c h a i n o f a p o l a r amino a c i d c o n t r i b u t e s t o t h e f o r m a t i o n o f h y d r o p h o b i c bonds, t h e i r a pproaches do n o t seem t o measure t h e e f f e c t i v e h y d r o p h o b i c i t i e s w h i c h a l s o depend on the f l e x i b i l i t y and c o n f o r m a t i o n o f t h e m o l e c l u e . M o r e over, t h e r e might be h y d r o p h o b i c amino a c i d s w h i c h do n o t t a k e p a r t i n i n t e r a c t i o n s due t o s t e r i c h i n d r a n c e o r o t h e r r e a s o n s . I n t h i s s t u d y h y d r o p h o b i c chromatography and a f f i n i t y p a r t i t i o n methods, w h i c h a r e w i d e l y used 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 macro-m o l e c u l e s , were s u c c e s s f u l l y m o d i f i e d t o d e t e r m i n e the e f f e c t i v e h y d r o p h o b i c i t y o f p r o t e i n s . S e r i e s o f l i g a n d s (amines, a l c o h o l s , o l e i c and p a l m i t i c m o i e t i e s ) were used i n t h i s s t u d y . I n t e r a c t i o n o f p r o t e i n w i t h t h e n o n i o n i c d e t e r g e n t , T r i t o n X-100, was a l s o employed t o d e t e r m i n e t h e h y d r o p h o b i c i t y o f p r o t e i n s . The e f f e c t i v e h y d r o p h o b i c i t y o f p r o t e i n s was c o r r e l a t e d w i t h t h e i r s u r f a c t a n t p r o p e r t i e s i n t h e hope o f r e a c h i n g a b e t t e r u n d e r s t a n d i n g 5 4 o f t h e c o m p l i c a t e d s y s t e m o f e m u l s i o n f o r m a t i o n , t h u s l a y i n g the groundwork f o r f u t u r e i n t e r p r e t a t i o n o f f o o d e m u l s i o n f o r m a t i o n i n terms o f p r o t e i n s t r u c t u r e . T h i s r e s e a r c h was i n i t i a t e d : 1) t o d e t e r m i n e t h e e f f e c t i v e h y d r o p h o b i c i t y o f p r o t e i n s as i n v o l v e d i n h y d r o p h o b i c i n t e r a c t i o n s ; and 2) t o c o r r e l a t e t h e e f f e c t i v e h y d r o p h o b i c i t i e s w i t h t h e s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s . The f o l l o w i n g methods were a p p l i e d t o d e t e r m i n e t h e e f f e c t i v e h y d r o p h o b i c i t y o f the p r o t e i n s : 1. Chromatography w i t h d e t e r g e n t s . 2. Chromatography on o l e i c - s u b s t i t u t e d Sepharose 4B. 3. Chromatography on a m i n o a l k y l - s u b s t i t u t e d Sepharose 4B: a. A l i p h a t i c amines: b u t y l - , h e x y l - and o c t y l a m i n e s ; b. A r o m a t i c amines: 4 - p h e n y l b u t y l a m i n e . 4. H y d r o p h o b i c chromatography on: a. B u t y l e p o x y - S e p h a r o s e 4B; b. H e x y l e p o x y - S e p h a r o s e 4B; c. O c t y l e p o x y - S e p h a r o s e 4B. 5. H y d r o p h o b i c p a r t i t i o n method. I n t e r f a c i a l t e n s i o n o f p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e was measured f o r t h e s u r f a c t a n t a c t i v i t y o f the p r o t e i n s . 6 LITERATURE SURVEY Knowledge o f t h e i n t e r r e l a t i o n s h i p s between c o m p o s i t i o n , s t r u c t u r e , and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s has been o b t a i n e d m o s t l y t h r o u g h b i o c h e m i c a l s t u d i e s c o n c e r n i n g enzyme r e a c t i o n , p r o t e i n -p r o t e i n i n t e r a c t i o n s , and l i g a n d - p r o t e i n b i n d i n g . These s t u d i e s p r o v i d e t h e f o o d c h e m i s t w i t h t h e t e c h n i q u e s and b a s i c i n f o r m a t i o n t h a t c o u l d be a p p l i c a b l e t o r e s e a r c h d e s i g n e d t o e l u c i d a t e p h y s i c o c h e m i c a l phenomena u n d e r l y i n g p r o t e i n f u n c t i o n s i n f o o d s . However, f o o d systems a r e g e n e r a l l y t o o h e t e r o g e n e o u s and complex t o p e r m i t a p p l i c a t i o n o f c l a s s i c a l p h y s i c o c h e m i c a l t e c h n i q u e s i n the s t u d y of t h e m o l e c u l a r changes ( c h e m i c a l o r c o n f o r m a t i o n a l ) t h a t o c c u r d u r i n g t e c h n i c a l p r o c e s s e s . I n f o o d s y s t e m s , t h e r e l a t i v e c o n t r i b u t i o n of s e v e r a l t y p e s o f f o r c e s and t h e i r v a r i a b l e b e h a v i o u r i n d i f f e r e n t e n v i r o n m e n t s i n f l u e n c e t h e f u n c t i o n a l b e h a v i o u r o f p r o t e i n s . These c o n t r i b u t i n g f o r c e s a r e h ydrogen and i o n i c bonds, e l e c t r o s t a t i c f o r c e s and h y d r o p h o b i c i n t e r -a c t i o n s ( S h e r a g a , 1961). The i n f l u e n t i a l e f f e c t s a r e e x e r c i s e d t h r o u g h c o n f o r m a t i o n a l changes, m o l e c u l a r ' f l e x i b i l i t y , and by i n f l u e n c i n g t h e e x t e n t o f i n t e r - m o l e c u l a r i n t e r a c t i o n s . A g g r e g a t i o n by h y d r o p h o b i c a s s o c i a t i o n o r f o r m a t i o n o f i n t e r f a c i a l membrane l a y e r s i n e m u l s i o n s and foams a r e examples o f t h e s e k i n d s o f i n t e r a c t i o n . 1. Types o f b o n d i n g : I s h i n o and Okamoto (1975) showed t h e i m p o r t a n c e of h y d r o p h o b i c i n t e r a c t i o n s on t h e p h y s i c a l b e h a v i o u r o f soy p r o t e i n . Thompson and F a r r e l l (1973) d i s c u s s e d t h e c o n t r i b u t i o n o f h y d r o p h o b i c and 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 , h ydrogen b o n d i n g and d i s u l f i d e bonds t o t h e s t r u c t u r e and b e h a v i o u r o f m i l k c a s e i n s . Hegg and L o f q u i s t (1974) adopted a new and p r a c t i c a l approach, t o q u a n t i f y t h e t h e r m a l a g g r e g a t i o n o f o v a l b u m i n . T h i s p r o t e i n was used as a model from w h i c h d a t a c o u l d be e x t r a p o l a t e d t o h e l p m i n i m i z e t h e r m a l d e n a t u r a t i o n o f p r o t e i n s d u r i n g i s o l a t i o n and r e f i n i n g . These w o r k e r s found t h a t i o n i c d e t e r g e n t s , a t v e r y low c o n c e n t r a t i o n s , m i n i m i z e t h e r m a l d e n a t u r a t i o n o f o v a l b u m i n p a r t i c u l a r l y a t a l k a l i n e pH. I t was c o n c l u d e d t h a t a n i o n i c d e t e r g e n t s s t a b i l i z e o v a l b u m i n m o l e c u l e s by i n t e r n a l s t a b i l i z a t i o n o f t h e i r p o l y p e p t i d e s t h r o u g h a c o m b i n a t i o n o f i o n i c and h y d r o p h o b i c i n t e r a c t i o n s . 2. S u r f a c e p r o p e r t i e s and e m u l s i f i c a t i o n : R e c e n t l y , F r a n k s (1975) and Graham and P h i l i p s (1975) s t u d i e d t h e r e l a t i o n s h i p s between the m o l e c u l a r s t r u c t u r e and s u r f a c e p r o p e r t i e s o f t h r e e p r o t e i n s h a v i n g d i f f e r e n t s t r u c t u r e s . These p r o t e i n s were: l y s o z y m e , w h i c h i s g l o b u l a r and r i g i d ; b o v i n e serum a l b u m i n , w h i c h i s g l o b u l a r and f l e x i b l e ; and 3 - c a s e i n , w h i c h i s a f l e x i b l e c o i l . F r a n k s (1975) used s u r f a c e c h e m i c a l t e c h n i q u e s t o m o n i t o r t h e k i n e t i c s o f s u r f a c e a d s o r p t i o n . I t was o b s e r v e d t h a t the f l e x i b i l i t y and r e l a t i v e h y d r o p h o b i c i t y o f p o l y m e r s , as t y p i f i e d by 3 - c a s e i n , r e s u l t e d i n r a p i d i n i t i a l a d s o r p t i o n . T h i s was f o l l o w e d by a p a r t i a l d e s o r p t i o n a s s o c i a t e d w i t h a s l o w r e a r r a n g e m e n t o f t h e polymer m o l e c u l e s , w h i l e a t t a i n i n g an e q u i l i b r i u m c o n f i g u r a t i o n . I n c o n t r a s t , l y s o z y m e a d s o r b e d s l o w l y on t h e s u r f a c e t o f o r m a monolayer o f n a t i v e , u n f o l d e d p r o t e i n , and t h e n a m u l t i l a y e r b u i l t up. B o v i n e serum a l b u m i n behaved i n an i n t e r m e d i a t e f a s h i o n . Heat i n d u c e d u n f o l d i n g o f t h e l a t t e r p r o t e i n caused i t t o p e r f o l i k e f l e x i b l e 3 - c a s e i n m o l e c u l e s . The r h e o l o g i c a l b e h a v i o u r o f t h e a d s o r b e d p r o t e i n f i l m w a s c l o s e l y c o r r e l a t e d w i t h t h e m o l e c u l a r s t r u c t u r e . T h e f l e x i b l e p - c a s e i n f o r m e d l i q u i d - l i k e f i l m s , w h e r e a s t h e b o v i n e s e r u m a l b u m i n a n d l y s o z y m e f i l m s p o s s e s s e d a m a r k e d v i s c o e l a s t i c i t y , w h i c h r e a c h e d a max imum v a l u e a t t h e m o n o l a y e r s t a g e . T h i s w a s i n t u r n r e l a t e d t o f o a m s t a b i l i t y . F r a n k s ( 1 9 7 5 ) a l s o s t a t e d t h a t t h e s o l u t i o n a n d a g g r e g a t i o n p r o p e r t i e s w e r e r e l a t e d t o s p e c i f i c a m i n o a c i d r e s i d u e s i n t h e 3 - c a s e i n m o l e c u l e . A n u m b e r o f t h e o r i e s h a v e b e e n s u g g e s t e d t o e x p l a i n e m u l s i f i c a t i o n c a p a c i t y o f t h e s u r f a c t a n t s . T h e m e c h a n i s m s o f e m u l s i f i c a t i o n c a p a c i t y a n d e m u l s i o n s t a b i l i t y s e e m t o b e d i f f e r e n t ( F r i b e r g , 1 9 7 6 ) . A l s o , f o r m a t i o n o f o i l / w a t e r a n d w a t e r / o i l e m u l s i o n s d o e s n o t f o l l o w t h e s ame p a t t e r n . H o w e v e r , p H , s a l t c o n c e n t r a t i o n , v i s c o s i t y , s t r u c t u r e a n d c o n c e n t r a t i o n o f t h e e m u l s i f i e r a n d t e m p e r a t u r e p l a y i m p o r t a n t r o l e s i n e m u l s i f i c a t i o n a c t i v i t i e s . V i s c o s i t y o f t h e c o n t i n u o u s p h a s e , a d s o r p t i o n o f s o l i d p a r t i c l e s t o t h e s u r f a c e o f t h e e m u l s i f i e d p h a s e , a n d f o r m a t i o n o f m u l t i l a y e r s a t t h e i n t e r f a c e b e t w e e n t h e e m u l s i f i e d p h a s e a n d t h e c o n t i n u o u s p h a s e a r e o f p r i m e i m p o r t a n c e i n d e t e r m i n i n g t h e s t a b i l i t y o f t h e e m u l s i o n ( L y n c h a n d G r i f f i n , 1 9 7 4 ) . A l t h o u g h t h e p r i m i t i v e v i e w w h i c h c o r r e l a t e d e m u l s i o n s t a b i l i t y w i t h l o w i n t e r f a c i a l t e n s i o n h a s l a r g e l y b e e n a b a n d o n e d , t h e e f f e c t o f t h e i n t e r f a c i a l f r e e e n e r g y o n e m u l s i o n f o r m a t i o n c a n n o t b e w h o l l y d i s r e g a r d e d . A l o w i n t e r f a c i a l t e n s i o n may b e c l a s s e d a s a " n e c e s s a r y b u t n o t s u f f i c i e n t " c o n d i t i o n f o r s t a b i l i t y ( R o s s a n d C h e n , 1 9 5 9 ) . S u r f a i a c t i v e a g e n t s r e d u c e t h e i n t e r f a c i a l t e n s i o n b e t w e e n t w o n o n - m i s c i b l e l i q u i d s . C o n s e q u e n t l y , t h e e n e r g y r e q u i r e d f o r e m u l s i f i c a t i o n o f t h e t w o l i q u i d s i s r e d u c e d . T h e r e f o r e , a r e d u c t i o n o f the i n t e r f a c i a l t e n s i o n t h r o u g h t h e a d d i t i o n o f an e m u l s i f i e r f a c i l i t a t e s t he f o r m a t i o n o f an e m u l s i o n . R e p u l s i o n f r o m t h e e l e c t r i c d o u b l e l a y e r , the l a t e r a l i n t e r a d h e s i v e e nergy of the s u r f a c t a n t m o l e c u l e s and t h e i r i n f l u e n c e on the s t r u c t u r e o f w a t e r a r e d e s c r i b e d by D a v i e s e_t a l . (1973) as t h e most i m p o r t a n t s t a b i l i z i n g f a c t o r s i n e m u l s i f i c a t i o n . S u p p o s e d l y , t h e s e f o r c e s s t a b i l i z e t h e " t h i c k s k i n " a t t h e i n t e r f a c e . I t has l o n g been known t h a t p r o t e i n s , as e m u l s i f i e r s , form a t h i c k s k i n around t h e s u r f a c e o f e m u l s i f i e d d r o p l e t s ( F r i b e r g 1976). Owing t o t h e i r a m p h i p h i l i c c h a r a c t e r , t h e s e s u b s t a n c e s a r e f o r c e f u l l y a d s o r b e d t o t h e i n t e r f a c e between o i l and w a t e r , c a u s i n g a pronounced r e d u c t i o n o f t h e i n t e r f a c i a l t e n s i o n . The a b i l i t y o f p r o t e i n s t o b i n d l i p i d s h o u l d be v e r y i m p o r t a n t f o r such a p p l i c a t i o n s as meat r e p l a c e r s and e x t e n d e r s (Rand 1976). G e n e r a l l y , more h y d r o p h o b i c p r o t e i n s , w h i c h a l s o l o w e r s u r f a c e t e n s i o n , show s u p e r i o r b i n d i n g o f l i p o p h i l i c m a t e r i a l . The p r o t e i n m o l e c u l e i s u n f o l d e d due t o s h e a r i n g i n v o l v e d i n the e m u l s i f i c a t i o n p r o c e s s . T h i s i s i n f l u e n c e d by h y d r o p h o b i c a s s o c i a t i o n of t h e p e p t i d e c h a i n s w i t h t h e l i p i d d r o p l e t s . The r e s u l t i s t h a t a much g r e a t e r v o l u m e / s u r f a c e a r e a o f p r o t e i n i s made a v a i l a b l e and t h e e m u l s i f y i n g e f f i c i e n c y o f the p r o t e i n i s enhanced ( K i n s e l l a 1976). H y d r o p h o b i c i n t e r a c t i o n s a r e n o t p r e c l u d e d by c o n d i t i o n s f a v o r i n g i o n i c b o n d i n g . M o r e o v e r , the l a t t e r may be n e c e s s a r y i n o r d e r t o g e t t h e p r o t e i n m o l e c u l e s c l o s e enough f o r subsequent s h o r t e r range 10 i n t e r a c t i o n s t o o c c u r ( R a n d , 1976). Weak i n t e r a c t i o n s o f t h e e m u l s i f i e r s w i t h t h e o i l a n d w a t e r m o l e c u l e s d e t e r m i n e t h e f o r m a t i o n o f a s s o c i a t i o n s t r u c t u r e s c a l l e d l y o t r o p i c l i q u i d c r y s t a l s ( F r i b e r g j2t al., 1969). T h e s e l i q u i d c r y s t a l s a p p e a r a s a t h i r d p h a s e i n t h e e m u l s i o n a n d , a t t h e e m u l s i f i e r c o n c e n t r a t i o n a t w h i c h t h i s p h a s e i s f o r m e d , a s u d d e n i n c r e a s e . i n e m u l s i o n s t a b i l i t y i s e x p e r i e n c e d . T h e p r e s e n t d e f i n i t i o n o f a n e m u l s i o n , t h e r e f o r e , a l s o i n c l u d e s l i q u i d c r y s t a l l i n e p h a s e s a s a p o s s i b l e p a r t o f t h e d i s p e r s i o n s y s t e m w h i c h c o n t r i b u t e s t o e m u l s i o n f o r m a t i o n ( F r i b e r g , 1976). F o o d e m u l s i o n s w h i c h a r e o f t e n c h a r a c t e r i z e d b y t h e p r e s e n c e o f s m a l l a m o u n t s o f n e u t r a l e m u l s i f i e r s a n d a m p h i p h i l i c s u b s t a n c e s h i g h l y h y d r o p h o b i c i n n a t u r e d e p e n d o n t h e s e p h e n o m e n a m o r e t h a n o t h e r e m u l s i o n s y s t e m s ( B e r g e r , 1976). T h e v i s c o s i t y o f t h e l i q u i d c r y s t a l i s a t l e a s t o n e h u n d r e d t i m e s t h a t o f w a t e r . T h e p r e s e n c e o f l i q u i d c r y s t a l l i n e m e s o p h a s e s i n e m u l s i o n s w a s r e c o g n i z e d s e v e n t e e n y e a r s a g o b y J a m e s a n d G o l d e m b e r g (1960). T h e s p e c i f i c i n f l u e n c e o f l i q u i d c r y s t a l l i n e p h a s e s o n t h e s t a b i l i t y o f e m u l s i o n s w a s d e m o n s t r a t e d b y F r i b e r g a n d c o - w o r k e r s (1969) w i t h t h e a i d o f t h e m e t h o d s f o r s t u d y i n g p h a s e e q u i l i b r i a . T h e e f f e c t o f l i q u i d c r y s t a l l i n e p h a s e s o n t h e s t a b i l i t y o f e m u l s i o n s a g a i n s t c o a l e s c e n c e i s a s s o c i a t e d w i t h t h e i r s o - c a l l e d s t e r i c h i n d r a n c e , w h i c h i s c o m p o s e d o f s e v e r a l i n d e p e n d e n t f a c t o r s a f f e c t i n g t h e o r d e r e d l a y e r s o f e m u l s i f i e r s a t t h e o i l - w a t e r i n t e r f a c e . T h e s e f a c t o r s c a n b e c o n s i d e r e d t o b e d u e t o t h e p a c k i n g c o n d i t i o n s o f t h e h y d r o c a r b o n c h a i n s a n d a l s o d u e t o t h e o v e r a l l p h a s e b e h a v i o u r o f t h e o i l - w a t e r - e m u l s i f i e r m i x t u r e ( K r o g a n d L a u r i d s e n , 1976). T h e s u r f a c e f i l m s u r r o u n d i n g t h e e m u l s i f i e d d r o p l e t s i s a n e m u l s i o n , w h e t h e r o f a n o i l / w a t e r o r w a t e r / o i l t y p e , a n d s h o u l d b e c o n s i d e r e d t o c o n s i s t o f a n a s s o c i a t i o n s t r u c t u r e between t h e t h r e e components: o i l , e m u l s i f i e r and w a t e r ( K r o g and L a u r i d s e n , 1976). B u l l (1947) s u g g e s t e d t h a t the i o n i c groups a r e r e s p o n s i b l e f o r s u r f a c e s p r e a d i n g and s t a b i l i t y o f t h e monomolecular f i l m . He found the b e s t s p r e a d i n g g e n e r a l l y o c c u r s a t pH c l o s e t o the i s o e l e c t r i c p o i n t o f p r o t e i n s . ' . A c o n s i d e r a b l e r e d u c t i o n i n f r e e energy was s u g g e s t e d t o be due t o p o l a r i n t e r a c t i o n s and hydrogen b o n d i n g between p o l a r groups and w a t e r ( F r i b e r g e t a l . , 1969). Even a weak h y d r o p h i l i c group s u c h as th e a l c o h o l i c - O H group l o s e s about 3.4 K c a l / m o l e when b e i n g a d s o r b e d f r o m a h y d r o c a r b o n t o t h e o i l / w a t e r i n t e r f a c e . A d s o r p t i o n t o the aqueous phase g i v e s about 12 K c a l / m o l e due t o t h e s t r u c t u r a l changes of t h e h y d r o c a r b o n c h a i n s when t h e y a r e d i s s o l v e d i n w a t e r . The pronounced r e d u c t i o n i n s u r f a c e energy has been s a i d t o i n f l u e n c e t h e s t a b i l i t y o f t h e e m u l s i o n s and t o f a c i l i t a t e the e m u l s i f i c a t i o n p r o c e s s ( F r i b e r g , 1976). I t seems t o be d i f f i c u l t t o d i s c e r n a d i r e c t c o n n e c t i o n between e m u l s i o n s t a b i l i t y and s u r f a c e t e n s i o n . F o r about two decades h y d r o p h i l e - l y p o p h i l e b a l a n c e (HLB) has been u s e d as t h e most u s e f u l c r i t e r i a f o r o p t i m a l s e l e c t i o n o f s t a b l e e m u l s i f i e r s f o r a g i v e n system. The HLB concept g i v e s i n f o r m a t i o n about the s o l u b i l i t y o f t h e e m u l s i f i e r i n e i t h e r the o i l o r the w a t e r phase and i t may be used as a g u i d e t o p r e d i c t w h i c h t y p e o f e m u l s i o n w i l l be formed, HLB number depends d i r e c t l y on t h e m o l e c u l a r s t r u c t u r e o f the e m u l s i f i e r . However, t h i s v a l u e h a s , i n f a c t , o n l y a l i m i t e d a p p l i c a t i o n w i t h i n t h e f o o d i n d u s t r y b e c a u s e s u c h a c l a s s i f i c a t i o n o f f o o d e m u l s i f i e r s i s t o o na r r o w , and i t does n o t a c c o u n t f o r o t h e r p r o p e r t i e s o f e m u l s i f i e r s s u c h as complex f o r m a t i o n w i t h s t a r c h components o r p r o t e i n s (Krog and L a u r i d s e n , 1976). M o r eover, HLB d e t e r m i n a t i o n cannot be used to e v a l u a t e e m u l s i f i c a t i o n p r o p e r t i e s o f p r o t e i n s . Ross and Chen (1959) s u g g e s t e d r e p l a c e m e n t o f t h e l a b o r i o u s and i m p r e c i s e d e t e r m i n a t i o n o f HLB w i t h t h e i r method. These w o r k e r s showed t h a t the s t a b i l i t y o f an e m u l s i o n may be r e l a t e d t o t h e s p r e a d i n g c o e f f i c i e n t o f w a t e r and o i l s o l u b l e s o l u t e s . The s p r e a d i n g c o e f f i c i e n t s were c a l c u l a t e d f r o m i n t e r f a c i a l and s u r f a c e t e n s i o n measurements and were shown t o c o r r e l a t e w i t h the HLB o f t h e e m u l s i f i e r s . Ross and Chen (1959) r e p o r t e d t h a t o i l / w a t e r e m u l s i o n s p r e p a r e d w i t h o i l s w h i c h had n e g a t i v e s p r e a d i n g c o e f f i c i e n t s appeared t o be most s t a b l e t o c r e a m i n g . Those o i l s w h i c h s p r e a d most e a s i l y on w a t e r a l s o had t h e g r e a t e s t s o l u b i l i t i e s i n w a t e r , and p o s s i b l y p o s i t i v e s p r e a d i n g c o e f f i c i e n t s . The more w a t e r s o l u b l e h y d r o c a r b o n s may t e n d t o p e n e t r a t e i n t o the a d s o r b e d s u r f a c t a n t l a y e r s , w h i c h s t a b i l i z e t h e o i l d r o p l e t s , and i n t o the a d j a c e n t i n t e r s t i t i a l f l u i d . T h i s would g i v e r i s e t o r a p i d c o a l e s c e n c e o f t h e o i l d r o p l e t s ( R e h f e l d , 1974). I t i s o f t e n d i f f i c u l t t o e s t a b l i s h w h e t h e r , upon m i x i n g l i p i d and p r o t e i n , an i n t e r a c t i o n o c c u r s o r the l i p i d s and p r o t e i n s s i m p l y c o e x i s t s e p a r a t e l y . The i n t e r f a c e between t h e b u l k phases must i n v o l v e m o l e c u l a r a s s o c i a t i o n s ( G u r r and James, 1971). G o n z a l e z and M a c R i t c h i e (1970) c o n s i d e r e d the o i l / w a t e r i n t e r f a c e as a "good s o l v e n t " f o r p r o t e i n i n w h i c h t h e p o l a r groups i n t e r a c t w i t h t h e p o l a r phase and t h e n o n p o l a r groups w i t h the n o n p o l a r phase. T h i s a l l o w s the p r o t e i n t o t a k e up a c o n f i g u r a t i o n o f v e r y low f r e e energy. I f a l i p i d - p r o t e i n i n t e r a c t i o n t o o k p l a c e , h y d r o p h o b i c b o n d s a r e t h e m o s t p r o b a b l e t y p e s o f b o n d t o b e i n v o l v e d ( R a n d , 1 9 7 6 ) . 