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Studies on the functions of disulfide linkages in k-casein Toma, Sadiq Jawad 1974

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c I S T U D I E S ON T H E F U N C T I O N S O F D I S U L F I D E L I N K A G E S I N K - C A S E I N B Y S A D I Q J A W A D T O M A B . S c . U n i v e r s i t y o f B a g h d a d 1 9 6 5 M . S c . 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 1 9 7 1 A T H E S I S S U B M I T T E D 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 T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n t h e D e p a r t m e n t o f F o o d S c i e n c e We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d 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 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r equ i r emen t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag r ee that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e 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 pu rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thou t my w r i t t e n p e r m i s s i o n . Department o f FOOD SCIENCE The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date January 10, 1974 i ABSTRACT ,v A three part investigation i s described i n which a method for measuring SH and SS contents of K-casein and some other food proteins, u t i l i z i n g Ellman's reagent, was developed. The role of d i s u l f i d e groups i n the s t a b i l i t y of heated K-casein , i was studied after modification of those groups by desulf u r i z a t i o n or a l k y l a t i o n . Sepharose gel chromatography was u t i l i z e d to separate a pure K-casein f r a c t i o n from whole casein or skimmilk. For the determination of SH groups i n food proteins urea (6.5 M), Na dodecylsulfate (0.5%), and a mixture of 6.5 M urea and 5 M guanidine-HCl were used as d i s s o c i a t i n g agents for skimmilk, egg white, f l o u r and gluten respectively. D i s u l f i d e groups were reduced with 1 to 2% mercaptoethanol i n the presence of the same dis s o c i a t i n g agents as for the SH determination and the proteins were precipitated by adding 8-11% trichloroace-t i c acid. Total SH (SH + reduced SS) was analyzed after dissolving the precipitates i n 8 M. urea or 0.5% Na dodecy l s u l f ate . (SDS) at pH 8.0. Values of SH. and SS for K-casein, a s5~casein, c t g^-casein, g-lactoglobulin, ovalbumin, egg white, skimmilk, fl o u r and gluten were i n good agreement with l i t e r a t u r e values. Recoveries of SH and SS ranged from 91 to 98% and from 89 to 102% respectively. '•' Modification of the d i s u l f i d e groups i n K-casein was performed by des u l f u r i z a t i o n with. Raney nic k e l under an atmos-phere of hydrogen or by a l k y l a t i o n with iodoacetamide. Approxi-mately 50% of the SS groups were removed by the d e s u l f u r i z a t i o n r e a c t i o n at pH 7.0 f o r 48 hours. The sedimentation c o e f f i c i e n t decreased from 16 S to 12 S by d e s u l f u r i z a t i o n and K - c a s e i n migrated as bands by polyacrylamide-gel e l e c t r o p h o r e s i s at pH 9.0 w i t h 4 M urea compared to a smear f o r the untreated c o n t r o l . These r e s u l t s i m p l i c a t e d the d i s s o c i a t i o n of K - c a s e i n by d e s u l f u r i z a t i o n . On heating f o r 30 minutes i n b o i l i n g water, both the d e s u l f u r i z e d and the a l k y l a t e d K - c a s e i n s c o n s i d e r a b l y decreased the a -c a s e i n s t a b i l i z i n g a b i l i t y whereas the c o n t r o l S l ^ JT revealed no s i g n i f i c a n t changes. The fluorescence p o l a r i z a t i o n of modified K - c a s e i n was a l s o decreased upon heating. Aggregated peaks at the v o i d volume were observed when the heated modified K - c a s e i n was e l u t e d on a Sepharose 2B column wi t h phosphate b u f f e r at pH 7.0, whereas untreated K - c a s e i n d i d not show those peaks upon heating. I n t e r a c t i o n between 3 - l a c t o g l o b u l i n and K - c a s e i n was observed by Sepharose g e l f i l t r a t i o n and poly a -crylamide g e l e l e c t r o p h o r e s i s , when the mixture was heated. However, t h i s i n t e r a c t i o n was not detected w i t h the modified K - c a s o i n s . These r e s u l t s i n d i c a t e the importance of d i s u l f i d e groups i n maintaining a s t r u c t u r a l i n t e g r i t y which c o n t r o l s heat s t a b i l i t y of the molecules. The a p p l i c a t i o n of Sepharose 6B i n the pre p a r a t i o n of K - c a s e i n d i r e c t l y from skimmilk or whole casein i s reported i n the l a s t chapter. Attempts were made to u t i l i z e the m i l d e s t p o s s i b l e c o n d i t i o n f o r the pre p a r a t i o n of K - c a s e i n by t h i s technique. The e f f e c t of temperature, i o n i c s t r e n g t h and pH was st u d i e d . The best r e s o l u t i o n and d i s s o c i a t i o n of the K - B - a ^ - c a s e i n complex were obtained when whole casein was eluted with phosphate buffer 0.005 M, pH 9.0 at 25°C. Complete elimination of a s^-casein was d i f f i c u l t . Introducing 3 M urea in-to the system eliminated a l l the a , - c a s e i n contaminant and s l produced a pure K-casein f r a c t i o n . Further improvement i n the resolution was obtained by increasing the urea concentration to 6.6 M and an a c - c a s e i n r i c h f r a c t i o n was obtained when s5 whole casein or d i r e c t l y skimmilk were applied to the column. A r e l a t i v e l y high y i e l d was obtained, about 200 mg K - c a s e i n from 2 gm whole casein from a single run on a column of 4X77 cm length. K - c a s e i n f r a c t i o n obtained by t h i s method was capable of s t a b i l i z i n g 95% of the a ^ - c a s e i n at a r a t i o of 0.13 K — to 1.0 of a ,-casein. The sedimentation c o e f f i c i e n t s l was 14.0 S. The molecular weight of a g^-casein and i t s sensi-t i v i t y to calcium was investigated and i t was found that only 2 mM C a C l 2 was required for p r e c i p i t a t i o n of 80% of the agg-casein compared to 8 mM for a s l - c a s e i n . The t u r b i d i t y of a g 5 - c a s e i n i n 10 mM C a C l 2 was 4.5 f o l d greater than that of a s l ~ c a s e i n . Calcium a s 5~caseinate was s t a b i l i z e d by K-casein to a lesser extent than Ca-a s^-caseinate. The molecular weight determined by the d i f f e r e n t i a l boundary method i n an u l t r a -centrifuge were 65,750 and 31,800 for a s 5 - c a s e i n and for the i mixture of a ^"and a g^-casein respectively. TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS L I S T OF TABLES L I S T OF FIGURES ACKNOWLEDGEMENTS INTRODUCTION SURVEY OF LITERATURE C a s e i n s i n M i l k C a s e i n M i c e l l e The D i s u l f i d e G r o u p s i n K - c a s e i n S e p a r a t i o n o f K - c a s e i n E f f e c t o f H e a t on K - c a s e i n CHAPTER I : SULFHYDRYL-DISULFIDE DETERMINATIONS SOME PROTEINS AND FOOD PRODUCTS INTRODUCTION EXPERIMENTAL Rea'gents, M a t e r i a l s a n d M e t h o d s F l o u r a n d g l u t e n Egg w h i t e S k i m m i l k SH and SS D e t e r m i n a t i o n i n P u r e P r o t e i n s C a l c u l a t i o n s RESULTS AND DISCUSSION R e d u c t i o n o f SS G r o u p s SH a nd SS o f P u r i f i e d P r o t e i n s SH a n d SS o f F o o d P r o d u c t s V TABLE OF CONTENTS ( C o n t i n u e d ) PAGE CHAPTER I I : EFFECT OF HEAT ON THE D I S U L F I D E MODIFIED K-CASEIN INTRODUCTION 33 MATERIALS AND METHODS 3 6 D e s u l f u r i z a t i o n o f K - c a s e i n 36 P r e p a r a t i o n o f Raney n i c k e l 36 U l t r a c e n t r i f u g a t i o n 37 S t a b i l i z a t i o n o f a , - c a s e i n w i t h K - c a s e i n 37 s l F l u o r e s c e n c e P o l a r i z a t i o n a n d A s s o c i a t i o n C o n s t a n t 38 D a n s y l a t i o n o f a , - c a s e i n 38 J s l A l k y l a t i o n o f K - c a s e i n w i t h I o d o a c e t a m i d e 39 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 39 H e a t i n g E x p e r i m e n t s 41 R e n n i n R e a c t i o n 41 RESULTS AND DISCUSSION 42 D e s u l f u r i z a t i o n R e a c t i o n 42 C h e m i c a l a n d P h y s i c a l P r o p e r t i e s o f D e s u l f u r i z e d K - c a s e i n 44 A c t i o n o f R e n n i n 50 E f f e c t o f H e a t i n g o n K - c a s e i n 50 Changes i n F l u o r e s c e n c e P o l a r i z a t i o n o f K - c a s e i n due t o SS M o d i f i c a t i o n and H e a t T r e a t m e n t 63 I n t e r a c t i o n o f K - c a s e i n a n d 3 - l a c t o g l o b u l i n 70 CHAPTER I I I : THE USE OF THE GEL CHROMATOGRAPHIC TECHNIQUE I N THE ISOLATION AND CHARACTERIZATION OF K-and a - c a s e i n s s 5 INTRODUCTION 83 v i TABLE OF CONTENTS ( C o n t i n u e d ) PAGE METHODS AND MATERIALS 87 P r e p a r a t i o n o f Whole C a s e i n 87 P r e p a r a t i o n o f K- and a ^ - c a s e i n s 87 S e p h a r o s e g e l F i l t r a t i o n 88 RESULTS AND DISCUSSION 9 0 SECTION 1: 9 0 The S e p a r a t i o n o f K - c a s e i n on S e p h a r o s e 6B 90 A. The e f f e c t s o f i o n i c s t r e n g t h on t h e e l u t i o n p a t t e r n 90 B. E f f e c t o f pH and t e m p e r a t u r e 94 C. E f f e c t o f u r e a c o n c e n t r a t i o n o n t h e e l u t i o n p a t t e r n 9 8 D. Y i e l d o f K - c a s e i n a n d c o l u m n l e n g t h 102 The P r o p e r t i e s o f K - c a s e i n p r e p a r e d b y S e p h a r o s e 109 G e n e r a l D i s c u s s i o n 113 SECTION 2: C a l c i u m S e n s i t i v i t y a n d m o l wt o f a - c a s e i n 115 s5 LITERATURE CITED 125 LIST OF TABLES S u l f h y d r y l and d i s u l f i d e v a l u e s f o r p u r i f i e d p r o t e i n s S u l f h y d r y l and d i s u l f i d e values f o r food products Recovery of s u l f h y d r y l and d i s u l f i d e groups of 3 - l a c t o g l o b u l i n from food products Amino A c i d composition of K - c a s e i n b e f o r e and a f t e r d e s u l f u r i z a t i o n E f f e c t of heat on the p o l a r i z a t i o n and a s s o c i a t i o n constant (ka) of K - c a s e i n and a l k y l a t e d K - c a s e i n Sedimentation r a t e and molecular weight of a - c a s e i n with and without m e r c a p t o e t h l n o l v i i i LIST OF FIGURES FIGURES PAGE Fi g . 1. E f f e c t of urea concentration on the t o t a l SH groups of several protein materials a f t e r incubation f o r 1 hour at 25°C i n the presence of 2-mercapto-ethanol 26 F i g . 2 The rate of de s u l f u r i z a t i o n reaction of K-casein i n the presence of 8 M urea or 0.5% SDS 43 F i g . 3 Ultracentrifuge pattern of K - c a s e i n and desulfurized K-casein obtained 10 minutes after reaching 59,100 RPM 45 F i g . 4 El u t i o n pattern of unmodified K-casein alkylated K-casein and desulfurized K-casein on Sepharose 6B column (43 X 2.5 cm) eluted with 0.05 M phosphate buffer pH 7.6 at 25°C (buffer contains 6.6 M urea) 46 F i g . 5 Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 4C i n the presence and absence of 2-mercaptoethanol 49 F i g . 6 E f f e c t of rennin on the polyacrylamide gel electrophoretic patterns of K-casein and desulfurized K-casein after 0, 5 and 30 minutes reaction time 51 F i g . 7 E f f e c t of d i f f e r e n t heating temperature (30 minutes) on the sedimentation pattern of 1% K-casein solution 52 F i g . 8 S t a b i l i z a t i o n of a s l ~ c a s e i n by K-casein before and afte r heating (100°C/30 minutes) against p r e c i p i t a t i o n by C a + + 54 F i g . 9 S t a b i l i z a t i o n of a s-,-casein by desul-furized K-casein before and after heating (100°C/30 minutes) against p r e c i p i t a t i o n by C a + + 55 ix FIGURES PAGE F i g . 10 S t a b i l i z a t i o n of a -casein by alkylated K-casein before ana after heating (100°C/ 30 minutes) against p r e c i p i t a t i o n by C a + + 56 Fi g . 11 El u t i o n patterns of K-casein before and after heating (1Q00C/3G minutes) on Sepharose 2B column (40 X 2.5 cm) eluted with 0.05 M phosphate buffer pH 7.6 at 25°C 57 Fi g . 12 E l u t i o n patterns of alkylated K-casein before and after heating (100°C/30 minutes) on Sepharose 2B column (40 X 2.5) eluted with 0.05 M phosphate buffer pH 7.6 at 25°C 58 F i g . 13 El u t i o n patterns of desulfurized K-casein before and after heating (100°C/ 30 minutes) on Sepharose 2B column (40 X 2.5 cm) eluted with phosphate buffer pH 7.6 at 25°C F i g . 14 E f f e c t of heat on polyacrylamide gel electrophoretic patterns of K-casin alkylated K-casein and desulfurized K-casein 61 F i g . 15 S t a b i l i z a t i o n of a^-^casein by heated, K-casein, desulfurized K-casein and alkylated K-casein, i n the presence of 5 M urea 62 Fi g . 16 P o l a r i z a t i o n of dansylated a s^-casein t i t r a t e d with K-casein and heated (100QC/30 minutes) K-casein 66 Fi g . 17 P o l a r i z a t i o n of dansylated a ^casein t i t r a t e d with, desulfurized 5 1 K-casein and heated (100°C/30 minutes) desulfurized K-casein 67 Fi g . 18 P o l a r i z a t i o n of dansylated a -^-casein t i t r a t e d with alkylated and heated (100°C/30 minutes) alkylated K-casein 59 68 Fi g . 19 E f f e c t of d e s u l f u r i z a t i o n of K-casein on i t s fluorescent i n t e n s i t y 71 E f f e c t of b o i l i n g temperature (30 minutes) on the e l u t i o n pattern of skimmilk ( 1 . 5 ml) eluted with 0.02 M phosphate buffer con-taining 6.6 M urea, pH 7.6 Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 20 E f f e c t of b o i l i n g temperature (10 min) on the e l u t i o n pattern of B-lactoglobulin and a mixture of 3-lactoglobulin and K-casein, eluted with 6.6 M urea i n phosphate buffer 0.0 2 M, pH 7.6 Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 22 E f f e c t of b o i l i n g temperature (10 min) on the e l u t i o n pattern of alkylated K-casein eluted with 0.02 M phosphate buffer containing 6.6 M urea, pH 7.6 Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 24 E f f e c t of b o i l i n g temperature (10 min) on the el u t i o n pattern of K-casein and a mixture of K-casein and 3-lacto-globulin, eluted with 0.02 M phosphate buffer containing 6.6 M urea, pH 7.6: Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 26 E f f e c t of i o n i c strength on the e l u t i o n patterns of whole casein on Sepharose 6B column (37 X 2 cm) eluted with phosphate buffer pH 7.6 and 4°C Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 28A Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 28B, 28C, 28D and from F i g . 3 1 B E f f e c t of pH and temperature on the elut i o n patterns of whole casein on Sepharose 6B column (37 X 2 cm) eluted with phosphate buffer 0.05 M x i FIGURES PAGE F i g . 32 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d from F i g . 31C 96 F i g . 3 3 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d from F i g . 31B and from F i g . 31D 97 F i g . 34 E f f e c t o f u r e a c o n c e n t r a t i o n on the e l u t i o n p a t t e r n s o f whole c a s e i n on Sepharose 6B column (.3 0 X 2 cm) , e l u t e d with, phosphate b u f f e r 0.05 M, pH 9.0 99 F i g . 35A P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d from F i g . 34B 100 F i g . 3 5 B P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s - o b t a i n e d from F i g . 34C and 34D 101' F i g . 36 E f f e c t o f column length, on the e l u t i o n p a t t e r n o f whole c a s e i n on Sepharose 6B, e l u t e d w i t h , phosphate b u f f e r 0.05 M c o n t a i n i n g 3 M u r e a , pH 9.0 a t room tem p e r a t u r e 103 F i g . 37 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d from F i g . 36A 104 F i g . 38 E l u t i o n p a t t e r n o f whole c a s e i n (150 mg) on Sepharose 6B column (43 X 2.5 cm) e l u t e d w i t h , phosphate b u f f e r 0.05 M, c o n t a i n i n g 6.6 M u r e a pH 7.6 a t 25°C 105 F i g . 39 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d from F i g . 38 106 F i g . 40 E l u t i o n p a t t e r n o f s k i m m i l k (2.5ml). on Sepharose 6B column (43 X 2.5 cm), e l u t e d w i t h phosphate b u f f e r 0.0 5 M c o n t a i n i n g . 6.6 M u r e a , pH 7.6 a t 25°C 107 F i g . 41 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d from F i g . 40 108 F i g . 42 E l u t i o n p a t t e r n o f K - c a s e i n r i c h f r a c t i o n ( Z i t t l e ' s method) on Sepharose 6B column (33 X 2 cm) e l u t e d w i t h phosphate b u f f e r 0.05 M, c o n t a i n i n g 6.6 M u r e a pH 7.6 110 x i i FIGURES PAGE F i g . 43 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 42 111 F i g . 44 S t a b i l i z a t i o n o f a - c a s e i n by K -c a s e i n p r e p a r e d f r o m w h o l e c a s e i n and f r o m K - c a s e i n r i c h f r a c t i o n o f Z i t t l e ' s b y S e p h a r o s e m e t h o d 112 F i g . 45 S o l u b i l i t y o f a ,- and a ^ - c a s e i n s i n _ ^ n s i S O C a C l 2 116 F i g . 46 S e n s i t i v i t y o f a n - and a ^ - c a s e i n s t o C a + + a s t u r b i S i t y m e a s u r e d a t 540 nm 117 F i g . 47 S t a b i l i z a t i o n o f o t ^ - a n c ^ a ^ - c a s e i n s b y K - c a s e i n 118. F i g . 48A E l u t i o n p a t t e r n o f s k i m m i l k o n S e p h a d e x G-100 c o l u m n (3.8 X 140 c m ) , e l u t e d w i t h 0.01 M p h o s p h a t e b u f f e r pH 10.8 and 5 mM EDTA, 4°C 122 F i g . 48B P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 48A. The g e l was r u n i n t h e a b s e n c e o f 2 - m e r c a p t o e t h a n o l 123 F i g . 48C P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 48 A, t h e g e l was r u n i n t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l 124 x i i i ACKNOWLEDGEMENTS The author uiiihzi to zxpnzii hii> dzzpzit gnatitudz to VH. SV Nakai, Aaociatz VHO izaoH, Ve.pa.ntme.nt oi food Sc.te.nce., Untve.ft.6tty oi B n i t i i h Columbia, undzn whoiz iupzn-v i i i o n thti, pnojzct wa.6 unde.Hta.kzn. Hii constant advicz and zncouHagzmznt thnoughout thz couHiz oi t h t i uionk and duning tkz u n i t i n g o & thti, thziii, wai> vzny much appnzctatzd. Thank* anz a l i o duz to thz mzmbzHA oi my committzz-VH. W.V. Powniz, Chaihman oi thz VzpaHtmznt oi Food Sctzncz, VH. J . F . Richandi and VH. J . VandzHAtozp, VzpaHtmznt oi Food Sctzncz, UntvzHAtty oi B n i t i i h Columbta and VH. W.J. P o l g l a i z , Acting Head, VzpaHtmznt oi Bio chzmiitny, Univzn&ity oi Bniti&h Columbia, ion thziH intznz&t in thz dii>cui>i>ion and Hzvizui oi tki& t h z i i * . Thz valuablz a*i>i*tancz oi UH6. J . Ruddick ii, g n a t z i u l l y acknowlzdgzd. 