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Chemical modification of carboxyl groups in porcine pepsin Ma, Ching-Yung 1979

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CHEMICAL MODIFICATION OF CARBOXYL GROUPS IN PORCINE PEPSIN by CHING YUNG|MA B.Sc, University of Hong Kong, 1970 M.Sc, University of Br i t i s h Columbia, 1975 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 SCIENCE We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA ©Febuary, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t . o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f F o o d Science T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 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 A p - r i i 9Ut 1070 11 ABSTRACT Carboxyl groups i n porcine pepsin were chemically modified "by the carbodiimide reaction using waterrsoluble l-ethyl-3-(3-dimethylaminopropyl) carbodiimide and amino acid esters as nucleophiles. The modification resulted in pro-found changes in the a c t i v i t i e s , specificity and.some physico-chemical properties of the enzyme. These include* (1) signi-ficant decrease in milk clotting activity without changes i n proteolytic activity against hemoglobin; (2) decrease i n peptidase activity against N-acetyl-L-phenylalanyl-diiodo-L-tyrosine; (3) increase in clotting activity against X-casein but decrease i n clotting activity against K-casein mixture; (k) s h i f t -in proteolytic pH profile with pH optimum increased from 2.0 to about 3*5; (5.) decrease i n relative electrophoretic mobility and a slight decrease in isoelectric point; (6) increase i n K m without much change i n k c a i ;; and (7) increase i n s t a b i l i t y at pH above 6.0. Results suggest that the drop i n milk clotting activity was due to a change i n the charge distribution on the enzyme affecting enzyme-micelle interaction. The presence of dipeptide substrates interfered with the carboxyl modification suggestive of the proximity of the modified groups to the enzyme active s i t e . i i i The m o d i f i e d enzyme r e m a i n e d r e a c t i v e t o s i t e - s p e c i -f i c i n a c t i v a t o r s b u t a t r a t e s s l o w e r t h a n t h e n a t i v e enzyme. The m o d i f i c a t i o n was n o t s p e c i f i c , c a u s i n g s i m i l a r changes i n p e p s i n o g e n and c h y m o s i n . The m o d i f i e d a n d n a t i v e p e p s i n s h a d s i m i l a r c a s e i n o -l y t i c p r o p e r t i e s and p r o d u c e d c o m p a r a b l e r a t e s o f s y n e r e s i s a n d c u r d t e n s i o n d e v e l o p m e n t on c u r d l e d m i l k . The i n c r e a s e i n pH s t a b i l i t y s u g g e s t e d t h a t t h e m o d i f i e d enzyme may be a b e t t e r c a l f r e n n e t s u b s t i t u t e t h a n n a t i v e p e p s i n f o r c h e e s e -m a k i n g . TABLE OF CONTENTS PAGE INTRODUCTION 1 LITERATURE REVIEW C h e m i c a l M o d i f i c a t i o n o f P r o t e i n s 5 M o d i f i c a t i o n o f C a r b o x y l G r o u p s 1. E s t e r i f i c a t i o n 9 2. C a r b o d i i m i d e r e a c t i o n 10 P e p s i n 13 ' C h e m i c a l M o d i f i c a t i o n o f P e p s i n 19 MATERIALS AND METHODS B i o c h e m i e a l s a n d S p e c i a l C h e m i c a l s 22 Methods 1. M o d i f i c a t i o n o f c a r b o x y l g r o u p s 24 2. D e t e r m i n a t i o n o f m i l k c l o t t i n g a c t i v i t y . . . . 25 3... D e t e r m i n a t i o n o f p r o t e o l y t i c a c t i v i t y 27 4. D e t e r m i n a t i o n o f p e p t i d a s e a c t i v i t y 28 5. A s s a y o f p e p s i n w i t h x - c a s e i n a n d < * g l - c a s e i n 29 6. D e t e r m i n a t i o n o f r a t e o f c a s e i n h y d r o l y s i s 30 7. A g a r o s e g e l e l e c t r o p h o r e s i s 31 8. D e t e r m i n a t i o n o f i s o e l e c t r i c p o i n t 32 9. D e t e r m i n a t i o n o f K f f l a n d k c a t 33 10. A s s a y o f p e p s i n o g e n 34 11. D e t e r m i n a t i o n o f p e p s i n s t a b i l i t y t o pH . . . . 34 12. D e t e r m i n a t i o n o f t h e r m a l s t a b i l i t y o f p e p s i n 35 13. D e t e r m i n a t i o n o f c u r d t e n s i o n 37 14. D e t e r m i n a t i o n o f r a t e o f s y n e r e s i s 38 RESULTS AND DISCUSSION C h o i c e o f Enzyme S o u r c e 40 C h o i c e o f C a r b o d i i m i d e s and N u c l e o p h i l e s E f f e c t o f N u c l e o p h i l e C o n c e n t r a t i o n on t h e E x t e n t o f C a r b o x y l M o d i f i c a t i o n A c t i v i t y o f N a t i v e a n d C a r b o x y l M o d i f i e d P e p s i n s 1 . M i l k c l o t t i n g a c t i v i t y 2. P r o t e o l y t i c a c t i v i t y 3 . M i l k c l o t t i n g : p r o t e o l y t i c a c t i v i t y r a t i o . . . 4 . P e p t i d a s e a c t i v i t y E f f e c t o f C a r b o x y l M o d i f i c a t i o n on pH P r o f i l e s 1 . M i l k c l o t t i n g pH p r o f i l e 2. P r o t e o l y t i c pH p r o f i l e 3 . p H - A c t i v i t y c u r v e s f o r t h e h y d r o l y s i s o f APDT E l e c t r o p h o r e t i c M o b i l i t y o f N a t i v e and C a r b o x y l M o d i f i e d P e p s i n s on A g a r o s e G e l I s o e l e c t r i c P o i n t o f N a t i v e a n d C a r b o x y l M o d i f i e d P e p s i n s C a r b o x y l M o d i f i c a t i o n o f P e p s i n B y O t h e r Amino A c i d M e t h y l . E s t e r s C a s e i n o l y t i c P r o p e r t i e s o f N a t i v e a n d . C a r b o x y l M o d i f i e d . P e p s i n s 1 . R a t e o f c a s e i n h y d r o l y s i s 2. E l e c t r o p h o r e t i c p a t t e r n s o f h y d r o l y s e d c a s e i n 3 . E l e c t r o p h o r e t i c p a t t e r n s o f m i l k c u r d s C o a g u l a t i o n o f K - C a s e i n a n d o < g l - C a s e i n b y N a t i v e and C a r b o x y l M o d i f i e d P e p s i n s S p e c i f i c i t y o f C a r b o x y l M o d i f i c a t i o n E f f e c t o f C a r b o x y l M o d i f i c a t i o n on K f f l and 'K ^ o f P e p s i n .<>»<. E f f e c t o f S y n t h e t i c D i p e p t i d e s on C a r b o x y l M o d i -f i c a t i o n v i PAGE Response of Native and Carboxyl Modified Pepsins to Inhibitors 91 S t a b i l i t y of Native and Carboxyl Modified Pepsins near neutral pH • 9^ Thermal S t a b i l i t y of Native and Carboxyl Modified Pepsins ... 103 E f f e c t of Carboxyl Modification on Curd Tension and Rate of Syneresis 112 GENERAL DISCUSSION 117 CONCLUSIONS 131 LITERATURE CITED 133 v i i L I S T OF TABLES TABLE PAGE 1 Some p h y s i c a l p r o p e r t i e s o f p o r c i n e p e p s i n and p e p s i n o g e n 17 2 A d j u s t m e n t o f pH o f m i l k u l t r a f i l t r a t e f o r s t a b i l i t y t e s t o f p e p s i n 36 3 C a r b o x y l m o d i f i c a t i o n o f p e p s i n w i t h d i f f e r -e n t n u c l e o p h i l e s 43 4 E f f e c t o f c a r b o x y l m o d i f i c a t i o n on p e p s i n a c t i v i t i e s 47 5 C a r b o x y l m o d i f i c a t i o n o f p e p s i n b y d i f f e r -e n t amino a c i d m e t h y l e s t e r s 67 6 E f f e c t o f c a r b o x y l m o d i f i c a t i o n on c l o t t i n g a c t i v i t y o f p e p s i n t o X - c a s e i n a n d - K - ^ g j " c a s e i n m i x t u r e 76 7 C o m p a r i s i o n o f a c t i v i t i e s o f c a r b o x y l m o d i f i e d p e p s i n t o d i f f e r e n t s u b s t r a t e s 78 8 E f f e c t o f c a r b o x y l m o d i f i c a t i o n on a c t i v i t i e s o f p e p s i n , p e p s i n o g e n a n d c h y m o s i n 81 9 K i n e t i c s o f t h e h y d r o l y s i s o f N - a c e t y l - L -p h e n y l a l a n y l - L - d i i o d o t y r o s i n e b y n a t i v e a n d c a r b o x y l m o d i f i e d p e p s i n s 86 10 E f f e c t o f d i p e p t i d e s on c a r b o x y l m o d i f i c a t i o n o f p e p s i n 89 v i i i TABLE PAGE 11 Response of native and carboxyl modified pepsins to inhibitors 93 12 Effect of carboxyl modification of pepsin on Q^0 values (milk clotting) 107 13 Effect of carboxyl modification of pepsin on Q 1 Q values (proteolytic) I l l 14 Tension of curds produced by pepsin, car-boxyl modified pepsin and chymosin 113 Ix-L I S T OF FIGURES FIGURE PAGE 1 The r e a c t i o n o f p r o t e i n c a r b o x y l group, w i t h w a t e r - s o l u b l e c a r b o d i i m i d e a n d n u c l e o -p h i l e 12 2 Dependence o f c h a r g e on n u c l e o p h i l e s on t h e m o d i f i c a t i o n o f p r o t e i n c a r b o x y l group, 14 3 E f f e c t o f n u c l e o p h i l e c o n c e n t r a t i o n on t h e e x t e n t o f c a r b o x y l m o d i f i c a t i o n o f p e p s i n . . . 44 4 E f f e c t o f c a r b o x y l m o d i f i c a t i o n o f p e p s i n on m i l k , . c l o t t i n g . a c t i v i t y 46 5 E f f e c t o f c a r b o x y l m o d i f i c a t i o n o f p e p s i n on p r o t e o l y t i c a c t i v i t y 49 6 E f f e c t o f c a r b o x y l m o d i f i c a t i o n o f p e p s i n on m i l k c l o t t i n g * p r o t e o l y t i c a c t i v i t y r a t i o . . 51 7 E f f e c t o f c a r b o x y l m o d i f i c a t i o n o f p e p s i n on p e p t i d a s e a c t i v i t y 53 8 M i l k c l o t t i n g pH p r o f i l e s o f n a t i v e a n d c a r b o x y l m o d i f i e d p e p s i n s 55 9 P r o t e o l y t i c pH p r o f i l e s o f c r y s t a l l i n e p e p s i n , c a r b o x y l m o d i f i e d c r y s t a l l i n e p e p s i n and c h y m o s i n 56 10 P r o t e o l y t i c pH p r o f i l e s o f c r u d e n a t i v e a n d c a r b o x y l m o d i f i e d p e p s i n s 58 X FIGURE PAGE 11 pH-Activity curves for the action of native and carboxyl modified pepsins on N-acetyl-L-phenylalanyl-diiodo-L-tyrosine 60 12 Effect of carboxyl modification of pepsin on relative electrophoretic mobility 63a 13 Determination of isoelectric points of native and carboxyl modified pepsins 65 14 Rate of casein hydrolysis by native and car-boxyl modified pepsins at pH 5*3 • 70 15 Rate of casein hydrolysis by native and car-boxyl modified pepsins at pH 6.5 71 16 Lineweaver-Burk plots of the hydrolysis of N-acetyl-L-phenylalanyl-diiodo-L-tyrosine by native and carboxyl modified pepsins at pH 2.0 84 17 Lineweaver-Burk plots of the hydrolysis of N-acetyl-L-phenylalanyl-diiodo-L-tyrosine by native and carboxyl modified pepsins at pH 4.5 85 o 18 Inactivation of native and carboxyl modified pepsins by EPNP 92 19 St a b i l i t y of crystalline native and carboxyl modified pepsins in 0.05 M phosphate buffer, pH 6.5 95 x i FIGURE PAGE 20 Stability of crude native and carboxyl modified pepsins i n 0.05 M phosphate buffer, pH 6.5 97 21 Sta b i l i t y of crystalline native and carboxyl modified pepsins i n milk u l t r a f i l t r a t e under cheese-making conditions 98 22 Stability of crude native and carboxyl modified pepsins in milk u l t r a f i l t r a t e under cheese-making conditions 99 23; Thermal profiles (milk clotting) of cryst-alline native and carboxyl modified pepsins 104 24 Thermal profiles (milk clotting) of crude native and carboxyl modified pepsins 105 25 Thermal profiles (proteolytic) of crystalline native and carboxyl modified pepsins 108 26 Thermal profiles (proteolytic) of crude native and carboxyl modified pepsins 109 27 Syneresis of curd by crystalline pepsin, car-boxyl modified crystalline pepsin and chy-mosin • 115 28 Syneresis of curd by crude native and car-boxyl modified pepsins 116 x i i L I S T OF PLATES PLATE PAGE I A g a r o s e g e l e l e c t r o p h o r e t i c p a t t e r n s o f n a t i v e and c a r b o x y l m o d i f i e d p e p s i n s 6l I I A g a r o s e g e l e l e c t r o p h o r e t i c . p a t t e r n s o f w h o l e c a s e i n h y d r o l y s e d b y n a t i v e a n d c a r -b o x y l m o d i f i e d p e p s i n s 72 I I I A g a r o s e g e l e l e c t r o p h o r e t i c p a t t e r n s o f m i l k c u r d s p r o d u c e d b y n a t i v e a n d c a r b o x y l m o d i -f i e d p e p s i n s 74 X l l l ACKNOWLEDGEMENTS I w o u l d l i k e t o e x p r e s s my g r a t i t u d e t o my a c a d e m i c s u p e r v i s o r , D r . S . N a k a i , f o r h i s e n t h u s i a s t i c s u p p o r t a n d u n f a i l i n g g u i d a n c e t h r o u g h o u t t h i s s t u d y , and i n t h e p r e p a r a t i o n o f t h i s t h e s i s . I w o u l d l i k e t o t h a n k M r s . V . S k u r a f o r h e r t e c h n i c a l a s s i s t a n c e i n t h e amino a c i d a n a l y s i s . F i n a l l y , I w i s h t o t h a n k my w i f e , S t e p h a n i e , f o r h e r h e l p , u n d e r s t a n d i n g a n d l o v e w i t h o u t w h i c h t h i s work c o u l d n o t be f i n i s h e d . 1 INTRODUCTION C h e m i c a l m o d i f i c a t i o n o f p r o t e i n s b y s p e c i f i c r e -a g e n t s i s a u s e f u l t e c h n i q u e i n t h e s t u d y o f t h e p h y s i c o -c h e m i c a l b a s i s a n d mechanisms o f p r o t e i n f u n c t i o n i n t h e b i o l o g i c a l s y s t e m s (Means a n d F e e n e y , 1971). I n t h e f o o d i n d u s t r y , c h e m i c a l m o d i f i c a t i o n i s p l a y i n g a n i n c r e a s i n g l y i m p o r t a n t r o l e . Many f o o d p r o t e i n s , p a r t i c u l a r l y t h o s e f r o m n o n - c o n v e n t i o n a l s o u r c e s , h a v e been m o d i f i e d t o i m p r o v e t h e i r f u n c t i o n a l p r o p e r t i e s . A l k a l i t r e a t m e n t and m o d i f i c a t i o n w i t h v a r i o u s a c y l a t i n g a g e n t s a r e t h e two most w i d e l y u s e d methods t o a l t e r t h e f u n c t i o n a l i t y o f f o o d p r o t e i n s ( K i n s e l l a , 1976i R y a n , 1977). A l a r g e number o f p a t e n t s have a p p e a r e d , a n d i n most p u b l i s h e d c a s e s , i m p r o v e m e n t s i n f u n c t i o n a l p r o p e r t i e s w e r e o b t a i n e d m a k i n g i t p o s s i b l e t o e x t e n d a n d r e p l a c e e x i s -t i n g f o o d p r o t e i n s w i t h n o v e l p r o t e i n s i n p r o c e s s e d f o o d a n d f o r t h e f a b r i c a t i o n o f new f o o d s . C h e m i c a l m o d i f i c a t i o n o f enzymes i s u s u a l l y u n d e r t a k e n t o e l u c i d a t e t h e a c t i o n mechanism and a c t i v e - s i t e r e s i d u e s o f t h e b i o l o g i c a l c a t a l y s t s . I m m o b i l i z a t i o n o f e n z y m e s , w h i c h i s one f o r m o f c h e m i c a l m o d i f i c a t i o n , h a s b e e n e x t e n s i v e l y u t i l i z e d i n b i o - m e d i c a l r e s e a r c h as w e l l as i n t h e f o o d i n -d u s t r y . The f e a s i b i l i t y o f m o d i f y i n g f o o d - r e l a t e d enzymes t o c o n f o r m t o p a r t i c u l a r p r o c e s s i n g r e q u i r e m e n t s o r t o a v o i d 2 u n d e s i r a b l e c h a r a c t e r i s t i c s h a s n o t b e e n e x p l o r e d , a l t h o u g h i t s h o u l d h o l d g r e a t p r o m i s e s i n e n h a n c i n g t h e a p p l i c a b i l i t y o f many enzymes i n f o o d m a n u f a c t u r e . The a i m o f t h e p r e s e n t i n v e s t i g a t i o n i s t o d e t e r m i n e w h e t h e r t h e p e r f o r m a n c e o f a f o o d - r e l a t e d enzyme c a n be i m -p r o v e d b y c h e m i c a l m o d i f i c a t i o n . P o r c i n e p e p s i n ( E . C . 3f4,23»l)» a n a c i d p r o t e a s e f o u n d i n t h e s t o m a c h mucosa o f p i g , i s c h o s e n f o r t h e f o l l o w i n g r e a s o n s t (1) P o r c i n e p e p s i n i s t h e m o s t w i d e l y a c c e p t e d m i l k c o a g u l a n t f r o m a n a n i m a l s o u r c e a s a s u b s t i t u t e f o r c a l f r e n n e t i n c h e e s e - m a k i n g , a n d has b e e n u s e d as 50*50 m i x t u r e s w i t h c a l f r e n n e t i n t h e c h e e s e i n d u s t r y ( P h e l a n , 1973; B o t t a z z i e t a l . , 1976; C a r b o n e a n d E m a l d i , 1976). (2) The u s e o f s w i n e p e p s i n a l o n e i s g e n e r a l l y c o n -s i d e r e d u n s u i t a b l e f o r c h e e s e - m a k i n g ( S a r d i n a s , 1972). T h i s h a s b e e n p a r t l y a t t r i b u t e d t o s l o w e r p r o t e o l y s i s i n c h e e s e s made w i t h s w i n e p e p s i n t h a n i n t h o s e made w i t h c a l f r e n n e t ( G r e e n a n d F o s t e r , 1974). P o r c i n e p e p s i n i s known t o be l e s s s t a b l e t h a n c h y m o s i n ( m a j o r enzyme i n c a l f r e n n e t ) a t pH above 6.0 ( H e r r i o t t , 1955; F o l t m a n n , 1966; A n t o n i n i a n d R i b a d e a u Dumas, 1971), s u g g e s t i n g t h a t i t may be i n a c t i v a t e d t o a g r e a t e r e x t e n t t h a n r e n n e t d u r i n g t h e c h e e s e - m a k i n g p r o c e s s . (3) P o r c i n e p e p s i n h a s b e e n t h e most t h o r o u g h l y s t u d i e d among a l l a c i d p r o t e a s e s . The p r i m a r y s t r u c t u r e was d e t e r m i n e d (Tang e t a l . , 1973), a n d i t s p r o p e r t i e s a n d modes o f a c t i o n were w e l l c h a r a c t e r i z e d . 3 The p r i m a r y o b j e c t i v e o f t h e p r o j e c t i s t o m o d i f y s e l e c t i v e l y t h e r e a c t i v e g r o u p s o f t h e amino a c i d r e s i d u e s i n p e p s i n w i t h s p e c i f i c c h e m i c a l r e a g e n t s s o as t o change t h e a c t i v i t y , s t a b i l i t y a n d p h y s i c o c h e m i c a l p r o p e r t i e s o f t h e enzyme. T h e s e a l t e r a t i o n s , i f b e n e f i c i a l , s h o u l d e n -h a n c e t h e p o t e n t i a l u t i l i z a t i o n o f p o r c i n e p e p s i n i n t h e m a n u f a c t u r e o f c h e e s e . P r e l i m i n a r y e x p e r i m e n t s h a d b e e n c a r r i e d o u t t o m o d i f y a r g i n i n e , t r y p t o p h a n , t y r o s i n e a n d c a r b o x y l g r o u p s i n p e p s i n . C a r b o x y l m o d i f i c a t i o n was f o u n d t o be most p r o m i s i n g and was f u r t h e r i n v e s t i g a t e d . The e n z y m a t i c c o a g u l a t i o n o f m i l k i s a c o m p l e x p r o -c e s s i n v o l v i n g a p r i m a r y p r o t e o l y t i c p h a s e and a s e c o n d a r y a g g r e g a t i o n p h a s e . The mechanism b y w h i c h m i c e l l e s a g g r e -g a t e t o f o r m c u r d i s s t i l l u n c l e a r ( E r n s t r o m , 1974). I t i s hoped t h a t t h e p r e s e n t s t u d y c a n c o n t r i b u t e t o t h e e l u c i -d a t i o n o f t h i s mechanism and h e l p t o d e f i n e t h e r e l a t i o n s h i p b e t w e e n m i l k c l o t t i n g a n d g e n e r a l p r o t e o l y s i s , two c l o s e l y s i m i l a r p r o c e s s e s . I n t h e p r e s e n t p r o j e c t , p o r c i n e p e p s i n was m o d i f i e d w i t h l i m i t e d q u a n t i t i e s o f r e a g e n t s t o y i e l d l i m i t e d m o d i f i c a t i o n o f t h e c a r b o x y l g r o u p s . E x t e n s i v e d e r i v a t i -z a t i o n o f c a r b o x y l g r o u p s i s d e t r i m e n t a l t o p e p s i n a c t i v i t y s i n c e t h e two a c t i v e - s i t e amino a c i d s were shown t o be a s p a r t i c a c i d ( B a y l i s s e t a l . , 1969$ Chen a n d T a n g , 1972). 