3 . H y d r o p h o b i c b o n d i n g : T h e h y d r o p h o b i c b o n d i s t h e t e r m u s e d b y K a u z m a n n (1959) t o d e s c r i b e t h e g a i n i n f r e e e n e r g y o n t h e t r a n s f e r o f n o n p o l a r r e s i d u e s f r o m a n a q u e o u s e n v i r o n m e n t t o t h e i n t e r i o r o f p r o t e i n . K a u z m a n n s u g g e s t e d t h a t t h e e n t r o p i c t e r m s t a b i l i z e s p r o t e i n a g g r e g a t i o n a n d t h a t i t i s m a i n l y a r e s u l t o f h y d r o p h o b i c b o n d i n g . T h e e x i s t e n c e o f t h e h y d r o p h o b i c b o n d s e e m s t o b e d u e b a s i c a l l y t o t h e e n t r o p y i n c r e a s e , w h i c h i s a c o n s e q u e n c e o f t h e i n c r e a s e d d i s o r d e r w i t h i n t h e c l u s t e r o f w a t e r m o l e c u l e s s u r r o u n d i n g t h e p r o t e i n m o l e c u l e ( H j e r t e n , 1975). T h e i n c r e a s e i n h e a t c a p a c i t y h a s b e e n a t t r i b u t e d t o t h e e x c e s s h e a t n e e d e d t o m e l t t h e i c e - l i k e w a t e r f o r m e d a r o u n d n o n p o l a r g r o u p s ( C r o t h e r s a n d R a t n e r , 1968). K a u z m a n n (1959) p o s t u l a t e d t h a t t h e n a t u r e o f t h e w a t e r -p r o t e i n i n t e r f a c e i s r e l a t i v e l y s i m p l e , a n d i s d o m i n a t e d b y t h e s o l v a t i o n o f c h a r g e d g r o u p s , w i t h t h e a c c o m o d a t i o n o f w a t e r o v e r n o n p o l a r a r e a s . T h i s o c c u r s i n s u c h a w a y s o a s n o t t o d i m i n i s h t h e n u m b e r o f h y d r o g e n b o n d s o r t h e f r e e v o l u m e , u p o n w h i c h t h e e n t r o p y o f t h e w a t e r s t r o n g l y d e p e n d s . K l o t z (1958) h a s b e e n t h e m a j o r s p o k e s m a n f o r a m o r e m o d e r a t e v i e w w h i c h p r o p o s e s t h a t t h e p r o t e i n i n d u c e s a g e n e r a l o r d e r i n g e f f e c t o n s m a l l s h e l l s o f w a t e r m o l e c u l e s a r o u n d p r o t e i n s . When a h y d r o p h o b i c r e s i d u e i s i n t r o d u c e d i n t o w a t e r the degree of s t r u c t u r e i n t h e l a y e r o f w a t e r n e x t t o t h e h y d r o c a r b o n i n c r e a s e s and becomes i c e - l i k e . I f two h y d r o p h o b i c c h a i n s , s u r r o u n d e d by i c e - l i k e s t r u c t u r e d w a t e r , a r e b r o u g h t c l o s e t o g e t h e r a p a r t o f t h i s s t r u c t u r e w h i c h i s between them must be d i s r u p t e d b e f o r e the h y d r o c a r b o n c h a i n s can c o a l e s c e . D i s r u p t i n g s t r u c t u r e i n w a t e r r e q u i r e s h e a t , j u s t l i k e m e l t i n g i c e . Thus, A H i s p o s i t i v e and h e a t must e n t e r t h e s y s t e m i n o r d e r t o m e l t the w a t e r and t h i s b r i n g s t h e h y d r o p h o b i c c h a i n s t o g e t h e r . A f t e r c o a l e s c e n c e , the q u a n t i t y o f s t r u c t u r e d w a t e r s u r r o u n d i n g them i s l e s s t h a n t h e t o t a l amount w h i c h p r e v i o u s l y s u r r o u n d e d t h e i n d i v i d u a l c h a i n s ( S t a u f f e r , 1 9 7 2 ) . T h i s d e c r e a s e i n the t o t a l amount o f o r d e r ( s t r u c t u r e d w a t e r ) i n the s y s t e m g i v e s an i n c r e a s e i n A S , t h e e n t r o p y o f t h e system. The two h y d r o p h o b i c r e s i d u e s come t o g e t h e r s p o n t a n e o u s l y because a l t h o u g h A H i s p o s i t i v e , T A S i s n u m e r i c a l l y even l a r g e r , and so A F has an o v e r a l l n e g a t i v e v a l u e . The i n t e r a c t i o n between w a t e r and the a p o l a r groups o f p r o t e i n i s g e n e r a l l y a g r e e d t o be a s i g n i f i c a n t f a c t o r c o n t r i b u t i n g t o t h e e x i s t e n c e and s t a b i l i t y o f the u n i q u e s t r u c t u r e o f most g l o b u l a r p r o t e i n s ( R i c h a r d s j 1 9 6 3 ) . The h y d r o p h o b i c bond i s s u g g e s t e d t o be t h e major d e t e r m i n i n g f e a t u r e of p r o t e i n s t r u c t u r e , i n d e e d p r o b a b l y much more i m p o r t a n t t h a n t h e hydrogen bond as a s t a b i l i z i n g f o r c e . S c h e l l m a n (1955) d e m o n s t r a t e d t h a t hydrogen b o n d i n g c o u l d n o t a c c o u n t f o r the s t a b i l i t y o f g l o b u l a r p r o t e i n s i n aqueous s o l u t i o n s . B i g e l o w (1967) found g e n e r a l l y low h y d r o p h o b i c i t y v a l u e s f o r f i b r o u s p r o t e i n s and h i g h e r v a l u e s f o r g l o b u l a r p r o t e i n s . He p o i n t e d out the n e c e s s i t y o f a c e r t a i n a v e r a g e h y d r o p h o b i c i t y f o r a p r o t e i n i n o r d e r f o r i t t o be s t a b l e i n the g l o b u l a r c o n f o r m a t i o n . F o r m a t i o n o f the h y d r o p h o b i c bond as d e s c r i b e d by Sheraga (1961) s t a r t s by two o r more s i d e c h a i n s a p p r o a c h i n g each o t h e r and e s t a b l i s h i n g as many c o n t a c t s as p o s s i b l e . The c o n t r i b u t i o n o f the h y d r o p h o b i c s i d e c h a i n s t o t h e h y d r o p h o b i c i t y o f t h e p r o t e i n s depends on the l e n g t h and s i z e o f the c h a i n s . I n w a t e r s o l u b l e p r o t e i n s about 25-30% o f t h e amino a c i d s i d e c h a i n s a r e g e n e r a l l y h y d r o p h o b i c and 45-50% a r e t y p i c a l l y h y d r o p h i l i c ( T a n f o r d , 1973). I n the n a t i v e c o n f o r m a t i o n o f such a p r o t e i n , a s u b s t a n t i a l f r a c t i o n o f t h e h y d r o p h o b i c s i d e c h a i n s i s u s u a l l y b u r i e d w i t h i n the i n t e r i o r of the m o l e c u l e (Mohamadzadeh e t a l . , 1969). Charged groups a r e n o t g e n e r a l l y f ound i n s i d e m a c r o m o l e c u l e s , p r e s u m a b l y because t h e "thermodynamic c o s t " o f b u r y i n g s u c h a group i n t h e n o n p o l a r i n t e r i o r i s too g r e a t . The h y d r o p h o b i c p a r t s o f t h e p r o t e i n m o l e c u l e must be t a k e n t o i n c l u d e n o t o n l y the f u l l y n o n p o l a r s i d e c h a i n s s u c h as l e u c i n e , b u t a l s o t h e n o n p o l a r p a r t s o f s i d e c h a i n s w h i c h c o n t a i n c h a r g e d o r p o l a r g r o u p s , e.g., t h e f o u r m ethylene groups o f the l y s i n e s i d e c h a i n ( T a n f o r d , 1962). The p r o p o r t i o n o f t h e p r o t e i n s u r f a c e t h a t becomes b u r i e d by f o l d i n g i n c r e a s e s w i t h i n c r e a s i n g m o l e c u l a r w e i g h t . B u t , a t t h e same t i m e , t h e p r o p o r t i o n o f p o l a r groups t h a t form i n t r a m o l e c u l a r hydrogen bonds i s e s s e n t i a l l y c o n s t a n t and i s i n d e p e n d e n t o f m o l e c u l a r w e i g h t ( C h o t h i a , 1976). D i s t r i b u t i o n o f p o l a r r e s i d u e s between the i n t e r i o r and s u r f a c e o f t h e m a c romolecule can be s e a r c h e d f o r r e l a t i o n s h i p between s t r u c t u r e and f u n c t i o n . P r o t e i n s s h o u l d be l e s s s o l u b l e i n t h e i r t h r e e d i m e n s i o n a l s t r u c t u r e where p o l a r r e s i d u e s a r e b u r i e d and n o n p o l a r ones a r e a c c e s s i b l e t o w a t e r . F o r a p r o t e i n t o be r e a s o n a b l y s o l u b l e i t must remove much o f t h i s h y d r o p h o b i c s u r f a c e from c o n t a c t w i t h w a t e r by b u r y i n g i t between the p i e c e s o f s e c o n d a r y s t r u c t u r e . However, K l o t z (1970) examined t e n p r o t e i n s f o r w h i c h t h e t h r e e d i m e n s i o n a l s t r u c t u r e was known and showed t h a t the h y d r o p h o b i c g r o u p s , i n c l u d i n g the l a r g e r h y d r o p h o b i c amino a c i d s i d e c h a i n s s u c h as t h o s e o f p h e n y l a l a n i n e and t r y p t o p h a n , o c c u r more f r e q u e n t l y on t h e s u r f a c e o f the p r o t e i n t h a n had been assumed. 4. P r o t e i n - l i g a n d i n t e r a c t i o n : I n the f o r m a t i o n o f a s t a b l e g l o b u l a r s t r u c t u r e , the complete r e m o v a l of h y d r o p h o b i c s i d e c h a i n s f r o m c o n t a c t w i t h w a t e r i s g e n e r a l l y n o t p o s s i b l e . I n most n a t i v e p r o t e i n s , some h y d r o p h o b i c groups r e m a i n exposed a t t h e m o l e c u l a r s u r f a c e o r i n c r e v i c e s ( T a n f o r d , 1972). I f s u f f i c i e n t l y l o n g h y d r o p h o b i c p a t c h e s a r e formed, t h e y may c o n s t i t u t e b i n d i n g s i t e s f o r h y d r o c a r b o n o r a m p h i p h i l i c m o l e c u l e s . W e t l a u f e r and L o v r i e n (1964) s u g g e s t e d two d i s t i n c t l y d i f f e r e n t modes o f i n t e r a c t i o n between a p r o t e i n and a s m a l l n o n p o l a r m o l e c u l e : a) a d i s s o l v e d n o n p o l a r m o l e c u l e c o u l d a t t a c h i t s e l f t o t h e a c c e s s i b l e s u r f a c e o f a n o n p o l a r c l u s t e r perhaps w i t h p a r t i a l p e n e t r a t i o n ; o r b) t h e n o n p o l a r m o l e c u l e might p e n e t r a t e i n t o t h e r e l a t i v e l y n o n p o l a r i n t e r i o r o f the p r o t e i n and l o d g e . An i n t e r a c t i o n o f the s o r t i n d i c a t e d i n a) seems more l o g i c a l because i t might o c c u r w i t h v e r y minor s t r u c t u r a l changes. On t h e o t h e r hand, i f b) i s t h e c a s e , i t w o u l d r e q u i r e s u b s t a n t i a l m o l e c u l a r rearrangement t o accomodate the n o n p o l a r m o l e c u l e o r m o l e c u l e s , s i n c e t h e p r e - e x i s t e n c e of h o l e s i n the p r o t e i n i n t e r i o r i s h i g h l y u n l i k e l y on e n e r g e t i c grounds. S i n c e t h e d r i v i n g f o r c e b e h i n d t h e h y d r o p h o b i c i n t e r a c t i o n i s t h o u g h t t o be e n t r o p i c (Kauzmann, 1959), t h e s e i n t e r a c t i o n s s h o u l d be s t r o n g e r a t h i g h e r t e m p e r a t u r e s because t h e t e r m T A S i n A F = A H - T A S becomes i n c r e a s i n g l y u n f a v o r a b l e towards d i s s o c i a t i o n o f n o n p o l a r b i n d i n g s . A t t h e same t i m e most o f t h e o t h e r bonds a r e weakened, s u c h t h a t h y d r o -p h o b i c b o n d i n g w o u l d p r e d o m i n a t e . H y d r o p h o b i c i n t e r a c t i o n s a r e a l s o f a v o u r e d by h i g h i o n i c s t r e n g t h ( H j e r t e n , 1975) and low pH. C e r t a i n i o n s a f f e c t t h e e s t a b l i s h m e n t o f t h i s t y p e o f b o n d i n g . T h i s e f f e c t i s d e c r e a s e d i n o r d e r o f SO. > P0,~ > C l " > B r " > I ~ > CIO ~ > and CNS~ 4 4 4 (von H i p p e l and S c h l e i c h , 1969a). Non i o n i c s o l u t e s l i k e d i o x a n e and a l c o h o l s weaken h y d r o p h o b i c i n t e r a c t i o n s ( T a n f o r d , 1973). U r e a , g u a n i d i n e h y d r o c h l o r i d e and some s u r f a c t a n t s d i s r u p t h y d r o p h o b i c i n t e r a c t i o n s , by p r o m o t i n g t h e s o l u b i l i z a t i o n o f h y d r o p h o b i c r e s i d u e s i n the aqueous phase (Gordon and Warren, 1968). D e t e r g e n t s m i n i m i z e h y d r o p h o b i c i n t e r a c t i o n s and may a c t u a l l y f o r m c h e m i c a l b r i d g e s between h y d r o p h o b i c and h y d r o p h i l i c p o r t i o n s o f p o l y p e p t i d e s , t h e r e b y f a c i l i t a t i n g d e n a t u r a t i o n ( S t e i n h a r d t , 1974). The a d d i t i o n o f a c i d o r a l k a l i i o n i z e s w e a k l y a c i d i c o r b a s i c g r o u p s , w h i c h a r e b u r i e d w i t h i n the i n t e r i o r , s u p p o s e d l y i n h y d r o p h o b i c r e g i o n s o f t h e p r o t e i n m o l e c u l e . Upon i o n i z a t i o n , t h e s e c h a r g e d groups a t t r a c t w a t e r m o l e c u l e s and f o r m h y d r a t i o n s h e l l s , w h i c h i n t u r n d i s r u p t h y d r o p h o b i c a s s o c i a t i o n s and cause u n f o l d i n g ( P e r u t z , 1974). 5. D e t e r m i n a t i o n o f h y d r o p h o b i c i t y : The i m p o r t a n c e o f h y d r o p h o b i c i n t e r a c t i o n s has a t t r a c t e d t h e a t t e n t i o n o f many r e s e a r c h e r s i n t h e a r e a o f p r o t e i n c h e m i s t r y . A n u m b e r o f i n v e s t i g a t o r s h a v e a t t e m p t e d t o e s t a b l i s h a " h y d r o p h o b i c s c a l e " f o r s e r i e s o f p r o t e i n s . I n t h e c a s e o f s m a l l m o l e c u l e s , h y d r o -p h o b i c i t y h a s b e e n d e t e r m i n e d m a i n l y i n t e r m s o f t h e i r s o l u b i l i t i e s i n o r g a n i c l i q u i d s o r t h e i r p a r t i t i o n b e t w e e n a q u e o u s a n d o r g a n i c p h a s e s . Waugh (1954) e s t i m a t e d t h e h y d r o p h o b i c n a t u r e o f p r o t e i n s s i m p l y b y d e t e r m i n i n g t h e f r a c t i o n o f n o n p o l a r r e s i d u e s . He c a l c u l a t e d n o n p o l a r s i d e c h a i n (NPS ) f r e q u e n c i e s f o r s e r i e s o f p r o t e i n s . T r p , l i e , T y r , P h e , P r o , L e u a n d V a l w e r e d e f i n e d a s n o n p o l a r r e s i d u e s . F i s h e r (1964) c a l c u l a t e d p o l a r i t y v a l u e s f o r p r o t e i n s b y d i v i d i n g e x t e r n a l v o l u m e ( V e ) b y i n t e r n a l v o l u m e ( V i ) o f a g l o b u l a r p r o t e i n . T h i s w a s o n l y a s i m p l e a r i t h m e t i c c a l c u l a t i o n a n d b a s e d o n t h e a s s u m p t i o n t h a t a l l n o n p o l a r a m i n o a c i d s a r e p r e s e n t i n s i d e a n d a l l p o l a r a m i n o a c i d s a r e o n t h e o u t e r s u r f a c e o f t h e m o l e c u l e . T h e t o t a l v o l u m e ( V t ) w a s e s t i m a t e d u s i n g s p e c i f i c v o l u m e s o f a m i n o a c i d r e s i d u e s . F i s h e r s u g g e s t e d t h a t h i g h l y p o l a r p r o t e i n s a r e l i k e l y n o n s p h e r i c a l b e c a u s e t h a t s o r t o f V e s h a p e g i v e s t h e m a h i g h e r r a t i o o f — t h a n a s p h e r e o f t h e same V t . H e n c e m o r e s p a c e w o u l d b e a v a i l a b l e f o r t h e e x c e s s o f t h e p o l a r r e s i d u e s . K l o t z ( 1 9 7 0 ) d i s a g r e e d w i t h t h e v i e w t h a t r e g a r d s p o l a r a m i n o a c i d s o u t s i d e a n d a p o l a r o n e s i n s i d e t h e m o l e c u l e . A l t h o u g h c r y s t a l o g r a p h i c s t u d i e s s h o w t h a t i n a l m o s t a l l p r o t e i n s i o n i c s i d e c h a i n s a r e f u l l y e x p o s e d t o t h e s o l v e n t , a s u b s t a n t i a l n u m b e r o f a p o l a r g r o u p s s e e m t o b e a c c e s s i b l e t o t h e s o l v e n t m o l e c u l e s i n a l l p r o t e i n s e x a m i n e d b y X - r a y d i f f r a c t i o n . F o r some o f t h e m , s u f f i c i e n t i n f o r m a t i o n i s a v a i l a b l e t o p r o c e e d o n e s t e p f u r t h e r : t o e s t i m a t e t h e e x t e n t o f e x p o s u r e o f e a c h o f t h e major groups o f a p o l a r r e s i d u e s ( K l o t z , 1970). T a n f o r d (1962) c o n s i d e r e d s o l u b i l i t y o f f r e e amino a c i d s as an i n d e x f o r t h e i r h y d r o p h o b i c i t i e s . He d e t e r m i n e d t h e f r e e energy r e q u i r e d t o t r a n s f e r one m o l e c u l e of amino a c i d from w a t e r t o e t h a n o l . N e g a t i v e v a l u e s were o b t a i n e d f o r t h e amino a c i d s w h i c h p r e f e r r e d w a t e r t o e t h a n o l . These a r e supposed t o be p o l a r s i d e c h a i n s and pr e s u m a b l y o c c u r i n t h e o u t e r s h e l l o f t h e p r o t e i n m o l e c u l e . T a n f o r d (1962) showed t h a t t h e c a l c u l a t e d v a l u e s were i n d e p e n d e n t o f t h e k i n d o f o r g a n i c s o l v e n t s . A c c o r d i n g t o T a n f o r d t h i s f r e e energy f o r each amino a c i d can be r e g a r d e d as t h e sum o f two te r m s : one f o r t h e backbone H-^N- c - COO J H and t h e o t h e r f o r t h e s i d e c h a i n . The backbone s t r u c t u r e i s s i m i l a r t o t h a t f o r g l y c i n e . W i t h s o l u b i l i t y d a t a f o r g l y c i n e , s u b t r a c t i o n t h e r e f o r e l e a d s t o a t r a n s f e r f r e e energy f o r t h e s i d e c h a i n i t s e l f . These t r a n s f e r f r e e e n e r g i e s c a l c u l a t e d by T a n f o r d were c a l l e d h y d r o p h o b i c i t i e s by D u n n i l l i n 1965. I t i s i n t e r e s t i n g t o n o t e t h a t T a n f o r d (1962) and Waugh (1954) r e g a r d g l y c i n e as an amino a c i d o f low h y d r o p h o b i c c h a r a c t e r , whereas F i s h e r (1964) c o n s i d e r s i t t o be n o n p o l a r . A l s o , t y r o s i n e was assumed p o l a r by F i s h e r (because o f i t s OH group) w h i l e n o n p o l a r by Waugh (1954) and T a n f o r d ( 1 9 6 2 ) . B i g e l o w (1967) c a l c u l a t e d an "average h y d r o p h o b i c " i n d e x from the r e s i d u e p e r c e n t a g e amino a c i d c o m p o s i t i o n o f the p r o t e i n and a f r e e energy f o r each r e s i d u e d e t e r m i n e d by T a n f o r d ( 1 9 6 2 ) . T o t a l h y d r o -p h o b i c i t y d i v i d e d by t h e number o f r e s i d u e s i n t h e m o l e c u l e y i e l d e d t h e "average h y d r o p h o b i c i t y " . B i g e l o w (1967) s u g g e s t e d t h a t low m o l e c u l a r w e i g h t p r o t e i n s have t h e l a r g e s t s p r e a d i n a l l o w e d h y d r o p h o b i c i t i e s w h i l e p r o t e i n s o f h i g h m o l e c u l a r w e i g h t t e n d t o o c c u r i n q u i t e a narrow range o f h y d r o p h o b i c i t i e s . The above mentioned r e s e a r c h e r s d e t e r m i n e d h y d r o p h o b i c i t i e s w i t h t h e i s o l a t e d p r o t e i n m o l e c u l e and t h e measurements were based on the number and p r o p e r t i e s o f t h e amino a c i d components. The v a l u e s do n o t r e p r e s e n t t h e a b i l i t y o f t h e p r o t e i n t o form h y d r o p h o b i c bonds beca u s e c o n f o r m a t i o n and f l e x i b i l i t y o f t h e p r o t e i n were n o t a c c o u n t e d f o r . The f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s m o s t l y depend on t h e degree of i n t e r a c t i o n s t h e y can t a k e p a r t i n , and n o t o n l y on t h e i r m o l e c u l a r s t r u c t u r e and amino a c i d c o m p o s i t i o n s . There i s a need f o r a method t h a t d e t e r m i n e s t h e e f f e c t i v e h y d r o p h o b i c i t y o f a p r o t e i n w h i c h a c c o u n t s f o r c o n f o r m a t i o n and f l e x i b i l i t y as w e l l as on i t s amino a c i d c o m p o s i t i o n . 