UBC Theses and Dissertations

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UBC Theses and Dissertations

Relationship between physicochemical properties of proteins and their foaming characteristics Townsend, Althea-Ann E. 1982

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RELATIONSHIP BETWEEN PHYSICOCHEMICAL PROPERTIES OF PROTEINS AND THEIR FOAMING CHARACTERISTICS \ ALTHEA-ANN E. TOWNSEND B . S c , U n i v e r s i t y of the West I n d i e s , 1 974-M.Sc, U n i v e r s i t y of Guelph, 1977 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 S c i e n c e ) We a c c e p t t h i s t h e s i s as co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA January ' 1 98 2 0 A l t h e a - A n n E. Townsend, 1982 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying 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 o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D E - 6 (2/79} i i ABSTRACT The r e l a t i o n s h i p b e t w e e n t h e f o a m i n g c h a r a c t e r -i s t i c s o f p r o t e i n s and some o f t h e i r . . p h y s i c o c h e m i c a l p r o p e r t i e s i n b u l k s o l u t i o n were e x a m i n e d . The p h y s i c o -c h e m i c a l p r o p e r t i e s w h i c h were i n v . e s t i g a t e d i n r e l a t i o n t o f o a m i n g were h y d r o p h o b i c i t y , c h a r g e d e n s i t y , s e c o n d a r y s t r u c t u r e , m o l e c u l a r f l e x i b i l i t y , , d i s p e r s i b i l i t y , v i s c o s i t y and s u r f a c e t e n s i o n , . , E l e v e n , m o d el p r o t e i n s . ( r i b o n u c l e a s e , o v o m u c o i d , t r y p s i n , l.ysozyme, p e p s i n , o v a l b u m i n , c o n a l b u -m i n , b o v i n e ser.um albumin., K - c a s e i n , 3 - l a c t o g l o b u l i n , 3 - c a s e i n ) and e i g h t f o o d p r o t e i n s . ( s o y , p e a , s u n f l o w e r and c a n o l a i s o l a t e s . , p r o - p u l s e , P r o m i n e - D , w h o l e c a s e i n , a c i d s o l u b i l i z e d g l u t e n ) were u s e d . I t was f o u n d t h a t t h e a v e r a g e h y d r o p h o b i c i t y o f p r o t e i n s c o u l d be m e a s u r e d u s i n g c i s - p a r i n a r i c a c i d as a p r o b e o f t h e h y d r o p h o b i c r e g i o n s , . . a f t e r t h e s a m p l e s had b e e n u n c o i l e d by h e a t i n g f o r . 1.0. min a t 1 00°C i n t h e p r e s e n c e o f 1.5% s o d i u m dodec.yl. s u l p h a t e . H y d r o p h o b i c i t y m e a s u r e d . i n t h i s way showed a'. s i g n i f i c a n t l i n e a r c o r r e l a -t i o n ( r = 0...820, P <0...01 ). . w i t h t h e c a l c u l a t e d a v e r a g e h y d r o p h o b i c i t y v a l u e s o f B i g e l o w . Two r e g r e s s i o n e q u a t i o n s . w e r e g e n e r a t e d , w h i c h a c c o u n t e d f o r a p p r o x i m a t e l y 77% o f t h e v a r i a b i l i t y i n t h e I l l foaming c a p a c i t i e s of the. p r o t e i n s ; one i n c l u d e d hydro-p h o b i c i t y and d i s p e r s i b i l i t y and the other i n v o l v e d h y d r o p h o b i c i t y and v i s c o s i t y as the independent v a r i a b l e s . High hydr o p h o b i c i t y and v i s c o s i t y . , and moderate d i s p e r s i -b i l i t y were a s s o c i a t e d .with optimum foaming c a p a c i t y . A l t h o u g h charge d e n s i t y i n f l u e n c e d . t h e foaming c a p a c i t y of p r o t e i n s i t s r o l e i n d e t e r m i n i n g t h i s p r o p e r t y seemed t o be a minor one. H i g h e s t foaming c a p a c i t y was e x h i b i t e d by the most f l e x i b l e p r o t e i n s . There was a s i g n i f i c a n t n e g a t i v e r e l a t i o n s h i p (r =-0.726, P<0.01) between foam s t a b i l i t y and the r e c i p r o c a l charge d e n s i t y . T h i s i n d i c a t e d t h a t e l e c t r o s t a t i c r e p u l s i o n between p r o t e i n m o l e c u l e s had an i m p o r t a n t d e s t a b i l i z i n g e f f e c t on foams. I t was demonstrated t h a t h y d r o p h o b i c i t y p l a y s an i m p o r t a n t r o l e i n de t e r m i n i n g , the, foaming b e h a v i o u r of p r o t e i n solutions... U n l i k e , e m u l s i f i c a t i o n , which i s dependent on s u r f a c e hydrophobicit.y,. the hydr o p h o b i c i t y of the u n c o i l e d p r o t e i n m olecule i s i m p o r t a n t f o r foaming. i v TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS x i i L I S T OF TABLES v i i L I S T OF FIGURES i x I . INTRODUCTION 1 I I . LITERATURE REVIEW 5 1. P r o t e i n Foams 5 2. P r o t e i n s a t t h e A i r - W a t e r I n t e r f a c e 13. 3. H y d r o p h o b i c i t y o f P r o t e i n s 18 4-. P r o t e i n C h a r g e 22 5. M o d e l P r o t e i n s 24. I I I . MATERIALS AND METHODS 34 1. M a t e r i a l s 34 2. M e t h o d s - - 35 2.1 A c i d S o l u b i l i z e d G l u t e n 35 2.2 B - C a s e i n . 35 2.3 K - C a s e i n 37 2.4 C a n o l a . P r o t e i n I s o l a t e 38 2.5 Enzyme H y d r o . l y z e d P r o t e i n s 38 2.6 F o a m i n g 39 2.7 H y d r o p h o b i c i t y 4.1 2.8 T o t a l P r o t e i n 42 2.9 D i s p e r s i b i l i t y 43 V PAGE 2.10 V i s c o s i t y 4-3 2.11 N o n - P r o t e i n N i t r o g e n 44. 2.12 Bound SDS 4-4-2.13 Simplex O p t i m i z a t i o n 4-5 2.14- S u r f a c e T ension 4-6 2.14- Charge D e n s i t y 4-6 2.16 E r r o r A n a l y s i s from R e p l i c a t e Measurements 4-8 IV. RESULTS AND DISCUSSION 4:9 1 . Choice of Model P r o t e i n s 4-9 2. L' .Choice of C i s - P a r i n a r i c A c i d f o r Hydro-p h o b i c i t y Measurement 4-9 3. Importance of P r o t e i n Secondary S t r u c t u r e , M o l e c u l a r S i z e , Shape and F l e x i b i l i t y For Foaming 50 4.. R e l a t i o n s h i p between P r o t e i n Hydro-p h o b i c i t y and Foaming P r o p e r t i e s 58 5. Importance of Charge D e n s i t y f o r Foaming 89 6. R e l a t i o n s h i p between S o l u t i o n V i s c o s i t y and Foaming P r o p e r t i e s 101 7. R e l a t i o n s h i p between S u r f a c e T ension and Foaming P r o p e r t i e s 109 8. Importance of M o l e c u l a r S i z e f o r Foaming •• 112 V. GENERAL DISCUSSION 116 V I . CONCLUSIONS 121 v i PAGE V I I . LITERATURE CITED 123 APPENDICES I Simplex O p t i m i z a t i o n 1 32; I I C a l c u l a t i o n of Net P r o t o n Charge 134. v i i LIST OF TABLES TABLE PAGE 1 The m o l e c u l a r w e i g h t , f r i c t i o n a l r a t i o and number of d i s u l p h i d e bonds of the model p r o t e i n s 25 2 P e r c e n t a g e s of a - h e l i x , 3-sheet and t u r n s i n the model p r o t e i n s 27 3 Amino a c i d c o m p o s i t i o n of the model p r o t e i n s 28 4. Foaming c a p a c i t y , and foam s t a b i l i t y of model p r o t e i n s 56 5 Percentage of h y d r o p h o b i c groups i n the model p r o t e i n s 59 6 R e s u l t s of the. s i m p l e x s e a r c h 81 7 Q u a n t i t y of SDS bound d u r i n g h e a t i n g f o r 10 min at. 100°C i n 1.5/6 SDS 82 8 M u l t i p l e . r e g r e s s i o n a n a l y s i s of foaming c a p a c i t y on h y d r o p h o b i c i t y and s o l u b i l i t y 86 9 Net p r o t o n charge on the model p r o t e i n s a t pH 7.0 91 10 E f f e c t of i o n i c s t r e n g t h on foaming c a p a c i t y 9A 11 ANOVA f o r the e f f e c t , of i o n i c s t r e n g t h on foaming c a p a c i t y 95 12 E f f e c t of pH on foaming c a p a c i t y 96 v i i i TABLE PAGE 13 ANOVA f o r the e f f e c t of pH on foaming c a p a c i t y 97 14- M u l t i p l e r e g r e s s i o n a n a l y s i s of foaming c a p a c i t y on h y d r o p h o b i c i t y and b u l k v i s c o s i t y 104-15 E f f e c t of enzyme h y d r o l y s i s on the l e v e l of n o n - p r o t e i n n i t r o g e n 113 16 E f f e c t . o f enzyme h y d r o l y s i s on foaming c a p a c i t y , h y d r o p h o b i c i t y and d i s p e r s i -b i l i t y 114-i x LIST OF FIGURES FIGURE PAGE 1 Diagram of foaming a p p a r a t u s 4-0 2 R e l a t i o n s h i p between quantum y i e l d of c i s - p a r i n a r i c a c i d and s o l v e n t p o l a r i t y 51 3 R e l a t i o n s h i p between foaming c a p a c i t y and m o l e c u l a r f l e x i b i l i t y measured as §SS bonds/molecular weight 57 4 R e l a t i o n s h i p between foaming c a p a c i t y and B i g e l o w s average h y d r o p h o b i c i t y (H0 ) 61 ave 5 R e l a t i o n s h i p between.foam s t a b i l i t y and B i g e l o w ' s . a v e r a g e h y d r o p h o b i c i t y (H0 ) 63 6 R e l a t i o n s h i p betwe.en foaming c a p a c i t y and s u r f a c e h y d r o p h o b i c i t y (S ) 65 7 R e l a t i o n s h i p between foaming c a p a c i t y and h y d r o p h o b i c i t y (S ) measured i n the presence of 0.005$ SDS 67 8 R e l a t i o n s h i p between foaming c a p a c i t y and h y d r o p h o b i c i t y (S ) measured i n the presence of 0.01$ SDS 68 9 R e l a t i o n s h i p between.foaming c a p a c i t y and h y d r o p h o b i c i t y (S ) measured i n the presence of 0.05$ SDS 69 X FIGURE PAGE 10 R e l a t i o n s h i p b e t w e e n . f o a m i n g c a p a c i t y and h y d r o p h o b i c i t y (S ) m e a s u r e d I n t h e p r e s e n c e o f 1M p h e n e t h y l g u a n i d i n e h y d r o c h l o r i d e 71 11 R e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y and h y d r o p h o b i c i t y ..(S.). m e a s u r e d a f t e r a l k a l i t r e a t m e n t 73 12 R e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y and h y d r o p h o b i c i t y (S ) m e a s u r e d a f t e r h e a t i n g a t 1 00.°C f o r 5 m i n u t e s 75 13 R e l a t i o n s h i p , .between .foaming c a p a c i t y ... and h y d r o p h o b i c i t y (S ) m e a s u r e d a f t e r e x p o s u r e t o 100°C f o r 20 min i n t h e p r e s e n c e o f m e r c a p t o e t h a n o l and SDS 77 14- R e l a t i o n s h i p b e t w e e n h y d r o p h o b i c i t y (S ) m e a s u r e d a f t e r e x p o s u r e t o 100°C f o r 10 m i n . i n t h e p r e s e n c e o f 1.5$ SDS and B i g e l o w h y d r o p h o b i c i t y (H0avg) 78 15 R e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y and h y d r o p h o b i c i t y (-S ). m e a s u r e d a f t e r e x p o s u r e t o 100°C f o r 10 min i n t h e p r e s e n c e o f 1.5% SDS 79 16 C o n t o u r d i a g r a m , o f t h e r e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y , h y d r o p h o b i c i t y and d i s p e r s i b i l i t y 87 x i FIGURE PAGE 17 T h r e e d i m e n s i o n a l p l o t o f t h e r e l a t i o n -s h i p b e t w e e n f o a m i n g c a p a c i t y , h y d r o -p h o b i c i t y and d i s p e r s i b i l i t y 88 18 R e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y and c h a r g e d e n s i t y 93 19 R e l a t i o n s h i p b e t w e e n foam s t a b i l i t y a nd c h a r g e d e n s i t y 1 00 20 R e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y and b u l k v i s c o s i t y 1 03 21 T h r e e d i m e n s i o n a l p l o t o f f o a m i n g c a p a c i t y a g a i n s t h y d r o p h o b i c i t y and b u l k v i s c o s i t y 106 22 C o n t o u r d i a g r a m o f t h e r e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y , , h y d r o p h o b i c i t y and d i s p e r s i b i l i t y 1 07 23 R e l a t i o n s h i p b e t w e e n foam s t a b i l i t y a nd b u l k v i s c o s i t y 108 2U R e l a t i o n s h i p b e t w e e n f o a m i n g c a p a c i t y and s u r f a c e t e n s i o n 110 25 R e l a t i o n s h i p b e t w e e n foam s t a b i l i t y and s u r f a c e t e n s i o n 111 x i i ACKNOWLEDGEMENTS I w i s h t o thank my s u p e r v i s o r , Dr. S. N a k a i , f o r h i s guidance a n d . i n t e r e s t i n t h i s p r o j e c t , and the oth e r members of my s u p e r v i s o r y committee Dr. D. D o l p h i n , Dr. W. Powrie and Dr... M. Tung f o r t h e i r s u g g e s t i o n s and a s s i s t a n c e i n the p r e p a r a t i o n of t h i s t h e s i s . I a l s o w ish t o ex p r e s s my g r a t i t u d e t o CIDA f o r f i n a n c i n g my d o c t o r a l s t u d i e s . 