4 The a c t i v i t i e s o f t h e m o d i f i e d p e p s i n a g a i n s t v a r -i o u s s u b s t r a t e s i n c l u d i n g s y n t h e t i c d i p e p t i d e s , h e m o g l o b i n , r e c o n s t i t u t e d s k i m m i l k , w h o l e c a s e i n and / c - a n d o e g l - c a s e i n s , w e r e m e a s u r e d . Some p h y s i c a l p r o p e r t i e s o f t h e c a r b o x y l m o d i f i e d enzyme s u c h as pH p r o f i l e s and e l e c t r o p h o r e t i c m o b i l i t y , and p r o p e r t i e s p e r t i n e n t t o t h e p e r f o r m a n c e o f p e p s i n i n c h e e s e - m a k i n g s u c h as pH s t a b i l i t y , c u r d t e n s i o n a n d r a t e o f s y n e r e s i s w e r e s t u d i e d . Some c h e m i c a l p r o p e r -t i e s o f t h e m o d i f i e d enzyme s u c h as t h e r e s p o n s e t o i n h i b i -t o r s a n d k i n e t i c s were a l s o s t u d i e d . The c h a r a c t e r i s t i c s o f t h e m o d i f i c a t i o n , i n c l u d i n g t h e s p e c i f i c i t y o f t h e r e -a c t i o n and t h e e f f e c t o f s u b s t r a t e s on t h e m o d i f i c a t i o n , w e r e i n v e s t i g a t e d . 5 LITERATURE REVIEW C h e m i c a l M o d i f i c a t i o n o f P r o t e i n s I n i t s b r o a d e s t s e n s e , a n y t r a n s f o r m a t i o n w h i c h i n v o l v e s t h e f o r m a t i o n o r r u p t u r e o f a c o v a l e n t b o n d may be r e g a r d e d as c h e m i c a l m o d i f i c a t i o n o f a p r o t e i n . T h i s w o u l d i n c l u d e p r o c e s s e s s u c h as p r o t o n t r a n s f e r , m e t a l c h e l a t i o n , e n z y m e - s u b s t r a t e i n t e r a c t i o n and e v e n h y d r o g e n b o n d i n g ( C o h e n , 1970). I n a more r e s t r i c t e d s e n s e , how-e v e r , c h e m i c a l m o d i f i c a t i o n i s g e n e r a l l y r e f e r r e d t o a s t h e i n t e n t i o n a l a l t e r a t i o n o f p r o t e i n s t r u c t u r e o r c o n -f o r m a t i o n b y c h e m i c a l a g e n t s (Means a n d F e e n e y , 1971). E s s e n t i a l l y , i t i n v o l v e s t h e d e r i v a t i z a t i o n b y s p e c i f i c r e a g e n t s o f some r e a c t i v e s i d e - c h a i n g r o u p s i n t h e p r o t e i n m o l e c u l e s u c h as c h a r g e d a n i o n i c and c a t i o n i c g r o u p s , h y d r o x y i , amide and t h i o l r e s i d u e s . C h e m i c a l m o d i f i c a t i o n o f p r o t e i n s h a s a l o n g h i s t o r y r e l a t e d t o t h e p h a r m a c e u t i c a l , d y e i n g a n d c l o t h i n g i n d u s -t r i e s . The t r e a t m e n t o f a n i m a l h i d e s o r h a i r s f o r human u s e as i n t h e t a n n i n g o f l e a t h e r i s one o f t h e o l d e s t p r o -c e s s e s u l t i l i z i n g c h e m i c a l m o d i f i c a t i o n . T h i s a n c i e n t p r o c e d u r e was r e c e n t l y i m p r o v e d b y t h e use o f g l u t a r a l d e -h y d e , a c r o s s - l i n k i n g r e a g e n t o f p r o t e i n s . S i m i l a r l y , s e v e r a l m o d i f i c a t i o n s were u s e d t o g i v e w o o l f i b r e s s u p e r i o r p e r f o r m a n c e f o r c l o t h i n g . F o r m a l d e h y d e h a d b e e n u s e d t o 6 m o d i f y b a c t e r i a l t o x i n s , r e n d e r i n g them i n c a p a b l e o f e l i -c i t i n g a t o x i c r e s p o n s e b u t s t i l l r e t a i n i n g i t s a b i l i t y t o p r o d u c e a n i m m u n o l o g i c a l r e s p o n s e when i n j e c t e d i n t o a n a n i m a l . W i t h t h e a v a i l a b i l i t y o f new, s p e c i f i c c h e m i c a l r e a g e n t s a n d more s o p h i s t i c a t e d a n a l y t i c a l t e c h n i q u e s , c h e m i c a l m o d i f i c a t i o n has become one o f t h e most p o w e r f u l t o o l s o f p r o t e i n c h e m i s t s f o r t h e s t u d y o f s t r u c t u r e s and f u n c t i o n s o f b i o l o g i c a l l y - a c t i v e p r o t e i n s ( C o h e n , 1968} S t a r k , 1970j Means and F e e n e y , 1971; K n o w l e s , 1974; G l a z e r e t a l . . , 1976). C h e m i c a l m o d i f i c a t i o n s a r e r o u t i n e l y u s e d t o i n v e s t i g a t e t h e r o l e s o f i n d i v i d u a l amino a c i d c h a i n s i n r e l a t i o n t o t h e p h y s i c a l , c h e m i c a l a n d b i o l o g i c a l p r o p e r t i e s o f p r o t e i n s and t o d e t e r m i n e t h e a c t i v e - s i t e r e s i d u e s i n e n z y m e s . P r o t e i n m o d i f i c a t i o n h a s w i d e a p p l i c a t i o n i n o t h e r a r e a s o f b i o c h e m i c a l r e s e a r c h i n c l u d i n g i m m u n o c h e m i s t r y , X - r a y c r y s t a l l o g r a p h y and p u r i f i c a t i o n o f p r o t e i n s . B y m o d i f i c a t i o n o f a n a n t i b o d y and d e t e r m i n a t i o n o f changes i n i t s a b i l i t y t o i n t e r a c t w i t h a n t i g e n , i n f o r m a t i o n c a n b e o b t a i n e d on t h e f u n c t i o n a l g r o u p s i n v o l v e d i n t h e b o n d i n g ( S i n g e r , 1965; H a b e r , 1968). D e t e r m i n a t i o n o f t h e c r y s t a l s t r u c t u r e o f a p r o t e i n a t h i g h r e s o l u t i o n r e q u i r e s t h e p r e p a r a t i o n o f h e a v y atom d e r i v a t i v e s w h i c h a r e i s o m o r p h o u s w i t h t h e p a r e n t c r y s t a l . Heavy atoms may be i n c o r p o r a t e d 7 by selective chemical modification which makes important contributions to X-ray crystallography (Benisek and Richards, 1968). Some proteins form reversible complexes with other substances and have properties which can be used to separate proteins by conventional methods. One example i s the separation of a group of closely related antifreeze glycoproteins from blood serum of Antarctic fishes by complexing with borate (Vandenheede, 1972). In the food industry, chemical modification is mainly used to improve functional properties of proteins. A l k a l i treatments have been used i n the solubilization of protein-containing materials i n preparation for extrusion processing (Van Beek et a l . . 1974). Hydrolysates of some proteins have improved flavor characteristics, better emulsifying property and improved foaming a b i l i t y (Richard-son, 1977). The other chemical approach to modify the function-a l i t y of proteins which has received much attention i s the use of various acylating agents. Egg white modified with 3»3-dimethyl-glutaric anhydride shows increased heat s t a b i l i t y while the foaming capacity i s not significantly affected (Gandhi et a l . . 1968). Vegetable proteins such as soy proteins have been modified with acetic and succinic anhydrides to be used i n coffee whitener formulations 8 ( M e l n y c h y n and S t a p l e y , 1973). The a c y l a t e d p r o t e i n s have d e c r e a s e d v i s c o s i t y and i s o e l e c t r i c p o i n t as w e l l a s m i l d f l a v o r a n d o d o r . U n l i k e c o n v e n t i o n a l v e g e t a b l e p r o t e i n i s o l a t e s , t h e y do n o t " f e a t h e r " o r p r e c i p i t a t e when added t o h o t c o f f e e o r t e a . N - S u c c i n y l a t e d egg y o l k p r o t e i n s a n d o v a l b u m i n have b e e n s u c c e s s f u l l y made f o r u s e i n may-o n n a i s e a n d s a l a d d r e s s i n g (Evans a n d I r o n s , 1971a: b ) . O t h e r a c y l a t e d p r o t e i n s i n c l u d i n g whey, c a s e i n , s e r u m a n d g e l a t i n a r e a l s o u s e d f o r s t a b i l i z i n g o i l - i n - w a t e r e m u l -s i o n s and i n i c e c r e a m m i x ( E v a n s , 1970a; b ) . F i s h myo-f i b r i l l a r p r o t e i n s have b e e n s u c c i n y l a t e d and f o u n d t o f o r m v i s c o u s aqueous d i s p e r s i o n s w i t h good h e a t s t a b i l i t y , a h i g h e m u l s i f y i n g c a p a c i t y , b l a n d f l a v o r and i m p r o v e d f o a m i n g s t a b i l i t y ( G r o n i n g e r , 1973; G r o n i n g e r and M i l l e r , 1975; Chen e t a l . , 1975). A p a r t f r o m i m p r o v i n g t h e f u n c t i o n a l p r o p e r t i e s , f o o d p r o t e i n s a r e m o d i f i e d b y c h e m i c a l methods t o b l o c k d e t e r i o r -a t i v e r e a c t i o n s and t o i m p r o v e n u t r i t i o n a l v a l u e . P r o t e i n amino g r o u p s h a v e b e e n m o d i f i e d b y a c y l a t i o n (B j a m a s on a n d C a r p e n t e r , 1970) a n d d i m e t h y l a t i o n (Galembeck e t a l . , 1977) t o b l o c k t h e M a i l l a r d r e a c t i o n . The n u t r i t i o n a l q u a l i t y o f f o o d p r o t e i n s c a n be i m p r o v e d b y i n c r e a s i n g t h e d i g e s t i b i l i t y o f t h e p r o t e i n s , i n a c t i v a t i n g t o x i c o r i n h i b i t o r y s u b s t a n c e s o r a t t a c h i n g e s s e n t i a l n u t r i e n t s t o t h e p r o t e i n s ; c o l o r i n g a n d f l a v o r i n g a g e n t s c a n a l s o be a t t a c h e d t o i m p r o v e t h e a c c e p t a b i l i t y o f f o o d p r o t e i n s ( F e e n e y , 1977). 9 M o d i f i c a t i o n o f C a r b o x v l G r o u p s Two r e a c t i o n s most commonly e m p l o y e d t o m o d i f y t h e c a r b o x y l g r o u p s o f p r o t e i n s a r e e s t e r i f i c a t i o n a n d c o u p l i n g w i t h n u c l e o p h i l e s m e d i a t e d b y a w a t e r - s o l u b l e c a r b o d i i r a i d e . 1. E s t e r i f i c a t i o n P r o t e i n c a r b o x y l g r o u p s may be e s t e r i f i e d b y a number o f p r o c e d u r e s . C a r b o x y l g r o u p s c a n be c o n v e r t e d t o m e t h y l e s t e r s i n m e t h a n o l c o n t a i n i n g s m a l l amounts o f h y d r o c h l o r i c a c i d ( W i l c o x , 1967). U n l e s s done u n d e r l i m i t i n g c o n d i t i o n s t h a t may r e s u l t i n i n c o m p l e t e r e a c t i o n , a c i d - c a t a l y s e d e s t e r i f i c a t i o n c a n l e a d t o s i d e r e a c t i o n s s u c h as N-*0 a c y l s h i f t o r d e a m i d a t i b n ( W i l c o x , 1967; C o h e n , 1968). The r e a c t i o n i s a l s o a c c o m p a n i e d b y c o n f o r m a t i o n a l changes w h i c h p r e c l u d e i t s u s e w i t h most p r o t e i n s . H o w e v e r , t h e p r o c e d u r e has b e e n u s e d s u c c e s s f u l l y t o s t u d y t h e r o l e o f c a r b o x y l g r o u p s i n more s t a b l e p r o t e i n s s u c h a s l y s o z y m e ( F r a e n k e l - C o n r a t a n d O l c o t t , 19451 F r i e d e n , 1956), chymo-t r y p s i n o g e n ( D o s c h e r a n d W i l c o x , 1961) a n d b o v i n e s e r u m a l b u m i n (Ram and M a u r e r , 1959). D i a z o compounds s u c h as d e r i v a t i v e s o f d i a z o a c e t i c a c i d a r e w i d e l y u s e d t o e s t e r i f y c a r b o x y l i c a c i d s . D i a z o -a c e t a t e s r e a c t w i t h w a t e r and many s i m p l e i n o r g a n i c a n i o n s , 10 l i m i t i n g m o d i f i c a t i o n t o t h e more a c c e s s i b l e c a r b o x y l g r o u p s . M o d i f i c a t i o n o f o n l y 20-30$ o f t h e c a r b o x y l g r o u p i s u s u a l l y p o s s i b l e w i t h a t y p i c a l p r o t e i n . D i a z o a c e t a t e s r e a c t o p t i m a l l y w i t h p r o t e i n ; ^ c a r b o x y l g r o u p s n e a r pH 5» A t l o w e r p H * s , h y d r o l y s i s o f r e a g e n t becomes a p p r e c i a b l e a n d l i m i t s t h e e x t e n t o f m o d i f i c a t i o n . T h i s p r o c e d u r e has b e e n u s e d t o m o d i f y p a n c r e a t i c r i b o n u c l e a s e (Riehm and S c h e r a g a , 1965)» c h y m o t r y p s i n o g e n A ( D o s c h e r and W i l c o x , 1961) a n d p e p s i n ( R a j a g o p a l a n e t a l . , 1966a? L u n d b l a d and S t e i n , 1969). A l k y l h a l i d e s a r e t o o u n s p e c i f i c f o r e s t e r i f i c a t i o n o f p r o t e i n c a r b o x y l g r o u p s u n l e s s t h e y a l s o behave as an a f f i n i t y l a b e l , as i n t h e e s t e r i f i c a t i o n o f t h e a c t i v e - s i t e a s p a r t i c a c i d o f p e p s i n b y p - b r o m o p h e n a c y l b r o m i d e ( G r o s s a n d M o r e l l , 19661 E r l a n g e r e t . a l . , 1966) and t h e a c t i v e -s i t e g l u t a m i c a c i d o f r i b o n u c l e a s e T i b y i o d o a c e t a t e ( T a k a h a s h i e t a l . , 1967)* T r i e t h y l o x o n i u m f l u o r o b o r a t e h a s b e e n u s e d t o e s t e r i f y s p e c i f i c a l l y a s p a r t i c a c i d 52 i n l y s o z y m e ( P a r s o n s a n d R a f t e r y , 1969). 2,. C a r b o d i i m i d e r e a c t i o n The most p o p u l a r method f o r m o d i f y i n g c a r b o x y l g r o u p s i n p r o t e i n s i n v o l v e s t h e u s e o f w a t e r - s o l u b l e c a r b o d i i m i d e s . The c a r b o d i i m i d e s . r e a c t w i t h c a r b o x y l g r o u p s a t s l i g h t l y a c i d i c pH t o g i v e a n 0 - a c y l i s o u r e a ( K h o r a n a , 1953)t an 11 a c t i v a t e d i n t e r m e d i a t e t h a t c a n e i t h e r r e a r r a n g e t o a n a c y l u r e a o r r e a c t w i t h a n u c l e o p h i l e as shown i n F i g . 1. I f t h e n u c l e o p h i l e i s an a m i n e , i t w i l l condense w i t h 0-a c y l i s o u r e a t o y i e l d t h e c o r r e s p o n d i n g a m i d e . The c a r b o d i i m i d e r e a c t i o n has b e e n w i d e l y u s e d i n t h e d e t e r m i n a t i o n o f t h e c a r b o x y l g r o u p c o n t e n t o f p r o t e i n s a s w e l l a s i n t h e s t u d y o f c a r b o x y l g r o u p f u n c t i o n . Under m i l d c o n d i t i o n s , o n l y t h e more a c c e s s i b l e o r r e a c t i v e c a r b o x y l g r o u p s r e a c t , w h i l e i n t h e p r e s e n c e o f d e n a t u r a n t s a n d e x c e s s r e a g e n t s , n e a r l y q u a n t i t a t i v e s u b s t i t u t i o n c a n be o b t a i n e d (Hoare a n d K o s h l a n d , 1967; C a r r a w a y a n d K o s h -l a n d , 1972). T h i s p r o c e d u r e o f f e r s c o n s i d e r a b l e f l e x i b i l i t y i n t h e c h o i c e o f c a r b o d i i m i d e a n d n u c l e o p h i l e . S e v e r a l w a t e r -s o l u b l e c a r b o d i i m i d e s have b e e n u s e d f o r p r o t e i n m o d i f i -c a t i o n . l - G y c l o h e x y l - 3 - ( 2 - m o r p h o l i n y l - 4 - e t h y l ) - c a r b o d i -i m i d e m e t h o - p - t o l u e n e s u l f o n a t e and 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 a r e c o m m e r c i a l l y a v a i l a b l e . l - B e n z 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 i s a l s o f r e q u e n t l y u s e d ( H o a r e and K o s h l a n d , 1966). A l l c a r b o d i -i m i d e s r e a c t s i m i l a r l y a l t h o u g h t h e s m a l l e r r e a g e n t s m i g h t b e e x p e c t e d t o be more a c c e s s i b l e t o p a r t i a l l y b u r i e d c a r b o x y l g r o u p s . D i f f e r e n t n u c l e o p h i l e s c a n be u s e d t o s u i t p a r t i c u l a r e x p e r i m e n t a l c o n d i t i o n s . The i o n i c c h a r a c t e r o f p r o t e i n s 12 R N Protein-COOH + C II N i R' R O NH Protein-C-O-C H N R' rearrange O R ' O Prote in-C-NHCNHR + R"Nu-H O Protein - C - N u - R O R N H C N H R ' FIGURE 1. THE REACTION OF PROTEIN CARBOXYL GROUP WITH WATER-SOLUBLE CARBODIIMIDE AND NUCLEOPHILE. Nu : NUCLEOPHILE. 13 c a n be v a r i e d c o n s i d e r a b l y d e p e n d i n g on t h e c h a r g e o f t h e n u c l e o p h i l e s ( F i g . 2). R a d i o a c t i v e o r c o l o r e d a m i n e s c a n b e e m p l o y e d t o f a c i l i t a t e q u a n t i t a t i o n and i d e n t i f i c a t i o n ( M a t y a s h e t a l . , 1973). C a r b o d i i m i d e a l s o r e a c t s w i t h b o t h s u l f h y d r y l and t y r o s y l - O H g r o u p s . T y r o s i n e c a n be r e g e n e r a t e d b y t r e a t -ment o f t h e d e r i v a t i v e w i t h h y d r o x y l a m i n e a t pH 7 ( G a r r a w a y a n d K o s h l a n d , 1968). S u c c e s s f u l r e g e n e r a t i o n o f t h i o l g r o u p s h a s n o t b e e n r e p o r t e d due t o e x c e s s i v e s t a b i l i t y o f t h e r e s u l t i n g p r o d u c t ( C a r r a w a y and T r i p l e t ! , 1970). P e p s i n The c a t a l y t i c a c t i v i t y o f p e p s i n i n t h e d i g e s t i v e f u n c t i o n o f s t o m a c h was f i r s t i d e n t i f i e d b y t h e I t a l i a n r e s e a r c h e r Abbe S p a l a n z a n i in,1783 ( c i t e d b y G i l l e s p i e , I 8 9 8 ) . T h i s was p e r h a p s t h e f i r s t s c i e n t i f i c d e m o n s t r a t i o n o f e n z y m a t i c a c t i v i t y . P e p s i n was t h e f i r s t enzyme t o be named (by T . Schwann i n 1825) a n d t h e s e c o n d enzyme t o be c r y s t a l l i z e d ( N o r t h r o p , 1930). The t e r m p e p s i n i s a p p l i e d t o t h e g a s t r i c p r o t e i n a s e s f o r m e d b y p a r t i a l p r o t e o l y s i s o f t h e i r i n a c t i v e zymogens, p e p s i n o g e n s . The n o m e n c l a t u r e o f p e p s i n i s c o m p l i c a t e d b y t h e m u l t i p l i c i t y o f t h e enzyme. H o w e v e r , p e p s i n ( E . C . 3»4 ,231 . )has b e e n r e f e r r e d t o b y most a u t h o r s and b y t h e 14 PROTEIN -C + NH 2- CH2CH2SO; R N - C ' N R . V 0 " //> TAURINE PROTEIN - C /^CH^O-j NNH u. fi - fi RN=C=NR' PROTEIN -G + NH2-CH2C VO" NOCH 3 . GLYCINE METHYL ESTER /fi ... /fi PROTEIN-C CH2C NNH / N 0CH 3 /fi + RN=C=NR' PROTEIN -C + NH 2-CH 2CH 2NH 3  V ETHYLENE-DIAMINE fi PROTEIN-C CH 2CH 2NH 3 NH FIGURE 2. DEPENDENCE OF CHARGE ON NUCLEOPHILES ON THE MODIFICATION OF PROTEIN CARBOXYL GROUP. 15 Enzyme C o m m i s s i o n as p o r c i n e p e p s i n A , t h e m a j o r g a s t r i c p r o t e i n a s e f r o m p i g . M o s t o f t h e a v a i l a b l e d a t a on t h e c h e m i c a l s t r u c t u r e and k i n e t i c p r o p e r t i e s o f p e p s i n a r e f r o m s t u d i e s on p o r c i n e p e p s i n A a n d i t s zymogen, p e p s i n o -g e n A . H i g h l y p u r i f i e d o r c r y s t a l l i n e p e p s i n p r e p a r a t i o n s have b e e n o b t a i n e d f r o m t h e g a s t r i c mucosa o f man, cow ( N o r t h r o p , 1933) t c h i c k e n ( L e v c h u k a n d O r e k h o v i c h , 1963) a n d f i s h ( N o r r i s and E l a m , 1940s S p r i s s l e r , 1942). P e p s i n i s a p r o t e i n a s e ( o r p r o t e a s e ) w h i c h c a t a l y s e s t h e c l e a v a g e o f p e p t i d e bonds i n t h e s u b s t r a t e p r o t e i n s . U n l i k e o t h e r t y p e s o f p r o t e a s e s ( s e r i n e , raetallo, a n d s u l f h y d r y l p r o t e a s e s ) , p e p s i n f u n c t i o n s o p t i m a l l y i n a v e r y a c i d i c medium o f a b o u t pH 2-3* P e p s i n a n d p e p s i n -l i k e enzymes a r e t h e r e f o r e r e f e r r e d t o as a c i d p r o t e a s e s . The p r i m a r y s t r u c t u r e o f p o r c i n e p e p s i n was d e t e r -m i n e d r e c e n t l y (Tang e t a l . , 1973; S e p u l v e d a e t a l . , 1975). I t c o n t a i n s 327 amino a c i d r e s i d u e s . P e p s i n s t r u c t u r e i s u n i q u e i n t h a t o n l y one l y s i n e a n d two a r g i n i n e s a r e f o u n d w i t h i n t h e t e r m i n a l 20 r e s i d u e s o f t h e c a r b o x y l end o f t h e p r o t e i n . The o t h e r 307 r e s i d u e s c o n t a i n o n l y one b a s i c amino a c i d , h i s t i d i n e - 9 7 . A l o n g t h e same s t r e t c h a r e 44 a c i d i c r e s i d u e s , i n c l u d i n g a p h o s p h o s e r i n e . The t h r e e - d i m e n s i o n a l s t r u c t u r e o f p o r c i n e p e p s i n h a s b e e n r e s o l v e d a t 0.27 nm r e s o l u t i o n ( N . A n d r e e v a , c i t e d b y T a n g , 1976). 