6. H y d r o p h o b i c chromatography: H y d r o c a r b o n s l i k e p ropane, b u t a n e , and pentane have sometimes been used t o s t u d y the n o n p o l a r r e g i o n s o f p r o t e i n s ( W i s h n i a , 1962). The a l k a n e s i n t e r a c t d i r e c t l y w i t h t h e h y d r o p h o b i c a r e a s ( W i s h n i a and Thomas, 1966). T h i s p r o p e r t y has been employed i n chromatography w i t h t h e h y d r o -c a r b o n c o a t e d a g a r o s e s . C u a t r e c a s a s and A n f i n s e n (1971) d e v e l o p e d a f f i n i t y chromatography by a t t a c h i n g h y d r o c a r b o n l i g a n d s a t v a r y i n g d i s t a n c e s f r o m g e l m a t r i x backbone. A g a r o s e i s t h e most w i d e l y used i n s o l u b l e m a t r i x f o r t h e p r e p a r a t i o n o f the a f f i n i t y a d s o r b e n t s . S e p h a r o s e , a "beaded" f o r m o f a g a r o s e , w i t h i t s h i g h l y porous s t r u c t u r e , i s v e r y s u i t a b l e f o r t h e p r e p a r a t i o n o f s u b s t i t u t e d g e l s . The p o l y s a c c h a r i d e m a t r i x i s a c t i v a t e d by the CNBr method and t h e h y d r o - • c a r b o n l i g a n d s a r e s u b s e q u e n t l y c o u p l e d t o i t v i a the f r e e amino group. These h y d r o c a r b o n c o a t e d a g a r o s e s o r a l k y l - S e p h a r o s e s were c a l l e d h y d r o p h o b i c g e l s by S h a l t i e l ert a l . (1973b) and were used e x t e n s i v e l y f o r p u r i f i c a t i o n p u r p o s e s by many w o r k e r s ( C u a t r e c a s a s and A n f i n s e n , 1970; C u a t r e c a s a s e t a l . , 1968; J e n i s s e n , 1975). H o f s t e e (1974) e s t i m a t e d t h e r e l a t i v e degree o f s u b s t i t u t i o n f r o m t h e dye b i n d i n g c a p a c i t i e s o f t h e r e s i n and showed t h a t t h e degree o f s u b s t i t u t i o n d e c r e a s e d upon s t o r a g e . T h i s i n a c t i v a t i o n t o o k p l a c e g r a d u a l l y even when the g e l was r e f r i g e r a t e d . F o r i n s t a n c e , i n one case a d e c r e a s e o f as much as 85 p e r c e n t o f t h e ponceau b i n d i n g c a p a c i t y had o c c u r r e d d u r i n g a p e r i o d o f a l m o s t f i v e months. I n a n o t h e r c a s e , a 40% d e c r e a s e i n 40 days s t o r a g e was o b s e r v e d ( H o f s t e e , 1974). B a s i c a l l y , t h e a d s o r b e n t s w i t h t h e h i g h e s t degree o f s u b s t i t u t i o n were t h e l e a s t s t a b l e ones. The a d j u s t m e n t o f t h e column h y d r o p h o b i c i t y so as t o ad s o r b b u t n o t d e n a t u r e t h e p r o t e i n s has been approached i n two ways: 1) E r - e l e t a l . ( 1 9 7 2 ) , H o f s t e e (1973b) and S h a l t i e l and E r - e l (1973) used a s e r i e s o f a l k y l - S e p h a r o s e s , o r e j - a m i n o a l k y l - S e p h a r o s e s o f v a r i o u s a l k y l c h a i n l e n g t h s , f r o m w h i c h t h e a p p r o p r i a t e one was found by t r i a l ; 2) by m a n i p u l a t i n g pH on s t r o n g l y h y d r o p h o b i c a l k y l - S e p h a r o s e g e l , Yon (1974) i n t r o d u c e d e l e c t r o s t a t i c r e p u l s i o n e n e r g i e s t o c o n t r o l t h e b i n d i n g f o r c e s and a d s o r p t i o n p r o p e r t i e s o f t h e g e l m a t e r i a l . S h a l t i e l e_t a l . (1973b) s u g g e s t e d t h a t t h e r e t e n t i o n power of a l k y l - a g a r o s e s i s d e r i v e d m a i n l y f r o m l i p o p h i l i c i n t e r a c t i o n s between h y d r o p h o b i c p a c k e t s o r r e g i o n s i n t h e p r o t e i n and l o n g c h a i n a l k y l groups on t h e a g a r o s e . H y d r o p h o b i c i n t e r a c t i o n s c o n t r i b u t e p r e d o m i n a n t l y t o t h e r e t e n t i o n and d i s c r i m i n a t i o n power o f t h e a l k y l - S e p h a r o s e s . However, a t t h e same t i m e , one s h o u l d be aware t h a t i o n i c i n t e r a c t i o n s a l s o a f f e c t t h e e l u t i o n p a t t e r n o f t h e s e columns ( H o f s t e e , 1973a). By p r e p a r i n g h y d r o c a r b o n c o a t e d Sepharoses t h r o u g h t h e CNBr a c t i v a t i o n p r o c e d u r e , i t w o u l d seem u n l i k e l y t o o b t a i n a bed m a t e r i a l t h a t e x h i b i t e d o n l y h y d r o -p h o b i c i n t e r a c t i o n . The amino groups o f t h e l i g a n d s r e t a i n t h e i r b a s i c p r o p e r t i e s a f t e r b i n d i n g t o Sepharose ( H j e r t e n , 1973). E l e c t r o s t a t i c , van d e r Waals, and hydrogen b o n d i n g f o r c e s g e n e r a l l y p l a y more o r l e s s i m p o r t a n t r o l e s i n c h r o m a t o g r a p h i c b e h a v i o u r o f p r o t e i n s on a l k y l -S e p h a r o s e s . Agar c o n t a i n s two t y p e s o f c h a r g e d g r o u p s , c a r b o x y l and s u l f a t e . I n t h e c r o s s l i n k e d a g a r , s u l f a t e groups a r e a l m o s t c o m p l e t e l y removed b u t t h e c a r b o x y l group i s p r e s e n t . Even h i g h l y p u r i f i e d a g a r o s e s c o n t a i n some n e g a t i v e c h a r g e s ( P o r a t h e t a l . , 1971) and p o s i t i v e c h a r g e s may be i n t r o d u c e d as a r e s u l t o f a c t i v a t i o n w i t h CNBr w h i c h a r e s u b s e q u e n t l y c o u p l e d w i t h a l k y l a m i n e . I t i s e v i d e n t t h a t t h i s t y p e o f h y d r o p h o b i c a d s o r b e n t s c o n t a i n s i g n i f i c a n t amounts o f i o n i c g r o u p s , m a i n l y c a t i o n i c ( H o f s t e e , 1973b; H j e r t e n , 1973). T h e r e f o r e , t h e y e x h i b i t a s u p e r i m p o s e d e l e c t r o s t a t i c e f f e c t . The p r e s e n c e of t h e s e i o n i c groups c o m p l i c a t e s and p o t e n t i a l l y l i m i t s the u s e f u l n e s s o f t h e s e s o r b e n t s when a s p e c i f i c i n t e r a c t i o n i s d e s i r e d ( H o f s t e e , 1974). The s i m u l t a n e o u s p r e s e n c e o f e x t r a n e o u s i o n i c and h y d r o p h o b i c groups has been found t o cause s u b s t a n t i a l n o n s p e c i f i c p r o t e i n b i n d i n g . These g e l s e x h i b i t b o t h e l e c t r o s t a t i c and h y d r o p h o b i c p r o p e r t i e s a t low i o n i c s t r e n g t h ( H j e r t e n , 1973; H o f s t e e , 1973a) and m a i n l y h y d r o p h o b i c i n t e r a c t i o n s a t h i g h i o n i c s t r e n g t h . An i d e a l uncharged g e l w i t h a h y d r o p h o b i c l i g a n d s h o u l d g i v e h y d r o p h o b i c i n t e r a c t i o n s a t a l l i o n i c s t r e n g t h s ( H j e r t e n e t a l . , 1974; Rosengren e_t a l . , 1975). However, the term " h y d r o p h o b i c chromatography" has u n f o r t u n a t e l y been used f o r a l l e x p e r i m e n t a l p r o c e d u r e s i n w h i c h an a d s o r b e n t w i t h a n o n p o l a r l i g a n d i s employed, i r r e s p e c t i v e o f whether t h e s e l i g a n d s a r e charged o r uncharged. 7. Uncharged r e s i n s : J o s t et_ a l _ . (1974) used h y d r a z i d e s t o p r e p a r e uncharged a g a r o s e d e r i v a t i v e s . Whereas a - l a c t a l b u m i n and o v a l b u m i n bound s t r o n g l y t o the a l k y l - s u b s t i t u t e d S e p h a r o s e , the b i n d i n g of t h e s e p r o t e i n s t o t h e h y d r a z i d e - s u b s t i t u t e d r e s i n was r e p o r t e d t o be weak. These w o r k e r s c o n c l u d e d t h a t no i o n i c i n t e r a c t i o n i s i n v o l v e d when chromatography i s p e r f o r m e d on h y d r a z i d e d e r i v a t i v e s o f Sepharose. N i s h i k a w a and B a i l o n (1975 ) employed a c y l - h y d r a z i d e m o i e t i e s i n t h e c o u p l i n g of f u n c t i o n a l groups t o cyanogen b r o m i d e - a c t i v a t e d Sepharose. T h e y s u g g e s t e d t h a t r e a g e n t s s u c h as c a p r y l y l h y d r a z i d e p r o v i d e s o r b e n t s t h a t were e s s e n t i a l l y f r e e o f charge a t pH 7.0. H j e r t e n e_t a l . (1974) p r e s e n t e d a method f o r a t t a c h i n g a l k y l o r a r y l m o i e t i e s w i t h o u t i n t r o d u c i n g c h a r g e d groups onto an a g a r o s e m a t r i x . 24 T h e g e l w a s p r e p a r e d t h r o u g h t h e r e a c t i o n o f a g a r o s e w i t h e p o x i d e s i n a n a p r o t i c s o l v e n t . T h e p r e p a r e d g e l s w e r e c l a i m e d t o be- d e v o i d o f i o n i c g r o u p s . T h e y w e r e i n t r o d u c e d f o r u s e i n s e p a r a t i o n a n d p u r i f i c a t i o n o f p r o t e i n s b a s e d o n t h e i r h y d r o p h o b i c i t i e s . 8. I n f l u e n t i a l f a c t o r s : I t i s g e n e r a l l y b e l i e v e d t h a t t h e p a r t i c u l a r " n a t i v e " s t r u c t u r e o f a p r o t e i n r e p r e s e n t s a s t a t e o f m i n i m u m f r e e e n e r g y , a n d f u r t h e r m o r e t h a t t h e f r e e e n e r g y o f t h i s s t a t e i s t h e a l g e b r a i c sum o f t h e f r e e e n e r g i e s o f t h e i n d i v i d u a l r e s i d u e - r e s i d u e a n d r e s i d u e - s o l v e n t c o n t a c t s i n v o l v e d i n t h e e q u i l i b r i u m c o n f o r m a t i o n . H o w e v e r , t h e n a t i v e c o n f o r m a t i o n o f m o s t ' p r o t e i n s i s o n l y r e l a t i v e l y s t a b l e . I t c a n b e d e s t a b i l i z e d e a s i l y b y t h e e f f e c t o f p e r t u r b a n t s s u c h a s s a l t s , t e m p e r a t u r e , o r g a n i c s o l v e n t s , e t c . ( T a n f o r d , 1973). P e r t u r b a n t s c a n b e c l a s s i f i e d a s p o t e n t i a l h y d r o g e n b o n d , h y d r o p h o b i c b o n d , a n d e l e c t r o s t a t i c b o n d d e s t a b i l i z e r s . H o w e v e r , i t i s i m m e d i a t e l y a p p a r e n t t h a t t h e r e a r e v e r y f e w s t r u c t u r a l p e r t u r b a n t s w h i c h a f f e c t e x c l u s i v e l y o n e o f t h e a b o v e t y p e s o f i n t e r a c t i o n s , a n d i t i s f u r t h e r a p p a r e n t t h a t i o n i c s p e c i e s c a n p o t e n t i a l l y a f f e c t a l l t h r e e t y p e s o f i n t e r a c t i o n s . I n h y d r o p h o b i c i n t e r a c t i o n c h r o m a t o g r a p h y , many f a c t o r s a f f e c t t h e i n t e r a c t i o n b e t w e e n p r o t e i n a n d t h e a d s o r b e n t , n a m e l y t h e i o n i c s t r e n g t h , t h e h y d r o p h o b i c i t y o f t h e s u b s t i t u e n t , t h e d e g r e e o f t h e s u b s t i t u t i o n , a n d t h e a m b i e n t t e m p e r a t u r e ( H j e r t e n e t a l . , 1974; R o s e n g r e n et^ a l _ . , 1975) . T h e a m o u n t o f t h e l i g a n d b o u n d t o t h e m a t r i x a n d t h e l e n g t h o f t h e s p a c e r a r m m a i n l y d e t e r m i n e t h e h y d r o p h o b i c i t y o f t h e g e l . T a n f o r d (1973) assumed t h a t t h e f i r s t two c a r b o n atoms i n an a l k y l c h a i n a t t a c h e d t o a p o l a r group g i v e o n l y a s m a l l c o n t r i b u t i o n t o the h y d r o p h o b i c i t y o f t h e m o l e c u l e . I n f a c t , m e t h y l , e t h y l and even p r o p y l a g a r o s e s have m i n o r o r no h y d r o p h o b i c p r o p e r t i e s . S a l t e f f e c t : C o n t r a r y t o i o n exchange chromatography, the a d s o r p t i o n i n h y d r o p h o b i c i n t e r a c t i o n chromatography d e c r e a s e s w i t h l o w e r i n g i o n i c s t r e n g t h ( H j e r t e n et_ a l . , 1974). von H i p p e l and S c h l e i c h (1969b) and H a t e f i and H a n s t e i n (1969) have f o u n d , f o r many sy s t e m s , t h a t t h e degree o f h y d r o p h o b i c i n t e r a c t i o n s i n f r e e s o l u t i o n d i f f e r s s i g n i f i c a n t l y a c c o r d i n g t o the t y p e o f t h e n e u t r a l s a l t s u s e d . B a s i c a l l y , s a l t i n g - o u t a gents such as PO^ , SO^ and C l i n t e n s i f y t h e s e k i n d s o f bonds w h i l e B r and SCN which' a r e known as s a l t i n g - i n a g e n t s d i m i n i s h t h e s e i n t e r a c t i o n s (Pahlman _et a l . , 1977). Phosphate and s u l f a t e a n i o n s a r e s t r u c t u r e f o r m i n g i o n s , w h i c h a r e known t o d e c r e a s e the s o l u b i l i t y o f p r o t e i n and s t a b i l i z e h y d r o p h o b i c bonds between n o n p o l a r m o l e c u l e s (von H i p p e l and S c h l e i c h , 1969a). Ammonium s u l f a t e and p o t a s s i u m phosphate b u f f e r s w i t h d e c r e a s i n g concen-t r a t i o n g r a d i e n t have been a p p l i e d i n h y d r o p h o b i c chromatography by Rimerman and H a t f i e l d (1973) t o p u r i f y p r o t e i n s on t h e b a s i s o f t h e i r h y d r o p h o b i c c h a r a c t e r . R e c e n t l y Pahlman e_t a l . (1977) used p e n t y l - a n d d e c y l - S e p h a r o s e s o f t h e s e s e r i e s t o measure t h e degree o f h y d r o p h o b i c i n t e r a c t i o n s based on t h e amount o f the p r o t e i n bound t o the a d s o r b e n t . These w o r k e r s m a i n l y s t u d i e d t h e e f f e c t o f d i f f e r e n t s a l t s on t h e a d s o r p t i o n o f t h r e e p r o t e i n s , namely o v a l b u m i n , BSA and p h y c o e r y t h r i n . They f o u n d t h a t 3 M sodium bromide and 3 M sodium t h i o c y a n a t e changed t h e c o n f o r m a t i o n o f BSA and o v a l b u m i n , w h i c h i n most c a s e s was accompanied by a d e c r e a s e i n p r o t e i n - a d s o r b e n t i n t e r a c t i o n . These w o r k e r s r e p o r t e d no c o n f o r m a t i o n a l change i n p r o t e i n i n the p r e s e n c e o f 3 M sodium c h l o r i d e o r 1 M sodium s u l f a t e . S a l t s i n d e e d p l a y an e x t r e m e l y i m p o r t a n t r o l e i n h y d r o p h o b i c b i n d i n g and, t h e r e f o r e , i n h y d r o p h o b i c chromatography. The h y d r o p h o b i c i n t e r a c t i o n i s c o n s i d e r e d t o be promoted by h i g h c o n c e n t r a t i o n s o f n e u t r a l s a l t s (Rosengren e t a l , , 1975). The n e u t r a l s a l t s a r e c a p a b l e o f a l t e r i n g t h e s t r u c t u r e and p r o p e r t i e s of p r o t e i n s and p o l y p e p t i d e s i n s o l u t i o n due t o t h e i r e f f e c t s on t h e s o l u b i l i t y , c o n f o r m a t i o n a l s t a b i l i t y , a s s o c i a t i o n - d i s s o c i a t i o n e q u i l i b r i a o r r a t e s o f t r a n s f o r m a t i o n a l r e a c t i o n s . I t i s c l e a r t h a t t h e v a r i o u s i o n s have s p e c i f i c and p r e d i c t a b l e e f f e c t s on t h e s t a b i l i t y o f a w ide v a r i e t y of m a c r o m o l e c u l a r s t r u c t u r e s and complexes. T h e i r e f f e c t s on p r o t e i n s have been s t u d i e d e x t e n s i v e l y by von H i p p e l and S c h l e i c h (1969b). When the e f f e c t s o f d i f f e r e n t s a l t s i n p r o m o t i n g h y d r o p h o b i c i n t e r a c t i o n o r a f f e c t i n g w a t e r s t r u c t u r e a r e t o be compared, t h e p r o b l e m a r i s e s o f comparing t h e e f f e c t s o f monovalent and p o l y v a l e n t i o n s . Comparison can be made, f o r example, a t c o n s t a n t s a l t c o n c e n t r a t i o n , c o n s t a n t i o n i c s t r e n g t h o r c o n s t a n t space charge d e n s i t y . C h l o r i d e s o f monovalent c a t i o n s have been found t o change c o n f o r m a t i o n and a f f e c t f l u o r e s c e n c e p r o p e r t i e s o f p r o t e i n s . P o t a s s i u m and sodium c h l o r i d e s have been shown to be r e s p o n s i b l e f o r c a u s i n g t h e d e s t a b i l i z a t i o n o f m a c r o m o l e c u l e s (von H i p p e l and S c h l e i c h , 1969b). I n the c o n c e n t r a t e d s a l t s o l u t i o n s , changes i n t h e s i z e o f t h e p r o t e i n c o u l d a l s o t a k e p l a c e ( S t e i n b e r g et_ a l . , 1960). A t low s a l t c o n c e n t r a t i o n s , 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 a r e d o m i n a n t . I n h i g h s a l t c o n c e n t r a t i o n s t h e s e i n t e r a c t i o n s w o u l d b e a b s e n t . I n c r e a s i n g t h e i o n i c s t r e n g t h b y a d d i t i o n o f ammon ium s u l f a t e o r s i m i l a r a d d i t i v e s t o t h e s o l v e n t i n c r e a s e s t h e s t a b i l i t y o f r e s i d u e - r e s i d u e c o n t a c t s r e l a t i v e t o r e s i d u e - s o l v e n t i n t e r a c t i o n s , c o n v e r t i n g some p r e v i o u s l y s t a b l e s u r f a c e r e s i d u e s i n t o s p e c i e s f o r w h i c h t h e f r e e e n e r g y b a l a n c e now f a v o u r s r e s i d u e - r e s i d u e i n t e r a c t i o n s . A g g r e g a t i o n o f n a t i v e p r o t e i n r e s u l t s ( s a l t i n g - o u t ) . T h i s e f f e c t i s o p p o s i t e t o t h e e f f e c t o f u r e a w h i c h s t a b i l i z e s r e s i d u e - s o l v e n t c o n t a c t r e l a t i v e t o r e s i d u e - r e s i d u e i n t e r a c t i o n . I n h y d r o p h o b i c c h r o m a t o g r a p h y , s a l t i n g - o u t a g e n t s e n h a n c e b i n d i n g o f p r o t e i n s t o t h e s o r b e n t , w h i l e s a l t i n g - i n c o m p o u n d s d i m i n i s h t h e s e b i n d i n g s . T h e s a l t i n g - o u t e f f e c t i s s u g g e s t e d t o b e i n v o l v e d i n t h e i n t e r a c t i o n o f p r o t e i n s w i t h a m p h i p h i l i c S e p h a r o s e d e r i v a t i v e s ( P o r a t h e t a l . , 1973; R imer -man a n d H a t f i e l d , 1973; M e m o l i a n d D o e l l g a s t , 1975). T h e e f f e c t o f i o n s o n t h e s t r u c t u r e o f w a t e r a r o u n d n o n p o l a r g r o u p s m u s t p l a y a r o l e i n t h e s a l t i n g - o u t o f n o n p o l a r c o m p o u n d s f r o m a q u e o u s s o l u t i o n s . T h e s a l t i n g o u t o f p r o t e i n s h a s b e e n s h o w n t o b e a c c o m p a n i e d b y c h a n g e s i n t h e i r c o n f o r m a t i o n a l s t r u c t u r e ( K a u z m a n n , 1959). P a h l m a n e t a l . (1977) r e p o r t e d t h a t b r o m i d e a n d t h i o c y a n a t e a n i o n s c h a n g e t h e c o n f o r m a t i o n o f p r o t e i n s w h i l e c h l o r i d e a n d s u l f a t e a n i o n s d o n o t . T e m p e r a t u r e e f f e c t : Some a d s o r p t i v e f o r c e s s u c h a s h y d r o g e n b o n d i n g o r v a n d e r W a a l s f o r c e s a r e w e a k e n e d b y a r i s e i n t e m p e r a t u r e b e c a u s e o f t h e e n t h a l p y d e c r e a s e . H y d r o p h o b i c b o n d i n g , b e i n g a n e n t r o p y e f f e c t , b e h a v e s i n t h e o p p o s i t e m a n n e r , a n d i n g e l c h r o m a t o g r a p h y a r e t e n t i o n o f t h e 28 s o l u t e s h o u l d o c c u r w i t h i n c r e a s e i n t e m p e r a t u r e . H j e r t e n (1973) s t u d i e d the e f f e c t o f t e m p e r a t u r e on t h e b e h a v i o u r o f the h y d r o p h o b i c columns. He found t h a t a d e c r e a s e i n t e m p e r a t u r e d i d n o t d i s t u r b t h e a d s o r p t i o n o f p r o t e i n s t o the h y d r o c a r b o n c o a t e d Sepharose w h i l e d e s o r p t i o n o f h y d r o p h o b i c p r o t e i n s f r o m t h e epoxy d e r i v a t i v e s o f Sepharose 4B was a c h i e v e d upon a d e c r e a s e i n t e m p e r a t u r e from 25°C t o 0°C ( H j e r t e n , 1973). T h i s f i n d i n g i s n o t s u r p r i s i n g , s i n c e e l e c t r o s t a t i c elements a r e p r e s e n t i n a l k y l - S e p h a r o s e s w h i c h i n t e n s i f y i n t e r a c t i o n s a t l o w e r t e m p e r a t u r e s . P u r e h y d r o p h o b i c b o n d i n g on the o t h e r hand i s d e s t a b i l i z e d when t h e t e m p e r a t u r e i s d e c r e a s e d . The e f f e c t o f t e m p e r a t u r e on h y d r o p h o b i c b o n d i n g i s due t o t h e f a c t t h a t t h e s e k i n d s o f bonds a r e e n t r o p y e f f e c t s . 9. D e t e r g e n t e f f e c t s : A n o t h e r i n t e r e s t i n g phenomenon w h i c h has p r o v e d t o be u s e f u l i n t h e s t u d y o f membrane s t r u c t u r e i s t h e e f f e c t o f d e t e r g e n t s on p r o t e i n s . H i g h d e t e r g e n t c o n c e n t r a t i o n d i s s o c i a t e s t h e l i p i d s f rom the p r o t e i n s i n membranes and l i p o p r o t e i n s (Engelman et_ a l . , 1967). D e t e r g e n t s b i n d t o h y d r o p h o b i c p r o t e i n s s p e c i f i c a l l y and the major d r i v i n g f o r c e f o r t h i s a s s o c i a t i o n i s h y d r o p h o b i c i n t e r a c t i o n s . I n p r o t e i n - d e t e r g e n t s y s t e m s , t h e i n t e r a c t i o n i s enhanced by i n c r e a s i n g the h y d r o p h o b i c c h a i n l e n g t h o f th e d e t e r g e n t . The s t a n d a r d f r e e energy change f o r t h e s e p r o c e s s e s has been found t o d e c r e a s e w i t h t h e a d d i t i o n o f - C ^ - r e s i d u e s (Yonath and B l a u e r , 1974). B i n d i n g o f T r i t o n X-100 ( n o n i o n i c d e t e r g e n t ) t o h y d r o p h o b i c p r o t e i n s t a k e s p l a c e i n i t s monomeric form w h i l e t h e b u l k o f d e t e r g e n t r e m a i n s as m i c e l l e s . H e l e n i u s and Simons (1972) s t u d i e d , w i t h t h e a i d o f r a d i o a c t i v e d e t e r g e n t , i n t e r a c t i o n s o f p r o t e i n s w i t h T r i t o n X-100 and d e s o x y c h o l a t e . L i p o p h i l i c p r o t e i n s bound l a r g e amounts o f t h e d e t e r g e n t , w h i l e t h e h y d r o p h i l i c p r o t e i n s bound l i t t l e o r no d e s o x y c h o l a t e o r T r i t o n X-100. These w o r k e r s c o n c l u d e d t h a t b o n d i n g was p r i m a r i l y h y d r o p h o b i c and h y d r o p h o b i c r e g i o n s on t h e p r o t e i n m o l e c u l e s were i n v o l v e d . 