1 I INTRODUCTION The impact which a p r o t e i n has on human n u t r i t i o n i s g r e a t l y i n f l u e n c e d by i t s f u n c t i o n a l p r o p e r t i e s eg. foaming a b i l i t y , g e l f o r m i n g a b i l i t y and e m u l s i f y i n g a c t i v i t y . These p r o p e r t i e s determine whether the p r o t e i n can be s u c c e s s f u l l y i n c o r p o r a t e d i n t o a p a r t i c u l a r food.. I t i s g e n e r a l l y agreed t h a t the f u n c t i o n a l i t y of a p r o t e i n i s a r e f l e c t i o n of i t s m o l e c u l a r p r o p e r t i e s ( K i n s e l l a , 1976; P h i l l i p s , 1977). T h e r e f o r e , once p r o t e i n s t r u c t u r e can be m e a n i n g f u l l y c o r r e l a t e d w i t h s p e c i f i c f u n c t i o n a l p r o p e r t i e s i t sh o u l d be p o s s i b l e t o p r e d i c t the e f f e c t of v a r i o u s p r o c e s s e s on p r o t e i n f u n c t i o n a l i t y . . . I t s h o u l d a l s o be p o s s i b l e t o d e s i g n t r e a t m e n t s f o r the i n t e n t i o n a l a l t e r a t i o n of p r o t e i n f u n c t i o n a l i t y . T h i s knowledge would i n c r e a s e the u s e f u l n e s s of many n o n - c o n v e n t i o n a l p r o t e i n s and pe r m i t m.ore e f f i c i e n t use of the w o r l d ' s p r o t e i n r e s o u r c e s . Foaming i s one of the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s which has been of i n t e r e s t f o r many y e a r s . There has been, c o n s i d e r a b l e s p e c u l a t i o n as t o the r o l e of v a r i o u s m o l e c u l a r p r o p e r t i e s of p r o t e i n s i n d e t e r -m i n i n g t h e i r foaming a b i l i t y ( K i n s e l l a , 1976). However, 2 t h e r e i s a need f o r e x p e r i m e n t a l work t o i d e n t i f y the m o l e c u l a r parameters which are i m p o r t a n t f o r d e t e r m i n i n g foaming a b i l i t y and to determine the r e l a t i v e importance of t h e s e parameters. Most of the s t u d i e s on p r o t e i n foams have examined the e f f e c t of changes i n pH,. i o n i c s t r e n g t h , t emperature and of the presence of v a r i o u s compounds on the foaming p r o p e r t i e s , of s p e c i f i c p r o t e i n s . Many of t h e s e s t u d i e s have been c a r r i e d out u s i n g complex m i x t u r e s of proteins... T h i s has made, i t d i f f i c u l t t o e l u c i d a t e t h e . r e l a t i o n s h i p between p r o t e i n s t r u c t u r e and foaming a b i l i t y . . . E xperiments s e t up to e x p l a i n the foaming phenomenon have c o n c e n t r a t e d on the r e l a -t i o n s h i p between foaming p r o p e r t i e s and the p h y s i c a l p r o p e r t i e s of s u r f a c e s eg. s u r f a c e p r e s s u r e , s u r f a c e e l a s t i c i t y (Graham and P h i l l i p s , . 1 976)... Work i n t h i s a r e a has been hampered by the d e a r t h of t e c h n i q u e s f o r s t u d y i n g the b e h a v i o u r of p r o t e i n s a t . i n t e r f a c e s w i t h r e s p e c t t o p r o t e i n c o n f o r m a t i o n and i n t e r - and i n t r a -m o l e c u l a r bonding. In o r d e r t o a v o i d such d i f f i c u l t i e s i n t h i s work, the s t r u c t u r e . o f some pure, p r o t e i n s was c o r r e l a t e d w i t h t h e i r foaming c h a r a c t e r i s t i c s by means of p r o p e r t i e s of the p r o t e i n s which c o u l d be .measured i n b u l k s o l u t i o n . 3 The r e s e a r c h was c a r r i e d out 1) t o measure and compare the foaming p r o p e r t i e s of a number of pure p r o t e i n s w i t h d i f f e r e n t s t r u c t u r e s 2) t o c o r r e l a t e the.foaming p r o p e r t i e s of t h e s e p r o t e i n s w i t h some of t h e i r p h y s i c o c h e m i c a l c h a r a c t e r i s t i c s , , which were measured i n b u l k s o l u t i o n and 3) t o a p p l y the procedures used i n model p r o t e i n s t u d i e s to. examine. the . r e l a t i o n s h i p , between the foaming p r o p e r t i e s of some f o o d p r o t e i n s and t h e i r p h y s i c o c h e m i c a l c h a r a c t e r i s t i c s . A l t h o u g h p r o t e i n s behave d i f f e r e n t l y at the a i r - w a t e r i n t e r f a c e and i n b u l k s o l u t i o n , b oth t y p e s of b e h a v i o u r are determined by p r o t e i n s t r u c t u r e . T h e r e f o r e , c h a r a c t e r i s t i c p r o p e r t i e s .of p r o t e i n s at i n t e r f a c e s must c o r r e l a t e w i t h b u l k phase p r o p e r t i e s . An important, advantage of . e x p l a n a t i o n of the foaming phenomenon i n terms of p h y s i c o c h e m i c a l p r o p e r t i e s of p r o t e i n s i n b u l k s o l u t i o n , i s . t h a t o n l y i n s t r u m e n t s n o r m a l l y used f o r p h y s i c o c h e m i c a l s t u d i e s on p r o t e i n s are r e q u i r e d f o r measuring b u l k p r o p e r t i e s . T h i s s h o u l d make i t f a i r l y easy t o a p p l y knowledge of the foaming phenomenon t o f o o d p r o c e s s i n g . In t h i s work, r e c e n t l y developed methods 4-f o r d e t e r m i n i n g p r o t e i n h y d r o p h o b i c i t y were used t o i n t r o d u c e a new i n t e r p r e t a t i o n of t h e foaming phenomena of f o o d p r o t e i n s . . T h i s approach has not been used b e f o r e f o r s t u d y i n g p r o t e i n foams.. 5 I I . .LITERATURE REVIEW 1. PROTEIN FOAMS T h r e e t e c h n i q u e s a r e commonly u s e d t o g e n e r a t e foams f o r s t u d y . . These methods i n v o l v e i n c o r p o r a t i o n o f a i r i n t o a s t a n d a r d v olume o f s o l u t i o n by w h i p p i n g , s h a k i n g , or. s p a r g i n g ( B i k e r m a n , 1 9 7 3 ; K i n s e l l a , 1 9 7 6 ; . W a n i s k a and K i n s e l l a , 1 9 7 9 ) . S h e a r i n g f o r c e s .are o f minor, i m p o r t a n c e i n s p a r g i n g b u t a r e v e r y i m p o r t a n t i n . b o t h w h i p p i n g and s h a k i n g and l e a d t o t h e . r u p t u r e o f many b u b b l e s i n t h e l a t t e r two p r o c e s s e s . . . Of t h e . t h r e e t e c h n i q u e s s p a r g i n g i s most s u i t a b l e when p r o t e i n s u p p l i e s a r e l i m i t e d . The r e a s o n f o r t h i s i s . t h a t . s p a r g i n g r e q u i r e s 0.01 - 2% p r o t e i n s o l u t i o n s w h i l e w h i p p i n g and s h a k i n g r e q u i r e 3 - 4-0$ and a p p r o x i m a t e l y 1 % . s o l u t i o n s r e s p e c t i v e l y . F o a m i n g a b i l