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Alpha sl-casein-k-carrageenan interaction : a physicochemical study Skura, Brenton James 1976

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aCASEIN-K-CARRAGEENAN INTERACTION: A PHYSICOCHEMICAL STUDY by BRENTON JAMES SKURA B.Sc. (Food S c i e n c e ) , U n i v e r s i t y of A l b e r t a , 1970 M.Sc, U n i v e r s i t y of A l b e r t a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1976 (c) Brenton Janes Skura, 1976 In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree t h a t permiss ion for e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date •• i t^^f ol rf , / •//'/, Department of 7 A B S T R A C T K - C a r r a g e e n a n , a s u l f a t e d p o l y g a l a c t a n , s t a b i l i z e s a . - c a s e i n , t h e c a l c i u m s e n s i t i v e p r o t e i n o f b o v i n e m i l k , s i f r o m p r e c i p i t a t i o n b y C a + + . T h e a ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n , i n c a l c i u m - f r e e s y s t e m s , was i n v e s t i g a t e d e m p l o y i n g s e d i m e n t a t i o n v e l o c i t y , s e d i m e n t a t i o n e q u i l i b r i u m , v i s c o s i t y , f r o n t a l c h r o m a t o g r a p h y a n 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 e x p e r i m e n t s . K - C a r r a g e e n a n s t a b i l i z a t i o n o f a ^ - c a s e i n was a l s o i n v e s t i g a t e d i n s y s t e m s c o n t a i n i n g 0 . 0 1 M C a + + . S c h l i e r e n p a t t e r n s o f a ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e s i n c a l c i u m f r e e s y s t e m s d u r i n g s e d i m e n t a t i o n v e l o c i t y u l t r a c e n t r i f u g a t i o n r e v e a l e d a l a r g e a ^ - c a s e i n c o n t a i n i n g i n t e r a c t i o n p e a k f o l l o w e d b y a s l o w e r s e d i m e n t i n g p e a k t h o u g h t t o b e r e s i d u a l t c - c a r r a g e e n a n . T h e S^Q w o f t h e i n t e r a c t i o n p e a k was g r e a t e r t h a n t h e s „ _ o f a . . - c a s e i n ^ ^ 2 0 , w s i a l o n e , u n d e r i d e n t i c a l c o n d i t i o n s . I t was c o n c l u d e d , o n t h e b a s i s o f t h e e f f e c t s o f i o n i c s t r e n g t h , t e m p e r a t u r e , p H , a n d 6 . 0 M u r e a o n t h e s__ a n d r e d u c e d v i s c o s i t i e s o f a , - c a s e i n -2 0 , w s i K - c a r r a g e e n a n i n t e r a c t i o n m i x t u r e s , t h a t h y d r o g e n b o n d i n g was i n v o l v e d i n t h e o b s e r v e d a ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n . H o w e v e r , f l u o r e s c e n c e p o l a r i z a t i o n , f r o n t a l g e l c h r o m a t o g r a p h y a n d 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 s c a l c u l a t e d f r o m s e d i m e n t a t i o n e q u i l i b r i u m p a t t e r n s b y a m u l t i p l e r e g r e s s i o n t e c h n i q u e r e v e a l e d t h a t a ^ - c a s e i n a n d K - c a r r a g e e n a n d i d n o t i n t e r a c t i n c a l c i u m - f r e e s y s t e m s . F l u o r e s c e n c e i i i p o l a r i z a t i o n s t u d i e s employed both D N S - c t ^ - c a s e i n and D N S - K -carrageenan as the l a b e l l e d component. a ^ - C a s e i n - K - c a r r a -geenan mixtures (pH 6.6, y = 0.08) e l u t e d from a c o n t r o l l e d o pore g l a s s (170 A pore diameter) column as the i n d i v i d u a l components w i t h e l u t i o n volumes s i m i l a r to those o b t a i n e d when a ^ - c a s e i n and K-carrageenan were chromatographed s e p a r a t e l y . The molecular weight d i s t r i b u t i o n s f o r a g ^ -ca s e i n - K - c a r r a g e e n a n mixtures (pH 6.6, u = 0.08) con t a i n e d a major peak i n the molecular weight range corresponding t o unreacted a ..-casein, s i The " i n t e r a c t i o n " r e v e a l e d by sedimentation v e l o c i t y and v i s c o s i t y data was not a chemical i n t e r a c t i o n , but, r a t h e r a p h y s i c a l entrapment of a ^ - c a s e i n by K - c a r r a -geenan. F l u i d flow through the c a p i l l a r y d u r i n g v i s c o s i t y measurements and the i n t e n s e g r a v i t a t i o n a l f i e l d s generated d u r i n g sedimentation v e l o c i t y u l t r a c e n t r i g u a t i o n induced p h y s i c a l entanglement of the K-carrageenan. As a consequence, a ^ - c a s e i n - K - c a r r a g e e n a n mixtures flowed as a "porous-plug" where c t ^ - c a s e i n , l a r g e r than the pores o f the K-carrageenan network, was trapped g i v i n g r i s e t o the observed " i n t e r a c t i o n " . Urea (6.0 M) d i s s o c i a t e d the a ^ - c a s e i n aggregates to the monomer s t a t e , w i t h a mol e c u l a r s i z e s m a l l e r than the pores i n the entangled carrageenan network, r a t h e r than d i s s o c i a t i n g an " a ^ - c a s e i n - K - c a r r a g e e n a n complex". P h y s i c a l entanglement o f a ^ - c a s e i n w i t h i n the K-carrageenan system was not induced and an " i n t e r a c t i o n " was i v not observed d u r i n g f r o n t a l g e l chromatography, se d i m e n t a t i o n e q u i l i b r i u m and f l u o r e s c e n c e p o l a r i z a t i o n measurements s i n c e an i n t e n s e f o r c e was not a p p l i e d to the system d u r i n g these a n a l y s e s . K-Carrageenan s t a b i l i z a t i o n o f a ^ - c a s e i n i n the presence of 0.01 M C a + + (pH 6.6) decreased w i t h i n c r e a s i n g g r a v i t a t i o n a l f i e l d s d u r i n g the c e n t r i f u g a t i o n step o f the s t a b i l i z a t i o n t e s t employed. I n c r e a s i n g i o n i c s t r e n g t h i n h i b i t e d the s t a b i l i z a t i o n o f a ^ - c a s e i n by K-carrageenan as d i d i n c r e a s i n g the a g ^ - c a s e i n c o n c e n t r a t i o n from 1.5 mg/ ml to 10 mg/ml (pH 6.6, y = 0.08). Thus, a g ^ - c a s e i n and K-carrageenan d i d not c h e m i c a l l y i n t e r a c t i n c a l c i u m - f r e e systems. S t a b i l i z a t i o n o f a g ^ - c a s e i n by K-carrageenan i n v o l v e d a p h y s i c a l entrapment of c a l c i u m - a ^ - c a s e i n a t e p a r t i c l e s by K-carrageenan. V TABLE OF CONTENTS Page ABSTRACT . . . i i TABLE OF CONTENTS . V LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x i INTRODUCTION . 1 SURVEY OF THE LITERATURE . . . . . . . . . . . . . . . . 2 a -CASEIN 2 s i G e n etic V a r i a n t s of a s l - C a s e i n 2 Mo l e c u l a r Weight 3 A s s o c i a t i o n of a s ^ - C a s e i n . 4 I n t e r a c t i o n of a ^ - C a s e i n 10 CARRAGEENAN . 14 Composition of Carrageenan . . . . 14 Conformation 16 Mo l e c u l a r Weight 20 CARRAGEENAN-CASEIN INTERACTIONS . . . 22 MATERIALS AND METHODS 28 TURBIDITY MEASUREMENTS 29 SEDIMENTATION VELOCITY 30 E f f e c t of I o n i c S trength and pH 31 E f f e c t o f Temperature 31 E f f e c t o f Urea 31 C a l c u l a t i o n o f s o n T I 32 Area C a l c u l a t i o n . . . . . . 34 FRONTAL CHROMATOGRAPHY . 35 FLUORESCENCE POLARIZATION 37 MOLECULAR WEIGHT DISTRIBUTION FROM SEDIMENTATION EQUILIBRIUM 39 v i Page S T A B I L I Z A T I O N OF a S L - C A S E I N WITH K-CARRAGEENAN . . 43 R E S U L T S AND D I S C U S S I O N . . 45 P H E N O L - S U L F U R I C A C I D T E S T FOR CARRAGEENAN . . . . . . . 47 T U R B I D I T Y FORMATION . 53 S E D I M E N T A T I O N V E L O C I T Y 55 M O L E C U L AR WEIGHT D I S T R I B U T I O N 75 F R O N T A L CHROMATOGRAPHY 87 F L U O R E S C E N C E P O L A R I Z A T I O N 96 S T A B I L I Z A T I O N T E S T S . . . . 1 0 8 G E N E R A L D I S C U S S I O N . 1 2 1 C O N C L U S I O N S 1 2 9 R E F E R E N C E S . 130 v i i LIST OF TABLES Table Page I E f f e c t of V a r i o u s C o n s t i t u e n t s on the C o l o r Y i e l d of the Phenol S u l f u r i c A c i d T e s t f o r K-Carrageenan (100 yg/ml) . . . . . 52 II Sedimentation C o e f f i c i e n t s f o r a s i - C a s e i n -K-Carrageenan I n t e r a c t i o n S t u d i e s . . . . . . 58 I I I Area Under S c h l i e r e n P a t t e r n s f o r a s i - C a s e i n -K-Carrageenan I n t e r a c t i o n S t u d i e s . . . . . . 60 IV Reduced V i s c o s i t i e s o f a s l - C a s e i n , K-Carrageenan and c t s i - C a s e i n - K - C a r r a g e e n a n Mixtures 64 V E l u t i o n Volumes of a s]_-Gasein and K-Carrageenan E l u t i o n P a t t e r n s on C o n t r o l l e d Pore Glass Columns (170 A) a t pH 6.6, y = 0.0 8 95 VI E f f e c t of pH on D a n s y l a t i o n of Carrageenan 99 VII E f f e c t o f I o n i c S t r e n g t h and Temperature on the S o l u b i l i t y o f a -Casein i n 0.01 M C a + + (pH 6.6). 112 V I I I V i s c o s i t y of K-Carrageenan i n the Presence of 0.01 M CaCl2 (pH 6.6, F i n a l I o n i c Strength 0.08) 116 v i i i L I S T OF F I G U R E S F i g u r e P a g e 1 . C o u r s e o f t h e f o r m a t i o n a n d d i s a p p e a r a n c e o f 5 - h y d r o x y l m e t h y l - 2 - f u r a l d e h y d e d u r i n g a n a l y s i s f o r 3 , 6 - a n h y d r o - D - g a l a c t o s e i n K - c a r r a g e e n a n 46 2 . I n f r a r e d s p e c t r u m o f K - c a r r a g e e n a n 48 3 . V i s i b l e s p e c t r u m f o r t h e c o l o r c o m p l e x f r o m t h e p h e n o l - s u l f u r i c a c i d m e t h o d o f c a r r a g e e n a n d e t e r m i n a t i o n 49 4 . S t a n d a r d c u r v e s f o r p h e n o l - s u l f u r i c a c i d m e t h o d o f c a r r a g e e n a n d e t e r m i n a t i o n . . . . . 50 5 . E f f e c t o f i o n i c s t r e n g t h o n t u r b i d i t y f o r m a t i o n i n a g ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e s a t pH 6 . 6 54 6 . E f f e c t o f c e n t r i f u g a t i o n a t 3 9 , 0 0 0 * G f o r 30 m i n o n a s ^ - c a s e i n a n d K - c a r r a g e e n a n c o n c e n t r a t i o n i n a s ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e s 56 7 . S e d i m e n t a t i o n v e l o c i t y s c h l i e r e n p a t t e r n s o f K - c a r r a g e e n a n , a s x - c a s e i n . a n d a s ^ - c a s e i n a n d K - c a r r a g e e n a n m i x t u r e s (pH 6 . 6 , y = 0 . 0 8 ) 57 8 . C o n c e n t r a t i o n d e p e n d e n c e o f S 2 0 , w f ° r K - c a r r a g e e n a n a n d a , - c a s e i n (pH 6 . 6 , . y = 0 . 0 8 ) . . . . 62 9 . E f f e c t o f c o n c e n t r a t i o n o n S 2 0 , w ° ^ a s i - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n c o m p l e x e s (pH 6 . 6 , y = 0 . 0 8 ) 63 1 0 . U l t r a v i o l e t s c a n s d u r i n g s e d i m e n t a t i o n v e l o c i t y a n a l y s i s o f o t s i - c a s e i n ( 0 . 5 m g / m l ) , K - c a r r a g e e n a n ( 0 . 5 m g / m l ) a n d a g ^ - c a s e i n ( 0 . 5 m g / m l ) + K - c a r r a g e e n a n ( 0 . 5 m g / m l ) , p H 6 . 6 , y = 0 . 0 8 66 1 1 . E f f e c t o f t e m p e r a t u r e o n S 2 Q W 70 1 2 . E f f e c t o f u r e a o n s c h l i e r e n p a t t e r n s f o r a s ] _ - c a s e i n (10 m g / m l ) + K - c a r r a g e e n a n ( 3 . 4 m g / m l ) a t pH 6 . 6 , y = 0 . 0 8 72 i x F i g u r e P a g e 1 3 . S c h l i e r e n s e d i m e n t a t i o n e q u i l i b r i u m p a t t e r n s f o r MWD c a l c u l a t i o n s . . . . . . . . 76 1 4 . P a r t i a l s p e c i f i c - v o l u m e s o f s e l e c t e d a s i - c a s e i n : K - c a r r a g e e n a n i n t e r a c t i o n c o m p l e x e s . 78 1 5 . - 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 f o r a s ] _ - c a s e i n (5 m g / m l ) a n d K - c a r r a g e e n a n (1 m g / m l ) m i x t u r e , pH 6 . 6 , y = 0 . 0 8 . . . . . . . . . . 82 1 6 . S e d i m e n t a t i o n e q u i l i b r i u m s c h l i e r e n p a t t e r n s f o r a s x - c a s e i n (5 m g / m l ) a n d K - c a r r a g e e n a n (1 m g / m l ) , pH 6 . 6 , u = 0 . 0 8 . 83 17. 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 f o r a s i - c a s e i n (5 m g / m l ) a t pH 6 . 6 , y = 0 . 0 8 84 1 8 . S e d i m e n t a t i o n e q u i l i b r i u m s c h l i e r e n p a t t e r n f o r a s i - c a s e i n (5 m g / m l ) , pH 6 . 6 , y = 0 . 0 8 . 85 1 9 . E l u t i o n p a t t e r n o f a s ; i - c a s e i n ( 1 . 2 m g / m l ) , pH 6 . 6 , y = 0 . 0 1 88 2 0 . E l u t i o n p a t t e r n s o f a s i - c a s e i n ( 1 . 2 m g / m l ) , + K - c a r r a g e e n a n ( 1 . 2 m g / m l ) , pH 6 . 6 , y = 0 . 0 1 89 2 1 . E l u t i o n p a t t e r n s o f a s i - c a s e i n (10 m g / m l ) a n d K - c a r r a g e e n a n (1 m g / m l ) , pH 6 . 6 , y = 0 . 0 8 91 2 2 . E l u t i o n p a t t e r n s f o r a s i - c a s e i n (10 m g / m l ) a n d K - c a r r a g e e n a n (1 m g / m l ) i n t e r a c t i o n m i x t u r e (pH 6 . 6 , y = 0 . 0 8) . . . . . . . . . 92 2 3 . D e r i v a t i v e p a t t e r n s o f a d v a n c i n g a n d t r a i l i n g e d g e s o f a g ^ - c a s e i n (10 m g / m l ) a n d K - c a r r a g e e n a n (1 m g / m l ) , pH 6 . 6 , y = 0 . 0 8 . . 93 2 4 . A b s o r p t i o n s p e c t r a o f a s ^ - c a s e i n a n d D N S -a s x ~ c a s e i n (pH 6 . 6 , y = 0 . 0 1 ) 97 2 5 . A b s o r p t i o n s p e c t r a o f K - c a r r a g e e n a n a n d D N S - K - c a r r a g e e n a n (pH 6 . 6 , y = 0 . 0 8 ) . . . . 100 2 6 . F l u o r e s c e n c e e x c i t a t i o n s p e c t r u m o f D N S -K - c a r r a g e e n a n (2 m g / m l ) , pH 6 . 6 , y = 0 . 0 8 . . 101 F i g u r e x Page 27. F l u o r e s c e n c e emission spectrum o f DNS-K-carrageenan and DNS - K-carrageenan + a s l ~ c a s e i n (pH 6.6, y = 0.08) . . . . . . . . 102 28. T i t r a t i o n of D N S - a s i - c a s e i n (1 mg/ml) wit h K-carrageenan (20 mg/ml), pH 6.6, y = 0.08 . 104 29. Fluo r e s c e n c e emission s p e c t r a of DNS-a , -c a s e i n (pH 6.6, y = 0.08) . . . . 105 30. T i t r a t i o n of DNS - K-carrageenan (2 mg/ml) wi t h a s i - c a s e i n (50 mg/ml), pH 6.6, y = 0.08 107 31. E f f e c t of c e n t r i f u g a t i o n on s t a b i l i z a t i o n of a s ^ - c a s e i n by K-carrageenan (pH 6.6, y = 0.04) 109 32. E f f e c t o f i o n i c s t r e n g t h on the s t a b i l i z a t i o n of a ^ - c a s e i n by K-carrageenan I l l 33. E f f e c t of temperature on s t a b i l i z a t i o n o f a s ^ - c a s e i n by K-carrageenan . . . . . . . . . 114 34. S t a b i l i z a t i o n o f a s ^ - c a s e i n (10 mg/ml) by K-carrageenan (pH 6.6, y = 0.08) 118 35. E f f e c t of a s i - c a s e i n c o n c e n t r a t i o n on s t a b i l i z a t i o n o f a , - c a s e i n by K-carrageenan (pH 6.6, y = 0.08) 119 x i ACKNOWLEDGEMENTS The author would l i k e to express his sincere gratitude to Br. S. Nakai, Professor 3 .Department of Food Science, for his advice and encouragement throughout the course of t h i s work and during the w r i t i n g of t h i s thesis. He i s also thankful to the members of his supervisory committee: Drs. W.D. Powrie, J.F. Richards, M.A. Tung and J. Vanderstoep of the Department of Food Science and to Dr. R.J. Dose, Fisheries and Marine, Environment Canada for t h e i r i n t e r e s t in and review of t h i s thesis. A special acknowledgement i s extended to my wife, V a l e r i e , for her valuable laboratory assistance and for her dedicated e f f o r t s during the preparation of t h i s thesis. The support of the National Research Council of Canada, the Ralston Purina Company and the H.R. MacMillan Foundation through post-graduate scholarships i s also acknowledged. 1 INTRODUCTION P o l y s a c c h a r i d e s a r e n a t u r a l l y p r e s e n t i n , a n d a r e u s e d i n a w i d e v a r i e t y o f f o o d p r o d u c t s . I n t e r a c t i o n s b e t w e e n p o l y s a c c h a r i d e s a n d p r o t e i n s may b e r e s p o n s i b l e f o r many o f t h e f u n c t i o n a l a n d p h y s i c a l c h a r a c t e r i s t i c s o f some f o o d s y s t e m s . K - C a r r a g e e n a n , a s u l f a t e d p o l y g a l a c t a n e x t r a c t e d f r o m m a r i n e , r e d a l g a e o f t h e c l a s s R h o d o p h y c e a e , i n t e r a c t s w i t h a ^ - c a s e i n , t h e c a l c i u m s e n s i t i v e p r o t e i n o f b o v i n e m i l k . T h e i n t e r a c t i o n b e t w e e n t h e s e m a c r o m o l e c u l e s i s s u c h t h a t c a l c i u m p r e c i p i t a t i o n o f a g ^ - c a s e i n i s p r e v e n t e d ( H a n s e n , 196 8 ) . K - C a r r a g e e n a n a l s o i n t e r a c t s i n a s i m i l a r m a n n e r w i t h a n u m b e r o f c a l c i u m s e n s i t i v e p l a n t p r o t e i n s ( C h a k r a b o r t y a n d R a n d o l p h , 1972) . S i n c e t h e s e p o l y s a c c h a r i d e - p r o t e i n i n t e r a c t i o n s may b e o f g r e a t i m p o r t a n c e i n t h e m a n u f a c t u r e o f f a b r i c a t e d f o o d s , i t i s o f i n t e r e s t t o i n v e s t i g a t e a n d c h a r a c t e r i z e t h e i n t e r -a c t i o n , b e t w e e n K - c a r r a g e e n a n a n d a - c a s e i n , t a k e n a s a s i m o d e l s y s t e m . A k n o w l e d g e o f t h e m e c h a n i s m s o f i n t e r a c t i o n w o u l d h o p e f u l l y s h e d i n s i g h t t o w a r d m e t h o d s w h i c h w o u l d b e o f i n t e r e s t f o r t h e m a n i p u l a t i o n o f p o l y s a c c h a r i d e - p r o t e i n i n t e r a c t i o n s i n o r d e r t o o b t a i n t h e d e s i r e d p h y s i c a l p r o p e r -t i e s i n a n u m b e r o f f a b r i c a t e d a n d e x i s t i n g f o o d s y s t e m s . 2 REVIEW OF T H E L I T E R A T U R E a , - C A S E I N Si B o v i n e m i l k c o n t a i n s a p p r o x i m a t e l y 3% p r o t e i n w h i c h c a n b e f r a c t i o n a t e d b y a c i d p r e c i p i t a t i o n i n t o t h e c a s e i n s ( i n s o l u b l e a t pH 4 . 5 ) a n d t h e whey p r o t e i n s ( s o l u b l e a t pH 4 . 5 ) . T h e c a s e i n s c a n b e s u b f r a c t i o n a t e d i n t o t h e a - , p - , a n d y c o m p o n e n t s . T h e a - c a s e i n s w h i c h c o n s t i t u t e a p p r o x i m a t e l y 50% o f t h e c a s e i n f r a c t i o n ( R o s e e t a l , 1969) a r e a f a m i l y o f p r o t e i n s w h i c h m i g r a t e i n t h e same g e n e r a l r e g i o n o f t h e g e l d u r i n g e l e c t r o p h o r e s i s i n s t a r c h o r p o l y a c r y l a m i d e g e l s a t a l k a l i n e pH 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 a n d u r e a ( A n n a n a n d M a n s o n , 1 9 6 9 ) . When t h e m o b i l i t y o f t h e a . - c a s e i n b a n d i s t a k e n a s 1 . 0 0 , t h e J s 4 ' r e l a t i v e m o b i l i t i e s o f t h e o t h e r c o m p o n e n t s a r e 0 ^ ( 1 . 1 8 ) , a ' ( 1 . 1 4 ) , a , ( 1 . 1 1 ) , a ( 1 . 0 6 ) , a n d a _ ( 0 . 8 8 ) . T h e m a j o r c o m p o n e n t i s a g ^ - c a s e i n . G e n e t i c V a r i a n t s o f a , - C a s e i n s i T h o m p s o n e t a l . (196 2 , 1969) a n d K i d d y e t a l . (1964) s h o w e d t h a t a g ^ - c a s e i n c o n s i s t s o f t h r e e g e n e t i c a l l y c o n t r o l l e d v a r i a n t s , named a . - c a s e i n A , B , a n d C i n o r d e r o f s i t h e i r d e c r e a s i n g e l e c t r o p h o r e t i c m o b i l i t y . K i d d y e t a l . (1964) o b s e r v e d t h a t t h e v a r i a n t s c o u l d o c c u r s i n g l y ( A , B , a n d C) o r i n p a i r s ( A B , A C , o r B C ) . S t u d i e s o n t h e o c c u r -r e n c e o f t h e s e v a r i o u s a . - c a s e i n s i n d i f f e r e n t b r e e d s o f s i c a t t l e h a v e d e m o n s t r a t e d t h a t a . . - c a s e i n B i s t h e m o s t 3 common w h i l e a -, - c a s e i n C a n d a , - c a s e i n A a r e r e l a t i v e l y s i s i J r a r e . ( K i d d y e t a l . , 1 9 6 4 ) . T h e d i f f e r e n c e s b e t w e e n a - c a s e i n s B a n d C a r e s i m i n o r w i t h t h e B v a r i a n t c o n t a i n i n g o n e m o r e g l u t a m i c a c i d t h a n a - c a s e i n C w h i l e a - c a s e i n C c o n t a i n s o n e m o r e s i s i g l y c i n e r e s i d u e t h a n t h e B v a r i a n t . a , - C a s e i n A c o n t a i n s 8 ^ •* s i f e w e r a m i n o a c i d r e s i d u e s t h a n t h e B v a r i a n t . F i n g e r p r i n t s t u d i e s o n t h e c h y m o t r y p t i c d i g e s t s o f a g ^ - c a s e i n A s u g g e s t t h a t t h e s e d i f f e r e n c e s a r o s e f r o m a m u t a t i o n a l e v e n t i n v o l v i n g a l y s i n e / a r g i n i n e s u b s t i t u t i o n a s w e l l a s t h e d e l e t i o n o f a s e q u e n c e o f e i g h t a m i n o a c i d r e s i d u e s > ( T h o m p s o n e t a l . , 1969) . A l l t h r e e v a r i a n t s h a v e a n N -t e r m i n a l a r g i n i n e r e s i d u e a n d t h e C - t e r m i n a l s e q u e n c e : l e u -l e u - t r p - O H ( K a l a n e t a l . , 1 9 6 4 ; Waugh e t a l . , 1 9 6 2 ) . T h e a g ^ - c a s e i n s a r e p h o s p h o p r o t e i n s c o n t a i n i n g a p p r o x i m a t e l y 1% p h o s p h o r u s ( T h o m p s o n a n d K i d d y , 1964) w h i c h a m o u n t s t o a p p r o x i m a t e l y 8 p h o s p h a t e s p e r m o l e o f p r o t e i n ( M e r c i e r e t a l . , 1 9 7 1 ) . I n a d d i t i o n , t h e s e p r o t e i n s l a c k c y s t e i n e o r c y s t i n e ( d e K o n i n g a n d v a n R o o i j e n , 1 9 6 5 ) . T h e monomer n e t c h a r g e a t pH 6 . 6 , t h e pH o f m i l k , h a s b e e n d e t e r m i n e d t o b e - 2 2 . 8 b y S c h m i d t (1969) a n d - 1 9 b y Waugh e t a l . ( 1 9 7 1 ) . M o l e c u l a r W e i g h t A m o l e c u l a r w e i g h t o f 2 6 , 9 0 0 d a l t o n s was o b t a i n e d b y S w a i s g o o d a n d T i m a s h e f f (1968) b y l i g h t s c a t t e r i n g e x p e r i m e n t s p e r f o r m e d i n 2 - c h l o r o e t h a n o l , a n h y d r o u s f o r m i c a c i d o r a w a t e r - m e t h a n o l m i x t u r e . T h e s e s o l v e n t s a r e 4 capable of d i s r u p t i n g hydrogen bonds and of opening hydrophobic groups to the s o l v e n t . Noelken (19 67) o b t a i n e d a m o l e c u l a r weight of 24,600 d a l t o n s f o r a - c a s e i n B from s i s edimentation e q u i l i b r i u m experiments i n 3M guanidine h y d r o c h l o r i d e . Waugh e t a l . (196 2) o b t a i n e d , by osmotic p r e s s u r e measurements i n 6.5M urea (pH 4.5), a v a l u e of 27,500 d a l t o n s for. a n - c a s e i n which was probably a mixture S J. | / of the B and C g e n e t i c v a r i a n t s . A monomer mole c u l a r weight of 23,000 ± 2000 d a l t o n s was o b t a i n e d by Schmidt e t a l . (1967) by l i g h t s c a t t e r i n g and sedimentation e q u i l i b r i u m methods f o r a g ^ - c a s e i n B a t pH 12.0. A m o l e c u l a r weight of 27,000 d a l t o n s was o b t a i n e d by D r e i z e n e t a l . (1962) f o r a . ..-caseins a t pH 12 by means of l i g h t s c a t t e r i n g measurements. Schmidt and van Markwijk (196 8) c a l c u l a t e d a molecular weight of 29,000 d a l t o n s f o r the a s s o c i a t i n g s u b u n i t of a . - c a s e i n B ^ s i a t pH 6.6 and i o n i c s t r e n g t h s ( y ) ranging from 0.01 to 0.21. Ono e t a l . (1974) observed t h a t a - c a s e i n e x i s t s as a s i monomer of m o l e c u l a r weight 23,000 d a l t o n s , a t c o n c e n t r a t i o n s below 0.4 g/1 and pH 6.9. Grosclaude e t a l . (1970) have c a l c u l a t e d a molecular weight of 23,600 d a l t o n s f o r a s ^ - c a s e i n B on the b a s i s of the primary s t r u c t u r e of the p r o t e i n . A s s o c i a t i o n of a .-Casein s 1 a g ^ - C a s e i n has a strongtendency to a s s o c i a t e . The C v a r i a n t a s s o c i a t e s to a g r e a t e r e x t e n t than a . - c a s e i n B s i under i d e n t i c a l c o n d i t i o n s (Schmidt and Payens, 1964). Hydrophobic bond formation i n p r o t e i n s i s an endothermic 5 p r o c e s s a c c o m p a n i e d b y a n i n c r e a s e i n e n t r o p y a n d e n t h a l p y o f t h e s y s t e m . T h e a ^ - c a s e i n s c o n t a i n a h i g h c o n t e n t o f a p o l a r a m i n o a c i d s , i n d i c a t i n g a g r e a t p o s s i b i l i t y o f h y d r o p h o b i c i n t e r a c t i o n s f o r s e l f - a s s o c i a t i o n ( M e r c i e r e t a l . , 1 9 7 1 ) . P a y e n s a n d S c h m i d t (1965) s t u d i e d t h e a s s o c i a t i o n o f a , -s i c a s e i n s B a n d C a n d o b s e r v e d t h a t e a c h o f t h e s e c a s e i n s u n d e r g o a r a p i d e n d o t h e r m i c p o l y m e r i z a t i o n . T h e p o l y m e r i z i n g s u b u n i t o f a ^ - c a s e i n s B was 8 7 , 5 0 0 d a l t o n s w h i l e t h a t o f C v a r i a n t was 1 1 3 , 0 0 0 d a l t o n s . U s i n g a m o d i f i c a t i o n o f t h e S t e i n e r p r o c e d u r e ( S t e i n e r , 1952) t h e y c a l c u l a t e d t h e a s s o c i a t i o n c o n s t a n t s f o r s t e p w i s e p o l y m e r i z a t i o n u p t o t h e p e n t a m e r a t 2 , 9 , a n d 1 4 ° C . T h e r e s u l t s s h o w e d t h a t , f o r a g ^ - c a s e i n C , AH a n d AS w e r e p o s i t i v e a n d t h e f r e e e n t h a l p y d e c r e a s e d b y a c o n s t a n t a m o u n t o f 3 . 