10. A f f i n i t y p a r t i t i o n : A l b e r t s s o n (1971) p r e s e n t e d a p a r t i t i o n method f o r the 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 m a c r o m o l e c u l e s and p r o t e i n s . The b a s i s f o r s e p a r a t i o n i s t h e s e l e c t i v e d i s t r i b u t i o n o f s u b s t a n c e s between th e two-phase system. F o r s o l u b l e s u b s t a n c e s , d i s t r i b u t i o n t a k e s p l a c e m a i n l y between t h e two b u l k p h a s e s , and t h e p a r t i t i o n i s c h a r a c t e r i z e d by the p a r t i t i o n c o e f f i c i e n t , K. I d e a l l y , K i s i n d e p e n d e n t o f c o n c e n t r a t i o n and a l s o i n d e p e n d e n t o f t h e volume r a t i o o f t h e p h a s e s . I t i s m a i n l y a f u n c t i o n o f t h e p r o p e r t i e s o f t h e two p h a s e s , t h e p a r t i t i o n e d s u b s t a n c e and t e m p e r a t u r e . D i s t r i b u t i o n o f t h e m a c r o m o l e c u l e s and p a r t i c l e s i s i n f l u e n c e d by f a c t o r s such as m o l e c u l a r w e i g h t o f t h e p o l y m e r s , concen-t r a t i o n o f t h e p o l y m e r s , i o n i c c o m p o s i t i o n o f t h e phase s y s t e m , n e t c h a r g e o f t h e p a r t i c l e s , a f f i n i t y o f t h e compounds t o t h e two p h a s e s , phase volume r a t i o and t h e q u a n t i t y o f p r o t e i n added. When s o l u b l e s u b s t a n c e s d i s s o l v e i n t h e phase system t h e y p a r t i t i o n i n a r e p r o d u c i b l e manner. The p a r t i t i o n c o e f f i c i e n t depends on s i z e , c h a r g e , c o n f o r m a t i o n and p r o b a b l y on t h e k i n d of groups exposed t o t h e p h a s e s . The s i z e e f f e c t i s s u c h t h a t s m a l l m o l e c u l e s t e n d t o p a r t i t i o n e v e n l y between t h e phases w h i l e l a r g e m o l e c u l e s c o n c e n t r a t e i n one o f t h e p h a s e s , w h i c h depends on f a c t o r s o t h e r t h a n s i z e . Shanbhag and A x e l s s o n (1975) e s t i m a t e d t h e e x t e n t o f h y d r o p h o b i c i n t e r a c t i o n s u s i n g t h e m o d i f i e d p a r t i t i o n method o f A l b e r t s s o n ( 1 9 7 1 ) . The method was based on p a r t i t i o n o f p r o t e i n s i n an aqueous two-phase s y s t e m c o n t a i n i n g d e x t r a n , p o l y e t h y l e n e g l y c o l and p o l y e t h y l e n e g l y c o l p a l m i t a t e . The p a r t i t i o n was measured under c o n d i t i o n s where c o n t r i b u t i o n s f r o m 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 were e l i m i n a t e d . W i t h t h i s t e c h n i q u e t h e y c o u l d r a n k p r o t e i n s based on t h e degree o f t h e i r i n t e r a c t i o n s w i t h t h e p a l m i t a t e group. These w o r k e r s s u g g e s t e d t h a t t h e a f f i n i t y o f p r o t e i n s toward t h e p a l m i t a t e e s t e r o f p o l y e t h y l e n e g l y c o l can d e t e r m i n e t h e h y d r o p h o b i c i t i e s o f t h e s e p r o t e i n s . 31 MATERIALS AND METHODS Materials: Albumin Bovine No.A-7511 essentially fatty acid free, cytochrome C No. C-4381 from Candida krusei-type VII, conalbumin No. C-0755 type I from chicken egg white,a -chyraotrypsin No. C-4129 from bovine pancreas type II-salt free, y-globulin BG-II-bovine Cohn fraction II approximately 9 9 %Y , B -lactoglobulin No. L-6879 from milk (contains B-lactoglobulins A and B and approximately 2% NaCl), Trypsin, pancreatic type II crude, were purchased from Sigma Chemical Company, St. Louis, Mo. Ovalbumin 17268, lot # 24427-A was from ICN Pharmaceuticals Inc., Cleveland, Ohio. Lysozyme, salt free 11.800, was purchased from Worthington Biochemical Corporation, Freehold, N.J. Myoglobin 47592 (horse skeletal muscle), B grade 98% pure, were obtained from Calbiochem, Sah Diego, Calif. Cyanogen bromide and 4-phenyl-butylamine (PBA) were purchased from Aldrich Chemical Company.Inc., Milwaukee, Wis.. Ethylene glycol was "Baker analysed" from J.T. Baker Chemical Co., Phillipsburg, N.J. Sephadex G-150, Sepharose 4B and 3 Dextran T70 (M^ = 70 x 10 ) were purchased from Pharmacia Fine Chemicals, Sweden. Polyethylene glycol was obtained from Applied Science Laboratories Inc., State College, Penna. Palmitoyl chloride, boron trifluoride ethylether, hexylalcohol and the n-alkylamines were from Eastman Organic Chemicals, Rochester, N.Y. Octanol was from Matheson, Coleman and Bell, Norwood, Ohio. Epichlorohydrin, dioxane, corn o i l , ethylene diamine and oleic acid were from Fisher Scientific Company, Fair Lawn, N.J. Butyl.alcohol was from American Scientific & Chemicals, Seattle, WA 98199. Biobeads SM-2 (20-50 mesh) was purchased from B i o - r a d L a b o r a t o r i e s , Richmond, C a l i f . P o l y v i n y l p y r r o l i d o n e K-30 No. 203 was o b t a i n e d from Pope S c i e n t i f i c I n c . , Menomonee F a l l s , Wis. T r i e t h y l a m i n e AC-9421 was p u r c h a s e d from Anachemia C h e m i c a l s L t d . , Mo. Methods: 1. A c t i v a t i o n o f Sepharose 4B w i t h cyanogen bromide: I n a w e l l v e n t i l a t e d hood 50 g o f Sepharose 4B was g e n t l y s t i r r e d w i t h 50 ml w a t e r and t o t h i s m i x t u r e 5 g of f i n e l y d i v i d e d cyanogen bromide was added a l l a t once. S i x N sodium h y d r o x i d e was used t o m a i n t a i n t h e pH a t 11. Temperature was m a i n t a i n e d a t 20°C by a d d i n g p i e c e s o f i c e as needed. When t h e r e was no f u r t h e r change i n pH ( p r o t o n r e l e a s e ) , a l a r g e amount of i c e was r a p i d l y added t o t h e s u s p e n s i o n and t h e r e a c t i o n m i x t u r e was q u i c k l y t r a n s f e r r e d t o a Buchner f u n n e l and e x t e n s i v e l y washed w i t h 0.1 M sodium b i c a r b o n a t e b u f f e r (pH 9.5) under s u c t i o n ( C u a t r e c a s a s , 1970). 2. C o u p l i n g amines t o Sepharose 4B: F i f t y ml o f c o l d 0.1 M sodium b i c a r b o n a t e (pH 9.5) c o n t a i n i n g 10 m l amine was a d j u s t e d t o pH 10 w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d and added t o the g e l i n t h e Buchner f u n n e l . The g e l was i m m e d i a t e l y mixed w i t h a g l a s s r o d , t r a n s f e r r e d t o a b e a k e r c o n t a i n i n g a m a g n e t i c s t i r r i n g b a r and g e n t l y s t i r r e d a t 4°C f o r 20 h r s . The g e l was t h e n t h o r o u g h l y washed w i t h b u f f e r b e f o r e p a c k i n g . D u r i n g the p r o c e d u r e f o r c o u p l i n g o c t y l a m i n e t o the a c t i v a t e d g e l , a p r e c i p i t a t e formed a f t e r t h e a d d i t i o n o f h y d r o c h l o r i c a c i d t o a d j u s t t h e pH t o 10.0. T h i s p r e c i p i t a t e was formed even i n t h e p r e s e n c e of d i o x a n e o r d i m e t h y l formamide, w h i c h were employed t o keep t h e amine i n s o l u t i o n . When f o r m i c a c i d was used i n s t e a d o f h y d r o c h l o r i c a c i d t h e c o u p l i n g r e a c t i o n t o o k p l a c e s u c c e s s f u l l y . 3. C o u p l i n g o f 4 - p h e n y l b u t y l a m i n e (PBA) t o Sepharose: A c t i v a t e d Sepharose 4B was i m m e d i a t e l y t r a n s f e r r e d t o a b e a k e r c o n t a i n i n g 10 ml o f 0.1 M sodium b i c a r b o n a t e (pH 9.5), 20 m l d i m e t h y l f o r m a m i d e : w a t e r V/V ( 5 0 : 5 0 ) , and 6 m l o f PBA. A f t e r t h e pH was a d j u s t e d t o 9.5 w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d , t h e m i x t u r e was s t i r r e d f o r 20 h o u r s a t 4°C. The s u b s t i t u t e d g e l was t h e n washed w i t h 100 m l o f d i m e t h y l -formamide:water V/V ( 5 0 : 5 0 ) . Washing was c o n t i n u e d w i t h c o l d w a t e r f o l l o w e d by 0.1 M sodium b i c a r b o n a t e b u f f e r (pH 9.5) and t h e n 0.05 M T r i s - h y d r o c h l o r i c a c i d b u f f e r (pH 8.0). 4. C o u p l i n g o f o l e i c a c i d t o Sepharose: A m i n o e t h y l a m i n o -Sepharose 4B was p r e p a r e d t h r o u g h c o u p l i n g of e t h y l e n e d i a m i n e t o t h e a c t i v a t e d Sepharose 4B a c c o r d i n g t o p r o c e d u r e ( 2 ) . The s u b s t i t u t e d g e l was aged f o r 5 days ( P e t e r s e_t a l _ . , 1973) . A s u s p e n s i o n o f 28.5 ml o l e i c a c i d i n 48 ml w a t e r was s a p o n i f i e d a t pH w i t h 6 N sodium h y d r o x i d e s o l u t i o n w h i l e s t i r r i n g v i g o r o u s l y . To t h i s soap, 50 ml o f a m i n o e t h y l a m i n o - S e p h a r o s e 4B and 1.5 g l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) c a r b o d i i m i d e were added and t h e m i x t u r e s t i r r e d a t 37°C f o r 3 d a y s . U n r e a c t e d amino groups were b l o c k e d by a c e t y l a t i o n w i t h a c e t i c a n h y d r i d e a t pH 7, 0°C, f o r one h o u r . The g e l was t h e n t h o r o u g h l y washed w i t h w a t e r b e f o r e p a c k i n g . 5. Chromatography on a l k y l a m i n o - S e p h a r o s e s : Chromatography was c a r r i e d out i n a column of 150 mm l e n g t h and 10 mm d i a m e t e r packed w i t h 34 20 ml a l k y l a m i n o - S e p h a r o s e . The p r o t e i n s were e l u t e d w i t h 0.05 M T r i s - h y d r o c h l o r i c a c i d , pH 8.0. Sodium c h l o r i d e (0.1 M) and 50% e t h y l e n e g l y c o l were s u c c e s s i v e l y added to the b u f f e r . One-ml f r a c t i o n s were c o l l e c t e d w i t h an I s c o Model 326 F r a c t i o n C o l l e c t o r . The absorbances were measured a t 280 nm w i t h an Unicam SP 800 Spectrophotometer. 6. S y n t h e s i s o f g l y c i d y l e t h e r s : B u t y l - , h e x y l - and o c t y l -g l y c i d y l e t h e r s were p r e p a r e d by r e a c t i o n o f the r e s p e c t i v e a l c o h o l s w i t h e p i c h l o r h y d r i n i n the p r e s e n c e o f b o r o n t r i f l u o r i d e (BF^) e t h y l e t h e r a t e as a c a t a l y s t ( U l b r i c h et. a l . - , 1963) . To a t h r e e neck d i s t i l l i n g f l a s k , equipped w i t h thermometer, condensor, s e p a r a t o r y f u n n e l and a s t i r r i n g b a r , c o n t a i n i n g 37 g of a l c o h o l , 2 ml of b o r o n t r i -f l u o r i d e (1.5% i n d i e t h y l e t h e r ) was added and the m i x t u r e warmed to 55°C i n a water b a t h . While s t i r r i n g the m i x t u r e i n t e n s i v e l y and m a i n t a i n i n g the temperature c o n s t a n t , 46.6 g e p i c h l o r h y d r i n was added dropwise over a two hour p e r i o d . The m i x t u r e was then l e f t o v e r n i g h t . While s t i r r i n g c o n s t a n t l y and m a i n t a i n i n g the temperature a t 20-25°C, a s o l u t i o n of 50 g sodium h y d r o x i d e i n 50 ml water was added dropwise w i t h i n a two hour p e r i o d . The m i x t u r e was f i l t e r e d through No. 1 Whatman f i l t e r paper. The f i l t r a t e was washed w i t h water i n a s e p a r a t o r y f u n n e l , d r i e d w i t h magnesium s u l f a t e , then f i l t e r e d a g a i n and vacuum d i s t i l l e d . The g l y c i d y l e t h e r s can be r e p r e s e n t e d by the g e n e r a l f o r m u l a : C C - CH. - 0 - CH. - CH„ - (CH-) - CH i. I / n 3 H. 2 H 7. Treatment of Sepharose 4B: Sepharose 4B (50 g)was washed successively with a series of solvents of decreasing polarity to exchange the water in the gel with dioxane. The washing procedure suggested by Hjerten et_ a l . (1974) was performed as follows: 1 - Once with 50 ml water-dioxane (4:1) 2 - Once with 50 ml water-dioxane (3:2) 3 - Once with 50 ml water-dioxane (2:3) 4 - Once with 50 ml water-dioxane (1:4) 5 - Seven times with 50 ml dioxane. After exchanging the water i n the Sepharose with dioxane, the coupling with the glycidyl ether was carried out. 8. Coupling glycidyl ethers to Sepharose: The coupling procedure introduced by Ellingboe et. a l . (1970) and modified by Hjerten et a l . (1974) was used to attach glycidyl ethers to the polysaccharide matrix. Fifty ml of gel was transferred to a round-bottomed reaction vessel equipped with a s t i r r e r . F i f t y ml dioxane and 1 ml of a 48% solution of borontrifluoride etherate in diethyl ether were then added. The mixture was gently stirred for 5 minutes, then a mixture of one ml glycidyl ether and 5 ml dioxane was added dropwise from a separatory funnel within a 40 minute period while stirring constantly at room temperature. Afterwards, the gel was washed with the same ratios of water-dioxane mixtures but in the reverse order and f i n a l l y was washed with 1 l i t e r of water. The alkylepoxy derivatives produced have the following formula: 0 Seph)- OH + CH 'CH - CH 2 - OR OH Seph)-0 - CH 2 - CH - CH 2 - OR where R r e p r e s e n t s the a l k y l group. These g e l s were c a l l e d a l k y l -epoxy-Sepharoses. 9. D e t e r m i n a t i o n o f the d r y weight o f Sepharose g e l s u s p e n s i o n s : Two ml g e l s u s p e n s i o n , w e l l homogenized by s t i r r i n g , were t r a n s f e r r e d to a g l a s s f i l t e r f u n n e l . The g e l s were deh y d r a t e d through s u c c e s s i v e washings w i t h acetone and f i n a l l y d r i e d to c o n s t a n t weight a t 90°C. The d r y we i g h t s were determined. 10. Hydrophobic column chromatography: G l a s s columns from Pharmacia F i n e Chemicals ( K 1 6 ( 2 6 ) , 16 ± 0.02mm dia m e t e r , 200mm l e n g t h ) were used f o r h y d r o p h o b i c chromatography. T h i r t y g o f a l k y l e p o x y -Sepharoses were packed i n the column. They were e q u i l i b r a t e d w i t h 0.002 M sodium phosphate b u f f e r (pH 6.9) c o n t a i n i n g 2 M sodium 2 c h l o r i d e . P r o t e i n s were e l u t e d a t a f l o w r a t e o f 3 ml/hr/cm a t room temperature. The e l u t i o n p r o f i l e s were mo n i t o r e d c o n t i n u o u s l y a t 280 nm u s i n g a LKB Bromma 8300 UVICORD I I . One ml f r a c t i o n s were c o l l e c t e d . The g e l was washed w i t h t h r e e bed volumes of b u f f e r b e f o r e each a p p l i c a t i o n o f sample. Hydrophobic a d s o r p t i o n o f p r o t e i n s to these columns was expres as t h e i r r e t e n t i o n c o e f f i c i e n t s . T h i s v a l u e was c a l c u l a t e d from R.C. Ve - Vt g dry g e l where Ve = e l u t i o n volume V t = t o t a l v o l u m e g d r y g e l = gram d r y g e l packed i n the column. 11. G e l chromatography w i t h d e t e r g e n t s : A column (2 x 35 cm) was packed w i t h Sephadex G-150. The b u f f e r employed was 0.025 M sodium p h o s p h a t e , pH 7.2, c o n t a i n i n g 0.05% T r i t o n X-100. P r o t e i n 2 samples (lOmg) were a p p l i e d t o t h e column. A f l o w r a t e o f 2ml/hr/cm was m a i n t a i n e d t h r o u g h o u t t h e a n a l y s i s . F r a c t i o n s o f 1.5 ml were c o l l e c t e d i n tube s w i t h an I s c o Model 326 f r a c t i o n c o l l e c t o r . The e l u t i o n p r o f i l e f o r t h e p r o t e i n - d e t e r g e n t complex was o b t a i n e d by m o n i t o r i n g t h e absorbance a t 280 nm w i t h a Unicam SP 800 S p e c t r o -p hotometer. 12. Removal o f the T r i t o n X-100: A column ( 1 x 8 cm) c o n t a i n i n g 5g o f m o i s t SM-2 B i o b e a d s was e q u i l i b r a t e d w i t h lOmM p o t a s s i u m phosphate (pH 7.2). The p r o t e i n - d e t e r g e n t complex c o l l e c t e d from Sephadex G-150 was c o n c e n t r a t e d by d i a l y s i s a g a i n s t 20% aqueous s o l u t i o n s o f p o l y v i n y l -p y r r o l i d o n e (PVP) t h e n a p p l i e d t o the B i o b e a d column. F o l l o w i n g e l u t i o n o f p r o t e i n , the beads were washed w i t h 50% e t h a n o l p l u s 10% e t h y l e n e g l y c o l i n lOmM p o t a s s i u m phosphate b u f f e r (pH 7.2) t o remove T r i t o n X-100 bound t o t h e beads. 13. Q u a n t i t a t i o n o f T r i t o n X-100: The method o f Garewal (1973) was u s e d . Ammonium c o b a l t o t h i o c y a n a t e was p r e p a r e d by d i s s o l v i n g 17.8 g of ammonium t h i o c y a n a t e and 2.8g o f c o b a l t n i t r a t e h e x a h y d r a t e i n w a t e r . The r e a g e n t was d i l u t e d t o 100ml w i t h d i s t i l l e d w a t e r . A 0.5mg/ml s t a n d a r d s o l u t i o n o f T r i t o n X-100 i n 50% e t h a n o l c o n t a i n i n g 10% e t h y l e n e g l y c o l was p r e p a r e d t o c o n s t r u c t the s t a n d a r d c u r v e . An a p p r o p r i a t e amount o f T r i t o n X-100 s t a n d a r d s o l u t i o n was t r a n s f e r r e d t o a t e s t tube and the volume was made t o 1 ml w i t h 50% e t h a n o l . C o n c e n t r a t i o n o f t h e d e t e r g e n t ranged from 50 t o 500 ygm. The c o n t e n t s of t h e tube s were mi x e d , t h e n 0.50 ml o f t h e ammonium c o b a l t o t h i o c y a n a t e r e a g e n t was added and t h e m i x t u r e was a l l o w e d t o s t a n d f o r f i v e m i n u t e s a t room t e m p e r a t u r e . 1.5 ml o f e t h y l e n e d i c h l o r i d e was t h e n added and the c o n t e n t s were mixed f o r 2 min w i t h a v o r t e x s h a k e r . The samples were t h e n c e n t r i f u g e d a t 1500 x G and t h e b o t t o m phase ( e t h y l e n e d i c h l o r i d e ) was removed w i t h a P a s t e u r p i p e t t e f o r t h e s p e c t r o p h o t o m e t r i c a n a l y s i s . The d i f f e r e n c e i n abs o r b a n c e a t 622 nm and 687 nm was r e c o r d e d w i t h a Unicam SP 800 S p e c t r o p h o t o m e t e r . The T r i t o n X-100 f r a c t i o n e l u t e d f r o m the B i o b e a d column was c o l l e c t e d i n a 5 ml v o l u m e t r i c f l a s k . I t was made up t o volume w i t h 50% e t h a n o l and 1 ml o f t h i s s o l u t i o n was used. The a n a l y s i s was c a r r i e d out as d e s c r i b e d i n p r e p a r a t i o n o f samples f o r the s t a n d a r d c u r v e . 14. H y d r o p h o b i c p a r t i t i o n : To an a p p r o p r i a t e amount o f t h e p r o t e i n (5-15 mg), i n 10 ml c e n t r i f u g e tubes ( c o n i c a l , p l a i n , P y r e x b r a n d ) , b u f f e r (100 mM K^SO^ and 2 mM K P h o s p h a t e , pH 7.1) was added t o make up t h e t o t a l w e i g h t o f 200 mg. The p r o t e i n was s o l u b i l i z e d by s h a k i n g w i t h t h e v o r t e x s h a k e r . Then 1.6 g o f 40% p o l y e t h y l e n e g l y c o l o r i t s p a l m i t a t e e s t e r and 3.2 g o f 20% d e x t r a n T70 were added. The c o n t e n t s o f each tube were mixed f o r 2 m i n u t e s on the v o r t e x s h a k e r . A f t e r warming a t 40°C i n a w a t e r b a t h f o r 10 m i n u t e s , the tubes were 39 c e n t r i f u g e d i n a c l i n i c a l c e n t r i f u g e at 1500x G f o r 10 minutes. An a l i q u o t of 2g was withdrawn from each phase and d i l u t e d w i t h water except f o r the polyethylene g l y c o l p a l m i t a t e phase which Was d i l u t e d w i t h a s o l u t i o n of 0.02% SDS i n 0.1 N sodium hydroxide. A l l d i l u t i o n s were made on a weight b a s i s . The absorbance of p r o t e i n was measured at 280 nm against a s o l u t i o n of the corresponding phase from a system c o n t a i n i n g no p r o t e i n handled i n an i d e n t i c a l manner. D i l u t i o n s from polyethylene g l y c o l p a l m i t a t e phases were warmed i n a 90°C water bath f o r 3 minutes before the reading was taken. The p a r t i t i o n c o e f f i c i e n t of p r o t e i n was determined from K = CU/CL where CU and CL are concentrations of the p r o t e i n at e q u i l i b r i u m i n the upper phase and lower phase r e s p e c t i v e l y . P a r t i t i o n c o e f f i c i e n t of a p r o t e i n i n polyethylene g l y c o l p a l m i t a t e and polyethylene g l y c o l systems were designated as and 1^ , r e s p e c t i v e l y . A l o g K was c a l c u l a t e d from A l o g K = l o g - l o g and was used as an index of hydrophobicity of the p r o t e i n s . 