i t y . i s .commonly . e v a l u a t e d i n t e r m s o f t h e v o l u m e o f . f o a m p r o d u c e d o r f o a m i n g c a p a c i t y and t h e l i f e t i m e , o f t h e . foam o r foam s t a b i l i t y ( K i n s e l l a , 1976.).. Foaming, c a p a c i t y may.be m e a s u r e d as t h e maximum, volume, o f foam., p r o d u c e d p e r u n i t v o l u m e o f s o l u t i o n o r as t h e r a t i o . o f . t h e v o l u m e o f g a s i n t h e foam t o t h e v o l u m e sparged.. ..Foam s t a b i l i t y i s o f t e n m e a s u r e d as t h e v o l u m e of., l i q u i d d r a i n e d f r o m t h e 6 foam i n a g i v e n time or as the time r e q u i r e d f o r a g i v e n volume of foam t o c o l l a p s e . The. i n f l u e n c e of fa.ctors such as pH, t e m p e r a t u r e , the presence of s a l t s and sugars on the foaming a b i l i t y has been s t u d i e d f o r a l a r g e number of f ood. p r o t e i n s . . I t i s d i f f i c u l t t o genera-l i z e about the, e f f e c t s of s p e c i f i c f a c t o r s on the foaming p r o p e r t i e s of p r o t e i n s from r e p o r t s i n the l i t e r a t u r e . T h i s i s due. t.o the . v a r i e t y of t e s t c o n d i t i o n s used, to. the. range of responses by d i f f e r e n t p r o t e i n s t o a g i v e n , set of c o n d i t i o n s and t o the p r e s e n c e , i n some of the samples., of n o n - p r o t e i n m a t e r i a l which may a f f e c t foaming, p r o p e r t i e s . However, i t i s c l e a r t h a t numerous f a c t o r s i n f l u e n c e the foaming p r o p e r t i e s of . p r o t e i n s . Foaming p r o p e r t i e s are a f f e c t e d by s o l u t i o n pH. At extremes of pH, where t h e r e . i s moderate d e n a t u r a t i o n of p r o t e i n s . , foaming i s good ( L i n d b l o m , 1 974-) . At moderate pH. v a l u e s a . v a r i e t y of e f f e c t s have been observed w i t h d i f f e r e n t . p r o t e i n s . . . Sunflower p r o t e i n had i t s minimum foaming c a p a c i t y at the i s o -e l e c t r i c p o i n t , w h i l e . i t s foam s t a b i l i t y was g r e a t e s t j u s t above the i s o e l e c t r i c p o i n t ( C a n e l l a , 1978). For a l f a l f a l e a f p r o t e i n the pH foaming c a p a c i t y curve 7 p a r a l l e l l e d i t s p H - s o l u b i l i t y p r o f i l e (Wang and K i n s e l l a , 1976). Both the foaming c a p a c i t y and foam s t a b i l i t y of c o t t o n s e e d . f l o u r were l o w e s t near the i s o e l e c t r i c p o i n t , , where i t s s o l u b i l i t y was a t a minimum (C h e r r y and McWalters., 198*1). Ovalbumin foams were r e p o r t e d t.o have best s t a b i l i t y j u s t below the i s o e l e c t r i c , p o i n t (Waniska and K i n s e l l a , 1979). D e n a t u r a t i o n of p r o t e i n s by m i l d h e a t i n g b e f o r e foaming t e s t s , g e n e r a l l y improves foaming a b i l i t y . G a n e l l a (1978) observed i n c r e a s e s i n foaming c a p a c i t y when . sunflower, p r o t e i n was heated at 50 - 60°C b e f o r e t e s t i n g . . . H i g her t e m p e r a t u r e s , which l e d t o e x t e n s i v e d e n a t u r a t i o n and l o s s of s o l u b i l i t y t h r o u g h c o a g u l a t i o n , , l owered foaming c a p a c i t y . , Riehert., e.t. a l . (1974) observed t h a t the foaming p r o p e r t i e s .of whey were improved by h e a t i n g t o 65 - 70°C. However,.. the. s t a b i l i t y of g l u t e n foams was found to, decrease g r a d u a l l y w i t h i n c r e a s i n g temperature. ( M i t a e_t a l . , 1 977). When they foamed ovalbumin s o l u t i o n s , at. d i f f e r e n t t e m p e r a t u r e s Waniska and K i n s e l l a (1 979) found ..that. the most s t a b l e foams were produced a t 15°C. They suggested t h a t the improved foam s t a b i l i t y at. low.temperature was due 8 i n p a r t t o i n c r e a s e d s o l u t i o n v i s c o s i t y . T h e o r y s u g g e s t s t h a t t h e r e s h o u l d be some r e l a t i o n s h i p b e t w e e n t h e f o a m i n g p r o p e r t i e s o f p r o t e i n s o l u t i o n s and t h e i r v i s c o s i t i e s ( B i k e r m a n , 1973). E x p e r i m e n t s t o d e m o n s t r a t e t h i s r e l a t i o n s h i p h a v e y i e l d e d c o n f l i c t i n g r e s u l t s . . S a t o and Hayakawa (1 979) f o u n d t h a t t h e f o a m i n g , c a p a c i t y , o f y e a s t p r o t e i n was e n h a n c e d when s o l u t i o n . v i s c o s i t y was i n c r e a s e d by a d d i t i o n o f p o l y e t h y l e n e g l y c o l o r . m e t h y l c e l l u l o s e b u t foam s t a b i l i t y was u n a f f e c t e d . M i t a e_t a l . (1 977) o b s e r v e d an i n c r e a s e i n t h e s t a b i l i t y . o f g l u t e n foams when s o l u t i o n v i s c o s i t y was i n c r e a s e d by a d d i t i o n o f s u c r o s e . The p r e s e n c e o f l i p i d s i n a. p r o t e i n - s o l u t i o n i n h i b i t s f o a m i n g ( B i k e r m a n , 1973; Wang and K i n s e l l a , 1 976) . The e f f e c t o f a d d e d s a l t s on f o a m i n g d e p e n d s on t h e p a r t i c u l a r s a l t , and p r o t e i n , c o n c e r n e d . S a t o and Hayakawa (1979) -found t h a t . s o d i u m c h l o r i d e and c a l c i u m c h l o r i d e e n h a n c e d foam s t a b i l i t y a t b o t h a c i d and a l k a l i n e pH. W a n i s k a . a n d . K i n s e l l a (1979) f o u n d t h a t t h e s t a b i l i t y . o f o v a l b u m i n foams was g r e a t e s t a t h i g h s o d i u m c h l o r i d e . c o n c e n t r a t i o n s and l o w pH. V a r i a t i o n o f t h e i o n i . c s t r e n g t h o f f i s h 9 p r o t e i n d i s p e r s i o n s w i t h sodium c h l o r i d e r e v e a l e d t h a t optimum foaming c a p a c i t y and foam s t a b i l i t y o c c u r r e d a t i o n i c . s t r e n g t h . 