1. INTRODUCTION The processing of milk products may depend on some changes i n the state of the milk proteins. The need to control these changes w i l l determine the manufacturing technique i n some cases. In other cases, the a l t e r a t i o n of milk proteins i s related to d i f f i c u l t i e s which occur i n the manufacture of milk. An understanding of these changes and the i n t e r a c t i o n of milk proteins under various conditions i s c l e a r l y desirable. Decreases i n protein s t a b i l i t y have been shown i n s t e r i l e concentrated milk and i n model systems of casein solu-tion during storage ( 7 6 , 7 7 , I U ) . Spontaneous gelation has been reported to occur i n condensed milk es p e c i a l l y when i t i s stored at elevated temperature ( ' • < « ) . A considerable study has been directed toward the cause of gelation which i s an undesir-able phenomenon i n processed milk. However as yet there i s no s a t i s f a c t o r y explanation for a l l facets of the phenomenon. K-casein being the casein f r a c t i o n which functions as a key mi c e l l a r s t a b i l i z i n g factor and the only major casein containing d i s u l f i d e groups has been suggested to be the major factor i n the i n s t a b i l i t y of processed milk (?s ( 7 9 ) . I t was the aim of t h i s work to investigate the role of d i s u l f i d e groups i n the heat s t a b i l i t y of K-casein. This thesis describes a three part study on <-casein: 1 ) A method for estimation of the d i s u l f i d e content u t i l i z i n g Ellman's reagent. Studies on the role of those groups i n the heat s t a b i l i t y of K-casein a f t e r modification by d e s u l f u r i z a t i o n or a l k y l a t i o n . Chemical and physical properties of the modified K-casein (heated and unheated) were studied by polyacrylamide gel electrophoresis, u l t r a c e n t r i f u g a t i o n , fluorescence p o l a r i z a t i o n , rennin assay and Sepharose gel f i l t r a t i o n techniques A Sepharose gel chromatographic technique was u t i l i z e d to i s o l a t e a pure K-casein f r a c t i o n from whole casein or skimmilk. A study was also conducted to characterize a c - c a s e i n ; one of the minor casein so components containing d i s u l f i d e groups. The molecular weight of a ^ - c a s e i n was determined by the d i f f e r e n t i a l boundary method. SURVEY OF THE LITERATURE C a s e i n s i n M i l k A c i d - p r e c i p i t a t e d c a s e i n was f i r s t c o n s i d e r e d t o be a p u r e homogeneous p r o t e i n when H a m m e r s t e i n ( 3 0 ) i n 1883 d e v e l o p e d a n i s o l a t i o n p r o c e d u r e f o r t h e p r e p a r a t i o n o f w h o l e c a s e i n . The e a r l y o b s e r v a t i o n on t h e h e t e r o g e n e i t y o f c a s e i n was made by O s b o r n e and Wakeman ( 8 e ) when a s m a l l amount o f c a s e i n w i t h u n i q u e p r o p e r t i e s was o b t a i n e d f r o m t h e a l c o h o l e x t r a c t s . T h i s f r a c t i o n d i f f e r e d f r o m o r d i n a r y c a s e i n i n i t s h i g h s o l u b i l i t y i n 50% a l c o h o l and i t s l o w p h o s p h o r u s c o n t e n t o f o n l y 0.1% as c o m p a r e d w i t h 0.85% i n u n f r a c t i o n a t e d c a s e i n . A s i m i a l r a l c o h o l s o l u b l e f r a c t i o n was o b t a i n e d by H i p p e t a l . ( 3 8 ) i n 1950. T h i s was d e s i g n a t e d y - c a s e i n . The e x t e n s i v e w o r k o f L i n d e r s t r 0 m - L a n g ( 55 ) demon-s t r a t e d t h e g r o s s h e t e r o g e n e i t y o f c a s e i n s i n 1929. He p o s t u l a t e d t h e e x i s t e n c e o f .a f r a c t i o n w h i c h was i n s e n s i t i v e t o t h e c a l c i u m i o n b u t s u s c e p t i b l e t o t h e a c t i o n o f r e n n i n , a f t e r w h i c h t h e p r o t e i n i s p r e c i p i t a t e d b y c a l c i u m . A l t h o u g h he d i d n o t s e p a r a t e t h i s f r a c t i o n a t t h a t t i m e , t h e d e v e l o p -ment o f t h e e l e g a n t m e thod o f e l e c t r o p h o r e t i c a n a l y s i s o f p r o t e i n s by T i s e l i u s ( 1 2 2 ) and i t s a p p l i c a t i o n t o t h e s t u d y o f c a s e i n by M e l l a n d e r Us) imade p o s s i b l e t h e e s t a b l i s h m e n t o f t h e n a t u r e o f t h e h e t e r o g e n e i t y o f c a s e i n . I t was f o u n d t h a t c a s e i n was composed o f t h r e e e l e c t r o p h o r e t i c c o m p o n e n t s d e s i g n a t e d by M e l l a n d e r Us) i n 1939 a s a-, 6 - and y- i n t h e o r d e r o f d e c r e a s i n g m o b i l i t y . F u r t h e r m o r e , t h e r e s u l t s o f 4. electrophoretic analysis conducted by Warner i n 1944 ( . 1 3 2 ) indicated that those compounds were not el e c t r o p h o r e t i c a l l y homogeneous i n acid pH. A method of fr a c t i o n a t i o n of casein with CaCl2 was developed by von Hippel and Waugh ( 1 3 0 , 1 3 7 ) and they were able to i d e n t i f y and separate a casein f r a c t i o n which f i t t e d the d e f i n i t i o n of a protein f r a c t i o n described by Linderstrjzfrn ( 5 S ) . This f r a c t i o n was designated K-casein. Waugh and von Hippel concluded also that this f r a c t i o n (K-casein) was the f r a c t i o n acted on by rennin and the one responsible for micelle s t a b i l i t y . These findings were reconciled with McKenzie and Wake's ( 6 6 ) reports, where they showed that K-casein i s concentrated along with a s-casein i n fr a c t i o n A during the alcohol f r a c t i o n a t i o n of casein ( 3 7 ) . Casein Mi c e l l e The casein components e x i s t i n milk as large spherical c o l l o i d a l complexes d i s t r i b u t e d as polydisperse stable m i c e l l a r aggregates, i n association with calcium and phosphate and lesser amounts of magnesium and c i t r a t e . The size of these spherical micelles have been estimated to range from 300 to 30 00 X i n diameter with a molecular weight of a single micelle between 1 X 10 7 to 3 X 10 9 ( . 117 ) . Formation of a stable micelle i n a model system requires an adequate amount of K-casein and also depends on the sequence of in t e r a c t i o n of the casein components and calcium, temperature and i o n i c strength of the system. C a l c i u m c o n c e n t r a t i o n o f 0 . 0 3 M i s r e q u i r e d a s a m i n i m u m f o r s p o n t a n e o u s f o r m a t i o n o f s t a b l e m i c e l l e s i n s o l u t i o n c o n t a i n i n g a - a n d K - c a s e i n w i t h r a t i o o f 4 : 1 ( 1 3 7 ) . A l t h o u g h i t i s w e l l d o c u m e n t e d t h a t a l l t h e c a s e i n s a r e i n c l u d e d w i t h i n t h e m i c e l l e , a s m a l l f r a c t i o n h a s b e e n o b s e r v e d a s s m a l l e r s o l u b l e u n i t s t h a t a r e i n e q u i l i b r i u m w i t h m i c e l l a r 1 ' 1 c o n s t i t u e n t s . I n a s t u d y o f m o d e l m i c e l l a r s y s t e m s c o n t a i n i n g c a l c i u m , K - a n d a , - c a s e i n s o r c a l c i u m , < - , a n - a n d ^ s l s l 1 3 - c a s e i n s , a r a p i d e q u i l i b r i u m f o r t h o s e s y s t e m s w a s o b s e r v e d ( 1 3 6 ) . T h e m i c e l l a r s i z e i n t h e s e m o d e l s y s t e m s w a s d e p e n d e n t o n l y o n t h e a , / K - r a t i o a t a c o n s t a n t c a l c i u m c o n c e n t r a t i o n . J s l S m a l l e r m i c e l l e s a l w a y s c o n t a i n l a r g e r p r o p o r t i o n s o f K-c a s e i n i n b o t h n a t i v e a n d m o d e l s y s t e m s o f m i c e l l e . M a n y m o d e l s h a v e b e e n p r o p o s e d t o d e s c r i b e t h e s t r u c t u r e o f t h e m i c e l l e . S o m e d i f f i c u l t i e s h a v e b e e n e n c o u n -t e r e d i n e x p l a i n i n g a l l a s p e c t s o f m i c e l l a r p r o p e r t i e s , b u t e a c h m o d e l c a n b e u s e d t o e x p l a i n m a n y f a c e t s o f t h e m i c e l l e b e h a v i o r . A c o r e - c o a t m o d e l w h i c h w a s d e v e l o p e d b y W a u g h a n d c o - w o r k e r s ( 136 8 6 135) v i s u a l i z e s t h e m i c e l l e a s a c o r e o f c s l ~ a n d B - c a s e i n s s u r r o u n d e d b y a c o a t c o m p o s e d o f m o n o -m o l e c u l a r l a y e r o f a ' u n i t m o l a r r a t i o K / a ^ - c a s e i n c o m p l e x . T h i s m o d e l i s c o n g r u e n t w i t h a l l t h e p r o p e r t i e s o f m o d e l m i c e l l a r s y s t e m s a n d e x p l a i n s a c o r r e l a t i o n o f t o t a l m i c e l l e s u r f a c e a r e a w i t h K - c a s e i n c o n t e n t , b u t i t d o e s n o t s e e m t o a c c o u n t a d e q u a t e l y f o r a c c e s s o f " c o r e " p o l y m e r s t o 6 . macromolecular reagents such, as carboxypeptidase. Payens ( .91) proposed a s i m i l a r model but emphasized more the role of hydrophobic interactions i n micelle formation. A d i f f e r e n t model was proposed by Rose ( . 1 0 0 ) which v i s u a l i z e s the micelle as polymer chains of 3-casein with a -casein coupling at hydrophobic s i t e s , and the S JL entire structure being interwoven with calcium apatite chains and on the surface, a s-^-casein i n t e r a c t i n g with K-casein. Another model having a porous framework was proposed by Garnier et a l . ( 2 7 ) which d i f f e r s considerably from other . models. In t h i s model K-casein trimers occupy nodes from which three branches of 0. ^ and 3-casein polymers emanate to form a large three dimensional network. The f a c t that calcium and phosphate ions are included i n the micelle structure, has led many authors to the conclus-ion that the i n t e r a c t i o n between caseins involves p a r t i a l l y , or exclusively, calcium and/or phosphate bridges. Removal of c o l l o i d a l calcium phosphate by- adjusting the pH of c h i l l e d milk to a pH of 4 . 8 - 5 . 0 , and d i a l y z i n g against a large excess of milk, would lead to tHe disruption and the i n s t a b i l i t y of micelle structure (.62) . i t has been found by electronmicros-copy that calcium i s responsible for large micelle formation by bridging smaller casein micelles together ( 1 0 9) • Bridges of phosphate between the hydrophobically interacted caseins have been suggested to lead to a stable micelle structure i n the 7. proposed model for the micelle by Garnier et a l . ( 2 7 ) . C o l l o i d a l calcium can be e a s i l y replaced by i o n i c calcium leading to a stable micelle structure ( 2 1 * ) . This suggests the p o s s i b i l i t y of a non-specific e l e c t r o s t a t i c i n t e r a c t i o n . However, Nakai et a_l. (so ) found that calcium bridges alone could not be responsible for the maintenance of micelle structure. The importance of calcium and phosphate i n maintaining the micelle structure was also suggested by Rose and Colvin (102) . They postulated that the calcium and phosphate are probably linked with the e-amino groups of lysines, i n the a , and g-caseins. A non-specific e l e c t r o s t a t i c i n t e r a c t i o n between casein com-ponents has been suggested by several investigators as a mechanism responsible for the s t a b i l i z a t i o n of casein micelle. H i s t i d i n e and tryptophan residues i n K-casein have also been suggested to play an important r o l e i n the s t a b i l i t y of a ,-casein against p r e c i p i t a t i o n by calcium (3 >* , 1 5 1 ) . S _L However, the r o l e of the h i s t i d i n e residue was recently denied by Nakai et al_. ( 7 **) . The modification of t h i s residue did not a f f e c t the s t a b i l i z i n g a b i l i t y of K-casein i n protecting a g l - c a s e i n . Others suggested that lysine residues i n K-casein are related to i t s a b i l i t y to s t a b i l i z e a ^-casein against calcium p r e c i p i t a t i o n ( 3 3 , 1 3 , 9 3 , 1 2 1 ) and v a r i a t i o n in the degree of l y s i n e modification was shown to be necessary to cause a loss or reduction of s t a b i l i z i n g a b i l i t y . This loss was interpreted to be due to s p e c i f i c charge e f f e c t s or 8 . conformational changes caused by an increase i n net negative charge on the protein. The i n t e r a c t i o n between a , - and K-casein has been s l suggested to be hydrophobic i n nature based on the temperature dependent, association and d i s s o c i a t i o n of these proteins (^s), Waugh ( 1 3 h) suggested that t h i s temperature e f f e c t could be due to a d i f f e r e n t type of calcium binding among the proteins. Understanding the type of bonding within the caseins becomes more d i f f i c u l t when i t i s r e a l i z e d that 3 - c a s e i n can leave the micelle structure and not re-enter i t ; and also- by the f a c t that the r a t i o of a g : P : K-caseins i n milk i s s K 3 : 2: 1; Z i t t l e ( i ^ 9) demonstrated that a r a t i o of a of 10 : 1 can be s t a b i l i z e d i n the presence of calcium ions. Waugh and von Hippel ( H 7 ) suggested that the i n t e r a c t i o n r a t i o of a g - : K-casein was 4 : 1 . However, Noble and Waugh ( 8 6 ) and Garnier et aJL. ( 2 6) suggested l a t e r that t h i s r a t i o was close to unity. Garnier ( 2 5 ) showed that the K-casein species involved i n the i n t e r a c t i o n was a trimer and that this combined with a monomer of a s~casein,.hence the i n t e r -action r a t i o of a ^- : K-casein was 1 : 3 on a monomer basis A weight r a t i o of unity has been shown more recently by Parry et a l . (89) and Clarke and Nakai (13) using gel f i l t r a t i o n and fluorescence p o l a r i z a t i o n techniques respec-t i v e l y . The d i s u l f i d e groups i n K-casein In 19 62 Waugh and co-workers ( i 3 6 ) indicated that 9. K-casein i s the only major casein f r a c t i o n possessing d i s u l f i d e bonds. Later, Swaisgood demonstrated that these bonds occurred intermolecularly giving a series of covalent polymers ranging i n molecular weight from 60,000 (trimer) to well above 150,000 daltons. Cleavage of these d i s u l f i d e bonds at pH 12 resulted i n reduction of the observed molecular weight to 23,000 and similar values were obtained for K-casein i n d i s s o c i a t i n g solvent after d i s u l f i d e reduction with 2-mercapto-ethanol. Swaisgood and Brunner ( 1 1 8) have reported values of 18,000 and 20,000 daltons for reduced K-casein i n urea and guanidine hydrochloride solutions respectively. Measurement i n 5 M guanidine-HCl for reduced K-casein containing no carbo-hydrate was performed by Woychik et a l . (1 "* •*) / and values between 17,800 and 18,400 daltons were observed. A l l those values were consistent with the calculated values from amino acid contents ( 9 6 ) . When urea i s incorporated into polyacrylamide gels for zone electrophoresis of ct ^ - and 3-caseins, a complete d i s s o c i a t i o n of these proteins to t h e i r monomeric forms w i l l r e s u l t allowing the i d e n t i f i c a t i o n of these polymorphic proteins. On the other hand, native K-casein y i e l d s a broad smeared zone near the o r i g i n due to the heterogeneous mixture of covalent polymers. Because of t h i s phenomenon, Neelin (8 7 ) , Woychik ( i t 2 ) and Schmidt ( H ? ) suggested the incorpora-tion of 2-mercaptoethanol i n the electrophoretic procedure 1 0 . f o r t h e c o m p l e t e s e p a r a t i o n o f t h e m o n o m e r i c f o r m s o f K - c a s e i n . T h e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f r e d u c e d K - c a s e i n f r o m a n i n d i v i d u a l c o w p r o d u c i n g a s i n g l e v a r i e n t r e v e a l s a l w a y s o n e i n t e n s e b a n d a n d 5 - 6 b a n d s w i t h l e s s i n t e n s i t y w h i c h d i s a p p e a r u p o n r e n n i n t r e a t m e n t ( 1 0 1 / i o e ) • I t i s b e l i e v e d t h a t t h e m o r e r a p i d l y m i g r a t i n g b a n d s d i f f e r f r o m t h e m o r e i n t e n s e l y s t a i n i n g a n d s l o w e s t b a n d o n l y i n t h e a m o u n t o f c a r b o h y d r a t e s . A r e c e n t r e p o r t ( 1 2 5 ) i n d i c a t e s t h a t t h e c a r b o h y d r a t e m o i e t y i s a t r i s a c c h a r i d e u n i t , a - N -a c e t y l n e u r a m i n y l (2 ->- 6 ) - 3 - g a l a c t o s y l - ( 1 + 3 o r 6 ) - N -a c e t y l g a l a c t o s a m i n e , t h a t i s a t t a c h e d t o t h e p e p t i d e c h a i n t h r o u g h OH g r o u p s o f s e r i n e o r t h r e o n i n e , b o t h o f w h i c h o c c u r f r e q u e n t l y i n t h e C - t e r m i n a l p o r t i o n o f K - c a s e i n . T h e n u m b e r o f t h e s e t r i s a c c h a r i d e r e s i d u e s h a s b e e n s u g g e s t e d t o v a r y f r o m 0 t o 5 p e r m o l e ( 1 0 1 ) a n d e a c h s u c h r e s i d u e i n c r e a s e s t h e m o n o m e r w e i g h t b y 6 5 7 d a l t o n s . M o s t o f t h e c o m m o n l y o c c u r r i n g a m i n o a c i d s a r e p r e s e n t i n K - c a s e i n . H o w e v e r , t h e p r o p o r t i o n o f p r o l i n e i s r e l a t i v e l y h i g h ( 8 . 