16 The a c t i v e - s i t e r e s i d u e s o f p e p s i n have b e e n i d e n t i -f i e d a s Asp-32 ( B a y l i s s e t a l . , 1969) and Asp-2l5, (Chen a n d T a n g , 1972). They a r e f o u n d t o be l o c a t e d i n a n a p p a r e n t b i n d i n g c l e f t i n t h e t h r e e - d i m e n s i o n a l s t r u c t u r e . T a b l e 1 s u m m a r i z e s some p h y s i c a l p r o p e r t i e s o f p o r c i n e p e p s i n a n d p e p s i n o g e n . P e p s i n o g e n , t h e p r e c u r s o r o f p e p s i n , i s a c t i v a t e d a t pH b e l o w 5 t o f o r m p e p s i n u p o n t h e r e m o v a l o f t h e 4 l - r e s i d u e a m i n o - t e r m i n a l p o r t i o n o f t h e zymogen. The mechanism- f o r p e p s i n o g e n a c t i v a t i o n i s s t i l l n o t k n o w n , a l t h o u g h s e v e r a l m o d e l s h a v e b e e n p r o p o s e d ( T a n g , 1970} A l - J a n a b i e t a l . . 1972; K a s s e l l and K a y , 1973). P e p s i n i s a p r o t e a s e w i t h b r o a d s i d e c h a i n s p e c i f i -c i t y ( H i l l , 1965). The s e n s i t i v e bonds a r e g e n e r a l l y p r e s e n t i n d i p e p t i d y l u n i t s c o n t a i n i n g a t l e a s t one h y d r o p h o b i c a m i n o a c i d r e s i d u e s u c h as p h e n y l a l a n i n e , t y r o s i n e , l e u c i n e a n d m e t h i o n i n e ( T a n g , 1963). P e p t i d e s c o n t a i n i n g p - n i t r o -p h e n y l a l a n i n e ( I n o u y e a n d F r u t o n , 1967)» 3,5 - d i n i t r © t y r o s i n e ( K n o w l e s e t a l . , 1969) a n d d i i o d o t y r o s i n e ( J a c k s o n e t a l . . 1965) a r e h y d r o l y s e d b y p e p s i n and have b e e n u s e d i n k i n e t i c i n v e s t i g a t i o n s . I n a d d i t i o n t o h y d r o l y s i n g p e p t i d e b o n d s , p e p s i n c a t a l y s e s t r a n s p e p t i d a t i o n o f t h e a m i n o - t r a n s f e r t y p e (Neumann e t a l . , 1959; F r u t o n e t . a l . , 1961). P e p s i n c a n a l s o a c t as a n e s t e r a s e , h y d r o l y s i n g e s t e r l i n k a g e s i n s u i t a b l e 17 Table 1. Some physical properties of porcine pepsin and pepsinogen. Pepsin Pepsinogen Reference s 2 0 , w (sec X 1 0 1 3 ) 2.96-3 .0 3 . 2 - 3 . 3 5 a) Blumenfeld and Perlmann, .1959. b) Arnon and Perl-mann, 1963. D 2 0 , w (cm 2/sec X 1 0 1 3 ) 8.70 7.54 c) Orekhovich et a l . , 1956. Molecular Weight 35,000 41,000 A c ( n m ) 216 236 d) Ryle, i 9 6 0 . e) Ryle, 1965. -M366 nm 232 212' „278 nm Tflol 50 ,990 1 (MW=35000) 51,300 1 f ) Ryle and Por-(MW=41000) t e r , 1959. 18 s u b s t r a t e s ( L o k s h i n a e t a l . . . , 1964; I n o u y e and F r u t o n , 1967). P e p s i n has b e e n d e m o n s t r a t e d t o p a r t i c i p a t e i n t h e " p l a s t e i n r e a c t i o n " (Wasteneys a n d B o r s o o k , 1930). I t c a t a -l y s e s p e p t i d e b o n d s y n t h e s i s b y d i r e c t c o n d e n s a t i o n o f « < -amino and »c - c a r b o x y l g r o u p s o f o l i g o p e p t i d e s a t h i g h s u b -s t r a t e c o n c e n t r a t i o n (Determann e t a l . , 1965). P e p s i n b e l o n g s t o a g r o u p o f enzymes c l o s e l y s i m i l a r i n s t r u c t u r e a n d a c t i v i t y . These i n c l u d e o t h e r g a s t r i c e n -zymes ( e . g . , g a s t r i c s i n a n d c h y m o s i n ) , p r o t e a s e s f r o m m i c r o -o r g a n i s m s , p r o t e a s e s f r o m p l a n t s , mammalian l y s o s o m a l p r o t e a s e s ( e . g . , c a t h e p s i n D a n d E ) a n d p r o t e a s e s i n b l o o d p l a s m a a n d s e m i n a l p l a s m a . . The amino a c i d s e q u e n c e s o f a l l t h e a c i d p r o t e a s e s s t u d i e d so f a r a r e homologous ( S e p u l v e d a e t a l . , 1975)» a p p a r e n t l y a r e s u l t o f d i v e r g e n t e v o l u t i o n , f r o m a common a n c e s t r a l p r o t e i n (Hofmann, 1974; T a n g , 1976). The a c t i v e s i t e s o f t h e a c i d p r o t e a s e s a r e a l s o a l i k e and thea'e enzymes a r e i n h i b i t e d b y t h e same s i t e - s p e c i f i c i n h i b i t o r s . T h e s e f a c t s s u g g e s t t h a t enzymes i n t h i s g r o u p have a s i m i l a r c a t a l y t i c m e c h a n i s m , a l t h o u g h t h e i r s p e c i f i c i t y may v a r y due t o d i f f e r e n c e s i n t h e t o p o g r a p h y o f t h e i r b i n d i n g s i t e s ( T a n g , 1976). 19 Chemical Modification of Pepsin Chemical modification has contributed greatly to the identification of active-site residues in pepsin as well as other amino acid side groups essential for pepsin activity. Diazoacetyl norleucine methyl ester, a derivative of diazoacetic acid, was found to inactivate pepsin by esteri-fication of only one carboxyl group (Rajagopalan et a l . , 1966a; Lundblad and Stein, 1969). Isolation and sequence analysis of a peptide containing the diazo modified residue indicates that the esterified aspartyl group i s located at residue 215 (Bayliss et.al., 1969). A second carboxyl group in the active site of pepsin was determined by the use of a substrate-like epoxide i n -activator, l,2-epoxy-3-(p-nitrophenoxy) propane (EPKP). Sequence determination of the EPNP-containing peptide placed the aspartyl residue at position 32!(Chen and Tang, 1972). A third carboxyl group i n pepsin was modified by bromophenacyl bromide (Erlanger et al... 1965). However, the fact that the f u l l y reacted enzyme retained about 20% of i t s activity and that other acid proteases were unaffected suggests that this modified group was not directly involved i n catalysis (Clement, 1973). The presence of an arginyl residue near the active centre of pepsin was demonstrated by modification with phenylglyoxal (Kitson and Knowles, 1971) and butanedione 20 (Huang and Tang, 1972), and was found to be Arg- 316. The f u l l y reacted enzyme retained about 20$ of a c t i v i t y . The reaction was retarded by the presence of peptide substrates i n d i c a t i n g the proximity of the modified residue to the active centre of the enzyme (Huang and Tang, 1972). Selective modification of carboxyl groups i n pepsin was ca r r i e d out using a colored amine, N-(2,4-dinitrophenyl)-hexamethylenediamine and a water-soluble carbodiimide (Mat-yash et a l , , 1973). Incorporation of one amine molecule per molecule of pepsin led to a drop of a c t i v i t y of about 40$. Three carboxyl groups were modified* /& -carboxyl group of aspartate, r-carboxyl group of glutamate. and the carboxyl group of C-terminal alanine. N-Bromosuccinimide has been used to modify tryptophan residues i n pepsin (Lokshina and Orekhovich, 1964; Green and Witkop, 1964). Pepsin i n a c t i v a t i o n by the reagent amounted to 85-90%. Dopheide and Jones (1968) used 2-hydroxyl-5-nitrobenzylbromide f o r tryptophan modification. Incorporation of two residues of the reagent was observed, r e s u l t i n g i n a loss of only 25-30$ of p r o t e o l y t i c and pep-tidase a c t i v i t y . Methionine residues, i n pepsin were alkylated by! iodo-acetic acid with no observable change i n a c t i v i t y , i n d i c a t i n g that methionine residues were not important f o r the action of pepsin (Lokshina and Orekhovich, 1964). The amino groups 21 o f p e p s i n were a l s o shown t o be n o n - e s s e n t i a l f o r enzyme a c t i v i t y . A c e t y l a t i o n o f t h e amino g r o u p s w i t h k e t e n e d i d n o t a f f e c t p r o t e a s e a c t i v i t y ( H e r r i o t t and N o r t h r o p , 1934). T h i s was f u r t h e r s u p p o r t e d b y d e a m i n a t i o n w i t h n i t r o u s a c i d ( P h i l p o t a n d S m a l l , 1938) a n d N - e t h o x y f o r m y l a t i o n ( M e l c h i o r and F a h r n e y , 1970) b o t h o f w h i c h c a u s e d n o a p p r e -c i a b l e change i n a c t i v i t y . T y r o s i n e r e s i d u e s have b e e n m o d i f i e d b y a c e t y l a t i o n a n d i o d i n a t i o n . A 95% i n h i b i t i o n i n p r o t e o l y t i c a c t i v i t y was o b s e r v e d a f t e r a c e t y l a t i o n o f 11-12 t y r o s i n e - O H g r o u p s i n t h e p e p s i n m o l e c u l e . The e s t e r a s e and p e p t i d a s e a c t i v i t y , h o w e v e r , was i n c r e a s e d ( L o k s h i n a and O r e k h o v i c h , 1966). I n c o m p a r i s o n , i o d i n a t i o n l e d t o a d e c r e a s e i n p r o t e a s e , p e p -t i d a s e a n d e s t e r a s e a c t i v i t i e s ( H o l l a n d and F r u t o n * 1968). Sequence a n a l y s i s o f t h e i o d i n a t e d p e p t i d e s l o c a t e d t h e m o d i f i e d t y r o s i n e r e s i d u e s a t p o s i t i o n s 9 and 175 ( M a i n s e t a l . , 1973). 22 MATERIALS AND METHODS B i o c h e m i c a l s and S p e c i a l C h e m i c a l s A l l c h e m i c a l s u s e d were o f r e a g e n t g r a d e u n l e s s s p e c i f i e d o t h e r w i s e . P o r c i n e p e p s i n ( E . C . 3.4.23,.l|from h o g s t o m a c h m u c o s a , 2X c r y s t a l l i z e d ) was p u r c h a s e d f r o m S i g m a C h e m i c a l C o . and W o r t h i n g t o n B i o c h e m i c a l C o r p . The l o t f r o m S i g m a h a d an a s s a y o f 3»520 u n i t s / m g p r o t e i n , w h i l e t h e l o t f r o m W o r t h i n g t o n h a d a n a s s a y o f 2,700 u n i t s / m g p r o t e i n . C r u d e p e p s i n (1»10,000), p e p s i n o g e n ( f r o m h o g s tomach) a n d chymo-s i n ( r e n n i n , - E . C . 3.4.23.4; f r o m c a l f s t o m a c h ) were p r o d u c t s o f S i g m a C h e m i c a l C o . R e n n e t (NF r e n n i n ) was p u r c h a s e d f r o m I C N P h a r m a c e u t i c a l s , I n c . The two w a t e r - s o l u b l e c a r b o d i i m i d e s u s e d i n t h i s p r o j e c t , l - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n y l - 4 - e t h y l ) c a r b o d i -i m i d e m e t h o - p - t o l u e n e s u l f o n a t e a n d 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 p u r c h a s e d f r o m S i g m a C h e m i c a l C o . G l y c i n e m e t h y l e s t e r h y d r o c h l o r i d e was a p r o d u c t o f A l d r i c h C h e m i c a l C o . , I n c . , w h i l e t h e m e t h y l e s t e r s o f a r g -i n i n e , l e u c i n e , l y s i n e , t y r o s i n e and t r y p t o p h a n w e r e f r o m S i g m a C h e m i c a l C o . E t h y l e n e d i a m i n e d i h y d r o c h l o r i d e and t a u r i n e (2 - a m i n o e t h a n e s u l f o n i c a c i d ) w e r e a l s o f r o m S i g m a C h e m i c a l C o . 23 The s y n t h e t i c d i p e p t i d e s , N - a c e t y l - L - p h e n y l a l a r i y l -d i i o d o - L - t y r o s i n e , N - a c e t y l - D - p h e n y l a l a n y l - L - t y r o s i n e a n d N - c a r b o b e n z o x y - L - g l u t a m y l - L - t y r o s i n e w e r e p r o d u c t s o f S i g m a C h e m i c a l C o . Two p e p s i n i n h i b i t o r s , l,2 - e p o x y-3-( p - n i t r o p h e n o x y ) p r o p a n e and b r o m o p h e n a c y l b r o m i d e were p u r c h a s e d f r o m E a s t m a n Kodak C o . The s u b s t r a t e f o r t h e d e t e r m i n a t i o n o f p r o t e o l y t i c a c t i v i t y , a c i d - d e n a t u r e d h e m o g l o b i n ( b o v i n e s e r u m ) , was f r o m W o r t h i n g t o n B i o c h e m -i c a l C o r p . 3-(2 - A m i n o e t h y l ) i n d o l e a n d p - t o l u e n e s u l f o n i c a c i d were p r o d u c t s o f M a t h e s o n C o l e m a n & B e l l , N o r w o o d , O h i o . N i n h y d r i n a n d h y d r i n d a n t i n were p u r c h a s e d f r o m E a s t m a n K o d a k C o . 24 M e t h o d s 1. M o d i f i c a t i o n o f c a r b o x y l g r o u p s The c a r b o x y l g r o u p s i n p e p s i n , p e p s i n o g e n a n d c h y -m o s i n were m o d i f i e d b y t h e c a r b o d i i m i d e - p r o m o t e d amide f o r m -a t i o n . The method was e s s e n t i a l l y t h a t d e s c r i b e d b y H o a r e a n d K o s h l a n d (1967) f o r t h e d e t e r m i n a t i o n o f c a r b o x y l c o n -t e n t i n p r o t e i n s . H o w e v e r , s u f f i c i e n t l y l o w e r c o n c e n t r a t i o n s o f c a r b o d i i m i d e and n u c l e o p h i l e were u s e d t o a c h i e v e s e l e c -t i v e m o d i f i c a t i o n . D e n a t u r a n t s s u c h a s 8 M u r e a o r 6 M g u a n i d i n e h y d r o c h l o r i d e , as s u g g e s t e d b y t h e above w o r k e r s f o r t o t a l c o v e r i n g o f c a r b o x y l g r o u p s , were o m m i t t e d i n t h e p r e s e n t i n v e s t i g a t i o n . The p r o t e i n (10 mg/ml) a n d n u c l e o p h i l e were d i s s o l v e d i n d i s t i l l e d w a t e r . The pH o f t h e s o l u t i o n was a d j u s t e d w i t h 1 N NaOH t o 5 . 5 . The w a t e r - s o l u b l e c a r b o d i i m i d e was a d d e d a s a s o l i d t o b r i n g i t s c o n c e n t r a t i o n t o t h e d e s i r e d l e v e l . The r e a c t i o n m i x t u r e was k e p t a t room t e m p e r a t u e a n d s t i r r e d c o n t i n u o u s l y f o r 90 m i n u t e s . The pH o f t h e r e a c t i o n m i x t u r e was k e p t c o n s t a n t a t 5 .5 b y a u t o m a t i c a d d i t i o n o f 0.1 N H C l , u s i n g a R a d i o m e t e r t y p e T T T I c t i t r -a t o r ( C o p e n h a g e n , D e n m a r k ) . The r e a c t i o n was t e r m i n a t e d b y t h e a d d i t i o n o f e x c e s s 3 M s o d i u m a c e t a t e b u f f e r a t pH 5*5 w h i c h r e a c t e d w i t h e x c e s s n u c l e o p h i l e . The r e s i d u a l r e a g e n t s were removed b y e x h a u s t i v e d i a l y s i s a g a i n s t d i s -25 t i l l e d water for 48 hours at 4° C and the modified protein was recovered by lyophilization. To determine the extent of carboxyl modification, the modified protein was subjected to amino acid analysis. The protein was hydrolysed for 24 hours at 110° C with 3-(2-aminoethyl) indole and p-toluenesulfonic acid, as des-cribed by Liu and Chang (1971). The hydrolysate was then neutralized and applied to an amino acid analyser column. From the chromatogram, the peak area of the particular amino acid which had been incorporated was measured and compared to that of the control. The increase i n peak area was pro-portional to the number of molecules of amino acid methyl ester incorporated into the protein, or the number of car-boxyl groups modified. 2. Determination of milk clotting activity The milk clotting activity of pepsin and chymosin was determined by the methods of Berridge (1945) and Folt-mann (1970) with some modifications. Commercial spray-dried skimmilk powder was used as substrate and was kept desiccated at 4°C. The substrate solution was pre-pared by reconstituting 12 g of skimmilk powder i n 100 ml of 0.01 M CaClg with vigorous s t i r r i n g for 5 minutes with-out foaming. The reconstituted skimmilk had a pH of about 26 6.3. The pH can be adjusted by varying the concentration of CaCl 2 from 0.001 M (pH=6.5) to 0.08 M (pH=5.7) and the addition of small amount of HCl or NaOH. The reconstituted skimmilk substrate was l e f t at room temperature for one hour to equilibrate, The skim-milk (10 ml) was pipetted into a stoppered test tube and incubated in a water bath at 30° C for 10 minutes. The enzyme was diluted with citrate buffer to an activity cor-responding to a clotting time of 4-5 minutes. The diluted enzyme solution (1 ml), pre-incubated to 30°C, was pipet-ted into the test tube with thorough mixing. A stopwatch was started. The milk was kept flowing from one end of the stoppered tube to the other. The moment when the thin film of milk broke into visible particles was recorded as the clotting time. As defined by Berridge (1945), one unit of milk clotting activity was the amount of enzyme which would clot 10 ml of reconstituted skimmilk i n 100 seconds at 30°C. The specific activity was expressed as milk clot-ting activity per mg protein. The enzyme concentrations were determined by the method of Lowry et a l . (1951) using bovine serum albumin as a protein standard. 27 3. Determination of proteolytic activity The assay method of Anson (1938) was followed with modifications. Acid-denatured hemoglobin was used as sub-strate. Substrate solution was freshly prepared by dis-solving hemoglobin powder in d i s t i l l e d water. Hydrochloric acid was added to yield the desired pH, and the concentra-tion was adjusted to 2.0% (w/v) protein by the addition of d i s t i l l e d water. The enzyme (0.5 ml), in suitable dilution, was added to a stoppered test tube containing 0.5 ml of 0.1 M KCl/HCl buffer (citrate buffer for pH's above 2.5) of appropriate pH. For routine analysis, the optimum pH for pepsin activity (pH 2.0) was used. The enzyme and substrate solutions were equilibrated i n a water bath at 37°C for 10 minutes. The substrate solution (0.5 ml) was added to the assay tubes with vigorous mixing using a vortex mixer. After incubation at 37° C for exactly 10 minutes, 10 ml of 5% (w/v) trichloroacetic acid (TCA) were added to the assay tubes. TCA stopped the enzyme reaction and precipitated the unhydrolysed substrate. The samples were f i l t e r e d through Whatman No. 2 papers and the absorbance at 280 nm of the f i l t r a t e was read with a Beckman DB spectrophotometer using 1 cm cuvettes. A blank was run i n which TCA was added before the enzyme solution. The absorbance of the blank was measured and deducted from the sample absorbance. A l l 2 8 d e t e r m i n a t i o n s were r u n i n d u p l i c a t e 1 and t h e a v e r a g e s were u s e d f o r c a l c u l a t i n g t h e p r o t e o l y t i c a c t i v i t y . One u n i t o f p r o t e o l y t i c a c t i v i t y was d e f i n e d a s t h e amount o f enzyme t h a t p r o d u c e d a n a b s o r b a n c e o f 0.001 p e r m i n u t e a t 37°C . The s p e c i f i c a c t i v i t y was e x p r e s s e d as t h e p r o t e o l y t i c a c t i v i t y p e r mg p r o t e i n . 