2 k c a l / m o l e a t e a c h a s s o c i a t i o n s t e p u p t o t h e p e n t a m e r . P a y e n s a n d S c h m i d t (1965) a n d P a y e n s (1966) s u g g e s t e d t h a t t h e p o l y m e r i z a t i o n was d u e t o h y d r o p h o b i c b o n d f o r m a t i o n b e t w e e n t h e p o l y m e r i z i n g u n i t s . S i n c e t h e e n t h a l p y c h a n g e a t e a c h s t e p was c o n s t a n t , t h e a u t h o r s c o n c l u d e d t h a t t h e same b o n d was f o r m e d b e t w e e n t h e h y d r o p h o b i c s i t e s o n t h e s u b u n i t s a n d t h a t t h e i n t e r a c t i n g s i t e s w e r e s u f f i c i e n t l y f a r a p a r t t o a v o i d m o r e t h a n o n e c o n t a c t b e t w e e n s u b u n i t s , t h e r e b y l i m i t i n g t h e n u m b e r o f p o s s i b l e c o n f i g u r a t i o n s f o r t h e p o l y m e r i z e d s p e c i e s . K a m i n o g a w a e t a l . (1974) o b s e r v e d t h a t a g l - c a s e i n e x i s t s a s a t e t r a m e r a t pH 7 . 1 , (y = 0 . 0 8 ) . A t pH 6 . 6 , a . - c a s e i n B e x i s t s a s a monomer a t an r s i i o n i c s t r e n g t h o f 0 . 0 1 , a s a d i m e r a t i o n i c s t r e n g t h 0 . 0 3 6 a n d a s a t e t r a m e r when t h e i o n i c s t r e n g t h i s 0 . 0 5 ( S c h m i d t , 1 9 7 0 ) . S c h m i d t a n d v a n M a r k w i j k (1968) c o n f i r m e d t h e d e p e n d e n c e o f a g ^ - c a s e i n B a s s o c i a t i o n o n i o n i c s t r e n g t h a n d c o n c l u d e d t h a t s e l f - a s s o c i a t i o n o f a - c a s e i n B was d u e n o t s i o n l y t o h y d r o p h o b i c i n t e r a c t i o n b u t a l s o d u e t o a n e l e c t r o -s t a t i c i n t e r a c t i o n a s w e l l . A t l o w c o n c e n t r a t i o n s ( l e s s t h a n 1 g/1) t h e m o l e c u l a r w e i g h t o f t h e a s s o c i a t i n g u n i t was f o u n d t o b e 2 9 , 0 0 0 d a l t o n s r e g a r d l e s s o f i o n i c s t r e n g t h . T h u s , a - c a s e i n B a t l o w c o n c e n t r a t i o n s e x i s t s a s t h e s i m o n o m e r , i n d e p e n d e n t o f i o n i c s t r e n g t h . A s i m i l a r o b s e r v a t i o n was made b y Ono e t a l . ( 1 9 7 4 ) , who o b s e r v e d t h a t a . - c a s e i n e x i s t s a s t h e monomer a t c o n c e n t r a t i o n s b e l o w s i 0 . 5 g / 1 (pH 6 . 9 , y = 0 . 0 9 ) w i t h s e l f - a s s o c i a t i o n o c c u r r i n g a t c o n c e n t r a t i o n s g r e a t e r t h a n 0 . 5 g / 1 . S c h m i d t a n d P a y e n s (197 2) c o n c l u d e d t h a t h y d r o p h o b i c b o n d i n g i s t h e m a i n d r i v i n g f o r c e i n t h e a s s o c i a t i o n o f a g ^ - c a s e i n B o n t h e b a s i s o f t h e r m o d y n a m i c d a t a o b t a i n e d f r o m l i g h t s c a t t e r i n g m e a s u r e m e n t s . T h e y a l s o f o u n d t h a t t h e i n t e n s i t y o f s c a t t e r e d l i g h t b y s o l u t i o n s o f a g ^ - c a s e i n B d e c r e a s e d w i t h i n c r e a s i n g p r e s s u r e . S i n c e h y d r o p h o b i c b o n d i n g i s a c c o m p a n i e d b y a n i n c r e a s e i n v o l u m e ( N e m e t h y a n d S c h e r a g a , 19 6 2 ) , t h e c a s e i n p o l y m e r s w o u l d b e e x p e c t e d t o d i s s o c i a t e u n d e r p r e s s u r e a s was o b s e r v e d . U s i n g s o l v e n t p e r t u r b a t i o n t e c h n i q u e s , Ono e t a l . (1974) o b s e r v e d t h a t d u r i n g s e l f - a s s o c i a t i o n o f c c g ^ - c a s e i n , t y r o s y l a n d t r y p t o p h a n y l r e s i d u e s w e r e b u r i e d , p o s s i b l y a s a r e s u l t o f i n t e r a c t i o n b e t w e e n h y d r o p h o b i c s e g m e n t s o f t h e 7 p r o t e i n c h a i n . M e r c i e r e t a l . (1971) presented evidence t h a t the t y r o s y l and t r y p t o p h a n y l r e s i d u e s of a . - c a s e i n are S _L l o c a l i z e d i n segments 90-110 and 140-175 of the primary s t r u c t u r e of the c a s e i n molecule. Ono e t a l . (1974) suggest-ed t h a t i n t e r c h a i n a s s o c i a t i o n of a . - c a s e i n occurs between s i segments 140-175 of i n t e r a c t i n g a g ^ - c a s e i n monomers. Ho and Chen (1967) s t u d i e d the p o l y m e r i z a t i o n of a g ^ - c a s e i n B by osmotic p r e s s u r e measurements at n e u t r a l pH and observed t h a t the degree of p o l y m e r i z a t i o n depended on the i o n i c strength. Thus, i n 0.01M potassium c h l o r i d e a t 4°C and 20°C mo l e c u l a r weights of 31,000 and 29,400 were obtained w h i l e molecular weights of 91,700 and 122,300 were obt a i n e d i n 0.1M potassium c h l o r i d e a t 4 and 20°C r e s p e c t i v e l y . The l a t t e r m o l e c u l a r weights would be expected to correspond to the t r i m e r and tetramer of the a g ^ - c a s e i n B monomer. Since the degree of p o l y m e r i z a t i o n was much more dependent on i o n i c s t r e n g t h than on temperature, they suggested t h a t e l e c t r o s t a t i c i n t e r a c t i o n s p l a y an important r o l e i n the p o l y m e r i z a t i o n of a ^ - c a s e i n B. These authors measured the v i s c o s i t y of the a . - c a s e i n B s o l u t i o n s s i a t the two i o n i c s t r e n g t h s to determine the shape of the molecule i n the monomeric and polymeric s t a t e s . The i n t r i n s i c v i s c o s i t i e s of the monomer v a r i e d from 11.8 ml/g to 10.2 ml/g as the temperature was i n c r e a s e d from 4.9 to 37°C i n 0.01M potassium c h l o r i d e . At pH 7.0 i n the presence of 0.IM potassium c h l o r i d e , the i n t r i n s i c v i s c o s i t i e s of the t r i m e r or tetramer v a r i e d from 9.3 ml/g to 7.7 ml/g from 4 8 to 20°C. A d i f f u s i o n c o e f f i c i e n t ( i n 0.IM sodium c h l o r i d e , -7 2 pH 7.08) D__ of 3.7 x 10 cm /sec was a f a c t o r of two l e s s . 20w than t h a t o f g l o b u l a r p r o t e i n s o f s i m i l a r m o l e c u l a r weight. I t i s a l s o w e l l known t h a t the i n t r i n s i c v i s c o s i t y of common g l o b u l a r p r o t e i n s vary from 3.3 to 4.0 ml/g. Thus i t was concluded t h a t a ^ - c a s e i n B i s not a s p h e r i c a l molecule. In a d d i t i o n , the i n t r i n s i c v i s c o s i t y o f a ,- c a s e i n B i n 6M 2 s i guanidine h y d r o c h l o r i d e was about twice t h a t o b t a i n e d i n the absence of guanidine h y d r o c h l o r i d e . Consequently, Ho and Chen (1967) concluded t h a t a - c a s e i n B does not e x i s t as a s i random c o i l , c h a r a c t e r i z i n g the s t r u c t u r e of a denatured p r o t e i n but r a t h e r i s r e l a t i v e l y compact as compared to a denatured p r o t e i n . Swaisgood and Timasheff (1968) , u s i n g l i g h t s c a t t e r i n g , u l t r a c e n t r i f u g a l and v i s c o m e t r i c techniques to study the a s s o c i a t i o n o f a ^ - c a s e i n C, confirmed the o b s e r v a t i o n s o f Ho and Chen (1967) t h a t the a s s o c i a t i o n i s s e n s i t i v e to i o n i c s t r e n g t h w i t h hi g h e r polymers being found at h i g h e r i o n i c s t r e n g t h . Swaisgood and Timasheff (1968) a l s o observed t h a t the dimer was the most s t a b l e u n i t between pH 8 and 9 while a t pH value s above 9 ( i o n i c s t r e n g t h 0.3) the monomer was pr e s e n t . They were unable to c l a s s i f y a g ^ - c a s e i n C as a s o l i d e l l i p s o i d o f r e v o l u t i o n or as a random c o i l as t h e i r v i s c o m e t r i c data were c o n s i s t e n t w i t h both models. The dimer however, c o u l d be d e s c r i b e d i n terms of a r i g i d g l o b u l a r compact s t r u c t u r e w h i l e a tetramer of mole c u l a r weight 110, 000 d a l t o n s c o u l d be d e s c r i b e d as a 9 p r o l a t e e l l i p s o i d of r e v o l u t i o n having an a x i a l r a t i o of o 20-25 or as a r i g i d rod of 560 A i n l e n g t h w i t h a c r o s s -o s e c t i o n a l area of 12.5 A. However, Kaminogawa e t a l . (1974), on the b a s i s of f r i c t i o n a l c o e f f i c i e n t c a l c u l a t i o n s and the Scheraga and Mandelkern constant,, suggested t h a t the o t g ^ -c a s e i n tetramer o f molecular weight 100,000 d a l t o n s i s g l o b u l a r i n shape. However, o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) measurements ( H e r s k o v i t s , 1966) and l i g h t s c a t t e r i n g data (Kresheck e t a l . 1964) i n d i c a t e d t h a t a , - c a s e i n i n s o l u t i o n a t s i n e u t r a l pH e x i s t s as a random c o i l almost completely devoid of a - h e l i c a l o r g a n i z a t i o n . H e r s k o v i t s (1966) r e p o r t e d the a - h e l i x content of a ^ - c a s e i n to be 4 - 15% depending on the procedure used i n c a l c u l a t i o n . a ,-Casein i s r e l a t i v e l y r i c h i n v a l i n e and s i J i s o l e u c i n e and c o n t a i n s 8.8 mole percent p r o l i n e ( H e r s k o v i t s , 1966). Szent-Gyorgi and Cohen (1957) observed t h a t 8.0 per-cent p r o l i n e u n i f o r m l y d i s t r i b u t e d along a p e p t i d e c h a i n w i l l deform the backbone of the p o l y p e p t i d e i n t o a random c o i l . In a d d i t i o n , Bloom e t a l . (1962) and K a t c h a l s k i e t a l . (1964) p o s t u l a t e d t h a t amino a c i d s such as v a l i n e and i s o l e u c i n e which have branched s i d e chains tend to r e s i s t h e l i x formation. I t has been observed t h a t the a - h e l i x content o f a , - c a s e i n s i s low i n s o l v e n t s such as 2 - c h l o r o e t h a n o l and s i a c i d i c methanol, which f a v o r a - h e l i x formation (Herskovits and Meschanti, 1965). I t has a l s o been observed t h a t 10 s t r u c t u r e - b r e a k i n g s o l v e n t s s u c h a s u r e a o r f o r m i c a c i d h a v e o n l y a s m a l l e f f e c t u p o n t h e o p t i c a l r o t a r y p r o p e r t i e s o f a ^ - c a s e i n , w i t h t h e M o f f i t - Y a n g p a r a m e t e r b Q c h a n g i n g f r o m - 3 0 i n p o t a s s i u m c h l o r i d e b u f f e r o f n e u t r a l pH t o 0 i n 8 . 0 M u r e a ( H e r s k o v i t s , 1 9 6 6 ) . T h e s e f i n d i n g s a r e c o n s i s t e n t w i t h a s s i g n m e n t o f a d i s c o r d e r e d , n o n - h e l i c a l , r a n d o m c o i l c o n f i g u r a t i o n t o a ^ - c a s e i n . T h e y may b e c o n t r a s t e d w i t h t h e s u g g e s t i o n s o f S w a i s g o o d a n d T i m a s h e f f ( 1 9 6 8 ) , Ho a n d C h e n (1967) a n d K a m i n o g a w a e t a l . (1974) w h i c h i n d i c a t e d t h a t a , - c a s e i n d o e s n o t e x i s t a s a r a n d o m c o i l b u t r a t h e r s i a s a r e l a t i v e l y c o m p a c t s t r u c t u r e . I n t e r a c t i o n o f a . . - C a s e i n w i t h C a + + s i A n i m p o r t a n t p r o p e r t y o f a ^ - c a s e i n i s i t s ' s e n s i t i v i t y t o p r e c i p i t a t i o n b y c a l c i u m i o n s , (Waugh e t a l . , 1 9 6 2 ) . C l a s s i c a l l y , a . - 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 . s i i s q u a n t i t a t i v e l y p r e c i p i t a t e d b y 8 mM c a l c i u m c h l o r i d e ( Z i t t l e , 1 9 6 3 ) . T h i s p r o p e r t y v a r i e s s o m e w h a t d e p e n d i n g o n t e m p e r a t u r e a n d t h e p h e n o t y p e . ++ T h e p r i m a r y s i t e s f o r C a b i n d i n g i n a g ^ - c a s e i n a r e t h e o r g a n i c p h o s p h a t e g r o u p s (Ho a n d W a u g h , 1 9 6 5 ) . H o w e v e r , t h e r e a r e a t l e a s t two c l a s s e s o f b i n d i n g s i t e s i n a ^ - c a s e i n . C a + - i o n s i n t e r a c t w i t h t h e e s t e r p h o s p h a t e g r o u p s o f s e r i n e a n d t h r e o n i n e w i t h w e a k e r i n t e r a c t i o n s o c c u r i n g w i t h t h e f r e e c a r b o x y l g r o u p s o f a s p a r t i c a n d ++ g l u t a m i c a c i d s ( D i c k s o n a n d P e r k i n s , 1 9 7 1 ) . B i n d i n g o f C a t o a , - c a s e i n B was o b s e r v e d t o i n c r e a s e w i t h t h e f r e e m e t a l s i i o n c o n c e n t r a t i o n a n d t o d e c r e a s e w i t h i n c r e a s i n g i o n i c 11 s t r e n g t h . The e f f e c t o f i o n i c s t r e n g t h i s most l i k e l y due to a supp r e s s i o n o f the a c t i v i t y o f the c a l c i u m i o n r a t h e r than by co m p e t i t i o n f o r b i n d i n g s i t e s ( Z i t t l e and Pepper, 1958; Dickson and P e r k i n s , 1971) . On the b a s i s o f i n f r a r e d s p e c t r o s c o p i c data, Ho and Waugh (1965) concluded t h a t the i n t e r a c t i o n of c a l c i u m w i t h the c a s e i n phosphate groups i s w e l l advanced j u s t p r i o r to the p o i n t where Ca- a^- c a s e i n a t e p r e c i p i t a t e s from s o l u t i o n . Noble and Waugh (1965) r e p o r t e d t h a t 6-8 moles of ca l c i u m are bound per mole of a g ^ - c a s e i n p r i o r to p r e c i p i t a -t i o n w i t h an a d d i t i o n a l 2 moles o f C a + + bound d u r i n g p r e c i p i t a t i o n . An average o f 11 moles of c a l c i u m were bound per mole o f p r e c i p i t a t e d a g ^ - c a s e i n . S i m i l a r r e s u l t s have been o b t a i n e d by Bingham e t a l . (1972) who concluded t h a t 7 moles of c a l c i u m are bound p r i o r to p r e c i p i t a t i o n w i t h an a d d i t i o n a l 3 moles bound d u r i n g the p r e c i p i t a t i o n p r o c e s s . The presence o f c a l c i u m - b i n d i n g s i t e s other than the phosphate r e s i d u e s i s suggested s i n c e a g ^ - c a s e i n has approximately 11 c a l c i u m - b i n d i n g s i t e s although there are only 8 phosphate r e s i d u e s per mole of p r o t e i n . These s i t e s c o u l d be the c a r b o x y l groups of glu t a m i c and a s p a r t i c a c i d s s i n c e dephosphorylated a g l - c a s e i n does not appear to b i n d any c a l c i u m p r i o r to p r e c i p i t a t i o n but does b i n d 2 moles of ca l c i u m per mole of p r o t e i n d u r i n g p r e c i p i t a t i o n (Dickson and P e r k i n s , 1971). Simple charge r e d u c t i o n by c a l c i u m b i n d i n g i s u n l i k e l y to be the s o l e f a c t o r f o r the c a l c i u m p r e c i p i t a t i o n 12 o f a s ^ - c a s e i n s i n c e o n l y a s l i g h t l y h i g h e r c a l c i u m c o n c e n t r a t i o n i s r e q u i r e d t o p r e c i p i t a t e d e p h o s p h o r y l a t e d a ^ - c a s e i n ( n e t c h a r g e - 1 0 t o - 1 4 ) t h a n i s r e q u i r e d f o r n a t i v e a g ^ - c a s e i n ( n e t c h a r g e - 2 2 ) . I n a d d i t i o n , d e p h o s p h o r y l a t e d a . - c a s e i n h a s a h i g h e r n e t c h a r g e b u t b i n d s S J-f e w e r c a l c i u m i o n s t h a n K - c a s e i n . U n l i k e t h e n a t i v e a n d d e p h o s p h o r y l a t e d a s ^ - c a s e i n s , K - c a s e i n d o e s n o t p r e c i p i t a t e i n t h e p r e s e n c e o f c a l c i u m ( B i n g h a m e t a l . , 1 9 7 2 ) . D a l g l e i s h (1973) i n v e s t i g a t e d t h e i n t e r a c t i o n o f c a l c i u m i o n s w i t h a ^ - c a s e i n b y f o l l o w i n g t h e c h a n g e i n f l u o r e s c e n c e o f t h e two t r y p t o p h a n r e s i d u e s i n a ^ - c a s e i n a s a c o n s e q u e n c e o f c a l c i u m b i n d i n g . A r e d s h i f t i n t h e f l u o r e s c e n c e e m i s s i o n s p e c t r u m o f a ^ - c a s e i n w i t h i n c r e a s i n g pH f r o m 5 . 6 t o 7 . 0 was i n t e r p r e t e d a s t h e t r y p t o p h a n r e s i d u e s b e i n g i n a m o r e h y d r o p h o b i c e n v i r o n m e n t a t pH 5 . 6 c o m p a r e d t o pH 7 . 0 a t w h i c h t h e p r o t e i n i s s l i g h t l y u n c o i l e d . R e m o v a l o f t h e N - t e r m i n a l t r y p t o p h a n w i t h c a r b o x y p e p t i d a s e A s u g g e s t e d t h a t 80% o f t h e o b s e r v e d f l u o r e s c e n c e was d u e t o t r y p t o p h a n -1 6 4 . C h a n g e s i n f l u o r e s c e n c e m a i n l y r e f l e c t c h a n g e s i n t h e m i c r o e n v i r o n m e n t a r o u n d t r y p t o p h a n - 1 6 4 . A n i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y was o b s e r v e d w i t h i n c r e a s i n g c a l c i u m i o n c o n c e n t r a t i o n a t a l l pH v a l u e s (pH 5 . 6 , 6 . 3 , 6 . 6 , a n d 7 . 0 ) . H o w e v e r , a b l u e s h i f t i n t h e e m i s s i o n s p e c t r u m o b s e r v e d a t pH 7 . 0 a n d 6 . 6 was n o t o b s e r v e d a t pH 5 . 6 a n d 6 . 3 . A t h r e e - s t e p a l t e r a t i o n i n t h e f l u o r e s c e n c e p r o p e r t i e s o f a g ^ - c a s e i n d u e t o c a l c i u m b i n d i n g was o b s e r v e d a t pH 7 . 0 w h i l e o n l y t h e l a t t e r two s t e p s w e r e 13 observed a t pH 5.6. The f i r s t step and the c o r r e s p o n d i n g b l u e s h i f t i n the emission spectrum, a t pH 7.0, may r e f l e c t a g e n e r a l t i g h t e n i n g of the p r o t e i n by n e u t r a l i z a t i o n of n e g a t i v e charges w i t h i n the p r o t e i n . B i n d i n g of 4 to 5 moles of c a l c i u m per mole of a g ^ - c a s e i n appears to occur i n the f i r s t s t e p / p r o d u c i n g a c o n f o r m a t i o n a l s t a t e t h a t may be s i m i l a r to the conformation of a . - c a s e i n a t pH 5.6 i n the s i absence of c a l c i u m i o n s . The second and t h i r d steps of the change i n f l u o r e s c e n c e w i t h i n c r e a s i n g c a l c i u m c o n c e n t r a t i o n at both pH 5.6 and 7.0 may correspond to f u r t h e r b i n d i n g o f C a + + i o n s which may induce aggregation e i t h e r by charge n e u t r a l i z a t i o n on by c a l c i u m mediated i o n i c bond formation between two p r o t e i n molecules. On the b a s i s of a c a l o r i m e t r i c study of the r e a c t i o n between c a l c i u m i o n s and a - c a s e i n , H o l t e t a l . s i (19 75) have concluded t h a t a t c a l c i u m i o n c o n c e n t r a t i o n s l e s s than 3 mM, r e a c t i o n of c a l c i u m w i t h a . - c a s e i n i s exothermic. s i T h i s was i n t e r p r e t e d to be due to c a l c i u m b i n d i n g with phosphate and c a r b o x y l groups accompanied by i n t r a m o l e c u l a r c r o s s l i n k i n g by Ca . T h i s b r i n g s together amino a c i d r e s i d u e s which can then form i n t r a m o l e c u l a r hydrogen bonds, r a t h e r than forming hydrogen bonds w i t h the s o l v e n t . A change i n the heat of r e a c t i o n from exothermic a t 3.3 mM C a C l 2 to endothermic a t 5 mM C a C l ^ w i t h a concomitant i n c r e a s e i n t u r b i d i t y was i n t e r p r e t e d to be due to a s s o c i a t i o n of the c a s e i n through the formation o f i n t e r m o l e c u l a r hydrophobic bonds. 14 T h u s i n t e r a c t i o n o f C a w i t h a . - c a s e i n c a u s e s a s i c o m p l e x s e r i e s o f c o n f o r m a t i o n a l c h a n g e s a n d a s s o c i a t i o n o f t h e p r o t e i n m o l e c u l e s . CARRAGEENAN C o m p o s i t i o n o f C a r r a g e e n a n C a r r a g e e n a n s a r e h i g h m o l e c u l a r w e i g h t , w a t e r s o l u b l e , s u l f a t e d g a l a c t a n s . T h e c a r r a g e e n a n s a r e f o u n d i n g e n e r a o f R h o d o p h y c e a e s u c h a s C h o n d r u s , G i g a r t i n a , a n d E u c h e u m a , w h e r e t h e y o c c u r i n t e r c e l l u l a r l y a n d i n t h e c e l l w a l l s ( S t o l o f f , 1 9 5 9 ) . T h e c a r r a g e e n a n s a r e w i d e l y u s e d i n t h e f o o d i n d u s t r y a s s t a b i l i z e r s , v i s c o s i t y b u i l d e r s a n d g e l l i n g a g e n t s . S m i t h a n d C o o k (1953) a n d S m i t h e t a l . (1954) f i r s t d e m o n s t r a t e d t h a t t h e c a r r a g e e n a n o f C h o n d r u s c r i s p u s was c o m p o s e d o f s e v e r a l d i s t i n c t p o l y s a c c h a r i d e s w h i c h c o u l d b e s e p a r a t e d o n t h e b a s i s o f d i f f e r i n g r e s p o n s e t o p o t a s s i u m i o n s . T h e K - c a r r a g e e n a n f r a c t i o n p r e c i p i t a t e s i n t h e p r e s e n c e o f p o t a s s i u m c h l o r i d e w h i l e t h e X - c a r r a g e e n a n f r a c t i o n r e m a i n s i n s o l u t i o n . T h e K - a n d X - c a r r a g e e n a n f r a c t i o n s w e r e f o u n d t o d i f f e r i n s u l f a t e c o n t e n t , g a l a c t o s e c o n t e n t a n d o p t i c a l r o t a t i o n . V i s c o s i t y a n d s e d i m e n t a t i o n v e l o c i t y s t u d i e s r e v e a l e d t h a t t h e X - f r a c t i o n was o f a l a r g e r m o l e c u l a r w e i g h t t h a n t h e K - c o m p o n e n t ( S m i t h e t a l . , 1 9 5 4 , 1 9 5 5 ) . K - C a r r a g e e n a n was s h o w n t o b e c o m p o s e d o f g a l a c t o s e a n d 3 , 6 - a n y d r o - D - G a l a c t o s e r e s i d u e s , w h i l e X - c a r r a g e e n a n 15 c o n t a i n e d m a i n l y D - g a l a c t o s e ( S m i t h e t a l . , 1 9 5 5 ; O ' N e i l l , 1 9 5 5 a , 1 9 5 5 b ) . O ' N e i l l (1955b) s u g g e s t e d t h a t K - c a r r a g e e n a n p o s s e s s e d a b a s i c s t r u c t u r e o f g a l a c t o s e u n i t s l i n k e d a l t e r n a t e l y a - 1 , 3 a n d 3 - 1 , 4 w i t h a l a r g e p r o p o r t i o n o f t h e 4 - l i n k e d u n i t o c c u r r i n g a s t h e 3 , 6 - a n y d r i d e . X-C a r r a g e e n a n h a s a s i m i l a r b a s i c s t r u c t u r e e x c e p t t h a t t h e 4 - l i n k e d u n i t o c c u r s a s t h e 2 , 6 - d i s u l f a t e ( R e e s , 1 9 6 3 ) . S u l f a t i o n i n K - c a r r a g e e n a n i s o n p o s i t i o n 4 o f t h e 3 - l i n k e d g a l a c t o s e u n i t s . A b o u t 2 0 - 2 5 % o f t h e 3 , 6 - a n h y d r o - D -g a l a c t o s e u n i t s a r e 2 - s u l f a t e d ( A n d e r s o n a n d R e e s , 1 9 6 5 ) . T h e r e i s a l s o e v i d e n c e t h a t some o f t h e a n h y d r i d e r e s i d u e s may b e r e p l a c e a t v a r i o u s p o i n t s i n t h e p o l y s a c c h a r i d e c h a i n b y 2 , 6 - d i s u l f a t e r e s i d u e s w h i c h a r e 4 - l i n k e d ( A n d e r s o n a n d R e e s , 1 9 6 5 ) . t - C a r r a g e e n a n p o s s e s s e s a s t r u c t u r e s i m i l a r t o t h a t o f K - c a r r a g e e n a n , e x c e p t t h a t t h e 3 , 6 - a n y d r o - g a l a c t o s e r e s i d u e i s 2 - s u l f a t e d . T h i s c a r r a g e e n a n , i s o l a t e d f r o m s p e c i e s o f E u c h e u m a , a l s o g e l s i n t h e p r e s e n c e o f p o t a s s i u m i o n s . P a i n t e r (1965) s u g g e s t e d t h a t t h e d i f f e r e n c e i n b e h a v i o r o f K - a n d A - c a r r a g e e n a n i n t h e p r e s e n c e o f K + may b e d u e i n p a r t t o h i g h c o n t e n t o f 3 , 6 - a n h y d r o - D - g a l a c t o s e i n K - c a r r a g e e n a n . 