8 % ) a n d t h i s p r o b a b l y a c c o u n t s f o r t h e a l m o s t c o m p l e t e a b s e n c e o f a - h e l i c a l o r o t h e r o r d e r e d s t r u c t u r e s ( 3 1 ) . S w a i s g o o d i n a r e c e n t r e v i e w ( 1 1 7 ) h a s i n d i c a t e d t h a t K - c a s e i n i s a f a i r l y r i g i d g l o b u l a r s t r u c t u r e , i n c l u d i n g r o u g h l y t w o t h i r d s o f t h e m o l e c u l e , t o w h i c h a f l e x i b l e , h i g h l y s o l v a t e d t a i l i s a t t a c h e d . O t h e r e v i d e n c e s i n d i c a t e t h a t t h e g l o b u l a r s e g m e n t s a r e l i n k e d t o g e t h e r b y d i s u l f i d e b o n d s f o r m i n g a h e t e r o g e n e o u s p o p u l a t i o n o f c o v a l e n t p o l y m e r s . S e p a r a t i o n o f K - c a s e i n S i n c e t h e d i s c o v e r y o f K - c a s e i n f r a c t i o n by Waugh and v o n H i p p e l ( 1 3 0 / 1 3 7 ) / a c o n s i d e r a b l e e f f o r t h a s b e e n made t o i s o l a t e a n d p u r i f y t h i s f r a c t i o n . Wake ( x 3 1 ) s u c c e s s f u l l y i s o l a t e d K - c a s e i n b u t c o n t a i n i n g s l i g h t i m p u r i t i e s f r o m t h e s e c o n d c y c l e c a s e i n f r a c t i o n S. An i m p r o v e m e n t f o r t h i s m ethod was s u b s e q u e n t l y s u g g e s t e d by i n c o r p o r a t i n g an a l c o h o l t r e a t m e n t i n t o the p r o c e d u r e ( 6 7 ) . A t t e m p t s w e r e a l s o made f o r t h e p r e -p a r a t i o n o f K - c a s e i n o f an i m p r o v e d p u r i t y u t i l i z i n g t h e s o l u -b i l i t y d i f f e r e n c e s i n c a s e i n f r a c t i o n s i n u r e a - t r i c h l o r o a c e t i c a c i d ( l i s ) - F r a c t i o n a t i o n o f a - c a s e i n w i t h u r e a s o l u t i o n a n d t h e r e m o v a l o f a g ^ - c a s e i n by t h e a d d i t i o n o f c a l c i u m w e r e p e r -f o r m e d b y Cheeseman ( 1 0 ) • One o f t h e most w i d e l y u s e d methods so f a r i s Z i t t l e and C u s t e r ' s m e thod ( 1 5 3 ) . T h i s m e thod i n v o l v e s a u r e a - s u l f u r i c a c i d t r e a t m e n t o f w h o l e c a s e i n (pH 1.3) w i t h a s u b s e q u e n t a l c o h o l f r a c t i o n a t i o n t o o b t a i n r e l a t i v e l y p u r e K - c a s e i n w i t h a h i g h y i e l d . M o s t o f t h e s e m e t h o d s s u f f e r f r o m one o r more o f t h e f o l l o w i n g : h i g h c o n t a m i n a t i o n , l e n g t h y and t e d i o u s p r o c e d u r e s , l o w r e c o v e r y a n d / o r d r a s t i c c o n d i t i o n s . I o n e x c h a n g e c h r o m a t o g r a p h y a nd g e l f i l t r a t i o n h a v e b e e n u s e d e x t e n s i v e l y i n t h e i s o l a t i o n and p u r i f i c a t i o n o f many p r o t e i n s y s t e m s . . I o n e x c h a n g e c h r o m a t o g r a p h y i n v o l v e s t h e e s t a b l i s h m e n t o f m u l t i p l e e l e c t r o s t a t i c b o n d s b e t w e e n t h e i o n i z e d g r o u p s o n t h e s u r f a c e o f t h e e x c h a n g e r a nd o p p o s i t e c h a r g e s on t h e p r o t e i n s , f o l l o w e d by a s e l e c t i v e r e l e a s e o f t h e s e b o n d s by c h a n g e s i n t h e c o n c e n t r a t i o n o r pH o f t h e e l u t i n g b u f f e r . T h i s t e c h n i q u e h a s b e e n u s e d w i d e l y i n f r a c t i o n a t i o n a n d p u r i f i c a t i o n o f K - c a s e i n ( 1 0 3 / 1 2 s r 1 4 1 * / 8 6 ) - B e c a u s e o f t h e s t r o n g t e n d e n c y o f c a s e i n s t o a g g r e g a t e , d i s s o c i a t i n g a g e n t s ( e . g . u r e a ) w i t h o r w i t h o u t r e d u c i n g a g e n t s h a v e b e e n n e c e s s a r y f o r e l u t i n g K - c a s e i n f r o m a n i o n e x c h a n g e c o l u m n . G e l f i l t r a t i o n i s t h e o t h e r c h r o m a t o g r a p h i c t e c h n i q u e w h i c h h a s b e e n u s e d f o r p u r i f i c a t i o n o f p r o t e i n s . T h i s m e t h o d d e p e n d s o n a m o l e c u l a r s i e v i n g e f f e c t o f a c r o s s - l i n k e d d e x t r a n a n d w a s f i r s t a p p l i e d t o m i l k b y d e K o n i n g ( 5 2 ) • U s i n g S e p h a d e x G - 5 0 h e i s o l a t e d m i l k p r o t e i n s f r o m o t h e r l o w m o l e c u l a r w e i g h t m i l k c o n s t i t u e n t s . F r a c t i o n a t i o n o f m i l k p r o t e i n b y g e l f i l t r a t i o n o n S e p h a d e x G - 1 0 0 r e s u l t e d i n f o u r f r a c t i o n s w h i c h w e r e i d e n t i f i e d e l e c t r o p h o r e t i c a l l y a s c a s e i n m i c e l l e , a - l a c t a l b u m i n , 3 - l a c t o g l o b u l i n a n d n o n - p r o t e i n n i t r o g e n i n t h e o r d e r o f e l u t i o n ( 3 6 / 7 0 ) - D i f f e r e n t s i z e r a n g e s o f i n t a c t m i c e l l e h a v e b e e n s e p a r a t e d o n a g a r o s e g e l ( S e p h a r o s e 2 B a n d 4 B ) w i t h c o m p l e x b u f f e r m i x t u r e s d e s i g n e d t o m i n i m i z e t h e d i a l y s i s e f f e c t ( 6 8 / 7 1 ) . I n a n a t t e m p t t o f r a c t i o n a t e s k i m m i l k p r o t e i n s o n S e p h a d e x G - 2 0 0 w i t h p h o s p h a t e b u f f e r , Y a g u c h i ( 1 4G ) o b t a i n e d s i x f r a c t i o n s . N e i t h e r o f t h e s e f r a c t i o n s w e r e p u r e . H o w e v e r , a h i g h l y p u r i f i e d K - c a s e i n w a s o b t a i n e d i n t h e p r e s e n c e o f 6 . 6 M u r e a o f p H 8 . 6 u s i n g S e p h a d e x G - 1 5 0 ( 1 * 5 ) . A t t h e s a m e t i m e C h e e s e m a n ( 1 1 ) p u r i f i e d K - c a s e i n f r o m w h o l e c a s e i n o n S e p h a d e x G - 2 0 0 , u s i n g 0 . 0 2 M S D S a s a d i s s o c i a t i n g a g e n t . Nakahori and Nakai ( 7 2 ) also p u r i f i e d a g^- and K-caseins on a Sephadex column using SDS solution. Pure K-casein was pre-pared by Nakai et a l . ( 7 5 ) i n the absence of any d i s s o c i a t i n g agents. However, the pH of the eluent buffer was r e l a t i v e l y high (pH 10.8, 4°C). More recently a gel chromatographic technique using Biogel A-15, was used for the i n t e r a c t i o n studies of casein ( 92 ) and a free K-casein f r a c t i o n was obtained when low concentration of a mixture of c t s ^ - and K-caseins were applied to the gel eluted with phosphate buffer without any d i s s o c i a t i n g agents. E f f e c t of Heat on K-casein A decrease i n protein s t a b i l i t y and the production of a gel l i k e structure have been shown i n s t e r i l e concentrated milk and i n model systems of casein solution during storage ( 7 6 , 7 7 , H I ) . S t a b i l i t i e s of caseins and of casein complexes from s t e r i l e concentrated milk have been shown to be affected by o x i d i z i n g and reducing agents (7 8 , 7 9 ) . A gel l i k e structure i n heated concentrated skimmilk has been recently reported by Kalab et 'al. ( 4 1, •* 2 , 4 4 ) . The firmness of the gel was shown to be dependent on the temperature, duration of heating and protein concentration. In a study of the role of sulfhydryl and d i s u l f i d e groups i n milk i n gel firmness, Kalab and Emmons ('•s) showed that divalent copper and mercury which i n t e r a c t with the sulfhydryl groups, remarkably reduce the gel firmness. Decrease i n the gel firmness was also o b s e r v e d when d i f f e r e n t s u l f h y d r y l c o n t a i n i n g compounds w e r e a d d e d a t c e r t a i n c o n c e n t r a t i o n s . R e d u c i n g a g e n t s s u c h as s o d i u m b o r o h y d r i d e a l s o d e c r e a s e d t h e g e l f i r m n e s s . The s t o r a g e o f a s e p t i c a l l y p a c k e d u l t r a - h i g h t e m p e r a t u r e (UHT) m i l k h a s b e e n shown t o be a c c o m p a n i e d by c h a n g e s i n t h e m o l e c u l a r w e i g h t d i s t r i b u t i o n o f c a s e i n s ( 2 ) . T h e s e c h a n g e s a r e b o t h t i m e and t e m p e r a t u r e d e p e n d e n t and p r o c e e d much more r a p i d l y a t 37°C t h a n a t 30°C. A r e m a r k a b l e i n c r e a s e i n t h e p r o p o r t i o n o f t h e h i g h m o l e c u l a r w e i g h t m a t e r i a l a f t e r s t o r a g e o f UHT m i l k a t 30°C f o r 11 months h a s b e e n o b s e r v e d u s i n g S e p h a d e x G-200 and s e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t s . The a u t h o r s a s c r i b e d t h i s phenomenon t o p h y s i c a l f o r c e s o f a s s o c i a t i o n s u c h as h y d r o p h o b i c b o n d i n g and t o t h e p r o d u c t i o n o f c o v a l e n t p o l y m e r s by t h e M a i l l a r d . r e a c t i o n . An i n c r e a s e i n t h e p r o p o r t i o n o f c a s e i n s e l u t e d a t t h e v o i d v o l u m e was a l s o o b s e r v e d a f t e r s t o r i n g h e a t e d o r f r o z e n m i l k ( e i , 8 2 ) . The e f f e c t o f d i r e c t s t e a m i n j e c t i o n UHT t r e a t m e n t o f c a s e i n m i c e l l e s and a m i x t u r e o f c a s e i n m i c e l l e s and 3-l a c t o g l o b u l i n on t h e e x p o s u r e o f SH g r o u p s and t h e number o f d i s u l f i d e b o n d s was r e c e n t l y i n v e s t i g a t e d b y S w a i s g o o d and Cho ( 1 2 0 ) . T h e i r r e s u l t s i n d i c a t e d t h e d e s t r u c t i o n o f d i s u l -f i d e b o n d s i n c a s e i n m i c e l l e s a c c o u n t e d f o r 75% o f t h e d i s u l -f i d e c o n t e n t o f c a s e i n m i c e l l e . I t was a l s o n o t i c e d t h a t t h e d i s u l f i d e b o n d s o f m i c e l l a r c a s e i n w e r e p r o t e c t e d when h e a t e d i n t h e p r e s e n c e o f 3 - l a c t o g l o b u l i n . 1 5 . The involvement of the d i s u l f i d e bonds of K-casein i n the heat s t a b i l i t y of the milk system has been postulated by many investigators (7 9 , i u ) , and more recently the S-S interchange reaction i n K-casein has been suggested to play an important role i n .the s t a b i l i z a t i o n of a ^ - c a s e i n against p r e c i p i t a t i o n by calcium ( 7 •*) . A d r a s t i c loss i n the s t a b i l i z i n g power of K-casein was observed when h i s t i d i n e residues i n K-casein were carbethoxylated with DEP (Diethylpyrocarbonate). However, blocking the SS groups i n the carbethoxylated K-casein prevented this loss almost completely. Z i t t l e ( 1 5 4 ) has shown that heating solutions of K-casein at pH 7.0 causes up to 60% reduction i n the rennin c l o t t i n g time, but heated K-casein s t i l l retained the a b i l i t y to protect a ^ - c a s e i n against p r e c i p i t a t i o n by calcium. Z i t t l e ( 1 5 2 ) also showed that the protective property was l a b i l e i f s a l t was included i n the system. Reducing agents such as mercaptoethanol or cysteine enhanced heat l a b i l i t y , but reduction and a l k y l a t i o n or i n c l u s i o n of a ,-casein i n s l the system tended to prevent heat l a b i l i t y . Decrease i n the s t a b i l i z a t i o n of K-casein has been reported upon heating a solution of this protein to 100-110°C for 30 minutes, and a complete loss was observed when the temperature was increased to 120°C or above ( 1 2 a , a t ) . Kresheck et a_l. ( 5 3 ) , studying the e f f e c t of heat on a 3- and K-caseins by l i g h t scatter-ing, observed that only K-casein showed any marked i n s t a b i l i t y 1 6 . on heating to 90°C. Increases i n t u r b i d i t y were noted for K-casein and the average molecular weight increased from 6 1 3.43 X 10 to 1.77 X 10 and the radius of gyration from 592 to 1330 A between 30°C and 90°C respectively. No data was presented by these authors to suggest the nature of bonds involved i n aggregate formation. The d i s u l f i d e interchange reaction has been used to explain a complex formation between K-casein and 3-lacto g l o b u l i n when milk i s heated. The denaturation of 3-lactoglobulin and the e f f e c t of heat was reviewed by Sawyer ( 1 0 5 ) . On heating 3-lactoglobulin f i r s t s p l i t s into two monomer ha l f - u n i t s and then association takes place pre-sumably through intermolecular d i s u l f i d e bridges. No aggregate formed when the protein was heated i n the presence of N-ethylmaleimide. A secondary reaction r e s u l t s i n the formation of a heavy component (29 S), the formation of which i s not affected by sulfhydryl reagent. In 1952, Tobias et a l . ( 1 2 3 ) reported the presence of a complex between 3-lacto-globulin and a-casein i n skimmilk heated to 300°F for a short time. Since that report, a large amount of data has accumulated suggesting that the complex i s formed between 3-lactoglobulin and K-casein. I t has also been suggested that t h i s complex i s caused by formation of intermolecular d i s u l f i d e bonds ( 1 5 4) . Yo.shino et a l . ( 1 7 ) reported that t h i o g l y c o l i c acid prevented i n t e r a c t i o n of K-casein with whey proteins i n heated milk, thereby enabling K-casein to s t a b i l i z e a -casein against calcium more e f f i c i e n t l y than the interacted proteins. On the other hand, forewarming milk to temperatures up to 90°C before concentration s t a b i l i z e s the concentrated product against gel formation. I t has been suggested (9 8) that t h i s e f f e c t i s due to the i n t e r a c t i o n of 3 - l a c t o g l o b u l i n and K-casein, the micelle being coated with i n t e r a c t i o n products thereby i n h i b i t i n g micellar association which i s thought to occur on gelation. C H A P T E R I S U L F H Y D R Y L - D I S U L F I D E D E T E R M I N A T I O N I N S O M E P R O T E I N S A N D F O O D P R O D U C T S 18. INTRODUCTION Sulfhydryl ( SH ) and d i s u l f i d e ( SS ) groups have been widely implicated as important functional groups i n many food proteins. The formation of 3 - l a c t o g l o b u l i n -K-casein complexes when milk i s heated may occur through SS interchange reaction (1o6) . Many investigators have postu-lated the involvement of d i s u l f i d e bonds of K-casein i n the heat s t a b i l i t y of milk system ( 7 9 , 1 4 1 ) . The thick gel of egg white i s considered to consist of large glycoprotein molecules crosslinked by SS bonds and a reductive mechanism has been proposed to explain the thinning reaction which occurs on aging of eggs ( 1 1 2 ) . Further, i t i s well documented that addition of SH compounds to fl o u r d r a s t i c a l l y weakens the rheological properties of the fl o u r ( 51*) . Interaction between SS and SH of fl o u r have been suggested to explain, i n part, this change. Many of the current procedures used for the determina-tion of SH and SS groups i n protein systems are lengthy, complicated or have limited applications to milk systems ( 9 ; 5 1 , 5 6 , 8 5 , 9 5 , 1 0 5 , 1 2 9 , 1 4 8 ) . A simple method for measurement of those groups, especially SS i n K-casein, i s c l e a r l y desirable to i n v e s t i -gate the role of d i s u l f i d e groups i n the heat s t a b i l i t y of K-casein. S e v e r a l i n v e s t i g a t o r s ( 1 7 , 2 1 ) have used Ellman's reagent; 5 , 5 1 - d i t h i o b i s - 2 - n i t r o b e n z o i c a c i d (DTNB) f o r the e s t i m a t i o n of f r e e SH groups i n some p r o t e i n systems. The DTNB has been found to be a s e n s i t i v e t o o l f o r ass a y i n g t h i o l groups i n t i s s u e , body f l u i d s and p r o t e i n s . I t i s an aromatic d i s u l f i d e , and s i n c e i t has a high e r standard o x i d a t i o n - r e d u c t i o n p o t e n t i a l than a l i p h a t i c analogs i t w i l l r e a c t w i t h a l i p h a t i c t h i o l s by an exchange r e a c t i o n to form a mixed d i s u l f i d e of the p r o t e i n p l u s one mole of 2 - n i t r o - 5 - t h i o b e n z o a t e per mole of p r o t e i n s u l f h y d r y l group ( r e a c t i o n 1 ) . C o l o r l e s s Y e l l o w T h e n i t r o m e r c a p t o b e n z o a t e a n i o n h a s a n i n t e n s e y e l l o w c o l o r w i t h a m o l a r a b s o r p t i v i t y o f 1 3 , 6 0 0 M 1 c m 1 a t 4 1 2 n m . A s s a y p r o c e d u r e s u t i l i z i n g E l l m a n ' s r e a g e n t h a v e t h e a d v a n t a g e o f b e i n g s i m p l e , r a p i d a n d d i r e c t ( 0 ) a n d 2 0 . have yielded values comparable with other a n a l y t i c a l methods for SH groups ( 21 ) and for SS groups ( 5 ) i n p u r i f i e d proteins. In this report a method for the determination of both SH and SS groups u t i l i z i n g Ellman's reagent was applied to pure protein systems (K-casein, 3-lactoglobulin, a -casein, ovalbumin and a ,-casein) as well as to some food s5 s l products (flour, egg white and milk). 2 1 . E X P E R I M E N T A L R e a g e n t s , M a t e r i a l s a r i d M e t h o d s G u a n i d i n e h y d r o c h l o r i d e ( G u H C l ) w a s t h e U l t r a P u r e r e a g e n t o b t a i n e d f r o m M a n n R e s e a r c h L a b o r a t o r i e s . B ' - L a c t o -g l o b u l i n w a s p u r c h a s e d f r o m N u t r i t i o n a l B i o c h e m i c a l s I n c . O v a l b u m i n w a s i s o l a t e d f r o m e g g w h i t e b y t h e m e t h o d o f K e k w i c k a n d C a n n a n ( •* 7) , a , - c a s e i n b y t h e m e t h o d o f Z i t t l e a n d C u s t e r s l J ( i § 3 ) , a - c a s e i n b y t h e m e t h o d o f A n n a n a n d M a n s o n ( 3 ) , a n d K - c a s e i n b y t h e m e t h o d o f N a k a i e t a _ l . ( 7 5) . F l o u r , i n s t a ' n -t i z e d s k i m m i l k a n d s p r a y d r i e d e g g w h i t e w e r e l o c a l c o m m e r c i a l p r o d u c t s a n d e g g s a n d m i l k w e r e o b t a i n e d f r o m t h e U n i v e r s i t y f a r m . R e a g e n t g r a d e c h e m i c a l s w e r e u s e d t o p r e p a r e t h e f o l l o w i n g : T r i s - g l y c i n e b u f f e r ( 1 0 . 4 g T r i s , 6 . 9 g g l y c i n e a n d 1 . 2 g E D T A p e r l i t e r , p H 8 . 0 , d e n o t e d a s T r i s - G l y ) , 0 . 5 % s o d i u m d o d e c y s u l f a t e ( S D S ) i n T r i s - G l y , 1% N a C l i n T r i s - G l y , E l l m a n ' s r e a g e n t ( 5 , 5 1 - d i t h i o b i s - 2 - n i t r o b e n z o i c a c i d ) i n T r i s -G l y (4 m g / m l ) , 12% t r i c h l o r o a c e t i c a c i d ( T C A ) , 8M a n d 1 0 M u r e a i n T r i s - G l y , a n d 8M u r e a c o n t a i n i n g 5M G u H C l i n T r i s - G l y ( U r e a - G u H C l ) . F l o u r a n d g l u t e n . A s a m p l e ( 7 5 mg) w a s s u s p e n d e d i n 1 m l o f T r i s - G l y , 4 . 7 g o f G u H C l w a s a d d e d , a n d t h e v o l u m e m a d e t o 1 0 m l . F o r S H , t o 1 m l o f t h i s s l i g h t l y t u r b i d s o l u t i o n w a s a d d e d 4 m l o f U r e a - G u H C l f o l l o w e d b y 0 . 0 5 m l o f E l l m a n ' s r e a g e n t . F o r S S , t o 1 m l o f t h e f l o u r o r g l u t e n 2 2 . s o l u t i o n w a s a d d e d 0 .0 5 m l o f 2 - m e r c a p t o e t h a n o l a n d 4 m l o f U r e a - G u H C l a n d t h e m i x t u r e w a s i n c u b a t e d f o r 1 h r a t 2 5 ° C . A f t e r a n a d d i t i o n a l 1 h r i n c u b a t i o n w i t h 1 0 m l o f 12% T C A , t h e t u b e s w e r e c e n t r i f u g e d a t 5 0 0 0 x g i n S o r v a l l - R C 2 B c e n t r i -f u g e f o r 10 m i n . T h e p r e c i p i t a t e w a s t w i c e r e s u s p e n d e d i n 5 m l o f 12% T C A a n d c e n t r i f u g e d t o r e m o v e 2 - m e r c a p t o e t h a n o l . T h e p r e c i p i t a t e w a s d i s s o l v e d i n 10 m l o f 8M u r e a i n T r i s -G l y a n d t h e c o l o r w a s d e v e l o p e d w i t h 0 . 0 4 m l o f E l l m a n ' s r e a g e n t . E g g w h i t e . T h o r o u g h l y b l e n d e d e g g w h i t e ( 1 . 5 g ) . w a s d i l u t e d t o 10 m l w i t h 1% N a C l i n T r i s - G l y . F o r S H , 2 . 9 m l o f o . 5 % S D S i n T r i s - G l y w a s a d d e d t o 0 , 1 m l o f d i l u t e d e g g w h i t e a n d 0 . 0 2 m l o f E l l m a n ' s r e a g e n t d e v e l o p e d c o l o r . F o r S S , 0 . 2 m l o f d i l u t e d s a m p l e , 1 m l o f 1 0 M u r e a a n d 0 . 0 2 m l o f 2 - m e r c a p t o -e t h a n o l w e r e m i x e d a n d a l l o w e d t o s t a n d f o r 1 h o u r . A f t e r p r e c i p i t a t i o n o f p r o t e i n w i t h 10 m l o f T C A a n d w a s h i n g a s d e s c r i b e d f o r f l o u r a n d g l u t e n , t h e p r e c i p i t a t e w a s d i s s o l v e d i n 3 m l o f 0.5% S D S o r 8M u r e a i n T r i s - G l y , t h e n 1 m l w a s d i l u t e d t o 1 0 m l w i t h t h e s a m e s o l v e n t . C o l o r w a s d e v e l o p e d b y a d d i n g 0J05 m l o f E l l m a n ' s r e a g e n t . F o r d r i e d e g g w h i t e , 2 0 0 mg w a s d i s s o l v e d i n 1 0 m l o f 1% N a C l i n T r i s - G l y . M i x i n g w a s g e n t l e t o a v o i d s u r f a c e d e n a t u r a t i o n . S k i m m i l k . F o r S H , 0 . 5 m l o f s k i m m i l k w a s a d d e d t o 2 . 5 m l o f 8M u r e a i n T r i s - G l y a n d a 0 2 m l o f E l l m a n ' s r e a g e n t . F o r S S , 0 .2 m l o f s k i m m i l k , 1 m l o f 1 0 M u r e a i n T r i s - G l y a n d 2 3 . 0 . 0 2 m l o f m e r c a p t o e t h a n o l w e r e i n c u b a t e d a t 2 5 ° C f o r 1 h r . A f t e r p r e c i p i t a t i o n a n d w a s h i n g o f p r o t e i n a s f o r f l o u r a n d g l u t e n , t h e p r e c i p i t a t e w a s d i s s o l v e d i n 3 m l o f 8M u r e a i n T r i s -G l y a n d 0 . 