4. D e t e r m i n a t i o n o f p e p t i d a s e a c t i v i t y The p e p t i d a s e a c t i v i t y o f p e p s i n was d e t e r m i n e d u s i n g N - a c e t y l - L - p h e n y l a l a n y l - d i i o d o - L - t y r o s i n e (APDT) as s u b -s t r a t e . The a s s a y method o f J a c k s o n e t a l . (19^5) was f o l -l o w e d w i t h some m o d i f i c a t i o n s a s d e s c r i b e d b y R y l e (1970). N i n h y d r i n r e a g e n t was p r e p a r e d b y d i s s o l v i n g 20 g n i n h y d r i n a n d 6 g h y d r i n d a n t i n i n 750 m l o f 2 - m e t h o x y e t h a n o l . To t h i s was added 250 m l o f a c e t a t e b u f f e r (544 g CH^COO. 3 H 20 + 100 m l o f g l a c i a l a c e t i c a c i d ) . The r e a g e n t was s t o r e d u n d e r n i t r o g e n i n a d a r k b o t t l e e q u i p p e d w i t h a d i s -p e n s e r . The s u b s t r a t e s o l u t i o n (1.0 mM) , p r e p a r e d b y d i s s o l v i n g APDT i n 0.01 N NaOH , was s t a b l e f o r s e v e r a l weeks when k e p t i n t h e c o l d r o o m . The enzyme s o l u t i o n (0.5 m l ) was added t o t u b e s w i t h 0.25 m l o f H C l o f a p p r o p r i a t e n o r m a l i t y a n d i n c u b a t e d i n a w a t e r b a t h a t 37°C . A f t e r 10 m i n u t e s , 0.25 m l o f APDT s o l u t i o n was added t o t h e r e a c t i o n t u b e s a n d 1.0 m l o f 29 n i n h y d r i n r e a g e n t was added t o t h e b l a n k s . A f t e r e x a c t l y 10 m i n u t e s , 1.0 m l o f n i n h y d r i n r e a g e n t was p i p e t t e d t o t h e r e a c t i o n t u b e s and a t a n y t i m e 0.25 m l o f APDT was added t o t h e b l a n k s . A l l t h e t u b e s , s t o p p e r e d b y m a r b l e s , were p l a c e d i n a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s t o d e v e l o p t h e c o l o r , a n d were t h e n c o o l e d w i t h c o l d w a t e r . The c o n t e n t s o f t h e t u b e s were d i l u t e d w i t h 5 m l o f 60$ ( v / v ) e t h a n o l . A f t e r m i x i n g , t h e a b s o r b a n c e o f t h e s o l u t i o n a t 570.nm was r e a d a g a i n s t t h e b l a n k s . D u p l i c a t e s were r u n f o r a l l s a m p l e s a n d b l a n k s , a n d t h e a v e r a g e s were u s e d t o c a l c u l a t e t h e p e p t i d a s e a c t i v i t y , e x p r e s s e d a s APDT u n i t s . One APDT u n i t i s d e f i n e d as t h e q u a n t i t y o f enzyme w h i c h l i b e r a t e s 1.0 m i c r o m o l e o f d i i o d o t y r o s i n e p e r m i n u t e a t 37° C . S i n c e E Q ^ f o r * n e c o l o r r e a c t i o n o f d i i o d o -t y r o s i n e i s 22.8, t h e v o l u m e o f t h e s o l u t i o n was 7 m l a n d t h e t i m e o f i n c u b a t i o n was 10 m i n u t e s , A £ 1 7 n was c o n v e r t e d prO nm t o APDT u n i t s b y m u l t i p l y i n g b y 0.0306. S p e c i f i c p e p t i d a s e a c t i v i t y was e x p r e s s e d as APDT u n i t s / m g p r o t e i n . 5. A s s a y o f p e p s i n w i t h x - c a s e i n and « s 3 l - c a s e i n P e p s i n a c t i v i t y was a s s a y e d b y a method u s i n g K-c a s e i n a s s u b s t r a t e ( D o u i l l a r d a n d R i b a d e a u Dumas, 1970). - O a s e i n m i x t u r e s were a l s o u s e d as s u b s t r a t e . 30 A : - C a s e i n a n d « : s l - c a s e i n were p r e p a r e d b y t h e method o f Z i t t i e a n d C u s t e r (1963). S o l u t i o n s o f J< - c a s e i n and o f g l -c a s e i n (0.2% w / v ) were made b y d i s s o l v i n g t h e p r o t e i n s i n 0.05 M c i t r a t e b u f f e r , pH 5*3. A m i x t u r e o f K - c a s e i n a n d * * g l - c a s e i n ( l i l ) was p r e p a r e d b y m i x i n g e q u a l v o l u m e s o f t h e 0.2% s o l u t i o n s , w h i c h gave a f i n a l c o n c e n t r a t i o n o f 0.195 X - c a s e i n a n d 0.1$ « * s l - c a s e i n . The s u b s t r a t e s o l u -t i o n s were i n c u b a t e d i n s t o p p e r e d t e s t t u b e s a t 37° C . Enzyme s o l u t i o n o f a p p r o p r i a t e c o n c e n t r a t i o n was added w i t h i m m e d i a t e m i x i n g . The t i m e a t w h i c h v i s i b l e p a r t i c l e s a p p e a r e d was t a k e n as t h e c l o t t i n g t i m e . One u n i t o f a c t i v i t y was a r b i t r a r i l y d e f i n e d as t h e amount o f enzyme w h i c h w o u l d c l o t 10 m l o f c a s e i n s o l u t i o n i n 100 s e c o n d s a t 37°C . S p e c i f i c a c t i v i t y was e x p r e s s e d as c l o t t i n g a c t i v i t y p e r mg p r o t e i n . 6. D e t e r m i n a t i o n o f r a t e o f c a s e i n h y d r o l y s i s The r a t e o f r e l e a s e o f n o n - p r o t e i n n i t r o g e n f r o m w h o l e c a s e i n b y p e p s i n was d e t e r m i n e d b y a method d e s c r i b e d b y G r e e n (1972). The s u b s t r a t e s o l u t i o n c o n t a i n e d 1% (w/v) w h o l e c a s e i n i n 0.1 M s o d i u m c i t r a t e b u f f e r , pH 5.3 o r 6.5. A f t e r i n c u b a t i o n a t 30° C f o r 10 m i n u t e s , enzyme was a d d e d . A t t i m e i n t e r v a l s , s a m p l e s were w i t h d r a w n a n d s u f f i c i e n t 10% (w/v) TCA was added t o g i v e a f i n a l TCA c o n c e n t r a t i o n 31 of 3$. After f i l t e r i n g through Whatman No. 2 papers, the nitrogen content of the f i l t r a t e was determined by the procedure of Lang (1958). The rate of liberation of TCA-soluble nitrogen was expressed as jig N released/mg casein. 7. Agarose gel electrophoresis Electrophoresis of enzymes and milk proteins was carried out with the agarose film cassette system of Analy-t i c a l Chemists, Inc. (Palo Alto, California) The agarose films contained 1.2$ agarose, 10$ sucrose and 0.035$ EDTA in 0.05 M barbital buffer, pH 8.6. The plate consisted of a thin layer of agarose gel adhered to a transparent plastic backing. Samples (1-2 jul) containing 20-40 jag proteins were pipetted into the pre-castrsample wells with a Hamilton microliter sample dispenser and a disposable t i p . The agarose plate was f i t t e d into a cas-sette cover. Electrophoresis was started by inserting the film-loaded cassette onto the electrophoresis c e l l /power supply unit. The electrophoresis cells contained 200 ml of 0.05 M barbital buffer, pH 8.6, with 0.035$ EDTA. Since the film carried a voltage gradient of 15 V/cm with negligible heat buildup, no cooling was necessary. After electrophoresis which took 35-50 minutes, the plate was removed from the cassette cover and stained with 32 0.2$ (w/v) amido b l a c k 10B i n 5$ ( v / v ) a c e t i c a c i d f o r 15 m i n u t e s i n a s t a i n i n g b a t h w i t h c o n t i n u o u s s t i r r i n g b y a m a g n e t i c s t i r r e r . The s t a i n e d f i l m was r i n s e d w i t h 5$ a c e t i c a c i d and d r i e d a t 7 2 ° C f o r 15 m i n u t e s i n a n o v e n . The d r i e d f i l m was d e s t a i n e d b y w a s h i n g t w i c e w i t h 5$ a c e t i c a c i d , a n d t h e d e s t a i n e d g e l p l a t e was d r i e d a g a i n i n t h e o v e n . F o r e l e c t r o p h o r e s i s o f m i l k c u r d s , t h e c u r d s were d i s s o l v e d i n t h e e l e c t r o p h o r e t i c b u f f e r c o n t a i n i n g 6 M u r e a . The a g a r o s e f i l m was e q u i l i b r a t e d f o r one m i n u t e w i t h t h e u r e a - c o n t a i n i n g b a r b i t a l b u f f e r b e f o r e sample a p p l i c a t i o n . 8. D e t e r m i n a t i o n o f i s o e l e c t r i c p o i n t The i s o e l e c t r i c p o i n t o f p e p s i n s a m p l e s was d e t e r -m i n e d b y g e l e l e c t r o p h o r e s i s a t d i f f e r e n t p H * s . The a g a r o s e f i l m was c u t up i n t o s t r i p s a n d e q u i l i b r a t e d i n 0.1 M b u f f e r o f a p p r o p r i a t e pH ( K C l / H C l b u f f e r f o r pH 1-2, c i t r a t e b u f f e r f o r pH 3-5)• A f t e r s a m p l e a p p l i c a t i o n , . e a c h s t r i p was c o n -n e c t e d b y b u f f e r c o n t a i n e d i n two 5 -ml b e a k e r s w h i c h w e r e p l a c e d i n t h e two e l e c t r o d e c o m p a r t m e n t s . E a c h compartment was f i l l e d w i t h s u f f i c i e n t 0,2 1 N a C l s o l u t i o n w h i c h was c o n n e c t e d t o t h e b u f f e r i n t h e b e a k e r s b y a s m a l l p a p e r s t r i p . F i v e s a m p l e s were r u n a t t h e same t i m e i n b u f f e r s w i t h p H ' s o f 1 .0, 2 .0 , 3 .0 , 4.0 a n d 5 . 0 . 33 A f t e r electrophoresis, the pH*s of the buffers were measured to detect pH change that may occur during the run. A f t e r staining, the m o b i l i t i e s of the enzyme bands were measured and plotted against the pH*s. The s t r a i g h t l i n e curve obtained by l i n e a r regression analysis was extraplot-ated to cut the pH axis which corresponded to zero mobility. This was the estimated i s o e l e c t r i c point of the enzyme. 9. Determination of and k. ^ The i n i t i a l v e l o c i t y (V q) of the pepsin-catalysed hydrolysis of APDT, expressed as jmoles diiodotyrosine l i b e r -ated per minute, was measured at d i f f e r e n t substrate concen-t r a t i o n s . The Michaelis constant, K , was determined from m the Lineweaver-Burk plot which i s the r e c i p r o c a l of substrate concentration against the r e c i p r o c a l of v Q . Linear regres-sion analysis was employed to y i e l d the b e s t - f i t curves. The molecular a c t i v i t y c o e f f i c i e n t or c a t a l y t i c constant, ^cat* was calculated by the Michaelis-Menten equation! v 0 - * c a t [sum / ( K m + rsj) where £E] was the t o t a l enzyme concentration and [S] was the substrate concentration at zero time. The concentration of pepsin was estimated from the absorbance at 280 nm, assuming a molar a b s o r p t i v i t y of 50,990. K m and k a.j. were determined at both pH 2.0 and 4.5. 34 10. Assay of pepsinogen The proteolytic and milk clotting activities of porcine pepsinogen were determined after activation of the zymogen. Pepsinogen was dissolved i n 0.01 M KCl/HCl buffer, pH 2.0 at a concentration of 1 rag/ml. The solution was kept at room temperature overnight when a l l the zymogen was converted to the active enzyme (Herriott, 1938). 11. Determination of pepsin s t a b i l i t y near neutral pH The pH s t a b i l i t y of both crystalline and crude pepsins was determined by the methods described by Lowenstein (1974) and Green (1972). The enzyme s t a b i l i t y i n buffer was measured by incu-bation of a suitable concentration of pepsin i n 0.05 M phos-phate buffer, pH 6.5, at 3 0 ° C . At time intervals, an a l i -quot of sample was removed and the milk clotting and proteo-l y t i c activities were determined. The changes in activities were followed for 2-4 hours. The s t a b i l i t y of pepsin i n milk u l t r a f i l t r a t e s was also studied. Reconstituted milk (from skimmilk powder) was passed through an u l t r a f i l t r a t i o n c e l l (Model 52, Amicon Corp., Lexington, MA.) equipped with a PM Diaflo ultra-f i l t e r . The f i l t r a t e collected was stored at 4°C and used within two days after preparation. 35 The enzyme was incubated at 30° C i n the milk ultra-f i l t r a t e which has been previously adjusted to pH 6.60 with 1 N lactic acid. The pH of the incubation mixture was lowered by adding l a c t i c acid at 15-minute intervals, at approximately the same rate at which i t was lowered during cheese-making. The scheme suggested by Green (1972) was followed (Table 2). Samples of enzyme were taken at various time intervals for assay of milk clotting and proteolytic a c t i v i t i e s . 12. Determination of thermal s t a b i l i t y of pepsin The thermal s t a b i l i t y of pepsin was determined by measuring both milk clotting and proteolytic a c t i v i t i e s of the enzyme at increasing temperatures. For proteolysis, the activity was measured at 30, 40, 50, 60 and 70° C. For milk clotting, the activity was determined at 25t 30, 35, 40, 45 and 50°C. The pH was 2.0 for proteolysis and 6.3 for milk coagulation. The Q 1 0 values were calculated which represent the ratio of the activity at temperature (T + 10)° to that at T°. 36 T a b l e 2. A d j u s t m e n t o f pH o f m i l k u l t r a f i l t r a t e f o r s t a b i l i t y t e s t o f p e p s i n . I n c u b a t i o n Time pH C o r r e s p o n d i n g ( m i n u t e ) C h e e s e - m a k i n g S t a g e 1 6 .60 Enzyme a d d i t i o n 50 6.50 C u t t i n g 120 6 .40 Maximum s c a l d 160 6.25 210 5.90 P i t c h i n g 37 13. D e t e r m i n a t i o n o f c u r d t e n s i o n The f i r m n e s s o r c u r d t e n s i o n o f t h e c o a g u l u m f r o m p e p s i n - c u r d l e d m i l k was d e t e r m i n e d b y a method d e v e l o p e d b y H e h i r (1968), l a t e r i m p r o v e d b y E l l i s (1972). T h i s t e c h n i q u e i n v o l v e s t h e measurement o f t h e w e i g h t t r a n s -f e r when a w e i g h t i s p l a c e d on t h e s u r f a c e o f a c o a g u l a t e d m i l k s a m p l e . P l a s t i c b e a k e r s (50 m l ) were u s e d f o r c u r d t e n s i o n d e t e r m i n a t i o n s a n d were r e d u c e d t o a s t a n d a r d w e i g h t b y s h a v i n g t h e r i m s . U s i n g t h e t a r e f a c i l i t y on a 1,200 g (+ 0.01 g) c a p a c i t y , d i g i t a l t o p - p a n b a l a n c e ( S a r t o r i u s 3704) t h e w e i g h t o f t h e b e a k e r s was r e d u c e d t o z e r o . A s t a n d a r d v o l u m e o f r e c o n s t i t u t e d s k i m m i l k (19.8 m l ) was p i p e t t e d i n t o a t a r e d b e a k e r . The b e a k e r was p l a c e d i n a w a t e r b a t h a t 3 0 ° C . A f t e r i n c u b a t i o n f o r 5 m i n u t e s , 0.2 m l o f a p p r o p r i a t e l y d i l u t e d enzyme was added t o t h e m i l k w i t h m i x i n g . The c o n c e n t r a t i o n o f enzyme was s u c h t h a t m i l k was c l o t t e d i n 4-5 m i n u t e s ( T ) . The c o a g u l a t e d m i l k s a m p l e was i n c u b a t e d a t 30° C f o r f u r t h e r 30 m i n u t e s w h i c h i s a c o n v e n i e n t t i m e t o g i v e c u r d f o r m a t i o n . A t t h e p r e c i s e t i m e (T +30 m i n u t e s ) , t h e b e a k e r o f c o a g u l u m was g e n t l y t a k e n f r o m t h e w a t e r b a t h , d r i e d on t h e o u t s i d e w i t h t i s s u e p a p e r a n d p l a c e d on t h e b a l a n c e p a n . E x a c t l y 15 s e c o n d s a f t e r r e m o v a l f r o m t h e b a t h , a f l a t b o t t o m e d 10 g w e i g h t s u p p o r t e d b y a m e t a l c h a i n was 38 placed on the surface of the curd* The scale was read to the nearest 10 mg (centigram) at exactly 15 seconds (to minimize surface tension effect) and 60 seconds after the weight was in contact with the curd, i.e., 30.5 minutes and 31.25 minutes after curd formation. The difference between the two readings, in centigrams, was taken as an arbitrary measure of the curd tension which was the amount of weight transferred from the curd to the chain i n 45 seconds. A constant displacement of the hanging weight was set by placing 20 ml d i s t i l l e d water in a standardized beaker. By adjusting the height of the point of support of the weight, the balance was set ar b i t r a r i l y at 20.70 g when about 70% of the weight's volume was immersed. This operation ensured a constant distance between the point of chain suspension and the curd surface. 14. Determination of the rate of syneresis The rate of exudation of whey from curd was measured by the discontinuous method of Lawrence (1959) with some modifications. Samples of reconstituted skimmilk (100 ml) at pH 6.3 were added to 250-ml beakers and incubated at 30° C in a water bath. An appropriate amount of enzyme was added to the 39 m i l k . The g e l was c u t i n t o b l o c k s o f u n i f o r m s i z e 30 m i n -u t e s a f t e r c l o t t i n g . The c u r d was t h e n h e l d a t 30° C w i t h -o u t s t i r r i n g . A t d i f f e r e n t t i m e i n t e r v a l s , e a c h b e a k e r was t i p p e d on t o a s i e v e p l a c e d o v e r a f u n n e l . The c u r d was r e t a i n e d b y t h e s i e v e a n d t h e whey was d r a i n e d i n t o a g r a d u a t e d c y l i n d e r u n d e r t h e f u n n e l . The v o l u m e o f whey i n t h e c y l i n d e r 30 s e c o n d s a f t e r t i p p i n g was r e c o r d e d . Volume o f t h e c u r d was c a l c u l a t e d b y s u b t r a c t i n g t h e vo lume o f t h e whey f r o m t h e i n i t i a l v o l u m e o f m i l k . The r e s u l t was e x p r e s s e d as p e r c e n t a g e s y n e r e s i s i P e r c e n t a g e s y n e r e s i s - ^ 1 ^ 0 1 ^ 1 ^ 1 1 * x " > ° 40 RESULTS AND DISCUSSION Choice of Enzyme Source The pepsin samples used i n the present investigation were 2X crystallized and lyophilized enzyme from hog stomach mucosa. Two lots, one from Sigma Chemical Co. (Lot No. 67C-8195) and the other from Worthington Biochemical Corp. (Lot No. PM 37C6?5)» were used. Both enzymes appeared as one sharp band when applied to agarose gel electrophoresis, and were eluted as one sharp peak on DEAE cellulose column with 0.1 M sodium acetate buffer (pH 4.2) and increasing concentration of NaCl as eluents. These observations sug-gest that the enzyme preparations used were essentially homogeneous and were therefore used directly without further purification. Rajagopalan et a l . (1966b) proposed a method to pre-pare homogeneous pepsin from pepsinogen. The procedure i n -volved activation of the zymogen at 14° C and pH 2 for 20 minutes and the separation of the enzyme from the peptide by passage through a column of sulfoethyl Sephadex C-25. The high cost of pepsinogen prohibited preparation of large amount of pepsin by this method. In some experiments, crude pepsin (1:10,000) was used. The industrial pepsin i s the starting material from which crystalline pepsin has traditionally been prepared. 41 This i s the enzyme preparation that can he used, together with calf rennet as 50*50 mixtures to make cheese. Hence, i n experiments designed to test the sui t a b i l i t y of modified pepsin for cheese-making, 1x10,000 pepsin i s a more appro-priate enzyme than the crystalline pepsin. In this thesis, the term pepsin refers to the cry-stalline porcine pepsin while the impure enzyme w i l l be specified as crude or 1x10,000 pepsin. Choice of Carbodiimides and Nucleophiles Two commercially available water-soluble carbodi-imides, l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide metho-p-toluenesulfonate (CMC) and l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) were used to modify the carboxyl groups in pepsin. Both carbodiimides were found to modify pepsin carboxyl groups with similar changes i n enzymatic activities and properties. However, EDC was found to be more effective than CMC at the same molar con-centration. This could be due to the smaller size of EDC (M.W.=191.7) than CMC (M.W..423.6) enabling EDC to react with p a r t i a l l y buried carboxyl groups in pepsin. EDC was therefore used subsequently. Three nucleophiles, ethylenediamine, methyl esters of various amino acids and 2-aminoethanesulfonic acid 42 (taurine) were used for carboxyl modification. The different charges carried by these nucleophiles caused considerable difference i n the ionic character of the modified enzymes (see Fig. 2). Ethylenediamine and taurine were found to cause extensive loss in milk clotting activity of pepsin even at low concentrations (Table 3 ) . Hence, methyl esters of various amino acids, mainly glycine, were used i n later experiments. Effect of Nucleonhile Concentration on the Extent of Carboxyl  Modification The number of pepsin carboxyl groups covered in the carbodiimide reaction was determined by amino acid analysis. EDC and glycine methyl ester at various concentrations were used. The number of carboxyl groups modified increased as the concentration of nucleophile was increased (Fig. 3 ) . The concentration of EDC was found to be less c r i t i c a l i n affecting the amount of modification. However, EDC concen-trations lower than 10 mM (33-fold excess to pepsin) were found to be ineffective i n modifying pepsin. For most of the subsequent experiments, unless speci-fied otherwise, the concentrations of reagents used were* 33-fold excess of EDC and 174-fold excess of glycine methyl T a b l e 3. C a r b o x y l m o d i f i c a t i o n o f p e p s i n w i t h d i f f e r e n t n u c l e o p h i l e s . * N u c l e o p h i l e C o n c n . (mM) P r o t e o l y t i c a c t i v i t y (% c o n t r o l ) M i l k c l o t t i n g a c t i v i t y (fo c o n t r o l ) G l y m e t h y l e s t e r 50 94 17 E t h y l e n e -d i a m i n e 50 85 3 T a u r i n e 50 90 5 P e p s i n (10 mg/ml) was m o d i f i e d w i t h 33 - f o l d e x c e s s o f EDC a n d 174 - f o l d e x c e s s o f n u c l e o p h i l e a t pH 5.5 f o r 90 m i n u t e s . 