3 , 6 - A n h y d r o - D - g a l a c t o s e i s much l e s s h y d r o p h i l i c t h a n g a l a c t o s e s i n c e 3 , 6 - a n h y d r o - D - g a l a c t o s e d i e t h y l m e r c a p t a l i s s o l u b l e i n e t h e r , w h i l e g a l a c t o s e d i e t h y l m e r c a p t a l i s n o t e t h e r s o l u b l e ( O ' N e i l l , 1 9 5 5 b ) . I n a d d i t i o n , a g a r o s e , w h i c h c o n t a i n s much 3 , 6 - a n h y d r o g a l a c t o s e 16 b u t l i t t l e s u l f a t e , i s i n s o l u b l e i n c o l d w a t e r w h e r e a s a l l n o n - s u l f a t e d h o m o g a l a c t a n s a r e r e a d i l y s o l u b l e i n w a t e r ( P a i n t e r , 1 9 6 5 ) . T h u s , t h e p r e s e n c e o f s u l f a t e i n K - c a r r a g e e n a n r e n d e r s i t m o r e w a t e r - s o l u b l e t h a n a g a r o s e . K - C a r r a g e e n a n i s p r o b a b l y i n t r i n s i c a l l y much l e s s s o l u b l e t h a n A . - c a r r a g e e n a n i n w a t e r a n d t h e f r a c t i o n a l d e c r e a s e i n s o l u b i l i t y t h a t o c c u r s o n c h a n g i n g some o f t h e c o u n t e r i o n s f r o m s o d i u m t o p o t a s s i u m i s s u f f i c i e n t t o p r e c i p i t a t e K - c a r r a g e e n a n b u t n o t t h e m o r e h y d r o p h i l i c A - c o m p o n e n t . P e r n a s e t a l . (196 7) d e m o n s t r a t e d a r e l a t i o n -s h i p b e t w e e n p o t a s s i u m c h l o r i d e r e q u i r e m e n t f o r p r e c i p i t a t i o n a n d t h e 3 , 6 - a n y d r o - D - g a l a c t o s e c o n t e n t o f K - c a r r a g e e n a n . C o n f o r m a t i o n M o d e l b u i l d i n g s t u d i e s b a s e d o n x - r a y d i f f r a c t i o n d a t a f o r L - a n d K - c a r r a g e e n a n s r e v e a l t h a t t h e s e p o l y s a c c h a r i d e s c a n f o r m d o u b l e h e l i c e s s t a b i l i z e d b y i n t e r s t r a n d h y d r o g e n b o n d i n g ( A n d e r s o n e t a l . , 1 9 6 9 ) . I n t h e p r o p o s e d m o d e l t h e s u l f a t e g r o u p s a r e l o c a t e d o n t h e o u t s i d e o f t h e d o u b l e h e l i x . T h e d o u b l e h e l i x may b e s t a b i l i z e d b y i n t e r a c t i o n s b e t w e e n t h e s u l f a t e g r o u p s a n d c a t i o n s i n a d d i t i o n t o h y d r o g e n b o n d i n g b e t w e e n t h e h y d r o x y l g r o u p o n C - 4 o f g a l a c t o s e a n d t h e h y d r o x y l o n C - 2 o f i t s p a r t n e r i n t h e s e c o n d s t r a n d . I t i s known t h a t b o t h K - a n d i-carrageenans o c c u r n a t u r a l l y i n t h e g e l s t a t e . G e l a t i o n i s known t o i n v o l v e t h e c r o s s - l i n k i n g o r c l o s e a s s o c i a t i o n b e t w e e n two o r m o r e 17 p o l y s a c c h a r i d e c h a i n s . B o t h i- a n d K - c a r r a g e e n a n s f o r m + + + + o r i e n t e d f i b e r s a n d g e l a s t h e K , NHit , Rb , a n d C s s a l t s b u t n o t i n t h e L i + a n d N a + s a l t f o r m ( A n d e r s o n e t a l . , 1 9 6 9 ) . S i n c e t h e c a t i o n r e q u i r e m e n t s f o r f i b e r o r i e n t a t i o n a n d g e l a t i o n a r e t h e s a m e , t h e same t y p e o f m o l e c u l a r o r i e n t a t i o n may o c c u r i n o r i e n t e d f i b e r s a n d g e l s o f K - a n d t - c a r r a g e e n a n s . O p t i c a l r o t a t i o n s t u d i e s o f s o l u t i o n s o f p o t a s s i u m K - c a r r a g e e n a t e s u g g e s t t h a t t h e p o l y s a c c h a r i d e e x i s t s a s a r a n d o m c o i l a t t e m p e r a t u r e s a b o v e 4 0 ° C . A t l o w e r t e m p e r a t u r e s t h e s e p o l y s a c c h a r i d e c h a i n s b e g i n t o a s s o c i a t e f o r m i n g d o u b l e h e l i c e s w h i c h s u b s e q u e n t l y b e g i n t o a g g r e g a t e u n t i l a c o n t i n u o u s , t h r e e - d i m e n s i o n a l n e t w o r k i s f o r m e d ( R e e s e t a l . , 1969) . F u r t h e r e v i d e n c e o f d o u b l e h e l i x f o r m a t i o n i n s o l u t i o n h a s b e e n p r o v i d e d b y M c K i n n o n e t a l . (1969) a n d R e e s e t a l . ( 1 9 7 0 ) . S e g m e n t s o f t - c a r r a g e e n a n w e r e o b t a i n e d b y s p l i t t i n g t h e p o l y s a c c h a r i d e b y t h e S m i t h d e g r a d a t i o n a t t h e p o i n t s i n t h e c h a i n w h e r e 3 , 6 - a n h y d r o - g a l a c t o s e r e s i d u e s a r e r e p l a c e d b y 2 , 6 - d i s u l f a t e r e s i d u e s . T h e s e g m e n t s w e r e f u r t h e r t r e a t e d w i t h a l k a l i n e b o r o h y d r i d e t o r e m o v e 6 - s u l f a t e g r o u p s . A q u e o u s s o l u t i o n s o f t h e p o t a s s i u m s a l t s o f t h e s e s e g m e n t s r e v e a l e d c h a n g e s i n o p t i c a l r o t a t i o n w i t h c h a n g e s i n t e m p e r a t u r e , a t t r i b u t e d t o d o u b l e h e l i x f o r m a t i o n o n c o o l i n g a n d d i s s o c i a t i o n o f t h e d o u b l e h e l i c e s t o r a n d o m c o i l s o n h e a t i n g . S i m i l a r r e s u l t s f o r n a t i v e a n d s e g m e n t e d 18 K - c a r r a g e e n a n w e r e o b t a i n e d b y D e a e t a l . ( 1 9 7 2 ) . J o n e s e t a l . (1973) d e m o n s t r a t e d b y l i g h t s c a t t e r i n g a n d o s m o t i c p r e s s u r e m e a s u r e m e n t s o f ^ - - c a r r a g e e n a n s e g m e n t s i n 0 . IM N a C l , t h a t t h e o p t i c a l r o t a t i o n s h i f t s f i r s t o b s e r v e d b y M c K i n n o n e t a l . (1969) w e r e a c c o m p a n i e d b y a d o u b l i n g o f m o l e c u l a r w e i g h t o n c o o l i n g . 13 T h e i n t e n s i t y o f t h e C - n u c l e a r m a g n e t i c r e s o n a n c e ( n . m . r . ) s p e c t r u m o f ^ - - c a r r a g e e n a n s e g m e n t s i n 0 . 1 M N a C l d e c r e a s e s w i t h c o o l i n g f r o m 8 0 ° C t o 1 5 ° C . A l l o f t h e p e a k s o f t h e s p e c t r u m d i s a p p e a r a t a p o i n t c o r r e s p o n d i n g t o c o m p l e t e c o n v e r s i o n t o d o u b l e h e l i c e s a s j u d g e d b y o p t i c a l r o t a t i o n m e a s u r e m e n t s . I t a p p e a r s t h a t t h e m o b i l i t y o f t h e r e s i d u e s i n t h e h e l i x i s s o r e s t r i c t e d t h a t t h e r e l a x a t i o n t i m e s f o r t h e c a r b o n n u c l e i a r e d e c r e a s e d , c a u s i n g b r o a d e n i n g 13 a n d t h u s l o s s o f p e a k s f r o m t h e h i g h r e s o l u t i o n C - n . m . r . s p e c t r u m ( B r y c e e t a l . , 1 9 7 4 ) . R e i d e t a l . (1974) p r o p o s e d a m o d e l f o r t h e a s s o c i a t i o n p r o c e s s . A s s o c i a t i o n p r o d u c e s j u n c t i o n z o n e s i n g e l a t i o n o f L-carrageenan a n d i s p r o p o s e d t o b e a s i m p l e t w o - s t a t e , a l l - o r - n o n e p r o c e s s i n w h i c h t h e two c h a i n s e g m e n t s a r e e i t h e r c o m p l e t e l y i n d e p e n d e n t o f o n e a n o t h e r ( t h e r a n d o m c o i l c o n f o r m a t i o n ) o r f u l l y a s s o c i a t e d ( t h e d o u b l e h e l i x ) . I n a d d i t i o n , t h e h y d r o g e n b o n d p e r s a c c h a r i d e r e s i d u e w h i c h s t a b i l i z e d t h e d o u b l e h e l i x i n t h e s o l i d s t a t e may a l s o c o n t r i b u t e t o t h e s t a b i l i z a t i o n o f t h e d o u b l e h e l i x i n s o l u t i o n . T h e r e p l a c e m e n t o f t h e a n y d r i d e g r o u p s i n t h e 19 p o l y s a c c h a r i d e c h a i n of - L - and K-carrageenans by a few 2 , 6 - d i s u l f a t e r e s i d u e s causes k i n k i n g of the h e l i c a l carrageenan c h a i n s . T h i s r e s u l t s from the i n v e r t e d r i n g conformation of the 2 , 6 - d i s u l f a t e r e s i d u e s r e l a t i v e to the 3,6-anhydrogalactose r e s i d u e s . These k i n k s serve as h e l i x b r e a k i n g i n t e r r u p t i o n s t h a t enable each c h a i n to enter i n t o double h e l i x formation w i t h more than one p o l y s a c c h a r i d e c h a i n , necessary f o r the formation of a t h r e e - d i m e n s i o n a l g e l network (Rees, 1972). Bryce e t a l . (1974) estimated t h a t there are s i x independent double h e l i c e s formed i n the i n t a c t t-carrageenan p o l y s a c c h a r i d e . Goring and Chepeswick.(1955) observed t h a t , above -3 a c r i t i c a l c o n c e n t r a t i o n (2.5 x 10 g/ml), the sedimentation r a t e s f o r s e v e r a l sodium carrageenates of w i d e l y v a r y i n g molecular weights a t pH 5.5, y = 0.2 were co n c u r r e n t . Below the c r i t i c a l c o n c e n t r a t i o n the p l o t s of sedimentation r a t e vs c o n c e n t r a t i o n separated to g i v e the a p p r o p r i a t e v a l u e s o f sedimentation r a t e at zero c o n c e n t r a t i o n . They concluded t h a t a t c o n c e n t r a t i o n s above the c r i t i c a l c o n c e n t r a t i o n , the sedimenting system was i n the form of an i n t e r c o n n e c t i n g f i b r o u s network moving through the s o l v e n t a t a r a t e of s e d i m e n t a t i o n independent of m o l e c u l a r weight. However, below the c r i t i c a l c o n c e n t r a t i o n i t was assumed t h a t the carrageenan molecules sediment s i n g l y and v a r i a t i o n s i n sedimentation r a t e w i t h c o n c e n t r a t i o n are observed. Goring and Chepeswick (1955) proposed t h a t the sodium carrageenate 20 exists as helices somewhat sim i l a r to those observed for deoxyribonucleic acid. That carrageenan can e x i s t as helices has been demonstrated (Reid et a l . , 1974). Masson and Gaines (1954) observed a l i n e a r increase i n the reduced v i s c o s i t y of sodium carrageenan solutions (O.l.M NaCl) with increasing carrageenan concentration. However, the reduced v i s c o s i t y decreased with increasing carrageenan concentration when the carrageenan was dissolved -4 in water or i n 2.3 x 10 M NaHSO^ . Similar observations were made by Masson and Goring (1955) and Goring and Chepeswick (1955). Thus, the carrageenan i n d i l u t e aqueous solutions of low i o n i c strength became r i g i d l y extended due to repulsive forces from neighboring sulfate groups. This phenomenon was r e f l e c t e d by the large increases i n reduced v i s c o s i t y with decreasing carrageenan concentration. Goring (1954a) observed that the i n t r i n s i c v i s c o s i t y of sodium carrageenate solutions was constant between pH 3.5 and 11.6 (y = 0.13). This suggested that the shape of the molecule remained the same within that pH range. Molecular Weight The molecular weights of carrageenans have been the subject of a number of investigations. Cook et a l . (1952) performed sedimentation v e l o c i t y , d i f f u s i o n and electrophoretic studies on carrageenan extracted from Chondrus crispus. The sedimentation behavior of the carrageenan (pH 6.6,.y = 0.15) was observed to be concentration dependent. Peak spreading during sedimentation 21 e x c e e d e d t h a t t o b e e x p e c t e d f r o m d i f f u s i o n , i n d i c a t i n g p o l y d i s p e r s i t y . S a m p l e s o f h i g h i n t r i n s i c v i s c o s i t y c o n t a i n -e d a m i n o r , m o r e r a p i d l y s e d i m e n t i n g c o m p o n e n t , l a t e r i d e n t i f i e d a s X - c a r r a g e e n a n ( S m i t h e t a l . , 1 9 5 4 ) . T h e m o l e c u l a r w e i g h t s o f a n u m b e r o f p u r i f i e d K -a n d X - c a r r a g e e n a n f r a c t i o n s w e r e d e t e r m i n e d b y S m i t h e t a l . ( 1 9 5 4 , 1955) who u s e d t h e M a n d e l k e r n - F l o r y e q u a t i o n b a s e d o n t h e a s s u m p t i o n o f a r a n d o m c o i l ( M a n d e l k e r n a n d F l o r y , 1 9 5 2 ) . 5 T h e m o l e c u l a r w e i g h t s o f K - c a r r a g e e n a n r a n g e d f r o m 1 .8 x 10 5 t o 3 . 2 x 10 d a l t o n s w i t h t h e X - f r a c t i o n h a v i n g m o l e c u l a r 5 5 w e i g h t s b e t w e e n 4 x 10 a n d 7 x 10 d a l t o n s . S u b f r a c t i o n a -t i o n s t u d i e s r e v e a l e d t h a t b o t h K - a n d X - c a r r a g e e n a n , e x t r a c t e d f r o m a s i n g l e s o u r c e w e r e p o l y d i s p e r s e . B o n t o u x e t a l . ( 1 9 6 8 , 1970) a l s o d e m o n s t r a t e d t h e p o l y d i s p e r s i t y o f K - a n d X - c a r r a g e e n a n s o f C h o n d r u s c r i s p u s b y means o f l i g h t s c a t t e r i n g a n d o s m o t i c p r e s s u r e m e a s u r e m e n t s a s w e l l a s 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 p r o f i l e s o b t a i n e d b y c h r o m a t o g r a p h y o n p o r o u s s i l i c a . T h e s e a u t h o r s o b t a i n e d w e i g h t a v e r a g e m o l e c u l a r w e i g h t s (Mw) o f 1 . 7 x 10 t o 2 . 1 x 6 6 6 10 d a l t o n s f o r K - c a r r a g e e n a n a n d 4 . 5 x 10 t o 6 x 10 d a l t o n s f o r X - c a r r a g e e n a n . N u m b e r a v e r a g e m o l e c u l a r w e i g h t s 5 5 (Mn) o f 1 x 10 t o 1 . 3 4 x 10 d a l t o n s f o r K - c a r r a g e e n a n a n d 5 5 2 . 0 5 x 10 t o 2 . 6 5 x 10 d a l t o n s f o r X - c a r r a g e e n a n (pH 6 . 6 , y = 0 . 1 5 ) w e r e o b t a i n e d b y o s m o m e t r y . G o r i n g a n d Y o u n g (1955) o b t a i n e d m o l e c u l a r w e i g h t s o f 1 .1 x 1 0 6 t o 1 .4 x 1 0 6 d a l t o n s f o r X - c a r r a g e e n a n a n d 8 x 10^ t o 1 x 10^ d a l t o n s f o r K - c a r r a g e e n a n (pH 5 . 5 , y = 22 0 . 0 5 ) b y a p p l i c a t i o n o f t h e M a n d e l k e r n a n d F l o r y e q u a t i o n t o s e d i m e n t a t i o n v e l o c i t y a n d v i s c o s i t y d a t a . On t h e b a s i s o f o s m o t i c p r e s s u r e m e a s u r e m e n t s , M a s s o n a n d G o r i n g (1955) c o n c l u d e d t h a t t h e Mn o f a h i g h m o l e c u l a r w e i g h t c a r r a g e e n a n g s a m p l e was i n t h e o r d e r o f 2 . 5 x 10 d a l t o n s . T h u s t h e r e a p p e a r s t o b e a v e r y l a r g e v a r i a t i o n i n t h e m o l e c u l a r w e i g h t o f c a r r a g e e n a n s , d e p e n d e n t o n t h e s o u r c e a n d m e t h o d s o f e x t r a c t i o n a n d f r a c t i o n a t i o n . C A R R A G E E N A N - C A S E I N I N T E R A C T I O N S C a r r a g e e n a n s a r e u t i l i z e d i n a n u m b e r o f d a i r y p r o d u c t s t o c o n t r o l v i s c o s i t y , m a i n t a i n a d e s i r a b l e b o d y a n d t e x t u r e , p r e v e n t s e p a r a t i o n o f c o n s t i t u e n t s a n d f o r t h e i r p r o t e i n r e a c t i v i t y ( T o w l e , 1 9 7 3 ) . I n c h o c o l a t e m i l k , c a r r a g e e n a n i s e m p l o y e d a s a s u s p e n d i n g a g e n t f o r t h e f i n e c o c o a p o w d e r i n t h i s b e v e r a g e . R o s e a n d C o o k (1949) d e m o n s t r a t e d t h a t c a r r a g e e n a n , a t c o n c e n t r a t i o n s s i m i l a r t o t h o s e r e q u i r e d t o s t a b i l i z e c h o c o l a t e m i l k , m a r k e d l y i n c r e a s e d t h e v i s c o s i t y o f m i l k . I t was s u g g e s t e d t h a t i n t e r a c t i o n o f c a r r a g e e n a n w i t h t h e c a s e i n c o m p l e x p r e s e n t i n m i l k was r e s p o n s i b l e f o r t h e l a r g e i n c r e a s e s i n v i s c o s i t y o b s e r v e d u p o n t h e i n c o r p o r a t i o n o f s m a l l q u a n t i t i e s o f c a r r a g e e n a n i n m i l k . U s e o f h i g h e r c o n c e n t r a t i o n s o f c a r r a g e e n a n l e d t o t h e f o r m a t i o n o f w e a k , b u t d i s t i n c t g e l s . L a r g e a m o u n t s o f c a r r a g e e n a n (0.1%) c a u s e d t h e m i l k t o s e t t o a g e l w h i c h , u p o n s t i r r i n g , 23 s e p a r a t e d i n t o whey a n d c u r d i n d i s t i n g u i s h a b l e f r o m r e n n e t c u r d . T h e c a r r a g e e n a n - c a s e i n g e l h o w e v e r , m e l t e d u p o n w a r m i n g t o 4 0 ° C , t h e t e m p e r a t u r e a t w h i c h a q u e o u s c a r r a g e e n a n g e l s m e l t . K - C a r r a g e e n a n e x e r t e d a g r e a t e r i n f l u e n c e o n m i l k v i s c o s i t y t h a n d i d A - c a r r a g e e n a n ( S m i t h , 1 9 5 3 ) . H a n s e n (1968) d e m o n s t r a t e d t h a t K - c a r r a g e e n a n c o u l d s t a b i l i z e t h e C a + + - s e n s i t i v e a . - c a s e i n f r a c t i o n o f s i m i l k 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 i o n s . S t a b i l i t y was a c h i e v e d a t a c a r r a g e e n a n / a g ^ - c a s e i n r a t i o o f 1:5 f o r K - c a r r a g e e n a n w h i l e t h e A - f r a c t i o n was f u l l y e f f e c t i v e o n l y a t r a t i o s i n e x c e s s o f 2 : 5 . T h e c a r r a g e e n a n s t a b i l i z e d c o m p l e x was c o m p a r a b l e t o t h e K - a g ^ - c a s e i n c o m p l e x i n t h a t b o t h s y s t e m s w e r e o p a q u e a t pH 6 . 5 t o 7 . 0 a n d s t a b l e a t r o o m t e m p e r a t u r e . A n i n c r e a s e i n a l k a l i n i t y a b o v e pH 8 . 0 d i s s o c i a t e d t h e c a r r a g e e n a n s t a b i l i z e d m i c e l l e s . S t a b i l i z a -t i o n o f a g l - c a s e i n b y c a r r a g e e n a n p r e v e n t e d t h e u s u a l i s o e l e c t r i c p r e c i p i t a t i o n a t pH 4 . 6 . T h e c a r r a g e e n a n s t a b i l i z e d c o m p l e x e x h i b i t e d an i n c r e a s e d r e s i s t a n c e t o w a r d s d e s t a b i l i z a t i o n b y i n c r e a s e d c a l c i u m i o n c o n c e n t r a t i o n s a n d h e a t t h a n t h e c o r r e s p o n d i n g K - c a s e i n s t a b i l i z e d c o m p l e x . P r e c i p i t a t i o n a n d e l e c t r o p h o r e t i c m e t h o d s w e r e u s e d b y G r i n d r o d a n d N i c k e r s o n (196 8) i n a s t u d y o f t h e i n t e r a c t i o n b e t w e e n c a r r a g e e n a n s a n d t h e v a r i o u s p r o t e i n ++ f r a c t i o n s o f b o v i n e m i l k i n t h e p r e s e n c e o f C a . a . - a n d ^ s i 3 - c a s e i n s w e r e b o u n d b y c a r r a g e e n a n b u t w e r e r e l e a s e d u n d e r c o n d i t i o n s e m p l o y e d f o r e l e c t r o p h o r e s i s (pH 8 . 3 ) . H o w e v e r , H a n s e n (196 8) o b s e r v e d t h a t t h e a . - c a s e i n - c a r r a g e e n a n 24 c o m p l e x d i s s o c i a t e d when t h e p H e x c e e d s 8 . 0 . K - C a s e i n f o r m e d a c o m p l e x w i t h c a r r a g e e n a n , w h i c h c o u l d o n l y b e d i s r u p t e d b y u r e a ( 9 . 0 M) a n d 0 . 2 % 2 - m e r c a p t o -e t h a n o l , o r b y a d j u s t i n g t h e pH a b o v e 10 ( G r i n d r o d a n d N i c k e r s o n , 1 9 6 8 ) . P a y e n s (1972) c o n c l u d e d t h a t K - c a s e i n was t h e o n l y c a s e i n c o m p o n e n t w h i c h s p e c i f i c a l l y i n t e r a c t e d w i t h K - c a r r a g e e n a n i n a m i l k - s a l t s o l u t i o n . T h i s s t u d y was b a s e d o n d e t e c t i o n , b y l i g h t - s c a t t e r i n g , o f g e l - f o r m a t i o n b y c a s e i n - c a r r a g e e n a n m i x t u r e s i n m i l k - s a l t s o l u t i o n s u p o n c o o l i n g f r o m 8 0 ° C . A c c o r d i n g t o P a y e n s ( 1 9 7 2 ) , a - c a s e i n S J-d o e s n o t i n t e r a c t w i t h K - c a r r a g e e n a n i n t h e p r e s e n c e o f C a + + . R e c e n t l y , S n o r e n e t a l . (19 75) p r o v i d e d f u r t h e r e v i d e n c e t h a t K - c a s e i n i n t e r a c t s w i t h K - c a r r a g e e n a n a t pH 5 . 9 i n a c a l c i u m - f r e e s y s t e m b y a n e l e c t r o s t a t i c m e c h a n i s m . I n t e r a c t i o n b e t w e e n K - c a r r a g e e n a n a n d a ^ - o r B - c a s e i n s c o u l d n o t b e d e t e c t e d . T h e p r o c e d u r e s e m p l o y e d i n v o l v e d r e m o v a l o f t h e p r o t e i n - c a r r a g e e n a n i n t e r a c t i o n p r o d u c t s f r o m s o l u t i o n b y s e d i m e n t a t i o n a t 9 0 , 0 0 0 x G f o r 60 m i n a n d a n a l y s i s o f : t h e p r o t e i n a n d c a r b o h y d r a t e i n t h e s u p e r n a t a n t a t t h e v a r i o u s i o n i c s t r e n g t h s t e s t e d . H a n s e n (1968) a l s o r e p o r t e d t h a t a n i n t e r a c t i o n b e t w e e n a g ^ - c a s e i n a n d K - c a r r a g e e n a n c o u l d n o t b e d e t e c t e d , i n a c a l c i u m f r e e s y s t e m , b y f r e e b o u n d a r y e l e c t r o p h o r e s i s . T h u s t h e r e e x i s t s a v a r i e t y o f d a t a a b o u t i n t e r -a c t i o n s o f a ^ - c a s e i n w i t h K - c a r r a g e e n a n . W h e t h e r a g ^ - c a s e i n t r u l y i n t e r a c t s c h e m i c a l l y w i t h K - c a r r a g e e n a n i s a m a t t e r 25 w h i c h h a s y e t t o b e s o l v e d , i n v i e w o f t h e c o n f l i c t i n g e v i d e n c e o b t a i n e d b y v a r i o u s r e s e a r c h g r o u p s . H o w e v e r , K - c a r r a g e e n a n d o e s h a v e t h e a b i l i t y t o f o r m s t a b l e m i c e l l e s w i t h a g ^ - c a s e i n , a n d o t h e r c a l c i u m - s e n s i t i v e p r o t e i n s i n c a l c i u m - c o n t a i n i n g s y s t e m s ( H a n s e n , 196 8 ; L i n a n d H a n s e n , 1 9 7 0 ; C h a k r a b o r t y a n d R a n d o l p h , 1 9 7 2 ) . T h e r e a c t i v i t y o f c a r r a g e e n a n t o w a r d s a g ^ - c a s e i n d o e s n o t r e s i d e s o l e l y w i t h t h e e s t e r s u l f a t e r e s i d u e s s i n c e A - c a r r a g e e n a n c o n t a i n s a p p r o x i m a t e l y 35% e s t e r s u l f a t e w h i l e t h e m o r e r e a c t i v e K - c a r r a g e e n a n c o n t a i n s o n l y 30% e s t e r s u l f a t e ( H a n s e n , 1 9 6 8 ; L i n a n d H a n s e n , 1 9 7 0 ) . O t h e r s u l f a t e d p o l y s a c c h a r i d e s s u c h a s c h o n d r o i t i n s u l f a t e a n d h e p a r i n f a i l -e d t o s t a b i l i z e a g ^ - 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 i o n s ( H a n s e n , 1 9 6 8 ; L i n a n d H a n s e n , 1 9 7 0 ) . T h e 3 , 6 - a n h y d r o - D - g a l a c t o s e r e s i d u e s o f K - c a r r a g e e n a n do n o t a p p e a r t o b e e s s e n t i a l f o r t h e a ^ - c a s e i n s t a b i l i z a t i o n b y t h i s p o l y s a c c h a r i d e . L i n a n d H a n s e n (1970) d e m o n s t r a t e d t h a t A - c a r r a g e e n a n e x t r a c t e d f r o m G i g a r t i n a  a c i c u l a r i s , c a p a b l e o f c o m p l e t e s t a b i l i z a t i o n o f a ^ - c a s e i n , i s d e v o i d o f 3 , 6 - a n h y d r o - D - g a l a c t o s e r e s i d u e s a n d s u l f a t e e s t e r s o n t h e C - 6 p o s i t i o n , b u t c o n t a i n s s u l f a t e e s t e r s o n t h e C - 2 o r t h e C - 4 p o s i t i o n o f t h e g a l a c t o s e r e s i d u e s . T h e s t a b i l i z i n g a b i l i t y o f C . c r i s p u s A - c a r r a g e e n a n was i m p r o v e d b y a l k a l i t r e a t m e n t ( L i n a n d H a n s e n , 19 70) w h i c h e l i m i n a t e s t h e C - 6 s u l f a t e v i a a r i n g c l o s u r e l e a d i n g t o 3 , 6 - a n h y d r o - D - g a l a c t o s e f o r m a t i o n ( R e e s , 1 9 6 3 ) . T h u s , s u l f a t e 26 on the C-6 p o s i t i o n i s a n t a g o n i s t i c t o carrageenan s t a b i l i z i n g a b i l i t y . Any carrageenan with a l a r g e p r o p o r t i o n o f 3,6-anhydrogalactose i s , as a consequence, low i n C-6 s u l f a t e content. Thus, w i t h i n the carrageenans, the m i c e l l e -b u i l d i n g p r o p e r t i e s are dependent upon the presence and l o c a t i o n of s u l f a t e groups, t h e i r l o c a t i o n w i t h i n the pyranose u n i t s as w e l l as on the m o l e c u l a r weight o f the polymer. Other c a l c i u m s e n s i t i v e p r o t e i n s such as soy, peanut and coconut p r o t e i n s are s t a b i l i z e d from c a l c i u m p r e c i p i t a t i o n by K-carrageenan (Chakraborty and Randolph, 1972). T r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c examination of the carrageenan s t a b i l i z e d p r o t e i n s o l u t i o n s l e d Chakraborty and Randolph (1972) to suggest t h a t s t a b i l i z a t i o n may be a t t a i n e d by entrapment o f s m a l l , calcium-aggregated p r o t e i n bodies w i t h i n the carrageenan s t r u c t u r e . T h i s would prevent these p r o t e i n aggregates from f u r t h e r agglomeration i n t o l a r g e , c o l l o i d a l l y u n s t a b l e p a r t i c l e s . R e l a t i v e l y p r o t e i n - f r e e f i b e r r e g i o n s o f the p o l y s a c c h a r i d e were observed. I t was suggested t h a t these p r o t e i n - f r e e r e g i o n s corresponded to ion-aggregated double h e l i x r e g i o n s of low p r o t e i n r e a c t i v i t y , c o n s i s t e n t w i t h the double h e l i x j u n c t i o n zone model (Anderson e t a l . , 1969). The double h e l i x j u n c t i o n zones may p r o v i d e e f f e c t i v e s e p a r a t i o n of the entrapped p r o t e i n p a r t i c l e s from one another, as w e l l as i m p a r t i n g s t a b i l i t y to the system due to adequate s o l v a t i o n of these p r o t e i n - f r e e r e g i o n s . Thus, Chakraborty and Randolph (1972) proposed t h a t the s p e c i f i c i t y o f the i n t e r a c t i o n of K-carrageenan w i t h c a l c i u m s e n s i t i v e p r o t e i n s l i e s not so much wit h the p r o t e i n - p o l y s a c c h a r i d e i n t e r a c t i o n phenomenon i t s e l f , but r a t h e r i n the s p e c i f i c c o n f o r m a t i o n a l f e a t u r e s unique to K-carrageenan. 28 MATERIALS AND METHODS A l l chemicals used were o f reagent grade u n l e s s otherwise s p e c i f i e d and g l a s s d i s t i l l e d water was used throughout. a ^ - C a s e i n B was i s o l a t e d , by the method o f Z i t t l e and Custer (1963), from m i l k o b t a i n e d from a cow i n the U n i v e r s i t y o f B r i t i s h Columbia d a i r y herd. The cow was demonstrated to be homozygous f o r t h i s g e n e t i c v a r i a n t by phenotyping s t u d i e s , on the c a s e i n f r a c t i o n , employing 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 . The i s o l a t e d a ^ - c a s e i n was f r e e z e d r i e d and s t o r e d at 4°C i n a d e s i c c a t o r . The sodium s a l t of K-carrageenan ( V i s c a r i n , l o t #101203) was obt a i n e d from Marine C o l l o i d s Inc., S p r i n g f i e l d , NJ. I t was s t o r e d i n a d e s i c c a t o r at 4°C. S o l u t i o n s o f a s ^ - c a s e i n were prepared by d i s s o l v i n g d i r e c t l y i n t o the b u f f e r s used u n l e s s otherwise s t a t e d . Carrageenan s o l u t i o n s were prepared by d u s t i n g the carrageenan on to the s u r f a c e o f r a p i d l y s t i r r e d b u f f e r , t o prevent clumping, f o l l o w e d by h e a t i n g at 50 t o 55°C f o r 30 min w i t h s t i r r i n g . The c a s e i n and carrageenan s o l u t i o n s were c e n t r i f u g e d a t 10,000 X G f o r 10 min to remove any u n d i s s o l v e d m a t e r i a l . a - C a s e i n c o n c e n t r a t i o n s were s i determined from absorbance a t 280 nm us i n g A 2 ° Q of 10.0 (Waugh e t a l . , 1970) . Carrageenan c o n c e n t r a t i o n s were 29 d e t e r m i n e d b y t h e p h e n o l - s u l f u r i c a c i d m e t h o d o f D u b o i s e t a l . ( 1 9 5 6 ) . M o i s t u r e c o n t e n t o f t h e c a r r a g e e n a n was d e t e r m i n e d a c c o r d i n g t o t h e s t a n d a r d A . O . A . C . p r o c e d u r e ( 1 9 7 0 ) . I n f r a r e d s p e c t r a o f c a r r a g e e n a n w e r e o b t a i n e d u s i n g t h i n f i l m s p r e p a r e d a c c o r d i n g t o L i n a n d H a n s e n ( 1 9 7 0 ) . S p e c t r a w e r e r e c o r d e d w i t h a P e r k i n - E l m e r M o d e l 457 G r a t i n g I n f r a r e d S p e c t r o p h o t o m e t e r a g a i n s t a i r a s a r e f e r e n c e . 