0 3 m l o f E l l m a n ' s r e a g e n t w a s a d d e d f o r c o l o r d e v e l o p -m e n t . SH a n d S S D e t e r m i n a t i o n i n P u r e P r o t e i n s T h e S H a n d S S l e v e l s o f t h e e l e c t r o p h o r e t i c a l l y p u r e p r o t e i n s ( K - c a s e i n , a ^ - c a s e i n , 8 - l a c t o g l o b u l i n , o v a l b u m i n a n d a ^ - c a s e i n ) w e r e d e t e r m i n e d o n 1% s o l u t i o n s u s i n g t h e s a m e p r o c e d u r e a s t h a t o f s k i m m i l k e x c e p t f o r o v a l b u m i n w h i c h w a s d e t e r m i n e d b y t h e s a m e p r o c e d u r e a s e g g w h i t e . T h e c o n c e n t r a -t i o n s - o f t h e s e p u r i f i e d p r o t e i n s w e r e d e t e r m i n e d f r o m t h e I s -a b s o r b a n c e s u s i n g t h e i r a b s o r b t i v i t i e s , A n ° : 9 . 5 a n d 7 . 3 5 1 c m a t 2 8 0 nm f o r B - l a c t o g l o b u l i n a n d o v a l b u m i n ( 8 0 ) , 1 1 . 7 a t 2 8 0 nm f o r K - c a s e i n a n d 1 0 . 7 a n d 1 0 . 1 a t 27 8 nm f o r a - a n d s l a - c a s e i n s ( 3 ) . s 5 A b s o r b a n c e w a s m e a s u r e d a t 4 1 2 n m , a f t e r c o l o r d e v e l o p m e n t f o r e a c h s a m p l e , o n a B e c k m a n DB s p e c t r o p h o t o m e t e r . D r y w e i g h t o f t h e f o o d p r o d u c t s w a s a n a l y z e d b y d r y i n g a t 1 0 5 ° C f o r 24 h o u r s . C a l c u l a t i o n s 7 3 . 5 3 A D y M o l e S H / g m p r o t e i n = -o r f o o d p r o d u c t w h e r e : A 4 1 2 = t ^ i e a ^ s o r b a n c e a t 4 1 2 nm C = t h e s a m p l e c o n c e n t r a t i o n i n mg s o l i d / m l 24. D = the d i l u t i o n factor, 5.02, 6.04, and 30.2 for SH and 10, 15 and 150 for t o t a l SH (SH + reduced SS) i n f l o u r or gluten, milk and egg white respec-t i v e l y 10 6 4 73.53: derived from — =- , 1.36 X 10 1.36 X 10 4 6 is the molar absorptivity ( i ? ) and 10 i s for conversions from the molar basis to the um/ml basis and from mg so l i d s to gm s o l i d s . R E S U L T S A N D D I S C U S S I O N R e d u c t i o n o f S S G r o u p s D i s u l f i d e g r o u p s i n p r o t e i n d o n o t r e a c t w i t h E l l m a n ' s r e a g e n t , b u t t h e y c a n b e d e t e r m i n e d a s S H g r o u p s a f t e r r e d u c t i o n . H o w e v e r , s e l e c t i o n o f a r e d u c i n g a g e n t i s l i m i t e d w h e n t h e d i r e c t s p e c t r o p h o t o m e t r y m e t h o d i s u s e d f o r t h e d e t e r m i n a t i o n . E l l m a n ' s r e a g e n t a l s o c o n t a i n s - a n S S l i n k a g e a n d w i l l b e s u b j e c t t o t h e s a m e r e d u c i n g r e a c t i o n s a s f o r t h e c o m p o u n d s u n d e r s t u d y a n d t h u s , e x c e s s r e d u c i n g , a g e n t s m u s t b e d e s t r o y e d o r r e m o v e d b e f o r e a d d i t i o n o f E l l m a n ' s r e a g e n t b y a p r o c e s s t h a t d o e s n o t a f f e c t t h e S H v a l u e s . R e d u c t i o n o f S S g r o u p s i n p r o t e i n p r i o r t o t h e i r e s t i m a t i o n a s S H h a s c o m m o n l y e m p l o y e d r e a g e n t s s u c h a s s u l f i t e o r b o r o h y d r i d e a s r e d u c i n g a g e n t s . A t t e m p t s t o u s e s o d i u m b o r o h y d r i d e f o r e s t i m a t i o n o f S S g r o u p s i n K - c a s e i n r e s u l t e d i n v a l u e s a p p r o x i m a t e l y o n e h a l f t h a t e x p e c t e d f r o m l i t e r a t u r e v a l u e s ( 59 ) . C o n s e q u e n t l y , t h e p o s s i b l e u s e o f 2 - m e r c a p t o e t h a n o l a s r e d u c i n g a g e n t w a s e x p l o r e d , a n d t h e e x c e s s o f t h i s r e a g e n t w a s w a s h e d c o m p l e t e l y w i t h t r i c h l o r o -a c e t i c a c i d s o l u t i o n b e f o r e a d d i t i o n o f t h e D T N B r e a g e n t . T h e e f f e c t o f u r e a c o n c e n t r a t i o n o n t h e t o t a l S H v a l u e s f o r 8 - l a c t o g l o b u l i n , o v a l b u m i n , K - c a s e i n , m i l k a n d e g g w h i t e a r e s h o w n i n F i g u r e 1 . I t i s a p p a r e n t t h a t o n e h o u r i n 2 6 . £ 200 O ^ 16 0!-U l o X O or UJ u_ I U 6 F i g . 1 2 0,-!' EGG WHITE MILK "OVALBUMIN G-LACTOGLOBUI.IN ;C-CASEIN 2 4 6 8 UREA CONCENTRATION- MOLAR E f f e c t o f u r e a c o n c e n t r a t i o n on the t o t a l SH groups of s e v e r a l p r o t e i n m a t e r i a l s a f t e r i n c u b a t i o n f o r 1 hour a t 25°C i n the presence of 2-mercaptoethanol M i l k and Egg White r e p o r t e d on d r y w e i g h t b a s i s . 27 . 8M u r e a i s s u f f i c i e n t t o r e d u c e S S g r o u p s i n a l l p r o t e i n s o l u t i o n s t e s t e d . F u r t h e r i n c u b a t i o n u p t o 3.5 h o u r s s l i g h t l y i n c r e a s e d t o t a l S H l e v e l s , b u t e x t e n d e d r e d u c t i o n f o r 8.5 h o u r s d e c r e a s e d t h e m p r e s u m a b l y d u e t o a i r o x i d a t i o n o f t h e S H g r o u p s . T h i s r e d u c i n g c o n d i t i o n w a s a l s o a p p l i c a b l e t o f l o u r a s i n c u b a t i o n b e y o n d 1 h o u r d i d n o t i n c r e a s e t o t a l S H v a l u e . S H a n d S S o f P u r i f i e d P r o t e i n s A s s h o w n i n T a b l e l , t h e v a l u e s f o r a l l o f t h e p r o t e i n s a g r e e d w e l l w i t h p u b l i s h e d v a l u e s . N o f a l s e p o s i t i v e v a l u e f o r a ^ - c a s e i n , f o r w h i c h n e i t h e r S H n o r S S h a s b e e n r e p o r t e d b e f o r e , i n d i c a t e s t h a t t h e w a s h i n g p r o c e d u r e w i t h T C A w a s s u f f i c i e n t . S H a n d S S o f F o o d P r o d u c t s S a t i s f a c t o r y a g r e e m e n t w a s o b t a i n e d b e t w e e n S H a n d S S o f f o o d p r o d u c t s a n d r e p o r t e d v a l u e s ( T a b l e I I ) . T h e v a l u e s f o r f r e s h s k i m m i l k a g r e e d w i t h m o s t l i t e r a t u r e v a l u e s a n d w i t h c a l c u l a t e d v a l u e s a s s u m i n g t h a t a l l S H a n d S S w e r e d e r i v e d f r o m K - c a s e i n , B - l a c t o g l o b u l i n a n d a - l a c t a l b u m i n . H o w e v e r , t h e v a l u e s r e p o r t e d b y P o f a h l a n d V a k a l e r i s ( 9 5 ) a s d e t e r m i n e d b y a f l u o r o m e t r i c p r o c e d u r e w e r e c o n s i d e r a b l y h i g h e r t h a n o u r v a l u e s . T h e v a l u e s o b t a i n e d f o r i n s t a n t i z e d s k i m m i l k p o w d e r a r e i n g o o d a g r e e m e n t w i t h p u b l i s h e d d a t a , e x c e p t f o r a b o u t a h a l f v a l u e f o r S S r e p o r t e d b y K a l a b ( •* 0 ) w h o u s e d s o d i u m b o r o h y d r i d e f o r r e d u c t i o n o f S S . N o s i g n i f i -T a b l e I S u l f h y d r y l and d i s u l f i d e v a l u e s f o r p u r i f i e d p r o t e i n s P r o t e i n SH (moles/mole SS (moles/mole p r o t e i n ) p r o t e i n )  Found L i t e r a t u r e Found L i t e r a t u r e ( 3 - L a c t o g l o b u l i n 0. 95 0.9 (21) 2. 0 2. 0 (65) O v a l b u m i n 4. 0 3.8-4 .1 (75) 1. 0 1. 0 (23) K - c a s e i n 0 0 (59) 1. 0 1. 0 (59) a - c a s e i n s 5 0 0 (39) 1. 9 1. 8 (39) a n - c a s e i n s l 0 0 ( 7) 0 0 ( 7) Assumed M o l e c u l a r W e i g h t 3 - L a c t o g l o b u l i n 18,000 (68) O v a l b u m i n 45,000 (23) K - c a s e i n 20,000 (59) a - c a s e i n 65,750 (124) s o a s l ~ c a s e i n 27,000 (64) 29. T a b l e I I S u l f h y d r y l and d i s u l f i d e v a l u e s f o r f o o d p r o d u c t s F o o d P r o d u c t SH (yM/g, d r y w e i g h t ) SS (yM/g d r y w e i g h t ) F o u n d L i t e r a t u r e F o u n d L i t e r a t u r e S k i m m i l k F r e s h I n s t a n t i z e d powder F r e e z e - d r i e d Egg w h i t e F r e s h P o w d e r e d F l o u r 1 2 C r u d e g l u t e n 2.13 1.33 2.20 2.65 3.1 2.06-2 . 4 5 . 8 1.78 2 . 0 a (148) 10.8 (95) (105) 0.98 (40) 11.3 1.02-1.38 (56) 1.22 (105) 10 .4 50.7 50.0 51.6 48.2 (20) 79.7 (60) 79.4 2.18 1.0-1.7 (127) 14.6 12.8 60.9 13.3 (148) 48.0 (95) 10.8 (105) 9 . 7 a 14.5 (61) 4.01 (40) 10.1 (105) 84.5 70 5-16.9 (127) (46) C a l c u l a t e d v a l u e s , s e e t e x t , D e p e n d i n g on v a r i e t y . 30. cant change was observed during freeze-drying of fresh skimmilk. With the c o e f f i c i e n t of v a r i a t i o n of 2% for t h i s method, i t i s d i f f i c u l t to detect differences i n t o t a l SH for fresh skimmilk, instantized skimmilk powder and freeze-dried skimmilk, although SH was s i g n i f i c a n t l y lower i n commercial instantized powder than i n fresh skimmilk. Sulfhydryl values of fresh and powdered egg white were i n good agreement with l i t e r a t u r e values for fresh egg white. The d i s u l f i d e values obtained agreed reasonably well with a value calculated from amino acid composition ( 2 2 ) and the protein concentration' i n the t o t a l s o l i d s of egg white ( 1 9 ) . No d i r e c t data was available i n the l i t e r a t u r e . As with milk products i t i s unlikely that dried and fresh egg white d i f f e r s i g n i f i c a n t l y i n t h e i r SH and SS values. The SH values for f l o u r were s l i g h t l y higher than the reported values. Some uncertainty i n the values was unavoidable because low absorbances had to be used for c a l c u l a t i o n , as high concentrations of f l o u r developed t u r b i d i t y . The values for SS i n flour agreed with the published values. The crude gluten used i n th i s study had approximately 90% purity so that the value obtained for t o t a l SH i s within the agreeable range ( 6 ) . A problem encountered during the course of these measurements was the f a i l u r e of the reduced proteins to develop color a f t e r d i s s o l v i n g i n urea or SDS and addition of Ellman's reagent. This was p a r t i c u l a r l y noticeable i n the 31. case of f l o u r where a c r y s t a l l i n e p r e c i p i t a t e , presumably of GuHCl, appeared after addition of TCA. This p r e c i p i t a t e was somewhat soluble i n TCA and almost disappeared aft e r the second washing. The f a i l u r e to develop color could be over-come by further d i l u t i o n of the reaction mixture with T r i s -Gly buffer. This d i f f i c u l t y appears to be due to the f i n a l pH. The pH of the reaction mixture has a marked e f f e c t on the rate of color development ( 8 ). Apparently, enough TCA sometimes clings to the test tube wall or i s absorbed i n the p r e c i p i t a t e , p a r t i c u l a r l y when c r y s t a l s of GuHCl are present, to lower the pH of the reaction mixture. This slows or prevents the color development. The problem was largely solved by increasing the buffer concentration. When the pH was maintained at about 8, color development was complete i n 5 min for a l l proteins tested, and was stable for at least 2 hours. The recoveries of SH and SS added as 8-lactoglobulin to food products ranged from 91 to 98.3% and from 89 to 102% for SH and SS, respectively (Table-'III) , 3 2 . T a b l e I I I R e c o v e r y o f S u l f h y d r y l a n d D i s u l f i d e G r o u p s o f 8 - l a c t o g l o b u l i n f r o m F o o d P r o d u c t s R e c o v e r y (%) P r o d u c t S H S £ R a w S k i m m i l k 9 8.3 10 2 F r e s h E g g W h i t e 91.0 101 C r u d e G l u t e n 91.0 89 C H A P T E R I I E F F E C T O F H E A T ON T H E D I S U L F I D E M O D I F I E D K - C A S E I N 3 3 . I N T R O D U C T I O N T h e c h a n g e s t h a t , o c c u r w h e n m i l k i s h e a t e d d u r i n g p r o c e s s i n g a n d t h e f a c t o r s a f f e c t i n g i t s h e a t s t a b i l i t y h a v e b e e n w i d e l y s t u d i e d . D e s p i t e t h e g r e a t v o l u m e o f d a t a r e p o r t e d i n t h e l i t e r a t u r e o v e r t h e p a s t 3 0 y e a r s c o n c e r n i n g t h e h e a t s t a b i l i t y o f m i l k , t h e r e i s , a s y e t , n o s a t i s f a c t o r y e x p l a n a t i o n f o r a l l f a c e t s o f t h e p r o b l e m . T h e c a s e i n s i n m i l k a r e g e n e r a l l y c o n s i d e r e d t o e x i s t i n s o l u t i o n a s r a n d o m c o i l s a n d s o a r e t h o u g h t t o h a v e l i t t l e o r d e r e d s t r u c t u r e t h a t c a n b e d i s r u p t e d b y h e a t . D e c r e a s e i n p r o t e i n s t a b i l i t y a n d p r o d u c t i o n o f a g e l l i k e s t r u c t u r e h a v e b e e n s h o w n i n s t e r i l e c o n c e n t r a t e d m i l k a n d i n m o d e l s y s t e m s o f c a s e i n s o l u t i o n s d u r i n g s t o r a g e ( 7 6 , 7 7 , i i ) • S t a b i l i t i e s o f c a s e i n a n d o f c a s e i n c o m p l e x f r o m s t e r i l e c o n c e n t r a t e d m i l k w e r e a f f e c t e d m o s t b y o x i d i z i n g a n d r e d u c i n g a g e n t s ( 7 8 , 7 9 ) . K - c a s e i n i s t h e c a s e i n f r a c t i o n w h i c h f u n c t i o n s a s a k e y m i c e l l a r s t a b i l i z i n g f a c t o r a n d i s t h e p r i m a r y s u b s t r a t e o f r e n n i n a c t i o n . U n l i k e t h e o t h e r m a j o r c a s e i n s , K - c a s e i n c o n t a i n s a s i g n i f i c a n t q u a n t i t y o f h a l f c y s t i n e (2 m o l e s p e r 2 0 , 0 0 0 m o l e c u l a r w e i g h t ) a n d i t i s l i k e l y t h a t a l l t h e c y s t e i n e s a r e p r e s e n t a s S S . a n d n o t a s S H ( 5 7 I 19) . T h e s e d i s u l f i d e b o n d s a r e i n t e r m o l e c u l a r b o n d s w h i c h l e a d t o t h e f o r m a t i o n o f l a r g e a g g r e g a t e s . T h i s c a s e i n f r a c t i o n c o n s t i t u t e s o n e f i f t h o f t h e t o t a l a - c a s e i n s a n d 1 2 - 1 5 % o f t h e t o t a l c a s e i n s . D e c r e a s e i n t h e s t a b i l i z i n g - a b i l i t y o f K . - ' ease ih h a s b e e n r e p o r t e d u p o n h e a t i n g a s o l u t i o n o f t h i s p r o t e i n t o 1 0 0 - 1 1 0 ° C f o r 30 m i n u t e s ( 1 2 s ) a n d a c o m p l e t e l o s s w a s o b s e r v e d w h e n t h e t e m p e r a t u r e i n c r e a s e d t o 1 2 0 ° C o r a b o v e ( 8 4 , 1 2 8 ) . Z i t t l e ( 1 5 1 +) h a s s h o w n t h a t h e a t i n g s o l u t i o n s o f K - c a s e i n a t a n e u t r a l p H c a u s e d u p t o 60% r e d u c t i o n i n t h e r e n n i n c l o t t i n g t i m e , b u t h e a t e d K - c a s e i n s t i l l r e t a i n e d t h e a b i l i t y t o p r o t e c t a ^ - c a s e i n a g a i n s t p r e c i p i t a t i o n b y c a l c i u m . H o w e v e r , i n a l a t e r p a p e r , Z i t t l e ( 1 5 2 ) s h o w e d t h a t t h e p r o t e c t i v e p r o p e r t y w a s l a b i l e i f s a l t ( 0 . 0 5 M ) w a s i n c l u d e d i n t h e s y s t e m . R e d u c i n g a g e n t s s u c h a s m e r c a p t o -e t h a n o l o r c y s t e i n e e n h a n c e d h e a t l a b i l i t y , b u t r e d u c t i o n a n d a l k y l a t i o n t e n d t o p r e v e n t h e a t l a b i l i t y . T h e e f f e c t o f s u l f h y d r y l r e d u c i n g a g e n t s s u g g e s t s t h a t s u l f h y d r y l g r o u p s m a y b e r e s p o n s i b l e f o r t h e p h e n o m e n o n . O t h e r i n v e s t i g a t o r s h a v e p o s t u l a t e d t h e i n v o l v e m e n t o f d i s u l f i d e b o n d s o f K - c a s e i n i n t h e h e a t s t a b i l i t y o f t h e m i l k s y s t e m ( 7 9 , 1 4 1 ) . M o r e r e c e n t l y , t h e f u n c t i o n s o f t h e S S i n t e r c h a n g e r e a c t i o n i n K - c a s e i n h a s b e e n s u g g e s t e d t o p l a y a n i m p o r t a n t r o l e i n t h e s t a b i l i z a t i o n o f a ^ - c a s e i n a g a i n s t p r e c i p i t a t i o n b y c a l c i u m ( 7 4 ) . A d r a s t i c l o s s i n t h e s t a b i l i z i n g a b i l i t y o f K - c a s e i n w a s o b s e r v e d w h e n h i s t i d i n e r e s i d u e s w e r e c a r b e t h o x y l a t e d w i t h D E P * . H o w e v e r , b l o c k i n g * D E P = D i e t h y l p y r o c a r b o n a t e 3 5 . t h e S S g r o u p s i n t h e c a r b e t h o x y l a t e d K - c a s e i n p r e v e n t e d a l m o s t c o m p l e t e l y t h i s l o s s ( 7 h ) . K r e s h e c k e t a _ l . ( s 3 ) , s t u d y i n g a , 3 a n d K - c a s e i n s b y l i g h t s c a t t e r i n g , o b s e r v e d t h a t o n l y K - c a s e i n s h o w e d a n y m a r k e d i n s t a b i l i t y o n h e a t i n g t o 9 0 ° C . I n c r e a s e s i n t u r b i d i t y w e r e n o t e d f o r K - c a s e i n a n d t h e w e i g h t a v e r a g e m o l e c u l a r 6 7 w e i g h t i n c r e a s e d f r o m 3 . 4 3 X 1 0 t o 1 . 7 7 X 1 0 a n d t h e r a d i u s o f g y r a t i o n f r o m 5 9 2 t o 1 , 3 3 0 8 b y h e a t i n g f r o m 3 0 ° C t o 9 0 ° C . N o d a t a w a s p r o v i d e d b y t h e s e a u t h o r s t o s u g g e s t t h e n a t u r e o f b o n d s i n v o l v e d i n a g g r e g a t e f o r m a t i o n . H o w e v e r , a r i n g p r e c i p i t a t e w a s o b s e r v e d a t t h e a i r - g l a s s - l i q u i d i n t e r f a c e i n t h e l i g h t s c a t t e r i n g c e l l w h i c h c o u l d s u g g e s t t h e o x i d a t i o n o f s u l f h y d r y l g r o u p s . I n t h i s c h a p t e r t h e r e s u l t s o f a?, 'study a r e p r e s e n t e d r e g a r d i n g t h e e f f e c t o f d e s u l f u r i z a t i o n a n d a l k y l a t i o n o f d i s u l f i d e g r o u p s o n t h e h e a t s t a b i l i t y o f K - c a s e i n . T h e d i s u l f i d e i n t e r c h a n g e r e a c t i o n w h i c h h a s b e e n u s e d t o e x p l a i n a c o m p l e x f o r m a t i o n b e t w e e n K - c a s e i n a n d 8 - l a c t o g l o b u l i n i n h e a t e d m i l k w a s i n v e s t i g a t e d u s i n g t h e S e p h a r o s e 6 B g e l c h r o m a t o g r a p h y . T h e e x t e n t o f d e s u l f u r i z a t i o n o f K - c a s e i n w a s m e a s u r e d b y t h e D T N B m e t h o d f o r S S d e t e r m i n a t i o n . P h y s i c a l a n d c h e m i c a l p r o p e r t i e s o f m o d i f i e d K - c a s e i n w e r e d e t e r m i n e d b y s e d i m e n t a t i o n a n a l y s i s , p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , f l u o r e s c e n c e p o l a r i z a t i o n , g e l f i l t r a t i o n a n d a m i n o a c i d a n a l y s i s . 3 6 . M A T E R I A L S A N D M E T H O D S D e s u l f u r i z a t i o n o f K - c a s e i n P r e p a r a t i o n o f R a n e y n i c k e l S o d i u m b o r o h y d r i d e , 1 g m , w a s a d d e d t o 3 0 0 m l o f 2% n i c k e l a c e t a t e . T h e b l a c k a m o r p h o u s n i c k e l p r e c i p i -t a t e w a s f i l t e r e d a n d w a s h e d o n t h e f i l t e r p a p e r w i t h d i s t i l l e d w a t e r u n t i l t h e w a s h i n g s b e c a m e n e u t r a l . T h e n i c k e l w a s n o t a l l o w e d t o d r y a n d i t w a s a l w a y s u s e d i m m e d i a t e l y a f t e r p r e p a r a t i o n . D e s u l f u r i z a t i o n r e a c t i o n w a s p e r f o r m e d a c c o r d i n g t o P e r l s t e i n e t a l . (9 ) w i t h s l i g h t m o d i f i c a t i o n a s f o l l o w s : K - c a s e i n w a s f i r s t r e d u c e d w i t h 2 - m e r c a p t o e t h a n o l i n t h e p r e s e n c e o f 8M u r e a , a n d t h e e x c e s s r e d u c i n g a g e n t w a s w a s h e d w i t h 10% T C A . E i g h t y m i l l i l i t e r s o f 0 . 2 - 0 . 