44 r\h i i l 1 .05 .10 .15 .20 Nucleophile Concn., M FIGURE 3. EFFECT OF NUCLEOPHILE CONCENTRATION ON THE EXTENT OF CARBOXYL MODIFICATION OF PEPSIN. 45 e s t e r , c o r r e s p o n d i n g t o a n i n c o r p o r a t i o n o f 5.2 m o l e s o f n u c l e o p h i l e p e r mole p e p s i n . A c t i v i t y o f N a t i v e a n d C a r b o x y l M o d i f i e d P e p s i n s 1. M i l k c l o t t i n g a c t i v i t y The e f f e c t o f c a r b o x y l m o d i f i c a t i o n on m i l k c l o t t i n g a c t i v i t y o f p o r c i n e p e p s i n i s shown i n F i g . 4. The a c t i v i t y was f o u n d t o d r o p r a p i d l y w i t h i n c r e a s e i n t h e e x t e n t o f c a r b o x y l m o d i f i c a t i o n . A d r o p o f 70-90% i n m i l k c l o t t i n g a c t i v i t y was o b s e r v e d . When t r e a t e d w i t h g l y c i n e m e t h y l e s t e r a l o n e , p e p s i n r e t a i n e d 100$ m i l k c l o t t i n g a c t i v i t y . When t h e c a r b o d i i m i d e c o n c e n t r a t i o n was l o w e r e d t o 5 mM ( t h e n u c l e o p h i l e c o n c e n -t r a t i o n was 50 mM), t h e m o d i f i e d enzyme h a d a b o u t 60% o f m i l k c l o t t i n g a c t i v i t y w i t h a c o r r e s p o n d i n g d e c r e a s e i n t h e number o f m o d i f i e d c a r b o x y l g r o u p s ( s e e T a b l e 4). When p e p s i n was t r e a t e d w i t h 10 mM EDC i n t h e a b s e n c e o f n u c l e o -p h i l e , t h e m i l k c l o t t i n g a c t i v i t y o f p e p s i n was a l s o d e c r -e a s e d b y a b o u t 35% ( s e e T a b l e 4). When c r u d e p e p s i n was m o d i f i e d w i t h a l o w c o n c e n t r -a t i o n o f g l y c i n e m e t h y l e s t e r ( 8 7 - f o l d e x c e s s t o p e p s i n ) a n d EDC ( 3 3 - f o l d e x c e s s t o p e p s i n ) , t h e r e was a d r o p o f a b o u t 5®?* i n m i l k c l o t t i n g a c t i v i t y . W i t h a h i g h e r n u c l e o -46 No. of COOH G p . Modified FIGURE 4. EFFECT OF CARBOXYL MODIFICATION OF PEPSIN ON MILK CLOTTING ACTIVITY. Table 4. Effect of carboxyl modification on pepsin a c t i v i t i e s . EDC Glv methvl ester No. C00H gp. Milk clotting Proteolytic activity (% control) Concn. Excess to pepsin (-fold) Concn. Excess to pepsin (-fold) modified/mole activity (mM) (mM) pepsin (# control) pH 2.0 PH3.5 10 33 0 •0 0 65 100 100 10 33 25 87 2.8 27 88 210 10 33 50 174 5.2 17 94 340 10 33 100 348 8.6 13 105 330 10 33 200 706 11.2 9 90 340 0 0 50 174 0 100 100 100 5 16.5 50 174 1.0 60 100 120 48 phile concentration (174-fold excess to pepsin), the drop i n a c t i v i t y was 70$. This indicates that compared to cry-stalline pepsin, crude pepsin retained higher milk clotting activity when modified with the same quantity of reagents. Amino acid analysis of the native and modified crude pepsin did not yield consistent results, probably due to impurities present in the enzymes. Hence, the extent of carboxyl modification was not determined for the crude pepsin. 2. Proteolytic activity The proteolytic activity of pepsin at pH 2.0 was found to decrease slightly after carboxyl modification (Fig. 5 ) » Even with extensive modification (11.2 moles of carboxyl groups covered/mole pepsin), the enzyme s t i l l retained 90% of i t s activity, showing that the carboxyl groups blocked were not directly involved i n proteolysis of hemoglobin. In contrast to the present result, carboxyl modi-fication of porcine pepsin with CMC and a colored amine caused about 40$ drop in activity against hemoglobin, a l -though only one amine molecule was incorporated per molecule of pepsin (Matyash et a l . , 1973). The carboxyl groups modi-fied by the colored amine could.be more directly related to pepsin catalysis. o 3 6 9 12 No. of COOH Gp . Modif ied EFFECT OF CARBOXYL MODIFICATION OF PEPSIN ON PROTEOLYTIC ACTIVITY, • , PH 2 . 0 ; o , pH 3 . 5 , 50 When t h e a c t i v i t y a g a i n s t h e m o g l o b i n was m e a s u r e d a t pH 3»5» t h e c a r b o x y l m o d i f i e d p e p s i n was f o u n d t o h a v e a c t i v i t y much h i g h e r t h a n t h e c o n t r o l ( F i g . 5 ) . T h i s was m a i n l y due t o t h e f a c t t h a t a t pH 3 . 5 t t h e c o n t r o l h a d s p e -c i f i c p r o t e o l y t i c a c t i v i t y much l o w e r t h a n t h a t a t pH 2 . 0 , w h i l e t h e m o d i f i e d enzyme h a d h i g h e r a c t i v i t y a t pH 3 . 5 . T h i s i n d i c a t e s t h a t t h e r e may be a s h i f t i n t h e pH p r o f i l e . S i m i l a r r e s u l t s were o b t a i n e d when c r u d e p e p s i n was m o d i f i e d w i t h 3 3 - f o l d e x c e s s o f EDC a n d 8 7 - f o l d e x c e s s o f g l y c i n e m e t h y l e s t e r . The m o d i f i e d enzyme r e t a i n e d a b o u t 9 0 $ o f i t s p r o t e o l y t i c a c t i v i t y a t pH 2 . 0 , w h i l e a t pH 3 . 5 , t h e a c t i v i t y o f t h e m o d i f i e d enzyme was a b o u t 2 . 5 t i m e s t h a t o f t h e c o n t r o l . T a b l e k s u m m a r i z e s t h e e f f e c t o f c a r b o x y l m o d i f i -c a t i o n o f p e p s i n on m i l k c l o t t i n g a n d p r o t e o l y t i c a c t i v i t y i n r e l a t i o n t o t h e e x t e n t o f m o d i f i c a t i o n . 3 . M i l k c l o t t i n g * p r o t e o l y t i c a c t i v i t y r a t i o W i t h a marked d e c r e a s e i n m i l k c l o t t i n g a c t i v i t y a n d n o s i g n i f i c a n t changes i n p r o t e o l y t i c a c t i v i t y , c a r b o x y l m o d i f i c a t i o n o f p e p s i n r e s u l t e d i n a d r a m a t i c d e c r e a s e i n m i l k c l o t t i n g : p r o t e o l y t i c a c t i v i t y r a t i o ( F i g . 6 ) . W i t h e x t e n s i v e m o d i f i c a t i o n ( 1 1 . 2 c a r b o x y l g r o u p s m o d i f i e d / m o l e p e p s i n ) , t h e r a t i o was o n l y 1 0 $ t h a t o f t h e c o n t r o l . FIGURE 6 , EFFECT OF CARBOXYL MODIFICATION OF PEPSIN ON MILK CLOTTING:PROTEOLYTIC ACTIVITY RATIO. 52 The milk clotting:proteolytic activity ratio is an indication of the spec i f i c i t y of proteases. Chymosin, the major enzyme component of calf rennet, was found to have the highest clotting to proteolytic activity ratio among other proteolytic enzymes (Ernstrom, 1 9 7 4 ) , indicating that chymosin has the highest specific milk clotting a c t i v i t y . The present data show that relative to general proteolysis, the specific activity of pepsin towards milk clotting was lowered after carboxyl modification. 4. Peptidase activity The peptidase activity of pepsin was measured using APDT as substrate. Result shows that the peptidase activity at pH 2.0 and 3 7 ° C was decreased by carboxyl modification (Pig. 7 ) . Unlike milk clotting, however, the decrease in peptidase activity was moderate. The modified pepsin re-tained about 40-60$ of activity against the dipeptide sub-strate . Pepsin modified with CMC and a colored amine also showed a drop of 50$ in peptidase activity, when N-acetyl-L-phenylalanyl-L-tyrosine was used as substrate (Matyash et a l . , 1 9 7 3 ) . 53 F I G U R E 7 . E F F E C T OF CARBOXYL M O D I F I C A T I O N OF P E P S I N ON P E P T I D A S E A C T I V I T Y . 54 E f f e c t o f C a r b o x y l M o d i f i c a t i o n on pH P r o f i l e s 1. M i l k c l o t t i n g PH p r o f i l e The pH p r o f i l e s o f m i l k c l o t t i n g f o r n a t i v e and c a r b o x y l m o d i f i e d p e p s i n s a r e shown i n F i g . 8. F o r b o t h e n z y m e s , t h e m i l k c l o t t i n g a c t i v i t y d r o p p e d r a p i d l y f r o m pH 6.0-to 6.5. The m o d i f i e d enzyme r e t a i n e d s l i g h t l y h i g h e r a c t i v i t y a t pH 6.3 and 6.5 t h a n t h e c o n t r o l . C r u d e p e p s i n a n d t h e c o r r e s p o n d i n g m o d i f i e d enzyme a l s o showed s i m i l a r pH p r o f i l e s , i n d i c a t i n g t h a t c a r b o x y l m o d i f i c a t i o n d i d n o t change t h e pH p r o f i l e o f m i l k c l o t t i n g i n p e p s i n . 2 . P r o t e o l y t i c PH p r o f i l e The e f f e c t o f c a r b o x y l m o d i f i c a t i o n o n t h e p r o t e o -l y t i c pH p r o f i l e o f p e p s i n i s i l l u s t r a t e d i n F i g . 9. N a t i v e p e p s i n h a d a pH optimum a t a b o u t 2.0 and t h e s p e c i f i c a c t i -v i t y a g a i n s t h e m o g l o b i n d e c r e a s e d s h a r p l y t o w a r d s h i g h e r p H ' s . A f t e r c a r b o x y l m o d i f i c a t i o n , t h e pH optimum was s h i f t e d t o a b o u t 3.5. The p l a t e a u o b s e r v e d a t pH 1.5-2.0 f o r t h e m o d i f i e d enzyme c o u l d be a t t r i b u t e d t o i n c o m p l e t e m o d i f i -c a t i o n . 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 c h y m o s i n h a d a p r o -t e o l y t i c p H - a c t i v i t y c u r v e c l o s e l y r e s e m b l i n g t h a t o f t h e m o d i f i e d p e p s i n , w i t h a pH optimum n e a r 3.5 ( P i g . 9). 55 FIGURE 8. M I L K CLOTTING PH P R O F I L E S OF N A T I V E AND CARBOXYL MODIFIED P E P S I N S . N A T I V E P E P S I N j O , CARBOXYL MODIFI ED P E P S I N 01 — i i I i • • i 1.5 2.0 2.5 3.0 3.5 4.0 4.5 p H F I G U R E 9. PROTEOLYTIC PH P R O F I L E S OF C R Y S T A L L I N E P E P S I N CARBOXYL MODIFIED C R Y S T A L L I N E P E P S I N o , AND CHYMOSIN A , 57 A similar s h i f t in proteolytic pH profiles was also observed when crude pepsin was modified with EDC and glycine methyl ester (Fig. 10). The native enzyme had a pH optimum at about 2.0, and after modification the optimum shifted to between pH 3.5-4.0. The modified crude pepsin had a wider pH optimum than the modified pure enzyme. A shift in pH profile had been noted in some immo-bil i z e d enzymes and was directly related to the "micro-environment" effect resulting from the embedment of enzymes within the carriers (Silman and Katchalski, 1968). Binding of charged carriers to enzymes may also produce changes in the distribution of charges on the enzyme molecules. Con-sequently, the pH in the domain of the enzymes w i l l be different from the external bulk solution, thus creating an apparent shift i n pH profile (Goldstein, 1970). Since the nucleophile (glycine methyl ester) attached to pepsin was of small molecular size when compared to the carriers used in enzyme immobilization, the shift in pH profile in the modified pepsin was unlikely due to "micro-environment" effect. It was probably related to changes in the charge distribution on the enzyme as a result of covering of the negatively-charged carboxyl groups by the neutral nucleo-phile. 58 F I G U R E 10. P R O T E O L Y T I C PH P R O F I L E S OF CRUDE N A T I V E AND C A R B O X Y L M O D I F I E D P E P S I N S . ®, NATIVE PEPSIN (1:10,000); O, CARBOXYL MODIFIED PEPSIN (1:10,000) 59 3' PH-Activitv curves for the hydrolysis of APDT When the synthetic dipeptide, APDT, was used as sub-strate, the pH-activity curves of both native and carboxyl modified pepsin were found to be similar except that the modified pepsin had slightly higher activity at more alka-line pH (Fig. 11). Both enzymes had a pH optimum around 2.0 and the activity decreased rapidly towards higher pH. However, the decline i n activity was more gradual in the modified enzyme. The result shows that unlike hemoglobin, the pH-activ i t y curve on dipeptide substrate was not significantly shifted by carboxyl modification. The pH-activity curves for APDT hydrolysis were not determined for the crude pepsins. Electrophoretic Mobility of Native and Carboxyl Modified  Pepsins on Agarose Gel Agarose gel electrophoresis was used to measure •the electrophoretic mobility of native pepsin and the carboxyl modified enzymes. Both native and modified enzymes app-eared as one band on the agarose film (Plate I ) . Hetero-geneity of chemically modified proteins, resulting from substitution of different numbers of carboxyl groups, might be observed in electrophoresis as a diffused band. The pH F I G U R E 11, PH A C T I V I T Y CURVES FOR THE ACTION OF N A T I V E AND CARBOXYL MODIFIED PEPSINSON N-ACETYL-L-PHENYLALANYL-D MODO-L-TYROSINE. • , NATIVE PEPSIN; A, CARBOXYL MODIFIED PEPSIN. P L A T E I . AGAROSE G E L E L E C T R O P H O R E T I C P ATT ERN S OF N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . NUMBER OF CARBOXYL GROUPS MODIFIED/MOLE ENZYME: lj 0 (CONTROL); 2, 5.2; 3, 8.7; ^ 11,2; 5, 15.6. 61 + • origin 62 sharpness of the enzyme bands shown i n the present result suggest that the derivatives were practically free of unmodified pepsin, and the modified products were essen-t i a l l y homogeneous. The electrophoretic mobility of pepsin was found to decrease progressively with increases i n the extent of carboxyl modification (Plate I ) . Electrophoretic separation of proteins on gel mat-rices such as starch and polyacrylamide i s based on both electric charge and molecular size difference of the pro-teins. However, owing to the low gel concentration and large pore size of the agarose gel used, the molecular sieving effect i s minimal. Furthermore, as glycine methyl ester has a molecular weight of 125» the incorporation of ten molecules of nucleophile would increase the molecular weight of pepsin by less than % . Gel f i l t r a t i o n chromato-graphy of the most extensively modified pepsin (11.2 carboxyl groups modified/mole pepsin) on Sephadex G-200 (Superfine) confirmed that the change i n molecular size was not marked, as the modified enzyme was eluted at the same elution volume as the native pepsin (0.1 M acetate buffer, pH 4.5). This result also indicates that there was no aggregation be-tween the modified pepsin molecules. Hence, the drop in electrophoretic mobility after carboxyl modification was 6 3 mainly attributed to a decrease in the net negative charge on the modified enzyme resulting from the blocking of nega-tively charged carboxyl groups.by glycine methyl ester. When the number of carboxyl groups modified/mole pepsin was plotted against logarithm of the relative electro-phoretic mobility, R^ , a linear relationship was obtained (Fig. 12). This indicates that the net charge of the pep-sin molecule i s directly proportional to the electrophoretic mobility. It has been shown that provided the molecular weight and molecular shape of a protein are known, the net charge of the protein can be calculated from i t s electro-phoretic mobility (Shaw, 1969). The present result i s i n agreement with the above finding. Presumably, the decrease in negative charge on pep-sin was not only a result of the blocking of carboxyl groups by nucleophile but also of the fixation of positively charged carbodiimide residues as N-acylurea on other carboxyl groups of the pepsin molecule. Matyash et a l . (1973) observed that pepsin treated with radioactive CMC alone contained 2-3 residues of labelled carbodiimide. In the present study, pepsin treated with 10 mM EDC i n the absence of nucleophile had a R f value of O.89 (native pepsin was assumed to have a R f of 1.0), showing that the contribution of carbodiimide to the decrease in net negative charge on pepsin was signi-ficant . 63a 0 5 1 0 1 5 NO. OF COOH GROUPS MODIFIED F I G U R E 1 2 . E F F E C T OF CARBOXYL M O D I F I C A T I O N OF P E P S I N ON R E L A T I V E E L E C T R O P H O R E T I C M O B I L I T Y , NATIVE PEPSIN WAS ASSUMED TO HAVE A RF VALUE OF 1.0. 64 Isoelectric Point of Native and Carboxyl Modified Pepsins The isoelectric point of native pepsin, determined by electrophoresis of the protein on agarose gel at d i f f e r -ent pH's, was found to be below 0.5. After extensive car-boxyl modification (11.2 carboxyl groups modified/mole pepsin), the isoelectric point was found to rise to about 0.7 (Fig. 1 3 ) . The present result is in accordance with that of other workers who observed that at pH 1.0, highly purified pepsin s t i l l migrated as an anion, indicating that the isoelectric point of porcine pepsin i s below 1.0 (Tiselius et a l . , 1 9 3 8 j Herriott et a l . , 1 9 4 0 ) . The isoelectric point of proteins determined by zone electrophoresis may be subject. to errors such as absorp-tion, capillary flow and electro-osmosis. More superior techniques would be moving boundary electrophoresis and isoelectrofocusing. However, while moving boundary electro-phoresis requires expensive instruments and complicated experimental procedures, the pH range of commercially avail-able ampholytes for isoelectrofocusing i s between 3«5 and 10.0, well above the isoelectric point of pepsin. Agarose has been shown to exhibit l i t t l e electro-osmosis and ad-sorption, and electrophoretic mobilities determined by agar-ose gel electrophoreis are found to be similar to those determined by moving boundary technique (Shaw, 1 9 6 9 ) . 65 FIGURE 13, DETERMINATION OF ISOELECTRIC POINTS OF NATIVE AND CARBOXYL MODIFIED PEPSINS. ®, NATIVE PEPSIN; O, CARBOXYL MODIFIED PEPSIN (11,2 GROUPS MODIFIED/MOLE ENZYME) CARBOXYL 66 Thus, agarose gel electrophoresis was employed to provide information on changes in the isoelectric point of pepsin after chemical modification. The increase in isoelectric point in the carboxyl modified pepsin further indicates that there was a decrease i n the net negative charge in the enzyme as a result of modification. Carboxyl Modification of Pepsin Bv other Amino Acid  Methyl Esters Methyl esters of amino acids other than glycine were used as nucleophiles to modify pepsin. The extent of car-boxyl modification, the activ i t i e s and the relative electro-phoretic mobility of the modified enzymes were measured and the results are presented in Table 5» A l l nucleophiles were found to incorporate into pepsin, but the extent of modification was variable. The milk clot-ting activity of a l l modified enzymes was markedly reduced, particularly those modified with tyrosine and tryptophan methyl esters. At pH 2.0, the proteolytic activity against hemoglobin was not changed i n pepsin modified with arginine and lysine methyl esters while pepsins treated with leucine, tyrosine and tryptophan methyl esters showed a drop in a c t i -v i t y . The specific proteolytic activity at pH 3 .5 was 67 Table 5. Carboxyl modification of pepsin by different amino acid methyl esters. a Methyl ester No. CGOH gp. modified/mole pepsin Milk clotting activity ($ control) Proteolytic activity (% control) pH2.0 pH3.5 Arg 4.7 14 100 300 0.50 Lys 5.1 12 100 330 0.48 Leu 7.3 10 75 200 0.68 Tyr 8.0 7 50 100 0.68 Try 3.5 4 40 25 0.70 Gly 5.2 17 94 340 0.56 a Pepsin (10 mg/ml) was modified with 33-fold excess of EDC and 174-fold excess of methyl ester at pH 5.5 for 90 minutes. Native pepsin was assumed to have a R_ value of 1.0. 