15. Synthesis of polyethylene g l y c o l p a l m i t a t e : Two hundred g polyethylene g l y c o l was d i s s o l v e d i n 1200ml toluene and 200ml of toluene was d i s t i l l e d out of the s o l u t i o n to remove traces of moisture. Four g t r i e t h y l a m i n e p u r i f i e d by d i s t i l l a t i o n over p h t h a l i c anhydride was added. Then, a s o l u t i o n of 5g p a l m i t o y l c h l o r i d e i n 50ml toluene was added dropwise under continuous s t i r r i n g . The mixture was gently r e f l u x e d f o r 15 minutes and then f i l t e r e d over Whatman No. 1 f i l t e r paper. Upon c o o l i n g to 3°C the polyethylene g l y c o l p a l m i t a t e p r e c i p i t a t e d and was c o l l e c t e d by vacuum f i l t r a t i o n . The p r o d u c t was r e c r y s t a l i z e d t w i c e f r o m a b s o l u t e e t h a n o l and has t h e f o l l o w i n g f o r m u l a : 0 II CH„ - ( C H J . . - C - 0 - CH - CH„(-.0 - CH 0 - C H j - OH P o l y e t h y l e n e g l y c o l 16. I n t e r f a c i a l t e n s i o n measurements: I n t e r f a c i a l t e n s i o n s were measured a t 25°C w i t h a F i s h e r S u r f a c e Tensiomat Model 21. The i n s t r u m e n t was c a l i b r a t e d a t 49 dynes/cm w i t h the a i d o f 600mg w e i g h t a c c o r d i n g t o the m a n u f a c t u r e r ' s recommendation. B e f o r e a n a l y s i s , t h e t e n s i o m e t e r r i n g was t h o r o u g h l y washed w i t h benzene and t h e n a c e t o n e f o l l o w e d by f l a m i n g o v e r an a l c o h o l b u r n e r . The r i n g was t h e n immersed i n t h e 0.2% p r o t e i n s o l u t i o n s and t h e c o r n o i l phase was s l o w l y added. A f t e r a l l o w i n g the m i x t u r e t o e q u i l i b r a t e f o r 2 m i n u t e s , t h r e e c o n s e c u t i v e r e a d i n g s were t a k e n a t 2 minu t e i n t e r v a l s . RESULTS AND DISCUSSION 1. Chromatography on Sepharoses s u b s t i t u t e d w i t h a l k y l a m i n e s : Chromatography on t h e s e g e l s c l a s s i f i e d p r o t e i n s i n t o t h r e e d i f f e r e n t g r o u p s : a. P r o t e i n s w h i c h had no a f f i n i t y towards t h e bed m a t e r i a l and were e l u t e d w i t h i n t h e t o t a l bed volume when e l u t e d w i t h 0.05 M T r i s - H C l b u f f e r , pH 8.0. b. P r o t e i n s w h i c h were bound t o the g e l m a t e r i a l , most p r o b a b l y by means o f e l e c t r o s t a t i c a t t r a c t i o n s between p r o t e i n and p r o t o n a t e d m a t r i x . These m o l e c u l e s were r e l e a s e d when t h e i o n i c s t r e n g t h o f t h e e l u e n t was i n c r e a s e d . c. P r o t e i n s w h i c h were t i g h t l y bound t o t h e l o n g c h a i n o f h y d r o c a r b o n l i g a n d and were n o t deso r b e d even a t h i g h i o n i c s t r e n g t h s . These m o l e c u l e s p r o b a b l y i n t e r a c t e d w i t h the l i g a n d by means o f h y d r o p h o b i c and i o n i c a s s o c i a t i o n s . These p r o t e i n s were n o t r e l e a s e d u n t i l p o l a r i t y r e d u c i n g agents s u c h as e t h y l e n e g l y c o l were i n c l u d e d i n the b u f f e r . The e l u t i o n p a t t e r n s o f some p r o t e i n s on a l k y l a m i n o -Sepharoses o f i n c r e a s i n g c h a i n l e n g t h a r e shown i n F i g . 1. I t s u g g e s t s t h a t lysozyme d i d n o t i n t e r a c t w i t h t h e h y d r o p h o b i c l i g a n d . B o v i n e serum a l b u m i n , o v a l b u m i n and g - l a c t o g l o b u l i n were a d s o r b e d on the g e l m a t e r i a l a p p a r e n t l y by means 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 as w e l l as by h y d r o p h o b i c b o n d i n g s . I t i s o b v i o u s t h a t t h e b i n d i n g p r o p e r t i e s o f t h e s e g e l m a t e r i a l s r e q u i r e t h a t t h e p r o t e i n and t h e a d s o r b e n t a r e o p p o s i t e l y 42 F i g u r e 1 - B e h a v i o u r o f p r o t e i n s o n 1 5 x1 cm c o l u m n s o f b u t y l - a n d o c t y l a m i n o - S e p h a r o s e 4 B . B u f f e r , 0 . 0 5 M T r i s h y d r o c h l o r i c a c i d (pH 8 . 0 ) . A r r o w s i n d i c a t e a d d i t i o n o f 0 . 1 M s o d i u m c h l o r i d e a n d 5 0% e t h y l e n e g l y c o l (EG) i n t h e b u f f e r . A b s o r b a n c e ( 2 8 0 nm) B - l a c t o - Y - g l o b u l i n O v a l b u m i n L y s o z y m e B o v i n e S e r u m g l o b u l i n A l b u m i n O 't—• O t—' ° i—' o c h a r g e d . T h e p r e d o m i n a n t r o l e o f e l e c t r o s t a t i c f o r c e s i n t h e b i n d i n g o f p r o t e i n s b y a l k y l a m i n o - S e p h a r o s e s i s made c l e a r b y t h e o b s e r v a t i o n t h a t t h e b i n d i n g , a t l e a s t t o t h e a d s o r b e n t s w i t h t h e s m a l l e r h y d r o -p h o b i c g r o u p s ( b u t y l a n d h e x y l ) , w a s r e a d i l y r e v e r s e d b y i n c l u s i o n o f s a l t i n t h e b u f f e r ( F i g . 1). I n v o l v e m e n t o f h y d r o p h o b i c i n t e r a c t i o n s s e e m e d t o b e m i n o r i n t h e c a s e o f o v a l b u m i n . T h i s p r o t e i n w a s d e s o r b e d , f r o m e v e n h i g h l y h y d r o p h o b i c g e l s , b y i n c r e a s i n g i o n i c s t r e n g t h . On t h e o t h e r h a n d , a m a j o r c o n t r i b u t i o n o f h y d r o p h o b i c b o n d s t o t h e a d s o r p t i o n o f b o v i n e s e r u m a l b u m i n w a s d e m o n s t r a t e d i n t h e c a s e o f h i g h l y h y d r o p h o b i c c o l u m n s . T h i s p r o t e i n w a s n o t r e l e a s e d f r o m t h e o c t y l a m i n o - S e p h a r o s e c o l u m n b y s i m p l y i n c r e a s i n g t h e i o n i c s t r e n g t h , t h u s s u g g e s t i n g t h a t t h e h y d r o p h o b i c b o n d s a r e r e s p o n s i b l e f o r t h i s i n t e r a c t i o n . F u r t h e r m o r e , b o v i n e s e r u m a l b u m i n w a s n o t e l u t e d b y d e c r e a s i n g t h e p o l a r i t y a l o n e . A c o n c o m i t a n t i n c r e a s e i n i o n i c s t r e n g t h w a s n e e d e d f o r d e s o r p t i o n o f t h e p r o t e i n t o o c c u r . I n t e r f e r e n c e o f i o n i c i n t e r a c t i o n s i n c h r o m a t o g r a p h y o n a l k y l a m i n o - S e p h a r o s e s r e l a t e s t o t h e m e t h o d o f t h e p r e p a r a t i o n o f t h e s e g e l s . P o s i t i v e c h a r g e s a r e i n t r o d u c e d t h r o u g h c y a n o g e n b r o m i d e a c t i v a t i o n a n d mono a m i n e s u b s t i t u t i o n . H e n c e , t h e e l e c t r o s t a t i c a t t r a c t i v e o r r e p u l s i v e f o r c e s c a n e x e r t g r e a t p o s i t i v e o r n e g a t i v e c o n t r i b u t i o n s t o t h e f o r m a t i o n o f t h e h y d r o p h o b i c b o n d s . T h e p r o t o n a t e d a m i n o g r o u p o n t h e g e l m a t r i x c a n p r e v e n t h y d r o p h o b i c b o n d f o r m a t i o n o f p o s i t i v e l y c h a r g e d p r o t e i n d u e t o e l e c t r o s t a t i c r e p u l s i o n s . T h i s 45 r e p u l s i o n p r o v i d e s l a r g e d i s t a n c e s b e t w e e n n o n p o l a r g r o u p s , t h u s l e a v i n g t h e m u n a b l e t o i n t e r a c t . T h e p o s i t i v e l y c h a r g e d p r o t e i n s w e r e a l l e l u t e d a t t h e f i r s t s t a g e o f e l u t i o n . C h r o m a t o g r a p h y o n b u t y l - , h e x y l - a n d o c t y l a m i n o - S e p h a r o s e s 4B ( F i g . 1 ) s u g g e s t s t h a t b o t h e l e c t r o s t a t i c a t t r a c t i o n s a n d r e p u l s i o n s i n t e r f e r e w i t h t h e b i n d i n g . L y s o z y m e w a s r e j e c t e d w h i l e o v a l b u m i n w a s a t t r a c t e d t o t h e g e l m a t e r i a l . T h i s i s p r o b a b l y d u e t o d i f f e r e n c e s i n t h e i r c h a r g e s . I n b i n d i n g o f n e g a t i v e l y c h a r g e d p r o t e i n s l i k e b o v i n e s e r u m a l b u m i n , 3 - l a c t o g l o b u l i n a n d o v a l b u m i n t o t h e s e g e l s , h y d r o p h o b i c i n t e r a c t i o n s s e e m t o b e a i d e d b y e l e c t r o -s t a t i c f o r c e s . I n c r e a s i n g t h e i o n i c s t r e n g t h o f t h e b u f f e r a t t h e s e c o n d s t a g e o f e l u t i o n c a u s e d d e s o r p t i o n o f o v a l b u m i n a n d 3 - l a c t o -g l o b u l i n . T h e s e p r o t e i n s w e r e n o t r e l e a s e d w h e n 5 0 % e t h y l e n e g l y c o l w a s a d d e d t o t h e o r i g i n a l b u f f e r . H o w e v e r , t h e y w e r e e l u t e d a f t e r a d d i t i o n o f s o d i u m c h l o r i d e t o 0 . 1 M c o n c e n t r a t i o n . I n t h e c a s e o f m o r e a p o l a r p r o t e i n s , l i k e b o v i n e s e r u m a l b u m i n , h y d r o p h o b i c b o n d s b e c o m e d o m i n a n t . A l t h o u g h t h i s p r o t e i n i s n e g a t i v e l y c h a r g e d a n d i s p r e s u m a b l y b o u n d p a r t l y t h r o u g h i o n i c i n t e r a c t i o n s , t h e c o n t r i b u t i o n o f h y d r o p h o b i c b o n d s i s i n t e n s i f i e d w h e n S e p h a r o s e h a s a l o n g e r a l k y l c h a i n a t t a c h e d t o i t . U n d e r t h e s e c i r c u m s t a n c e s , t h e p r o t e i n w a s n o t e l u t e d s i m p l y b y i n c r e a s i n g t h e i o n i c s t r e n g t h . E l u t i o n t o o k p l a c e o n l y a f t e r i n c l u d i n g 5 0% e t h y l e n e g l y c o l a n d 0 . 1 M s o d i u m c h l o r i d e i n t h e T r i s - H C l b u f f e r . T h e r e f o r e , i t a p p e a r s t h a t t h e s t r o n g b i n d i n g o f p r o t e i n t o t h i s t y p e o f a d s o r b e n t d e p e n d s , t o a l a r g e d e g r e e , o n a c o o p e r a t i v e a n d p r e s u m a b l y m u t u a l l y r e i n f o r c i n g e f f e c t o f h y d r o p h o b i c a n d e l e c t r o -s t a t i c f o r c e s . No d e t e c t a b l e a m o u n t o f t h e p r o t e i n w a s r e l e a s e d f r o m i t s c o m p l e x w i t h t h e a d s o r b e n t w h e n t h e i n t e r a c t e d b e d w a s w a s h e d e x t e n s i v e l y w i t h t h e T r i s - H C l b u f f e r . D i s r u p t i o n o f t h e c o m p l e x o f Y - g l o b u l i n o r b o v i n e s e r u m a l b u m i n w i t h t h e o c t y l a m i n o - S e p h a r o s e AB r e q u i r e d a n i n c r e a s e i n t h e i o n i c s t r e n g t h a s w e l l a s t h e p r e s e n c e o f p o l a r i t y r e d u c i n g a g e n t s . E l u t i o n o f o v a l b u m i n a n d 3 - l a c t o b l o b u l i n t h a t o c c u r r e d u p o n i n c r e a s i n g t h e i o n i c s t r e n g t h d o e s n o t m e a n t h a t t h e s e p r o t e i n s a r e i n c a p a b l e o f f o r m i n g h y d r o p h o b i c b o n d s ; h y d r o p h o b i c i t y o f t h e s e p r o t e i n s m u s t h a v e some e f f e c t . I n a c o m p l i c a t e d s y s t e m l i k e t h e p r o t e i n m o l e c u l e , t h e c o n f o r m a t i o n a n d a g g r e g a t i o n s t a t e s o f w h i c h a r e s t r o n g l y d e p e n d e n t o n i o n i c i n t e r a c t i o n s , i t i s q u i t e c o n c e i v a b l e t h a t t h e a v a i l a b i l i t y a n d s i z e o f h y d r o p h o b i c p o c k e t s may d e p e n d u p o n i o n i c s t r e n g t h , p H a n d e v e n o n t h e n a t u r e o f t h e i o n s p r e s e n t . I t i s q u i t e p o s s i b l e t h a t h y d r o p h o b i c i n t e r a c t i o n s a r e r e i n f o r c e d b y s t r o n g e r i o n i c f o r c e s . F o r m a t i o n o f c o o p e r a t i v e e l e c t r o s t a t i c a n d h y d r o p h o b i c i n t e r a c t i o n s c o u l d a l s o b e o b s e r v e d w h e n c h r o m a t o g r a p h y w a s p e r f o r m e d w i t h 4 - p h e n y l b u t y l a m i n e ( P B A ) s u b s t i t u t e d S e p h a r o s e 4B. B i n d i n g o f p r o t e i n s t o P B A - S e p h a r o s e may b e a s s u m e d t o o c c u r t h r o u g h i n t e r a c t i o n o f t h e h y d r o p h o b i c s i t e o f p r o t e i n w i t h t h e p h e n y l g r o u p o n t h e s u b s t i t u t e d l i g a n d . W i t h t h i s g e l , t h e r e s u l t s s u g g e s t e d t h a t t h i s t y p e o f i n t e r a c t i o n i s s i m i l a r t o o t h e r a l k y l a m i n o - S e p h a r o s e s . T h i s g e l r e t a i n e d t h e h y d r o p h o b i c p r o t e i n s b u t d i d n o t r e l e a s e t h e m w h e n w a s h e d w i t h T r i s - H C l b u f f e r . 47 1.0 h E f f l u e n t v o l u m e ( m l ) F i g u r e 2- B e h a v i o u r o f b o v i n e s e r u m a l b u m i n on S e p h a r o s e 4B ( a ) a n d 4 - p h e n y l b u t y l a m i n o - S e p h a r o s e 4B ( b ) . B u f f e r , 0.05 M T r i s h y d r o c h l o r i d e ( P H 8 . 0 ) . A r r o w s i n d i c a t e a d d i t i o n o f 0.1 M s o d i u m c h l o r i d e a n d 5 0 % e t h y l e n e g l y c o l i n t h e b u f f e r . 48 F i g . 2 shows the e l u t i o n p a t t e r n o f b o v i n e serum a l b u m i n on PBA-Sepharose 4B compared t o u n s u b s t i t u t e d Sepharose 4B. The s t r o n g i n t e r a c t i o n o f PBA w i t h serum a l b u m i n r e q u i r e d t he p r e s e n c e o f e t h y l e n e g l y c o l t o r e l e a s e t h e h y d r o p h o b i c p r o t e i n . y-globulin and BSA were bound s t r o n g l y t o t h i s g e l m a i n l y by h y d r o p h o b i c i n t e r a c t i o n s w h i l e o v a l b u m i n and 3 - l a c t o g l o b u l i n were r e l e a s e d a f t e r t h e i o n i c s t r e n g t h o f the b u f f e r was i n c r e a s e d t o 0.1 M. When a m i x t u r e o f 3 - l a c t o g l o b u l i n and y - g l o b u l i n was a p p l i e d t o a PBA-Sepharose column ( F i g . 3 ) , the p r o t e i n s were e l u t e d by b u f f e r s o f d i f f e r e n t c o m p o s i t i o n i n t o two d i s t i n c t i v e p e a k s . 3 - l a c t o g l o b u l i n was r e l e a s e d a f t e r i n c r e a s i n g t he i o n i c s t r e n g t h w i t h sodium c h l o r i d e t o 0.1 M, whereas y - g l o b u l i n remained bound under t h i s c o n d i t i o n and d e s o r b e d o n l y upon t h e a d d i t i o n o f 50% e t h y l e n e g l y c o l t o t h e b u f f e r . However, PBA s u b s t i t u t e d Sepharose was found u n s u i t a b l e f o r t h e d e t e r m i n a t i o n o f t h e e f f e c t i v e h y d r o p h o b i c i t y o f p r o t e i n s . Inhomogeneity o f t h e b i n d i n g s i t e s i n a l k y l a m i n o - S e p h a r o s e s o f t e n caused inhomogeneous a d s o r p t i o n and d e s o r p t i o n o f p r o t e i n s ; Thus p r o t e i n m o l e c u l e s bound t o b o t h e l e c t r o s t a t i c and h y d r o p h o b i c s i t e s . W h i l e t h e main p o r t i o n o f t h e p r o t e i n was d e s o r b e d a t e l e v a t e d i o n i c s t r e n g t h , some m o l e c u l e s remained bound, a p p a r e n t l y t o t h e h y d r o p h o b i c s i t e s on the g e l . These m o l e c u l e s were r e l e a s e d l a t e r when t h e h y d r o p h o b i c bonds were d e s t a b i l i z e d by e t h y l e n e g l y c o l . T h i s e f f e c t a ppeared as t h e f r a c t i o n a l e l u t i o n o f the p r o t e i n s ( F i g . 1 ) . The main f a c t o r r e s p o n s i b l e f o r t h i s b e h a v i o u r seems t o be in h o m o g e n e i t y o f t h e b i n d i n g s i t e s on t h e a d s o r b e n t w h i c h had b o t h i o n i c and h y d r o p h o b i c s i t e s a v a i l a b l e f o r the p r o t e i n m o l e c u l e . When a l a r g e amount o f p r o t e i n i s a p p l i e d t o t h e column, some o f t h e p r o t e i n 49 1.0 e c § 0 . 8 U c XI f-l in < 0 . 4 0 . 2 ^ N a C l E t h y l e n e Y g l y c o l 4 0 60 80 100 120 140 160 180 E f f l u e n t v o l u m e ( m l ) F i g u r e 3 - h l u t i o n o f a m i x t u r e o f 3 - l a c t o g l o b u l i n and y - g l o b u l i n f r o m p h e n y l b u t y l a m i n o _ S c p h a r o s e 4B b y s u b s e q u e n t a d d i t i o n o f 0 1 M s o d i u m c h l o r i d e a n d 50% e t h y l e n e g l y c o l t o b u f f e r B u f f e r 0 . 0 5 M T r i s h y d r o c h l o r i d e ( P H 8 . 0 ) . ' m o l e c u l e s b i n d t o t h e h y d r o p h o b i c s i t e s a n d some m o l e c u l e s b i n d t o t h e i o n i c s i t e s d e p e n d s o n t h e a v a i l a b i l i t y a n d p r o x i m i t y o f t h e i n t e r a c t i n g r e s i d u e s . T h e g e l s u r f a c e s h o u l d c o n t a i n t h e c e r t a i n s t r o n g b i n d i n g s i t e s w h i c h a r e o c c u p i e d f i r s t a n d i f m o r e p r o t e i n i s p r e s e n t , t h e y w o u l d t a k e s e v e r a l o t h e r s i t e s o f l o w e r a f f i n i t y f o r i n t e r a c t i o n . A s t h e p r o t e i n / a d s o r b e n t r a t i o i s d e c r e a s e d , b i n d i n g w o u l d b e c o m e s p e c i f i c d u e t o t h e l i m i t e d n u m b e r o f t h e m o s t f a v o r a b l e b i n d i n g s i t e s . R e d u c i n g t h e a m o u n t o f a p p l i e d p r o t e i n c a u s e s o n e t y p e o f s i t e o n t h e a d s o r b e n t t o p r e d o m i n a t e i n t h e b i n d i n g . L o w e r i n g t h e d e g r e e o f s u b s t i t u t i o n t o t h e p o i n t t h a t t h e d i s t a n c e b e t w e e n s u b s t i t u t e d g r o u p s i s l a r g e r t h a n t h e s i z e o f p r o t e i n m o l e c u l e i s a n o t h e r w a y t o e l i m i n a t e t h e s e k i n d s o f b i n d i n g s . U s e o f a n u n c h a r g e d a r m , a s w i l l b e d i s c u s s e d i n t h e s e c t i o n o n p r e p a r a t i o n o f a l k y l e p o x y S e p h a r o s e s , b a s i c a l l y m i n i m i z e s t h e p o s s i b i l i t y o f t h e s e k i n d s o f w e a k i n t e r a c t i o n s a n d p r o v i d e s c o n d i t i o n s f a v o r a b l e f o r p r o t e i n t o i n t e r a c t m a i n l y t h r o u g h h y d r o p h o b i c b o n d f o r m a t i o n . I n i d e a l h y d r o p h o b i c i n t e r a c t i o n c h r o m a t o g r a p h y , t h e a d s o r p t i o n s h o u l d b e e n t i r e l y d e p e n d e n t u p o n h y d r o p h o b i c a s s o c i a t i o n b e t w e e n t h e b e d a n d t h e s u b s t a n c e s t o b e a n a l y s e d . I n o r d e r t o i n c r e a s e t h e c o n t r i b u t i o n o f h y d r o p h o b i c i n t e r a c t i o n s , e l e c t r o s t a t i c a t t r a c t i o n s m u s t b e m i n i m i z e d . 2 . C h r o m a t o g r a p h y o n o l e i c - S e p h a r o s e 4 B : O l e i c a c i d w a s a n o t h e r h y d p h o b i c g r o u p w h i c h w a s c o u p l e d t o S e p h a r o s e 4B. I n t e r a c t i o n o f p r o t e i n w i t h o l e i c g r o u p s w a s t h o u g h t t o b e s i m i l a r t o p r o t e i n - l i p i d i n t e r a c t i o n s i n a s y s t e m s u c h a s a n e m u l s i o n i n t h a t o l e i c a n d s i m i l a r f a t t y a c i d s w i t h t h e i r h y d r o p h o b i c h y d r o c a r b o n c h a i n s c o n s t r u c t t h e m a j o r p a r t o f t h e l i p i d s t r u c t u r e . H o w e v e r , c h r o m a t o g r a p h y o n t h e o l e i c s u b s t i t u t e d S e p h a r o s e w a s u n s u c c e s s f u l a s a m e t h o d f o r d e t e r m i n a t i o n o f t h e e f f e c t i v e h y d r o p h o b i c i t y o f p r o t e i n s . T h i s w a s d u e t o t h e o c c u r r e n c e o f s t r o n g i n t e r a c t i o n s b e t w e e n t h e g e l a n d many o f t h e a p p l i e d p r o t e i n s . B o v i n e s e r u m a l b u m i n , y - g l o b u l i n , 3 - l a c t o g l o b u l i n a n d o v a l b u m i n i n t e r -a c t e d w i t h t h e b e d m a t e r i a l a n d r e m a i n e d i n t h e c o l u m n . F i f t y p e r c e n t e t h y l e n e g l y c o l i n 0.1 M N a C l w e r e u s e d i n v a i n t o e l u t e t h e p r o t e i n . 