0*5. (Hermansson et a l . , 1971). S t u d i e s by Cooney (1974) w i t h a v a r i e t y of d i f f e r e n t s a l t s rev.ealed t h a t the s t a b i l i t y of whey p r o t e i n foams decre a s e d l i n e a r l y w i t h the square r o o t 3 + of i o n i c s t r e n g t h . A l t h o u g h A l i o n s improved foam volume and s t a b i l i t y a t l e v e l s as low as 100 ppm, 2 + 2+ d i v a l e n t c a t i o n s such as. Ca and Ba de c r e a s e d foam s t a b i l i t y . I n o r d e r . f o r a l i q u i d t o foam i t must c o n t a i n a s o l u t e , such as a p r o t e i n , .which can adsorb at the a i r - l i q u i d i n t e r f a c e and lower the s u r f a c e t e n s i o n of t h e l i q u i d (Bikerman, 1973; Cherry and McWatters ,., 1 981).. A c c o r d i n g t o t h e o r y , t h i s l o w e r i n g of s u r f a c e t e n s i o n f a c i l i t a t e s de-f o r m a t i o n of t h e l i q u i d d u r i n g bubble f o r m a t i o n . Once the bubbles are formed t h e . l a y e r of p r o t e i n s at the i n t e r f a c e f u n c t i o n s l i k e an e l a s t i c membrane, which p r o t e c t s the bub b l e s a g a i n s t r u p t u r e and c o a l e s c e n c e . I t appears t h a t the l a m e l l a e or f i l m s between t h e b u b b l e s of a. foam have a. s a n d w i c h - l i k e s t r u c t u r e (Bikerman,. 1 973). Bulk . s o l u t i o n forms the 1 0 innermost l a y e r . w h i l e each o u t e r l a y e r c o n s i s t s of a t h i n c o a t i n g of pr o t e i n . , h a v i n g a v e r y h i g h v i s c o s i t y . T h i s s u r f a c e v i s c o s i t y i s o f t e n used as an e x p l a n a t i o n of f i l m s t a b i l i t y . I n i n s t a n c e s , where s u r f a c e v i s c o s i t y i s r e l a t i v e l y low, f i l m . s t a b i l i t y i s f r e q u e n t l y e x p l a i n e d i n terms of the Mar.angoni e f f e c t (Bikerman, 1 973). A c c o r d i n g t o t h i s t h e o r y , . a f i l m u s u a l l y has t o be deformed b e f o r e b u r s t i n g . . D e f o r m a t i o n of a f i l m r e s u l t s i n an i n c r e a s e i n . s u r f a c e a r e a and l o w e r i n g of the c o n c e n t r a t i o n of . p r o t e i n i n the extended s u r f a c e , s i n c e d i f f u s i o n of. p r o t e i n from the b u l k s o l u t i o n t o the s u r f a c e i s . f a i r l y slow. The s u r f a c e t e n s i o n of the ext.ended area, exceeds t h a t of the s u r r o u n d i n g l a m e l l a * . T h i s causes the s u r f a c e l a y e r of p r o t e i n i n . s u r r o u n d i n g a r e a s t o mi g r a t e t o the deformed s e c t i o n . I n d o i n g . t h i s . t h e m i g r a t i n g p r o t e i n drags some of the l i q u i d , between the l a y e r s t o the t r o u b l e s p o t . T h u s , t h i s s e c t i o n of the l a m e l l a , which was made t h i n n e r by d e f o r m a t i o n , , i s made t h i c k e r a g a i n . M u tual r e p u l s i o n of e l e c t r i c double l a y e r s has a l s o been . c i t e d ,.as one of . the. r e a s o n s f o r the s t a b i l i t y of foam l a m e l l a e (Bikerman, 1973). Charge 11 s e p a r a t i o n at. the i n t e r f a c e r e s u l t s . i n l a m e l l a e i n which the sur f a c e , l a y e r s are made up of the charged p o l y p e p t i d e c h a i n s and the middle l a y e r c o n t a i n s the c o u n t e r - i o n s . When the l a m e l l a i s v e r y t h i n , the charged o u t e r l a y e r s r e p e l each o t h e r . T h i s r e p u l s i v e f o r c e p r o v i d e s r e s i s t a n c e t o f u r t h e r t h i n n i n g of the f i l m and t h e r e f o r e , r e s i s t s the. r u p t u r e and c o a l e s c e n c e of b u b b l e s i n a foam. An i m p o r t a n t r e a s o n . f o r the e v e n t u a l c o l l a p s e of a foam i s l o s s of l i q u i d from the l a m e l l a e which l e a d s t o t h i n n i n g of the f i l m (Bikerman, 1973). Ano-t h e r i m p o r t a n t f a c t o r i n . f o a m . c o l l a p s e i s c o a g u l a t i o n , the f o r m a t i o n of. ag g r e g a t e s which show l i t t l e s u r f a c e a c t i v i t y ( C h e r r y and McWat.ter.S;, 1 981 )..., C o a g u l a t i o n weakens the f i l m , and l e a d s t o bubble r u p t u r e . C o a g u l a t i o n p r o b a b l y . r e s u l t s from n o n - c o v a l e n t i n t e r a c t i o n between .  m o l e c u l e s at • the i n t e r f a c e . U-sing t h r e e s t r u c t u r a l l y d i f f e r e n t p r o t e i n s , lysozyme, b o v i n e serum .albumin and 3 - c a s e i n , Graham and P h i l l i p s . (1 976). s t u d i e d , the. r e l a t i o n s h i p between foaming, c h a r a c t e r i s t i c s , and.some p r o p e r t i e s of the p r o t e i n s . They concl u d e d t h a t m o l e c u l e s l i k e 6 - c a s e i n , which have a f l e x i b l e r a n d o m . c o i l . s t r u c t u r e can adsorb r a p i d l y at. the air-.water i n t e r f ac e . and t h e r e f o r e foam 12 r e a d i l y . M o l e c u l e s l i k e bovine serum albumin and lysozyme which have a more r i g i d s t r u c t u r e are unable t o u n f o l d as r e a d i l y a t the a i r - w a t e r i n t e r f a c e . These m o l e c u l e s are t h e r e f o r e . l e s s e f f e c t i v e at s t a b i l i z i n g a i r b u bbles and. do not foam .as r e a d i l y . . Graham and P h i l l i p s (1-976) suggested t h a t l a r g e volumes of foam were produced by p r o t e i n s , which adsorb r a p i d l y at the i n t e r f a c e and t h e r e f o r e lower, s u r f a c e t e n s i o n r a p i d l y . I n c r e a s i n g the net charge on p r o t e i n m o l e c u l e s t h r o u g h a c y l a t i o n l e a d s t o an i n c r e a s e i n the r e p u l s i v e f o r c e s ..within the molecule which r e s u l t s i n u n f o l d i n g and. molecular., e x p a n s i o n (Means and Feeney, 1971). The foaming p r o p e r t i e s of p r o t e i n s are improved by a c y l a t i o n . Sato and Nakamura (197.7) observed t h a t the foaming cap a c i t y . . o f eg.g w h i t e p r o t e i n s was enhanced by s u c c i n y l a t i o n . . . S i m i l a r , improvements i n foaming a b i l i t y have been r e p o r t e d by. M i l l e r and G r o n i n g e r J r . (1 976) f o r f i s h p r o t e i n .and ...Franzen and K i n s e l l a (I 976a,b) f o r soy and l e a f p r o t e i n s . . . .The e f f e c t of a c y l a t i o n on foaming i s . p r o b a b l y l a r g e l y due t o u n f o l d i n g of. the m o l e c u l e . which a i d s a d