3 , 6 - A n h y d r o g a l a c t o s e c o n t e n t o f t h e c a r r a g e e n a n was d e t e r m i n e d u s i n g t h e p r o c e d u r e o f O ' N e i l l (1955a) a s m o d i f i e d b y P e a t e t a l . ( 1 9 6 1 ) . T U R B I D I T Y MEASUREMENTS T u r b i d i t y f o r m a t i o n i n a g ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e s was m e a s u r e d a s t h e t u r b i d a n c e a t 545 nm f o l l o w i n g t h e m e t h o d o f J o n e s a n d E r l a n d e r ( 1 9 6 7 ) . To a 5 m l t e s t t u b e c o n t a i n i n g 1 m l a g ^ - c a s e i n (20 m g / m l ) was a d d e d a s u f f i c i e n t v o l u m e o f b u f f e r a n d K - c a r r a g e e n a n (8 m g / m l ) t o y i e l d K - c a r r a g e e n a n / a s ^ - c a s e i n r a t i o s r a n g i n g f r o m 0 t o 0 . 3 5 (w/w) a n d a t o t a l v o l u m e o f 2 m l . C a r r a g e e n a n a n d c a s e i n b l a n k s w e r e a l s o p r e p a r e d . F o l l o w i n g i n c u b a t i o n a t 2 5 ° C f o r 1 h r t u r b i d i t y m e a s u r e m e n t s w e r e made w i t h a 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 u s i n g a 1 - c m m i c r o c u v e t t e ( 1 . 5 - m l v o l u m e ) . B u f f e r was u s e d a s a r e f e r e n c e s o l u t i o n . M e a s u r e m e n t s w e r e made 10 m i n a f t e r f i l l i n g t h e c u v e t t e . I t was o b s e r v e d t h a t 30 t h e a b s o r b a n c e d e c r e a s e d i m m e d i a t e l y a f t e r f i l l i n g t h e c u v e t t e , r e a c h i n g a s t e a d y l e v e l w i t h i n 10 m i n . T h e t u r b i d a n c e a t 545 nm was p l o t t e d a g a i n s t t h e K - c a r r a g e e n a n / a . , - c a s e i n r a t i o , s i S E D I M E N T A T I O N V E L O C I T Y A B e c k m a n 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 a l a c c e s s o r y a n d t h e P r e p . U . V . S c a n n e r was u s e d . U n l e s s s t a t e d o t h e r w i s e , a s p e e d o f 5 9 , 0 0 0 r p m a n d a t e m p e r a t u r e o f 2 5 ° C w e r e u s e d . A c h a r c o a l - f i l l e d e p o n d o u b l e s e c t o r c e l l (12 mm) was u s e d . T h e s a m p l e s e c t o r was f i l l e d w i t h 0 . 4 5 . m l s a m p l e w h i l e t h e s o l v e n t s e c t o r c o n t a i n e d 0 . 4 6 m l o f b u f f e r . A pH 6 . 6 p h o s p h a t e b u f f e r ( 0 . 0 0 2 1 M N a 2 H P 0 4 , 0 . 0 0 3 6 M N a H 2 P 0 4 / 0 . 0 7 M N a C l ) o f i o n i c s t r e n g t h 0 . 0 8 was u s e d , u n l e s s o t h e r w i s e s t a t e d . S t o c k s o l u t i o n s o f a - c a s e i n s i a n d K - c a r r a g e e n a n w e r e p r e p a r e d d a i l y . C o n c e n t r a t i o n s r a n g e d f r o m 0 . 1 mg t o 3 . 3 6 m g / m l f o r K - c a r r a g e e n a n a n d 0 . 5 mg t o 10 m g / m l f o r a ^ - c a s e i n when s c h l i e r e n o p t i c s w e r e u s e d . P r o t e i n c o n c e n t r a t i o n s o f 0 . 5 m g / m l a n d c a r r a g e e n a n c o n c e n t r a t i o n s o f 0 . 0 5 t o 1 m g / m l w e r e . e m p l o y e d i n c o n j u n c t i o n w i t h t h e u l t r a v i o l e t s c a n n e r . F o r e a c h s e t o f c o n d i t i o n s , i n d i v i d u a l s a m p l e s o f a ^ - c a s e i n a n d K - c a r r a g e e n a n a n d t h e r e s p e c t i v e i n t e r a c t i o n m i x t u r e s w e r e s u b j e c t e d t o s e d i m e n t a t i o n v e l o c i t y a n a l y s i s . a ^ - C a s e i n a n d K - c a r r a g e e n a n w e r e d i s s o l v e d s e p a r a t e l y , a t d o u b l e t h e d e s i r e d 31 c o n c e n t r a t i o n , i n t h e b u f f e r u t i l i z e d . T h e i n d i v i d u a l s o l u t i o n s w e r e t h e n m i x e d t o p r o v i d e t h e i n t e r a c t i o n m i x t u r e . T h e a ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n m i x t u r e s w e r e i n c u b a t e d f o r 1 h r a t 2 5 ° C p r i o r t o u l t r a c e n t r i f u g a t i o n . E f f e c t . o f I o n i c S t r e n g t h a n d pH I n o r d e r t o d e t e r m i n e t h e e f f e c t o f i o n i c s t r e n g t h o n t h e a s ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n , d e s i r e d i o n i c s t r e n g t h s w e r e o b t a i n e d b y i n c o r p o r a t i o n o f t h e r e q u i r e d q u a n t i t i e s o f s o d i u m c h l o r i d e i n t h e b u f f e r s . A pH 9 . 0 , 0 . 0 1 M g l y c i n e - N a O H b u f f e r c o n t a i n i n g 0 . 0 7 M N a C l was e m p l o y e d t o a s s e s s t h e e f f e c t o f a l k a l i n e pH o n t h e i n t e r a c t i o n . E f f e c t o f T e m p e r a t u r e T h e e f f e c t o f t e m p e r a t u r e o n t h e . a g ^ - c a s e i n -K - c a r r a g e e n a n i n t e r a c t i o n a t pH 6 . 6 , y = 0 . 0 8 was i n v e s t -i g a t e d b y c o n d u c t i n g s e d i m e n t a t i o n v e l o c i t y r u n s a t 4 , 2 5 , a n d 3 6 ° C . T h e r o t o r a n d a s s e m b l e d c e l l s w e r e e q u i l i b r a t e d t o 4 o r 36°C i n c o n t r o l l e d t e m p e r a t u r e r o o m s , p r i o r t o f i l l i n g t h e c e l l s . E f f e c t o f U r e a I n o r d e r t o i n v e s t i g a t e t h e e f f e c t o f d i s s o c i a t i n g a g e n t s , s a m p l e s o f a s ^ - c a s e i n , K - c a r r a g e e n a n a n d a s ^ - c a s e i n -K - c a r r a g e e n a n i n t e r a c t i o n m i x t u r e s w e r e p r e p a r e d i n p h o s p h a t e b u f f e r pH 6 . 6 , y = 0 . 0 8 . T h e s a m p l e s w e r e made up t o v o l u m e w i t h b u f f e r f o l l o w i n g t h e a d d i t i o n o f s o l i d u r e a t o a c o n c e n t r a t i o n o f 6 . 0 M a n d i n c u b a t e d a t 2 5 ° C f o r 1 h r 32 p r i o r to u l t r a c e n t r i f u g a t i o n . A s o l u t i o n o f phosphate b u f f e r made 6.0 M wit h r e s p e c t to urea was i n t r o d u c e d i n t o the s o l v e n t s e c t o r o f the c e l l . C a l c u l a t i o n o f s 0 _ 20,w At l e a s t s i x photographs were taken d u r i n g each run a t s e l e c t e d time i n t e r v a l s when s c h l i e r e n o p t i c s were used, w h i l e scans were a u t o m a t i c a l l y recorded by the U.V. scanner. The sedimentation c o e f f i c i e n t s were c a l c u l a t e d by the f o l l o w i n g r e l a t i o n s h i p (Chervenka, 1969): s 9 n = s , n t ^ s o l P )20,w 20,w obs n20 nw ( l - V ' p ) t , s o l where: s 0 r , = sedimentation c o e f f i c i e n t c o r r e c t e d to the value i t would have i n a s o l v e n t w i t h the v i s c o s i t y and d e n s i t y o f water at 20°C. s = l d r = 2.30 3 d l o g x obs — 2 2 to r d t 60 co ^ t , = observed sedimentation c o e f f i c i e n t to = angular v e l o c i t y i n r a d i a n s / s e c 2frrpm = 0.104 7 rpm 60 r = the r a d i a l d i s t a n c e between s c h l i e r e n peak and the c e n t r e of r o t a t i o n , t = time i n seconds t ' = time i n minutes 33 x = r a d i a l d i s t a n c e measured on photographic p l a t e s o r u l t r a v i o l e t scans b e f o r e c o r r e c t i o n s f o r l e n s m a g n i f i c a t i o n . T) = v i s c o s i t y o f water a t t degrees (temperature o f c e n t r i f u g e run) n20 = v i s c o s i t y o f water a t 20°C n s o l = v i s c o s i t y o f the sample s o l u t i o n a t t degrees n t r e l a t i v e to t h a t o f water a t t degrees. p„_ = d e n s i t y o f water a t 20°C 20, w 2 p. . = d e n s i t y o f sample s o l u t i o n a t t degrees 3 v =. p a r t i a l s p e c i f i c volume (cm /g) V i s c o s i t y measurements were made wit h an Ostwald c a p i l l a r y v iscometer having a flow time, f o r d i s t i l l e d water a t 25°C, of 197.7 sec when a sample volume of 1 ml was used. Temperature c o n t r o l , f o r v i s c o s i t y measurements, was achieved by means of a c i r c u l a t i n g water bath having an accuracy o f ±0.1°C. The v i s c o s i t i e s of the sample s o l u t i o n s were determined a t the same time as the sedimentation v e l o c i t y experiments were conducted. P a r t i a l s p e c i f i c volumes o f 0.51 ml/g (Smith e t a l . , 1954) and 0.73 ml/g (McKenzie and Wake, 1959) were used f o r K-carrageenan and ..c t g^-casein r e s p e c t i v e l y . S o l u t i o n d e n s i t i e s were c a l c u l a t e d from standard t a b l e s (Weast, 1973). 34 I n a d d i t i o n , t h e a s s u m p t i o n was made t h a t t h e v i s c o s i t y o f t h e s o l u t i o n s d u r i n g u l t r a c e n t r i f u g a t i o n w a s t h e same a s t h e b u l k s o l u t i o n v i s c o s i t y d e t e r m i n e d w i t h t h e O s t w a l d v i s c o m e t e r . A r e a C a l c u l a t i o n T r a u t m a n ' s Z - s c a l e m e t h o d ( T r a u t m a n , 1956) was e m p l o y e d t o c a l c u l a t e t h e w e i g h t a v e r a g e c e n t e r o f t h e s c h l i e r e n p e a k s , f o r s e d i m e n t a t i o n c o e f f i c i e n t c a l c u l a t i o n s a s w e l l a s t h e a r e a u n d e r t h e s c h l i e r e n p e a k s . I n t h i s 3 p r o c e d u r e , r a t h e r t h a n u s i n g a c t u a l v a l u e s o f x , t h e r a d i u s c u b e d s c a l e was i n d e x e d b y t h r e e d i g i t i n t e g e r s p r o p o r t i o n a l 3 t o x . T h u s Z , t h e " c u b e d - s c a l e n u m b e r i n d e x " was d e f i n e d 3 a s Z = ( 1 0 x / x r ) w h e r e t h e r e f e r e n c e , x ^ , was c h o s e n t o b e j u s t r a d i a l l y b e y o n d t h e b o t t o m o f t h e e n l a r g e d i m a g e ( 1 7 . 4 X ) o f t h e u l t r a c e n t r i g u g e c e l l . T h u s x r i n t h i s c a s e was 1300 mm. When x = 1300 mm, Z was e q u a l t o 1 0 0 0 . T h e w i d t h o f t h e Z - s c a l e i n t e r v a l s (AZ) w e r e s e l e c t e d t o p r o v i d e 15 p o i n t s a t w h i c h t h e h e i g h t o f t h e s o l u t i o n s c h l i e r e n p a t t e r n a b o v e t h e s o l v e n t s c h l i e r e n p a t t e r n (Ay) was m e a s u r e d . T h e Ay v a l u e was m e a s u r e d a t t h e c e n t r e o f t h e s u c c e s s i v e e q u a l AZ i n c r e m e n t s . T h e c e n t r e o f t h e s c h l i e r e n p a t t e r n was c a l c u l a t e d 2 f r o m E ( x / x . ) w h e r e x . was t h e v a l u e (mm) o n t h e a b s c i s s a o f r i l t h e e n l a r g e d s c h l i e r e n p a t t e r n c o r r e s p o n d i n g t o Z . D i v i s i o n 2 -b y £Ay y i e l d s ( x ^ / x ^ ) , f r o m w h i c h x , t h e c e n t r e p o s i t i o n o f t h e p e a k , w a s o b t a i n e d . D i v i s i o n o f x b y t h e m a g n i f i c a t i o n f a c t o r y i e l d s r , t h e r a d i a l d i s t a n c e o f t h e p e a k c e n t r e f r o m 35 the c e n t r e o f r o t a t i o n . The area under the s c h l i e r e n p a t t e r n was c a l c u l a t e d from the f o l l o w i n g : area = F tan 0AZ ZAy x •* where: F = x /3000 M _ x r 0 x = 1300 mm r M Q = m a g n i f i c a t i o n f a c t o r = m a g n i f i e d c e l l l e n g t h (cm) 1.6 cm tan 6 = tangent of the angle the s c h l i e r e n phase p l a t e makes wi.th the l i g h t source AZ = spacing i n t e r v a l on the Z s c a l e Ay = h e i g h t (mm) between the s o l u t i o n and s o l v e n t s c h l i e r e n p a t t e r n s a t each Z value C o r r e c t i o n s f o r r a d i a l d i l u t i o n were made by m u l t i p l y i n g the •o' 2 area by (T-) • T n e r a d i a l d i s t a n c e to the peak a t zero time (r n) was o b t a i n e d as the i n t e r c e p t from l i n e a r r e g r e s s i o n o f r on time. To c a l c u l a t e the area a t zero time a l i n e a r r e g r e s s i o n r 2 of F tan GAZ £y / _ t \ was performed on time. u 0 ; FRONTAL CHROMATOGRAPHY F r o n t a l chromatography was performed u s i n g a 0.9 x 75 cm column f i t t e d w i t h flow adapters (Chromatronix Inc; Berkeley, CA.). V o i d volume (VQ) was determined u s i n g Blue 36 Dextran 2000 (Pharmacia F i n e Chemicals; Uppsala, Sweden) whi l e sucrose was used to determine the t o t a l f l u i d volume (V^). The p r o t e i n e l u t i o n p r o f i l e s were monitored by absorbance a t 2 80 nm w h i l e carrageenan was monitored w i t h the phenol s u l f u r i c a c i d method (Dubois e t a l . , 1956). F r a c t i o n s of 0.5 ml were c o l l e c t e d . Sephadex G-200 (Pharmacia Fine Chemicals; Uppsala, Sweden) was used a t f i r s t . The b u f f e r was 0.01M phosphate, pH 6.6 c o n t a i n i n g 0.02% NaN^ as p r e s e r v a t i v e . A h y d r o s t a t i c head o f 14 cm of water was used, and the column was operated i n the upward flow mode. Samples of c t ^ - c a s e i n and a s ^ - c a s e i n -x-carrageenan i n t e r a c t i o n mixtures were a p p l i e d to the column i n volumes s u f f i c i e n t to form a p l a t e a u equal to the s t a r t i n g sample c o n c e n t r a t i o n p r i o r t o e l u t i o n w i t h b u f f e r . The a c t u a l volumes c o l l e c t e d were determined g r a v i m e t r i c a l l y by c o l l e c t i n g the f r a c t i o n s i n tared.tubes, assuming a s o l u t i o n d e n s i t y o f 1 g/ml (Winzor and Scheraga, 1963). C o n t r o l l e d pore g l a s s beads ( E l e c t r o n u c l e o n i c s Inc.; F a i r f i e l d , N J . ) , 200/400 mesh, having an average pore o diameter of 170 A were a l s o used. The g l a s s was t r e a t e d w i t h p o l y e t h y l e n e g l y c o l (Carbowax 20M; A p p l i e d Science L a b o r a t o r i e s ; S t a t e C o l l e g e , PA.) f o l l o w i n g the procedure of Hawk e t a l . (19 72). The t r e a t e d g l a s s was packed, i n the column d e s c r i b e d above, f o l l o w i n g the manufacturers d i r e c t i o n s . A constant flow r a t e of 0.5 ml/min was maintained 37 w i t h a B u c h l e r M i c r o - p u m p # 2 - 6 0 0 1 ( B u c h l e r I n s t r u m e n t s I n c . ; F o r t L e e , N J . ) . E x p e r i m e n t s w e r 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 ( 2 2 - 2 5 ° C ) . A p h o s p h a t e b u f f e r (pH 6 . 6 , y = 0 . 0 8 ) c o n t a i n i n g 0 . 0 2 % NaN^ was u s e d . T h e o p e r a t i n g c o n d i t i o n s w e r e s i m i l a r t o t h o s e d e s c r i b e d f o r t h e S e p h a d e x G - 2 0 0 c o l u m n . T h e e l u t i o n p r o f i l e s w e r e p l o t t e d a s c o n c e n t r a t i o n ( y g / m l ) a g a i n s t v o l u m e ( V e ) o f e f f l u e n t w h i c h f l o w e d t h r o u g h t h e c o l u m n , s i n c e t h e i n i t i a l a p p l i c a t i o n o f t h e s a m p l e o r f r o m t h e p o i n t a t w h i c h e l u t i o n w i t h b u f f e r c o m m e n c e d . F i r s t d e r i v a t i v e c u r v e s w e r e o b t a i n e d f r o m t h e e l u t i o n p r o f i l e b y p l o t t i n g t h e c h a n g e i n c o n c e n t r a t i o n f o r a 0 . 5 m l i n c r e m e n t i n v o l u m e / A C % a g a i n s t t h e mean v a l u e o f t h e e f f l u e n t v o l u m e (V) i n t h e i n t e r v a l A V . 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 ^ - c a s e i n was l a b e l l e d w i t h 1 - d i m e t h y l a m i n o n a p h -t h a l e n e - 5 - s u l f o n y l c h l o r i d e ( D N S - c h l o r i d e ) a c c o r d i n g t o R a w i t c h a n d W e b e r (19 7 2 ) . T h e f r e e z e d r i e d d a n s y l a t e d a - c a s e i n was s t o r e d i n a d e s i c c a t o r a t 4 ° C . s i C a r r a g e e n a n was d a n s y l a t e d e s s e n t i a l l y b y t h e p r o c e d u r e o f E r n s t a n d S c h i l l (1965) w i t h t h e f o l l o w i n g m o d i f i c a t i o n s . T h e e f f e c t o f pH o n t h e d e g r e e o f d a n s y l a t i o n was d e t e r m i n e d b y d a n s y l a t i n g a t pH 7 . 0 , 8 . 0 , 9 . 2 , a n d 1 0 . 2 u s i n g 0 . 1 M p h o s p h a t e b u f f e r s f o r pH 7 . 0 a n d 8 . 0 a n d 0 . 1 M N a H C O ^ - N a ? C O ^ b u f f e r s f o r pH 9 . 2 a n d 1 0 . 2 . T h u s , t o 350 mg 38 K - c a r r a g e e n a n d i s s o l v e d i n 25 m l o f t h e a p p r o p r i a t e b u f f e r , 2 5 mg D N S - c l o r i d e i n 5 m l a c e t o n e was a d d e d . T h e r e a c t i o n m i x t u r e was s t i r r e d i n t h e d a r k f o r 15 h r a t 4 ° C , f o l l o w e d b y d i a l y s i s a g a i n s t d i s t i l l e d w a t e r u n t i l t h e d i f f u s a t e was f r e e f r o m f l u o r e s c e n c e . F u r t h e r d e s a l t i n g was a c h i e v e d b y u l t r a f i l t r a t i o n u s i n g a n A m i n c o n P M - 3 0 m e m b r a n e u n t i l f l u o r e s c e n c e c o u l d n o t b e d e t e c t e d i n t h e u l t r a f i l t r a t e . F o l l o w i n g f u r t h e r d i a l y s i s a g a i n s t d i s t i l l e d w a t e r , t h e d a n s y l a t e d c a r r a g e e n a n was f r e e z e d r i e d a n d s t o r e d i n a d e s i c c a t o r a t 4 ° C . T h e d e g r e e o f l a b e l l i n g o f b o t h a ^ - c a s e i n a n d c a r r a g e e n a n was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y u s i n g a m o l a r e x t i n c t i o n c o e f f i c i e n t f o r t h e c o n j u g a t e d d a n s y l g r o u p s o f 3 . 4 x 1 0 3 M ~ 1 c m ~ 1 ( C h e n , 1 9 6 8 ) . F l u o r e s c e n c e e x c i t a t i o n a n d e m i s s i o n s p e c t r a w e r e r e c o r d e d w i t h a n A m i n c o Bowman s p e c t r o f l u o r o m e t e r 4 - 8 2 0 2 w i t h x e n o n l a m p a n d 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 a n d a M o s e l e y 7004A x - y R e c o r d e r . F l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t s w e r e made u s i n g P o l a c o a t UV 105 a n d P o l a r o i d HN 38 a s 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 . T h e s l i t s w e r e a s f o l l o w s : # 2 , 5 / 4 mm; # 3 , 4 / 2 mm a n d #7/1 mm. T h e e m i s s i o n was f i l t e r e d t h r o u g h 4 mm o f 1 M N a N 0 2 . D a n s y l a t e d a g ^ - c a s e i n (1 m g / m l ) i n p h o s p h a t e b u f f e r (pH 6 . 6 , y = 0 . 0 8 ) was t i t r a t e d w i t h K - c a r r a g e e n a n (20 m g / m l ) . E x c 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 350 a n d 508 nm r e s p e c t i v e l y . F l u o r e s c e n c e i n t e n s i t i e s w e r e r e c o r d e d w i t h a V a r i c o r d M o d e l 4 3 S t r i p C h a r t R e c o r d e r 39 (Photovolt Corp.; New York, NY.). Dansylated carrageenan (2 mg/ml) was t i t r a t e d with ct^-casein (50 mg/ml) . E x c i t a t i o n and emission wavelengths were 362 and 50 8 nm respectively. Fluorescence p o l a r i z a t i o n was calculated according to the method of Nakai and Kason (1974) . MOLECULAR WEIGHT DISTRIBUTION  FROM SEDIMENTATION EQUILIBRIUM Sedimentation equilibrium runs were performed using a Beckman L2-65B Ultracentrifuge equipped with schlieren optics. A c h a r c o a l - f i l l e d epon double sector centerpiece was used. One side of the centerpiece was f i l l e d with 0.18 ml of buffer (phosphate pH 6.6, y = 0.08), while the other side was f i l l e d with 0.12 ml of solution and 0.0 3 ml of fluorocarbon o i l . The sample solutions were dialyzed against 100 volumes of buffer for 24 hr. The dialysate was used i n the solvent channel. When a ,-casein-K-carrageenan in t e r a c t i o n s i ^ mixtures were used, the a ^ - c a s e i n and K-carrageenan were dialyzed separately i n the same d i a l y s i s chamber, then mixed and incubated at 25°C for 1 hr before u l t r a c e n t r i f u g a t i o n . Sedimentation equilibrium runs were conducted at 25°C using a speed of 5600 rpm. Photographs were taken a f t e r 24 hr. I n i t i a l solute concentrations were determined, using a synthetic boundary c e l l , on the same solutions 40 prepared f o r sedimentation e q u i l i b r i u m . The sample s e c t o r was f i l l e d w i t h 0.15 ml s o l u t i o n w h i l e 0.45 ml of d i a l y s a t e was added t o the s o l v e n t s e c t o r . A speed of 4000 rpm was used and the f i r s t photograph was taken when l a y e r i n g o f the s o l v e n t on the s o l u t i o n was complete. A bar angle o f 60 degrees was used. The area under the s c h l i e r e n peaks was c a l c u l a t e d by g r a p h i c a l i n t e g r a t i o n (Chervenka, 1969). M o l e c u l a r weight d i s t r i b u t i o n (MWD) w i t h i n the samples a t e q u i l i b r i u m were c a l c u l a t e d u t i l i z i n g the equations o f S c h o l t e (1969) t o c a l c u l a t e the MWD over a number of mol e c u l a r weight ranges, each range being r e p r e s e n t e d by i t s mean M^. Thus: U(A,5) = £ f.K.. x 1] 1 dc where U(A,£) = c Q d ? dc = the e q u i l i b r i u m c o n c e n t r a t i o n g r a d i e n t a t dC r a d i a l d i s t a n c e r . The h e i g h t o f the e q u i l i b r i u m s o l u t i o n s c h l i e r e n p a t t e r n (h^) above the s o l v e n t p a t t e r n was a measure o f dc d£ C Q — c o n c e n t r a t i o n o f the o r i g i n a l s o l u t i o n . C - was taken as the average h of the 0 r e q u i l i b r i u m pattern d i v i d e d by the r a t i o of the area under the e q u i l i b r i u m s c h l i e r e n p a t t e r n to the area under the 41 s y n t h e t i c b o u n d a r y s c h l i e r e n p a t t e r n T h e r e f o r e : h j :0 U = r f ^ = t h e w e i g h t f r a c t i o n o f m o l e c u l e s o f a g i v e n m o l e c u l a r w e i g h t , IVL . 2 2 A . M . e x p ( - £ . A . M . ) K. . = 1 i 3 1 -^1 - e x p ( - A . M . ) 3 1 w i t h A = ( 1 - v p ) co2 ( r 2 2 - r 1 2 ) / 2RT v = p a r t i a l s p e c i f i c v o l u m e o f t h e d i s s o l v e d s u b s t a n c e ( m l / g ) p = d e n s i t y o f t h e s o l u t i o n ( g / m l ) co = r o t o r s p e e d ( r a d i a n s / s e c ) R = u n i v e r s a l g a s c o n s t a n t T = a b s o l u t e t e m p e r a t u r e r ^ = r a d i a l d i s t a n c e f r o m t h e m e n i s c u s t o t h e c e n t r e o f r o t a t i o n r 2 = r a d i a l d i s t a n c e f r o m t h e b o t t o m o f t h e s o l u t i o n c o l u m n t o t h e c e n t r e o f r o t a t i o n 5 = ( r 2 2 - r 2 ) / ( r 2 2 - r 1 2 ) M^ = m o l e c u l a r w e i g h t o f c o m p o n e n t i T h u s , w i t h a c e r t a i n n u m b e r o f e x p e r i m e n t a l d a t a , U ( A , £) . w i t h A . a n d £ . , a s m a l l e r n u m b e r o f m o l e c u l a r 3 3 3 w e i g h t s w e r e c h o s e n . A s e r i e s o f m o l e c u l a r w e i g h t s M^ w i t h a r a n g e l a r g e e n o u g h t o i n c l u d e t h e w h o l e MWD was c h o s e n . T h u s , f o u r m o l e c u l a r w e i g h t s e r i e s w e r e s e l e c t e d , e a c h s e r i e s 1/4 s e p a r a t e d b y a f a c t o r o f 2 . W i t h i n e a c h m o l e c u l a r w e i g h t 42 s e r i e s , e a c h two s u c c e s s i v e t e r m s d i f f e r e d b y a f a c t o r o f 2. O n c e t h e m o l e c u l a r w e i g h t s e r i e s w e r e c h o s e n , t h e f o l l o w i n g s e t o f e q u a t i o n s c o u l d b e w r i t t e n : U ( X , ? ) l = f ^ + f 2 K 2 1 + f 3 K 3 1 + U ( X , C ) 2 = f ^ + f 2 K 2 2 + f 3 K 3 2 + U ( X , ? ) 3 = f x K 1 3 + f 2 K 2 3 + f 3 K 3 3 + U ( A , 5 ) = f , K _ + f_K„ + f , K _ + n 1 I n 2 2n 3 3n T h e c a l c u l a t i o n s w e r e p e r f o r m e d u s i n g a c o m p l e t e m u l t i p l e l i n e a r r e g r e s s i o n p r o g r a m w i t h a M o n r o e 1880 C a l c u l a t o r , e n t e r i n g t h e v a l u e s o f U 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 s w e r e e q u i v a l e n t t o f ^ . H o w e v e r , n e g a t i v e c o e f f i c i e n t s c a n b e o b t a i n e d . T h u s t h e a d o p t e d p r o c e d u r e i n v o l v e d t h e s e l e c t i o n o f m o l e c u l a r w e i g h t s e r i e s s u c h t h a t a n y n e g a t i v e c o e f f i c i e n t s o b t a i n e d w e r e a s c l o s e t o z e r o a s p o s s i b l e . S u b s e q u e n t l y , w i t h i n e a c h m o l e c u l a r w e i g h t s e r i e s , t h e m o l e c u l a r w e i g h t s c o r r e s p o n d i n g t o t h e n e g a t i v e c o e f f i c i e n t s w e r e d i s c a r d e d . T h e d a t a f o r t h e m o l e c u l a r w e i g h t s h a v i n g p o s i t i v e c o e f f i c i e n t v a l u e s w e r e r e e n t e r e d i n t h e m u l t i p l e r e g r e s s i o n p r o g r a m u n t i l a l l c o e f f i c i e n t s o b t a i n e d w e r e p o s i t i v e . T h e a b o v e p r o c e d u r e was f o l l o w e d f o r e a c h o f t h e f o u r m o l e c u l a r w e i g h t s e r i e s . T h e MWD was o b t a i n e d b y 43 p l o t t i n g f r a g a i n s t l o g molecular weight, STABILIZATION OF a -CASEIN WITH <-CARRAGEENAN S t a b i l i z a t i o n t e s t s were performed as d e s c r i b e d by Hansen (1968) w i t h a few m o d i f i c a t i o n s . The volume of the i n t e r a c t i o n mixtures, a f t e r a d d i t i o n o f a l l reagents, was 5 ml. A pH 6.6, 0.01 M imidazo l e - H C l b u f f e r was used. S u f f i c i e n t carrageenan (1.5 mg/ml), b u f f e r , and 1 ml of a . - c a s e i n (7.5 mg/ml) were added t o 15 ml c e n t r i f u g e tubes and incubated a t 25°C f o r 15 min. Then 0.5 ml of 0.1 M C a C l 2 i n i m i d a z o l e - H C l b u f f e r was s l o w l y added down the s i d e o f the c e n t r i f u g e tube followed by mixing w i t h a s p a t u l a ( f i n a l i o n i c s t r e n g t h , 0.04). A f u r t h e r i n c u b a t i o n o f 15 min a t 25°C was f o l l o w e d by c e n t r i f u g a t i o n a t 1000 *.G f o r 5 min. The s o l u b l e c a s e i n i n the supernatant was determined s p e c t r o p h o t o m e t r i c a l l y a t 280 nm a f t e r c l a r i f i c a t i o n by the a d d i t i o n o f 1 drop o f 5N NaOH. Absorbance due t o the presence o f the carrageenan was c o r r e c t e d by the f o l l o w i n g method (Ta l b o t and Waugh, 1970): A 2 8 0 c o r r ~ A280obs " 1 - 7 ( A 3 2 0 ) where: & 2 S Q c o r r = c o r r e c t e d absorbance a t 2 80 nm A280obs = o b s e r v e d absorbance at 280 nm A320 = a b s o r b a n c e at 320 nm 44 I o n i c s t r e n g t h s o t h e r than 0.04 were achieved by the a d d i t i o n o f NaCl to the b u f f e r such t h a t a f t e r a d d i t i o n o f CaCl„ the d e s i r e d i o n i c s t r e n g t h was a t t a i n e d . 