68 significantly increased in most modified enzymes, indicating a shift i n pH profile. However, tyrosine methyl ester-treated pepsin did not show a change in proteolytic activity at pH 3.5 and the tryptophan derivative even showed a marked decrease i n activity at this pH. The difference in the response of pepsin to different types of amino acid methyl esters could be attributed to solu b i l i t y . Incorporation of hydrophobic amino acids such as tyrosine and tryptophan into pepsin would increase the hydrophobicity and decrease the solubility of the enzyme, leading to an apparent drop i n activity. Alternatively, the discrepancy could be due to the covering of the hydrophobic binding site in pepsin by the hydrophobic methyl esters. Apart from the catalytic site, several investigators advocated a hydrophobic binding site i n pepsin which plays an important role in pepsin activity (Tang, 1965$ Jackson et. a l . , 1 9 6 6 ) . The hydrophobic methyl esters would have a greater tendency to bind to this secon-dary substrate binding site, thus rendering i t unavailable for further interaction with other substrates and resulting i n a loss of a c t i v i t i e s . The electrophoretic mobility of a l l modified enzymes was found to decrease when compared to the native enzyme. Arginine and lysine methyl esters, being cationic, decreased the electrophoretic mobility to a greater extent. 6 9 Caseinolytic Properties of Native and Carboxyl Modified  Pepsins 1 . Rate of casein hydrolysis The rate of hydrolysis of 1 $ (w/v) whole casein by pepsin, determined by the release of non-protein nitrogen (NPN) soluble in 3% (w/v) TCA, was not significantly affected by carboxyl modification. As shown in Fig. 14 and Fig. 1 5 , at both pH 5»3 and 6 . 5 » there was an i n i t i a l rapid rise in the release of nitrogen followed by a gradual increase. The extent of casein hydrolysis was considerably lower at pH 6 . 5 than at pH 5 * 3 . At pH 5 » 3 t the carboxyl modified enzyme hydrolysed casein at a rate slightly higher than that of the control (Fig. 14). At pH 6 . 5 , however, the rate of casein hydrolysis was sl i g h t l y decreased after modification (Fig. 1 5 ) . 2 . Electrophoretic patterns of hydrolysed casein At time intervals when the release of NPN was deter-mined, casein samples were also withdrawn simultaneously, solubilized in 6 M urea and subjected to agarose gel electro-phoresis. The resulting patterns were essentially similar (Plate I I ) . They a l l showed an i n i t i a l conversion of Jt-casein to para-x-casein. This was followed by some break-down of ^-casein, the rate being faster with the modified pepsin. There was no perceptible breakdown of e>col-casein. 70 60 120 Time, min. 180 F I G U R E 1 4 . RATE OF C A S E I N HYDROLYSIS BY N A T I V E AND CARBOXYL MODIFIED P E P S I N S AT PH 5.3. •, NATIVE PEPSIN; o, CARBOXYL MODIFIED PEPSIN. « NPN> NON-PROTEIN NITROGEN 71 3 0 r o !20 180 Time, min. F I G U R E 1 5 . RATE OF C A S E I N HYDROLYSIS BY N A T I V E AND CAR-BOXYL MODIFIED P E P S I N S A T PH 6.5. •, NATIVE PEPSIN; o, CARBOXYL MODIFIED PEPSIN. NPN, NON-PROTEIN NITROGEN. 71a P L A T E I I . AGAROSE G E L E L E C T R O P H O R E T I C PATTERNS OF WHOLE C A S E I N HYDROLYSED BY N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . SAMPLE: 1, 1% (W/V) WHOLE C A S E I N ; 2, CASEIN HYDROLYSED BY NATIVE PEPSIN (30 M I N . ) ; 3, CASEIN HYDROLYSED BY NATIVE PEPSIN (180 M I N . ) ; CASEIN HYDROLYSED BY CARBOXYL MODIFIED PEPSIN (30 M I N . ) ; 5, CASEIN HYDROLYSED BY CARBOXYL MODIFIED PEPSIN (180 M IN . ) 72 73 3. Electrophoretic patterns of milk curds Reconstituted skimmilk was clotted with both native and carboxyl modified pepsins at pH 6.3. At time intervals after clotting, a sample of curd was withdrawn, solubilized i n 6 M urea and applied to agarose plate for electrophoresis. The electrophoretic patterns, shown in Plate III, appeared to be identical for both native and modified enzymes. In both instances, there was a decline i n the amount of x-casein together with the appearance of a para-,£-casein band. This indicates that the nature of the clotting reaction was not changed by the modification. The above results indicate that the caseinolytic properties of pepsin were not significantly affected by carboxyl modification. The decrease i n milk clotting a c t i -v i t y was therefore not due to the blocking of carboxyl group(s) essential for the hydrolysis of caseins in milk, since the rate of release of nitrogen from caseins was not decreased after modification. In the present investigation, chymosin was found to have caseinolytic properties very similar to those of pepsin, since both the rate of casein hydrolysis and the electro-phoretic patterns of hydrolysed. caseins and curdled milk were similar. The result i s consistent with the reports of other workers who compared the proteolysis of whole casein P L A T E I I I . AGAROSE G E L E L E C T R O P H O R E T I C P A T T E R N S OF M I L K CURDS PRODUCED BY N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . SAMPLES 1 , 2 , 3 , & 4 : DISSOLVED CURDS FORMED WITH NATIVE PEPSIN; SAMPLES 5 , 6 , 7 , & 8 : DISSOLVED CURDS FORMED WITH MODIFIED PEPSIN. TIME AFTER CLOTTING: 1 & 5 , 0 HR.; 2 & 6 , 1 / 2 HR.; 3 & 7 , 2 HR.; 4 & 8 , 24 HR. para-x-casein 75 and individual casein fractions by porcine pepsin and calf rennet under different conditions. In most instances, pepsin was found to have approximately the same activity as rennet (Mickelsen and Fish, 1970; Hansen, 1970; Itoh and Thomasow, 1971; Green, 1 9 7 2 ) . Coagulation of X--Casein and gc^-Casein by Native and Carboxyl  Modified Pepsins Since X-casein has been known to be the protein com-ponent responsible for stabilization of the caseinate mi-celles (Waugh and von Hipp el, 1956; Z i t t l e , .1961; Z i t t l e and Walter, 1 9 6 3 ) , an experiment was designed to test whether the diminished clotting activity of the carboxyl modified pepsin was due to a decreasejin i t s a b i l i t y to coagulate X-casein. The result shows that 0.2$ (w/v) x-casein at pH 5-3 and an ionic strength of 0.05 was coagulated by both native and modified pepsins. Omission of calcium in the reaction solution did not affect the results. The specific activity was markedly increased after modification (Table 6 ) . A 1:1 (w/w) mixture of X-casein and <*gl-casein was also used as substrate since -casein i s the ealcium-s l sensitive component which i s normally protected by X-casein against coagulation (Zittle, 1 9 6 1 ) . In this case, carboxyl 76 Table 6. Effect of carboxyl modification on clotting activity of pepsin to Jc-casein and * - * g ] _ -casein mixture. Enzyme Substrate Specific activity K Pepsin COOH-modi-fied pepsin 0.2$ K-casein pH 5.3 0.2$ /c-casein pH 5.3 1.2 3.0 Pepsin COOH-modi-fied pepsin x-«s-^-casein ( 1 : 1 ) , pH 5.3 /c-<*s ^ c a s e i n ( 1 : 1 ) , pH 5.3 14.3 1.5 11.9 0.5 The casein clotting activity is defined as described under Method. Specific activity i s expressed as clotting activity per mg protein. 77 modification greatly reduced the a b i l i t y of pepsin to coa-gulate the casein mixture (Table 6 ) . The result also shows that native pepsin had a much higher specific a c t i v i t y against the casein mixture than against X-casein, with an activity ratio (K+<*/K) of about 12. In contrast, the carboxyl modified pepsin had an activity ratio of only 0.5, indicating that the enzyme had greater specificity towards X-casein than X-cx^^-casein mixture (Table 6 ) . When the specific activities against casein fractions were compared to those against skimmilk and hemoglobin, i t can be seen that the rise in specific activity against K-casein coincided with an increase i n proteolytic activity at pH 3'5t with the exception of the most extensively modi-fied enzyme which showed a drop of 70% in x-casein clotting a c t i v i t y . The specific activity against X-«:g^-casein mix-ture was found to decrease with a trend very similar to the drop i n milk clotting activity (Table 7). The results obtained from the above experiments sug-gest that the increase i n specific activity against X-casein was probably attributed to a shift i n pH optimum of the enzyme similar to that observed when hemoglobin was used as substrate. Since the activity was measured at a pH (5.3) not far from the optimum pH (3*5) of the modified enzyme for proteolysis, the modified pepsin would hydrolyse x-casein Table 7. Comparisionof activities of carboxyl modified pepsin to different substrates. No. of COOH Specific activity ($ c ontrol) Enzyme groups modified x-casein pH 5.3 K-PC " -casein S|5H 5.3 skimmilk pH 6.3 hemoglobin pH2.0 pH3.5 Pepsin 0 100 100 100 100 100 COOH modi-fied pepsin 2.8 320 20 27 88 210 t» 5.2 250 12 17 94 340 n 8.6 200 10 13 105 330 n 11.2 30 5 9 90 340 CO 79 at a rate faster than the native enzyme which has low pro-teolytic activity at higher pHfs (see Fig. 9). The coa-gulation of isolated x-casein by an enzyme has been demon-strated to be due to the aggregation of insoluble para -x-casein s p l i t off from x>casein (Cheeseman, 1962) and i s therefore essentially proteolytic in nature. An increase i n proteolytic activity against X-casein would therefore increase the rate of x-casein coagulation. The dramatic drop in X-casein clotting activity .when pepsin was extensively modified (11.2 moles of glycine methyl ester incorporated/mole pepsin) .suggests that a carboxyl group(s) essential for x-casein hydrolysis may be blocked. This group(s), however, was not essential for hydrolysing hemoglobin since the specific activity of this derivative against hemoglobin s t i l l remained high when compared to the native enzyme. The high activity ratio (K+et/x) observed for "the native pepsin may suggest that interaction between X-casein and <?i i -casein i n the micelles could lead to changes that s l enhance the a f f i n i t y of the native enzyme for the substrate. Activation of X-casein hydrolysis by <*-casein and ,g-casein was reported in chymosin (Kanamori et a l . , 1977) and was attributed to the association of x-casein with other pro-teins . 80 The extensive loss in pepsin activity against K-casein/c<: g l-casein mixture after carboxyl modification suggests that interaction between the two caseins on the micelles may retard the binding between the micelles and the modified enzyme. Changes i n the charge distribution on the modified enzyme may interfere with substrate-enzyme interaction through electrostatic or ionic repulsion. This would affect the primary (enzymatic) phase of milk coagulation and slow down the secondary phase of micellar aggregation. The observed drop i n milk clotting activity i n the carboxyl modified pepsin could therefore be a t t r i -buted to a change in the ionic characteristic of the enzyme. Specificity of Carboxyl Modification To investigate whether the carboxyl modification by the carbodiimide reaction is specific for pepsin, pepsino-gen and chymosin were modified under similar conditions (33-fold excess of EDC, 7 l 6 - f o l d excess of glycine methyl ester, pH 5.5, 90 minutes incubation). The result, presented in Table 8, indicates that the carbodiimide reaction was not specific for pepsin. Both pepsinogen and chymosin were modified to about the same extent as pepsin with a significant decrease i n milk clotting a c t i v i t y . The decrease in proteolytic activity Table 8. Effect of carboxyl modification on activities of pepsin, pepsinogen and chymosin. No. of COOH Specific activity (% control) Enzyme ^ m o d i f i e d M i l k clotting Proteolytic pH 6.3 pH2.0 pH3.5 Pepsin 11.2 9 90 340 Pepsinogen 12.1 30 70 450 Chymosin 13.0 15 50 50 The enzymes (10 mg/ml) were modified with 33-fold excess of EDC and 706-fold excess of glycine methyl ester at pH 5.5 for 90 minutes. 82 of the modified zymogen and chymosin at pH 2.0 was not as extensive as the drop in milk clotting activity. Like the modified pepsin, the proteolytic activity of the modi-fied pepsinogen at pH 3*5 was significantly increased, suggesting a shift i n pH profile . In contrast, modified chymosin only had 50$ proteolytic activity at pH 3.5 when compared to the native enzyme. Native chymosin has a pro-teolytic pH optimum near 3»5 (Berridge, 1 9 ^ 5 ) , a shift in pH optimum, i f occurred in the modified chymosin, would be detected at pH lower or higher than 3«5« Both carboxyl modified pepsinogen and chymosin ex-hibited a marked retardation in electrophoretic mobility in agarose gels with R f values of 0.70 and 0.63 respectively (compared to the native proteins which were assumed to have a R^, value of 1 . 0 ) . This indicates a decrease in net nega-tive charge on the modified protein molecules. The results show that the carbodiimide reaction was not specific for pepsin. Both the pepsin precursor and other acid proteases such as chymosin, when modified, showed similar responses. In contrast to the present finding, modification with CMC and a colored amine was found to be specific to pepsin (Matyash et a l . , 1 9 7 3 ) . No amine was incorporated into pepsinogen and an acid protease from Aspergillus awamori. The zymogen and fungal protease retained 100$ proteolytic activity. This suggests that 83 CMC and the colored amine are only effective i n modifying the carboxyl groups in pepsin. On the other hand, EDC and glycine methyl ester are able to modify the carboxyl groups in pepsin and related enzymes, showing that they are less selective than the CMC-colored amine system. Effect of Carboxyl Modification on 1^ and i r c a t < of Pepsin For kinetic studies, the synthetic dipeptide APDT was used as substrate. The Michaelis constant ( 1 ^ ) and molecular activity coefficient (k c a^) were determined at pH 2.0 and 4.5. APDT was chosen because i t i s among the most sensitive substrates for pepsin (Fruton, 1970) and the kinetic parameters of pepsin on this substrate have been determined (Jackson et a l . , 1 9 6 5 ) . Fig. 16 and Fig. 17 depict the Lineweaver-Burk plots for native and carboxyl modified pepsins at pH 2.0 and 4.5 respectively. The K M values determined from the inter-cepts are presented i n Table 9. The kca^. values, calculated from the Miehaelis-Menten equation, are also shown in Table 9. The KJJJ and k.:cat values determined for native pepsin were found to compare favorably with the published data (Jackson et a l . , 1 9 6 5 ) . The result shows that after car-boxyl modification, 1 ^ was markedly increased at both pH -15 S]~ X 1 0 3 , fVI 1F I G U R E 1 7 . LINEWEAVER-BURK PLOTS OF THE.HYDROLYSIS OF N - A C E T Y L - L - P H E N Y L A L A N Y L -D I I O D O - L - T Y R O S I N E BY NATIVE (*) AND CARBOXYL M O D I F I E D (A) P E P S I N S AT PH 4,5. CO 86 Table 9« Kinetics of the hydrolysis of N-acetyl-L-,phenyl-alanyl-L-diiodotyrosine by native and carboxyl modified pepsins. Enzyme Concn. pH m K c a t M X 106 min."1 COOH modi-fied pepsin Pepsin 0.51 2.0 0.?8 12.0 0.76 2.0 1.33 13.8 Pepsin 0.76 4.5 12.0 4.1 ™ « 7 „ 1-52 4.5 37.0 4.2 fied pepsin 87 2 . 0 and 4 . 5 , while k c a t was not markedly altered. The increase i n K m suggests that carboxyl modifi-cation of pepsin decreased the a f f i n i t y of the enzyme for the dipeptide substrate, i.e., the enzyme-substrate binding step was hindered. On the other hand, the catalysis of the enzyme-substrate complex was not affected, as the kc&^. values were not changed markedly after carboxyl modification. The partial loss in peptidase activity in the modified pepsin could be attributed to a blocking of the active site on the enzyme. A change i n kinetic parameters was also reported in arginine modified pepsin which has 45$ of the proteolytic activity of the native pepsin (Kitson and Knowles, 1971) . In this case, however, K m was not changed while kca^. was significantly lowered, indicating that the inactivation took place not by the blocking of the enzyme*s active site, but by an effect on the catalytic process subsequent to enzyme-substrate binding. 88 Effect of Synthetic Dipeptides on Carboxyl Modification Carboxyl modification of pepsin was carried out in the presence of some synthetic dipeptides. These include a sensitive substrate (APDT), a poor substrate (N-carbo-benzoxy-L-glutamyl-L-tyrosine, Z-glu-tyr) and a non-sub-strate (N-acetyl-D-phenylalanyl-L-tyrosine, APT). Modi-fications were carried out under the same conditions ( 3 3 -fold excess EDC, 174-fold excess glycine methyl ester, pH 5 « 5 and 90 minutes). The extent of modification, milk clotting activity and electrophoretic mobility of pepsins after treatments were determined (Table 10). Result shows that in the presence of APDT, a sen-sit i v e pepsin substrate, the extent of carboxyl modification was significantly decreased when compared to pepsin modi-fied i n the absence of substrate. The milk clotting a c t i -v i t y and electrophoretic mobility, markedly decreased by carboxyl modification, were only moderately lowered when modification took place in the presence of APDT. In the presence of a poor substrate and a non-sub-strate, pepsin was also modified, but to a lesser extent than the f u l l y modified control. The milk clotting a c t i -v i t y and R^ value were increased relative to the f u l l y modified pepsin. However, the changes were not as marked as those that occurred in the presence of APDT. 89 Table 10. Effect of dipeptides on carboxyl modification of pepsin. Addition Concn. mM No. of COOH gp. modified Milk clotting activity (% control) 1. None - 0 100 1.00 2. EDC + gly methyl ester 10 50 5.2 17 0.56 3. (2) + APDT a 0.3 2.3 50 0.90 4. (2) + Z-glu-tyr ^ 2 3.6 35 0.77 5. (2) + APT C 2 4.8 22 0.65 a N-acetyl-L-phenylalanyl-diiodo-L-tyrosine. N-carbobenzoxy-L-glutamyl-L-tyrosine. c N-acetyl-D-phenylalanyl-L-tyrosine. 90 The present result indicates that the effect of carboxyl modification on pepsin properties were less prominent i n the presence of a dipeptide substrate. This could be due to one of the following: . F i r s t l y , the bin-ding of the dipeptide substrate to the enzyme may protect the carboxyl groups, mainly those in the v i c i n i t y of the pepsin active centre from modification by carbodiimide and nucleophile. Secondly, substrate-enzyme binding may lead to conformational changes in the pepsin molecule which indirectly affect subsequent modification. Finally, the substrate may interact with carbodiimide and nucleophile and less reagents would be available for modifying pepsin. The result shows that even non-substrate can hinder carboxyl modification, though to a lesser extent than the two dipeptide substrates. This suggests that dipeptides can react with the reagents. APDT caused more pronounced changes than Z-glu-tyr probably because i t i s a better substrate and can bind to pepsin more effi c i e n t l y . Hence, substrate-enzyme binding would be the major factor in causing such moderation i n carboxyl modification. 