s o r p t i o n at the i n t e r f a c e . However,, i t s h o u l d be borne i n mind t h a t i n a l l cases a c y l a t i o n was accompanied by an i n c r e a s e i n the s o l u b i l i t y of. the samples which must 13 have p l a y e d a r o l e i n enhancing the foaming a b i l i t y of the p r o t e i n s . Her.mansson et a l . ( 1 974-) observed t h a t a l t h o u g h e x t e n s i v e h y d r o l y s i s improved the s o l u b i l i t y of c a n o l a p r o t e i n c o n c e n t r a t e , h y d r o l y s i s d e c r e a s e d the s t a b i l i t y of i t s foams.. K u e h l e r and S t i n e (1974) r e p o r t e d t h a t enzyme h y d r o l y s i s of.whey p r o t e i n decreased the s t a b i l i t y of i t s foams.. There was an improvement i n foaming c a p a c i t y a f t e r l i m i t e d h y d r o l y s i s but e x t e n s i v e h y d r o l y s i s d e c r e a s e d foaming c a p a c i t y . . . These r e s u l t s i n d i c a t e t h a t low m o l e c u l a r weight- compounds are not as e f f e c t i v e as compounds h a v i n g l a r g e r m o l e c u l a r w e i g h t s f o r promo-t i n g foam f o r m a t i o n . • • • U s i n g f i v e enzyme hydrolyze.d f o o d p r o t e i n s , H o r i u c h i e t a l . ( 1 9 7 6 ) were a b l e t o c o r r e l a t e foam s t a b i l i t y , w i t h the. content., of. s u r f a c e h y d r o p h o b i c r e g i o n s on the .molecules.. They, found t h a t the a d d i t i o n of 2-mereapto.ethanol t.o. t h e i r s a m p l e s . d i d not a f f e c t foam s t a b i l i t y , . w h i c h suggested .that d i s u l p h i d e bonds are not i n v o l v e d i n the maintenance. .of foam s t a b i l i t y . 2. PROTEINS AT THE AIR-WATER INTERFACE P r o t e i n a d s o r p t i o n at the a i r - w a t e r i n t e r -f a c e i s thought to. i n v o l v e t h r e e s t e p s ; 1) d i f f u s i o n u of the molecules to., the interface.; 2) a d s o r p t i o n at the i n t e r f a c e and 3). molecular rearrangement i n the i n t e r f a c e (Graham.and P h i l l i p s , 1979a; MacRitchie, 1978; Tornberg, 197.8)., The k i n e t i c s . o f p r o t e i n a d s o r p t i o n has been .studied by means of surface t e n s i o n measurements as w e l l as by means of r a d i o -t r a c e r experiments with l a b e l e d p r o t e i n s (Graham and P h i l l i p s , 1 979a,b; MacRitchie and. Alexander, 1 963a>t>>c; Tornberg, 1 978).. These s t u d i e s i n d i c a t e t h a t i n i t i a l l y the r a t e of a d s o r p t i o n at the i n t e r f a c e i s d i f f u s i o n c o n t r o l l e d . At t h i s stage the molecules are i r r e v e r -s i b l y adsorbed. The number of molecules which are i r r e v e r s i b l y adsorbed d i f f e r . . , f r o m p r o t e i n to p r o t e i n . I t i s commonly b e l i e v e d ..that i n . order to adsorb at the i n t e r f a c e a molecule must c l e a r a space f o r i t s e l f by compressing other molecules a l r e a d y i n the i n t e r -f a ce (Graham and P h i l l i p s , 1979a: MacRitchie, 1978). Once the c r i t i c a l ar.ea has been .cleared the molecule becomes anchored to the I n t e r f a c e and slowly unfolds to i t s i n t e r f a c i a l c o n f i g u r a t i o n * The r e a c t i o n i s d r i v e n by the lowering of the. f r e e energy of the system which i s a s s o c i a t e d with the o r i e n t a t i o n of hydrophobic groups of the molecule towards the a i r and the p o l a r groups towards. the. water... The work which must be 15 done to c l e a r a space i n the i n t e r f a c e i s e q u i v a l e n t to an energy b a r r i e r , to. a d s o r p t i o n . .As the surface becomes s a t u r a t e d -this energy, b a r r i e r i n c r e a s e s and consequently, the r a t e of .adsorption decreases. Recently, Saraga (1981) suggested that t h i s theory i s not correct... Water. molecules at the i n t e r -f a ce have a higher f r e e energy than molecules i n the l i q u i d bulk. Saraga suggested that as a r e s u l t of t h i s high f r e e energy, i n t e r f a c i a l water i s . a b l e to a s s o c i a t e with the p o l a r groups of a p r o t e i n and promote "micro-u n f o l d i n g " through r u p t u r e of hydrogen and i o n i c bonds. The p r o t e i n i s uns.tab.le i n . t h i s s t a t e because of the l o c a t i o n s of h y d r o p h i l i c . and hydrophobic groups r e l a t i v e to the a i r and water phases r e s p e c t i v e l y . The molecule t h e r e f o r e u n f o l d s to ..it s . i n t e r f a c i a l c o n f i g u r a t i o n i n which ..hydrophobic groups are o r i e n t e d towards the air . . Denaturation . of.. p r o t e i n s at the i n t e r f a c e is. t h e r e f o r e , thought t o be d r i v e n by a combination of i n t e r n a l h y d r a t i o n and hydrophobic e f f e c t s . As the number of. p r o t e i n s i n the i n t e r f a c e i n c r e a s e s the. amount, of water . a v a i l a b l e f o r i n t e r a c t i o n decreases, t h e r e f o r e a d s o r p t i o n decreases. A d s o r p t i o n at the i n t e r f a c e i s a l s o a f f e c t e d by the charge on the p r o t e i n molecules.. I f molecules 16 i n the i n t e r f a c e are charged, an e l e c t r i c a l p o t e n t i a l i s set up and incoming molecules must do work ag a i n s t t h i s p o t e n t i a l (MacRitchie, 1 978).. MacRitchie and Alexander (1963c) have, demonstrated the e x i s t e n c e of t h i s b a r r i e r to. a d s o r p t i o n by measuring the r a t e of a d s o r p t i o n of ly.soz.yme i n t o . d i f f erent monolayers at pH 6.5. They showed that as.the p o t e n t i a l of the i n t e r f a c e became more p o s i t i v e the r a t e of a d s o r p t i o n of lysozyme, which i s . p o s i t i v e l y charged at pH 6.5, decreased. They demonstrated that as p r o t e i n adsorbed at the i n t e r f a c e the surface p o t e n t i a l changed and a l t e r e d the r a t e of adsorption.. Graham and P h i l l i p s (1979b) observed t h a t while the primary l a y e r of p r o t e i n was being adsorbed, the. p o t e n t i a l of the s u r f a c e increased, c o n t i n u o u s l y . The p o t e n t i a l a t t a i n e d when the i n t e r f a c e was s a t u r a t e d was not a l t e r e d by m u l t i l a y e r f o r m a t i o n . I t i s thought t h a t at low surface concen-t r a t i o n s , p r o t e i n s at. the a i r - w a t e r i n t e r f a c e are as completely unfolded as .disulphide bridges w i t h i n the molecule w i l l permit..