45 RESULTS AND DISCUSSION A temperature of 50-55°C was used f o r s o l u b i l i z a -t i o n o f the K-carrageenan s i n c e d egradation of K-carrageenan i n s o l u t i o n a t n e u t r a l pH i s minimal a t temperatures below 60°C (Masson, 1955). Hansen and R e n o l l (1974) a p p l i e d a heat treatment o f 60°C f o r 30 min f o r the s o l u b i l i z a t i o n o f carrageenan u t i l i z e d i n s t u d i e s on the thermal degradation o f carrageenan d u r i n g food p r o c e s s i n g o p e r a t i o n s . Use o f temperatures below 50°C d i d not p r o v i d e complete s o l u b i l i z -a t i o n o f the carrageenan. The moisture content o f the carrageenan was 10.06 ±0.07%. In order to o b t a i n the r e q u i r e d dry weight, a c o r r e c t i o n f a c t o r o f 1.11 was a p p l i e d t o c o r r e c t f o r the moisture content of the carrageenan. The K-carrageenan c o n t a i n e d 16.1 ±0.19% 3,6-anhydro-D-galactose. During a c i d h y d r o l y s i s , 3,6-anhydro-D-galactose was c o n v e r t e d to 5-hydroxymethyl-2-furaldehyde (H.M.F.) which i n t u r n underwent a f i r s t o r d e r decomposition to formic and l e v u l i n i c a c i d s ( O ' N e i l l , 1955a). The p l o t of the l o g of the absorbance at 2 85 nm as a f u n c t i o n of time and e x t r a p o l a t i o n back to zero time accounted f o r the decomposition of H.M.F. ( F i g . 1). 3,6-anhydro-D-galactose content of K-carrageenan e x t r a c t e d from numerous samples o f C. c r i s p u s ranged from 46 Or - 0 . 2 0-4 00 CN < CO o -0.6 0.8 -l.Oh 0 8 16 H Y D R O L Y S I S T I M E , hr 2 4 F i g u r e 1. Course of the formation and disappearance of 5-hydroxymethyl-2-furaldehyde d u r i n g a n a l y s i s f o r 3,6-anhydro-D-galactose i n K-carrageenan. Each data p o i n t i s the average of 3 r e p l i c a t i o n s . 47 19.9% t o 28.4% w i t h a n a v e r a g e v a l u e o f 23.5% ( B l a c k e t a l . , 1965). T h u s , o n t h e b a s i s o f a n a s s u m e d c o n t e n t o f 23.5% 3 , 6 - a n h y d r o - D - g a l a c t o s e f o r p u r e K - c a r r a g e e n a n , t h e s a m p l e o f V i s c a r i n u s e d i n t h i s s t u d y w o u l d b e j u d g e d t o b e 68.5 ±0.7% p u r e w i t h r e s p e c t t o K - c a r r a g e e n a n . T h e i n f r a r e d s p e c t r u m o f t h e c a r r a g e e n a n ( F i g . 2) c o n t a i n s m a j o r a b s o r p t i o n b a n d s c e n t e r e d a t 930 cm 1 a n d a t 840 cm 1 . T h e b a n d a t 930 cm 1 h a s b e e n a t t r i b u t e d t o 3 , 6 - a n h y d r o - D - g a l a c t o s e . A b s o r p t i o n a t 840 cm 1 i s d u e t o t h e C-4 s u l f a t e . T h e f a c t t h a t t h i s a b s o r p t i o n b a n d i s :. . r a t h e r b r o a d i s . p r o b a b l y a n i n d i c a t i o n o f t h e p r e s e n c e o f some C-2 a n d C-6 s u l f a t e g r o u p s . B y d e f i n i t i o n p u r e K - c a r r a g e e n a n s h o u l d c o n t a i n o n l y C-4 s u l f a t e a n d some C-2 s u l f a t e e s t e r s ( M u e l l e r and. R e e s , 1967). P H E N O L - S U L F U R I C A C I D T E S T FOR CARRAGEENAN T h e e f f e c t o f i o n i c s t r e n g t h , a - c a s e i n a n d s i s o d i u m a z i d e o n t h e s e n s i t i v i t y a n d l i n e a r i t y o f t h e p h e n o l -s u l f u r i c a c i d m e t h o d f o r c a r r a g e e n a n a n a l y s i s was i n v e s t i g a t e d . T h e w a v e l e n g t h o f maximum a b s o r p t i o n o f t h e c o l o r c o m p l e x was 484 nm ( F i g . 3). A v a l u e o f 485 nm was r e p o r t e d b y P e r n a s e t a l . (1967). P l o t s o f a b s o r b a n c e a g a i n s t c o n c e n t r a t i o n o b e y e d B e e r ' s l a w w i t h i n t h e r a n g e o f 0 t o 175 y g K - c a r r a g e e n a n / m l t e s t e d ( F i g . 4). C h a n g e s i n i o n i c s t r e n g t h f r o m 0.01 t o 0.2 4 d i d n o t a p p r e c i a b l y a f f e c t t h e s e n s i t i v i t y o f t h e p h e n o l - s u l f u r i c F i g u r e 2. I n f r a r e d s p e c t r u m o f K - c a r r a g e e n a n CO 49 l . O r 350 400 450 500 550 600 WAV E L E N G T H , nm Figure 3. V i s i b l e spectrum of the color-complex from the phenol-sulfuric acid method for carrageenan determination. Spectrum a, 160 ug K-carrageenan/ml; Spectrum b, 160 ug K-carrageenan and 660 ug a s j - c a s e i n / ml; Spectrum c, 160 ug K-carrageenan/ml +0.02% NaN3; Spectrum d, 160 ug K -carrageenan and 660 ug a s]_-casein/ml +0.02% NaN_,. 0 50 100 150 / c - C A R R A G E E N A N ,/ i .g/ml F i g u r e 4. S t a n d a r d c u r v e s f o r p h e n o l - s u l f u r i c a c i d m e t hod o f c a r r a g e e n a n q u a n t i t a t i o n . L i n e a, K - c a r r a g e e n a n ; l i n e b, K - c a r r a g e e n a n + 660 yg a s ] _ - c a s e i n / m l ; l i n e c , K - c a r r a g e e n a n +0.02% N a ^ ; l i n e d, K - c a r r a g e e n a n +0.02% N a N 3 + 660 yg a s ] _ - c a s e i n / m l . D a t a r e p r e s e n t t h e a v e r a g e o f t h r e e t r i a l s . 51 a c i d method ( F i g . 4, T a b l e . I ) . The presence of a . - c a s e i n a t c o n c e n t r a t i o n s o f ^ s i 6 60 ug/ml d i d not have any e f f e c t on the c o l o r y i e l d per u n i t c o n c e n t r a t i o n o f carrageenan ( F i g . 4) . T h i s i s i n agreement with the o b s e r v a t i o n s o f Montgomery (1957) t h a t amino a c i d s and p r o t e i n s devoid o f carbohydrate d i d not i n t e r f e r e w i t h the p h e n o l - s u l f u r i c a c i d method. However, i n c l u s i o n o f 0.02% sodium a z i d e i n the b u f f e r markedly decreased the s e n s i t i v i t y of the phenol-s u l f u r i c a c i d method ( F i g . 4). I n c l u s i o n of 660, yg a - c a s e i n ^ 3 s i + 0.02% sodium a z i d e produced a standard curve i d e n t i c a l t o t h a t o b t a i n e d when o n l y 0.02% sodium a z i d e was present i n the b u f f e r . The presence o f 660 yg c t g ^ - c a s e i n , 0.02% sodium a z i d e o r 660 yg a ..-casein + 0.02% sodium a z i d e d i d not a l t e r S JL the wavelength o f maximum a b s o r p t i o n f o r the p h e n o l - s u l f u r i c a c i d method ( F i g . 3). Sodium a z i d e i n h i b i t s the anthrone method f o r carbohydrate a n a l y s i s by i n t e r f e r i n g w i t h the r e a c t i o n s l e a d i n g to formation o f the c o l o r e d complex (Wales, 1953). I t i s p o s s i b l e t h a t a s i m i l a r mechanism may be r e s p o n s i b l e f o r the d e p r e s s i o n i n s e n s i t i v i t y o f the p h e n o l - s u l f u r i c a c i d method by the az i d e i o n noted i n t h i s study. Thus, f o r a n a l y s i s o f carrageenan s o l u t i o n s prepared w i t h b u f f e r s c o n t a i n i n g sodium a z i d e , as a p r e s e r v a t i v e , a standard curve was prepared u t i l i z i n g standard carrageenan s o l u t i o n s prepared i n the same b u f f e r . 52 Table I EFFECT OF VARIOUS CONSTITUENTS ON THE COLOR YIELD OF THE PHENOL SULFURIC ACID TEST FOR K-CARRAGEENAN (100 yg/ml) C o n d i t i o n s A K - C B (y = 0.01) 0. 552 + 0. 006 K - C (y = 0.24) 0. 548 + 0. 002 K - C + 660 yg a s l C / m l (y = 0.01) 0 . 548 + 0. 007 K - C + 660 yg a ^ / m l (y = 0.24) 0. 547 + 0. 004 K - C + 0.02% NaN 3 (y = 0.01) 0. 459 + 0. 010 K - C + 0.02% NaN 3 (y = 0.24) 0. 451 + 0. 003 K - C + 0.02% NaN 3 + 660 yg a ^ / m l ( P = 0. 01) 0. 462 + 0. 004 K - C + 0.02% NaN 3 + 660 yg a s l / m l (y = 0. 08) 0. 456 + 0. 003 a. A l l v a l u e s are means ± standard d e v i a t i o n o f th r e e r e p l i c a t e s b. K-Carrageenan c. a n - C a s e i n 53 TURBIDITY FORMATION T u r b i d i t y f ormation o c c u r r e d on mixing a ..-casein J s i and K-carrageenan s o l u t i o n s a t pH . 6.6. a g ^ - C a s e i n (10 mg/ml) at pH 6.6, alone d i d not b r i n g about an i n c r e a s e i n the tu r b i d a n c e a t 545 nm r e g a r d l e s s o f the i o n i c s t r e n g t h . A s l i g h t i n c r e a s e i n tu r b i d a n c e w i t h i n c r e a s i n g c o n c e n t r a t i o n was noted f o r K-carrageenan alone ( F i g . 5). However, K - c a r r a g e e n a n - a g ^ - c a s e i n mixtures e x h i b i t e d i n c r e a s i n g t u r b i d i t y w i t h i n c r e a s i n g K-carrageenan c o n c e n t r a t i o n a t a constant a n - c a s e i n c o n c e n t r a t i o n . I o n i c s i s t r e n g t h had a marked e f f e c t on t u r b i d i t y f ormation upon c t g^-casein - K-carrageenan i n t e r m i x i n g . The t u r b i d i t y i n c r e a s e d w i t h i n c r e a s i n g i o n i c s t r e n g t h at each K-carrageenan - c t g^-casein r a t i o ( F i g . . 5) . However, dextran s u l f a t e (Mw = 500,000) appeared t o r e a c t o n l y s l i g h t l y w i t h a g ^ - c a s e i n ( F i g . 5 ) . L i n and Hansen (1970) observed t h a t dextran s u l f a t e d i d not s t a b i l i z e a . - c a s e i n from s i p r e c i p i t a t i o n by Ca , while K-carrageenan was an e f f e c t i v e s t a b i l i z e r . Since t u r b i d i t y formation may be an i n d i c a t i o n o f d e c r e a s i n g s o l u b i l i t y of one o f the components or o f the formation o f very l a r g e a g ^ - c a s e i n - K - c a r r a g e e n a n complexes, an attempt was made to c l a r i f y the i n t e r a c t i o n mixtures (y = 0.08) by c e n t r i f u g a t i o n a t 39000 x G f o r 30 min a t 25°C. A n a l y s i s o f the supernatant a f t e r c e n t r i f u g a t i o n r e v e a l e d 54 0 0.2 0.4 0.6 / C - C A R R A G E E N A N / C J , - C A S E I N , w/w Figure 5. E f f e c t of ion i c strength on t u r b i d i t y formation in a si-casein - K-carrageenan mixtures at pH 6.6. a s l ~ C a s e i n concentration (10 mg/ml). Line a, vi = 0.01; l i n e b, y = 0.08; l i n e c, y = 0.24; l i n e d, K-carrageenan alone at concentrations i d e n t i c a l to those used i n a s ^ - c a s e i n - K - c a r r a -geenan mixtures; l i n e e, dextran sulfate at concentrations i d e n t i c a l to those used for K-carrageenan, y = 0.08. 55 t h a t there was no change i n the c o n c e n t r a t i o n o f e i t h e r component due to c e n t r i f u g a t i o n ( F i g . 6). Thus, t u r b i d i t y f o rmation was probably not due t o a change i n s o l u b i l i t y o f e i t h e r a g ^ - c a s e i n o r K-carrageenan. T u r b i d i t y formation may be r e l a t e d t o the extent of aggregation o f c t ^ - c a s e i n and/or K-carrageenan. a ^ - C a s e i n undergoes s e l f - a s s o c i a t i o n w i t h i n c r e a s i n g i o n i c s t r e n g t h a t pH 6.6 (Derechin and Johnson, 1964; Ho and Chen, 1967; Schmidt, 1969). I o n i c s t r e n g t h a l s o appears t o a f f e c t the shape of K-carrageenan as the reduced v i s c o s i t y decreases w i t h i n c r e a s i n g i o n i c s t r e n g t h due to s h i e l d i n g o f r e p u l s i v e f o r c e s between n e i g h b o r i n g s u l f a t e groups (Masson and Gaines, 1954) . I t was d i f f i c u l t , however, to determine by t u r b i d i t y formation whether t h e r e was an a c t u a l i n t e r a c t i o n between a g ^ - c a s e i n and K-carrageenan o r whether the t u r b i d i t y observed was due to a change i n the s i z e of mol e c u l a r aggregates o f a ^ - c a s e i n or K-carrageenan as a consequence of the presence o f the o t h e r component. SEDIMENTATION VELOCITY T y p i c a l sedimentation p a t t e r n s f o r a g ^ - c a s e i n (10 mg/ml), K-carrageenan (1 mg/ml) and an a g ^ - c a s e i n - K -carrageenan i n t e r a c t i o n mixture (10 mg/ml; 1 mg/ml r e s p e c t -i v e l y ) a t pH 6.6, y = 0.08 are shown i n F i g . 7 and Table I I . 56 F i g u r e 6. E f f e c t o f c e n t r i f u g a t i o n a t 39,000 * G f o r 30 min on a s ] _ - c a s e i n ( l i n e a) and K - c a r r a g e e n a n ( l i n e b) c o n c e n t r a t i o n i n a s ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e s , (u = 0.08). S E D I M E N T A T I O N -> F i g . 7 S e d i m e n t a t i o n v e l o c i t y p a t t e r n s a . K - c a r r a g e e n a n (1 m g / m l , n r e ^ = 3 . 2 5 ) b. a . - c a s e i n (10 m g / m l , n n = 1 . 1 4 ) s i r e l c . a g ^ - c a s e i n (10 m g / m l ) + K - c a r r a g e e n a n (1 mg/ml) n r e l = 4 . 0 6 T i m e : 65 m i n a f t e r r e a c h i n g s p e e d ( 5 8 , 8 4 0 rpm) B a r a n g l e 70 d e g r e e s ; pH 6 . 6 , y = 0 . 0 8 . Table I I S E D I M E N T A T I O N C O E F F I C I E N T S F O R a . - C A S E I N - K - C A R R A G E E N A N I N T E R A C T I O N S T U D I E S C o n d i t i o n s Concentration s „ n (Svedbergs) (mg/ml) 2 0 ' w pH y T ( ° C ) a . a K-C° a n K - C I n t e r a c t i o n Mixture c s i s i Resid. I n t e r -K - C a c t i o n 6.6 0.08 25 10.0 0.1 4.51 n.d. n.d. 6. 04 6.6 0.08 25 10.0 0.5 4.51 8.42 7.64 9.66 6.6 0.08 25 10.0 1.0 4.51 9.61 9. 87 14.27 6.6 0.08 25 10.0 3.4 4.51 40.74 48.53 81.38 6.6 0.08 25 5.0 1.0 4. 36 9.61 10.45 14.01 6.6 0.08 4 10.0 1.0 4.90 11. 87 13.64 20.14 6.6 0.08 36 10.0 1.0 4.26 10. 06 9.32 12.88 6.6 0.24 25 10.0 1.0 7.61 11.26 10.87 18.67 9.0 0.08 2 5 d 10.0 1.0 3.12 8. 01 7.65 9.90 f 6.6 0.08 25 d 10.0 3.4 1.14 34.11 45.22 e 1.23 a. a .-Casein s i b. K-Carrageenan c. Not determined d. Contains 6.0 M urea e. K-Carrageenan peak of mixture c o n t a i n i n g 6.0 M urea f. a -Casein peak of mixture c o n t a i n i n g 6.0 M urea 59 I t can be observed t h a t a . - c a s e i n alone had an s„_ of s i 20,w 4.5S while K-carrageenan alone sedimented as a 9.6S peak. However, i n the s c h l i e r e n p a t t e r n of the i n t e r a c t i o n mixture a 4.5S peak was not observed. Instead, a l a r g e peak (14.3S) was observed sedimenting ahead of a smal l peak (9.9S). On the b a s i s of the absence of a 4.5S peak and the appearance o f the 14.3S peak i t was assumed t h a t the 14.3S peak r e p r e s e n t s an a ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n complex. I t would appear t h a t the a n - c a s e i n had i n t e r a c t e d w i t h a s i p o r t i o n o f the K-carrageenan s i n c e a s m a l l peak (9.9S) e x h i b i t i n g the s e l f - s h a r p e n i n g c h a r a c t e r i s t i c s (Cook e t a l . , 1952) o f the carrageenan was s t i l l observed. The area under the 9.9S peak ( F i g . 7) of the i n t e r a c t i o n mixture was l a r g e r than the area o f K-carrageenan of the same c o n c e n t r a t i o n (Table I I I ) . The i n c r e a s e i n area observed f o r the r e s i d u a l carrageenan peak, r a t h e r than the decrease expected due to i n t e r a c t i o n , may be due to the Johnston-Ogston e f f e c t (Johnston and Ogston, 1946). As a consequence o f the Johnston-Ogston e f f e c t the area under the slow peak i s i n c r e a s e d w h i l e t h a t o f the f a s t peak i s reduced. T h i s anomaly a r i s e s from the f a c t t h a t the molecules of the slow component move more s l o w l y i n the r e g i o n of the f a s t component than i n the r e g i o n behind the f a s t component. Thus, the molecules of the slow component p i l e up behind those o f the slow component i n the f a s t r e g i o n producing an i n v e r s e g r a d i e n t t h a t decreases the s i z e Table I I I AREA UNDER SCHLIEREN PATTERNS FOR a .-CASEIN-K-CARRAGEENAN INTERACTION STUDIES C o n d i t i o n s C o n c e n t r a t i o n Area (mm 2) a (mg/ml) pH y T ( ° C ) a . B K - C C a . K-C I n t e r a c t i o n Mixture ( a , + K - C ) D s i s i s i Res i d . I n t e r - (a . ) + (K-C) K-C a c t i o n s 6.6 0.08 25 10.0 0.1 7.64 . e n.d. n.d. 7.63 n.d. 6.6 0.08 25 10.0 0.5 7.64 0. 31 1.67 5. 84 0. 95 6.6 0.08 25 10.0 1.0 7.64 0.57 1.26 5.47 0.82 6.6 0.08 25 10.0 3.4 7.64 n.d. 2.03 4.85 n.d. 6.6 0.08 25 5.0 1.0 3.70 0.57 1.36 2.69 0. 95 6.6 0.08 4 10.0 1.0 7.63 0.67 1.10 5. 38 0.78 6.6 0.08 36 10.0 1.0 7. 30 0.60 1.94 5.95 1.00 6.6 0.24 25 10.0 1.0 7.66 0.60 1.25 6. 31 0. 92 9.0 0.08 25 10.0 1.0 7.54 0.65 2.21 5.33 0. 92 6.6 0.08 25 f 10.0 3.4 6.26 1.54 3.32 g 4.72 n 1.03 a. E x t r a p o l a t e d t o zero time of c e n t r i f u g a t i o n b. a .-Casein s i c. K-Carrageenan d. R a t i o of area under p a t t e r n f o r a s ^ - c a s e i n and K-carrageenan mixture to the sum o f the area under p a t t e r n s when a g ^ - c a s e i n and K-carrageenan were c e n t r i f u g e d s e p a r a t e l y . e. Not determined f. Contains 6.0 M urea g. K-Carrageenan peak of mixture c o n t a i n i n g 6.0 M urea h. a s j - C a s e i n peak of mixture made c o n t a i n i n g 6.0 M urea 61 of the f a s t s c h l i e r e n peak observed. The e f f e c t s o f changing carrageenan c o n c e n t r a t i o n while m a i n t a i n i n g a ^ - c o n c e n t r a t i o n a t 10 mg/ml are presented i n Table I I . For K-carrageenan, S 2 Q w i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n ( F i g . 8). T h i s may be an i n d i c a t i o n o f c o n c e n t r a t i o n dependent ag g r e g a t i o n . L i k e w i s e , the i n t e r a c t i o n peak d i s p l a y s a r a p i d i n c r e a s e i n s„_ from 2U,w 6.OS at a K - c a r r a g e e n a n / a ^ - c a s e i n weight r a t i o o f 0.01 to a value o f 81.4S a t a weight r a t i o o f 0.34 ( F i g . 9). The s e p a r a t i o n between the r e s i d u a l carrageenan and the i n t e r -a c t i o n peak decreased as the carrageenan c o n c e n t r a t i o n i n the i n t e r a c t i o n mixture decreased. However, i n a l l of the above cases a 4.5S peak, corresponding to unreaeted a g ^ - c a s e i n , was not observed. I f a . - c a s e i n and.K-carrageenan d i d not i n t e r a c t , s i one would expect t o observe a carrageenan peak f o l l o w e d by the slower sedimenting a n - c a s e i n . Since the r a t e of s i s e dimentation f o r a g i v e n molecular weight p a r t i c l e decreases w i t h i n c r e a s i n g s o l u t i o n v i s c o s i t y , the peak sedimenting ahead o f the r e s i d u a l K-carrageenan must be of a much high e r m o l e c u l a r weight than the K-carrageenan alone. In a d d i t i o n , the observed reduced v i s c o s i t i e s (Table IV) f o r the i n t e r a c t i o n mixtures are l a r g e r than those c a l c u l a t e d by the method of Mark and Whitby (1946). I f a s ^ - c a s e i n and K-carrageenan d i d not i n t e r a c t the observed and c a l c u l a t e d reduced v i s c o s i t i e s should have been the same oi 0 4 / c - c a r r a g e e n a n _L 0 6 8 C O N C E N T R A T I O N , m g / m | 10 0 f s l - c a s e i n F i g u r e 8 . C o n c e n t r a t i o n dependence o f S 2 Q W ( P H 6 . 6 , ii = 0 . 08 , 25 C) ; l i n e a, a s ] _ - c a s e i n ; l i n e b, K - c a r r a g e e n a n . D a t a r e p r e s e n t s t h e a v e r a g e o f two r u n s . cn M 80h 60 40 20 0' 0 0.2 0.4 K - C A R R A G E EN A N / a s - C A S E I N , w/w F i g u r e 9. E f f e c t o f c o n c e n t r a t i o n on S2o,w °f t n e a s ] _ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n complex a s i - c a s e i n c o n c e n t r a t i o n ( 1 0 mg/ml); pH 6 . 6 , ii = 0 . 0 8 , 2 5 ° C . D a t a r e p r e s e n t s t h e a v e r a g e o f two r u n s . T a b l e I V REDUCED V I S C O S I T I E S OF a . - C A S E I N , K-CARRAGEENAN AND a _ - C A S E I N - K - C A R R A G E E N A N M I X T U R E S C o n d i t i o n s C o n c e n t r a t i o n Reduced V i s c o s i t y (ml/g) (mg/ml) pH y T ( ° C ) a . A K-C* 3 a . . K - C I n t e r a c t i o n M i x t u r e s i s i c d Obs. C a l . 6.6 0.08 25 10.0 0.1 13.9 1450 35.6 28.1 6.6 0.08 25 10.0 0.5 13.9 1882 143.2 102.9 6.6 0.08 25 10.0 1.0 13.9 2257 278.4 217. 8 6.6 0.08 25 10.0 3.4 13.9 6643 2113.7 1695.9 6.6 0.08 25 5.0 1.0 10.4 2257 498.3 384.8 6.6 0.08 4 10.0 1.0 11.9 2564 345.4 243.9 6.6 0.08 - 36 10.0 1.0 9.8 2343 266.2 221.9 6.6 0.24 25 10.0 1.0 16.1 1855 193.3 183.4 9.0 0.08 25 10.0 1.0 12.7 1968 215.4 190.5 6.6 0.08 2 5 e 10.0 3.4 20. 3 9851 2493.0 2514.0 6.6 0.08 25 0.5 0.5 6.1 1882 945.2 944.0 6.6 0.01 25 0.5 0.5 3.6 2802 1403.0 1402.0 a. a . - C a s e i n s i b. K - C a r r a g e e n a n c. Reduced v i s c o s i t y o b s e r v e d d. C a l c u l a t e d r e d u c e d v i s c o s i t y a s s u m i n g no i n t e r a c t i o n e. C o n t a i n s 6.0 M u r e a 65 (Takeuchi, 1969). On the b a s i s o f the d i f f e r e n c e i n c a l c u l a t e d and observed reduced v i s c o s i t i e s the extent o f a g ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n appears t o have i n c r e a s e d w i t h . i n c r e a s i n g carrageenan c o n c e n t r a t i o n (Table I V ) . M a i n t a i n i n g K-carrageenan a t 1 mg/ml and lowering the a - c a s e i n c o n c e n t r a t i o n to 5 mg/ml y i e l d e d a 14S i n t e r -s i a c t i o n peak. a g ^ - C a s e i n (5 mg/ml) sedimented a t a r a t e o f 4.4S (Table I I ) . Thus, d e c r e a s i n g the p r o t e i n c o n c e n t r a t i o n i n the i n t e r a c t i o n mixture to 5 mg/ml d i d not produce any l a r g e changes i n the number of peaks observed or i n the sedimentation r a t e of the i n t e r a c t i o n peak. However, by lowering the a g ^ - c a s e i n c o n c e n t r a t i o n t o 0.5 mg/ml and use of the u l t r a v i o l e t scanner r e v e a l e d o n l y one boundary (5.5S) f o r a mixture o f a g ^ - c a s e i n (0.5 mg/ml) and K-carrageenan (0.5 mg/ml) ( F i g . 10). a g ^ - C a s e i n (0.5 mg/ml) has an s o f 3.2S compared t o 4.5S at 10 mg/ml. 2. (J f w Thus, a t a c o n c e n t r a t i o n o f 0.5 mg/ml (pH 6.6, y = 0.0 8) a . - c a s e i n e x i s t s i n an a s s o c i a t e d s t a t e s i n c e s»„ o f the s i 20,w a g l ~ c a s e i n monomer i s approximately IS (Schmidt e t a l . , 1967). T h i s i s i n agreement with the f i n d i n g s o f Ono e t a l . (1974). K-Carrageenan d i d not absorb a t 2 80 nm and thus, c o u l d not be d e t e c t e d with the U.V. o p t i c s ( F i g . 10). However, on the b a s i s o f sedimentation v e l o c i t y a n a l y s i s u s i n g s c h l i e r e n o p t i c s , K-carrageenan (0.5 mg/ml, pH 6.6, y = 0.08) sedimented at a r a t e o f 8.42S (Table I I ) . Thus, a t low a - c a s e i n c o n c e n t r a t i o n s (0.5 mg/ml) 66 S E D I M E N T A T I O N * Figure 10. U l t r a v i o l e t scans of sedimentation v e l o c i t y patterns (pH 6.6, y = 0.08). Scan A, us±-casein (0.5 mg/ml), S20 w = 3.07S; scan B, K-carrageenan (0.5 mg/ml); scan C, a s]_-casein (0.5 mg/ml) + K-carrageenan (0.5 mg/ml), s20,w = 3.12S. Time, 55 min a f t e r reaching speed (59,140 rpm). an i n t e r a c t i o n between a g ^ - c a s e i n and K-carrageenan d i d not appear to take p l a c e s i n c e only one sedimenting boundary wi t h a S 2 Q w l e s s than t h a t o f K-carrageenan (0.5 mg/ml) was observed. In a d d i t i o n , the observed reduced v i s c o s i t y was equal t o the c a l c u l a t e d reduced v i s c o s i t y i n d i c a t i n g t h a t an i n t e r a c t i o n d i d not occur under these c o n d i t i o n s (Table I V ) . For these d i l u t e s o l u t i o n s , c o r r e c t i o n o f the observed sedimentation c o e f f i c i e n t f o r the bulk s o l u t i o n v i s c o s i t y d i d not appear t o be v a l i d s i n c e the a g ^ - c a s e i n component of the mixture sedimented at a slower r a t e than the carrageenan component and'was t h e r e f o r e probably i n a medium having the v i s c o s i t y o f the a ^ - c a s e i n . Thus, without c o r r e c t i o n f o r the bulk s o l u t i o n v i s c o s i t y , the s„. u 20 ,w of the sedimenting boundary f o r the. i n t e r a c t i o n mixture remained unchanged from t h a t o f a s ^ - c a s e i n (0.5 mg/ml). The e f f e c t o f hig h pH and h i g h i o n i c s t r e n g t h was i n v e s t i g a t e d t o asses the p o s s i b i l i t y t h a t the a g ^ - c a s e i n - i c -carrageenan i n t e r a c t i o n i s e l e c t r o s t a t i c i n nature. Hansen (1968) observed t h a t the c a l c i u m mediated i n t e r a c t i o n between a ^ - c a s e i n and K-carrageenan was d i s r u p t e d by an i n c r e a s e i n pH above 8.0. A s i m i l a r phenomenon was noted by G r i n d r o d and N i c k e r s o n (196 8). A 0.01 M glycine-NaOH b u f f e r (pH 9.0, y = 0.08) was used. a -Casein (10 mg/ml) e x h i b i t e d a decreased sedimentation r a t e (3.IS) compared to 4.5S a t pH 6.6 (Table II) Schmidt e t a l . (1967) r e p o r t e d a value o f 3.6S f o r - c a s e i n 68 at pH 9.0, y•= 0.2, while Swaisgood and Timasheff (1968) r e p o r t e d a value o f 3.7S f o r a . - c a s e i n C (pH 9.0, y = 0.1). These authors observed t h a t the sedimentation r a t e o f a g ^ - c a s e i n decreased w i t h i n c r e a s i n g pH, r e a c h i n g a v a l u e o f IS a t pH 11.0. K-carrageenan (1 mg/ml) e x h i b i t e d a s l i g h t l y decreased sedimentation r a t e of 8.OS a t pH 9.0 compared to 9.6S at. pH 6.6, y = 0.08 (Table I I ) . T h i s may be an i n d i c a t i o n t h a t K-carrageenan was l e s s aggregated a t pH 9.0 than a t pH 6.6. However, Goring (1954a) observed t h a t the i n t r i n s i c v i s c o s i t y o f carrageenan was constant between pH 3.6 and pH 11.6, sugges t i n g t h a t the molecular s i z e and shape i s r e l a t i v e l y constant over t h i s pH range. An i n t e r a c t i o n peak was s t i l l observed a t pH 9.0 although the S 2 Q w was onl y 9.9S compared to 14.3S a t pH 6.6, y = 0.08. Thus, a t pH 9.0 the molecular weight of the complex appears t o be s m a l l e r than t h a t a t pH 6.6. However, the i n t e r a c t i o n d i d not appear to be d i s r u p t e d by a pH o f 9.0. I n c r e a s i n g i o n i c s t r e n g t h from 0.08 to 0.24 at pH 6.6 promoted aggregation o f a ^ - c a s e i n which sedimented at a r a t e o f 7.6S. The s c h l i e r e n p a t t e r n was very broad compared t o t h a t when y = 0.0 8 i n d i c a t i n g t h a t the p r o t e i n was p o l y d i s p e r s e at hig h i o n i c s t r e n g t h s . Derechin and Johnson (1964) demonstrated t h a t the mol e c u l a r weight o f a ^ - c a s e i n (10 mg/ml, pH 7.8) i n c r e a s e s with i n c r e a s i n g 69 i o n i c s t r e n g t h . These authors a l s o noted i n c r e a s e d peak spreading w i t h i n c r e a s i n g i o n i c s t r e n g t h d u r i n g sedimentation v e l o c i t y a n a l y s i s . K-Carrageenan e x h i b i t e d an S 2 Q w of 11.3S (pH 6.6, y = 0.24) compared t o 9.6S a t pH 6.6, y = 0.08 (Table I I ) . The i n c r e a s e d c o n c e n t r a t i o n o f co u n t e r i o n s (Na +) may reduce the r e p u l s i o n between the s u l f a t e groups on the p o l y s a c c h a r i d e c h a i n a l l o w i n g i n c r e a s e d i n t e r m o l e c u l a r a s s o c i a t i o n r e f l e c t e d by the l a r g e r S^Q w - A s i m i l a r e f f e c t of i o n i c s t r e n g t h was r e p o r t e d by Cook e t a l . (1952) and Goring and Chepeswick (1956). An i n t e r a c t i o n peak (18.7S) was observed a t pH 6.6, y = 0.24 (Table I I ) . T h i s peak spread very r a p i d l y as d i d the peak f o r a ^ - c a s e i n alone. Since the i n t e r a c t i o n was not d i s r u p t e d by hig h pH o r hig h i o n i c s t r e n g t h , i t would appear t h a t i n t e r a c t i o n between a g ^ - c a s e i n and K-carrageenan was not e l e c t r o s t a t i c i n nature. The same c o n c l u s i o n can be made on the b a s i s o f t u r b i d i t y formation d i s c u s s e d p r e v i o u s l y ( F i g . 