91 Response of Native and Carboxyl Modified Pepsins to Inhibitors The reactions of the native and carboxyl modified pepsins with inhibitors were studied. Two pepsin inhibitors, l,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) and bromophen-acyl bromide were used. EPNP i s a substrate-like epoxide inactivator modifying Asp-76 in pepsin with almost complete loss of proteolytic activity (Chen and Tang, 1 9 7 2 ) , while bromophenacyl bromide reacts with a carboxyl group in pepsin not directly involved in catalysis (Erlanger et a l . , 1965* Clement, 1 9 7 3 ) . Fig. 18 shows the inactivation of native and carboxyl modified pepsins by EPNP. Native pepsin was inactivated more rapidly and the treated enzyme retained only 10$ of proteolytic activity after 120 hours of incubation. The modified enzyme reacted less promptly with EPNP, and retained 30$ of i t s activity at the end of incubation. Table 11 shows the reaction of pepsin with EPNP and bromophenacyl bromide. Bromophenacyl bromide produced a loss of 70 $ proteolytic activity in the native pepsin, but only 35$ in the modified enzyme. The result indicates that carboxyl modified pepsin was s t i l l reactive to site-specific inactivators, confirming that the carboxyl groups blocked were not the active-site residues of pepsin. However, the modified pepsin was less reactive to the inactivators than the native enzyme. This suggests 9 2 24 48 ° 7 2 96 ™~120 T i m e , h o u r F I G U R E 1 8 . I N A C T I V A T I O N OF N A T I V E AND CARBOXYL M O D I F I E D PEPS INSBY E P N P . • , NATIVE PEPSIN j o, CARBOXYL MODIFIED P E P S I N . Table 11. Response of native and carboxyl modified pepsins to inhibitors. Enzyme Inhibitor Concn. Proteolytic activity ($ remaining) Pepsin EPNP a 1 mg/mg protein 10 COOH modi-fied pepsin EPNP 1 mg/mg protein 27 Pepsin BPB b 200 joM 30 COOH modi-fied pepsin BPB 200 JJM 65 a 1,2-epoxy-3-(p-nitrophenoxy) propane. bromophenacyl bromide. 94 that the carboxyl groups reactive to the inhibitors were protected i n the modified enzyme, probably as a result of conformational change or steric hindrance. Stability of Native and Carboxyl Modified Pepsins Near  Neutral pH The s t a b i l i t y of pepsin near neutral pH was studied. In the f i r s t series of experiments, pepsin was incubated in dilute buffer (0.05 M phosphate buffer) at pH 6.5 and 30 C. Both milk clotting and proteolytic activities of pepsin were determined at selected time intervals. The percentage changes in milk clotting and proteolytic a c t i v i t i e s were found to be similar. Subsequently, only milk clotting activity was determined. Fig. 19 shows the s t a b i l i t y curves of native and carboxyl modified crystalline pepsins in buffer. The milk clotting activity of native pepsin dropped rapidly, following a f i r s t order kinetic with a h a l f - l i f e ( t - ^ ) f o r i n a c t i -vation of about 15 minutes. Less than 10% activity was retained after one hour of incubation. With mild modifi-cation (incorporation of 2.8 moles of nucleophile /mole pepsin), the s t a b i l i t y was markedly improved, and the en-zyme retained 20% of milk clotting activity after 4 hours. When pepsin was more extensively modified (incorporation of 5.2 moles of nucleophile), the s t a b i l i t y was improved further. 95 120 T I M E , M S N , F I G U R E 1 9 . S T A B I L I T Y OF C R Y S T A L L I N E N A T I V E AND CARBOXYL M O D I F I E D PEPSINS IN 0 . 0 5 M PHOSPHATE B U F F E R , P H 6 . 5 . A , NATIVE P E P S I N ; A, PEPSIN MODIFIED WITH 87-FOLD EXCESS OF NUCLEOPHILE; • , PEPSIN MODIFIED WITH 174-FOLD EXCESS OF NUCLEOPHILE. 96 The curve started to level off after 2 hours and the loss i n activity was only about 50$ after k hours of incuba-tion. The s t a b i l i t y of crude pepsin in dilute buffer was also studied. As shown in Fig. 20, the crude pepsin pre-paration had a low s t a b i l i t y at pH 6.5. The time-dependent decrease i n milk clotting activity also followed a f i r s t order kinetic. The h a l f - l i f e of inactivation was calcu-lated to be 10 minutes. The s t a b i l i t y was again found to increase markedly after carboxyl modification. The degree of improvement was also dependent on the extent of modifi-cation. The less extensively modified enzyme retained 25$ acti v i t y after 2 hours incubation while the more extensively modified pepsin retained about 5Q$ activity. In the second series of experiments, pepsins were incubated in milk u l t r a f i l t r a t e at 3 0 ° C with progressive adjustment of pH according to that found i n cheese-making. The result for crystalline pepsins i s presented in Fig. 21 and that for crude pepsins i s shown in Fig. 22. In both cases, the s t a b i l i t y of the native enzyme was much higher i n milk u l t r a f i l t r a t e than i n dilute buffer. The milk clotting activity decreased rapidly in the f i r s t hour but started to level off after 2 hours. For both crystalline and crude pepsins, about 20$ activity was found to remain after incubation. 97 T i m e , m i n . F I G U R E 2 0 . S T A B I L I T Y OF CRUDE N A T I V E AND CARBOXYL MODIFIED P E P S I N S IN 0 . 0 5 M PHOSPHATE BUFFER, PH 6 . 5 . A , NATIVE PEPSIN; A , PEPSIN MODIFIED WITH 87-FOLD EXCESS OF NUCLEOPHILE; • , PEPSIN MODIFIED WITH 174-FOLD EXCESS OF NUCLEOPHILE. 98 O SR ° ' 60 I20 J80 240 T i m e , m i n . F I G U R E 2 1 . S T A B I L I T Y OF C R Y S T A L L I N E NATIVE AND CARBOXYL MODIFIED P E P S I N S IN M I L K U L T R A F I L T R A T E UNDER SIMULATED CHEESE-MAKING CONDITIONS. 9 , NATIVE PEPSIN; o, CARBOXYL MODIFIED PEPSIN,•' 99 F I G U R E 2 2 . S T A B I L I T Y OF CRUDE N A T I V E AND CARBOXYL M O D I F I E D PEPSINS IN M I L K U L T R A F I L T R A T E UNDER S I M U L A T E D C H E E S E - M A K I N G C O N D I T I O N S . • , NATIVE P E P S I N ; o, CARBOXYL MODIFIED P E P S I N . 100 After carboxyl modification, the s t a b i l i t y of both crystalline or crude pepsin was markedly improved. The rate of decline in activity was more gradual when compared to the control. For both enzymes, the decrease i n activity was about 40$. The results show that the s t a b i l i t y of pepsin near neutral pH was much improved by carboxyl modification. Pepsin was shown to be unstable at pH 6.0 (Herriott, 1955) and was rapidly denatured at pH 7.0 at 30°C (O'Leary and Fox, 1974). It was suggested that an electrostatic expan-sion of the negatively charged polypeptide chains may lead to denaturation of pepsin at pH values higher than 6 (Low-enstein, 1974). The decrease i n net negative charge on the carboxyl modified pepsin molecule may lower the extent of electrostatic expansion and subsequent denaturation. An improvement of enzyme s t a b i l i t y at pH 6.3-6.5 was also reported in porcine pepsin covalently bound to a soluble polyanionic carrier, ethylene maleic anhydride (Lowenstein, 1974). By incubating the enzyme in 0.05 M phosphate buffer of jM* s 6.3 and 6.5 at 30°C, i t was observed that the s t a b i l i t y against the irreversible de-naturation was 3-5 times better after modification. The enhanced s t a b i l i t y was attributed to different microenviron-mental states of the enzymes. 101 The higher s t a b i l i t y of native pepsin in milk u l t r a f i l t r a t e than in dilute buffer was probably due to the protection of the enzyme by other milk solids against denaturation. The estimation of the amount of i n a c t i -vation of pepsin i n milk u l t r a f i l t r a t e under simulated cheese-making conditions would therefore give a more valid assessment of the s u i t a b i l i t y of the modified enzyme as a milk coagulant for cheese manufacture. However, there were differences between the simulated test and conditions that actually exist during cheese-making. F i r s t l y , acid was added at intervals in the simu-lation while i n the actual process, acid was produced con-tinuously, although this probably has l i t t l e influence on the result. Secondly, the temperature was kept constant at 30°C i n the simulation but is; raised for scalding in cheese-making, although the rise i n temperature would be expected to reduce the amount of active enzyme to a greater extent. Thirdly, the environment of the enzyme i n the simulation was different from that in cheese-making. The concentrations of enzyme were lower and other proteins were present in higher concentrations in the curd, which might increase the s t a b i l i t y of the enzyme. Finally, the results may not represent the amount of active enzyme retained in the curd, since the distribution of enzyme between whey and curd was not known. 1 0 2 However, in spite of these provisions, the present results indicate that native pepsin was extensively i n a c t i -vated under cheese-making, conditions while the carboxyl modified pepsin was a much more stable enzyme. Consistent with the present finding, Green ( 1 9 7 2 ) reported that crude porcine pepsin ( 1 0 . 0 0 0 ) was almost completely denatured i n milk dialysate under simulated cheese-making conditions while rennet was found to be f a i r l y stable, retaining about 6 0 $ of proteolytic activity after treatment. In another report, Holmes and Ernstrom ( 1 9 7 3 ) were unable to recover any active enzyme from Cheddar curd made with pig pepsin whereas 5f» of the added calf rennet was recovered. It has been observed that cheese made with porcine pepsin has a harder body, developed flavor slowly and re-quired an aging period longer than was necessary with calf rennet (Maragoudakis et §X., 1961$ Melachouris and Tuckey, 1 9 6 4 $ Emmons et a l . , 1971$ Green, 1 9 7 2 ) . It was suggested that in cheese made with calf rennet, the active enzyme retained i n the curd would aid the starter enzymes in the ripening of cheese, while in pepsin cheese, the proteolytic breakdown essential for ripening would be almost entirely dependent on starter activity (Green, 1972$ Lawrence ,et a l . . 1 9 7 2 ) . Hence, the long aging period required in pepsin cheese has been attributed to a slow rate of proteolysis as a result of extensive inactivation of the enzyme during 103 cheese-making (Thomasow, 1971; Green and Foster, 1 9 7 4 ) . The carboxyl modified pepsin might be a better milk coagulant than the native enzyme in cheese manufacture. Since the s t a b i l i t y at pH above 6 was significantly im-proved, a greater amount of active enzyme would be retained i n the curd to aid in the ripening of cheese, with a shortening in the aging period and a reduction i n pro-duction cost. A more valid and complete comparision be-tween native and carboxyl modified pepsins in their per-formance in cheese manufacture would await further investi-gations with small scale cheese-making t r i a l s . Thermal Stability of Native and Carboxyl Modified Pepsins The s t a b i l i t y of pepsin at elevated temperatures was studied. Both milk clotting and proteolytic ac t i v i t i e s were measured. Fig. 23 and Fig. 24 show the thermal profiles (milk clotting) of crystalline and crude pepsins respectively. For crystalline pepsin, both native and modified enzymes showed a maximum milk clotting activity at about 40° C, and a rapid drop from 45 to 5 0 ° C. The percentage maximum acti v i t y was higher i n the native pepsin than in the modi-fied enzyme at a l l temperatures except at 5 0 ° C (Fig. 23). Temperature, °C F I G U R E 2 3 . THERMAL P R O F I L E S ( M I L K C L O T T I N G ) OF C R Y S T A L L I N E N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . • , NATIVE P E P S I N ; o , CARBOXYL MODIFIED P E P S I N . F I G U R E 2 4 . THERMAL P R O F I L E S ( M I L K C L O T T I N G ) OF CRUDE N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . • , NATIVE P E P S I N ; o, CARBOXYL MODIFIED P E P S I N . 106 The thermal profiles for crude pepsin were similar to those of the crystalline enzymes. The native crude pepsin had a maximum activity at 40° C. After modification, the maximum was shifted to 4 5 ° C . The percentage maximum a c t i -v i t y was considerably higher i n the native pepsin than i n the modified enzyme at temperatures between 25-40° C but lower at 4 5 - 5 0 ° C (Fig. 24). The Q 1 Q values (milk clotting) for both crystalline and crude pepsins are presented i n Table 12. The results indicate that the Q 1 Q values increased after carboxyl modification. The increases were particularly marked at the higher temperature range (40-50° C). This shows that modified pepsin had higher heat s t a b i l i t y than the native enzyme with regard to milk clotting activity. The thermal profiles for proteolysis are shown i n Fig. 25 and Fig. 26. For crystalline pepsins, the percen-tage maximum activity was higher i n carboxyl modified enzyme at lower temperatures ( 3 0 - 5 0 ° C ) . At higher temperatures ( 5 0 - 7 0 ° C ) , however, the proteolytic activity of the native pepsin was greater than that of carboxyl modified pepsin. Both enzymes showed a rapid decrease in activity from 6 0 - 7 0 ° C (Fig. 2 5 ) . For crude pepsins, the thermal profiles of the native and modified enzymes were almost identical. Both enzymes showed a gradual increase i n proteolytic activity with increasing temperature up to 6 0 ° C . From 6 0 ° C to 107 Table 12. Effect of carboxyl modification of pepsin on Q 1 Q values (milk clotting). o * Enzyme Temperature ( C) Q 1 0 Pepsin (crystalline) 30-40 1.6 40-50 0.4 C00H modified 30-40 1 Q pepsin (crystalline) ^ y 40-50 0.6 Pepsin (1:10,000) 30-40 1.3 40-50 0.4 COOH modified -,n ,,n -. ^ pepsin (1:10,000) 3 0 " 4 0 1 ' 6 40-50 0.7 * Q 1 0 v a l u e s represent the ratio of milk clotting activity at (T+10)° to that at T°. 108 F I G U R E 2 5 . THERMAL P R O F I L E S ( P R O T E O L Y T I C ) OF C R Y S T A L L I N E N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . • , NATIVE P E P S I N ; o , CARBOXYL MODIFI ED P E P S I N . 109 a. 01 • L_ 30 40 50 60 Temperature, °C FIGURE 26. THERMAL PROFILES (PROTEOLYTIC) OF CRUDE NATIVE AND CARBOXYL MODIFIED PEPSINS. o, NATIVE PEPSIN; CARBOXYL MODIFI ED PEPSIN. 110 7 0 ° C , the activity decreased sharply to less than 20$ of the maximum level (Fig. 2 6 ) . The Q^0 values for proteolysis are presented in Table 13. For both crystalline and crude pepsins, the native enzymes had higher Q^Q values than the modified enzymes at a l l temperature ranges. The difference was not marked except at 6 0 - 7 0 ° C when the Q^Q valuerof native crystalline pepsin was three times higher than that of the modified enzyme. The result shows that carboxyl modi-fication caused a slight drop i n thermal s t a b i l i t y of pepsin when the acti v i t y was measured against hemoglobin. The rapid decline i n milk clotting and proteolytic a c t i v i t i e s of pepsin at high temperatures was due to de-naturation. At pH 6.3 at which milk clotting activity was measured, denaturation occurred at temperatures above 4 5 ° C At pH 2.0 at which proteolytic activity was measured, denaturation did not occur u n t i l the temperature exceeded . 6 0 ° C . This indicates that pepsin was more stable against heat at lower pH's. Similar phenomena were reported by O'Keeffe et a l . (1977) who showed that denaturation of pepsin was c r i t i c a l l y dependent on pH and temperature within very narrow limit s . The improvement i n heat s t a b i l i t y of carboxyl modi-fied pepsin at milk;clotting pH may be of some practical I l l Table 13. Effect of carboxyl modification of pepsin on Q,n values-(proteolytic). o * Enzyme Temperature ( C) Q 1 0 Pepsin (crystalline) 30-40 1.43 40-50 1.10 50-60 1.21 60-70 0.54 C00H modified 30-40 1.27 pepsin (crystalline) 40-50 1.07 50-60 0.90 60-70 0.17 Pepsin (1*10,000) 30-40 1.37 40-50 1.04 50-60 1.11 60-70 0.16 COOH modified 30-40 1.31 pepsin (1*10,000) J+0-50 1.00 50-60 1.09 60-70 0.15 * o Q 1 Q values represent the proteolytic activity at (T +10) to that at T°. 112 importance. In the making of Cheddar cheese, the curd i s normally heated to temperatures near 40° C during cooking, while the pH remains above 6.0. An increase in enzyme s t a b i l i t y under these conditions would ensure that a higher proportion of active enzyme to be retained i n the curd and be available for aiding the ripening of cheese. Effect of Carboxyl Modification on Curd Tension and Rate  of Syneresis The firmness of curds from milk clotted by native and carboxyl modified pepsins was measured by the method of Hehir ( 1 9 6 8 ) . The result, shown in Table 14 indicates that for both crystalline and crude pepsins, there was no significant difference between the firmness of curds from native and modified enzymes. For crystalline pepsins,the native enzyme produced a slightly firmer curd than the modified enzyme, while the reverse was demonstrated for the crude pepsins. According to the comparision of curd tension measurements with subjective assessment of curd firmness (Hehir, 1 9 6 8 ) , a curd tension higher than 75 units (eg) corresponds to a firm curd. Hence, both cry-stalline and crude pepsins produced a firm curd and the curd tension was comparable to that of chymosin which gave a firmer curd (Table 14). 113 Table 14. Tension of curds produced by pepsin, carboxyl modified pepsin and chymosin. TTviQtrmA Clotting time Curd tension E n z y m e (min.) (cg) Pepsin (crystalline) 3.5 125 + 5* COOH modified pepsin (crystalline) COOH modified pepsin (1:10,000) 3.7 118+4 Pepsin (1:10,000) 4.5 98 +4 4.5 107 + 6 Chymosin 3.5 1 3 5 + 6 Curd tension values are presented as averages of five determinations + S.E. 114 The effect of carboxyl modification of pepsin on the rate of syneresis was studied. The results are pre-sented i n Fig. 27 and Fig. 28. For crystalline pepsins, the rate of syneresis was not markedly affected by modi-fic a t i o n . The native and modified enzymes produced almost identical curves (Fig. 27) . The percentage syneresis i n -creased rapidly in the f i r s t two hours and then levelled off. The modified pepsin produced syneresis at a rate faster than the control, but the f i n a l percentage syneresis was slightly lower than that of the native enzyme. Chymosin was found to yield syneresis at a rate very similar to that of pepsin (Fig. 27). For crude pepsins, the percentage syneresis was considerably lower than that of the crystalline pepsins. Syneresis . ; developed at a slower rate, and the curves did not level off u n t i l after three hours. There was no significant change in the syneresis rate after carboxyl modification. The modified enzyme produced syneresis at a rate faster than the control, while the f i n a l percentage syneresis was again sl i g h t l y lower than that of the native enzyme (Fig. 28). The above results show that i n the i n i t i a l stages of cheese-making, carboxyl modification of pepsin did not affect the quality of the curd such as curd tension and the rate of exudation of whey. 115 Time, hour FIGURE 27, SYNERESIS OF CURD BY CRYSTALLINE PEPSIN, CARBOXYL MODIFIED CRYSTALLINE.PEPSIN AND CHYMOSIN, A , NATIVE P E P S I N ; A , CARBOXYL MODIFIED P E P S I N ; • ,CHYMOSIN. 70 T i m e , hour F I G U R E 2 8 , S Y N E R E S I S OF CURD BY CRUDE N A T I V E AND CARBOXYL M O D I F I E D P E P S I N S . A , NATIVE P E P S I N ; A, CARBOXYL MODIFIED P E P S I N . 117 GENERAL DISCUSSION In the present investigation, selective modification of the carboxyl groups in porcine pepsin was carried out at pH 5'5 using a water-soluble carbodiimide, EDC and an amino acid methyl ester as nucleophile. The number of nucleophile molecules incorporated per molecule of pepsin ranged from 2.8 to 11.2. Profound changes in the a c t i v i -ties, s p e c i f i c i t y and physicochemical properties were observed i n the carboxyl modified pepsin. There was a significant decrease in milk clotting a c t i v i t y while the proteolytic activity against hemoglobin was not affected. The clotting activity against x-casein was increased by two to three-fold while the rate of coagulation of x-f< g l-casein mixture was decreased markedly to 10-20$ of the control. There was a shift i n proteolytic pH profile with the pH optimum increased from 2.0 to about 3 . 