(Graham..and P h i l l i p s , 1 979c). P r o t e i n s adopt t h i s . c o n f o r m a t i o n u n t i l there are enough molecules, i n the i n t e r f a c e to form a close-packed arrangement. At higher surface c o n c e n t r a t i o n s some 17 segments of the molecules are pushed out of the I n t e r f a c e . There i s c o n s i d e r a b l e c o n t r o v e r s y as to whether proteins.. r e t a i n t h e i r secondary s t r u c t u r e at the i n t e r f a c e . . Examination. of p r o t e i n f i l m s by hydrogen exchange.:.and i n f r a r e d spectroscopy, a f t e r they were removed .from, the i n t e r f a c e suggest that much of a protein's, secondary s t r u c t u r e , p a r t i c u l a r l y a - h e l i x , i s r e t a i n e d .at . the. ..interface. (Malcolm, 1973). In t h i s work the. f i l m s were compressed before removal from the interface..... This treatment would d i s p l a c e some segments of the molecules from...the i n t e r f a c e and i n t o the bulk phase.. . .MacRitchie (197.8) suggested t h a t , s i n c e the d i s p l a c e d . segments would probably form h e l i c e s , the conclusion.that the ..a-helix i s p a r t i c u l a r l y s t a b l e at the i n t e r f a c e . i s ..questionable . However, i n e a r l i e r work using a. s i m i l a r , technique Loeb and Baier (1968) observed considerable, d i f f e r e n c e s i n the con-formations, of.polymethyl glutamate monolayers spread on water with v a r i o u s s o l v e n t s . The u n f o l d i n g of p r o t e i n molecules at the a i r - w a t e r i n t e r f a c e should r e s u l t i n exposure of many r e a c t i v e , groups, thus i n c r e a s i n g i n t e r m o l e c u l a r i n t e r a c t i o n s . Hydrogen bond for m a t i o n and i o n i c and 18 hydrophobic i n t e r a c t i o n s are most l i k e l y to occur. Experimental evidence supports the i d e a t h a t i n t e r -molecular hydrogen bonding occurs at the i n t e r f a c e . Measurements of. the ..sur.f.ace v i s c o s i t y . , o f s o l u t i o n s of s e v e r a l polyamin© a c i d s and p r o t e i n s r e v e a l e d that only p o l y - L - p r o l i n e , which i s in c a p a b l e of hydrogen bonding, had no d e t e c t a b l e surface v i s c o s i t y (MacRitchie, 1970). 3. HYDROPHOBICITY OF PROTEINS Non-polar groups a l t e r the p a t t e r n of hydrogen bonding of water causing an i n c r e a s e i n the f r e e energy of the system (Richards, 1963). Since t h i s e f f e c t on.water s t r u c t u r e i s e n e r g e t i c a l l y unfavourable, .the hydrophobic or non-polar groups of p r o t e i n s i n aqueous s o l u t i o n tend to c l u s t e r together to a v o i d c o n t a c t with.. water..,. This a s s o c i a t i o n c o n s t i t u t e s the. hydrophobic e f f e c t . , . T y p i c a l l y , water-s o l u b l e p r o t e i n s c o n t a i n 25 - 30% hydrophobic and 4-5 -50% h y d r o p h i l i c amino a c i d s (Tanford, 1973). The hydrophobic r e s i d u e s l i e mainly i n the i n t e r i o r of the p r o t e i n and .are thought...to play an important r o l e i n determining p r o t e i n s t a b i l i t y and s t r u c t u r e (Ponnuswamy. et al., 1 980; Tanford, 1 973). Hydrophobic 19 patches on the surface of p r o t e i n molecules are thought to be important as b i n d i n g s i t e s f o r other p r o t e i n s or hydrophobic l i g a n d s . A number of methods hav.e. been proposed f o r e s t a b l i s h i n g the h y d r o p h o b i c i t y of p r o t e i n s . One of the f i r s t methods, was. proposed by Waugh i n 1 954-. He d e f i n e d v a l i n e , l e u c i n e , i s o l e u c i n e , . p r o l i n e , phenyl-a l a n i n e , tryptophan and t.yro.sine as nonpolar r e s i d u e s . He then c a l c u l a t e d the. .nonpolar . side chain f r e q u e n c i e s f o r a s e r i e s o f . p r o t e i n s as t h e . f r a c t i o n of nonpolar r e s i d u e s i n these p r o t e i n s . The f r e e energy r e q u i r e d to t r a n s f e r one molecule of amino a c i d from water to an organic solvent was found to.be independent of the s o l v e n t (Tanford, 1962). By equating the pr.otein i n t e r i o r with the organic s o l v e n t , i t was p o s s i b l e to c a l c u l a t e the f r e e energy of t r a n s f e r of an amino a c i d from an aqueous environment to t h e . p r o t e i n i n t e r i o r and t h e r e f o r e to e s t a b l i s h . a...hydrophobicity s c a l e (Nozaki and Tanford, 1971; Tanford, 1962). Two terms were regarded, as c o n t r i b u t i n g to t h i s f r e e energy f o r each amino, a c i d : one.for the backbone and the other f o r the s i d e , chain.. Since the backbone s t r u c t u r e i s s i m i l a r to. g l y c i n e , the t r a n s f e r f r e e 20 energy of each side chain was c a l c u l a t e d by s u b t r a c t i o n of the energy, measured f o r g l y c i n e . Bigelow (1967) c a l c u l a t e d the average h y d r o p h o b i c i t i e s of a l a r g e number of p r o t e i n s . He d i d t h i s by summing the f r e e energies f o r the r e s i d u e s determined by. Tanford (1962) and d i v i d i n g t h i s f i g u r e by the number of r e s i d u e s , i n the molecule. Using measurements of the surface t e n s i o n s of amino a c i d s i n . 0.1 OM ...sodium c h l o r i d e as a f u n c t i o n of t h e i r c o n c e n t r a t i o n , .Bull, and Breese (1974) were able to c a l c u l a t e the. f r e e energy of t r a n s f e r of amino a c i d r e s i d u e s . f r o m , s o l u t i o n to the s u r f a c e . The h y d r o p h o b i c i t y scale.which they d e r i v e d was i n f a i r l y good agreement with. that., of Nozaki and Tanford (1971). However, t h e i r measurements showed that tryptophan was l e s s hydro/phobic and l e u c i n e more hydrophobic than.reported.by. Nozaki and Tanford. The degree to which.a p r o t e i n i s r e t a i n e d by a hydrophobic g e l i n c r e a s e s as i t s a b i l i t y to p a r t i c i p a t e i n hydr o.phobic . i n t e r a c t i o n s i n c r e a s e s . By means of chromatography, on butylepoxy- or h e x y l -epoxy-Sepharose ,. Keshavarz and Nakai.