5). Temperature d i d not a p p r e c i a b l y a f f e c t the S 2 Q w of a ^ - c a s e i n or K-carrageenan a t pH 6.6, y = 0.08 (Table I I , F i g . 11). A s i m i l a r o b s e r v a t i o n f o r carrageenan was made by Goring and Chepeswick (1956). However, S 2 Q w of the i n t e r -a c t i o n peak i n c r e a s e d from 12.9S a t 36°C to 20.IS a t 4°C. T h i s may be i n d i c a t i v e o f hydrogen-bonding p l a y i n g an important r o l e i n the a o 1 - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n . o! 1 -J " 1 0 10 20 30 40 T E M P E R A T U R E , °C F i g u r e 11. E f f e c t o f t e m p e r a t u r e on W ( P H 6.6, p = 0.08) l i n e a, a s ^ - c a s e i n (10 mg/ml); l i n e b, K - c a r r a g e e n a n (1 mg/ml); l i n e c , a s ] _ - c a s e i n (10 mg/ml) + K - c a r r a g e e n a n (1 mg/ml). D a t a r e p r e s e n t s t h e a v e r a g e o f two r u n s . 71 Since hydrogen bond formation i s an exothermic process i t i s favored a t low temperatures and d i s r u p t e d by h i g h temperatures (Vinogradov and L i n n e l l , 1971). The e f f e c t of 6.0 M urea was i n v e s t i g a t e d to f u r t h e r i n v e s t i g a t e the p o s s i b i l i t y t h a t hydrogen bonding was i n v o l v e d i n a ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n . A mixture c o n t a i n i n g 3.4 mg K-carrageenan/ml and 10 mg a ^ - c a s e i n / m l was used. T u r b i d i t y formation o c c u r r e d upon mixing the K-carrageenan and a ^ - c a s e i n ( F i g . 5). However, upon a d d i t i o n of urea the t u r b i d i t y began to d i m i n i s h u n t i l a water c l e a r s o l u t i o n was o b t a i n e d w i t h i n s e v e r a l minutes. Sedimentation v e l o c i t y a n a l y s i s r e v e a l e d a small peak (45.2S) sedimenting ahead o f a l a r g e peak (1.2S), assumed to be a ^ - c a s e i n on the b a s i s o f i t s s i z e and shape (Table I I , I I I , F i g . 12). The s m a l l peak was assumed to be K-carrageenan on the b a s i s of. i t s s i z e . Thus, the, a s ^ - c a s e i n - K - c a r r a g e e n a n complex was d i s s o c i a t e d by 6.0 M urea. Urea i s known t o d i s r u p t hydrogen and hydrophobic bonds (Haschemeyer and Haschemeyer, 19 73). On the b a s i s o f the e f f e c t s of temperature and o f urea on the i n t e r a c t i o n , i t appears t h a t hydrogen bond formation may be a mechanism by which a ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n i n C a + + - f r e e systems proceeds. Hydrogen bond formation c o u l d occur by i n t e r a c t i o n of the f r e e h y d r o x y l groups of carrageenan w i t h the l y s i n e e-NH2 groups, n o n i o n i z e d c a r b o x y l s o f a s p a r t i c and glutamic a c i d s as w e l l as the amide groups of 72 a b S E D I M E N T A T I O N > F i g . 12 E f f e c t of urea on s c h l i e r e n p a t t e r n s f o r a . - c a s e i n (10 mg/ml) - K-carrageenan (3.4 mg/ml) at s pH 6.6, u sa 0.08. Bar angle 65 degrees. a. minus urea (85 min at 58,464 rpm). b. c o n t a i n s 6.0 M urea (320 min at 58,539 rpm). 73 a s p a r a g i n e and g l u t a m i n e r e s i d u e s o f a s ^ - c a s e i n . a s ^ - C a s e i n and K - c a r r a g e e n a n were a l s o t r e a t e d s e p a r a t e l y w i t h u r e a . The u r e a d i s s o c i a t e d a ^ - c a s e i n , t h e s„ d e c r e a s i n g from 4.5S (pH 6.6, y = 0.08) t o 1.1S (pH 6.6, y = 0.08, 6.0 M u r e a ) . A v a l u e o f IS c o r r e s p o n d s t o t h e monomer s t a t e o f a . - c a s e i n (Schmidt e t a l . , 1967). Ono e t s i a l . (1974) p r e s e n t e d e v i d e n c e t h a t a 1 - c a s e i n was f u l l y d e n a t u r e d i n t h e presence o f 6.0 M u r e a . I t was assumed i n t h i s s t u d y t h a t . u r e a d i d n o t a f f e c t t h e p a r t i a l s p e c i f i c volume o f a . - c a s e i n . Sakura and R e i t h e l (1972) have shown s i t h a t , f o r many p r o t e i n s , u r e a a f f e c t s the p a r t i a l s p e c i f i c volume o n l y s l i g h t l y . A s i m i l a r . a s s u m p t i o n was made r e g a r d i n g t h e e f f e c t o f u r e a on t h e p a r t i a l s p e c i f i c volume o f c a r r a g e e n a n . The s„ n o f the K - c a r r a g e e n a n s o l u t i o n d e c r e a s e d z. u, w from 40.7S t o 34.IS upon the a d d i t i o n o f 6.0 M u r e a . One would e x p e c t a l a r g e r d e c r e a s e i n t h e s 2 Q W S - * - N C E T ^ I E U R E A s h o u l d d i s r u p t t h e hydrogen bonds s t a b i l i z i n g the c a r r a g e e n a n double h e l i x and reduce t h e m o l e c u l a r w e i g h t t o o n e - h a l f t h e o r i g i n a l w e i g h t . A c o n c e n t r a t i o n o f 6.0 M u r e a , however, may have been i n s u f f i c i e n t t o d i s r u p t a l l o f t h e hydrogen bonds s t a b i l i z i n g t h e c a r r a g e e n a n h e l i x , t h u s , g i v i n g r i s e t o a s m a l l e r d e c r e a s e i n t h e s„,„ t h a n e x p e c t e d . 20,w c A r e a c a l c u l a t i o n s (Table I I I ) r e v e a l t h a t , i n t h e case o f a s ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e s (10 mg/ml, 1 mg/ml r e s p e c t i v e l y ) a t 25 and 4°C, t h e a r e a under t h e s c h l i e r e n 74 p a t t e r n was l e s s than the combined areas o f a . - c a s e i n and s i i K-carrageenan when s u b j e c t e d to the sedimentation v e l o c i t y a n a l y s i s s e p a r a t e l y . T h i s was p o s s i b l y due to the l o s s o f v e r y high m o l e c u l a r weight components formed i n the i n t e r a c t i o n , mixture, although r a p i d l y sedimenting peaks were not observed d u r i n g a c c e l e r a t i o n . I t was not p o s s i b l e t o determine whether the sedimented m a t e r i a l was composed of one or both components of the mixture s i n c e the observed areas under each peak o f the i n t e r a c t i o n mixtures were not the t r u e v a l u e s , due t o the Johnston-Ogston e f f e c t . As a r e s u l t of the Johnston-Ogston e f f e c t , a s s o c i a t i o n c onstants f o r the i n t e r a c t i o n c o u l d not be e v a l u a t e d s i n c e the peak areas o f the i n t e r a c t i o n mixtures were not r e p r e s e n t a t i v e o f the a c t u a l c o n c e n t r a t i o n s pre s e n t . In the absence o f the Johnston-Ogston e f f e c t i t would be expected t h a t the carrageenan peak would be d i m i n i s h e d i n s i z e as a r e s u l t of sedimentation o f complexed carrageenan w i t h i n the i n t e r a c t i o n peak. Methods f o r c o r r e c t i o n of the Johnston-Ogston e f f e c t i n n o n - i n t e r a c t i n g systems have been proposed by Trautman e t a l . (1954), Trautman (1956) and by Janando e t a l . (1972). The. s i t u a t i o n , .however, i s much more complex i n i n t e r a c t i n g mixtures s i n c e changes i n c o n c e n t r a t i o n o f one o r both components may l e a d to changes i n S^Q w and area under the peaks which cannot be r e a d i l y separated from those a l t e r a t i o n s due o n l y to the Johnston-Ogston e f f e c t . 75 H o w e v e r , o n t h e b a s i s o f s e d i m e n t a t i o n v e l o c i t y , a n d v i s c o s i t y d a t a , i t a p p e a r s t h a t i n t e r a c t i o n b e t w e e n a g ^ - c a s e i n a n d K - c a r r a g e e n a n o c c u r r e d v i a h y d r o g e n b o n d f o r m a t i o n a n d t h a t a d e c r e a s e i n . a . - c a s e i n c o n c e n t r a t i o n s i t o 0 . 5 m g / m l p r e v e n t e d t h e i n t e r a c t i o n f r o m t a k i n g p l a c e . MOLECULAR WEIGHT D I S T R I B U T I O N C o n d i t i o n s f o r t h e s e d i m e n t a t i o n e q u i l i b r i u m r u n s w e r e s e l e c t e d o n t h e b a s i s o f s „ _ d a t a f o r t h e a n - c a s e i n -2 0 , w s i K - c a r r a g e e n a n (5 m g / m l , 1 m g / m l r e s p e c t i v e l y ) i n t e r a c t i o n m i x t u r e ( T a b l e I I ) . A n s „ _ o f 1 4 S , f o r t h e r a p i d l y 2 0 , w ^ 2 s e d i m e n t i n g i n t e r a c t i o n p e a k , c o r r e s p o n d s .to a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 3 0 0 , 0 0 0 d a l t o n s w h i c h w o u l d , t h u s , r e q u i r e a r u n s p e e d o f 5 , 0 0 0 r p m ( C h e r v e n k a , 1 9 6 9 ) . T y p i c a l e q u i l i b r i u m p a t t e r n s a f t e r 24 h r a r e s h o w n i n F i g . 1 3 . I n c r e a s i n g t h e d u r a t i o n o f t h e r u n d i d n o t l e a d t o a n y o b s e r v a b l e c h a n g e s i n t h e e q u i l i b r i u m p a t t e r n . D a t a f o r K - c a r r a g e e n a n (1 m g / m l ) w e r e n o t r e l i a b l e d u e t o t h e f a c t t h a t a s o l u t i o n s c h l i e r e n p a t t e r n s u f f i c i e n t l y r e s o l v e d f r o m t h e b a s e l i n e c o u l d n o t b e o b t a i n e d a t t h i s l o w c o n c e n t r a t i o n . T h e m e n i s c u s p o s i t i o n was t a k e n a s 1/3 o f t h e w i d t h o f t h e s o l u t i o n m e n i s c u s p a t t e r n f r o m t h e a i r s i d e ( E r l a n d e r a n d B a b c o c k , 1 9 6 1 ) . F o r c a l c u l a t i o n . o f t h e MWD o f t h e a . . - c a s e i n - K -s l c a r r a g e e n a n m i x t u r e , a r a n g e o f c a l c u l a t e d p a r t i a l s p e c i f i c v o l u m e s was u s e d . T h e a s s u m p t i o n was m a d e , o n t h e b a s i s o f a b F i g . 13 Sedimentation equilibrium patterns f o r MWD c a l c u l a t i o n s . Bar angle 60 degrees. Time-24 hr. a. a s l ~ c a s e i n (5 mg/ml), pH 6.6, u = 0.08. b " a s l - c a s e i n (5 mg/ml) + K-carrageenan (1 mg/ml), pH 6.6, y = 0.08. 77 s e d i m e n t a t i o n v e l o c i t y d a t a , t h a t t h e i n t e r a c t i o n o b s e r v e d d u r i n g s e d i m e n t a t i o n v e l o c i t y c e n t r i f u g a t i o n p r o b a b l y i n v o l v e d t h e r e a c t i o n o f n a . - c a s e i n m o l e c u l e s ( n £ 1) w i t h s i e a c h i n t e r a c t i n g K - c a r r a g e e n a n m o l e c u l e . A l t h o u g h u n r e a c t e d K - c a r r a g e e n a n was o b s e r v e d , a l l o f t h e a g ^ - c a s e i n was l o c a t e d w i t h i n t h e i n t e r a c t i o n p e a k p r e s u m a b l y a s a n a g ^ - c a s e i n - K - c a r r a g e e n a n c o m p l e x . T h u s , p a r t i a l s p e c i f i c v o l u m e s w e r e c a l c u l a t e d f o r a n u m b e r o f i n t e r a c t i o n r a t i o s u s i n g t h e m e t h o d o f H o w l e t t a n d N i c h o l (1973) w h i c h a s s u m e s t h a t n o v o l u m e c h a n g e o c c u r s u p o n i n t e r a c t i o n ( F i g . 1 4 ) . C a l c u l a t i o n o f p a r t i a l s p e c i f i c v o l u m e s w e r e b a s e d 5 5 o n m o l e c u l a r w e i g h t s o f 1 . 0 6 x 10 a n d 2 . 9 x 10 d a l t o n s f o r a g ^ - c a s e i n a n d K - c a r r a g e e n a n r e s p e c t i v e l y . T h e s e v a l u e s w e r e c a l c u l a t e d f r o m s e d i m e n t a t i o n - d i f f u s i o n d a t a ( C h e r v e n k a , 1 9 6 9 ) . D i f f u s i o n c o e f f i c i e n t s w e r e c a l c u l a t e d f r o m s y n t h e t i c - 7 2 b o u n d a r y d a t a a n d v a l u e s o f D^Q w o f 3 . 7 7 x 10 cm / s e c f o r - 7 2 a g l - c a s e i n (5 m g / m l ) a n d 1 . 6 7 . x 10 cm / s e c f o r K - c a r r a g e e n a n (1 m g / m l ) w e r e o b t a i n e d . S m i t h e t a l . (1954) r e p o r t e d a - 7 2 d i f f u s i o n c o e f f i c i e n t o f 0 . 8 2 x 10 cm / s e c f o r K - c a r r a g e e n a n (pH 6 . 6 , .y = 0 . 1 5 ) . D i f f u s i o n c o e f f i c i e n t s r e p o r t e d f o r - 7 2 a g ^ - c a s e i n w e r e 2 . 9 0 x 10 cm / s e c a t pH 7 . 7 8 , y = 0 . 1 - 7 2 ( S u l l i v a n e t a l . , 1 9 5 5 ) ; 3 . 7 x 10 cm / s e c a t pH 7 . 0 8 , y = - 7 2 0 . 1 (Ho a n d C h e n , 1 9 6 7 ) ; 4 . 5 3 x 10 cm / s e c a t pH 7 . 1 , y = 0 . 0 8 (Kaminogawa e t a l . , 1 9 7 4 ) . 5 A m o l e c u l a r w e i g h t o f 1 . 0 x 10 d a l t o n s f o r a . - c a s e i n s i (pH 7 . 1 , y = 0 . 0 8) h a s b e e n r e p o r t e d b y K a m i n o g a w a e t a l . F i g u r e 14. P a r t i a l s p e c i f i c volume of s e l e c t e d a s ^ - c a s e i n K - c a r r a g e e n a n i n t e r a c t i o n complexes. 79 ( 1 9 7 4 ) . T h u s t h e m o l e c u l a r w e i g h t o f 1 . 0 6 x 10 d a l t o n s o b t a i n e d i n t h i s s t u d y i s i n a g r e e m e n t w i t h r e p o r t e d d a t a . M o l e c u l a r w e i g h t s o f 1 . 8 x 10^ t o 2 x 1 0 6 d a l t o n s h a v e b e e n r e p o r t e d f o r K - c a r r a g e e n a n ( G o r i n g a n d Y o u n g , 1 9 5 5 ; S m i t h e t a l . , 1 9 5 4 , 1 9 5 5 ; B o n t o u x e t a l . , 1 9 7 0 ) . T h e m o l e c u l a r w e i g h t o b t a i n e d i n t h i s s t u d y f a l l s w i t h i n t h e r a n g e o f r e p o r t e d v a l u e s . D u r i n g c a l c u l a t i o n o f t h e MWD b y t h e m u l t i p l e r e g r e s s i o n m e t h o d , n e g a t i v e r e g r e s s i o n c o e f f i c i e n t s c a n b e o b t a i n e d . S i n c e a n e g a t i v e c o m p o n e n t o f t h e MWD d o e s n o t e x i s t , s e v e r a l m e t h o d s f o r c o r r e c t i o n o f t h e n e g a t i v e r e g r e s s i o n c o e f f i c i e n t s w e r e i n v e s t i g a t e d . I n o r d e r t o e v a l u a t e t h e c o r r e c t i o n m e t h o d s f o r c a l c u l a t i o n o f t h e MWD, a n e q u i l i b r i u m p a t t e r n r e p r e s e n t a t i v e o f t h e c o r r e c t e d 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 was o b t a i n e d b y u s i n g K^.. a n d t h e c o r r e c t e d f ^ v a l u e s t o c a l c u l a t e U ( A , £ ) f r o m w h i c h v a l u e s o f h r w e r e o b t a i n e d . T h e sum o f t h e a b s o l u t e d e v i a t i o n s (Ed) o f t h e c a l c u l a t e d e q u i l i b r i u m p a t t e r n f r o m t h e e x p e r i m e n t a l l y o b s e r v e d p a t t e r n w e r e c a l c u l a t e d . T h e s u i t a b i l i t y o f e a c h c o r r e c t i o n m e t h o d was j u d g e d o n t h e m a g n i t u d e o f E d . A " m o s t n e g a t i v e c o r r e c t i o n m e t h o d " was a t t e m p t e d , w h e r e b y t h e m o s t n e g a t i v e f ^ w i t h i n a m o l e c u l a r w e i g h t g r i d was a d d e d t o t h e o t h e r f . v a l u e s w i t h i n t h a t g r i d s u c h t h a t t h e m o l e c u l a r w e i g h t r e p r e s e n t e d b y t h e m o s t n e g a t i v e f ^ now h a d f . = 0 . T h i s p r o c e d u r e was n o t s u c c e s s f u l s i n c e t h e 8 0 c a l c u l a t e d e q u i l i b r i u m p a t t e r n e x h i b i t e d l a r g e d e v i a t i o n s ( E d = 5 4 . 6 7 ) f r o m t h e e x p e r i m e n t a l l y o b s e r v e d e q u i l i b r i u m p a t t e r n f o r t h e a g ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e . A n i m p r o v e m e n t i n t h e c l o s e n e s s o f f i t b e t w e e n t h e c a l c u l a t e d a n d o b s e r v e d e q u i l i b r i u m p a t t e r n s was o b t a i n e d b y a s s u m i n g t h e n e g a t i v e r e g r e s s i o n c o e f f i c i e n t s t o b e e q u a l t o z e r o ( E d = 1 4 . 6 0 ) . T h e b e s t f i t b e t w e e n o b s e r v e d a n d e x p e r i m e n t a l d a t a was r e a l i z e d b y a s s u m i n g n e g a t i v e r e g r e s s i o n c o e f f i c i e n t s t o b e z e r o f o l l o w e d b y r e - e n t e r i n g m o l e c u l a r w e i g h t s w i t h p o s i t i v e r e g r e s s i o n c o e f f i c i e n t s i n t o t h e m u l t i p l e r e g r e s s i o n p r o g r a m . I n a d d i t i o n , t h e m o l e c u l a r w e i g h t r a n g e was e x p a n d e d i n t h e d i r e c t i o n i n w h i c h t h e d i s t r i b u t i o n a p p e a r e d t o b e h e a d i n g . F o r e x a m p l e , i f t h e r e g r e s s i o n c o e f f i c i e n t o f t h e l o w e s t m o l e c u l a r w e i g h t i n t h e g r i d was p o s i t i v e , a m o l e c u l a r w e i g h t e q u a l t o o n e - h a l f o f t h e l o w e s t m o l e c u l a r w e i g h t was a l s o e n t e r e d i n t o t h e p r o g r a m . T h i s p r o c e d u r e was c o n t i n u e d , s e t t i n g a l l n e g a t i v e r e g r e s s i o n c o e f f i c i e n t s t o z e r o , u n t i l a l l o f t h e r e g r e s s i o n c o e f f i c i e n t s o b t a i n e d w e r e p o s i t i v e . T h i s p r o c e d u r e g a v e a c l o s e n e s s o f f i t ( E d = 0 . 1 9 ) s u c h a s t h a t i l l u s t r a t e d i n F i g . 1 6 . T h e l e a s t s q u a r e s m e t h o d o f c a l c u l a t i n g t h e MWD was u s e d s i n c e Ma ( 1 9 7 5 ) f o u n d t h a t t h e l i n e a r p r o g r a m m i n g m e t h o d o f S c h o l t e ( 1 9 6 9 ) was u n a b l e t o r e s o l v e s y s t e m s c o n t a i n i n g m o r e t h a n t w o c o m p o n e n t s . N a k a i ( 1 9 7 5 , u n p u b l i s h e d d a t a ) o b s e r v e d t h a t t h e r e s o l v i n g p o w e r o f t h e m u l t i p l e r e g r e s s i o n 81 method was superior to that of Scholte's (1969) l i n e a r programming method. The MWD for the a ^ - c a s e i n (5 mg/ml) -K-carrageenan (1 mg/ml) mixture contained a major peak ranging from 5.2 x 4 5 10 to 1.24 x 10 daltons with minor peaks located i n the 5 5 molecular weight range of 3 x 10 to 4.1 x 10 daltons and 9.9 x 10 5 to 1.7 x 10 6 daltons (Fig. 15). The calculated equilibrium pattern was a close f i t to the observed pattern (Fig. 16). a g^-Casein (5 mg/ml) had a MWD with a major peak 5 4 centered at 1.04 x 10 daltons ranging from 7.5 x 10 to 5 2.1 x 10 daltons with a very minor peak ranging from 8.3 x 10 5 to 1.4 x 10 6 daltons (Fig. 17). Using Scholte's (1969) l i n e a r programming method, a MWD very si m i l a r to that derived by application of the multiple regression method was obtained for a g^-casein (5 mg/ml, pH 6.6, y = 0.08; Fi g . 17). A d i s t i n c t i v e feature of the MWD for the a n - c a s e i n -s i K-carrageenan int e r a c t i o n mixture (Fig. 15) was the major 4 peak centered at 7 x 10 daltons. On the basis of s„. 20 ,w values for t h i s mixture the presence of the large peak i n the MWD i n the v i c i n i t y of a g^-casein was not expected. Instead 5 a peak centered at 2.9 x 10 corresponding to unreacted K-carrageenan and another centered at a much higher molecular weight corresponding to the a g^-casein-K-carrageenan i n t e r -action product were expected. It does not appear as though the K-carrageenan 82 F i g u r e 15. M o l e c u l a r weight d i s t r i b u t i o n f o r a s]_-casein (5 mg/ml) + K-carrageenan (1 mg/ml) mixture, pH 6.6, ii = 0.08. 4 r u 0 6.75 6.85 6.95 7.05 c m F i g u r e 16. S e d i m e n t a t i o n e q u i l i b r i u m s c h l i e r e n p a t t e r n s f o r a s ] _ - c a s e i n (5 mg/ml) + K - c a r r a g e e n a n (1 mg/ml), pH 6.6, u = 0.08; c u r v e a, o b s e r v e d s c h l i e r e n p a t t e r n ; curve b, s c h l i e r e n p a t t e r n c a l c u l a t e d from the MWD o f F i g u r e 15. (Ed = 0.19) . 00 84 0.8 0.6 0.4 a-0.2 0 J L J L ' i i ' 10 M - x 10 100 F i g u r e 17. 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 f o r a s ] _ - c a s e i n (5 m g / m l ) a t pH 6 . 6 , ii = 0 . 0 8 ; c u r v e a , c a l c u l a t e d b y m u l t i p l e r e g r e s s i o n m e t h o d ; c u r v e b , c a l c u l a t e d b y l i n e a r p r o g r a m m i n g m e t h o d . 3 r , cm F i g u r e 18. S e d i m e n t a t i o n e q u i l i b r i u m s c h l i e r e n p a t t e r n s f o r a s l - c a s e i n (5 mg/ml), pH 6.6, y = 0.0 8; curve a, observed s c h l i e r e n p a t t e r n ; c u r v e b, s c h l i e r e n p a t t e r n c a l c u l a t e d from t h e m u l t i p l e r e g r e s s i o n MWD (curve a) of F i g u r e 17. (Ed = 0.12). CO 8 6 c o n t r i b u t e d t o t h e c a l c u l a t e d MWD, p o s s i b l y b e c a u s e o f t h e l o w c o n c e n t r a t i o n u s e d . H o w e v e r , t h e p e a k l o c a t e d i n t h e 5 5 m o l e c u l a r w e i g h t r a n g e o f 3 x 1 0 t o 5 . 6 x 1 0 d a l t o n s ( F i g . 1 5 ) may b e K - c a r r a g e e n a n . M o r e o v e r , t h e s i z e o f t h e p e a k i s m u c h s m a l l e r t h a n w o u l d b e e x p e c t e d o n t h e b a s i s o f t h e K - c a r r a g e e n a n / a g ^ - c a s e i n r a t i o i n t h e s a m p l e . T h e p e a k l o c a t e d i n t h e m o l e c u l a r w e i g h t r a n g e o f 5 6 9 . 8 x 1 0 t o 2 x 1 0 d a l t o n s ( F i g . 1 5 ) may b e d u e t o a s m a l l a m o u n t o f i n t e r a c t i o n p r o d u c t . H o w e v e r , . t h e p r e s e n c e o f a s m a l l p e a k i n t h e r a n g e o f 7 . 9 x 1 0 ^ t o 1 x 1 0 ^ d a l t o n s ( F i g . 1 7 ) f o r a g ^ - c a s e i n w o u l d a p p e a r t o i n d i c a t e t h a t t h e p e a k i n t h i s r a n g e , f o r b o t h s a m p l e s , may m o r e l i k e l y b e a r e s u l t o f t h e c u r v a t u r e a n d u n c e r t a i n t y i n v o l v e d i n t h e l o c a t i o n o f t h e c e n t e r o f t h e s a m p l e s c h l i e r e n p a t t e r n n e a r t h e b o t t o m o f t h e s o l u t i o n c o l u m n . T h u s , o n t h e b a s i s o f t h e p r e s e n c e o f a l a r g e p r o p o r t i o n o f t h e a g ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e h a v i n g a n MWD s i m i l a r t o t h a t o f a g ^ - c a s e i n , i t w o u l d a p p e a r t h a t a ^ - c a s e i n a n d K - c a r r a g e e n a n d i d n o t i n t e r a c t a t pH 6 . 6 , y = 0 . 0 8 . I t i s p o s s i b l e t h a t t h e m o l e c u l a r w e i g h t r a n g e , r e p r e s e n t e d b y t h e m a j o r p e a k o f t h e m i x t u r e c o v e r e d a l o w e r m o l e c u l a r w e i g h t r a n g e t h a n t h a t f o r a g ^ - c a s e i n a s a c o n s e q u e n c e o f s e l e c t i o n o f p a r t i a l s p e c i f i c v o l u m e s o n t h e b a s i s o f a n i n t e r a c t i o n b e t w e e n a ^ n - c a s e i n a n d K - c a r r a g e e n a n . 87 F R O N T A L CHROMATOGRAPHY E l u t i o n p a t t e r n s o f a s l - c a s e i n (1.2 m g / m l ) ( F i g . 19) a n d a g ^ - c a s e i n (1.2 m g / m l ) - K - c a r r a g e e n a n (1.2 m g / m l ) ( F i g . 20) w e r e o b t a i n e d u s i n g a c o l u m n o f S e p h a d e x G-200 (pH 6.6, y = 0.01). T h e v o i d v o l u m e was 16.0 m l w h i l e t h e t o t a l i n t r u s i o n v o l u m e was 30.0 m l . U n d e r t h e s e c o n d i t i o n s , a . - c a s e i n e x i s t s a s a monomer ( S c h m i d t , 1970) w i t h a s i m o l e c u l a r w e i g h t o f 23,600 d a l t o n s a n d e l u t e s a s u n i m o d a l b o u n d a r y (Ve 28.9 m l ; F i g . 19). C h r o m a t o g r a p h y o f t h e a ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e r e s u l t e d i n t h e K - c a r r a g e e n a n e l u t i n g i n t h e v o i d v o l u m e . T h e a ^ - c a s e i n c o m p o n e n t o f t h e m i x t u r e e l u t e d i n a v o l u m e i d e n t i c a l t o t h a t o b t a i n e d when a . - c a s e i n was s i c h r o m a t o g r a p h e d s e p a r a t e l y . No a s ^ - c a s e i n e l u t i n g w i t h K - c a r r a g e e n a n was o b s e r v e d ( F i g . 