5 . The relative electrophoretic mobility was decreased and the isoelectric point was slightly increased. There was a drop in peptidase activity and the K m was increased while K c a^ was not changed. Finally, the pH s t a b i l i t y of the enzyme was significantly increased. Several lines of evidence suggest that the drop in milk clotting activity of the modified pepsin was not 118 directly related to the modification of specific carboxyl group(s) involved i n the enzymatic clotting of milk. F i r s t l y , the rate of casein hydrolysis was not affected by carboxyl modification, and both native and modified pepsin showed similar caseinolytic properties. This indicates that the chemical procedure did not modify the carboxyl group(s) involved i n the enzymatic breakdown of casein which is the fi r s t step in the coagulation of milk mediated by an enzyme. Secondly, pepsin treated with EDC alone showed a slight drop i n milk clotting activity although the carboxyl residues were not modified. The treated enzyme had a sligh t l y lower electrophoretic mobility than the control, suggesting a decrease i n net negative charge on the enzyme molecule. The incorporation of the positively charged carbodiimide on the pepsin might lead to a change i n the charge distribution of the enzyme which could affect the milk clotting activity. Finally, the carboxyl modification was not specific to pepsin. It caused similar drop in milk clotting a c t i -v i t y of pepsinogen and chymosin. If the decrease i n milk clotting a c t i v i t y of the modified pepsin was due to the blocking of specific carboxyl group(s) responsible for the enzymatic coagulation of milk, modification of other enzymes may not cause similar changes. This was illustrated by 119 Matyash et a l . (1973) who found that modification of pepsin carboxyl groups by CMC and a colored amine resulted in a drop in proteolytic activity, while similar treatment on pepsinogen and another acid protease did not cause changes in activity. In order to explain the decrease in milk clotting a c t i v i t y of the carboxyl modified pepsin, one has to understand the mechanism of milk c l o t t i n g process. Cur-dling of milk by clotting enzymes is a complex phenomenon. The f i r s t step or primary phase involves a highly specific action of enzyme on x-casein to destroy the micelle-sta-b i l i z i n g power of the protein. X-Casein, a glycoprotein, i s s p l i t specifically at the Phe-Met bond. This results i n the release of an insoluble para-x-casein and a soluble glycomacropeptide. The sequence of events following the enzymatic action on x-casein to destabilize the micelles i s poorly understood. This i s partly due to an incomplete under-standing of the structure of the caseinate micelle. Sev-eral models have been proposed (Waugh and Noble, 1965; Gamier and Ribadeau Dumas, 1970j Parry and Carrell, 1969; Slattery and Evard, 1973) but no one i s absolutely satis-factory. However, from the available experimental data, some facts about the secondary phase are now known. Apparently, 120 coagulation requires the conversion of a minimum amount of X-casein to para-X-casein (Green and Marshall, 1 9 7 7 ) . The release of the highly negatively charged glycomacro-peptides from micelles decreases the negative charge on the micelles (Green and Crutchfield, 1971; Pearce, 1976). This results < in the reduction of electrostatic repulsion between micelles and promotes aggregation (Green and Crutchfield, 1971). Para-x-casein, present on the micellar surface, increases the hydrophobicity of the particles, again promoting aggregation through hydro-phobic interactions (Payens, 1966). It has been suggested that coagulation results from specific interaction be-tween micelles through enzymatically-modified areas on their surfaces (Waugh', 1971; Knoop and Peters, 1975; Green and Marshall, 1977). From the above observations, one may speculate that the decrease in the rate of milk coagulation by carboxyl modified pepsin i s attributed to a slow down in the release of para-x-casein resulting from an interference with the enzyme-micelle interaction. Since a defined amount of para-x-casein i s required to i n i t i a t e coagulation (Green and Marshall, 1977)» a drop i n the rate of para-x-casein release would directly affect the aggregation of the micelles. 121 Alternatively, the decrease in milk clotting a c t i -v i t y could be related to the secondary stage of coagulation, i.e., interactions of the enzymatically modified micelles and the subsequent aggregation to form curd. The carboxyl modified pepsin may not be able to produce the suitable types of micelles for micelle-micelle interaction, which i s viewed as a highly specific reaction (Knoop and Peters, 1975; Green and Marshall, 1977). Results from casein coagulation experiments indicate that K.-casein was clotted by the modified pepsin at a rate faster than the native enzyme, while the K-o( ^-casein mixture was much more resistant to clotting by the modified enzyme. The clotting activity against the casein mixture was decreased to the same extent as the drop in milk clot-ting activity after carboxyl modification (see Table 7). The data suggest that X - o C g^-casein interaction in the micelles may cause alterations that increase the a f f i n i t y of native pepsin for i t s substrate, but hinder the binding between the micelles and the modified enzyme. This i s consistent with the view that the decrease in milk clotting activity i s related to an interference with the enzyme-micelle interaction. The present results suggest that the clotting of milk i s a process i n which charge may play an important role. At least part of the forces involved in micelle-122 enzyme interaction i n the primary phase would be electro-static i n nature, and changes i n the charge distribution on the enzyme molecule could cause profound alteration i n the v i t a l substrate-enzyme binding process. This i s consistent with the work of Green and Marshall (1977) who observed an increase in the a f f i n i t y of calf rennet for the micelles and an acceleration i n the aggregation of caseinate micelles by the addition of cationic materials, indicating that charge plays an important role in the clotting of milk. In contrast to milk clotting, hydrolysis of proteins such as denatured hemoglobin by pepsin did not seem to be significantly influenced by charge effect. Although the pH profile was shifted, the specific proteolytic activity at the optimum pH was relatively unchanged after carboxyl modifi-cation. The result suggests that coagulation of milk by pepsin, although involving a v i t a l proteolytic step, i s different from general proteolysis, probably i n the mode of binding between enzyme and different substrates. Apart from the decrease i n milk clotting activity, changes in some physicochemical properties in the carboxyl modified pepsin are also attributed to an alteration in the net charge carried by the enzyme. The shift in pH acti v i t y curve when hemoglobin was used as substrate, the decrease in relative electrophoretic mobility on agarose 123 gel and the slight increase in isoelectric point could a l l be related to a change i n the net ionic charge on the modi-fied enzyme. Modification of individual charged groups of proteins usually affect the net ionic charge in a way which may be disruptive to their characteristic properties. Decreased sol u b i l i t y and changes in conformation or in the state of aggregation frequently result from such modifications (Means and Feeney, 1 9 7 1 ) . These changes were not observed i n carboxyl modified pepsin, except when tyrosine and try-ptophan methyl esters were incorporated into the enzyme, which resulted in a significant decrease in s o l u b i l i t y . This, however, was probably not attributed to the charge effect but to an increase i n the hydrophobicity of the enzyme upon the incorporation of hydrophobic groups. Kinetic study on the modified pepsin suggests that the drop i n peptidase activity was possibly due to a de-crease in the a f f i n i t y of the modified enzyme to the d i -peptide substrate, as indicated by the increase i n Km. This may be due to either a change i n the conformation or the charge distribution on the enzyme, both of which could affect the binding step between the enzyme and the subst-rate. The fact that Kca^. remained unchanged indicates that the decrease in peptidase activity was not due to a change i n the catalytic step. 124 The kinetic data cannot he applied directly to ex-plain the drop i n milk clotting activity in the modified pepsin since the substrates used were different. If the K m for milk coagulation can be calculated and found to increase after carboxyl modification, one can conclude that the drop in milk clotting activity was due to an interference with the binding between the enzyme and the micelles. Results from the present investigation show that carboxyl modification of pepsin greatly improves the sta-b i l i t y of the enzyme at pH around 6.5. This may have im-portant practical implication related to the use of this enzyme as a milk coagulant i n cheese-making. The traditional coagulant used for cheese-making since pre-historic time i s rennet extract from the abomasa of 10 to 30-day-old milk-fed calves. With a rapid rise i n world-wide cheese consumption, coupled with a decrease i n the practice to slaughter newly born calves, there has been a chronic world shortage of rennet during the last two decades. This has stimulated interest in finding suit-able substitutes for rennet to use as coagulants i n the making of cheese. The basic function of a clotting enzyme i s the con-version of liquid milk to a gel. This process can be 125 catalysed by most proteases. Apart from clotting milk, a suitable coagulant has to survive through cheese-making, and the residual enzyme incorporated in the curd should contribute to proteolysis of the cheese during ripening (Lawrence et al.., 1972; Green and Foster, 1 9 7 4 ) . However, many proteases are too proteolytic at the customary pH values of milk and cheese, hence reducing the yield of cheese and retention of fat by the curd (Veringa, 1961; Ritter, 1 9 7 0 ) , and generating bitter peptides and poor body (Green and Foster, 1 9 7 4 ) . Thus, the most useful milk coagulants have a high clotting to proteolytic en-zyme ratio (Ernstrom, 1 9 7 4 ) . Proteolytic enzymes from animals, higher plants and micro-organisms have been studied for their suita-b i l i t y to replace calf rennet in making cheese, but only a few were successful commercially. These are swine pepsin, used as 5 0 « 5 0 rennet-pepsin mixtures, and fungal rennets from Endothia .paras i t i c a (Sardinas,1968), Mucor  pusillus Lindt (Oka et a l , , 1973; Arima et a l . , 1976) and Mucor meihei (Sternberg, 1 9 7 2 ) . Recent literature indicates that good quality cheese can sometimes be produced using fungal rennets. In some cases, however, cheeses manufactured with micro-b i a l rennets were of a slightly lower quality than calf rennet cheeses (Martens and Naudts, 1 9 7 6 ) . 126 The use of pepsin, particularly porcine and bovine pepsins in cheese-making was considered a long time ago. In recent years, the use of calf rennet mixed with swine pepsin became wide-spread, and has been found to produce good quality cheeses of various types including Cheddar (Phelan, 1 9 7 3 ) . Pharmigiano-Reggiano (Corradini et a l . . 1 9 7 6 ) , Grana, Mozzarella and Taleggiano cheeses (Bottazzi et a l . , 1976"). Recently, pure bovine pepsin was used on an industrial scale i n the production of Cheddar (Emmons et a l . , 1974$ 1976) and other cheeses (Bottazzi et a l . , 1976; Corradini et.al., 1 9 7 6 ) . As calf rennet substitute, porcine pepsin has some distinct advantages. It i s considerably cheaper than other rennet substitutes (Green, 1972} 1 9 7 7 ) . In fact, the commercial success of the rennet-pepsin blends i s mainly due to the low cost of pepsin advantageously re-flected in the product price (Sardinas, 1 9 7 6 ) . Porcine pepsin i s commercially available, and the supply i s more stable than calf rennet and some rennet substitutes. However, when used alone i n cheese-making, porcine pepsin suffers from a number of disadvantages. The curd formed i s not as firm as calf rennet's, and there i s some loss of fat in the whey. Organoleptic quality of pepsin cheese i s inferior to that of rennet cheese (Green, 1 9 7 2 ) . Furthermore, a longer ripening peroid i s required i n cheese made with pepsin alone (Melachouris and Tuckey, 1964). The last drawback i s due to the in s t a b i l i t y of porcine pepsin under cheese-making conditions, mainly high pH* s. Green and Foster (1974) observed that coa-gulating enzymes retained in the cheese curd contribute significantly to casein hydrolysis, and that starter enzymes and rennet are synergistic in their action on caseins and their breakdown products (Ohimiya and Sato, 1972). In spite of some controversy (0*Keeffe et a l . . 1 9 7 7 ) , the majority of available experimental evidence indicates that porcine pepsin i s almost completely de-natured during cheese manufacture while at least some added calf rennet i s recovered and contributes to pro-teolysis i n cheese ripening (Green, 1972; Holmes and Ernstrom, 1973; Green and Foster, 1974). Consistent with the above reports, the present results also show that porcine pepsin was rapidly i n -activated i n buffer at pH 6.5 and i n milk u l t r a f i l t r a t e under simulated cheese-making conditions. After car-boxyl modification, however, the s t a b i l i t y of pepsin was significantly improved. This could greatly enhance the u l t i l i z a t i o n of porcine pepsin in cheese manufacture. If some active pepsin i s retained i n cheese curd, the ripening period could be shortened thus lowering the 128 production cost. In addition, some defects associated with pepsin cheese such as harder body and slow flavor development (Maragoudakis et a l . , 1961; Melachouris and Tuckey, 1964; Emmons et a l . , 1971) may also be overcome since these defects are probably attributed to a slow rate of proteolysis i n the cheese. Although the ultimate conclusion on the s u i t a b i l i t y of a milk coagulant should be derived from large scale cheese manufacture, some simple screening procedures have been described. These include non-protein nitrogen test (DeKoning, 1972), measurement of the rate of firming of the milk gel after coagulant addition (Stavlund and Kier-meier, 1973) and measuring the s t a b i l i t y of enzyme in a simulation of the early stages of cheese-making (Green and Stackpoole, 1975). The present results indicate that the carboxyl modified pepsin has increased pH and heat s t a b i l i t y under cheese-making conditions. It also has caseinolytic properties similar to those of the native pepsin or chymosin. The rate of syneresis and curd ten-sion development were also similar to those observed i n native pepsin and chymosin. One drawback of the modified pepsin i s the decreased milk clotting activity. However, modified 1«10,000 pepsin seems to retain higher milk clotting activity than the crystalline enzyme while the increase in s t a b i l i t y i s s t i l l 129 significant. The degree of loss of milk clotting activity can be controlled by the extent of modification. A loss of 50$ activity would double the clotting time which should not be a c r i t i c a l factor i n the making of cheese. Since porcine pepsin has relatively high proteolytic act i v i t y , the active enzyme retained i n the curd may lead to extensive breakdown of proteins resulting i n bitterness and other textural defects. However, as the s t a b i l i t y of porcine pepsin i s dependent on the extent of carboxyl modi-fication, the amount of active pepsin retained can be ad-justed by controlling the degree of modification on the enzyme. A principle deterrent in ut i l i z i n g chemical modi-fication i n food proteins i s the cost associated with proving to regulatory agencies that the products are non-toxic. Carbodiimides have been known to be toxic, but as they are just used as a coupling reagent in the reaction and are removed by dialysis, the quantity retained in the modified enzyme would be very small. Furthermore, since the clotting enzymetmilk ratio i s generally low in cheese-making, and a large percentage of coagulant i s lost i n the whey, the concentration of carbodiimide in the cheese, even i f bound to the enzyme, would be too low to be of any great significance. The reagents used in carboxyl modification are relatively inexpensive and the reaction i s simple and mild. Hence, the cost of producing carboxyl modified pepsin would not be too high, and the price of the en-zyme should be at least competitive to that of the calf rennet. The present data show the possibility'of modi-fying the activity, specificity and physical properties of an enzyme by chemical derivatization. This could widen the scope of food-related enzymes i n the food i n -dustry. Problems associated with enzyme-catalysed pro-cesses may be solved i f the undesirable characteristics of the enzyme are identified and can be corrected by chemical modifications. The present finding also i n -dicates the possibility of chemically modifying the properties of an enzyme to imitate another enzyme. It may be feasible to substitute enzymes that are expensive and/or i n short supply with cheaper modified enzymes i f their properties are compatible with the substituted enzymes. 131 CONCLUSIONS Selective modification of carboxyl groups in porcine pepsin by water-soluble EDC and amino acid methyl esters caused significant changes i n a c t i v i t i e s , s p e c i f i c i t y and physicochemical properties of the enzyme. (1) The milk clotting activity was significantly decreased while the proteolytic activity against hemoglobin was not altered. Consequently, the milk clotting:proteo-l y t i c activity ratio was markedly decreased. The peptidase acti v i t y against APDT was decreased by about 50$. (2) The charge density of pepsin was altered by carboxyl modification. This was shown by a decrease in relative electrophoretic mobility, a slight increase in isoelectric point and a shift i n the proteolytic activity pH profi l e . (3) The caseinolytic properties were not affected. However, the clotting activity against /c-casein was incr-eased while the clotting activity against /c-otg^-casein was significantly decreased. Results suggest that the drop i n milk clotting activity may be attributed to a change i n the charge distribution on the modified enzyme, thus hindering the interaction between pepsin and micelles. (4) Kinetic study using dipeptide substrate shows that K m was increased while k c a + < was not significantly 132 altered. This indicates that the lowering in peptidase activity was caused by an interference with the enzyme-substrate binding process, and the catalysis of the enzyme-substrate complex was not affected. (5) The presence of dipeptide substrates interfered with the carboxyl modification, suggesting that the modi-fi e d carboxyl groups were located near the enzyme-substrate binding s i t e . (6) The carboxyl modification was not specific to pepsin. 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