3. C h r o m a t o g r a p h y o n a l k y l e p o x y - S e p h a r o s e s : C o u p l i n g a l c o h o l s i n s t e a d o f a m i n e s t o t h e g e l m a t r i x p r o v i d e d n e u t r a l a d s o r b e n t s . I n t h i s s u b s t i t u t i o n p r o c e d u r e c y a n o g e n b r o m i d e a c t i v a t i o n w a s o m i t t e d a n d b e c a u s e a m i n e w a s n o t i n t r o d u c e d f o r t h e c o u p l i n g r e a c t i o n , t h e p r e p a r e d g e l s w e r e d e v o i d o f i o n i c s i t e s . T h i s k i n d o f g e l e x h i b i t e d s t r o n g h y d r o p h o b i c i t y e s p e c i a l l y w h e n l o n g e r c a r b o n c h a i n s w e r e i n t r o d u c e d t o S e p h a r o s e . A l c o h o l s m u s t b e c o u p l e d t o S e p h a r o s e i n t h e f o r m o f g l y c i d y l e t h e r s . B u t y l - , h e x y l - a n d o c t y l - g l y c i d y l e t h e r s w e r e s y n t h e s i z e d f r o m t h e i r r e s p e c t i v e a l c o h o l s . I n o r d e r t o b i n d a g l y c i d y l e t h e r t o S e p h a r o s e 4 B , t h e l a t t e r s h o u l d b e s w o l l e n i n o r g a n i c s o l v e n t s . A s e r i e s o f w a s h i n g s w e r e c a r r i e d o u t f o r S e p h a r o s e b y s t a r t i n g f r o m p u r e w a t e r a n d b y a d d i n g i n c r e a s i n g c o n c e n t r a t i o n s o f d i o x a n e t o t h e s o l v e n t up t o 1 0 0 % d i o x a n e . A f t e r t h i s a d a p t a t i o n p r o c e s s , g l y c i d y l e t h e r s w e r e c o u p l e d t o S e p h a r o s e 4B i n a n o n p o l a r e n v i r o n m e n t . When t h e c o u p l i n g r e a c t i o n w a s t e r m i n a t e d , a n o t h e r s e r i e s o f w a s h i n g s w e r e a p p l i e d t o t h e g e l , t h i s t i m e f r o m 1 0 0 % d i o x a n e b a c k t o p u r e w a t e r . The prepared gels were washed extensively with water to remove any unbound residues. The gels were packed in 30 ml columns and were equilibrated with 0.002 M sodium phosphate buffer (pH 6.9). Hydrophobic chromatography on alkylepoxy-Sepharoses provided a technique to classify proteins based on their a f f i n i t y towards the hydrophobic gel material. Considering that the gel contains only hydrophobic sites, the interaction must be apolar in nature. Those molecules without any aff i n i t y for the hydrophobic ligand would pass through the column, while those molecules having a binding a f f i n i t y are retarded. The degree of retardation would depend on the binding constant of the macromolecule. A molecule with a weak binding a f f i n i t y might be retarded enough to prevent i t s emergence at the void volume when the column is eluted with the buffer. A molecule with a slightly greater binding af f i n i t y may require several void volumes before emerging from the column, while a molecule with a very high binding af f i n i t y may never be eluted from the column. In the case of molecules tightly bound to the gel, i t is necessary to change the eluent composition to promote desorption of those proteins. The hydrophobic residues or side chains which are available on the surface of the protein molecule are able to form hydrophobic bonds with the carbon chain present on the Sepharose gel. The nonpolar amino acids which are buried inside the protein molecule are unable to participate in the interactions. This relationship seems to be similar to the function of proteins which interact mildly during emulsification. I n a l l chromatography p r o c e d u r e s t h e r e i s a r i s k t h a t the s u b s t a n c e s t o be chromatographed undergo d e n a t u r a t i o n . T h i s r i s k a l s o e x i s t s i n h y d r o p h o b i c i n t e r a c t i o n chromatography, b u t i s r e l a t i v e l y s m a l l i f the bed m a t e r i a l i s chosen t o g i v e o n l y m o d e r a t e l y s t r o n g i n t e r a c t i o n s w i t h h y d r o p h o b i c p r o t e i n s . The e x t e n t o f d e n a t u r a t i o n p r o b a b l y i s comparable w i t h d e n a t u r a t i o n o f p r o t e i n s i n an o i l / w a t e r i n t e r f a c e d u r i n g e m u l s i f i c a t i o n . However, t h e s e k i n d s o f d e n a t u r a t i o n a r e r e v e r s i b l e ( G o n z a l e z and M a c R i t c h i e , 1970). The d i f f i c u l t t a s k w i t h the h y d r o p h o b i c epoxy g e l was t o choose t h e l i g a n d w h i c h does n o t b i n d t o p r o t e i n i r r e v e r s i b l y . The most s u i t a b l e g e l i s the one w h i c h o n l y r e t a r d s the h y d r o p h o b i c p r o t e i n s and e v e n t u a l l y r e l e a s e s them s i m p l y by f l o w o f the o r i g i n a l b u f f e r . The more h y d r o p h o b i c p r o t e i n s were found t o be more r e s i s t a n t t o d e s o r p t i o n . P r e p a r a t i o n o f t h e g e l s w i t h v a r y i n g c a r b o n c h a i n l e n g t h s p r o v i d e d a s e r i e s o f a d s o r b e n t s w i t h d i f f e r e n t h y d r o p h o b i c i t i e s . Chromatography on o c t y l e p o x y - Sepharose r e s u l t e d i n s t r o n g b i n d i n g w i t h p r o t e i n s . T h i s v e r y h y d r o p h o b i c l i g a n d p r e v e n t e d e l u t i o n o f p r o t e i n s upon w a s h i n g the g e l w i t h t h e b u f f e r . Most o f t h e samples t i g h t l y bound t o the g e l i n 0.002 M sodium phosphate ( P H 6.9 ) c o n t a i n i n g 2 M N a C l . E l i m i n a t i o n o f sodium c h l o r i d e f r o m t h e b u f f e r d i d n o t cause d e s o r p t i o n o f t h e bound p r o t e i n f r o m o c t y l e p o x y - S e p h a r o s e . E l u t i o n was a c h i e v e d o n l y by i n c l u d i n g e t h y l e n e g l y c o l i n t h e b u f f e r . N a t u r a l l y , such a s t r o n g b i n d i n g i s u n d e s i r a b l e and a c c o r d i n g l y o c t y l e p o x y - Sepharose i s u n s u i t a b l e f o r d e t e r m i n a t i o n o f the e f f e c t i v e 54 h y d r o p h o b i c i t y . L e s s h y d r o p h o b i c g e l s were found more s u i t a b l e f o r t h i s o b j e c t i v e . However, t h e o c t y l e p o x y - Sepharose was used w i t h s u c c e s s i n o t h e r e x p e r i m e n t s . The g e l was e f f e c t i v e i n removing b i t t e r p e p t i d e s f r o m a c a s e i n h y d r o l y s a t e . The b i t t e r p e p t i d e s w h i c h were r e p o r t e d t o be h y d r o p h o b i c i n n a t u r e (Matoba and H a t a ,1972) bound to the o c t y l e p o x y - Sepharose g e l and the d e b i t t e r e d h y d r o l y s a t e was c o l l e c t e d f r o m the column. I t i s known t h a t the a d s o r p t i o n o f p r o t e i n s on h y d r o p h o b i c g e l s i n c r e a s e s w i t h t h e degree o f s u b s t i t u t i o n and h y d r o p h o b i c i t y o f the s u b s t i t u e n t s (Pahlman e_t a l _ . ,1977). However, the c r i t i c a l h y d r o -p h o b i c i t y needed f o r a p p r o p r i a t e r e t a r d a t i o n o f p r o t e i n s on the g e l i s n o t o n l y a f u n c t i o n o f t h e h y d r o p h o b i c i t y o f t h e a d s o r b e n t b u t a l s o depends on t h e s a l t e n v i r o n m e n t i n w h i c h t h e p r o t e i n i s chromatographed ( F i g . 4 ) . I t i s shown t h a t the r e t e n t i o n volume of b o v i n e serum a l b u m i n i n c r e a s e d when chromatography was c a r r i e d out i n the p r e s e n c e o f a h i g h e r c o n c e n t r a t i o n of s a l t s . T h i s e f f e c t was o b s e r v e d f o r o t h e r p r o t e i n s as w e l l . S a l t c o n c e n t r a t i o n up t o 3 M sodium c h l o r i d e i n t h e b u f f e r has been used w i t h o u t any d e n a t u r a t i o n e f f e c t on p r o t e i n (Pahlman e_t a l . ,1977) . A d d i t i o n o f s a l t s a l s o o b v i a t e s any p o s s i b i l i t y o f i n v o l v e m e n t o f i o n i c f o r c e s . S a l t s a r e w e l l known t o d e c r e a s e the s o l u b i l i t y o f n o n p o l a r s u b s t a n c e s i n w a t e r . However, the e f f e c t i s a p p r e c i a b l e a t h i g h s a l t c o n c e n t r a t i o n o n l y . A p r o t e i n w h i c h p r e c i p i t a t e s a t a low s a l t c o n c e n t r a t i o n s h o u l d l i k e w i s e be r e t a i n e d on h y d r o p h o b i c s u p p o r t s a t a low i o n i c s t r e n g t h . S i m i l a r l y , the p r o t e i n s w h i c h p r e c i p i t a t e a t h i g h 55 s a l t c o n c e n t r a t i o n s h o u l d r e q u i r e r e l a t i v e l y h i g h e r i o n i c s t r e n g t h s t o a f f e c t t h e i r r e t e n t i o n . Sodium s u l f a t e and sodium c h l o r i d e were found t o be more e f f e c t i v e i n r e t a i n i n g p r o t e i n s on h y d r o p h o b i c a d s o r b e n t s as compared w i t h c o r r e s p o n d i n g ammonium s a l t s . S u l f a t e and c h l o r i d e a n i o n s a r e known as s a l t i n g - o u t agents w h i c h r e d u c e s o l u b i l i t y o f p r o t e i n s . The h y d r o p h o b i c i t y o f e i t h e r g e l , p r o t e i n , o r b o t h r e a c t a n t s must be i n f l u e n c e d by the c o n c e n t r a t i o n o f s a l t s . The u n d e r l y i n g p r i n c i p l e o f s a l t i n g _ o u t a d s o r p t i o n i s n o t c l e a r l y u n d e r s t o o d , b u t presumably t h e d r i v i n g f o r c e i s the e n t r o p y g a i n a r i s i n g f rom s t r u c t u r e changes i n t h e w a t e r s u r r o u n d i n g t h e i n t e r a c t i n g h y d r o p h o b i c g r o u p s . No 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 m o l e c u l e o c c u r s i n t h e p r e s e n c e o f up t o 1 M sodium s u l f a t e o r 3 M sodium c h l o r i d e (Pahlman e_t a l . 1977). I n c r e a s i n g s a l t c o n c e n t r a t i o n makes the g e l s h r i n k . A l s o , h i g h l y s u b s t i t u t e d g e l s s h r i n k because o f t h e i r h y d r o p h o b i c i t i e s . The r e a s o n has been a t t r i b u t e d t o t h e f a c t t h a t t h e w a t e r m o l e c u l e s a r r a n g e d i n a c e r t a i n o r d e r around the s o l v a t e d g e l a r e d i s t u r b e d by t h e g r e a t numbers o f i o n s s e e k i n g h y d r a t i o n . The c o m p e t i t i o n f o r w a t e r m o l e c u l e s between s o l v a t e d polymer c h a i n s and i o n s ends up i n f a v o u r o f the i o n s and r e s u l t s i n s h r i n k i n g of t h e g e l a n a l o g o u s . W i t h an i n c r e a s e i n i o n i c s t r e n g t h , l i p o p h i l i c s o l u t e s a r e pushed towards the g e l m a t r i x . T h i s may be compared w i t h the w e l l - k n o w n s a l t i n g - o u t e f f e c t . An i n c r e a s e i n i o n i c s t r e n g t h f r o m 0.5 t o 1.0 M sodium s u l f a t e i n c r e a s e d the r e t e n t i o n volume o f b o v i n e serum a l b u m i n a p p r e c i a b l y ( F i g . 4 ) . 5 6 H e x y l - a n d b u t y l e p o x y - S e p h a r o s e s w e r e q u i t e c a p a b l e o f f o r m i n g m i l d h y d r o p h o b i c i n t e r a c t i o n s w i t h t h e p r o t e i n s i n t h e p r e s e n c e o f s u l f a t e a n d c h l o r i d e a n i o n s . T h e i n t e n s i t y o f t h e h y d r o p h o b i c i n t e r a c t i o n s a n d t h u s t h e r e t e n t i o n o f p r o t e i n s i n t h e c o l u m n w a s m a n i p u l a t e d b y u s i n g d i f f e r e n t i o n i c s t r e n g t h s . A f t e r c h o o s i n g t h e p r o p e r i o n i c s t r e n g t h , i d e n t i c a l c o n d i t i o n s w e r e u s e d i n t h e c h r o m a t o g r a p h y o f a l l p r o t e i n s o f t h e s e r i e s . Two M s o d i u m c h l o r i d e w a s f o u n d t o b e s u i t a b l e a n d c a u s e d m i l d r e t e n t i o n o f p r o t e i n s i n t h e c o l u m n . T h e p r e s e n c e o f s a l t i n d u c e d s h r i n k a g e o f t h e g e l a n d a c o n s i d e r a b l e d e c r e a s e i n f l o w r a t e . A s a r e s u l t o f t h i s , t h e c o l u m n h a d t o b e r e p a c k e d f r o m t i m e t o t i m e . T h e s a l t c o n c e n t r a t i o n w a s c h o s e n s o a s n o t t o p r e c i p i t a t e o r d e n a t u r e p r o t e i n s i n t h e g e l m a t r i x . T h e d i f f i c u l t p r o b l e m i n t h e d e t e r m i n a t i o n o f h y d r o p h o b i c i t y b y h y d r o p h o b i c c h r o m a t o g r a p h y i s t o s e l e c t t h e c o r r e c t h y d r o p h o b i c g r o u p . O u r e x p e r i e n c e s h o w e d t h a t a h i g h l y h y d r o p h o b i c g e l i s n o t s u i t a b l e t o d e t e r m i n e t h e e f f e c t i v e h y d r o p h o b i c i t y o f p r o t e i n s b a s e d o n t h e i r r e t e n t i o n v o l u m e . W i t h t h e s e g e l s t h e p r o t e i n s a r e n o t e l u t e d o r t h e r e t e n t i o n v o l u m e b e c o m e s s o l a r g e t h a t t h e e x p e r i m e n t i s i m p r a c t i c a l . S u b s t i t u t i n g s h o r t e r c a r b o n c h a i n l e n g t h s s u c h a s t h e b u t y l - o r h e x y l e p o x y g r o u p o n S e p h a r o s e p r e s e n t e d i n t e r e s t i n g r e s u l t s . B y a d j u s t i n g i o n i c s t r e n g t h r e a s o n a b l e r e t e n t i o n v o l u m e s f o r p r o t e i n s w e r e o b t a i n e d i n c o m p a r a t i v e l y s h o r t e r t i m e . H y d r o p h i l i c s u b s t a n c e s s u c h a s g l u c o s e s h o w e d n o i n t e r a c t i o n w i t h t h e h y d r o p h o b i c l i g a n d ( F i g . 5). E l u t i o n o f h y d r o p h o b i c p r o t e i n s r e q u i r e d a v o l u m e o f b u f f e r g r e a t e r t h a n t h e t o t a l v o l u m e o f t h e b e d . 0.71 20 30 4 0 50 60 70 80 1 E f f l u e n t v o l u m e ( m l ) F i g u r e 5 - R e t e n t i o n v o l u m e o f b o v i n e s e r u m a l b u m i n (BSA ) a n d g l u c o s e o n h e x y l e p o x y - S e p h a r o s e 4 B . B u f f e r , 0 . 0 0 2 M s o d i u m p h o s p h a t e c o n t a i n i n g 1 M s o d i u m s u l p h a t e (pH 6 . 9 ) . The r e t e n t i o n volumes of the p r o t e i n s were used to c a l c u l a t e the r e t e n t i o n c o e f f i c i e n t s . The r e s u l t s obtained from hydrophobic chromatography of the s e l e c t e d s e r i e s of p r o t e i n s showed that bovine serum albumin had the greatest a b i l i t y to form hydrophobic i n t e r a c t i o n s . I t has been suggested that i n g l o b u l a r p r o t e i n s the r a t i o of nonpolar/polar amino acid s increases w i t h i n c r e a s i n g molecular weights ( F i s h e r , 1964). This was shown to be the r e s u l t of an increase i n i n t e r n a l volume which i s composed mostly of apolar amino a c i d s . However, apparently not a l l nonpolar groups are able to p a r t i c i p a t e i n hydrophobic i n t e r a c t i o n s . This idea i s supported by the f a c t that there i s no c o r r e l a t i o n between the e f f e c t i v e hydrophobicity and molecular weights of the p r o t e i n s ( F i g . 6). This r e s u l t also suggests that no molecular s i e v i n g e f f e c t has been i n v o l v e d i n r e t e n t i o n of the p r o t e i n s on the hydrophobic columns. Thus, the r e t e n t i o n must be a r e s u l t of hydrophobic i n t e r a c t i o n s . I t i s i n t e r e s t i n g to i n v e s t i g a t e whether the e f f e c t i v e hydrophobicity i s the same parameter as the average hydrophobicity c a l c u l a t e d by Bigelow (1967) , based on the nonpolar amino a c i d content of p r o t e i n s . Figure 7 shows that there i s no c o r r e l a t i o n between these two expressions of hydrophobicity i n the experimental p r o t e i n s . I t i s apparent that hydrophobic chromatography does not determine the t o t a l content of the nonpolar amino acids i n the molecule, but rather measures the a b i l i t y of p r o t e i n to take p a r t i n hydrophobic i n t e r a c t i o n s whether p o l a r or nonpolar amino acids are i n v o l v e d . © 60 7 5 , 0 0 0 6 0 , 0 0 0 4 5 , 0 0 0 © • H CO * 3 0 , 0 0 0 3 O 1 5 , 0 0 0 © 0 © 0 2 4 6 8 10 12 14 16 18 R e t e n t i o n c o e f f i c i e n t o n HS4B F i g u r e 6 - C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y ( r e t e n t i o n c o e f f i c i e n t ) d e t e r m i n e d o n a h e x y l e p o x y - S e p h a r o s e 4B c o l u m n a n d m o l e c u l a r w e i g h t o f p r o t e i n s , o : o v a l b u m i n , a : a - l a c t a l b u m i n , c : c o n a l b u m i n , m: m y o g l o b u l i n , 8 : B - l a c t o g l o b u l i n , 1: l y s o z y m e , b : b o v i n e s e r u m a l b u m i n a n d c*2: a - c h y m o t r y p s i n . 61 1240 1210 *1180 o 1 150 r H <D bfl •H CQ 1120 4-> o 1 090 OU o >>1060 bO <u > 1030. 1 000 9 7 0 I © © © © © 0 16 18 2 4 6 8 10 12 14 R e t e n t i o n c o e f f i c i e n t o n HS4B F i g u r e 7 - C o r r e l a t i o n b e t w e e n e f f e c t i v e h y d r o p h o b i c i t y m e a s u r e d o n a h e x y l e p o x y - S e p h a r o s e 4 B ( H S 4 B ) c o l u m n a n d t h e a v e r a g e h y d r o -p h o b i c i t y c a l c u l a t e d b y B i g e l o w ( 1 9 6 7 ) . a : a - l a c t a l b u m i n , o.: o v -a l b u m i n , c : c o n a l b u m i n , m: m y o g l o b i n , fS: ( 3 - l a c t o g l o b u l i n , b : b o v i n e s e r u m a l b u m i n , 1: l y s o z y m e . 62 C h r o m a t o g r a p h y o n b u t y l - o r h e x y l e p o x y - S e p h a r o s e s c l a s s i f i e d p r o t e i n s b a s e d o n t h e i r h y d r o p h o b i c i t i e s . B o t h m e t h o d s a p p e a r t o p r o v i d e c o m p a r a b l e r e s u l t s ( s e e T a b l e s I a n d I I ) . T h i s i s e x p e c t e d s i n c e t h e m e c h a n i s m o f t h e i n t e r a c t i o n s i n b o t h c o l u m n s s h o u l d b e s i m i l a r . H o w e v e r , c o m p a r i s o n o f e l u t i o n p a t t e r n s i n F i g . 8 a n d F i g . 9 r e v e a l e d a n i n t e r e s t i n g e f f e c t w h i c h s h o w s t h e m o r e h y d r o p h o b i c p r o t e i n s h a v e a g r e a t e r a f f i n i t y t o w a r d s a m o r e a p o l a r l i g a n d . T h u s h i g h l y h y d r o p h o b i c p r o t e i n s s h o w e d g r e a t e r i n t e r a c t i o n w i t h t h e h e x y l m o i e t y . T h i s i s n o t s u r p r i s i n g c o n s i d e r i n g t h a t h y d r o p h o b i c i n t e r a c t i o n s a r e m u t u a l e f f e c t s . T h e m o r e h y d r o p h o b i c p r o t e i n s s h o u l d h a v e m o r e a f f i n i t y f o r h i g h l y n o n p o l a r l i g a n d s . T h e r e w a s a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n t h e t w o m e t h o d s ( F i g . 1 0 ) . A v e r y h i g h c o r r e l a t i o n c o e f f i c i e n t ( r = . 9 7 ) w a s o b t a i n e d w h e n t h e two l e a s t h y d r o p h o b i c p r o t e i n s w e r e e x c l u d e d f r o m t h e c a l c u l a t i o n . T h e r e s u l t s s u g g e s t t h a t a l t h o u g h b o t h m e t h o d s a r e a b l e t o d e t e r m i n e a w i d e r a n g e o f h y d r o p h o b i c i t y h e x y l e p o x y - S e p h a r o s e i s m o r e s u i t a b l e f o r a n a l y s i s o f t h e m o r e a p o l a r p r o t e i n s , w h e r e a s t h e b u t y l e p o x y - S e p h a r o s e c a n b e u s e d e f f i c i e n t l y f o r l e s s a p o l a r p r o t e i n s . 4 . C h r o m a t o g r a p h y w i t h d e t e r g e n t s : A c o m p a r i s o n o f t h e r e s u l t s o b t a i n e d f r o m h y d r o p h o b i c c h r o m a t o g r a p h y a n d o t h e r m e t h o d s w a s a t t e m p t e d . C h r o m a t o g r a p h y o n S e p h a d e x G - 1 5 0 w a s c a r r i e d o u t i n t h e p r e s e n c e o f T r i t o n X - 1 0 0 , a n o n - i o n i c d e t e r g e n t . Due t o t h e f a c t t h a t b i n d i n g o f T r i t o n X - 1 0 0 t o t h e p r o t e i n i s a h y d r o p h o b i c p r o c e s s i n w h i c h t h e a p o l a r r e g i o n s o f p r o t e i n s a r e i n v o l v e d , m o r e d e t e r g e n t m o l e c u l e s w e r e t h o u g h t t o b i n d w i t h t h e m o r e h y d r o p h o b i c p r o t e i n s t h a n t h e l e s s h y d r o p h o b i c o n e s . 00 0 . 8 0 . 7 0 . 6 •-0.5 o CO rsi 0 . 4 rt X> ' 0 . 3 X < 0 . 2 0 .1 30 c • H c • H .—1 • H 1/) 3 e PH X) 3 >, O c c x ( H c «-H • H • H 4-> •rH CO i—1 e rt O X) O 3 £ 3 •M E o <-> X O r-H o O N r-H rt i— CO rt -—t O Oj i— i O O H° ;>-l / l Ov i a i a X . > * V \ > ( 7 < V < < < < < < < < < < < •> > o in o 3 C X > rt o A T I / V / V I_3H. 32 34 36 38 E f f l u e n t v o l u m e ( m l ) F i g u r e 8 - R e t e n t i o n v o l u m e o f p r o t e i n s o n b u t y l e p o x y -s o d i u m p h o s p h a t e c o n t a i n i n g 2M s o d i u m c h l o r i d e . 40 42 44 46 4 8 S e p h a r o s e 4 B . B u f f e r , 0 . 0 0 2 M c • H 6 c c 3 • H • H 43 E e r - H 3 3 rt • Xi r - H i—i O rt rt rt > r—( o O 1 a \ • H 3 in 43 &, o X C .—1 r H - H OO +-> 43 o O O E O X 00 rt 4= O r - H O X 1 • s o , 'I l l j j J i i l H i >M 11 ' i i / / / /' / / i i \ \ V ; \ \ > > > > > i > > > 3 43 O i—i CO I >-> < > < W 32 34 36 38 48 40 42 . 44 46 E f f l u e n t v o l u m e ( m l ] F i g u r e 9 - R e t e n t i o n v o l u m e o f p r o t e i n s o n h e x y l e p o x y - S e p h a r o s e 4B s o d i u m p h o s p h a t e c o n t a i n i n g 2M s o d i u m c h l o r i d e . 50 52 B u f f e r , T a b l e I - R e t e n t i o n c o e f f i c i e n t s o f p r o t e i n s d e t e r m i n e d b y h y d r o p h o b i c c o l u m n c h r o m a t o g r a p h y o n b u t y l e p o x y - S e p h a r o s e 4B O v a l - a - l a c t a l - a-chymo- M y o g l o - B - l a c t o - y - g l o - L y s o - B o v i n e s e r u m b u m i n b u m i n t r y p s i n b i n g l o b u l i n b u l i n zyme a l b u m i n M e an, S t a n d a r d d e v i a t i o n , S t a t i s t i c a l , , d i f f e r e n c e 1.33 1.33 1.33 1.33 1.33 0.00 2.00, 1.33 1.33 2.00 1.66 0.38 2.66 2.66 2.66 2.00 2.49 0.33 2.66 2.66 2.66 4.00 2.99 0.67 4.66 4.00 4.00 5.00 4.41 0.49 5.33 4.66 4.66 4.66 6.66 8.00 8.00 7.33 4.82 7.49 0.33 0.64 10.00 10.66 10.00 10.66 10.33 0.38 _** S o u r c e o f v a r i a t i o n D e g r e e s o f f r e e d o m Sum o f s q u a r e s -Mean s q u a r e s Sx Among, 7 270.2 W i t h i n , - 24 4.9 0.20 0.225 T o t a l , 31 2 6 5.3 *) D e t e r m i n e d b y t h e D u n c a n ' s New M u l t i p l e R a n ge t e s t ; a =0,05; a, b', c , d a n d e a r e s i g n i -f i c a n t l y d i f f e r e n t g r o u p s . **) S t a n d a r d e r r o r o f t h e t r e a t m e n t , S x = y W i t h i n mean s q u a r e / n . T a b l e I I - R e t e n t i o n c o e f f i c i e n t s o f p r o t e i n s d e t e r m i n e d b y h y d r o p h o b i c c o l u m n c h r o m a t o g r a p h y o n h e x y l e p o x y - S e p h a r o s e 4 B . O v a l - a - l a c t a l - C o n a l - a - c h y m o - M y o g l o - 3 - l a c t o - y - g l o - L y s o - B o v i n e b u m i n b u m i n b u m i n t r y p s i n b i n g l o b u l i n b u l i n zyme s e r u m a l b u m i n m e a n , S t a n d a r d d e v i a t i o n , S t a t i s t i c a l * d i f f e r e n c e , 1 . 3 3 1 . 3 3 2 . 0 0 1 .55 0 . 3 8 1 . 3 3 2 . 0 0 2 . 0 0 1 . 7 8 0 . 3 8 a b -2 . 0 0 3 . 3 3 3 . 3 3 2 . 8 9 0 . 7 6 1 1 . 3 3 1 0 . 