20). H o w e v e r , d u e t o t h e v i s c o s i t y o f t h e a . - c a s e i n - K -J s i c a r r a g e e n a n m i x t u r e t h e g e l b e d was c o m p a c t e d d u r i n g t h e c o u r s e o f t h e c h r o m a t o g r a p h y i n s p i t e o f t h e u s e o f t h e u p w a r d f l o w m o d e . A s a c o n s e q u e n c e o f c h a n g e s i n t h e c o l u m n d i m e n s i o n s d u r i n g t h e r u n , t h e t r a i l i n g p r o f i l e s w e r e n o t a s s h a r p a s t h o s e o b s e r v e d f o r t h e a d v a n c i n g p r o f i l e . T h e t r a i l i n g p r o f i l e f o r a ^ - c a s e i n a l o n e was s h a r p , l i k e t h a t o f t h e a d v a n c i n g p r o f i l e ( F i g . 19). A s e a r c h was made f o r a c o l u m n p a c k i n g m a t e r i a l p o s s e s s i n g m e c h a n i c a l s t a b i l i t y a s w e l l a s s e p a r a t i o n p r o p e r t i e s s i m i l a r t o S e p h a d e x G-200. C o n t r o l l e d p o r e g l a s s -* trail in g. E L U T I O N V O L U M E , ml F i g u r e 1 9 . E l u t i o n p a t t e r n o f a s ] _ - c a s e i n ( 1 . 2 m g / m l ) . pH 6 . 6 , y = 0 . 0 1 ; c o l u m n , S e p h a d e x G - 2 0 0 . E L U T I O N V O L U M E , ml F i g u r e 20. E l u t i o n p a t t e r n s o f a s ] _ - c a s e i n (1.2 mg/ml) + K - c a r r a g e e n a n (1.2 mg/ml). pH 6.6, y = 0.01; c u r v e a, K - c a r r a g e e n a n component; c u r v e b, a s ] _ - c a s e i n component; curve c, sum o f e l u t i o n p a t t e r n s a and b. Column, Sephadex G-200. 90 o beads having an average pore diameter o f 170A were s e l e c t e d . The v o i d volume of the packed column was 22.8 ml which was 4 7% o f the envelope volume. C o l l i n s and H a l l e r (197 3) r e p o r t e d t h a t the v o i d volume of columns packed w i t h v a r i o u s c o n t r o l l e d pore g l a s s beads was approximately 4 3% o f the envelope volume. The t o t a l i n t r u s i o n volume of the column was 37.4 ml. Samples o f . a g ^ - c a s e i n (10 mg/ml), K-carrageenan (1 mg/ml) ( F i g . 21) and an a 1 - c a s e i n (10 mg/ml) - K - c a r r a g e e n a n (1 mg/ml) mixture ( F i g . 22) were a p p l i e d t o the column. The advancing p r o f i l e s i n a l l cases were sharp but the t r a i l i n g p r o f i l e s were d i f f u s e (Figs.. 21, 22 and.23). A s i m i l a r phenomenon was observed by Crone e t a l . (19 75) who concluded t h a t although treatment o f the g l a s s beads wi t h p o l y e t h y l e n e g l y c o l reduced a d s o r p t i o n phenomena, the r e s u l t i n g chromatographic s e p a r a t i o n s were c h a r a c t e r i z e d by a sharp l e a d i n g edge and a r a p i d i n i t i a l f a l l on the t r a i l i n g edge f o l l o w e d by a subsequent slow decrease t o the base l i n e . T h i s was i n t e r p r e t e d t o be a r e s u l t of i n t e r a c t i o n o f the samples w i t h the p o l y e t h y l e n e g l y c o l . However, on the b a s i s o f the r e s u l t s o b t a i n e d ( F i g s . 21 and 22) i t would appear t h a t a g ^ - c a s e i n and K-carrageenan d i d not r e a c t to form an i n t e r a c t i o n complex. I f o c g ^ - c a s e i n and K-carrageenan. i n t e r a c t e d , the complex would possess a molecular weight g r e a t e r than t h a t o f the K-carrageenan and would e l u t e i n the v o i d volume. In 0 F i g u r e 21. E l u t i o n p a t t e r n s from a column of c o n t r o l l e d pore g l a s s (170 A pore d i a m e t e r ) , pH 6.6, y = 0.08; p r o f i l e a, K - c a r r a g e e n a n (1 mg/ml); p r o f i l e b, a s ] _ - c a s e i n (10 mg/ml); p r o f i l e c, sum o f p r o f i l e s a and b wh i c h were o b t a i n e d from s e p a r a t e r u n s . T h i s p r o f i l e r e p r e s e n t s t h e type o f e l u t i o n p a t t e r n e x p e c t e d from an a s ] _ - c a s e i n - K - c a r r a g e e n a n m i x t u r e assuming the absence o f an i n t e r a c t i o n . F i g u r e 22. E l u t i o n p a t t e r n s f o r a s ^ - c a s e i n (10 mg/ml) + K - c a r r a g e e n a n (1 mg/ml) i n t e r a c t i o n Q m i x t u r e (pH' 6.6, y = 0.08) from a column o f c o n t r o l l e d pore g l a s s (170 A pore d i a m e t e r ) . p r o f i l e a, K - c a r r a g e e n a n component; p r o f i l e b, a s ^ - c a s e i n component; p r o f i l e c, sum o f p r o f i l e s a and b. to V, m I F i g u r e 23. F i r s t - d e r i v a t i v e c u r v e s of a d v a n c i n g and t r a i l i n g p r o f i l e s o f the e l u t i o n p a t t e r n s from the c o n t r o l l e d pore g l a s s column (170 A pore d i a m e t e r ) . pH 6.6, vi = 0.08: A, c x s l - c a s e i n (10 mg/ml); B, K - c a r r a g e e n a n (1 mg/ml); C, a -L-casein (10 mg/ml) + K - c a r r a g e e n a n (1 mg/ml) i n t e r a c t i o n m i x t u r e . VD 94 a d d i t i o n , t h e e l u t i o n p r o f i l e s o b s e r v e d f o r t h e a . - c a s e i n -s i K - c a r r a g e e n a n m i x t u r e w e r e t h e same a s a p r o f i l e , b a s e d o n t h e a s s u m p t i o n o f n o i n t e r a c t i o n , c a l c u l a t e d f r o m t h e d a t a f o r ct. , - c a s e i n a n d K - c a r r a g e e n a n c h r o m a t o g r a p h e d s e p a r a t e l y ( F i g . 1 8 , p r o f i l e C ) . D i f f i c u l t y was e n c o u n t e r e d i n o b t a i n i n g v a l u e s o f V e f r o m t h e f i r s t - d e r i v a t i v e c u r v e s o f t h e t r a i l i n g p r o f i l e s d u e t o t h e a b s e n c e o f w e l l d e f i n e d i n f l e c t i o n s . T h e v o l u m e c o r r e s p o n d i n g t o t h e m i d - p o i n t o f t h e c o n c e n t r a t i o n p r o f i l e s was u s e d a s V e ( T a b l e V ) . A t l o w a , - c a s e i n c o n c e n t r a t i o n s , s i some r e t e n 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 s i n d i c a t e d b y V e v a l u e s g r e a t e r t h a n 3 7 . 4 m l ( T a b l e V ) . I t i s p o s s i b l e t h a t t h i s p h e n o m e n o n was a c c e n t u a t e d a t l o w p r o t e i n c o n c e n t r a t i o n s s i n c e t h e c t g ^ - c a s e i n i s l e s s a g g r e g a t e d a t l o w c o n c e n t r a t i o n s a t pH 6 . 6 , y = 0 . 0 8 . a n d t h u s e l u t e d b e y o n d t h e V t p o s i t i o n d u e t o i n t e r a c t i o n w i t h p o l y e t h y l e n e g l y c o l ( C r o n e e t a l . , 1 9 7 5 ) . T h u s , t h e o b s e r v e d V e f o r a , - c a s e i n a t a l l s i c o n c e n t r a t i o n s e m p l o y e d was p r o b a b l y g r e a t e r t h a n i t w o u l d h a v e b e e n i n t h e a b s e n c e o f t h e s e e f f e c t s . H o w e v e r , t h e s e d a t a s u p p o r t t h e o b s e r v a t i o n s t h a t a g ^ - c a s e i n a t pH 6 . 6 , y = 0 . 0 8 e x i s t s a s a l a r g e r m o l e c u l a r w e i g h t s p e c i e s a s t h e c o n c e n t r a t i o n i n c r e a s e s ( T a b l e V ) . T h e r e d o e s n o t a p p e a r t o b e a n a g ^ - c a s e i n - K - c a r r a -g e e n a n i n t e r a c t i o n a t p H 6 . 6 , y = 0 . 0 8 t h a t c a n b e d e t e c t e d b y f r o n t a l c h r o m a t o g r a p h y . T h i s s u p p o r t s t h e o b s e r v a t i o n s made o n t h e b a s i s o f MWD c a l c u l a t i o n s , b u t , i s i n c o n f l i c t 95 Table V E L U T I O N VOLUMES OF a S L - C A S E I N AND K-CARRAGEENAN E L U T I O N P A T T E R N S ON C O N T R O L L E D PORE G L A S S COLUMNS ( 1 7 0 A) A T pH 6.6, y = 0.08 C o n c e n t r a t i o n Ve (ml) (mg/ml) Advancing P r o f i l e T r a i l i n g P r o f i l e b s l K - C C a -1 s l K - C a . s l K - C 1.0 1.0 36. 5 2 2 . 8 36.2 2 2 . 8 0.5 0.5 37.4 2 2 . 8 43 . 7 2 4 . 8 0.5 - 41.6 - 47.2 -- 0.5 - 2 2 . 8 - 2 2 . 8 5.0 • 1.0 28 . 5 2 2 . 8 2 8 . 9 2 2 . 8 5.0 - 2 9 . 5 - 2 7 . 9 -- 1.0 - 2 2 . 8 - 2 2 . 8 10. 0 - 27.2 - 2 7 . 0 -1 0 . 0 1.0 28. 0 2 2 . 8 26.4 2 5 . 0 a. Volume corresponding to the mid-point o f the c o n c e n t r a t i o n p r o f i l e s b. a -C a s e i n s l c. K-Carrageenan 96 w i t h t h e r e s u l t s o b t a i n e d f r o m t h e s e d i m e n t a t i o n v e l o c i t y s t u d i e s . F r o n t a l c h r o m a t o g r a p h y was s e l e c t e d s i n c e p a t t e r n s a n a l o g o u s t o t h o s e o b t a i n e d w i t h s e d i m e n t a t i o n v e l o c i t y may b e o b t a i n e d f r o m t h e f i r s t - d e r i v a t i v e c u r v e s o f t h e e l u t i o n p r o f i l e s ( W i n z o r a n d S h e r a g a , 1 9 6 3 ) . I n a d d i t i o n , q u a n t i t a t i o n o f a ^ - c a s e i n a n d K - c a r r a g e e n a n b y s e p a r a t e a n a l y t i c a l m e t h o d s w o u l d e n a b l e c o r r e c t i o n s t o b e made f o r t h e J o h n s t o n - O g s t o n e f f e c t p r i o r t o c a l c u l a t i o n o f a s s o c i a t i o n c o n s t a n t s - A s s o c i a t i o n c o n s t a n t s f o r i n t e r a c t i n g s y s t e m s c a n b e d e t e r m i n e d f r o m t h e t r a i l i n g e l u t i o n p r o f i l e b y t h e m e t h o d o u t l i n e d b y N i c h o l a n d W i n z o r (196 4) b a s e d o n t h e d e t e r m i n a t i o n o f u n r e a c t e d a . - c a s e i n i n t h e 3 - p h a s e o f s i t h e t r a i l i n g p r o f i l e ( N i c h o l a n d W i n z o r , 1964) a n d k n o w l e d g e o f t h e i n i t i a l c o n c e n t r a t i o n s o f a ^ - c a s e i n a n d K - c a r r a g e e n a n a p p l i e d t o t h e c o l u m 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 S T U D I E S 1.1 m o l e s o f d a n s y l c h l o r i d e i n t e r a c t e d w i t h o n e 4 m o l e o f a ^ - c a s e i n , a s s u m i n g a m o l e c u l a r w e i g h t o f 2 . 3 x 10 d a l t o n s f o r a , - c a s e i n ( G r o s c l a u d e e t a l . , 1970) a n d a m o l a r s i 3 - 1 - 1 a b s o r p t i v i t y o f 3 . 4 x 10 M cm a t 34 0 nm f o r t h e b o u n d d a n s y l m o i e t y ( C h e n , 1 9 6 8 ) . A v a l u e o f 1 m o l e D N S - c h l o r i d e p e r m o l e o f a g ^ - c a s e i n was r e p o r t e d b y C l a r k e a n d N a k a i ( 1 9 7 1 ) . T h e u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f D N S - a g ^ -c a s e i n a n d u n l a b e l l e d a n - c a s e i n i s s h o w n i n F i g . 2 4 . T h e F i g u r e 2 4 . A b s o r p t i o n s p e c t r a o f a s l - c a s e i n ( c u r v e a) a n d D N S - a s j _ - c a s e i n ( c u r v e b ) , pH 6 . 6 , u = 0 . 0 1 . P r o t e i n c o n c e n t r a t i o n = 1.5 m g / m l . 98 p r e s e n c e o f a n a b s o r b a n c e p e a k a t 340 nm, d u e t o t h e d a n s y l g r o u p , i s e v i d e n t f o r DNS ' -ct ^ - c a s e i n . . I n a d d i t i o n , t h e a b s o r p t i o n s p e c t r u m o f D N S - a g ^ - c a s e i n c o m p a r e d t o t h e u n l a b e l l e d p r o t e i n i s a l s o m a r k e d l y d i f f e r e n t b e l o w 280 nm. D a n s y l a t i o n o f K - c a r r a g e e n a n was pH d e p e n d e n t ( T a b l e V I ) w i t h a maximum d e g r e e o f l a b e l l i n g o c c u r r i n g a t pH 9 . 2 . T h e a b s o r p t i o n s p e c t r u m o f D N S - K - c a r r a g e e n a n i s 3 - 1 - 1 shown i n F i g . 2 5 . A m o l a r a b s o r b a n c e o f 3 . 4 x 10 M cm a t 340 nm was a l s o a s s u m e d f o r t h e D N S - g r o u p c o n j u g a t e d t o K - c a r r a g e e n a n a l t h o u g h C h e n (1968) s t a t e d t h a t t h e w a v e l e n g t h o f maximum a b s o r p t i o n a n d m o l a r e x t i n c t i o n may b e i n f l u e n c e d b y t h e e n v i r o n m e n t o f t h e b i n d i n g s i t e . A m o l e c u l a r w e i g h t 5 o f 2 . 9 x 10 d a l t o n s , o b t a i n e d f r o m s e d i m e n t a t i o n - d i f f u s i o n d a t a , was u s e d f o r K - c a r r a g e e n a n . F l u o r e s c e n c e e x c i t a t i o n a n d e m i s s i o n s p e c t r a o f K - c a r r a g e e n a n , d a n s y l a t e d a t pH 9 . 2 , a r e s h o w n i n F i g s . 26 a n d 2 7 . T h e e x c i t a t i o n w a v e l e n g t h was 362 nm w h i l e t h e w a v e l e n g t h o f maximum e m i s s i o n - w a s 508 nm. N e i t h e r D N S - a g ^ - c a s e i n n o r D N S - K - c a r r a g e e n a n a p p e a r e d t o c o n t a i n u n r e a c t e d , a d s o r b e d D N S - c h l o r i d e . W e b e r (1952) o b s e r v e d t h a t f r e e D N S - c h l o r i d e h a s a n a b s o r p t i o n maximum a t 369 nm. A s e c o n d a b s o r p t i o n maximum a t 369 nm f o r D N S - a g ^ - c a s e i n o r D N S - K - c a r r a g e e n a n was n o t o b s e r v e d ( F i g s . 24 a n d 2 5 ) . T i t r a t i o n o f D N S - a ^ - c a s e i n (1 m g / m l ) w i t h K - c a r r a g e e n a n (20 m g / m l , pH 6 . 6 , i i = 0 . 0 8 ) d i d n o t p r o d u c e Table VI EFFECT OF pH ON DANSYLATION OF CARRAGEENAN pH D a n s y l a t i o n 7.0 1.07 8.0 1.72 9.2 3.10 10.2 2.68 a. Mole DNS-chloride/mole carrageenan. M o l e c u l a r weight of K-carrageenan = 290,000 d a l t o n s . Each value i s the average of d u p l i c a t e d e t e r m i n a t i o n s . 100 b a 250 300 350 400 W A V E L E N G T H, n m F i g u r e 25. Ab s o r p t i o n s p e c t r a of K-carrageenan (curve a) and DNS-K-carrageenan (curve b ) , pH 6.6, v = 0.08. Carrageenan c o n c e n t r a t i o n = 4 mg/ml. > 320 340 360 380 400 E X C I T A T I O N W A V E L E N G T H , n m F i g u r e 2 6 . F l u o r e s c e n c e e x c i t a t i o n s p e c t r u m o f D N S -K - c a r r a g e e n a n (2 m g / m l ) . pH 6 . 6 , y = 0 . 0 8 A E m i s s i o n , 508 nm. 102 F i g u r e 2 7 . F l u o r e s c e n c e e m i s s i o n s p e c t r a o f D N S - K -c a r r a g e e n a n (pH 6 . 6 , u = 0 . 0 8 ) . C u r v e a , D N S - K - c a r r a g e e n a n (2 m g / m l ) ; c u r v e b , D N S -K - c a r r a g e e n a n (2 m g / m l ) + a s i - c a s e i n (25 m g / m l ) . X E x c i t a t i o n , 362 nm. 10 3 a n y c h a n g e s 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 ( F i g . 2 8 ) . I n t e r -a c t i o n o f K - c a r r a g e e n a n w i t h D N S - a ^ - c a s e i n w o u l d b e e x p e c t e d t o r e s u l t i n a n i n t e r a c t i o n p r o d u c t p o s s e s s i n g a much l a r g e r m o l e c u l a r w e i g h t t h a n t h e D N S - a g ^ - c a s e i n . T h e v e l o c i t y o f B r o w n i a n m o t i o n a n d t h u s t h e a m o u n t o f r o t a t i o n o c c u r r i n g d u r i n g t h e e x c i t e d l i f e t i m e o f t h e c o n j u g a t e d D N S - m o i e t y d e c r e a s e s a s t h e m o l e c u l a r s i z e i n c r e a s e s , t h u s l e a d i n g t o a n i n c r e a s e 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 ( D a n d l i k e r e t a l . , 1 9 6 4 ) . A l t h o u g h t h e f l u o r e s c e n c e i n t e n s i t y i n c r e a s e d s o m e w h a t , t h e p o l a r i z a t i o n o f f l u o r e s c e n c e d i d n o t i n c r e a s e . I n a d d i t i o n , t h e s h a p e o f t h e D N S - a g ^ - c a s e i n f l u o r e s c e n c e e m i s s i o n s p e c t r u m was n o t a l t e r e d b y t h e a d d i t i o n o f 1 1 . 2 mg K - c a r r a g e e n a n . . ( F i g . 29) . T h u s t h e i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y o b s e r v e d u p o n a d d i t i o n o f K - c a r r a g e e n a n t o D N S - a ^ -c a s e i n may b e i n d i c a t i v e o f c o n f o r m a t i o n a l c h a n g e s i n d u c e d i n D N S - a g ^ - c a s e i n w i t h o u t a n a c t u a l a g ^ - c a s e i n - K - c a r r a g e e n a n c o m p l e x b e i n g f o r m e d . A c h a n g e i n t h e c o n f o r m a t i o n o f D N S -a g l - c a s e i n c o u l d p r o d u c e a c h a n g e i n t h e m i c r o - e n v i r o n m e n t i n t h e v i c i n i t y o f t h e D N S - m o i e t y l e a d i n g t o a n i n c r e a s e i n f l u o r e s c e n c e . A d d i t i o n o f K - c a r r a g e e n a n p r o b a b l y d i d n o t i n d u c e a g g r e g a t i o n o f a . - c a s e i n s i n c e t h e V e o f a . - c a s e i n • s l s l i n t h e a g ^ - c a s e i n - K - c a r r a g e e n a n m i x t u r e was t h e same a s t h e V e o f a . - c a s e i n c h r o m a t o g r a p h e d s e p a r a t e l y ( T a b l e V ) . T h u s a t t h e c t g - ^ - c a s e i n c o n c e n t r a t i o n u s e d (1 m g / m l ) a n i n t e r a c t i o n b e t w e e n D N S - a o l - c a s e i n a n d K - c a r r a g e e n a n d i d n o t o c c u r a t hd 02 c hi fD CO F L U O R E S C E N C E P O L A R I Z A T I O N 3* (D rt CTi hj CD • ft) < CTI rt fD - H-Hj 0 ft) •c 3 iQ fD II 0 L—k 0 "ItO Hi • D o 2 rt CO r_n 1 p fD >^ cn fD M M I rt X O ri n cu H-ft) rt fD h-1 ft) h1-rt 3 • H-0 — 3 h-1 3 3 — 3 >- rt 3* fD 3 * H- 1 cn o cn cu H- t-i 0 hj 3 0J - iQ fD Ul (D O 3 CO ft> 3 3 3 ~ • M O O 3 ft) in rt \ ft) 3 h-1 o > 70 > o m rn Z > z \ 1/1 n > CO m v. O CO p o Vo o p Vo co h{ fD 105 8r 5 0 0 6 0 0 E M I S S I O N W A V E L E N G T H , nm F i g u r e 29. F l u o r e s c e n c e e m i s s i o n s p e c t r a o f DNS-a s]_-casein (pH 6.6, y = 0.08). Spectrum a, D N S - a s i - c a s e i n (1 mg/ml); spectrum b, DNS-ctg^-casein (1 mg/ml) + K - c a r r a g e e n a n (10 mg/ml). X E x c i t a t i o n , 365 nm. 106 pH 6 . 6 , y = 0 . 0 8 . S i m i l a r r e s u l t s w e r e o b t a i n e d u s i n g f r o n t a l c h r o m a t o g r a p h y e m p l o y i n g t h e same a ^ - c a s e i n c o n c e n t r a t i o n 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 e x p e r i m e n t s w e r e c o n d u c t e d b y t i t r a t i n g D N S - K - c a r r a g e e n a n w i t h a ^ - c a s e i n , s i n c e c o n c e n t r a t i o n s o f e a c h r e a c t a n t s i m i l a r t o t h o s e u s e d i n 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 , w i t h s c h l i e r e n o p t i c s , c o u l d b e u s e d . A 2 m g / m l D N S - K - c a r r a g e e n a n s o l u t i o n was t h e l o w e s t c o n c e n t r a t i o n t h a t c o u l d b e u s e d s i n c e t h e s p e c t r o -f l u o r o m e t e r h a d t o b e s e t a t maximum s e n s i t i v i t y i n o r d e r t o d e t e c t t h e f l u o r e s c e n c e . T i t r a t i o n o f t h e D N S - K - c a r r a g e e n a n w i t h c t ^ - c a s e i n d i d n o t p r o d u c e a n y i n c r e a s e s 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 ( F i g . 3 0 ) . H o w e v e r , t h e f l u o r e s c e n c e i n t e n s i t y d i d i n c r e a s e w i t h a d d e d c a s e i n a n d may r e f l e c t a c h a n g e i n e n v i r o n m e n t d u e t o t h e a d d e d p r o t e i n r a t h e r t h a n f r o m c h a n g e s a r i s i n g f r o m a n a c t u a l i n t e r a c t i o n s i n c e f l u o r e s c e n c e p o l a r i z a t i o n r e m a i n e d u n c h a n g e d ( F i g . 3 0 ) . A l t h o u g h t h e s h a p e o f t h e D N S -K - c a r r a g e e n a n f l u o r e s c e n c e e m i s s i o n s p e c t r u m was a l t e r e d b y t h e a d d i t i o n o f 25 mg o f a g ^ - c a s e i n , t h e w a v e l e n g t h o f maximum e m i s s i o n was u n a f f e c t e d ( F i g . 2 7 ) . T h u s , a t c o n c e n t r a t i o n s s i m i l a r t o t h o s e u t i l i z e d f o r s e d i m e n t a t i o n v e l o c i t y s t u d i e s , a g ^ - c a s e i n a n d K - c a r r a g e e n a n d i d n o t i n t e r a c t o n t h e b a s i s o f t h e f l u o r e s c e n c e p o l a r i z a t i o n d a t a . F l u o r e s c e n c e p o l a r i z a t i o n h a s b e e n s u c c e s s f u l l y u s e d b y T e i c h b e r g a n d S h i n i t z k y (1973) f o r t h e t h e r m o d y n a m i c c h a r a c t e r i z a t i o n o f t h e i n t e r a c t i o n o f l y s o z y m e w i t h a n u m b e r vQ C t-i CD co o F L U O R E S C E N C E P O L A R I Z A T I O N r t V (D fl> < CD fl) vQ CD O •-3 H-r t CD cn rt * H-O c 3 rt s: o rt fl> O • a o 3 00 CO • I I n M w X o ft CD ft) CD ft 3 . H- fl) 0 3 M U l 3 3 CD rt 3 3" H-cn p cn cn 0 I 3 O > fl) cn Cn CD O H-00 3 3 — 3 cn • O 3 D 03 0) \ rt 3 fl) r - 1 p Q in i n > CO I o > 73 73 > o m m Z > z oo o Vo co ~T~ o ko O A) hi fl) CD rt 108 of glucosamine s u b s t r a t e s . J o l l e y and Glaudemans (19 74) used f l u o r e s c e n c e measurements t o determine the a s s o c i a t i o n constants f o r the i n t e r a c t i o n o f myeloma immunoglobulins with carbohydrate l i g a n d s . STABILIZATION TESTS Z i t t l e (196 5) developed a method f o r the study o f the a b i l i t y o f K - c a s e i n t o prevent p r e c i p i t a t i o n o f ® s ^ -c a s e i n by C a + + . Hansen (1968) m o d i f i e d Z i t t l e s ' (1965) method i n order t o i n v e s t i g a t e the s t a b i l i z a t i o n a c t i o n of K-carrageenan i n p r e v e n t i n g the p r e c i p i t a t i o n o f a ^ - c a s e i n by C a + + . Hansen (1968) employed c e n t r i f u g a t i o n a t 3000 x G f o r 5 min i n order to sediment the i n s o l u b l e Ca-a , - c a s e i n a t e . s i However, c e n t r i f u g a t i o n a f f e c t e d the q u a n t i t y o f s o l u b l e a n - c a s e i n recovered d u r i n g s t u d i e s of the s t a b i l i z -s l 3 i n g a b i l i t y of K-carrageenan toward c t ^ - c a s e i n a t pH 6.6, y = 0.04 ( F i g . 31). S t a b i l i z a t i o n v a l u e s i n c r e a s e d w i t h d e c r e a s i n g c e n t r i f u g a l f o r c e w i t h maximum s t a b i l i z a t i o n o b t a i n e d by f i l t r a t i o n . However, upon s t a n d i n g a t room temperature a f i n e p r e c i p i t a t e developed i n the f i l t r a t e which i n d i c a t e s t h a t o n l y p a r t of the i n s o l u b l e Ca - c t ^ -c a s e i n a t e was r e t a i n e d by the f i l t e r paper. At 3000 x G and 6000 * G a q u a n t i t y of white f l u i d m a t e r i a l c o l l e c t e d between the sediment and the mi l k y supernatant. T h i s f l u i d flowed o u t w i t h the supernatant d u r i n g d e c a n t a t i o n . A much s m a l l e r q u a n t i t y of t h i s m a t e r i a l 109 1 0 0 / C - C A R R A G E E N A N / a s i - C A S E I N , w / w F i g u r e 31. E f f e c t o f c e n t r i f u g a t i o n on s t a b i l i z a t i o n of a s i - c a s e i n by K - c a r r a g e e n a n (pH 6.6, p = 0.04). Curve a, f i l t r a t i o n ; c u r v e b, 1000 x G; c u r v e c, 300 0 x G; c u r v e d, 6 00 0 x G. Data are the average o f t h r e e t r i a l s . 110 was n o t e d when a c e n t r i f u g a t i o n o f - 1 0 0 0 x G was u s e d . P r e c i p i t a t e f o r m a t i o n i n t h e s u p e r n a t a n t o n s t a n d i n g was n o t n o t e d a f t e r a c e n t r i f u g a t i o n o f 1000 x G f o r 5 m i n was u s e d . T h e a m o u n t o f a . . - c a s e i n s o l u b l e i n 0 . 0 1 M C a C l „ s i 2 a n d t h e l i g h t s c a t t e r i n g c o n t r i b u t i o n o f t h e c a r r a g e e n a n was s u b t r a c t e d f r o m t h e a b s o r b a n c e . o f t h e c l a r i f i e d s u p e r n a t a n t s d u r i n g c a l c u l a t i o n o f t h e a m o u n t o f c t g ^ - c a s e i n s t a b i l i z e d b y K - c a r r a g e e n a n . I o n i c s t r e n g t h h a d a m a r k e d e f f e c t o n t h e K - c a r r a g e e n a n s t a b i l i z a t i o n o f a ^ - c a s e i n . A s t h e i o n i c s t r e n g t h o f t h e m e d i u m i n c r e a s e d , t h e s t a b i l i z a t i o n a t e a c h K - c a r r a g e e n a n / a s ^ - c a s e i n r a t i o d e c r e a s e d ( F i g . 3 2 ) . A s i m i l a r p h e n o m e n o n was o b s e r v e d f o r t h e K - c a s e i n - a . - c a s e i n s i s y s t e m ( G r e e n , 1 9 7 1 ) , a l t h o u g h t h e e f f e c t s o f i o n i c s t r e n g t h w e r e n o t a s s e v e r e a s t h o s e o b s e r v e d f o r t h e s t a b i l i z a t i o n o f a g ^ - c a s e i n b y K - c a r r a g e e n a n . H o w e v e r , H a n s e n (1968) o b s e r v e d t h a t t h e K - c a r r a g e e n a n - a ^ - c a s e i n s y s t e m was n o t a s ++ s e n s i t i v e t o i n c r e a s e d C a c o n c e n t r a t i o n s a s t h e K - c a s e m -a ^ - c a s e i n s y s t e m . T h e q u a n t i t y o f a ^ - c a s e i n s o l u b l e i n t h e p r e s e n c e o f 0 . 0 1 M C a C l „ i n c r e a s e d , w i t h i n c r e a s i n g i o n i c s t r e n g t h ( T a b l e V I I ) . I t h a s b e e n o b s e r v e d t h a t c a l c i u m b i n d i n g b y a g ^ - c a s e i n d e c r e a s e s w i t h i n c r e a s i n g i o n i c s t r e n g t h a t pH 6 . 6 (Waugh e t a l . , 1971) a n d a t pH 7 . 4 ( D i c k s o n a n d P e r k i n s , 1 9 7 1 ) . A t a n i o n i c s t r e n g t h o f 0 . 2 4 , t h e p r e s e n c e o f 100 r 0.1 0.2 0.3 0.4 0.5 /C - C A R R A G E E N A N / CL S I - C A S E I N , w/w F i g u r e 32. 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 s t a b i l i z a t i o n . o f a s ] _ - c a s e i n by K-carrageenan. Curve a, y = 0.04; cur v e b, y = 0.08; c u r v e c, y - 0.24. Data are the average o f t h r e e t r i a l s . Table VII EFFECT OF IONIC STRENGTH AND TEMPERATURE ON THE SOLUBILITY OF a .-CASEIN IN 0.01 M C a + + (pH 6.6) S o l u b l e a - C a s e i n 3 s l T(°C) I o n i c S t r e n g t h 0.04 0.08 0.24 4 17.38 ± 0.64 47.51 ± 1.05 25 2.14 ± 0.57 5.22 ± 0.77 43.2 ± 2.1 37 1.79 ± 0.26 4.47 ± 0.45 a. A l l v a l u e s are the means of three r e p l i c a t e s ± standard d e v i a t i o n . 113 K - c a r r a g e e n a n a p p e a r s t o h a v e i n c r e a s e d t h e c a l c i u m s e n s i t i v i t y o f a ^ - c a s e i n ( F i g . 32) . I n a d d i t i o n , a t b o t h y = 0 . 0 4 a n d 0 . 0 8 , K - c a r r a g e e n a n i n a r a t i o g r e a t e r t h a n 0 . 3 0 d e s t a b i l i z e d a . - c a s e i n i n t h e p r e s e n c e o f 0 . 