5 % r e d u c e d K - c a s e i n s o l u t i o n c o n t a i n i n g 8M u r e a o r 0 . 5 % S D S w a s p r e p a r e d i n a 1 0 0 m l E r l e n m e y e r f l a s k . A b o u t 2 g m s o f t h e f r e s h l y p r e p a r e d R a n e y n i c k e l w a s a d d e d t o t h e f l a s k . T h e r e a c t i o n m i x t u r e w a s e v a c u a t e d u n t i l t h e p r e s s u r e r e a c h e d 50 - 1 0 0 m i c r o n s o f H g a f t e r f r e e z i n g i n a d r y i c e - a c e t o n e b a t h , t h e n t h a w e d a t . 2 5 ° C . F r e e z i n g a n d e v a c u a t i o n w e r e r e p e a t e d t w o m o r e t i m e s a n d t h e r e a c t i o n w a s s t a r t e d u n d e r a n a t m o s p h e r e o f h y d r o g e n a t 2 5 ° C a n d p H 7 . 0 . T h e p H o f t h e r e a c t i o n m i x t u r e w a s k e p t c o n s t a n t i n a p H s t a t w i t h I N H C 1 . F o u r t e e n m i l l i l i t e r s o f h y d r o c h l o r i c a c i d w e r e r e q u i r e d d u r i n g t h e r e a c t i o n t i m e o f 4 8 h o u r s . S a m p l e s 37. w e r e w i t h d r a w n p e r i o d i c a l l y , c e n t r i f u g e d a n d t h e s u p e r n a t a n t s w e r e d i a l y z e d a g a i n s t r u n n i n g w a t e r f o r 4 8 h o u r s , t h e n f r e e z e d r i e d . U l t r a c e n t r i f u g a t i o n S c h l i e r e n p a t t e r n s o f c a s e i n s o l u t i o n s w e r e o b t a i n e d i n a B e c k m a n L 2 - 6 5 B p r e p a r a t i v e u l t r a c e n t r i f u g e a t 2 0 ° C . A f i l l e d e p o n s i n g l e s e c t o r c e l l h a v i n g 2 ° s e c t o r a n g l e a n d 1 2 mm t h i c k n e s s w a s u s e d . O n e p e r c e n t s o l u t i o n w a s m a d e i n 0 . 0 1 M i m m i d a z o l e - H C l b u f f e r c o n t a i n i n g 0 . 0 7 M K C l , p H 6 . 8 . S t a b i l i z a t i o n o f a , - c a s e i n w i t h K - c a s e i n s l Z i t t l e ' s m e t h o d i15 °) w a s u s e d t o e s t i m a t e t h e s t a b i l i z a t i o n o f a s ^ - c a s e i n b y K - c a s e i n w i t h a s l i g h t m o d i f i c a -t i o n a s f o l l o w s : 1 . 5 m l o f 0 . 5 % a , - c a s e i n a n d 1 m l o f 0 . 0 2 M i m m i d a -s l z o l e b u f f e r p H 8 . 0 w e r e m i x e d i n a 1 5 - m l c e n t r i f u g e t u b e . S u f f i c i e n t w a t e r w a s a d d e d t o m a k e a f i n a l v o l u m e o f 5 . ' 0 m l a f t e r s u b s e q u e n t a d d i t i o n s . T h e d e s i r e d a m o u n t o f 0 . 2 % K - c a s e i n s o l u t i o n w a s a d d e d . T o d i s s o c i a t e K - c a s e i n a g g r e g a t e s p r i o r t o t h e s t a b i l i t y t e s t , 0.1 m l o f I N s o d i u m h y d r o x i d e w a s a d d e d t o 5 m l o f t h e i c e - c o o l e d c a s e i n s o l u t i o n a n d i m m e d i a t e l y n e u t r a l i z e d w i t h 0 . 1 m l o f 1 N h y d r o c h l o r i c a c i d . F i n a l l y 1 m l o f 0 . 0 5 M c a l c i u m c h l o r i d e w a s a d d e d t o g i v e a f i n a l p H o f a p p r o x i m a t e l y 7 . 0 . A f t e r s t i r r i n g , t h e m i x t u r e w a s k e p t i n a 3 0 ° C w a t e r b a t h f o r 1 5 m i n u t e s , t h e n c e n t r i f u g e d a t a b o u t 3 , 0 0 0 g f o r 5 m i n u t e s . T h e s u p e r -n a t a n t w a s c l a r i f i e d w i t h o n e d r o p o f 1 N s o d i u m h y d r o x i d e 3 8 . a n d t h e c a s e i n c o n c e n t r a t i o n w a s d e t e r m i n e d f r o m t h e a b s o r b a n c e a t 2 8 0 n m . B y s u b t r a c t i n g t h e a m o u n t o f K - c a s e i n u s e d i n t h e t e s t , t h e p e r c e n t a - c a s e i n s t a b i l i z e d a n d s u s p e n d e d i n t h e s u p e r n a t a n t w a s c a l c u l a t e d . F l u o r e s c e n c e P o l a r i z a t i o n a n d A s s o c i a t i o n C o n s t a n t P o l a r i z a t i o n w a s m e a s u r e d u s i n g a n A m i n c o - B o w m a n s p e c t r o p h o t o f l u o r o m e t e r # 4 - 8 2 0 2 w i t h z e n o n l a m p a n d a b l a n k s u b t r a c t p h o t o m u l t i p l i e r m i c r o p h o t o m e t e r 1 0 - 2 6 7 . S l i t s w e r e a s f o l l o w : # 2 , 5 , 4 m m , # 3 , 4 , 2 m m , # 7 , 1 m m . P o l a c o a t U V 1 0 5 a n d p o l a r o i d H N 3 8 w e r e u s e d f o r t h e p o l a r i z e r a n d • a n a l y z e r r e s p e c t i v e l y . E x i t a t i o n a n d e m i s s i o n w a v e l e n g t h s w e r e 3 5 0 nm a n d 5 0 5 nm r e s p e c t i v e l y . T h e a s s o c i a t i o n c o n s t a n t ( K a ) w a s c a l c u l a t e d a c c o r d i n g t o a p r o c e d u r e o u t l i n e d b y N a k a i ( 7 3 ) . A l l c a l c u l a t i o n s w e r e p r o g r a m m e d i n M o n r o e c a l c u l a t o r 1 8 8 0 . D a n s y l a t i o n o f a s ^ - c a s e i n D a n s y l a t e d a g l - c a s e i n w a s p r e p a r e d b y r e a c t i n g a s i ~ c a s e i n , 2 8 0 m g / 4 0 m l , i n c a r b o n a t e b u f f e r ( N a H C 0 3 , 0 . 4 M -N a 2 C 0 3 0 . 1 M , p H 9 . 2 ) w i t h a s u s p e n s i o n o f 6 mg d i m e t h y l -a m i n o n a p h t h a l e n e s u l f o n y l c h l o r i d e , i n 1 m l o f t h e s a m e b u f f e r . T h e r e a c t i o n w a s c a r r i e d o u t i n a n i c e b a t h f o r 20 m i n u t e s w i t h c o n s t a n t s t i r r i n g . T h e r e a c t i o n m i x t u r e w a s t h e n c e n t r i f u g e d a t 4 ° C a n d t h e s u p e r n a t a n t d i a l y z e d a g a i n s t d i s t i l l e d w a t e r a t 4 ° C f o r 4 8 h o u r s a n d l y o p h i l i z e d . O n e d a n s y l r e s i d u e w a s i n t r o d u c e d p e r m o l e o f a s l - c a s e i n ( m o l e c u l a r w e i g h t , 2 7 , 0 0 0 ) . 39. A l k y l a t i o n o f K - c a s e i n w i t h i o d o a c e t a m i d e D i s u l f i d e g r o u p s i n K - c a s e i n (0.2 gm/20 m l 0.1 M T r i s - g l y c i n e , 8 M u r e a , p H 7.5) w e r e f i r s t r e d u c e d b y a d d i n g 0.4 m l 2 - m e r c a p t o e t h a n o l . A f t e r o n e h o u r 200 m l o f 12% T C A w a s a d d e d a n d t h e p r o t e i n w a s c e n t r i f u g e d t h e n w a s h e d w i t h T C A t h r e e t i m e s a n d d i s s o l v e d i n 50 m l o f t h e s a m e b u f f e r • T h e p H w a s a d j u s t e d t o 7.5 a n d t h e r e a c t i o n s t a r t e d b y a d d i n g 2 m l o f i o d o a c e t a m i d e s o l u t i o n i n w a t e r (4 m g / m l ) . T h e r e m a i n i n g f r e e S H w a s m e a s u r e d p e r i o d i c a l l y b y t h e m e t h o d s t a t e d i n C h a p t e r I ( p a g e 23). P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s w a s p e r f o r m e d b y a s l i g h t m o d i f i c a t i o n o f A s c h a f f e n b u r g 1 s m e t h o d ( k ) . T h e f o l l o w i n g p r o c e d u r e w a s u s e d f o r t h e g e l p r e p a r a t i o n : 10.5 gm a c r y l a m i d e , 0.52 gm N , N 1 - m e t h y l e n e b i s a c r y l a m i d e a n d 40 gm u r e a w e r e d i s s o l v e d i n 0.1 M T r i s - g l y c i n e b u f f e r p H 9.1 a n d m a d e u p t o a f i n a l v o l u m e o f 150 m l . T h i s s o l u -t i o n w a s f i l t e r e d t h r o u g h N o . l f i l t e r p a p e r i n t o a v a c u u m f l a s k . A f t e r t h e d e a e r a t i o n , 0.42 m l o f 2 - m e r c a p t o e t h a n o l , 1.25 m l T M E D (30% N , N , N 1 , N 1 - t e t r a m e t h y l e n e e t h y l e n e -d i a m i n e i n 95% a l c o h o l ) a n d 1.25 m l o f 10% a m m o n i u m p e r s u l -f a t e w e r e a d d e d t o t h e s o l u t i o n i n t h i s o r d e r . T h e m i x t u r e w a s m i x e d g e n t l y a n d p o u r e d i m m e d i a t e l y i n t o a m o l d , c o v e r e d w i t h a p e r s p e x p l a t e a n d w e i g h e d d o w n t o e x p e l a l l t h e a i r . T h e g e l w a s a l l o w e d t o s t a n d f o r 30-60 m i n u t e s a 4 0 . t o p e r m i t c o m p l e t i o n o f t h e p o l y m e r i z a t i o n . A h o r i z o n t a l e l e c t r o p h o r e s i s a p p a r a t u s , w i t h d o u b l e c o n t a i n e r s a t e i t h e r e n d o f t h e g e l w a s u s e d . A s o d i u m c h l o r i d e s o l u t i o n , 0 . 1 M a n d 0 . 1 7 5 M T r i s - g l y c i n e b u f f e r , p H 9 . 1 w e r e p l a c e d i n t h e o u t e r a n d t h e i n n e r c o n t a i n e r s , r e s p e c t i v e l y . T h e c o n t a i n e r s w e r e c o n n e c t e d w i t h e i g h t f o l d s o f c h e e s e c l o t h . T h e g e l p l a t e w a s p l a c e d o n t h e e l e c t r o p h o r e s i s a p p a r a t u s a n d e q u i l i b r a t e d b y r u n n i n g t h e c u r r e n t t h r o u g h t h e g e l f o r 20 h o u r s a t 4 ° C . T h e c u r r e n t w a s m a i n t a i n e d a t 20 mA o n a p o w e r s u p p l y . S a m p l e s w e r e p r e p a r e d a s f o l l o w s : t o 1 0 mg o f d r i e d c a s e i n s , 2 d r o p s o f 0 . 1 7 5 M T r i s - g l y c i n e b u f f e r , 1 d r o p o f 2 - m e r c a p t o e t h a n o l a n d 1 5 mg o f u r e a w e r e a d d e d a n d t h e s a m p l e s d i s s o l v e d c o m p l e t e l y . A s t r i p o f W h a t m a n 3MM f i l t e r p a p e r 1 . 5 X 1 0 m m , w a s s o a k e d i n e a c h s a m p l e , t h e e x c e s s b l o t t e d a n d t h e n i n s e r t e d i n t h e s l o t o n t h e e q u i l i -b r a t e d g e l p l a t e . T h e g e l p l a t e w a s c o v e r e d w i t h s a r a n f i l m t o p r e v e n t d r y i n g . E l e c t r o p h o r e s i s w a s c a r r i e d o u t u n d e r t h e s a m e c o n d i t i o n s a s t h e e q u i l i b r a t i o n o f t h e g e l f o r 24 h o u r s a t 4 ° C . T h e g e l r e m o v e d f r o m t h e m o l d / w a s s t a i n e d i n 1% a m i d o b l a c k d y e i n 10% a c e t i c a c i d f o r 5 m i n u t e s . T h e g e l w a s d e s t a i n e d b y w a s h i n g i n 10% a c e t i c a c i d s o l u t i o n u n t i l a c l e a r g e l b a c k g r o u n d w a s o b t a i n e d . H e a t i n g E x p e r i m e n t s Two t o f i v e m i l l i l i t e r s o f 0.1-1% K - c a s e i n s o l u t i o n pH 7.0 i n a 15-ml t u b e w e r e i m m e r s e d i n a b o i l i n g w a t e r b a t h t o a 10 ml mark and h e a t e d f o r 30 m i n u t e s . The t u b e s t h e n w e r e c o o l e d i m m e d i a t e l y i n i c e d w a t e r f o r 2 m i n u t e s and t h e p r o t e i n s o l u t i o n was u s e d f o r s t a b i l i t y t e s t , u l t r a c e n t r i -f u g a t i o n o r g e l f i l t r a t i o n . R e n n i n R e a c t i o n i R e n n i n powder (0.5 mg) was d i s s o l v e d i n 1 ml o f 0.1 M p h o s p h a t e b u f f e r , pH 7.5, a n d 20 mg o f K - c a s e i n was d i s s o l v e d i n 0.5 m l o f t h e same b u f f e r . The K - c a s e i n s o l u t i o n (0.25 ml) was m i x e d w i t h 0.1 m l o f t h e enzyme s o l u t i o n . The r e a c t i o n was c a r r i e d o u t a t 37°C f o r 5 and 30 m i n u t e s and was s t o p p e d by t h e a d d i t i o n o f 0.2 gm o f u r e a . The r e a c t i o n p r o d u c t was t h e n a p p l i e d t o p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . R E S U L T S A N D D I S C U S S I O N D e s u l f u r i z a t i o n R e a c t i o n I t h a s b e e n r e p o r t e d t h a t d e s u l f u r i z a t i o n o f c y s t e i n e r e s i d u e s i n p r o t e i n s c a n e f f e c t i v e l y b e p e r f o r m e d a t r o o m t e m p e r a t u r e a n d n e u t r a l p H . H i g h e r o r l o w e r p H d e c r e a s e s t h e r a t e o f r e a c t i o n . I t w a s a l s o s h o w n t h a t t h e d i s u l f i d e g r o u p s i n l y s o z y i r e a r e n o t a v a i l a b l e f o r d e s u l f u r i z a t i o n u n l e s s t h o s e g r o u p s w e r e f i r s t r e d u c e d w i t h 2 - m e r c a p t o e t h a n o l i n t h e p r e s e n c e o f 8 M u r e a . K - c a s e i n s h o w e d a s i m i l a r b e h a v i o u r , t h u s t h e r e a c t i o n w a s p e r f o r m e d i n t h e p r e s e n c e o f d i s s o c i a t i n g a g e n t s , u r e a o r S D S a t p H 7.0 a n d r o o m t e m p e r a t u r e . U s i n g b o t h d i s s o c i a t i n g a g e n t s , t h e r e a c t i o n w a s r a p i d d u r i n g t h e f i r s t 24 h o u r s a n d s l o w e d d o w n o v e r t h e n e x t 36 h o u r s ( F i g u r e 2 ) . D e s u l f u r i z a t i o n g r e a t e r t h a n 5 0 % c o u l d n o t b e o b t a i n e d e v e n a f t e r 96 h o u r s r e a c t i o n t i m e . D e c r e a s e i n t h e r e a c t i o n r a t e a f t e r 24 h o u r s m a y b e d u e t o t h e r e o x i d a t i o n o f t h e s u l f h y d r y l g r o u p s i n K - c a s e i n . R e a c t i o n t i m e s l o n g e r t h a n 4 8 h o u r s w e r e n o t a t t e m p t e d b e c a u s e p a r t i a l i n s o l u b i l i z a t i o n o f t h e p r o t e i n o c c u r r e d a f t e r 60 h o u r s . U r e a w a s c h o s e n a s a d e n a t u r e n t f o r t h e b u l k p r e p a r a -t i o n o f d e s u l f u r i z e d K - c a s e i n a s S D S s o t i g h t l y b i n d s t o K - c a s e i n t h a t t h e r e m o v i n g o f b o u n d S D S r e q u i r e s a c h r o m a -t o g r a p h i c p r o c e d u r e t h r o u g h D o w e x A G - 1 X 2 ( n o ) . 12 24 36 48 60 REACTION TIME ( HOURS ) F i g . 2. T h e r a t e o f d e s u l f u r i z a t i o n r e a c t i o n o f K - c a s e i n i n t h e p r e s e n c e o f 8 M u r e a ( ®-«-©) a n d 0 . 5 % S D S ( A-A-A ) . CO 4 4 . Chemical and Physical Properties of Desulfurized K-casein K-casein exists i n an aggregated form i n the absence of reducing agents or d i s s o c i a t i n g agents with a sedimentation c o e f f i c i e n t of 16S ( I H ) . However, desulfurized K-casein may have a lower S-value since the intermolecular d i s u l f i d e linkages have been disrupted. This i s shown i n Figure 3 where A represents the sedimentation pattern of unmodified K-casein with an S-value of 16, and B for desulfurized K-casein having two peaks with S-values of 16 and 12., representing the unmodified and modified K-casein respectively. Attempts were made to purify the modified portion by gel f i l t r a t i o n . Using Sephadex G-150, G-200, Sepharose 6B i n the presence or absence of 6.6 M urea, the best separation was obtained with Sepharose 6B i n the presence of 6.6 M urea. Figure 4 shows the e l u t i o n patterns 'of unmodified K-casein (A), alkylated K-casein (B) and desulfurized K-casein (C) on Sepharose 6B eluted with 0.05 M phosphate buffer, pH 7.6 con-taining 6.6 M urea. In the presence of urea, K-casein dissociates p a r t i a l l y but remains at a high molecular weight of 90-125 X 10 as aggregates through intermolecular d i s u l f i d e bridges ( i 1 9 ) . Due to t h i s aggregation, the unmodified K-casein was almost completely excluded from the gel matrix and began eluting very near to the void volume (Figure 4A). On cleavage of the d i s u l f i d e bonds with 2-mercaptoethanol or 3 strong bases, K-casein dissociates to monomers of 20 X 10 daltons ( 7 5 ). Thus alkylated K-casein i s retarded due to 44 . Chemical and Physical Properties of Desulfurized K-casein K-casein exists i n an aggregated form i n the absence of reducing agents or d i s s o c i a t i n g agents with a sedimentation c o e f f i c i e n t of 165 ( I H ) . However, desulfurized K-casein may have a lower S-value since the intermolecular d i s u l f i d e linkages have been disrupted. This i s shown i n Figure 3 where A represents the sedimentation pattern of unmodified K-casein with an S-value of 16, and B for desulfurized K-casein having two peaks with S-values of 16 and 12., representing the unmodified and modified K-casein respectively. Attempts were made to p u r i f y the modified portion by gel f i l t r a t i o n . Using Sephadex G-150, G-200, Sepharose 6B i n the presence or absence of 6.6 M urea, the best separation was obtained with Sepharose 6B i n the presence of 6.6 M urea. Figure 4 shows the e l u t i o n patterns ' of unmodified K-casein (A), alkylated K-casein (B) and desulfurized K-casein (C) on Sepharose 6B eluted with 0.05 M phosphate buffer, pH 7.6 con-taining 6.6 M urea. In the presence of urea, K-casein dissociates p a r t i a l l y but remains at a high molecular weight of 90-125 X 10 as aggregates through intermolecular d i s u l f i d e bridges ( I I 9 ) . Due to this aggregation, the unmodified K-casein was almost completely excluded from the gel matrix and began el u t i n g very near to the void volume (Figure 4A). On cleavage of the d i s u l f i d e bonds with 2-mercaptoethanol or 3 strong bases, K-casein dissociates to monomers of 20 X 10 daltons ( 7 5 ). Thus alkylated K-casein i s retarded due to F i g . 3 . U l t r a c e n t r i f u g e p a t t e r n o f K - c a s e i n (A) a n d d e s u l f u r i z e d K - c a s e i n (B) o b t a i n e d 1 0 m i n u t e s a f t e r r e a c h i n g 5 9 , 1 0 0 r p m . 46. FRACTION NUMBER Fiq 4 E l u t i o n pattern of unmodified K - c a s e i n CA), alkylated K-casein (B) and desulfurized K-casein (C) on Sepharose 6B column ( 4 3 X 2.5 cm) eluted with 0.05 M phosphate buffer pH 7.6 at 25°C* containing 6.6 M urea. disaggregation of the molecule (Figure 4B). Figure 4C shows the el u t i o n pattern of desulfurized K-casein revealing a p a r t i a l d i s s o c i a t i o n of the aggregated protein by d e s u l f u r i -zation. E l e c t r o p h o r e t i c a l l y , K - c a s e i n behaves as an unresolved zone i n the absence of reducing agents such as 2-mercaptoethanol (slot C of Figure 5B), whereas reducing with 2-mercaptoethanol, gives discrete components of greater m o b i l i t i e s (slot C of Figure 5A). This phenomena can be explained by a molecular sieving e f f e c t of polyacrylamide gels on K - c a s e i n aggregates of high molecular weight. The electrophoretic behaviour of d i f f e r e n t fractions obtained by eluting the desulfurized K-casein on Sepharose 6B (Figure 4C) i s shown i n Figure 5, A and B. I t was found that the greater the e l u t i o n volume of the desulfurized K - c a s e i n , the larger the electrophoretic mobility and that f r a c t i o n 4 con-tained f u l l y dissociated K - c a s e i n . However, a value of 0.4 mole SH as SS per 20,000 daltons was s t i l l detected i n this f r a c t i o n . As i t has'been reported that alanine i s the only reaction product of d e s u l f u r i z a t i o n of the cystiene residues i n protein ( 9 4 ) , an increase i n alanine content of K - c a s e i n besides a decrease i n half cystiene was expected upon de s u l f u r i z a t i o n . Table IV shows the amino acid composition of K-casein before and a f t e r d e s u l f u r i z a t i o n . The half cysteine was s i g n i f i c a n t l y decreased from 2.06 to 1.24 residues per 48 . T a b l e I V . Amino A c i d C o m p o s i t i o n o f K - c a s e i n b e f o r e and a f t e r D e s u l f u r i z a t i o n ( m o l e s / 2 0 0 0 0 mo l wt K - c a s e i n ) K - c a s e i n D e s u l f u r i z e d ( c o n t r o l ) K - c a s e i n L y s i n e 10. .79 + 0. .33* 11. 16 + 0. ,56* H i s t i d i n e 3. .7 + 0 . .26 3 . ,86 + 0 . ,39 A r g i n i n e 4. .99 + 0. .71 4. .99 + 0. ,23 A s p a r t i c a c i d 12 . .15 + 1. . 5 12 . . 79 + 0 . ,91 T h r e o n i n e 12 . .98 + 0. . 65 ] 12. .80 + 0 . .56 S e r i n e 12 . . 