6 6 1 2 . 6 6 1 3 . 3 3 1 6 . 0 0 1 8 . 0 0 1 0 . 6 6 1 0 . 0 0 1 2 . 6 6 ' 1 5 . 3 3 1 4 . 6 6 1 8 . 0 0 9 . 3 3 1 1 . 6 6 1 1 . 3 3 1 4 . 6 6 1 4 . 6 6 1 8 . 6 6 1 0 . 4 4 1 0 . 7 7 1 2 . 2 1 1 4 . 4 3 1 5 . 1 0 1 8 . 2 2 1.01 0 . 8 5 0 . 7 6 1.01 0 . 7 7 0 . 3 8 S o u r c e o f v a r i a t i o n D e g r e e s o f f r e e d o m Sum o f s q u a r e s Mean s q u a r e s Sx ** A m o n g , 8 9 3 3 . 7 W i t h i n , 18 1 0 . 0 0 . 5 5 0 . 4 3 T o t a l , 26 9 2 3 . 7 * ) D e t e r m i n e d b y t h e D u n c a n ' s New M u l t i p l e R a n g e t e s t ; a = 0 . 0 5 ; a , b , c , d , e a n d f a r e s i g n i -f i c a n t l y d i f f e r e n t g r o u p s . * * ) S t a n d a r d e r r o r o f t h e t r e a t m e n t , S x = / W i t h i n mean s q u a r e / n . © © © © © © © 0 2 4 6 8 10 12 14 16 18 R e t e n t i o n c o e f f i c i e n t o n HS4B F i g u r e 1 0 - C o r r e l a t i o n b e t w e e n r e t e n t i o n c o e f f i c i e n t s m e a s u r e d b y t w o h y d r o p h o b i c c h r o m a t o g r a p h y t e c h n i q u e s . O r d i n a t e a n d a b s c i s s a a r e r e t e n t i o n c o e f f i c i e n t s o f p r o t e i n s on b u t y l - a n d h e x y l e p o x y - S e p h a r o s e 4 B , r e s p e c t i v e l y . r = 0 . 8 6 , s i g n i f i c a n t a t a = 0 . 0 5 . b : b o v i n e s e r u m a l b u m i n , 1: l y s o z y m e , m : m y o g l o b i n , o : o v a l b u m i n , a: a - l a c t a l b u m i n , a : a - c h y m o t r y p s i n , 8: 8 -l a c t o g l o b u l i n a n d y: y - g l ° b u l i n . 68 Thus the c o n c e n t r a t i o n of T r i t o n X-100 i n the p r o t e i n - d e t e r g e n t complex s h o u l d be a good c r i t e r i o n o f h y d r o p h o b i c i t y o f p r o t e i n s . Based on t h i s assumption, chromatography o f p r o t e i n s on Sephadex G-150 was c a r r i e d out i n the pres e n c e o f phosphate b u f f e r c o n t a i n i n g m i c e l l a r c o n c e n t r a t i o n o f T r i t o n X-100. The f r a c t i o n o f the e f f l u e n t which c o n t a i n e d the p r o t e i n - d e t e r g e n t complex was c o l l e c t e d . A f t e r c o n c e n t r a t i o n by d i a l y s i s a g a i n s t 20% p o l y v i n y l p y r r o l i d o n e s o l u t i o n , i t was a p p l i e d t o a Biobead SM-2 column. As a r e s u l t o f d e s t a b i l i z a t i o n , d e t e r g e n t was bound to the bed m a t e r i a l and p r o t e i n e l u t e d . The d e t e r g e n t was r e c o v e r e d from the bed by a p p l i c a t i o n o f 50% e t h a n o l c o n t a i n i n g 10% e t h y l e n e g l y c o l . The ammonium c o b a l t o t h i o c y a n a t e method was used to determine the c o n c e n t r a t i o n o f the d e t e r g e n t s p e c t r o p h o t o m e t r i c a l l y . The method i s based on the p r e s e n c e o f the p o l y ( e t h y l e n e o x i d e ) group i n T r i t o n X-100 ( o c t y l p h e n o x y p o l y e t h o x y e t h a n o l ) . Ammonium c o b a l t o t h i o c y a n a t e forms a b l u e p r e c i p i t a t e by complexing w i t h these groups. T h i s b l u e p r e c i p i t a t e i s then e x t r a c t e d i n t o the e t h y l e n e d i c h l o r i d e phase. The method i s c l a i m e d to be s e n s i t i v e f o r the assay o f microgram amounts o f T r i t o n X-100 (Garewal, 1973). However, our r e s u l t s showed poor r e p e a t a b i l i t y . T h i s c o u l d be due to the f o l l o w i n g r e a s o n s : 1 - The amount o f the d e t e r g e n t bound to p r o t e i n s was not s p e c i f i c . 2 - The method i s not a d e q u a t e l y s e n s i t i v e . 3 - The p o l y e t h y l e n e g l y c o l which was used to s e p a r a t e the d e t e r g e n t from i t s complex w i t h the Biobead SM-2 bed i n t e r f e r e s w i t h the c o b a l t o t h i o c y a n a t e r e a c t i o n . 4 - Complexity o f the pro c e d u r e f o r c l e a v a g e and s e p a r a t i o n o f the p r o t e i n - d e t e r g e n t complex. The 69 d i r e c t c o b a l t o t h i o c y a n a t e r e a c t i o n a p p l i e d t o the d e t e r g e n t - p r o t e i n f r a c t i o n e l u t e d f r o m Sephadex G-150 d i d n o t improve the s i t u a t i o n . 5. H y d r o p h o b i c p a r t i t i o n : The h y d r o p h o b i c p a r t i t i o n method r e v e a l e d d i f f e r e n t v a l u e s o f A l o g K f o r d i f f e r e n t p r o t e i n s ( T a b l e I I I ) . H y d r o p h o b i c i t y as w e l l as c o n f o r m a t i o n o f t h e p r o t e i n w h i c h d e t e r m i n e s the n a t u r e o f groups exposed t o t h e s u r r o u n d i n g s a r e e x p e c t e d t o a f f e c t t h e p a r t i t i o n o f p r o t e i n s . P a r t i t i o n c o e f f i c i e n t was d e t e r m i n e d p h o t o m e t r i c a l l y a t 280 nm. The d e x t r a n and p o l y e t h y l e n e g l y c o l s o l u t i o n s s l i g h t l y a b s o r b e d the r a d i a t i o n w h i c h was e l i m i n a t e d by s u b t r a c t i n g the v a l u e s measured a t t h e i d e n t i c a l c o n c e n t r a t i o n s o f p o l y m e r s . The d i f f e r e n c e i n t h e non-p o l a r i t y o f t h e two phases i s c o m p a r a t i v e l y s m a l l , and i s m a i n l y due t o t h e low p olymer c o n c e n t r a t i o n and t h e h y d r o p h i l i c n a t u r e o f t h e p o l y m e r s . However, p o l y e t h y l e n e g l y c o l i s s l i g h t l y more h y d r o p h o b i c t h a n d e x t r a n . D e l i c a t e changes i n the phase c o m p o s i t i o n can cause d r a s t i c changes i n t h e d i s t r i b u t i o n o f the p r o t e i n s . The g e n e r a l t r e n d i n p a r t i t i o n o f p r o t e i n s and p a r t i c l e s i s t h a t when t h e m o l e c u l a r w e i g h t o f one p olymer i s l o w e r e d , t h e a f f i n i t y o f t h e p r o t e i n s towards t h a t phase i s i n c r e a s e d . N e i t h e r p o l y e t h y l e n e g l y c o l n o r d e x t r a n a r e c h a r g e d . N e v e r t h e -l e s s , i o n s i n f l u e n c e d i s t r i b u t i o n o f p r o t e i n s w i t h i n the two phases. P r o t e i n s w h i c h a r e p o s i t i v e l y o r n e g a t i v e l y c h a r g e d can be pushed toward t h e upper o r l o w e r phase, d e p e n d i n g on t h e i o n i c c o m p o s i t i o n of t h e s y s t e m . When a l a r g e amount o f s a l t i s added t o t h e d e x t r a n - p o l y e t h y l e n e T a b l e I I I - H y d r o p h o b i c i t y o f p r o t e i n s d e t e r m i n e d b y t h e h y d r o p h o b i c p a r t i t i o n t e c h n i q u e . A l o g K was c a l c u l a t e d f r o m A l o g K = l o g k / k 2 w h e r e , k 1 i s t h e p a r t i t i o n c o e f f i c i e n t o f p r o t e i n s i n t h e p o l y e t h y l e n e g l y c o l p a l m i t a t e / d e x t r a n s y s t e m a n d i s t h e p a r t i t i o n c o -e f f i c i e n t o f p r o t e i n s i n t h e p o l y e t h y l e n e g l y c o l / d e x t r a n s y s t e m . L y s o - C y t o - C o n a l - O v a l - T r y p - a - c h y m o - 3 - l a c t o - M y o - B o v i n e s e r u m zyme c h r o m e C b u m i n b u m i n s i n t r y p s i n g l o b u l i n g l o b i n a l b u m i n k * 0 . 3 0 5 0 . 1 2 0 0 . 1 1 5 0 . 1 6 4 0 . 1 9 5 0 . 8 7 7 0 . 1 2 3 0 . 1 5 6 0 . 0 1 0 — i t * CO .010 ) ( 0 . 0 0 4 ) ( 0 . 0 0 2 ) ( 0 . 0 0 3 ) ( 0 . 0 0 4 ) ( 0 . 0 0 4 ) ( 0 . 0 0 2 ) ( 0 . 0 0 3 ) ( 0 . 0 0 4 ) k 2 , * 0 . 3 9 1 0 . 1 1 6 0 . 0 7 4 0 . 0 9 4 0 . 1 0 5 0 . 4 2 3 0 . 5 0 1 0 . 0 4 7 0 . 0 0 1 ( 0 . 0 0 7 ) ( 0 . 0 0 3 ) ( 0 . 0 0 1 ) ( 0 . 0 0 5 ) ( 0 . 0 0 3 ) ( 0 . 0 0 5 ) ( 0 . 0 0 1 ) ( 0 . 0 0 1 ) ( 0 . 0 0 0 ) k /k 0 . 7 7 1 . 03 1 .55 1 .75 1 .85 2 . 0 7 2 . 4 6 3 . 3 9 8 . 9 3 A l o g K - 0 . 1 0 0 . 0 1 0 . 1 9 0 . 2 4 0 . 2 6 0 . 3 1 0 . 3 9 0 . 5 3 0 . 9 5 * ) M e a n s o f 3 t o 4 d e t e r m i n a t i o n s . * * ) S t a n d a r d d e v i a t i o n s o f t h e means i n p a r e n t h e s i s . g l y c o l s y s t e m , a l l p r o t e i n s t e n d t o p a r t i t i o n i n f a v o u r o f t h e t o p phase. Sodium c h l o r i d e can be added a t c o n c e n t r a t i o n s up t o 5 M t o push p r o t e i n s t o t h e top phase. The main e f f e c t o f i n c r e a s i n g t h e i o n i c s t r e n g t h would be t h e i n c r e a s e i n t h e K v a l u e . The i n c r e a s e d a f f i n i t y o f the p r o t e i n s f o r the upper phase, w h i c h has a l e s s p o l a r c h a r a c t e r t h a n the b ottom phase, w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n may be compared w i t h t h e e f f e c t known as t h e " s a l t i n g - i n " phenomenon. D i - and t r i v a l e n t a n i o n s as w e l l as Rb > K > Cs > Na > L i c a t i o n s enhance t h i s phenomenon ( A l b e r t s s o n , 1971). The p a r t i t i o n o f a p r o t e i n depends b o t h on i t s r e l a t i v e a t t r a c t i o n t o the two phases and on i t s n e t c h a r g e ( J o h a n s s o n , 1974). I t has been s u g g e s t e d t h a t t h e dependence on the l a t t e r c o u l d be removed by i n c l u d i n g an a p p r o p r i a t e s a l t i n the system. I n the p r e s e n t work p o t a s s i u m s u l f a t e was used i n t h e b u f f e r and the p a r t i t i o n was d e t e r m i n e d s o l e l y on t h e b a s i s o f t h e g e n e r a l s o l v a t i o n a b i l i t y o f the p hases. M o r e o v e r , by making b o t h systems i d e n t i c a l i n c o m p o s i t i o n b u t d i f f e r i n g o n l y i n the h y d r o p h o b i c i t y o f one p h a s e , t h e o t h e r e f f e c t s i n c l u d i n g c h a r g e e f f e c t s were b a l a n c e d . B l a n k s d e t e r m i n e d under c o n d i t i o n s o f i d e n t i c a l c o m p o s i t i o n b u t d e v o i d o f p r o t e i n were used to c a n c e l the c o n t r i b u t i o n o f e f f e c t s such as polymer c o n c e n t r a t i o n and phase v i s c o s i t y . The two p o l y m e r s employed t o o b t a i n t h e b i p h a s i c system were c o n c e n t r a t e d i n t o the o p p o s i t e p h a s e s : p o l y e t h y l e n e g l y c o l i n t h e upper phase and d e x t r a n i n the l o w e r phase. W i t h t h e s t r o n g h y d r o p h o b i c group w h i c h i s bound t o p o l y e t h y l e n e g l y c o l , a consequent i n c r e a s e i n t h e s o l v a t i n g c a p a c i t y f o r h y d r o p h o b i c m o l e c u l e s o c c u r s i n the phase 72 r i c h i n t h i s p o l y m e r . T h e r e f o r e m o r e h y d r o p h o b i c p r o t e i n s w i l l b e c o n c e n t r a t e d i n t h e p h a s e c o n t a i n i n g t h e h y d r o p h o b i c l i g a n d . T h e p a l m i t a t e e s t e r o f p o l y e t h y l e n e g l y c o l w a s d i f f i c u l t t o d i s s o l v e i n t h e b u f f e r a n d t h e v i s c o s i t y o f a q u e o u s s o l u t i o n s o f e v e n l o w c o n c e n t r a t i o n w a s v e r y h i g h , p o s s i b l y d u e t o s t r o n g i n t e r a c t i o n s b e t w e e n p o l y e t h y l e n e g l y c o l m o l e c u l e s . A l t h o u g h s h a k i n g f o r 30 s e c w a s s u f f i c i e n t t o p r o d u c e a f i n e d i s p e r s i o n o f t h e t w o p h a s e s i n t h e p o l y e t h y l e n e g l y c o l / d e x t r a n s y s t e m , d u e t o t h e h i g h v i s c o s i t y o f t h e p o l y e t h y l e n e g l y c o l p a l m i t a t e e x t e n s i v e s h a k i n g w a s r e q u i r e d t o a c h i e v e u n i f o r m d i s t r i b u t i o n o f t h e m a t e r i a l s . T h i s v i s c o s i t y c a u s e d a d e l a y i n p h a s e s e p a r a t i o n a n d c r e a t e d p r o b l e m s i n t h e s p e c t r o p h o t o m e t r i c a n a l y s i s o f s a m p l e s . To e l i m i n a t e a l l t h e s e d i f f i c u l t i e s , t h e s a m p l e s w e r e t a k e n b y w e i g h t a n d a f t e r d i l u t i o n t h e s o l u t i o n s i n t h e t e s t t u b e s w e r e m i x e d w i t h a v o r t e x s h a k e r . S e v e r e s h a k i n g o f t u b e s m o s t p r o b a b l y c a u s e d m e c h a n i c a l d e n a t u r a t i o n o f p r o t e i n b u t t h i s m a k e s t h e s y s t e m s e v e n c l o s e r t o t h e c o n d i t i o n u s e d i n t h e e m u l s i f i c a t i o n p r o c e s s . C e n t r i f u g a l f o r c e o f 1500 x G w a s u s e d t o f a c i l i t a t e s e p a r a t i o n o f t h e p h a s e s . A l t h o u g h t h e K v a l u e ( p a r t i t i o n c o e f f i c i e n t ) w a s f a i r l y c o n s t a n t o v e r a r a n g e o f p r o t e i n q u a n t i t y t h e same c o n c e n t r a t i o n o f p r o t e i n w a s u s e d i n t h i s s t u d y . 0 . 2 % SDS i n 0 . 1 N NaOH w a s u s e d t o d i l u t e t h e p o l y e t h y l e n e g l y c o l p a l m i t a t e p h a s e s . T h e s a m p l e s w e r e t h e n w a r m e d up b e f o r e t h e s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s . T h i s w a s n e c e s s a r y t o d e c r e a s e t h e s o l u t i o n v i s c o s i t y a n d t o e l i m i n a t e t h e t u r b i d i t y p r o d u c e d p o s s i b l y d u e t o t h e p a l m i t a t e - p r o t e i n i n t e r a c t i o n . T h e c o n c e n t r a t i o n o f p r o t e i n i n t h e s y s t e m w a s a p p r e c i a b l y h i g h e r i n t h e b o t t o m p h a s e , t h u s r e s u l t i n g i n a n d v a l u e s l o w e r t h a n u n i t y . C o n s i d e r a b l e d i f f e r e n c e s i n t h e d i s t r i b u t i o n p a t t e r n o f p r o t e i n s w e r e o b s e r v e d w h e n t h e s y s t e m s c o n t a i n i n g p o l y e t h y l e n e g l y c o l a n d p o l y e t h y l e n e g l y c o l p a l m i t a t e w e r e c o m p a r e d . S i n c e h y d r o p h o b i c p r o t e i n s s h o w e d g r e a t a f f i n i t y t o w a r d s t h e p a l m i t a t e g r o u p , i n c r e a s e d , t h u s r e s u l t i n g i n l a r g e r A l o g K. B e c a u s e t h i s a f f i n i t y w a s c a u s e d o n l y b y h y d r o p h o b i c i n t e r a c t i o n b e t w e e n p r o t e i n a n d t h e p a l m i t a t e l i g a n d , A l o g K r e p r e s e n t s p r o t e i n h y d r o p h o b i c i t y d e s i g n a t e d a s h y d r o p h o b i c c o e f f i c i e n t ( T a b l e I I I ) . A s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n w a s f o u n d b e t w e e n t h e e f f e c t i v e h y d r o p h o b i c i t i e s d e t e r m i n e d b y h y d r o p h o b i c p a r t i t i o n a n d h y d r o p h o b i c c h r o m a t o g r a p h y m e t h o d s ( F i g s . 11 a n d 1 2 ) . H o w e v e r , f o r t h e s e c o r r e l a t i o n a n a l y s e s l y s o z y m e w a s e x c l u d e d . T h i s p r o t e i n , w h i c h h a d b e e n f o u n d h y d r o p h o b i c b y t h e h y d r o p h o b i c c h r o m a t o g r a p h y m e t h o d s , s h o w e d n o a f f i n i t y t o w a r d t h e h y d r o p h o b i c p a l m i t a t e l i g a n d a n d y i e l d e d a n e g a t i v e A l o g K v a l u e w i t h t h e h y d r o p h o b i c p a r t i t i o n m e t h o d . T h e l y s o z y m e m o l e c u l e c o n s i s t s o f a s i n g l e p o l y p e p t i d e c h a i n c o n t a i n i n g 129 a m i n o a c i d r e s i d u e s , a n d t h e h y d r o p h o b i c a n d h y d r o p h i l i c s i d e c h a i n s t e n d t o o c c u r i n l a r g e c l u s t e r s r a t h e r t h a n b e i n g r a n d o m l y d i s t r i b u t e d t h r o u g h o u t t h e p o l y p e p t i d e . A s i z e a b l e p r o p o r t i o n o f t h e f i r s t t h i r t y -n i n e r e s i d u e s , s t a r t i n g f r o m t h e a m i n o t e r m i n a l e n d , a r e h y d r o p h o b i c r e s i d u e s , t h e n e x t f o r t y r e s i d u e s a r e p r i m a r i l y h y d r o p h i l i c , a n d t h e t e r m i n a l t h i r d o f t h e s e q u e n c e i s o n c e a g a i n h y d r o p h o b i c ( B e r n h a r d , 1 9 6 8 ) 10 74 © / / / / / / / / / / / / 0 / / © / 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 .0 H y d r o p h o b i c c o e f f i c i e n t F i g u r e 1 1 - C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t i e s m e a s u r e d b y h y d r o p h o b i c c h r o m a t o g r a p h y o n b u t y l e p o x y - S e p h a r o s e 4B a n d h y d r o p h o b i c p a r t i t i o n . O r d i n a t e a n d a b s c i s s a a r e r e t e n t i o n c o e f f i c i e n t o n b u t y l e p o x y - S e p h a r o s e 4 B ( B S 4 B ) a n d h y d r o p h o b i c c o e f f i c i e n t d e t e r m i n e d b y h y d r o p h o b i c p a r t i -t i o n m e t h o d r e s p e c t i v e l y . r= 0 . 9 5 , s i g n i f i c a n t a t a= 0 . 0 5 . b : b o v i n e s e r u m a l b u m i n , m : m y o g l o b i n , o : o v a l b u m i n , a : a - c h y m o t r y p s i n a n d 6 : 6 -l a c t o g l o b u l i n . 75 18 16 to c 14 o c <u o 12 • - H 4-1 4-1 <U O ° 10 c o 0) • 0) © / / / / © /> / / © 0 0 . 2 0 . 4 0 . 6 0 . 8 H y d r o p h o b i c c o e f f i c i e n t F i g u r e 1 2 - C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t i e s m e a s u r e d b y h y d r o p h o b i c c h r o m a t o g r a p h y o n h e x y l e p o x y - S e p h a r o s e 4B a n d h y d r o p h o b i c p a r t i t i o n . O r d i n a t e a n d a b s c i s s a a r e r e t e n t i o n c o e f f i c i e n t o n h e x y l e p o x y - S e p h a r o s e 4B (HS4B ) a n d h y d r o p h o b i c c o e f f i c i e n t d e t e r m i n e d b y t h e h y d r o p h o b i c p a r t i t i o n m e t h o d , r e s p e c t i v e l y . r = 0 . 8 7 , s i g n i f i c a n t a t a = 0 . 0 S . b : b o v i n e s e r u m a l b u m i n , c : c o n a l b u m i n , o : o v a l b u m i n , m: m y o g l o b i n , a : a - c h y m o t r y p s i n a n d 8: 3 - l a c t o g l o b u l i n . L y s o z y m e a p p e a r s a s a h y d r o p h o b i c p r o t e i n s i n c e s u c h a n a r r a n g e m e n t o f n o n p o l a r r e s i d u e s p r o v i d e s h y d r o p h o b i c p a t c h e s w h i c h a r e a b l e t o i n t e r a c t w i t h h e x y l a n d b u t y l g r o u p s . T h e s e h y d r o p h o b i c s i t e s , h o w e v e r , may n o t h a v e t h e s u i t a b l e s i z e o r o r i e n t a t i o n t o i n t e r a c t w i t h t h e p a l m i t a t e l i g a n d . T h e l i m i t a t i o n o f t h e h y d r o p h o b i c p a r t i t i o n m e t h o d i s t h a t n o t a l l p r o t e i n s a r e s o l u b l e i n t h e p h a s e s y s t e m s u s e d i n t h e m e t h o d . Many h i g h m o l e c u l a r w e i g h t p r o t e i n s do n o t d i s s o l v e i n t h e d e x t r a n o r p o l y e t h y l e n e g l y c o l p h a s e s . T h e s e m o l e c u l e s t e n d t o d i s t r i b u t e a t t h e i n t e r f a c e a n d o n l y a s m a l l a m o u n t o f p r o t e i n i s d i s s o l v e d i n t h e p h a s e s ; a p r e c i p i t a t e u s u a l l y f o r m e d a t t h e i n t e r f a c e . T h e i n t e r f a c e h a s a c e r t a i n c a p a c i t y o f a d s o r p t i o n . A l t h o u g h t h i s d o e s n o t p l a y a n y s i g n i f i c a n t r o l e w i t h r e s p e c t t o s o l u b l e p r o t e i n s t h e i n t e r f a c e may a d s o r b a r e l a t i v e l y l a r g e q u a n t i t y o f s u s p e n d e d p a r t i c l e s . P a r t i c l e s d i s t r i b u t e t h e m s e l v e s b e t w e e n t h e l i q u i d p h a s e s a n d t h e i n t e r f a c e . T h e l a r g e r t h e s i z e o f t h e p a r t i c l e s , t h e g r e a t e r t h e a d s o r p t i o n a t t h e i n t e r f a c e . W i t h i n s o l u b l e f o o d p r o t e i n s t h e r e a r e i n f a c t t h r e e " p h a s e s " t o c o n s i d e r , t h e u p p e r , t h e i n t e r a n d t h e l o w e r p h a s e s . T h e l a r g e r a d i u s o f t h e p a r t i c l e s , h i g h m o l e c u l a r w e i g h t s o f p r o t e i n s a n d i n s o l u b i l i t y o f p r o t e i n s i n e i t h e r o f t h e two p h a s e s c a u s e p r o t e i n s t o l o c a t e a t t h e i n t e r f a c e . S o m e t i m e s g r a v i t a t i o n a l f o r c e s o v e r c o m e i n t e r a c t i o n f o r c e s ; t h e n t h e p a r t i c l e s a r e p u l l e d d o w n b y t h e f o r m e r . T h e i n f l u e n c e o f g r a v i t a t i o n a l f o r c e i s a f f e c t e d b y t h e s i z e a n d d e n s i t y o f t h e p a r t i c l e s as w e l l as d e n s i t y of the phases. T h i s was the case when v e g e t a b l e food p r o t e i n s were examined by the h y d r o p h o b i c p a r t i t i o n method. I n the case of Y - g l o b u l i n and i n s u l i n , p r o t e i n s were p r e c i p i t a t e d when p o l y e t h y l e n e g l y c o l was added. T h i s i n s o l u b i l i z a t i o n phenomenon was thought to be the r e s u l t o f i n t e r a c t i o n between p o l y e t h y l e n e g l y c o l and the p r o t e i n s . P r e l i m i n a r y r e s u l t s w i t h some s o l u b i l i z e d f o o d p r o t e i n s showed t h a t because o f the t u r b i d i t y o f the p a r t i t i o n e d phases s p e c t r o p h o t o m e t r i c a n a l y s i s i s n o t a p p l i c a b l e to the measurement of p a r t i t i o n c o e f f i c i e n t . The B i u r e t and Lowry methods were a p p l i e d f o r the d e t e r m i n a t i o n of p r o t e i n c o n c e n t r a t i o n i n the s e p a r a t e d phases. However,' d e x t r a n made a 2+ complex w i t h Cu and a p r e c i p i t a t e was formed. P o l y e t h y l e n e g l y c o l formed a p r e c i p i t a t e upon a d d i t i o n of F o l i n r e agent i n the B i u r e t and Lowry method of Layne (1957). S i n c e n e i t h e r p o l y e t h y l e n e g l y c o l nor d e x t r a n i s p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d (TCA) and HCIO^, i t i s p r e d i c t e d t h a t the most c o n v e n i e n t way to a v o i d the f o r e g o i n g problems would be as f o l l o w s : A f t e r phase s e p a r a t i o n , the p r o t e i n i s p r e c i p i t a t e d by TCA and i s washed f r e e o f the polymers. C h l o r o f o r m and d i e t h y l e t h e r a r e s u ggested to be used f o r washing away p o l y e t h y l e n e g l y c o l and d e x t r a n r e s p e c t i v e l y . T C A - p r e c i p i t a t e d p r o t e i n i s then d i s s o l v e d i n 0.5 M sodium h y d r o x i d e and p r o t e i n c o n c e n t r a t i o n i s measured a c c o r d i n g to the B i u r e t and Lowry method (Layne, 1957). The p o s i t i v e c o r r e l a t i o n s which were found between the h y d r o -p h o b i c i t i e s determined by h y d r o p h o b i c chromatography and h y d r o p h o b i c p a r t i t i o n t e c h n i q u e ( F i g . 