0 1 M C a + + . I t s i i s p o s s i b l e t h a t t h e K - c a r r a g e e n a n a t a h i g h i o n i c s t r e n g t h l e v e l i n d u c e d c o n f o r m a t i o n a l c h a n g e s i n a g ^ - c a s e i n s i n c e t h e a d d i t i o n o f K - c a r r a g e e n a n t o D N S - a ^ - c a s e i n p r o d u c e d a n i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y ( F i g . 2 9 ) . T e m p e r a t u r e h a d a m a r k e d e f f e c t o n t h e s t a b i l i z a -t i o n o f a g ^ - c a s e i n b y K - c a r r a g e e n a n w i t h t h e e f f e c t b e i n g m o r e p r o n o u n c e d a t a n i o n i c s t r e n g t h o f 0 . 0 8 c o m p a r e d t o a n i o n i c s t r e n g t h o f 0 . 0 4 ( F i g . 3 3 ) . T h e q u a n t i t y o f c a s e i n s o l u b l e i n 0 . 0 1 M C a C ^ i n c r e a s e d w i t h d e c r e a s i n g t e m p e r a t u r e ( T a b l e V I I ) . Z i t t l e a n d J a s e w i c z (1962) o b s e r v e d t h a t 0 . 1 5 M N a C l d i s r u p t e d t h e s t a b i l i z a t i o n o f c t ^ - c a s e i n b y K - c a s e i n a t 7 ° C . No e f f e c t was o b s e r v e d b y t h e s e a u t h o r s a t 3 0 ° C . T h e s t a b i l i z a t i o n o f a g ^ - c a s e i n b y K - c a s e i n (y = 0 . 0 6 ) was r e d u c e d b y a d e c r e a s e i n t e m p e r a t u r e f r o m 3 0 ° C t o 7 ° C ( Z i t t l e a n d J a s e w i c z , 1 9 6 2 ) . A l t h o u g h s i m i l a r p h e n o m e n a w e r e o b s e r v e d w i t h t h e a . - c a s e i n - K - c a r r a g e e n a n a n d t h e a . - c a s e i n -s i ^ s i K - c a s e i n s y s t e m s t h e m e c h a n i s m s i n v o l v e d i n s t a b i l i z a t i o n a n d d e s t a b i l i z a t i o n may n o t n e c e s s a r i l y b e t h e s a m e . S i n c e t h e s e d i m e n t a t i o n v e l o c i t y r u n s w e r e p e r f o r m e d a t a ^ - c a s e i n c o n c e n t r a t i o n s o f 10 m g / m l w i t h K - c a r r a g e e n a n c o n c e n t r a t i o n s u p t o 3 . 4 m g / m l (pH 6 . 6 , y = 0 . 0 8 ) , 100 R UJ CO o UJ < I 1/1 a /C-CARRAGE ENAN / a s,-CASEIN , w/w Figure 33. Effect of temperature on s t a b i l i z a t i o n of a s]_-casein by K-carrageenan. Ionic strength 0.04, curve a, 37°C; curve b, 25°C, curve c, 4°C. Ionic strength 0.08, curve d, 37°C; curve e, 25°C; curve f, 4°C. Average of three t r i a l s . I—1 115 i t was o f i n t e r e s t t o d e t e r m i n e t h e s t a b i l i z a t i o n a b i l i t y o f K - c a r r a g e e n a n t o w a r d 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 v i s c o s i t y o f t h e c a r r a g e e n a n a t t h e s e l e c t e d c o n c e n t r a t i o n s was d e t e r m i n e d i n a 0 . 0 1 M i m i d a z o l e b u f f e r c o n t a i n i n g 0 . 0 1 M C a C l 2 , a n d 0 . 0 4 M N a C l a t pH 6 . 6 , :y = ,0.08' (Table V I I I ) . S t o k e ' s l a w was u t i l i z e d t o c a l c u l a t e t h e g r a v i t a t i o n a l f o r c e r e q u i r e d t o s e d i m e n t t h e i n s o l u b l e C a - a . ^ - c a s e i n a t e a t t h e same r a t e u n d e r e a c h t e s t c o n d i t i o n i n o r d e r t o o v e r c o m e a n y p o s s i b l e e f f e c t s o f t h e i n c r e a s e d v i s c o s i t y i n m a i n t a i n i n g t h e i n s o l u b l e c a l c i u m c a s e i n a t e p a r t i c l e s i n s u s p e n s i o n , d u r i n g c e n t r i f u g a t i o n o f t h e s t a b i l i z a t i o n t e s t m i x t u r e s . A r e a c t i o n v o l u m e o f 2 m l was u s e d a n d c e n t r i f u g a -t i o n t i m e was 5 m i n . C o n t r o l s w e r e u t i l i z e d i n w h i c h t h e f i n a l a d d i t i o n o f 0 . 1 M C a C l 2 was r e p l a c e d b y a n a l i q u o t o f 0 . 3 M N a C l s u c h t h a t t h e f i n a l i o n i c s t r e n g t h was 0 . 0 8 . T h e c o n t r o l s w e r e t r e a t e d i d e n t i c a l l y t o t h e t e s t s a m p l e s a n d w e r e u t i l i z e d t o c o r r e c t f o r a n y c a s e i n a n d / o r c a r r a g e e n a n t h a t may h a v e b e e n s e d i m e n t e d o u t b y t h e h i g h c o n t r i f u g a l f o r c e s u s e d . T h e pH o f t h e c o n t r o l s a n d t e s t s a m p l e s was 6 . 5 t o 6 . 6 a f t e r ' c e n t r i f u g a t i o n . U p o n a d d i t i o n o f c a l c i u m t o t h e t e s t s a m p l e s , t h e s o l u t i o n s b e c a m e v e r y v i s c o u s . T h e m o s t e f f e c t i v e m e t h o d o f m i x i n g was f o u n d t o b e i n v e r s i o n o f t h e r e a c t i o n t u b e s t e n t i m e s . V e r y p o o r s t a b i l i z a t i o n v a l u e s w e r e o b t a i n e d b y i n c r e a s i n g t h e a g ^ - c a s e i n t o 10 m g / m l a n d u s e o f c a r r a g e e n a n / 116 T a b l e V I I I V I S C O S I T Y O F K-CARRAGEENAN I N T H E P R E S E N C E OF 0 . 0 1 M C a C l 2 (pH 6 . 6 , F I N A L I O N I C S T R E N G T H 0 . 0 8 ) K - C a r r a g e e n a n (mg/ml) R e l a t i v e V i s c o s i t y R e l a t i v e C e n t r i f u g a l F o r c e " ^ 0 . 5 1 . 5 5 1 , 9 5 0 1 . 0 2 . 5 6 3 , 1 4 0 1.5 3 . 87 4 , 7 0 0 2 . 0 5 . 6 2 6 , 8 5 0 2 . 5 7 . 9 7 9 , 7 0 0 3 . 0 1 1 . 2 3 1 3 , 7 0 0 3 . 5 1 6 . 0 4 1 9 , 5 0 0 C a l c u l a t e d u s i n g S t o k e ' s L a w . 117 c a s e i n r a t i o s s i m i l a r to those used when an a . - c a s e i n s i c o n c e n t r a t i o n o f 1.5 mg/ml was used ( F i g . 34). Only a ^ - c a s e i n was removed by c e n t r i f u g a t i o n s i n c e the superna-t a n t carrageenan c o n c e n t r a t i o n s were the same as those at the beginning o f the experiments. C e n t r i f u g a t i o n was not r e s p o n s i b l e f o r the poor s t a b i l i z a t i o n observed s i n c e c e n t r i f u g a t i o n a t 1000 x G of samples a t a carrageenan-casein r a t i o o f 0.35 d i d not i n c r e a s e the s o l u b l e c a s e i n t o any gr e a t extent ( F i g . 34). The p r o t e i n c o n c e n t r a t i o n o f the c o n t r o l s was u n a l t e r e d by the c e n t r i f u g a t i o n s used. M a i n t a i n i n g the K - c a r r a g e e n a n / a ^ - c a s e i n r a t i o a t 0.2 and v a r y i n g the a g ^ - c a s e i n c o n c e n t r a t i o n from 0.5 mg/ml to 10 mg/ml l e a d to decreased s t a b i l i z a t i o n w i t h i n c r e a s i n g a . - c a s e i n c o n c e n t r a t i o n ( F i g . 35). Thus, i t appears t h a t r e s u l t s of s t a b i l i z a t i o n data o b t a i n e d a t low p r o t e i n c o n c e n t r a t i o n s cannot be e x t r a p o l a t e d t o hi g h e r p r o t e i n c o n c e n t r a t i o n s as evidenced by the d e c r e a s i n g s t a b i l i z a t i o n o b t a i n e d w i t h i n c r e a s i n g a - c a s e i n c o n c e n t r a t i o n s ( F i g s . 34 and 35). s i ^ ' Chakraborty and Randolph (19 72) s t a t e d t h a t e x t r a p o l a t i o n o f s t a b i l i z a t i o n data obtained a t r e l a t i v e l y low p r o t e i n c o n c e n t r a t i o n s probably c o u l d not be c a r r i e d out to h i g h e r p r o t e i n l e v e l s due to the f a c t t h a t p r o p o r t i o n a l i n c r e a s e s i n K-carrageenan content l e a d to a h i g h l y v i s c o u s system. However, as demonstrated i n F i g s . 34 and 35 the i n c r e a s e i n v i s c o s i t y w i t h i n c r e a s i n g K-carrageenan content H-C ro t-3 O 7s O cn Cu • 1 • rt cr o o o cu H-1 CU co tr ro hi — H-s hi • h-1 < CU H-H N H fD o CD H n fD c rt h-1 3 hi H-CU < 0 0 3 fD 3 hi fu CU 0 n cn O htl fD c 3 hi hi cn R rt fD < C cn hj TJ fD TJ H- M fD 1 Hi Cu o hi 0 c Cl 3 Cu fD CU CO CD Cb cn rt fD rt c Cu H-H- rj"C 3 3 0 <^ fD rt 3 hi o 3 R cn • CU cn o TJ o r t M (D CU I 3 fD 3 O fu \ cn cn 3 R fD h-1 fD Cu cn H- ^ — 3 f i n 3 T! f—• 1 cr h-1 O 0 . — . CU o •< TJ cn c fD fD hi 1 H- < n • cn 3 fD ar CTi H- cr hj 3 ag 0 cn fD 0 c fD II 3 TJ 3 rt fD Cu o hi hi 3 • 0 3 o h-> Cu co rtTJ — Cu • 3" 3 fD rt cn hi • cn fD cn fD % a s | - C A S E I N S O L U B L E — SJ CO o o o S U P E R N A T A N T C A R R A G E E N A N b mg/ml 8TT 119 6 0 h CO ZD o CO Z CO < U 40\-7 2 0 0 0 a Sl C A S E I N , i ' 6 m g / m l 10 F i g u r e 35. E f f e c t o f a s ^ - c a s e i n c o n c e n t r a t i o n on s t a b i l i z a t i o n o f a s ] _ - c a s e i n by K - c a r r a g e e n a n (pH 6.6, u = 0.08). K - C a r r a g e e n a n / a s i - c a s e i n = 0.2 (w/w). Average o f d u p l i c a t e t r i a l s . C e n t r i f u g a t i o n ; 1000 x G, 5 min. 120 d i d not enhance the s t a b i l i z a t i o n o b t ained a t a g i v e n K - c a r r a g e e n a n / a s ^ - c a s e i n r a t i o . Instead, the presence of h i g h e r c o n c e n t r a t i o n s of carrageenan r e s u l t e d i n decreased s t a b i l i z a t i o n . I f K-carrageenan - p r e f e r e n t i a l l y bound C a + + , hi g h e r K-carrageenan c o n c e n t r a t i o n s would be expected to have l e a d to c o r r e s p o n d i n g l y l a r g e i n c r e a s e s i n s o l u b l e a g ^ - c a s e i n due to removal of C a + + from the system by the carrageenan. However, t h a t d i d not occur. Hansen (1968) a l s o observed t h a t i t was u n l i k e l y t h a t K-carrageenan s t a b i l i z e d a g ^ - c a s e i n by b i n d i n g C a + + . In a d d i t i o n Schachat and Morawetz (1957) observed, on the b a s i s of e q u i l i b r i u m d i a l y s i s experiments, t h a t K-carrageenan does not b i n d C a + + any more t i g h t l y than i t does Na + o r K +. The decrease i n s t a b i l i z a t i o n of a n - c a s e i n by s l 2 K-carrageenan observed a t h i g h c o n c e n t r a t i o n s of K-carrageenan may p o s s i b l y a r i s e from an a l t e r a t i o n i n the conformation of a g ^ - c a s e i n due to the presence of l a r g e amounts of K-carrageenan. Since a s s o c i a t i o n of K-carrageenan i s c o n c e n t r a t i o n dependent ( F i g . 8) the p o s s i b i l i t y a l s o e x i s t s t h a t at h i g h e r c o n c e n t r a t i o n s the K-carrageenan aggregates and as a r e s u l t e x i s t s i n a conformation t h a t i s i n e f f e c t i v e as a s t a b i l i z e r . 121 GENERAL D I S C U S S I O N O b s e r v a t i o n s b a s e d o n d a t a o b t a i n e d f r o m t u r b i d i t y , s e d i m e n t a t i o n v e l o c i t y a n d v i s c o m e t r i c m e a s u r e m e n t s s u b s t a n t i a t e d t h e p r e s e n c e o f a c h e m i c a l i n t e r a c t i o n b e t w e e n a g ^ - c a s e i n a n d K - c a r r a g e e n a n . H o w e v e r , 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 f r o n t a l g e l c h r o m a t o g r a p h y d a t a a s w e l l a s 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 s b a s e d o n s e d i m e n t a t i o n e q u i l i b r i u m e x p e r i m e n t s w e r e t o t h e c o n t r a r y . A n e x p l a n a t i o n o f t h e c o n f l i c t i n g r e s u l t s o b t a i n e d b y t h e v a r i o u s e x p e r i m e n t a l p r o c e d u r e s may b e t h a t a p h y s i c a l i n t e r a c t i o n r a t h e r t h a n a c h e m i c a l i n t e r a c t i o n was o b s e r v e d d u r i n g s e d i m e n t a t i o n v e l o c i t y a n d v i s c o s i t y e x p e r i m e n t s . K - C a r r a g e e n a n c a n e x i s t a s a d o u b l e h e l i x ( B r y c e e t a l . , 1974) w h i c h i s i n t e r r u p t e d b y t h e p o s i t i o n i n g o f h e l i x - d i s r u p t i n g g a l a c t o s e - 2 , 6 - d i s u l f a t e r e s i d u e s a t v a r i o u s p o i n t s a l o n g t h e p o l y s a c c h a r i d e c h a i n . T h u s , e a c h p o l y s a c c h a r i d e c h a i n c a n e n t e r i n t o d o u b l e h e l i c a l c o m p l e x e s w i t h o t h e r p o l y s a c c h a r i d e c h a i n s t o f o r m a t h r e e - d i m e n s i o n a l , e n t a n g l e d n e t w o r k ( R e e s e t a l . , 1 9 6 9 ) . N e t w o r k f o r m a t i o n i n s o d i u m c a r r a g e e n a t e . s o l u t i o n s h a s b e e n d e m o n s t r a t e d ( R e e s e t a l . , 1 9 6 9 ; A n d e r s o n e t a l . , 1 9 6 9 ) . G o r i n g (1954b) o b s e r v e d a n a n o m a l y , d u r i n g f r e e -b o u n d a r y e l e c t r o p h o r e s i s o f a n u m b e r o f s a m p l e s o f s o d i u m c a r r a g e e n a t e , w h i c h h e a t t r i b u t e d t o t h e i n d u c e m e n t o f n e t w o r k f o r m a t i o n d u r i n g e l e c t r o p h o r e s i s s u c h t h a t t h e 122 carrageenan would migrate as a porous p l u g . T h i s phenomenon was observed o n l y d u r i n g the a p p l i c a t i o n of a p o t e n t i a l across the e l e c t r o p h o r e s i s c e l l . The occurrence of a s i m i l a r phenomenon d u r i n g sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n was i n v e s t i g a t e d by Goring and Chepeswick (1955). They suggested t h a t sodium carrageenate, above a c r i t i c a l c o n c e n t r a t i o n , sedimented as an i n t e r c o n n e c t i n g f i b r o u s network moving through the s o l v e n t . Schachman (1959) and Johnson (1968) s t a t e d t h a t , i n s o l u t i o n s of elongated, f l e x i b l e macromolecules, a network i s l i k e l y to be formed i n which there i s l i t t l e freedom o f motion of the molecules forming the network. T h i s network of i n t e r f e r i n g macro-molecules would flow as a whole (porous plug) i n the d i r e c t i o n o f an a p p l i e d c e n t r i f u g a l f i e l d (Johnson, 1968). Thus, such a s i t u a t i o n probably e x i s t e d f o r the sodium carrageenate s o l u t i o n s used i n t h i s study even though most o f the s o l u t i o n c o n c e n t r a t i o n s were below the c r i t i c a l l e v e l o f 2.5 mg carrageenan/ml observed by Goring and Chepeswick (1955). During sedimentation v e l o c i t y experiments i t i s p o s s i b l e t h a t an entangled, t h r e e - d i m e n s i o n a l K-carrageenan network entrapped the a ^ - c a s e i n , pushing i t ahead, g i v i n g r i s e t o the a ^ - c a s e i n c o n t a i n i n g peak f o l l o w e d by the " r e s i d u a l " carrageenan observed f o r a ^ - c a s e i n - K - c a r r a g e e n a n mixtures. On the b a s i s of the above assumption i t would appear t h a t the pore s i z e of the carrageenan network was s m a l l e r than the s i z e of the a g ^ - c a s e i n aggregates p r e s e n t under the 123 conditions of pH, i o n i c strength, temperature and concentra-tions employed. Thus, as the carrageenan concentration increased, the pore size of the network decreased, excluding more a g^-casein from the K-carrageenan network giving r i s e to what appeared to be an "interaction" peak characterized by an S 2 Q w value that increased with increasing carrageenan concentration. Urea (6.0 M) disrupted the a .-casein into s i monomeric units smaller than the pore size of the K-carrageenan network. This enabled the slower sedimenting a .-casein to 3 s i pass through the pores of the carrageenan network during sedimentation giving r i s e to the a ^ - c a s e i n peak sedimenting behind the K-carrageenan peak. Thus, the action of urea was not a disruption of an a^-casein-K-carrageenan complex, but, rather a d i s s o c i a t i o n of the a s^-casein aggregates to a size smaller than the pore size of the K-carrageenan network. A si m i l a r mechanism would operate during v i s c o s i t y measurements. A physical entanglement of a ^ - c a s e i n within the K-carrageenan network would create a greater resistance to flow since the entangled network would behave sim i l a r to a molecule with a larger hydrodynamic volume than would be expected for a non-interacting system. Thus, the reduced v i s c o s i t i e s of most a .-casein-K-carrageenan mixtures were s i greater than the reduced v i s c o s i t i e s expected i n the absence of chemical i n t e r a c t i o n . During gel chromatography, however, a physical 124 i n t e r a c t i o n would be d i s r u p t e d by the column packing g i v i n g r i s e to the e l u t i o n of a g ^ - c a s e i n - K - c a r r a g e e n a n mixtures as the separate components. A p r e r e q u i s i t e f o r the passage of the a g ^ - c a s e i n - K - c a r r a g e e n a n mixtures through the column would be d i s r u p t i o n of the carrageenan network thus p e r m i t t i n g the a g ^ - c a s e i n t o enter the pores o f the g l a s s matrix w h i l e the h i g h molecular weight K-carrageenan e l u t e d i n the v o i d volume. Only i n the case o f a chemical i n t e r a c t i o n i n v o l v i n g e i t h e r c o v a l e n t or secondary bonds (e.g., hydrogen bonding) between a - c a s e i n and K-carrageenan, would the a . - c a s e i n s l ^ s l component e l u t e i n the v o i d volume as an a g ^ - c a s e i n - K - c a r r a -geenan complex under the experimental c o n d i t i o n s used. Sedimentation e q u i l i b r i u m , u n l i k e sedimentation v e l o c i t y , d i d not i n v o l v e i n t e n s e g r a v i t a t i o n a l f i e l d s . During sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n , entanglement of the carrageenan m a t r i x was probably induced by the i n t e n s e g r a v i t a t i o n a l f i e l d (253,417 * G, a t 59,000 rpm; r = 6.50 cm). A "porous p l u g " type o f carrageenan network probably d i d not e x i s t d u r i n g sedimentation e q u i l i b r i u m c e n t r i f u g a t i o n . a t 5600 rpm (2,283 x G; r = 6.50 cm) s i n c e low g r a v i t a t i o n a l f o r c e s were i n v o l v e d . Thus, the d i s t r i b u t i o n of each component of the mixture proceeded. The r e s u l t i n g m o l e c u l a r weight d i s t r i b u t i o n r e v e a l e d t h a t the a g ^ - c a s e i n component of the mixture was not c h e m i c a l l y o r p h y s i c a l l y complexed with the K-carrageenan. I f a chemical i n t e r a c t i o n between a n - c a s e i n 125 a n d K - c a r r a g e e n a n d i d o c c u r , a s s u g g e s t e d b y t h e s e d i m e n t a t i o n v e l o c i t y s c h l i e r e n p a t t e r n s o f t h e a g ^ - c a s e i n (5 m g / m l ) - K -c a r r a g e e n a n (1 m g / m l ) m i x t u r e , a v e r y h i g h m o l e c u l a r w e i g h t c o m p l e x w o u l d h a v e e x i s 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 m e a s u r e m e n t s s h o w e d t h e a b s e n c e o f a n a g ^ - c a s e i n - K - c a r r a g e e n a n i n t e r a c t i o n . S i n c e a p p l i e d f i e l d s s u c h a s t h o s e e n c o u n t e r e d i n s e d i m e n t a t i o n v e l o c i t y u l t r a c e n t r i f u g a t i o n o r d u r i n g f l o w t h r o u g h a c a p i l l a r y e x p e r i e n c e d d u r i n g v i s c o m e t r y w e r e n o t p r e s e n t d u r i n g f l u o r e s c e n c e m e a s u r e m e n t s , a " p o r o u s - p l u g " t y p e o f c a r r a g e e n a n n e t w o r k d i d n o t e x i s t . T h u s , a m i x t u r e o f D N S -a g ^ - c a s e i n a n d K - c a r r a g e e n a n w o u l d n o t b e e x p e c t e d t o l e a d t o a n i n c r e a s e 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 s i n c e t h e r a t e o f r o t a t i o n o f D N S - a . - c a s e i n w o u l d n o t b e h i n d e r e d b y t h e s i •* K - c a r r a g e e n a n u n d e r t h e s e c o n d i t i o n s i n t h e a b s e n c e o f a n a p p l i e d f o r c e . T h u s , o n l y i n t h e c a s e o f a c h e m i c a l i n t e r -a c t i o n w o u l d t h e r e b e a n i n c r e a s e 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 s i n c e t h e a g ^ - c a s e i n - K - c a r r a g e e n a n c o m p l e x w o u l d p o s s e s s a l a r g e r m o l e c u l a r w e i g h t a n d t h u s , a l o w e r r a t e o f B r o w n i a n m o t i o n t h a n t h a t o f t h e DNS-ct . - c a s e i n o r s i t h e D N S - K - c a r r a g e e n a n . T u r b i d i t y f o r m a t i o n u p o n m i x i n g o f K - c a r r a g e e n a n w i t h a g ^ - c a s e i n u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d a r o s e f r o m a c h a n g e i n t h e s o l u b i l i t y a n d / o r i n t h e s i z e o f a ^ - c a s e i n a g g r e g a t e s d u e t o t h e p r e s e n c e o f K - c a r r a g e e n a n . I t h a s b e e n d e m o n s t r a t e d t h a t d e x t r a n s r e d u c e t h e s o l u b i l i t y 126 o f a number of p r o t e i n s p o s s i b l y by removal o f water from the p r o t e i n - s o l v e n t system by the p o l y s a c c h a r i d e (Laurent, 1 9 6 3 ) . On the other hand, Jones and E r l a n d e r ( 1 9 6 7 ) noted a resemblance of g l u t e n - d e x t r a n i n t e r a c t i o n s t o a n t i g e n - a n t i b o d y r e a c t i o n s . Since n e i t h e r a g ^ - c a s e i n nor K-carrageenan c o u l d be removed from the system by c e n t r i f u g a t i o n ( F i g . 6 ) a t 3 9 , 0 0 0 x G the f a c t o r ( s ) producing the t u r b i d i t y were not of a molecular s i z e t h a t c o u l d be i s o l a t e d by t h i s procedure. The f a c t t h a t the t u r b i d i t y a t a g i v e n K - c a r r a g e e n a n / a g ^ - c a s e i n combination i n c r e a s e d w i t h i n c r e a s i n g i o n i c s t r e n g t h may r e l a t e to an i n c r e a s e i n aggregation of a ^ - c a s e i n by the K-carrageenan as w e l l as an i n c r e a s e i n the s i z e o f the a ^ - c a s e i n aggregates as a r e s u l t of i n c r e a s i n g i o n i c s t r e n g t h . Snoren e t a l . ( 1 9 7 5 ) have presented evidence t h a t the s i z e of a ^ - c a s e i n aggregates i n c r e a s e d with i n c r e a s i n g i o n i c s t r e n g t h . In a d d i t i o n , t h e i r r e s u l t s appear to s u b s t a n t i a t e the o b s e r v a t i o n s t h a t K-carrageenan may i n c r e a s e the s i z e of a g ^ - c a s e i n aggregates or decrease the s o l u b i l i t y o f a ^ - c a s e i n . They observed t h a t a g r e a t e r q u a n t i t y of a g ^ - c a s e i n c o u l d be i s o l a t e d , by c e n t r i f u g a t i o n a t 9 0 , 0 0 0 x G , from a g ^ - c a s e i n - K - c a r r a g e e n a n mixtures a t pH 5 . 9 than from a s o l u t i o n of a . - c a s e i n under i d e n t i c a l s l c o n d i t i o n s . Thus, i t would appear t h a t t u r b i d i t y f ormation i n a g ^ - c a s e i n - K - c a r r a g e e n a n mixtures ( a ^ - c a s e i n 1 0 mg/ml) was 127 not the r e s u l t o f a chemical i n t e r a c t i o n between these macromolecules. Rather, t h i s , phenomenon may have been a consequence of a change i n the molecular s i z e of a g ^ - c a s e i n a r i s i n g from the presence of K-carrageenan. T h e r e f o r e , on the b a s i s of t h i s study, the mechanism(s) o f K-carrageenan s t a b i l i z a t i o n of a ^ - c a s e i n from p r e c i p i t a t i o n by C a + + d i d not a r i s e from chemical i n t e r -a c t i o n between a -^.-casein and K-carrageenan p r i o r to a d d i t i o n o f C a + + . Thus, K-carrageenan probably s t a b i l i z e d a ^ - c a s e i n through the formation o f C a + + - s a l t b r i d g e s w i t h c a l c i u m -c a s e i n a t e p a r t i c l e s trapped w i t h i n the K-carrageenan matrix. Chakraborty and Randolph (1972) suggested t h a t K-carrageenan entraps c a l c i u m aggregated p r o t e i n p a r t i c l e s p r e v e n t i n g t h e i r agglomeration i n t o l a r g e r , c o l l o i d a l l y u n s t a b l e p a r t i c l e s by keeping them separated w h i l e p r o v i d i n g adequate s o l v a t i o n by means of p r o t e i n - f r e e r e g i o n s o f the h y d r o c o l l o i d . The reduced s t a b i l i z a t i o n observed at h i g h a ,-^ s i c a s e i n c o n c e n t r a t i o n s (e.g., 10 mg/ml) may have r e s u l t e d from i n c r e a s e s i n the molecular s i z e o f . a . - c a s e i n i n the s i absence o f C a + + as _a consequence of the i n c r e a s e d K-carrageenan c o n c e n t r a t i o n when compared to the s t a b i l i z a t i o n observed a t s i m i l a r K - c a r r a g e e n a n / a ^ - c a s e i n combinations when the c t ^ - c a s e i n c o n c e n t r a t i o n was 1.5 mg/ml. The c a l c i u m mediated i n t e r a c t i o n of l a r g e a .^-casein aggregates w i t h one another probably l e a d t o the formation of c o l l o i d a l l y u n s t a b l e p r o t e i n aggregates, which were too l a r g e to r e a d i l y 128 r e a c t w i t h the K-carrageenan and thus, p r e c i p i t a t e d from s o l u t i o n . 129 CONCLUSIONS a .-Casein and K-carrageenan d i d not i n t e r a c t s l c h e m i c a l l y i n a c a l c i u m - f r e e system. The " i n t e r a c t i o n " r e v e a l e d by sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n and v i s c o s i t y measurements arose from a p h y s i c a l entrapment o f a g ^ - c a s e i n aggregates w i t h i n a sodium-K-carrageenate network. Since techniques such as f l u o r e s c e n c e p o l a r i z a t i o n , f r o n t a l g e l chromatography and sedimentation e q u i l i b r i u m u l t r a c e n t r i -f u g a t i o n d i d not i n v o l v e the a p p l i c a t i o n o f a f o r c e such t h a t the formation o f an entangled K-carrageenan network and movement as a "porous p l u g " was fa v o r e d , an i n t e r a c t i o n was not observed. S t a b i l i z a t i o n o f a g ^ - c a s e i n by K-carrageenan was di m i n i s h e d by i n c r e a s i n g g r a v i t a t i o n a l f o r c e d u r i n g the c e n t r i f u g a t i o n step o f the s t a b i l i z a t i o n t e s t as w e l l as by i n c r e a s i n g the c t ^ - c a s e i n c o n c e n t r a t i o n from 1.5 mg/ml to 10 mg/ml. 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