50 + 0. .75 12. . 5 + 0. .74 G l u t a m i c a c i d 27, .03 + 0. . 10 26 . .98 + 0. .06 P r o l i n e 18, .07 + 0. . 95 17 . .54 + 0. .99 G l y c i n e 3. .89 + 0 . .14 3. . 6 + 0 . .41 A l a n i n e 12. .61 + 0. . 55 12. .88 + 0. .27 V a l i n e 11. .48 + 0 . .61 12. .48 + 0. .95 M e t h i o n i n e 2, .44 + 0. .13 2. .44 + 0. .23 I s o l e u c i n e 11, .00 + 0, .85 11, .01 + 0, .65 L e u c i n e 9, .67 + 0. .6 9. .32 + 0, .64 T y r o s i n e 8, .39 + 0. .55 9, .46 + 0, .75 P h e n y l a l a n i n e 4 . 52 + 0 , .86 4 , . 00 + 0, .57 C y s t e i n e * * 2 .06 + 0, .10 1, .24 + 0 , .07 * S t a n d a r d d e v i a t i o n ** D e t e r m i n e d a c c o r d i n g t o i t h e method d e s c r i b e d i n C h a p t e r 1, (page 2 3 ) . C o n d i t i o n o f H y d r o l y s i s : 6NHC1 24, 48 and 72 h r s a t 110°C. t -[ f l t i l w c 1 PRESENCE OF 2-MERCAPTOETHANOL 4 B-K-w c 2 3 ABSENCE OF 2 -MERCAPTOETHAMOL F i g , A B P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m s o f f r a c t i o n s o b t a i n e d from F i g . 4 C , W and C r e p r e s e n t s whole c a s e i n and u n m o d i f i e d K-casein r e s p e c t i v e l y . A - The g e l i n the pr e s e n c e o f 2-mercaptoethanol B - The g e l i n the absence o f 2-mercaptoethanol. 50 . 20,000 d a l t o n s . H o w e v e r , t h e i n c r e a s e i n a l a n i n e w a s o n l y s l i g h t a n d n o s i g n i f i c a n t c h a n g e s i n o t h e r a m i n o a c i d s w e r e o b s e r v e d . A c t i o n o f R e n n i n T h e a c t i o n o f r e n n i n o n K - c a s e i n f o r m s a p r e c i p i t a t e , p a r a K - c a s e i n , a n d a n o n d i a l y z a b l e g l y c o m a c r o p e p t i d e w h i c h r e m a i n s i n s o l u t i o n ( 1 3 1 ) . i t h a s b e e n r e p o r t e d t h a t t h e a c t i o n o f r e n n i n o n d i s u l f i d e r e d u c e d K - c a s e i n r e s u l t s i n t h e a p p e a r a n c e o f t w o p a r a - K - c a s e i n d e r i v a t i v e s , a m a j o r a n d a m i n o r b a n d ( s e ) . T h i s c a n b e s e e n i n F i g u r e 6 f o r b o t h t h e c o n t r o l (A) a n d t h e a l k y l a t e d K - c a s e i n s (B) a s w e l l a s t h e d e s u l f u r -i z e d K - c a s e i n s (c j Vrennin ' t r e a t e d f o r 5 a n d 30 m i n u t e s . T h e r a t e o f r e a c t i o n a p p e a r s t o b e s l o w e r f o r t h e d e s u l f u r i z e d K-c a s e i n l e a v i n g u n r e a c t e d b a n d s o n t h e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m b y 5 m i n r e n n i n r e a c t i o n . E f f e c t o f H e a t i n g o n K - c a s e i n I t h a s b e e n s h o w n t h a t h e a t i n g s o l u t i o n s o f K - c a s e i n d i d n o t a f f e c t t h e a b i l i t y t o p r o t e c t a ^ - c a s e i n f r o m p r e c i p i t a t i o n b y c a l c i u m u n t i l s a l t (0.05 M) w a s i n c l u d e d i n t h e s y s t e m C 1 5 2 ) . H o w e v e r , t h e s e d i m e n t a t i o n p a t t e r n o f h e a t e d K - c a s e i n (60^100°C f o r 30 m i n u t e s ) i n d i c a t e d t h e a p p e a r a n c e o f a s m a l l f a s t m o v i n g p e a k a f t e r 70°C h e a t t r e a t m e n t o r a b o v e i n a d d i t i o n t o t h e o r i g i n a l p e a k . T h e m i g r a t i o n o f t h i s p e a k w a s f a s t e r a t h i g h e r t e m p e r a t u r e s ( F i g u r e 7). A l m o s t n o d e t e c t a b l e c h a n g e i n t h e s i z e o f t h i s K - C A S A B C A B C A B C TIME(min.) o 5 30 E f f e c t o f r e n n i n o n t h e p o l y a c r y l a m i d 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 K - c a s e i n ( A ) , a l k y l a t e d K - c a s e i n ( B ) a n d d e s u l -f u r i z e d K - c a s e i n (C) a f t e r 0 , 5 a n d 30 m i n u t e s r e a c t i o n t i m e . 52. peak was observed. Z i t t l e (151*) and McKenzie ( 6 3 ) also observed a small aggregated peak upon heating K-casein solution to 90°C for 15 minutes or 74°C for 6 hours respec-t i v e l y . The nature of t h i s association i s not c l e a r . The e f f e c t of b o i l i n g temperature on K-casein and desulfurized K-casein i s shown i n Figures 8 and 9 respectively. I t i s clear that heat treatment decreased by 40r50% of the s t a b i l i z i n g a b i l i t y of desulfurized K-casein. The same was true for the alkylated K-casein (Figure 10). However, almost no s i g n i f i c a n t change i n the s t a b i l i z i n g a b i l i t y of unmodified K-casein was observed upon heating (Figure 8). Decrease i n the a b i l i t y of heated modified K-casein to s t a b i l i z e Ca-a^-caseinate may be caused by cross-linkings or an association of K-casein molecules, rendering t h e i r i n t e r a c t i n g s i t e with a g^-casein inaccessible. An aggregation phenomenon for those heated modified K-caseirswas observed on gel f i l t r a t i o n . Sepharose 2B which i s capable of fra c t i o n a t i n g proteins within a molecular weight range from 100,000 to 40 m i l l i o n eluted the heated and unheated unmodified K-casein at the same el u t i o n volume (Figure 11) implicating that the heat treatment did not cause any detectable changes i n the molecular size of K-casein. On the other hand, an aggregation phenomenon of the alkylated or desulfurized K-casein a f t e r heat treatment was demonstrated as peaks very close to the void volume (Figures 12 and 13). A p a r t i a l aggregation was also observed when heated desulfurized or alkylated K-casein (100°C/15 min) were eluted on Sepharose F i g . 9 . S t a b i l i z a t i o n o f a - c a s e i n b y d e s u l f u r i z e d K - c a s e i n b e f o r e a n d a f t e r h e a t i n g ( 1 0 Q ° C / 3 0 m i n u t e s ) a g a i n s t p r e c i p i t a t i o n b y C a + + , - H E A T E D A L K Y L A T E D K - C A S E I N 0.1 0-2 S t a b i l i z a t i o n o f a - ^ - c a s e i n b y a l k y l a t e d K - c a s e i n b e f o r e a n d . a f t e r h e a t i n g ( 1 0 Q ° C / 3 0 m i n u t e s ) a g a i n s t p r e c i p i t a t i o n b y C a " 1 " ^ . 0 - 4 J E c o °° 0-2 | CM I -< UJ o z < CO cc o CO CO < 0-4 0-2 10 20 30 40 FRACTION NUMBER 50 60 F i g . 1 1 . E l u t i o n p a t t e r n s o f K - c a s e i n b e f o r e h e a t i n g (A) a n d a f t e r h e a t i n g (B) ( 1 0 0 ° C / 3 Q m i n ) , o n S e p h a r o s e 2 B c o l u m n ( 4 0 X 2 . 5 cm) e l u t e d w i t h 0 . 0 5 M p h o s p h a t e b u f f e r p H 7 . 6 ^ a t 2 5 ° C , 10 20 30 40 50 60 FRACTION NUMBER F i g . 1 2 . E l u t i o n p a t t e r n s o f a l k y l a t e d K - c a s e i n b e f o r e (A) and a f t e r (B) h e a t i n g (100°C/30 min) on S e p h a r o s e 2B c o l u m n (40X2.5 cm) e l u t e d w i t h 0.05 M p h o s p h a t e b u f f e r pH 7.6 a t 25°C. CO o z < CQ CC 10 20 30 40 50 60 FRACTION NUMBER F i g . 1 3 . E l u t i o n p a t t e r n s o f d e s u l f u r i z e d K - c a s e i n b e f o r e (A) and a f t e r (B) h e a t i n g (100°C/30 min) on S e p h a r o s e 2B c o l u m n (40X2.5 cm) e l u t e d w i t h 0.05M p h o s p h a t e b u f f e r pH 7.6 a t 25°C. 60. 6B with 6.6 M urea (Figures 2 4B and 2 6B). I t i s evident therefore that the d i s u l f i d e groups i n K-casein are playing an important role i n keeping the protein molecule r e s i s t a n t to heat treatment. The electrophoretic behaviour of the modified and unmodified K-casein before and aft e r heating i s shown i n Figure 14. Both desulfurized and alkylated K-casein when heated did not resolve completely and smears near the o r i g i n were observed on polyacrylamide gel i n the presence of 4.5 M urea and 2-mercaptoethanol in d i c a t i n g a p a r t i a l aggregation. Whereas, the heated unmodified K-casein showed a single band without a smear. When those proteins were heated i n the presence of 5 M urea, the modified (alkylated and desulfurized) and unmodified K-caseins completely l o s t their s t a b i l i z i n g a b i l i t y (Figure 15), and an increase i n th e i r migration on polyacrylamide gel electrophoretogram was observed (Figure 14C). This was probably due to a reaction between the amino groups i n K-casein and the decomposition product of urea (probably cyanate). Modification of e-amino groups according to fluorescence studies ( 1 8 ) led to decreased a b i l i t y to s t a b i l i z e a -casein. These r e s u l t s suggest that the aggrega-s l t i on i s not due to hydrophobic or hydrogen bonding but may be due to a cross - l i n k i n g of the molecules. The fact that residual s t a b i l i z i n g power remains aft e r heating the alkylated K-casein suggests that the aggregates themselves may have some a b i l i t y to s t a b i l i z e a s l ~ c a s e i n . The d i s u l f i d e 2 3 1 2 3 2 3 B C F i g . 1 4 . E f f e c t o f h e a t i n g (100 C/30 min) on t h e p o l y a c r y l a m i d 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 o f K - c a s e i n ( 1 ) , a l k y l a t e d K - c a s e i n (2) and d e s u l f u r i z e d K - c a s e i n ( 3 ) . A - u n h e a t e d K - c a s e i n s o l u t i o n s B - h e a t e d K - c a s e i n s o l u t i o n s i n d i s t i l l e d w a t e r pH 7 . 0 C - h e a t e d K - c a s e i n s o l u t i o n s i n t h e p r e s e n c e o f 5 M u r e a . K-CASEIN DESULFURISED ALKYLATED S t a b i l i z a t i o n o f a ^ - c a s e i n by h e a t e d K-casein (•-•-•) d e s u l f u r i z e d K-casein (©-«-©) and a l k y l a t e d K-casein ( «-»-«) i n t h e p r e s e n c e o f 5 M u r e a . groups therefore are playing an important role i n maintaining a s t r u c t u r a l i n t e g r i t y which controls heat s t a b i l i t y of the molecule. Changes i n Fluorescence P o l a r i z a t i o n of K-casein due to  SS Modification and Heat Treatment Fluorescence p o l a r i z a t i o n of a macromolecule i s mainly dependent on the geometry and r i g i d i t y of the molecular structure. Changes i n molecular conformation due to aggrega-ti o n and i n t e r a c t i o n would therefore r e s u l t i n changes i n p o l a r i z a t i o n , because these changes modify the molecular geometry and volume i n the molecule ( 2 9, 4 9 , 1 1 3 , 1 3 8 , 1 3 9 ) . The p o l a r i z a t i o n technique has been used to study the i n t e r -action between a ^- and K-casein by l a b e l l i n g the former protein with dansyl chloride and following the changes i n p o l a r i z a t i o n . An experiment was conducted to investigate the changes i n the i n t e r a c t i o n between the dansylated a sj_-casein and K-casein upon de s u l f u r i z a t i o n or a l k y l a t i o n of K-casein and the e f f e c t of heating on the i n t e r a c t i o n . Modification of the procedure of Rawitch and Weber ( 9 7) for c a l c u l a t i o n of association constant (ka) of the reaction A + B + C made by Nakai et a l . ( 7 3 )was used. 1. Substituting the following formula with an assumed value for P^, the observed P^ at a r a t i o (B0)/(A0) , the r a t i o of fluorescence y i e l d s R and the p o l a r i z a t i o n of DNS-labelled protein P f, K was calculated. (A. - A f ) [ ( A b - A i ) R + A i - A f ] 6 4 [1] K . = [2] ( 1 - B ) ( [ B Q ] - [ A Q ] B i ) w h e r e K . = a s s o c i a t i o n c o n s t a n t f o r t h e r e a c t i o n 1 A + B t C A = f l u o r e s c e n c e a n i s o t r o p y . A _ 1 (1 I ) " 1  A = 3 - 3 ; 8 = f r a c t i o n o f l a b e l l e d p r o t e i n i n t e r a c t e d [ A ] , [ B Q ] = t h e c o n s t i t u e n t m o l a r c o n c e n t r a t i o n o f ° p r o t e i n A a n d B R = i s t h e q u a n t u m y i e l d r a t i o ' q ^ / q f ^ a n < ^ i s c a l c u l a t e d a s a n a s y m p t o t e R o f Y = R + a b x i u s i n g s i x R ^ a t s i x r a t i o s o f t h e s a m e i n t e r v a l . R . = ^ 1 1 + 2 J l ) b ( I u + 2 I 1 ) f ' W h e r e 1^^ a n d 1^ a r e t h e p o l a r i z e d f l u o r e s c e n c e i n t e n s i t i e s a t p a r a l l e l a n d v e r t i c a l c o m b i n a t i o n s o f p o l a r i z e r s . P. = ( I n - V b ( I 1 1 + V f 2. T h e c o e f f i c i e n t o f v a r i a t i o n ( C V ) o f K i m e a s u r e d a t t h e d i f f e r e n t [ B Q 1 / [ A ] a n d t h e r e g r e s s i o n c o e f f i c i e n t b o f Kj_ = a + b ( [ B 0 ] / [ A o ] ) i w e r e c a l c u l a t e d . 3 . D i f f e r e n t w e r e a p p l i e d i n s t e p 1 f o r c a l c u l a t i o n o f t h e a v e r a g e K u n t i l C V b e c o m e s m i n i m u m a n d b a p p r o a c h e s z e r o . F i g u r e s 1 6 , 1 7 , a n d 1 8 s h o w t h e c h a n g e s i n t h e p o l a r i z a t i o n o f d a n s y l a t e d a s ^ - c a s e i n u p o n t i t r a t i o n w i t h h e a t e d ( A ) a n d u n h e a t e d (B) K - c a s e i n , d e s u l f u r i z e d K - c a s e i n a n d a l k y l a t e d K - c a s e i n r e s p e c t i v e l y . H e a t i n g K - c a s e i n ( 1 0 0 ° C / 3 0 m i n ) d i d n o t c a u s e a n y s i g n i f i c a n t c h a n g e s i n p o l a r i z a t i o n , t h u s , t h e c a l c u l a t e d a s s o c i a t i o n c o n s t a n t ( K a - v a l u e ) o f t h e a , - K - c a s e i n i n t e r a c t i o n w a s a l m o s t t h e s l s a m e ( T a b l e V). A s l i g h t i n c r e a s e i n K a - v a l u e w a s o b s e r v e d w h e n t h e S S g r o u p s i n K - c a s e i n w e r e a l k y l a t e d w i t h i o d o -a c e t a m i d e a n d a s i g n i f i c a n t d e c r e a s e i n p o l a r i z a t i o n a n d a s s o c i a t i o n c o n s t a n t w a s d e t e c t e d u p o n h e a t i n g t h e a l k y l a t e d K - c a s e i n ( F i g u r e 1 8 , T a b l e v)• T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e r e s u l t s o f s t a b i l i t y t e s t i n t h e p r e s e n c e o f C a + + ( F i g u r e s 8 a n d 1 0 ) . A s l i g h t d e c r e a s e i n t h e p o l a r i z a t i o n o f d e s u l f u r i z e d K - c a s e i n w a s a l s o o b s e r v e d o n h e a t i n g ( F i g u r e 1 7 ) . H o w e v e r , c a l c u l a t i o n o f t h e a s s o c i a t i o n c o n s t a n t f o r b o t h h e a t e d a n d u n h e a t e d d e s u l f u r i z e d K - c a s e i n w a s n o t p o s s i b l e a c c o r d i n g t o t h e m e t h o d o u t l i n e d b e c a u s e o f t h e m a r k e d i n c r e a s e i n R - v a l u e u p o n d e s u l f u r i z a t i o n o f K - c a s e i n , s i n c e K a i s i n v e r s e l y p r o p o r t i o n a l t o R , ( e q u a t i o n 1 a n d 2 ) . T h e R - v a l u e r e p r e s e n t s t h e r a t i o o f f l u o r e s c e n t i n t e n s i t y o f t h e i n t e r a c t i o n p r o d u c t o f a - K - c a s e i n c o m p l e x t o t h e r e m a i n i n g c o n j u g a t e d a s ^ - c a s e i n w i t h f l u o r e s c e n t d y e . 16 5 10 15 K-'*s_ CASEIN F i g . 1 6 . P o l a r i z a t i o n o f d a n s y l a t e d a s ^ - c a s e i n t i t r a t e d w i t h K - c a s e i n («>^©-©) and h e a t e d ( 1 0 0 ° C / 3 0 m i n u t e s ) K - c a s e i n ( A - A - i . ) . <7l 16 • 5 10 15 *-/x s_ CASE IN F i g . 17. P o l a r i z a t i o n o f d a n s y l a t e d a ^ - c a s e i n t i t r a t e d w i t h d e s u l f u r i z e d K - c a s e i n («-«>-©) and h e a t e d (100°C/30 m i n u t e s ) d e s u l f u r i z e d K - c a s e i n ( A - A - A ) . CASEIN 1 5 F i g . 18. P o l a r i z a t i o n of dansylated a ^-casein t i t r a t e d with alkylated K-casein (•-•-•) and heated (100°C/30 minutes) alkylated K-casein {k-k-A) . CO TABLE V E f f e c t of heat on the p o l a r i z a t i o n and a s s o c i a t i o n constant (ka) of K - c a s e i n and a l k y l a t e d K - c a s e i n . K - c a s e i n ( c o n t r o l Pb Ka CV% 69 R Unheated 0.288 4.711X10 21.3 +0.13 2.32 Heated 0.294 3.697X10 20.5 +0.001 2.92 A l k y l a t e d K - c a s e i n Unheated 0.289 8.8X10 30.5 -0.036 1.71 Heated 0.267 3 . 9 X 1 0 3 3 . 6 - 0 . 0 2 5 1.82 pb = p o l a r i z a t i o n Ka = a s s o c i a t i o n c o n s t a n t CV% = c o e f f i c i e n t of v a r i a t i o n b = r e g r e s s i o n c o e f f i c i e n t R = the quantum y i e l d r a t i o . 70 . B e c a u s e o f t h e s h o r t l i f e t i m e o f t h e e x c i t e d s t a t e o f p r o t e i n s (1-5 n s e c . ) ( i s ) , K - c a s e i n s h o u l d h a v e a v e r y l o w f l u o r e s c e n t i n t e n s i t y i n t h e a b s e n c e o f f l u o r e s c e n t d y e . T h i s i s s h o w n i n F i g u r e 19. H o w e v e r , t h e d e s u l f u r i z e d K - c a s e i n s h o w e d a r e m a r k a b l y h i g h f l u o r e s c e n t i n t e n s i t y ( a b o u t 6 f o l d ) . A r e l a t i o n s h i p b e t w e e n t h e p o l a r i t y o f t h e e n v i r o n m e n t a n d t h e f l u o r e s c e n t y i e l d h a s b e e n d e m o n s t r a t e d b y S t r y e r ( i i •*) . T h e v a r i a t i o n s i n f l u o r s c e n t y i e l d c a n , t h e r e f o r e , g i v e a n i n d i c a -t i o n o f c o n f o r m a t i o n a l c h a n g e s , a g g r e g a t i o n , d i s a g g r e g a t i o n , s i z e a n d r i g i d i t y o f t h e m o l e c u l e . H i g h e r f l u o r e s c e n t i n t e n s i t y o n d e s u l f u r i z a t i o n m a y i n d i c a t e a n i n c r e a s e i n t h e r i g i d i t y o f t h e m o l e c u l e s a n d a m o r e c o m p a c t s t r u c t u r e m a y r e s u l t o n d e s u l f u r i z a t i o n d u e t o a p o s s i b l e i n c r e a s e i n t h e h y d r o p h o b i c i t y o f t h e m o l e c u l e ( c o n v e r t i n g a c y s t e i n e r e s i d u e t o a l a n i n e r e s i d u e o n d e s u l f u r i z a t i o n ) . I n t e r a c t i o n o f K - c a s e i n a n d B - l a c t o g l o b u l i n I n 1952, T o b i a s e t a _ l . ( 1 2 3 ) r e p o r t e d t h e p r e s e n c e o f a c o m p l e x b e t w e e n 8 - l a c t o g l o b u l i n a n d a - c a s e i n i n s k i m -m i l k h e a t e d f o r a s h o r t p e r i o d o f t i m e a t 3 0 0 ° F . S i n c e t h e n a l a r g e b o d y o f d a t a h a s b e e n a c c u m u l a t e d s u g g e s t i n g t h a t a c o m p l e x i s f o r m e d b e t w e e n B - l a c t o g l o b u l i n a n d K - c a s e i n . When . < - c a s e i n ' w a s h e a t e d w i t h 8 - l a c t o g l o b u l i n , a s e d i m e n t a t i o n v a l u e o f 45 S w a s o b s e r v e d f o r a c o m p l e x f o r m e d b e t w e e n t h e t w o p r o t e i n s ('is 4 ) . T h e s t a b i l i z i n g a b i l i t y o f 300 400 500 600 E M I S S I O N W A V E L E N G T H nm F i g . 