11) suggest t h a t b o t h methods are u s e f u l i n d e t e r m i n i n g the e f f e c t i v e h y d r o p h o b i c i t i e s . 6. I n t e r f a c i a l t e n s i o n m e a s u r e m e n t s : I n t e r f a c i a l t e n s i o n w a s t a k e n a s a r e p r e s e n t a t i v e p a r a m e t e r f o r s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s , s i n c e t h e s e p r o p e r t i e s a r e c l o s e l y r e l a t e d - t o :- c h a n g e s i n i n t e r f a c i a l t e n s i o n ( K i n s e l l a , 1 9 7 6 ) . I n t e r f a c i a l t e n s i o n s w e r e m e a s u r e d i n 0.2% p r o t e i n s o l u t i o n s / c o r n o i l i n t e r f a c e ( T a b l e I V ) . T h e r e s u l t s s h o w e d s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n w i t h t h e e f f e c t i v e h y d r o p h o b i c i t i e s o f p r o t e i n s ( F i g s . 13, 14 a n d 15). T h i s mean s t h a t t h e m o r e h y d r o p h o b i c i s t h e p r o t e i n , t h e g r e a t e r t h e d e p r e s s i o n i n t h e i n t e r f a c i a l t e n s i o n . T h i s c o u l d b e e x p e c t e d a s t h e h y d r o p h o b i c p r o t e i n s s h o u l d b e a b l e t o i n t e r a c t e a s i l y w i t h o i l a n d w a t e r m o l e c u l e s , d e n a t u r e o n t h e o i l s u r f a c e a n d b r i n g t h e i n t e r f a c i a l e n e r g y d o w n . C o n s e q u e n t l y m o r e h y d r o p h o b i c p r o t e i n s a r e e x p e c t e d t o b e a b l e t o i n c r e a s e ' t h e e m u l s i o n c a p a c i t y . When a p r o t e i n m o l e c u l e a r r i v e s a t t h e b o u n d a r y o f a t w o -p h a s e s y s t e m s u c h a s a n o i l / w a t e r i n t e r f a c e , o r i e n t a t i o n o f t h e s i d e c h a i n s i s a l o g i c a l r e s u l t b e c a u s e i t a l l o w s t h e p r o t e i n m o l e c u l e t o t a k e up a c o n f i g u r a t i o n w i t h l o w f r e e e n e r g y i n w h i c h p o l a r g r o u p s i n t e r a c t w i t h t h e p o l a r p h a s e ( w a t e r ) a n d t h e n o n p o l a r g r o u p s w i t h t h e n o n p o l a r p h a s e ( o i l ) . A s a c o n s e q u e n c e o f t h i s m e c h a n i s m i n t e r f a c i a l p r o t e i n d e n a t u r a t i o n o c c u r s a n d a p r o t e i n f i l m a r i s e s . T h i s d e n a t u r a t i o n n e e d n o t b e i r r e v e r s i b l e . I t s e e m s o b v i o u s t o a s s u m e t h a t t h e p r o p e r t i e s o f s u c h i n t e r f a c i a l f i l m s a r e i m p o r t a n t w i t h r e s p e c t t o t h e s t a b i l i t y o f t w o p h a s e s y s t e m s . I t h a s b e e n e m p h a s i z e d , i n d e e d ( P e a r s o n a n d A l e x a n d e r , 1 9 6 8 ) , t h a t t h e m e c h a n i c a l s t r e n g t h o f t h e p r o t e i n f i l m a n d i t s r e s i s t a n c e a g a i n s t e x t e r n a l f o r c e s d e t e r m i n e s t o a g r e a t e x t e n t t h e T a b l e I V - I n t e r f a c i a l t e n s i o n o f 0 . 2 % p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e . B o v i n e O v a l - L y s o - y - g l o - M y o g l o - 8 - l a c t o - T r y p - C o n a l - a - c h y m o t r y p -s e r u r a b u m i n zyme b u l i n b i n g l o b u l i n s i n b u m i n s i n a l b u m i n M e a n , S t a n d a r d d e v i a t i o n , S t a t i s t i c a l d i f f e r e n c e , * 6 .0 6 .5 9 .0 17 .5 18 .0 21 .5 21 .5 21 , .5 2 2 . 5 6, .0 7. .0 9 .5 17 .5 18 .0 20 .0 21 .5 2 1 . ,5 2 2 . 0 6. .0 6, .5 9. .5 18 .0 18 . .5 20 .0 21 .0 2 2 . 0 2 2 . 0 6 . .0 6 . .6 9 . .3 1 7 . .6 1 8 . 1 2 0 . .5 2 1 , .3 2 1 . 6 2 2 . 1 0 . 00 0 . 29 0 . 28 0 . 29 0 . 29 0 . 86 0 . .29 0 . 29 0 . 2 9 S o u r c e o f v a r i a t i o n D e g r e e s o f f r e e d o m ; Sum o f s q u a r e s M e a n o f s q u a r e s S x * * A m o n g , 8 1071 W i t h i n , 18 21 1.2 0 . 6 2 T o t a l , 26 1092 * ) D e t e r m i n e d b y t h e D u n c a n ' s New M u l t i p l e R a n g e t e s t ; a = 0 . 0 5 ; a , b , c a n d d a r e s i g n i f i c a n t l y d i f f e r e n t g r o u p s . * * ) S t a n d a r d e r r o r o f t h e t r e a t m e n t , Sx= J W i t h i n mean s q u a r e / n . 80 22 20 18 e u e X T3 O • H to c • a> 12 rt •H o £ i o ID \ 9 X © \ \ © © 0 1 2 3 4 5 6 7 8 9 10 Retention c o e f f i c i e n t on BS4B Figure 13- C o r r e l a t i o n between hydrophobicity determined on butylepoxy-Sepharose 4B (BS4B) and i n t e r f a c i a l t e n sion of 0.2% p r o t e i n s o l u t i o n / corn o i l i n t e r f a c e . r= -0.95, s i g n i f i c a n t at a= 0.05. b:bovine serum albumin, 1: lysozyme, m: myoglobin, a: a-chymotrypsin, 8: 3-lactoglobu-l i n and y: y - g l o b u l i n . ovalbumin i s shown by "*". 81 0 2 4 6 8 10 12 14 16 18 R e t e n t i o n c o e f f i c i e n t F i g u r e 14.- C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y d e t e r m i n e d o n h e x y l e p o x y - S e p h a r o s e 4B (HS4B ) a n d i n t e r f a c i a l t e n s i o n o f 0 . 2 % p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e . r = - 0 . 7 7 , s i g n i f i c a n t a t a= 0 . 0 5 . b : b o v i n e s e r u m a l b u m i n , c : c o n a l b u m i n , 1: l y s o z y m e , m: m y o g l o b i n , a : a - c h y m o t r y p s i n , 8 : 8 - l a c t o g l o b u l i n a n d y : y -g l o b u l i n . e o CO c C O •rH +-> c +J O rt <4H rH <D •P C 22 20 18 16 14 12 10 \ 82 \ \ \ © \ \ \ \ \ \ \ \ \ ©\ 0 0 . 2 0 . 3 0 . 8 0 . 9 0 . 4 0 . 5 0 . 6 0 . 7 H y d r o p h o b i c c o e f f i c i e n t F i g u r e 1 5 - C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y ( h y d r o p h o b i c c o -e f f i c i e n t ) o f p r o t e i n s d e t e r m i n e d b y t h e h y d r o p h b i c p a r t i t i o n m e t h o d a n d i n t e r f a c i a l t e n s i o n o f 0 . 2 % p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e . r = - 0 . 9 7 , s i g n i f i c a n t a t a = 0 , 0 5 . b : b o v i n e s e r u m • a l b u m i n , c : c o n a l b u m i n , m: m y o g l o b i n , t : t r y p s i n , a : a -c h y m o t r y p s i n a n d 8: B - l a c t o g l o b u l i n . O v a l b u m i n i s s h o w n b y " * " 1.0 s t a b i l i t y o f a t w o p h a s e s y s t e m . F u r t h e r m o r e , i t a p p e a r s t o b e m a i n l y t h e v i s c o e l a s t i c i t y o f t h e p r o t e i n f i l m w h i c h w a s d e c i s i v e f o r i t s m e c h a n i c a l s t r e n g t h . I t w a s a l s o s h o w n t h a t t h e m e c h a n i c a l p r o p e r t y o f t h e f i l m i s , i n g e n e r a l , n e i t h e r a p u r e v i s c o u s n o r a p u r e e l a s t i c o n e , b u t r a t h e r a c o m p l e x o n e , c h a r a c t e r i z e d b y a s u p e r -p o s i t i o n o f b o t h v i s c o s i t y a n d e l a s t i c i t y . S c h u t ( 1 9 7 6 ) a t t r i b u t e d t h e s e r h e o l o g i c a l p r o p e r t i e s o f t h e i n t e r f a c i a l l a y e r t o t h e h y d r o p h o b i c b o n d f o r m a t i o n i n t h e p r o t e i n f i l m w h i c h s u r r o u n d s t h e o i l d r o p l e t s . T h i s i n f o r m a t i o n a g r e e s w i t h o u r r e s u l t s w h i c h s u g g e s t t h e h y d r o p h o b i c i t y o f p r o t e i n a s a n o u t s t a n d i n g f a c t o r f o r i t s s u r f a c t a n t p r o p e r t i e s . One e x c e p t i o n t o t h i s h y p o t h e s i s w a s f o u n d i n t h e c a s e o f o v a l b u m i n . T h i s p r o t e i n , w h i c h h a d b e e n f o u n d t o b e o f l o w h y d r o -p h o b i c i t y , d e p r e s s e d t h e i n t e r f a c i a l t e n s i o n c o n s i d e r a b l y . T h i s i s s u g g e s t e d t o b e d u e t o t h e o t h e r p r o p e r t i e s o f t h i s p r o t e i n w h i c h w e r e not c o v e r e d i n t h i s s t u d y . T h i s e f f e c t m i g h t b e a t t r i b u t e d t o t h e p r e s e n c e o f c o n t a m i n a n t s s u c h a s p h o s p h o l i p i d . SUMMARY AND CONCLUS ION To i n v e s t i g a t e t h e r e l a t i o n s h i p b e t w e e n t h e h y d r o p h o b i c i t y a n d t h e s u r f a c t a n t p r o p e r t i e s o f p r o t e i n s , d i f f e r e n t m e t h o d s w e r e a p p l i e d : 1 . C h r o m a t o g r a p h y o n o l e i c s u b s t i t u t e d S e p h a r o s e 4 B : T h e s u b s t i t u t e d g e l d e m o n s t r a t e d v e r y h i g h h y d r o p h o b i c i t y a n d r e t a i n e d many p r o t e i n s s o t i g h t l y t h a t t h e e l u t i o n c o u l d b e d o n e o n l y a f t e r u s i n g p o l a r i t y r e d u c i n g a g e n t s i n t h e b u f f e r . S u c h a h i g h h y d r o p h o b i c i t y p r o h i b i t e d t h e m e t h o d f r o m b e i n g u t i l i z e d f o r d e t e r m i n a t i o n o f h y d r o p h o b i c i t y o f p r o t e i n s . 2 . C h r o m a t o g r a p h y o n S e p h a d e x G - 1 5 0 i n t h e p r e s e n c e o f d e t e r g e n t s : T r i t o n X - 1 0 0 a t i t s m i c e l l e c o n c e n t r a t i o n w a s i n c l u d e d i n s o d i u m p h o s p h a t e b u f f e r . P r o t e i n b o u n d t o t h e d e t e r g e n t a n d t h e c o m p l e x w a s e l u t e d . T h e a m o u n t o f t h e d e t e r g e n t i n t h e p r o t e i n - d e t e r g e n t c o m p l e x w a s d e t e r m i n e d t o e x p r e s s h y d r o p h o b i c i t y o f p r o t e i n s . T h i s m e t h o d s u f f e r e d f r o m a p o o r r e p e a t a b i l i t y i n a n a l y s i s o f t h e T r i t o n X - 1 0 0 . T h e m e t h o d w a s f o u n d u n s u i t a b l e f o r d e t e r m i n a t i o n o f t h e h y d r o p h o b i c i t y . 3 . C h r o m a t o g r a p h y o n a l k y l a m i n o S e p h a r o s e s : C N B r a c t i v a t e d S e p h a r o s e 4B w a s s u b s t i t u t e d w i t h n - a l k y l a m i n e s o f d i f f e r e n t c h a i n l e n g t h s ( C 4 - C 6 - C 8 ) . A l s o , b y c o u p l i n g P h e n y l b u t y l a m i n e t o S e p h a r o s e 4B t h e h y d r o p h o b i c g e l w i t h a r o m a t i c r i n g w a s o b t a i n e d . T h e a l k y l a m i n o S e p h a r o s e s c o n t a i n e d m i x e d h y d r o p h o b i c a n d i o n i c g r o u p s . T h e p r e s e n c e o f p o s i t i v e l y c h a r g e d n i t r o g e n r e n d e r e d t h e s e g e l s u n d e s i r a b l e d u e t o e l e c t r o s t a t i c r e p u l s i o n o r a t t r a c t i o n f o r c e s w h i c h made, t h e s e g e l s i n c a p a b l e o f m e a s u r i n g p u r e h y d r o p h o b i c i t y . H o w e v e r , c h r o m a t o g r a p h y w i t h a l k y l a m i n o S e p h a r o s e s p r o v i d e d i n f o r m a t i o n o n w h e t h e r e l e c t r o s t a t i c o r h y d r o p h o b i c i n t e r a c t i o n s a r e d o m i n a n t i n t h e r e a c t i o n s o f a p r o t e i n . M o r e o v e r , a l k y l a m i n o S e p h a r o s e s s h o w e d t h e c a p a b i l i t y o f d i s c r i m i n a t i o n b e t w e e n p r o t e i n s ( F i g . 3 w a s p r e s e n t e d a s a n e x a m p l e ) . . 4. H y d r o p h o b i c c h r o m a t o g r a p h y o n a l k y l e p o x y - S e p h a r o s e s : A l k y l e p o x y -S e p h a r o s e s c o n t a i n o n l y h y d r o c a r b o n g r o u p s . P r e p a r a t i o n o f t h e a d s o r b e n t s was c a r r i e d o u t i n t h e f o l l o w i n g o r d e r : s y n t h e s i s o f g l y c i d y l e t h e r s f r o m a l c o h o l s , c o n d i t i o n i n g o f S e p h a r o s e 4B, a n d c o u p l i n g g l y c i d y l e t h e r s t o S e p h a r o s e . B u t y l - , h e x y l - a n d o c t y l e p o x y - S e p h a r o s e s w e r e p r e p a r e d . O c t y l e p o x y - S e p h a r o s e , b e c a u s e o f i t s h i g h h y d r o p h o b i c i t y , i n t e r a c t e d t i g h t l y w i t h p r o t e i n s a n d w a s f o u n d u n s u i t a b l e f o r d e t e r m i n a t i o n o f h y d r o p h o b i c i t y . H o w e v e r , t h i s g e l w a s u s e d s u c c e s s f u l l y i n o t h e r e x p e r i m e n t s t o r e m o v e b i t t e r p e p t i d e s f r o m c a s e i n h y d r o l y s a t e . B u t y l - a n d h e x y l e p o x y - S e p h a r o s e s w e r e f o u n d s u i t a b l e f o r d e t e r m i n a t i o n o f h y d r o p h o b i c i t y . I n t e r a c t i o n o f n o n p o l a r r e g i o n s o f p r o t e i n w i t h t h e a l k y l g r o u p s a v a i l a b l e i n t h e s e n o n - i o n i c a m p h i p h i l i c g e l s w e r e m e d i a t e d w i t h s a l t . T h e u s e o f 2 M N a C l p r o v i d e d s u i t a b l e i o n i c s t r e n g t h a ) t o s t i p u l a t e c o n d i t i o n s f o r m i l d h y d r o p h o b i c i n t e r a c t i o n s , a n d b ) t o o b v i a t e a n y p o s s i b i l i t y o f i o n i c i n t e r a c t i o n s . T h e r e t e n t i o n v o l u m e s o f t h e p r o t e i n s w e r e u s e d t o c a l c u l a t e h y d r o p h o b i c i t i e s . S i g n i f i c a n t p o s i t i v e c o r r e l a t i o n was f o u n d b e t w e e n t h e h y d r o -p h o b i c i t i e s d e t e r m i n e d b y b u t y l - a n d h e x y l e p o x y - S e p h a r o s e c o l u m n c h r o m a -t o g r a p h y . H o w e v e r , t h e m o r e h y d r o p h o b i c p r o t e i n s s h o w e d g r e a t e r a f f i n i t y toward t h e h e x y l m o i e t y compared t o t h e b u t y l c o u n t e r p a r t . No c o r r e l a t i o n was found between the e f f e c t i v e h y d r o p h o b i c i t i e s and t h e "average h y d r o p h o b i c i t i e s " o f B i g e l o w (1967) c a l c u l a t e d f r o m t h e amino a c i d c o m p o s i t i o n o f p r o t e i n s . A p p a r e n t l y , o n l y exposed h y d r o p h o b i c r e s i d u e s t a k e p a r t i n h y d r o p h o b i c i n t e r a c t i o n s . No c o r r e l a t i o n was found between t h e e f f e c t i v e h y d r o p h o b i c i t y and t h e m o l e c u l a r w e i g h t o f p r o t e i n . T h i s c o n t r a d i c t s the p r i m i t i v e v i e w t h a t h i g h e r m o l e c u l a r w e i g h t g l o b u l a r p r o t e i n s a r e more h y d r o p h o b i c . I n f a c t , h i g h m o l e c u l a r w e i g h t p r o t e i n s c o n t a i n a g r e a t e r r a t i o o f n o n p o l a r amino a c i d s w h i c h a r e n o t n e c e s s a r i l y i n v o l v e d i n h y d r o p h o b i c i n t e r a c t i o n s . 5. H y d r o p h o b i c p a r t i t i o n : D e t e r m i n a t i o n o f t h e e f f e c t i v e h y d r o p h o b i c i t y was a l s o c a r r i e d o u t e m p l o y i n g a two phase system. One o f them c o n t a i n e d a h y d r o p h o b i c l i g a n d . The p a r t i t i o n c o e f f i c i e n t s o f p r o t e i n s were d e t e r m i n e d and t h e n the h y d r o p h o b i c c o e f f i c i e n t s were c a l c u l a t e d f r o m them. P o s i t i v e c o r r e l a t i o n was found between h y d r o p h o b i c i t i e s d e t e r m i n e d by h y d r o p h o b i c p a r t i t i o n and by h y d r o p h o b i c chromatography. A p p l i c a t i o n o f t h e h y d r o p h o b i c p a r t i t i o n method t o s o l u b i l i z e d f o o d p r o t e i n s needs f u r t h e r s t u d y t o s o l v e i n s o l u b i l i t y p roblems w h i c h o c c u r i n t h e p r e s e n c e o f t h e p o l y m e r s . 6. I n t e r f a c i a l t e n s i o n s : The i n t e r f a c i a l t e n s i o n s o f v a r i o u s p r o t e i n s o l u t i o n s / o i l showed a good c o r r e l a t i o n w i t h t h e e x p e r i m e n t a l l y d e t e r m i n e d v a l u e s o f h y d r o p h o b i c i t y . The n e g a t i v e c o r r e l a t i o n w h i c h was f o u n d between t h e i n t e r f a c i a l t e n s i o n s and t h e e f f e c t i v e h y d r o p h o b i c i t i e s s u g g e s t s t h a t m o r e h y d r o p h o b i c p r o t e i n s r e d u c e t h e i n t e r f a c i a l t e n s i o n m o r e e f f i c i e n t l y . T h e s e p r o t e i n s a r e e x p e c t e d t o h a v e h i g h e r e m u l s i f y i n g c a p a c i t y t h r o u g h r e d u c t i o n o f t h e i n t e r f a c i a l e n e r g y . I n s h o r t , p r o t e i n s w e r e f o u n d t o d i f f e r s i g n i f i c a n t l y i n t h e i r h y d r o p h o b i c i t y a s m e a s u r e d b y t h e i r r e t e n t i o n o n h y d r o p h o b i c g e l s o r t h e d e g r e e o f t h e i r i n t e r a c t i o n t o a h y d r o p h o b i c m o i e t y ( p a l m i t a t e ) o f t h e p a r t i t i o n s y s t e m . T h e h y d r o p h o b i c i t y o f a p o l a r p r o t e i n s i s n e g a t i v e l y c o r r e l a t e d w i t h t h e i r i n t e r f a c i a l t e n s i o n s a n d t h u s w i t h t h e i r s u r f a c t a n t p r o p e r t i e s T h i s i m p l i e s t h a t t h e m o r e h y d r o p h o b i c t h e p r o t e i n , t h e b e t t e r t h e s u r f a c t a n t p r o p e r t i e s . I n f o r m a t i o n o n h y d r o p h o b i c i t y o f p r o t e i n w o u l d e n a b l e u s t o p r e d i c t i t s f u n c t i o n a l i t y . BIBLIOGRAPHY A l b e r t s s o n , P.A., " P a r t i t i o n o f C e l l P a r t i c l e s and M a c r o m o l e c u l e s " , Second E d i t i o n , W i l e y I n t e r s c i e n c e , New Y o r k (.1971). B a k e r , B.R., in " D e s i g n o f A c t i v e S i t e D i r e c t e d I r r e v e r s i b l e Enzyme I n h i b i t o r s " , John W i l e y & Sons I n c . , New Y o r k (1967). B a r e t , J . F . , J . C o l l o i d I n t e r f a c e S c i . 3 0 ( 1 9 6 9 ) 1 . B e r g e r , K.G., i n "Food E m u l s i o n s " , Ed.: F r i b e r g , S., M a r c e l Dekker I n c . , New Y o r k and B a s e l ( 1 9 7 6 ) , Ch.4. B e r n h a r d , S.A., "The S t r u c t u r e and F u n c t i o n o f Enzymes", Benjamin I n c . , New Y o r k (1968). B i g e l o w , C C . , J . T h e o r e t . B i o l . 16(1967)187. B u l l , H.B., Adv. P r o t e i n Chem. 3 ( 1 9 4 7 ) 9 5 . C h o t h i a , C , N a t u r e (London) 248(1974)338. C h o t h i a , C , J . M o l . B i o l . 105(1976)1. C r o t h e r s , D.M., and R a t n e r , D.I., B i o c h e m i s t r y 7(1968)1823. C u a t r e c a s a s , P., W i l c h e k , M., and A n f i n s e n , C.B., P r o c . Nat..Acad. S c i . U.S.A. 61(1968)636. C u a t r e c a s a s , P., J . B i o l . Chem. 245(1970)3059. C u a t r e c a s a s , P., and A n f i n s e n , C.B., Ann. Revs. Biochem. 40(1971)753. 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W i s h n i a , A., and Thomas, W.P.J., B i o c h e m i s t r y 5(1966)1534. Yon, R . J . , Biochem. J . 137(1974)127. Y o n a t h , J . , and B l a u e r , G., i n " P r o t e i n - L i g a n d I n t e r a c t i o n s " , Eds.: Sund, H., and B l a u e r , G., W a l t e r de G r u y t e r , B e r l i n and New Y o r k (1974)427. 93 A P P E N D I X cn SEPHAROSE : .OH •OH" C N B r SEPHAROSE-O-C S E P H A R O S E - 0 - C NH 4 - P H E N Y L B U T Y L A M I N E ». ETHYLAM INE > BUTYLAM INE >• HEXYLAM INE >. OCTYLAM INE SEPH-NH-<^_3 S E P H - N H - C 2 H 5 S E P H - N H - ( C H 2 ) 3 - C H 3 S E P H - N H - ( C H 2 ) 5 - C H 3 S E P H - N H - ( C H 2 ) y - C H 3 ( C H 2 ) 3 - C H 3 T h e s c h e m e o f t h e C N B r a c t i v a t i o n a n d c o u p l i n g o f S e p h a r o s e 4 B . C 1 - C H 2 - C H - - C H 2 + H O . R B F 3 . E t 2 0 OH C l - C H 2 - C H - C H 2 - O R OH H 2 C : H - C H 2 - O R G l y c i d y l E t h e r / ' ^ S F P H A R O S F - O H 9^  H - C — C H - C H „ - O R Z Z v n Z " , > S E P H - 0 - C H o - C H - C H o - 0 R 2 2 B F ^ • E t O H I I A l k y l e p o x y - S e p h a r o s e T h e s c h e m e o f t h e r e a c t i o n s i n v o l v e d i n the p r e p a r a t i o n o f a l k y l -e p o x y - S e p h a r o s e s . 

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