19. E f f e c t o f d e s u l f u r i z a t i o n o f K - c a s e i n on i t s f l u o r e s c e n t i n t e n s i t y . A = D e s u l f u r i z e d K - c a s e i n B = C o n t r o l K - c a s e i n . i—• complexed K-casein for a ^ - c a s e i n against p r e c i p i t a t i o n by C a + + was considerably reduced. I t has been suggested that the complex formation takes place through the formation of intermolecular d i s u l f i d e bonds (15k ) . Most attempts to demonstrate the presence of B-lactoglobulin - K-casein complex i n heated skimmilk have r e l i e d on electrophoretic evidence ( 10 6) . Sepharose 6B gel chromatography i n addition to polyacrylamide gel electrophoresis was used i n thi s study to investigate the role of d i s u l f i d e groups i n K-casein on the complex formation of those two proteins. When skimmilk was applied to a column of Sepharose 6B and eluted with a buffer containing 6.6 M urea, K-casein was almost t o t a l l y excluded from the gel matrix and eluted very close to the void volume and a shoulder was observed af t e r the a s ~ 3-casein peak representing the whey proteins (Figure 20(A), and Figures 40 and 41 i n Chapter I I I ) . The size of K-casein peak increased remarkably when heated skim-milk (100"C/30 min) was applied to the gel under the same condition (Figure 20(B)). Polyacrylamide gel electrophoreto-gram r e s u l t s indicate that the constituent of t h i s peak was (3-lactoglobulin as well as K-casein (Figure 21, s l o t 2). This would indicate an in t e r a c t i o n between B-lactoglobulin and K-casein or an aggregation of B-lactoglobulin by i t s e l f which was not dissociated i n the presence of 6.6 M urea. Elu t i n g heated skimmilk with buffer containing 2-mercapto-ethanol would disrupt the B-lactoglobulin - K-casein complex 7 3 . FRACTION NUMBER F i g . 20. E f f e c t o f b o i l i n g t e m p e r a t u r e (30 m i n u t e s ) on the e l u t i o n p a t t e r n o f s k i m m i l k (150 mg), e l u t e d w i t h 0.02 M p h o s p h a t e b u f f e r c o n t a i n i n g 6.6 M u r e a , pH 7.6. A - S k i m m i l k ( u n h e a t e d ) B - S k i m m i l k ( h e a t e d ) C - S k i m m i l k ( h e a t e d ) , e l u t e d w i t h t h e a b o v e b u f f e r c o n t a i n i n g 0.3% m e r c a p t o e t h a n o l . S i z e o f c o l u m n = 36 X 2 cm, e a c h f r a c t i o n c o n t a i n s 1.6 m l e l u e n t . w = w h o l e c a s e i n . and i n i t i a t e the d i s s o c i a t i o n of each protein aggregate (and t h e i r i n t e r a c t i o n product) i f i t was caused by the d i -s u l f i d e interchange reaction. As B-lactoglobulin was the only component of the peak at the void volume (Figure 20(c)) when 2-mercaptoethanol was introduced to the e l u t i o n buffer (Figure 21, s l o t 3), th i s peak could be a product of thermo-denaturation of B-lactoglobulin ( 1 0 6 ) . On heating B-lactoglobulin solution alone, an aggregated peak was observed at the void volume (Figure 22(c)) i n addition to the o r i g i n a l peak of B-lactoglobulin. On the other hand, the o r i g i n a l peak of B-lactoglobulin disappeared completely when a mixture of K-casein and B-lactoglobulin were heated, and a single peak with a shoulder at larger mol wt size was observed at the void volume (Figure 22(D) and Figure 23). This implicates that an i n t e r -action occurred between the heat undenatured B-lactoglobulin (second peak on Figure 22(c)) and K-casein on heating. The di s s o c i a t i o n of the complex to i t s o r i g i n a l constituent was observed (Figure 22(E) and Figure 23, s l o t s 6 and 7) when 2-mercaptoethanol was introduced into the el u t i o n buffer suggesting that the complex was the r e s u l t of intermolecular d i s u l f i d e bonding. E l u t i o n patterns of heated desulfurized or alkylated K-casein i n the presence or absence of B-lactoglobulin are shown i n Figures 24 and 26. Modified K-casein was p a r t i a l l y aggregated on heating i n the absence of B-lactoglobulin 76 20 40 60 FRACTION NUMBER i11 . F i g . 2 2 . E f f e c t o f b o i l i n g t e m p e r a t u r e (10 m i n u t e s ) on t h e e l u t i o n p a t t e r n o f B - l a c t o g l o b u l i n and a m i x t u r e o f B - l a c t o g l o b u l i n and K - c a s e i n , e l u t e d w i t h 6.6 M u r e a i n p h o s p h a t e b u f f e r 0.02 M, pH 7.6. A - 1 m l o f 12.0 mg/ml K - c a s e i n ( n o t h e a t e d ) B - 1 m l o f 17 mg/ml B - l a c t o g l o b u l i n ( n o t h e a t e d ) C - 1 m l o f 17 mg/ml B - l a c t o g l o b u l i n ( h e a t e d ) D - m i x t u r e o f B - l a c t o g l o b u l i n and K - c a s e i n ( h e a t e d ) E - m i x t u r e o f B - l a c t o g l o b u l i n and K - c a s e i n ( h e a t e d ) e l u t e d w i t h t h e ab o v e b u f f e r c o n t a i n i n g 0.3%'; m e r c a p t o e t h a n o l . S i z e o f c o l u m n = -• (36 X 2 cm) E a c h f r a c t i o n c o n t a i n s 1.6 m l e l u e n t . 1 2 3 4 5 6 7 F i g . 2 3 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 2 2 A (1) , 22 B ( 2 ) , 22 C (3 and 4 ) , 22 D (5) a n d 22 E ( 6 and 7 ) . 78. A F R A C T I O N N U M B E R F i g . 24. E f f e c t of b o i l i n g temperature (10 minutes) on the elution pattern of alkylated K-casein (10 mg/ml) eluted with 0.02 M phosphate buffer containing 6.6 M urea pH 7.6. A - unheated alkylated K-casein B - heated alkylated K-casein C - heated mixture of alkylated K-casein and B-lactoglobulin Size.. tof column - 36 X 2 cm, each f r a c t i o n contains 1.6 ml eluent. F i g . 2 5 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 2 4 A ( 1 ) , 24 B (2 a n d 3) a n d 24 C (4 a n d 5 ) . 8 0 . F R A C T I O N N U M B E R F i g . 2 6 . E f f e c t o f b o i l i n g t e m p e r a t u r e ( 1 0 m i n u t e s ) o n t h e e l u t i o n p a t t e r n o f d e s u l f u r i z e d K - c a s e i n ( 1 0 m g / m l ) e l u t e d w i t h 0 . 0 2 M p h o s p h a t e b u f f e r , c o n t a i n i n g 6 . 6 M u r e a p H 7 . 6 . A - D e s u l f u r i z e d K - c a s e i n B - H e a t e d d e s u l f u r i z e d K - c a s e i n C - H e a t e d m i x t u r e o f d e s u l f u r i z e d K - c a s e i n a n d 8 - l a c t o g l o b u l i n ( 1 m l e a c h ) S i z e " o f . c o l u m n = 3 6 X 2 c m E a c h f r a c t i o n c o n t a i n s 1 . 6 m l e l u e n t . F i g . 2 7 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 2 6 A ( 1 ) , 26 B (2 a n d 3) a n d 26 C (4 •*• 7 ) . (Figure 24(B) and 2 6 ( B ) ) . Those aggregated peaks were not resolved f u l l y on polyacrylamide gel electrophoretogram (Figure 25, s l o t 2 and 27, s l o t 2 ) . No in t e r a c t i o n was observed between (3-lactoglobulin and the modified K-caseins when a mixture of any one of the modified K-casein was heated with 8-lactoglobulin, since the peak at the void volume for the heated mixture of the two proteins (3-lacto-globulin and modified K-casein) contained only denatured 3-lactoglobulin, and the modified K-casein was eluted at i t s o r i g i n a l p o s i t i o n (Figure 2 4 ( C ) , 2 6 ( C ) , Figure 25, s l o t s 4 and 5 and Figure 27, s l o t s 4->7) . &L<L CHAPTER III The Use of the Gel Chromatographic Technique i n the I s o l a t i o n and Characterization of K-and a^-Caseins. 83. INTRODUCTION Recent advanced techniques i n protein chemistry have enabled us to carry out a more det a i l e d study of the physico-chemical and b i o l o g i c a l properties of milk proteins. The discovery of K-casein i n 1956 brought about great progress i n the understanding of micelle structure. Since then, a great deal of i n t e r e s t has been shown i n the i s o l a t i o n and p u r i f i c a t i o n of t h i s p a r t i c u l a r protein i n the more natural state allowing greater study of the functional c h a r a c t e r i s t i c s of K-casein i n the casein micelle. A number of methods have been proposed for the f r a c t i o n a t i o n of the casein complex i n milk. Many reports have appeared giving methods for obtaining K-casein free from other components of whole casein. Generally most methods involve one of the following two approaches: a) An i n i t i a l step i s used to separate ct s- and K-casein; for example, a treatment with a concentrated solution of calcium s a l t s , followed by further p u r i f i c a t i o n of material corresponding to the f r a c t i o n S of Waugh and von Hippel ( 1 3 7) . b) a-casein i s f i r s t i s o l a t e d and K-casein i s then separated from a -casein, s l The most widely u t i l i z e d methods are the ones of Z i t t l e and Custer (*53) and of McKenzie and Wake ( 6 7 ) . Both methods produce nearly pure K-casein of' high yields..' 84 . The important step i n Z i t t l e ' s method i s a strong acid t r e a t -ment at a high urea concentration. An alcohol f r a c t i o n a t i o n i s used i n both McKenzie's and Z i t t l e ' s methods for further p u r i f i c a t i o n . Nakanishi and Ito (s3) reported that excessive mixing or agi t a t i o n during the p u r i f i c a t i o n of K-casein with alcohol and ammonium acetate used i n Z i t t l e ' s method ( 1 5 3 ) resulted i n gelation of K-casein. This i s probably due to the oxidation of sulfhydryl groups. Repeated p u r i f i c a t i o n s increased the degradation of casein and produced bands s i m i l a r to para K-casein on the gel electrophoretogram. Milder con-d i t i o n s , therefore, for the preparation of K-casein are required. Gel f i l t r a t i o n introduced i n 1959 has been increas-ingly used for both a n a l y t i c a l and preparative work and for chemical processings. Using the appropriate type and grade of Sephadex (a highly hydrated polymeric carbohydrate material), separation of molecules according to siz e can be performed rapidly and simply. Under certa i n conditions, K-casein remains as r e l a t i v e l y large aggregates caused by intermolecular d i s u l f i d e bonding, while other caseins are converted into t h e i r monomeric forms. Relatively pure K-casein has been obtained at the void volume when whole casein or skimmilk was applied to Sephadex G-100 at pH 10.8 ( 7 5 ) . K-casein was also prepared using Sephadex gel chromatography when whole casein was eluted with buffer containing 85. d i s s o c i a t i n g agents such, as SDS (.7 2 ) or urea (.ll*7). These observations suggest the possible u t i l i z a t i o n of Sepharose gel for the preparation of K-casein from whole casein or skimmilk at lower pH values or urea concentrations. The Sepharose types are, by- vi r t u e of the i r greater porosity, an important complement to the available series of Sephadex types. Whereas Sephadex allows the fr a c t i o n a t i o n of spherical molecules such as globular proteins having molecular weights of up to 800,000, or of randomly c o i l e d polymers such as dextran with molecular weights of up to 20 0,000, Sepharose may be used to separate molecules or p a r t i c l e s with molecular weights of up to several m i l l i o n s . The application of Sepharose 6B for the preparation of K-casein d i r e c t l y from skimmilk or whole caseins i s reported i n thi s chapter. I t was observed i n our laboratory ( 1 2 O that the a ^ - c a s e i n f r a c t i o n always accompanied both the K-casein and the a ,-casein fractions when casein solution was eluted with s l phosphate buffer at pH 10.8 on a Sephadex G-100 column. A d i s t i n c t peak for a -casein was also obtained when whole s5 casein or skimmilk was eluted on Sepharose 6B with a buffer containing 6.6 M urea. We then decided to investigate some of the main c h a r a c t e r i s t i c s of thi s f r a c t i o n . Physical and chemical properties of the minor proteins i n the a g-casein complex have been the subject of recent studies ( 3, . Hoagland et a^. ( 39 ) suggested that ct^-casein i s composed 86 . of equal numbers of molecules of a and a .-caseins, linked by d i s u l f i d e bonds, with a tentative molecular weight of 67,500 for a S5~casein and 33,700 for each of c t ^ - and c t ^ -caseins. No data for the molecular weight determined by physical methods has been reported for these proteins. The results of studies on the calcium s e n s i t i v i t y and the molecular weight of these caseins as determined by the d i f f e r e n t i a l boundary method i n an ultracentrifuge are reported i n t h i s chapter. 87 . METHODS AND MATERIALS Preparation of whole casein Whole casein was prepared from milk of a single Ayrshire cow from the University herd after centrifuging the fat at 4°C. Acid casein was then prepared by adding hydro-c h l o r i c acid (IN) at 20°C with mechanical s t i r r i n g over a period of 40-45 minutes u n t i l the pH value reached 4.5-4.6. S t i r r i n g was continued for a further 30 minutes and the pre c i p i t a t e was sedimented at 1300 X g for 15 minutes. The sediment was washed twice by suspending i t i n water and recentrifugiijg as previously. The pr e c i p i t a t e was then dissolved i n water by t i t r a t i n g with 1 N NaOH to pH 7.0. After the pr e c i p i t a t e dissolved completely, the solution was freeze-dried and stored at 4°C. Preparation of K - and a s^-casins K — and c< s^-caseins were prepared from whole casein by a procedure of Z i t t l e and Custer Q- 5 3) • a g^-casein was prepared by the sulphoethyl Sephadex C-50 procedure of Annan and Manson ( 3 ). Protein concentrations were calculated 1% using the absorbance at 280 nm using A of 10.7 and 10.1 lcm ( 3 ) for a - and a ^-casein respectively. The s o l u b i l i t y of a -caseins i n CaCl was determined according to a method s 2 outlined by Beveridge and Nakai ( 7 ) . A procedure described e a r l i e r (Chapter II) was used to determine s t a b i l i z a t i o n of ct c l -casein by K-casein i n the presence of CaC^. 8 8 . S e p h a r o s e g e l f i l t r a t i o n S e p h a r o s e 6 B p u r c h a s e d f r o m P h a r m a c i a F i n e C h e m i c a l s I n c . w a s e q u i l i b r a t e d i n 0 . 0 5 M p h o s p h a t e b u f f e r p H 7 . 6 c o n t a i n i n g 0 . 0 2 % s o d i u m a z i d e a s p r e s e r v a t i v e a n d w i t h ( p u b u f f e r ) a n d w i t h o u t 6 . 6 . M u r e a (P b u f f e r ) . A s l u r r y o f t h e e q u i l i b r a t e d S e p h a r o s e w a s p a c k e d i n t o a l a b o r a t o r y c o l u m n ( K 2 4 / 4 5 P h a r m a c i a , U p p s a l a , S w e d e n ) a s d e s c r i b e d b y P h a r m a c i a F i n e C h e m i c a l s I n c . ( 1 1 0 ) . T w o m i l l i l i t e r s o f c a s e i n s o l u t i o n ( 5 0 - 1 0 0 m g / m l b u f f e r ) w e r e a p p l i e d t o t h e c o l u m n a n d e l u t e d w i t h p u o r P b u f f e r s u n d e r a p r e s s u r e h e a d o f a b o u t 4 0 c m . T w o h u n d r e d a n d s i x t y m i l l i l i t e r s o f e l u e n t w e r e c o l l e c t e d i n 2 m l f r a c t i o n s . T h e o p t i c a l d e n s i t y o f t h e e f f l u e n t s w a s m o n i t o r e d a t 2 8 0 nm w i t h I S C O c o l u m n m o n i t o r o r B e c k m a n D B s p e c t r o -p h o t o m e t e r . F r a c t i o n s c o n t a i n i n g p r o t e i n s o l u t i o n w e r e c o l l e c t e d , d i a l y z e d a g a i n s t t a p w a t e r a t 4 ° C f o r 4 8 h o u r s a n d t h e n f r e e z e - d r i e d . A S p i n c o L 2 - 6 5 B u l t r a c e n t r i f u g e , e q u i p p e d w i t h t h e s c h l i e r e n o p t i c s w a s u s e d t o d e t e r m i n e m o l w t b y t h e d i f f e r e n t i a l b o u n d a r y m e t h o d o f C h e r n y a k e t al_. ( 1 1 8 ) . T h e f o l l o w i n g f o r m w a s d e r i v e d f o l l o w i n g C h e r n y a k ' s p r o c e d u r e : M m ( i-vp) w 2 r . RT 1 m ( h - l ) r * E r r y . A x 0 , D 1 P i 8 9 . where M = the molecular weight calculated at the m . ^ meniscus R = the gas constant T = the absolute temperature v = the p a r t i a l s p e c i f i c volume p = the density. The values for aqueous solutions of urea ( 2 8 ) were used co = the angular v e l o c i t y h = the s t a r t i n g concentration r a t i o of two segments of the sample protein ( C Q ^ / Q 1 > 1^ For the distances on the magnified schlieren patterns r ,r , = the coordinates of the meniscus and the m 0 D s t a r t i n g d i f f e r e n t i a l boundary 2 ' r 2y.Ax.: the trapezoidal approximation of r 2yAx x pz i J 1 1 x , ^ for the peak between the plateaus p i and p2 corresponding to the concentrations C , and C , °- 1 o. 2 y : the height; Ax: the scan spacing x 1 r?y^Ax^: the trapezoidal approximation of r 2yAx x for the peak between the meniscus and the m c plateau p i , F^ : the magnification factor of the o p t i c a l system i n the r a d i a l d i r e c t i o n . A programmable calculator, Monroe 1670, was used for calcula-t i o n . Gel electrophoresis was performed as described previously (Chapter I I ) . 90 RESULTS AND DISCUSSION SECTION 1 The separation of K-casein on Sepharose 6B A. The e f f e c t of i o n i c strength on the el u t i o n pattern: Preliminary tests for determining the e f f e c t of i o n i c strength on the resolution were performed on a 2 X 37 cm column eluted with P buffer using a water jacket for c o n t r o l l i n g temperature. Since the i n t e r a c t i o n between K - and a -casein decreases as the temperature s l c decreases ( I 3 ) 4°C was chosen as the e l u t i o n temperature. The e f f e c t of d i f f e r e n t i o n i c strengths on the e l u t i o n pattern i s shown i n Figure 28. Peak I i n Figure 28 i s the i n t e r a c t i o n product of K , 3 and a g^-casein, and the constituents of t h i s peak are shown i n Figure 29, slo t s 1 4. A decrease i n the size of t h i s peak would indicate d i s s o c i a t i o n of the i n t e r a c t i o n product of the casein component thus increasing the i n t e n s i t y of K-casein content of peak I. Increasing the concentration of phosphate buffer from 0.05 to 0.2 M d i d not cause any detectable changes i n the s i z e of peak I (Figure 28(B)) neither d i d i t change the i n t e n s i t y of K-casein i n t h i s peak (Figure 30, s l o t 1 -»- 3B) . However, decreasing the phosphate concentration to 0-005 M gave a s i g n i f i c a n t decrease i n the size of peak I and an increase i n peak 2 (Figure 28(C)). The i n t e n s i t y of K-casein i n t h i s peak was increased (Figure 30, s l o t s I C ) . Addition of 0.4 M 91. F R A C T I O N N U M B E R F i g . 28. E f f e c t o f i o n i c s t r e n g t h on t h e e l u t i o n p a t t e r n o f w h o l e c a s e i n o n S e p h a r o s e 6B c o l u m n (37 X 2 cm) e l u t e d w i t h p h o s p h a t e b u f f e r pH 7.6 and 4°C. A = 0.05 M p h o s p h a t e b u f f e r B = 0.2 M p h o s p h a t e b u f f e r C = 0.005M p h o s p h a t e b u f f e r D = 0.05 M p h o s p h a t e b u f f e r + 0.4 M N a C l . W 1 2 3 4 5 6 7 8 F i g . 2 9 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 2 8 A. W = w h o l e c a s e i n . 2 3 1 2 3 1 2 1 2 B C D E Polyacrylamide gel electrophoretogram of fractions obtained from F i g . 28 B (1 -»• 3B) , 28 C (1 - 3C) 28 D (1 and 2D) and from Fig.31 B (1 and 2E). W = whole casein. L O NaCl to the phosphate buffer (0.05 M) also caused a similar d i s s o c i a t i o n e f f e c t on the composition of peak 1 (Figure 28 D, and sl o t s 1, 2 D of Figure 30). Increase i n io n i c strength decreased the association constant (Ka) of a s ^ - K - c a s e i n i n t e r a c t i o n (7 3 ). However, concentration of the phospahte ions seems to be playing an important role i n separation of the complex. E f f e c t of pH and temperature: Results of fluorescence p o l a r i z a t i o n have indicated a decrease i n the d i s s o c i a t i o n constant of a n - K - c a s e i n s i i n t e r a c t i o n with increasing pH or decreasing temperatures (Nakai and Kason ( 73 ) ) . However, when the pH of the elutio n buffer i n the Sepharose gel f i l t r a t i o n was increased from 7.6 to 9.0, neither change i n the size of peak 1 (Figure 31, A and B) nor change i n the i n t e n s i t y of K-casein band on electrophoretogram was observed (Figure 30, s l o t s 1 and 2 E, and Figure 33, s l o t s 1 -> 4) . However, increasing the temperature from 4°C to 25°C improved the separation of K-casein from other caseins. As shown in Figure 31 C and D the size of the f i r s t peak remarkably decreased by increasing the temperature from 4 to 25°C. The electrophoretic patterns of peak 1 i n these elu t i o n patterns are shown i n Figures 32 and 33. The best resolu-t i o n and d i s s o c i a t i o n of the K-g-a s l-casein complex were obtained when whole casein was eluted with phosphate buffer 0.0 5 M pH 9.0 at 25°C. However, complete elimina-t i o n of a_ n-casein was d i f f i c u l t (Figure 33 D l ) . ~ 1 .I , . 20 40 60 80 FRACTION NUMBER E f f e c t of pH and temperature on the elut i o n patterns of whole casein on Sepharose 6B column (37 X 2 cm) eluted with phosphate buffer 0.05 M. A = pH 7.6, 4°C B = pH 9.0, 4°C C = pH 7.6, 25°C D = pH 9.0, 25°C 96 W 1 2 3 4 Fig.32. P o l y a c r y l a m i d e g e l electrophoretogram of f r a c t i o n s obtained from F i g . 31 C (1 •*• 4) . W = whole c a s e i n . W 1 2 3 4 5 6 1 B D F i g . 33. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 31 B (1 -> 6B) and f r o m F i g . 31 D (ID) . W = w h o l e c a s e i n . 9 8 . E f f e c t of urea concentration on the elu t i o n pattern: The tendency of K-casein to i n t e r a c t with a s ^ and 8-casein can be minimized by introducing d i s s o c i a t i n g agents such as urea ( 9 0 ) . An experiment was conducted to investigate the minimum urea concentration required to obtain a pure K-casein by the Sepharose gel f i l t r a t i o n . Figure 34 shows the e l u t i o n pattern of whole casein eluted with 0, 1, 2, and 3 M urea i n phosphate buffer pH 9.0. The size of K-casein r i c h peak (peak 1) decreased as the urea concentration increased to 3 M in d i c a t i n g d i s s o c i a -t i o n of the complex. Further increase i n urea concentra-tion did not change the si z e of t h i s peak. The i n t e r a c t i o n s t i l l exists when one molar urea i s used and the as^ and 8-caseins bands are c l e a r l y shown to be present i n peak 1 (Figure 35 A). However, using 2 molar urea, the early part of peak 1 was almost completely free of any ot s-casein contaminant. On the other hand, traces of B-casein were present and the amount increased as the e l u t i o n proceeded (Figure 35 B). E l u t i o n with 3 molar or higher concentration of urea yielded K-casein free of both a s ^ and B-casein at the early part of peak 1 (Figure 35 B). Some contamination of B-casein was observed at the second half of t h i s peak when eluted with the buffer containing 3 M urea. These contami-nants were almost completely eliminated when the column was 9 9 . FRACTION NUMBER F i g . 34. E f f e c t of urea concentration on the elut i o n pattern of whole casein on Sepharose 6B column (30 X 2.cm), elut i o n buffer = phosphate buffer 0.05 M, pH . 9.0 containing A = z e r o cone, u r e a B = 1 M urea C = 2 M urea D = 3 M urea at 4°C. 100. W 1 2 B Fig.35A. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m Fig.34 B (1 and 2B) . W = whole c a s e i n . a s _ • P -K_ w Fig.35B 2 C 2 3 D P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 34 C (1 3C) and f r o m F i g . 34 D (1 -* 4D) . W = w h o l e c a s e i n . 102. D, run at room temperature (Figure 36 A, and 37). Increasing the urea concentration to 6.6 M at room temperature improved the resolution remarkably (Figure 38) so that an a s 5 ~ r i c h f r a c t i o n i s c o l l e c t e d as well as a highly pure K-casein f r a c t i o n (slot 5 and s l o t s 1 + 3 of Figure 39). When skimmilk was applied d i r e c t l y to a column under these conditions, f i v e d i s t i n c t peaks were obtained i n which peak 1 contained pure K-casein, peak I I , an a - r i c h f r a c t i o n , peak IIIa o 1+B-casein peak, peak iv, s 5 i 3 ± whey proteins, and V was a dialyzable non-protein nitrogen (Figure 40). Polyacrylamide gel electrophoreto-grams of a l l these f r a c t i o n s are shown i n Figure 41. Yi e l d of K-casein and column length: To obtain K-casein rapidly by gel f i l t r a t i o n , the use of a wide and a short column (5 X 26 cm) was attempted using a phosphate buffer containing 3 M urea at room temperature. However, the res o l u t i o n of thi s column was poor (Figure 36B). When a longer column (4 X 77 cm) was used under the same conditions, good resolution with a r e l a t i v e l y high y i e l d of pure K-casein was obtained 200 mg K-casein from 2 gm whole casein on a single run) (Figure 36A). The purity of the early and l a s t part of peak 1 i n this run i s shown i n Figure 37. This column i s capable of separating K-casein from a 2 gm sample at each run successively without repacking the column i n order to ^ 40 60 < CQ CC o CO CO < 1.0-•5 10 20 30 40 50 FRACTION NUMBER F i g . 36. E f f e c t of column length on the elution pattern of whole casein on Sepharose 6B eluted with phosphate buffer 0.05 M, containing 3 M urea, pH 9.0 at room temperature. A - 2 gm whole casein applied to column 4 X 77 cm B - 650 mg whole casein applied to column 5 X 26 cm. o CO 80 100 120 B a S-K_ . 104 w F i g . 3 7 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 3 6 A (1 a n d 2) . W = w h o l e c a s e i n . 20 40 60 80 100 FRACTION NUMBER F i g . 3 8 . E l u t i o n p a t t e r n o f w h o l e c a s e i n (150 mg) o n S e p h a r o s e 6 B c o l u m n ( 4 3 X 2.5 cm) e l u t e d w i t h p h o s p h a t e b u f f e r 0.05 M c o n t a i n i n g 6 . 6 M u r e a , p H 7 . 6 a t r o o m t e m p e r a t u r e . o P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m f r a c t i o n s o b t a i n e d f r o m F i g . 3 8 . W = w h o l e c a s e i n . 20 40 60 80 FRACTION NUMBER 100 120 F i g . 4 0 . E l u t i o n p a t t e r n o f s k i m m i l k ( 2 ?-5 ml) o n S e p h a r o s e 6B c o l u m n ( 4 3 X 2 . 5 cm) e l u t e d w i t h p h o s p h a t e b u f f e r 0 . 0 5 M c o n t a i n i n g 6 . 6 . M u r e a , p H 7 . 6 a t r o o m t e m p e r a t u r e . i—• o 1 2 3 4 5 6 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o -gram o f f r a c t i o n s o b t a i n e d f r o m F i g . 4 0 . W = w h o l e c a s e i n . 109 . obtain larger quantities of K - c a s e i n . The y i e l d can further be increased by applying a K - c a s e i n r i c h f r a c t i o n i . e . the supernatant obtained from the urea s u l f u r i c acid treatment of whole casein &. s 3) before f i n a l ethanol treatment. Such a f r a c t i o n contains ^ 65% K - c a s e i n , thus 650 mg pure K-casein can be obtained by applying 1 gm of t h i s K-casein r i c h f r a c t i o n to a long column. Figure 42 shows the e l u t i o n pattern of t h i s f r a c t i o n on Sepharose 6B. A polyacrylamide gel electrophoretogram of peak 1 and 2 of this figure indicates that 1 contains highly pure K - c a s e i n and 2 contains mainly a , and 8-casein with 2 s l l i t t l e K-casein (Figure 43). The properties of K - c a s e i n prepared by Sepharose The s t a b i l i z i n g power of Sepharose-purified K-casein from whole casein, skimmilk or Z i t t l e ' s K-casein r i c h f r a c t i o n was equal to that prepared by other -methods. Application of s t a b i l i z i n g t e s t of Z i t t l e ( 1 5 0 ) indicated that these K-caseins were capable of s t a b i l i z i n g 95% of a ^ - c a s e i n at a r a t i o of 0.13 K-to 1.0 a g^-casein (Figure 44). Sedimentation v e l o c i t y data have been reported to be i n the range of 13-19 . S for K-casein depending on the method of preparation. These values suggest an average molecu-l a r weight of 650,000 daltons for the aggregated form of K - c a s e i n . In our previous report ( 7 5 ) an S-value of 13 was obtained for K - c a s e i n prepared by Sephadex G-100 eluted with 10 20 30 40" ou FRACTION NUMBER F i g . 42. E l u t i o n p a t t e r n o f K - c a s e i n r i c h f r a c t i o n ( Z i t t l e ' s m e t h o d b e f o r e a l c o h o l f r a c t i o n a t i o n ) on S e p h a r o s e 6B c o l u m n (33 X 2 cm) e l u t e d w i t h p h o s p h a t e b u f f e r 0.05 M; c o n t a i n i n g 6.6 M u r e a , pH 7.6. M i—1 o I a 11 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f f r a c t i o n s o b t a i n e d f r o m F i g . 4 2 . W = w h o l e c a s e i n K - r i c h w a s p r e p a r e d a c c o r d i n g t o Z i t t l e m e t h o d (153 ) w i t h o u t a l c o h o l f r a c t i o n a t i o n . K-rich I n i K _ / C A S E I N S t a b i l i z a t i o n o f a - c a s e i n by K - c a s e i n s l J p r e p a r e d by S e p h a r o s e method f r o m whole c a s e i n (•-•-•) and c r u d e K - c a s e i n o f Z i t t l e (4-A-A) . 113. phosphate buffer pH 10.8. This value was increased to 15.3 upon treatment with urea-sulfuric acid step of Z i t t l e ' s procedure and further to 16.7 upon application of the alcohol f r a c t i o n a t i o n step. This i s a t y p i c a l value for Z i t t l e ' s K-casein. However, on treatment with McKenzie's procedure (alcohol treatment) the S-value increased only to 15.3. The increase i n S-value upon treatment with alcohol or urea-s u l f u r i c acid (pH 1.3) would indicate a tendency towards aggregation of K-casein. The S-value of K-casein prepared by the Sepharose method from the K-casein r i c h f r a c t i o n using Z i t t l e ' s method was 14.4, s l i g h t l y higher than that of K-casein prepared d i r e c t l y from whole casein (14.0 S). General Discussion The separation of K-casein from other caseins by the Sepharose method may be due to molecular size differences among dissociated and unreduced K, a g^- and (3-caseins. In the presence of d i s s o c i a t i n g agents such as urea, a s]_- and 8-caseins d i s s o c i a t e completely to the monomoric forms 3 with a molecular weight of 20 to 27 X 10 whereas K-casem 3 remains at a higher molecular weight of 90 to 125 X 10 ( 1 1 9 ) . The aggregated forms of K-casein i n the presence of urea are linked by intermolecular d i s u l f i d e bonding (6 7 , 1 1 9 , 8 7 ) . The Sepharose chromatography method described i n this chapter provides ,means* for i s o l a t i n g e l e c t r o p h o r e t i c a l l y pure K-casein from whole casein or d i r e c t l y from skimmilk i n a single step, and i s thus simpler than the other available methods ( 1 5 3 , 1 1 8 , 8 6 , 6 7 , 3 5 ) . The advantage of using Sepharose 114 . over using Sephadex gel i s i n faster flow rate. The flow rate of Sepharose gel i s almost double that of Sephadex (3-4 ml/min for Sepharose 6B compared to 1.8 ml/min for Sephadex G150 packed i n a 5 X 100 cm column) ( 1 1 1 ) . The Sepharose method can also be used for the preparation of whey protein and an a ^ - r i c h f r a c t i o n . This method i s suitable for the preparation and p u r i f i c a t i o n of K-casein d i r e c t l y from skimmilk. 115. SECTION 2 Calcium s e n s i t i v i t y and mol wt of a --casein 2 s5 The purity of the a g^-casein preparation used i n this study was assessed by polyacrylamide gel electrophoresis. A single band corresponding to a ^ - c a s e i n was obtained i n the absence of 2-mercaptoethanol. On reduction with 2-mercaptoethanol the single band was replaced by two bands corresponding to as2>~ a n ( ^ ct^-caseins, as reported by Hoagland et a l . ( 3 9) . The d i s u l f i d e content of t h i s a ^ - c a s e i n was 1.95 moles per 65,750 grams (s). The s o l u b i l i t y of c t s ^ - and a s,--caseins i n C a C l 2 i s shown i n Figure 45. a g^-casein was considerably more sensi t i v e to CaCl2 than a ^ - c a s e i n requiring only 2 mM CaC^ for 80% p r e c i p i t a t i o n of protein as compared to 8 mM for ot^-casein. The t u r b i d i t y was determined as the absorbance at 540 nm for a and a^r-caseins i n 10 mM CaCl~ over a concentration range s l sb 2 3 of the caseins from 0.001 to 0.02%. The t u r b i d i t y was l i n e a r against the casein concentration and was 4.5 f o l d greater for a - than a c l - c a s e i n (Figure 46). No s i g n i f i c a n t difference s5 ° x i n a c i d i c amino acid and phosphate contents has been found between a„,- and a -casein to explain t h e i r marked difference s i s5 i n Ca s e n s i t i v i t y . The s t a b i l i z a t i o n by K-casein of a - and a c -1 s l s5 caseins i n the presence of C a + + was compared (Figure 47). The s t a b i l i t y of Ca a g ^ - K-caseinate was about one half that 116. mM C a C l 2 F i g . 45.;: S o l u b i l i t y of a s]_- and a 5- caseins i n C a C l 2 solutions. - k -: a ^ - c a s e i n ; - © - : ocs5_ casein 1 ml of .5% casein was added to 3 ml of .001 M imidazole (pH 7.5), d i f f e r e n t amount of 50 mM C a C l 2 and water to make a f i n a l v o l . of 5 ml, centrifuged after 30 min and the absorbance at 280 nm of the supernatant was measured. .4 .8 1.2 1.6 2 <*s- CASEIN CONCENTRATION mg/-|Om|. F i g . 46. S e n s i t i v i t y o f a s l ~ ( B-»-* ) and a s 5 ~ c a s e i n ( ) to C a + + as t u r b i d i t y measured a t 54 0 nm. 118. 100 GO <c o I CO \ so 60 UJ «J r> _l O W 40 20 \ \ \ \ \ \ \ \ \ 70 C*s/ 75 20 K F i g . 47-. S t a b i l i z a t i o n o f a s l ^ and a s 5 - c a s e i n s by K-T c a s e i n - A a s l - c a s e i n ; - • a s 5 - c a s e i n 1 ml o f .1% K - c a s e i n w i t h d i f f e r e n t amounts o f cx -,- o r a c - c a s e i n i n .01 M i m i d a z o l e b u f f e r s l s5 (pH 7.0) was d i l u t e d t o 5 m l , the m i x t u r e was i n c u b a t e d f o r 30 min a t 37°C a f t e r a d d i n g 10 mM C a C l ^ , and the absorbance a t 280 nm o f the s u p e r n a t a n t was measured a f t e r c e n t r i f u g a t i o n . 119 . of Ca a ^ - K - c a s e i n a t e on weight b a s i s (a quarter on molar b a s i s ) . I t i s apparent t h a t K - c a s e i n does i n t e r a c t w i t h a s 5 ~ c a s e i n so as to p r o t e c t i t from calcium p r e c i p i t a t i o n , although to a l e s s e r extent than w i t h a s-^-casein. The lower s t a b i l i t y of the a -K-caseinate may be due to the high calcium s e n s i t i v i t y of the a -c a s e i n or due to a reduced J s5 degree of i n t e r a c t i o n between c t g , - - and K - c a s e i n as compared to the degree of i n t e r a c t i o n of K - c a s e i n w i t h a g ^ - c a s e i n . The sedimentation r a t e s of 1% p r o t e i n at 25C were measured ( T a b l e V I ) . Without urea, both a ^ - c a s e i n and reduced a --casein (the equimolar mixture of a and so ^ ctg^-caseins) were i n aggregated forms as evidenced by the r e l a t i v e l y l a r g e sedimentation r a t e s . I n view of t h i s , Q.05 M phosphate b u f f e r (pH 6.8) c o n t a i n i n g 6.6 M urea was chosen f o r the mol wt determination. The p a r t i a l s p e c i f i c volumes of a„ c- and a^-3-caseins were c a l c u l a t e d from amino s5 s J a c i d composition ( 3 9 ) asO.727 andO.729, r e s p e c t i v e l y . A value of 0.7 28 was used f o r a l l c a s e i n p r e p a r a t i o n f o r the purpose of mol wt c a l c u l a t i o n . I t was assumed th a t urea d i d not a f f e c t the p a r t i a l s p e c i f i c volume si n c e McKenzie and Wake ( 6 6) and Sakura and R e i t h e l ( 1 0 *•) have shown t h a t f o r many p r o t e i n s , urea a f f e c t s the p a r t i a l s p e c i f i c volume only s l i g h t l y . Under these c o n d i t i o n s , a mol wt value of 27,000 was obtained f o r a g ^ - c a s e i n B, which agrees w i t h published values ( 6 1 t) . As shown i n T a b l e V I , the values of 120 . TABLE V I Sedimentation rate and molecular weight of a .--casein with and without mercapto-s5 ^ ethanol. Casein Sedimentation rate Molecular weight (1%, pH 6.8 and at 25°C a --casein s5 Reduced Without urea With 6.6M urea M^ L i t e r a t u r e 1 — X l O 1 3 s e c — 22.0 1.72 65,750 67,500 a ,-casein 2 6.9 1.21 31,800 33,700 s b 1 Hoagland et a l . (39) 2 a ..-casein with .2% 2-mercaptoethanol s5 1 2 1 . 6 5 , 7 5 0 a n d 3 1 , 8 0 0 o b t a i n e d f o r a r - c a s e i n a n d r e d u c e d a -' ' s 5 s 5 c a s e i n a r e i n g o o d a g r e e m e n t w i t h t h e v a l u e s c a l c u l a t e d b y H o a g l a n d e t a l . f r o m a m i n o a c i d c o m p o s i t i o n s . A n c t g ^ - c a s e i n r i c h f r a c t i o n m a y b e c o l l e c t e d f r o m a S e p h a d e x G - 1 0 0 c o l u m n a t t h e e l u t i o n v o l u m e b e t w e e n t h e K - c a s e i n r i c h p e a k a n d t h e 6- a n d a ^ - c a s e i n r i c h p e a k ( F i g u r e 4 8 A , f r a c t i o n s d a n d e ) d u r i n g f r a c t i o n a t i o n o f c a s e i n s b y g e l c h r o m a t o g r a p h y e l u t e d w i t h p h o s p a h t e b u f f e r s a t p H 1 0 . 8 a n d 4 ° C a s p r e v i o u s l y r e p o r t e d ( 7 5 ) . T h e t r e a t m e n t w i t h 2 - m e r c a p t o e t h o n a l d u r i n g g e l e l e c t r o p h o r e s i s d e c r e a s e d t h e a ^ - c a s e i n b a n d ( F i g u r e 4 8 C ) a n d e x c l u s i o n o f 2 - m e r c a p t o -e t h a n o l i n c r e a s e d t h e i n t e n s i t y o f t h i s b a n d ( F i g u r e 4 8 © ) w h i c h i s t h e t y p i c a l b e h a v i o u r o f c t ^ - c a s e i n ( 3 9 ) . S e p a r a t i o n o f c t g ^ - c a s e i n f r o m a ^ - c a s e i n b y g e l c h r o m a t o g r a p h y m a y b e d u e t o e a s i e r d i s s o c i a t i o n o f a s ^ - c a s e i n t h a n o f a s 5 ~ c a s e i n u n d e r t h e c o n d i t i o n s o f t h i s g e l c h r o m a t o g r a p h y . I f a-s$~ c a s e i n e x i s t s a s a d i m e r ( c tg^ + c t ^ - c a s e i n ) u n d e r t h e s e c o n d i t i o n s , t h e s e p a r a t i o n f r o m a s ] _ - c a s e i n i s e x p l a i n a b l e o n t h e b a s i s o f m o l e c u l a r s i z e . H o w e v e r , t h e p o s s i b i l i t y o f d i s u l f i d e b o n d r e d u c t i o n o c c u r r i n g u n d e r t h e a l k a l i n e c o n d i -t i o n s o f e l u t i o n ( 1 6 ) c a n n o t b e i g n o r e d . S u c h r e a c t i o n w o u l d c o n t r i b u t e t o s p r e a d i n g a n d a d m i x i n g o f a ^ - c a s e i n w i t h t h e K - c a s e i n r i c h p e a k o r w i t h t h e a ^ - a n d 3 - c a s e i n r i c h p e a k . —* E L U T I O N V O L U M E F i g . 48. 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J., Vol. 5, No. 4, 1972. Toma, S. and S. Nakai. Calcium sensitivity and molecular weight of as^-casein. J. Dairy Science (in press). Beveridge, T., S.J. Toma and S. Nakai. Determination of SH and SS groups in fcod proteins using Ellman's reagent. J. Fcod Science (in press). Toma, S.J. and Nakai, S. Heat stability of desulfurized K-casein. Paper No. 17, presented at the 16th National Conference of the C.I.F.S.T., May 30, 1973. Abid A. Mahdi, Sadiq J. Toma, Selwa L. Aziz and Nouria A. Dhiaddin. Evaluation of tomato pastes and recommended methods for their commercial production. The Iraqi Journal of Agricultural Science, Vol. 11, No. 1, 32-41, 1967. 

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