(1979) used t h i s p r i n c i p l e to., assess . the. e f f e c t i v e h y d r o p h o b i c i t i e s of nine p r o t e i n s . The r e t e n t i o n c o e f f i c i e n t of a 21 p r o t e i n on the hydrophobic g e l served as an index of i t s h y d r o p h o b i c i t y . They.also assessed the e f f e c t i v e h y d r o p h o b i c i t y o f . p r o t e i n s from t h e i r p a r t i t i o n c o e f f i c i e n t s , between two .phases. Keshavarz and Nakai measured•the . p a r t i t i o n c o e f f i c i e n t s (k) f o r each p r o t e i n between p o l y e t h y l e n e g l y c o l and dextran and between polyethylene, g l y c o l mixed with p o l y e t h y l e n e g l y c o l . p a l m i t a t e and. dextran. They used Alog k, obtained as a d i f f e r e n c e of, the l o g of the two p a r t i t i o n c o e f f i c i e n t s , measured,, as an index of p r o t e i n hydrophobicity... .. There was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between .the e f f e c t i v e hydropho-b i c i t i e s determined .by.the. hydrophobic p a r t i t i o n and the hydrophobic chromatography methods. L i n e a r .polyenes, such as c i s - p a r i n a r i c a c i d f l u o r e s c e on b i n d i n g to ,the hydrophobic s i t e s of p r o t e i n s . Eato. and. Nakai (1980). used t h i s p r o p e r t y of c i s - p a r i n a r i c a c i d to .determine.the e f f e c t i v e hydrophobicity. of . some . proteins... They measured the f l u o r e s c e n c e of a. s e r i e s of s o l u t i o n s containing' c i s - p a r i n a r . i c a c i d and. v a r y i n g c o n c e n t r a t i o n s of p r o t e i n s . The . . i n i t i a l slope of. t h e . p l o t of f l u o r e s c e n c e i n t e n s i t y vs.. protein, .concentration was used as an index of p r o t e i n . h y d r o p h o b i c i t y . 22 4. PROTEIN CHARGE The net charge on a p r o t e i n , a t any pH, i s d e t ermined by the .degree of i o n i z a t i o n of the s i d e -c h a i n s of i t s c o n s t i t u e n t amino a c i d s . P o s i t i v e charges are c o n t r i b u t e d by e-amino groups, i m i d a z o l e , i n d o l e and g u a n i d y l groups w h i l e the c a r b o x y l r e s i d u e s c o n t r i b u t e n e g a t i v e charges... At v e r y h i g h pH, s u l p h y d r y l and p h e n o l i c groups may a l s o c o n t r i b u t e n e g a t i v e c h a r g e s . Ionic, i n t e r a c t i o n s are. thought t o be. of l i t t l e i m p o r t a n c e i n d e t e r m i n i n g p r o t e i n , s t r u c t u r e .  and s t a b i l i t y , s i n c e most charged groups occur at the. p r o t e i n s u r f a c e (Hasche-meyer and Haschemeyer.,, 1 973)... However,, the net charge on p r o t e i n s • s h o u l d i n f l u e n c e t h e i r a d s o r p t i o n a t i n t e r f a c e s ( M a c R i t c h i e , 1978). Measurements of. the r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y (R^) of a p r o t e i n i n a s e r i e s of g e l s of v a r y i n g c o n c e n t r a t i o n (T) have been used t o determine the net charge of p r o t e i n . (Chrambach and Rodbard, 1971). The a n t i l o g of the y - i n t e r c e p t of a p l o t of l o g R^ v e r s u s T i s a measure of the f r e e e l e c t r o -p h o r e t i c m o b i l i t y of the p r o t e i n and t h e r e f o r e of i t s net charge. . . The z e t a p o t e n t i a l or the e l e c t r i c p o t e n t i a l at the s u r f a c e of a p a r t i c l e c o a t e d w i t h p r o t e i n can 23 a l s o serve as an index of i t s net.charge (MacRitchie, 1978). The zeta p o t e n t i a l . o f p r o t e i n coated p a r t i c l e s can be c a l c u l a t e d from, measurements of the e l e c t r o -p h o r e t i c m o b i l i t i e s , of t h e . p a r t i c l e s i n a b u f f e r and the p o t e n t i a l .gradient of the e l e c t r i c f i e l d , the v i s c o s i t y and.the d i e l e c t r i c constant of the b u f f e r used (Shaw, 1 96 9). A v a r i e t y of p a r t i c l e s i n c l u d i n g g l a s s or p o l y s t y r e n e beads, and o i l . d r o p l e t s have been used as c a r r i e r s ( W i l k i n s and Myers, 1970). S i m i l a r l y the de t e r m i n a t i o n of the zeta p o t e n t i a l of b u b b l e s . i n s u r f a c t a n t s o l u t i o n s have been made ( C o l l i n s et a l . , 1-978). Usui and Sasaki (1 978) suggested the calculation., of zeta p o t e n t i a l from measurements of the, sedimentation p o t e n t i a l of the bubbles. For t h i s c a l c u l a t i o n i t . i s . necessary to know the height, and cross, s e c t i o n a l area of the column i n which the bubbles are made, as w e l l as the s o l u t i o n v i s c o s i t y , d i e l e c t r i c constant and s p e c i f i c conductance, the d e n s i t y d i f f e r e n c e between the gas and the s o l u t i o n , bubble s i z e and.the d i s t a n c e between the electrodes.. Since zeta p o t e n t i a l Increases with d e c r e a s i n g bubble s i z e the. procedure i s complicated by the f a c t t h a t due to the decreasing, h y d r o s t a t i c pressure the bubbles expand as they move up the 24 c o l u m n ( U s u i e t a l . , 1 9 8 1 ) . I t i s a l s o p o s s i b l e t o d e t e r m i n e t h e n e t c h a r g e on a p r o t e i n a t any pH by a t i t r a t i o n method ( N o z a k i and T a n f o r d , 1 967..).. The p r o c e d u r e I n v o l v e s t h e a d d i t i o n o f h y d r o g e n o r h y d r o x y l i o n s t o an i s o i o n i c s o l u t i o n o f t h e p r o t e i n u n t i l t h e d e s i r e d pH i s r e a c h e d . From t h e amount o f a c i d o r b a s e added and t h e m o l a r c o n c e n t r a t i o n o f p r o t e i n i n s o l u t i o n , t h e c h a r g e on t h e p r o t e i n i s c a l c u l a t e d as t h e number o f , m o l e s , o f h y d r o g e n i o n s bound o r d i s s o c i a t e d p e r mole o f p r o t e i n . 5. MODEL PROTEINS The p r o t e i n s r i b o n u c l e a s e , o v o m u c o i d , t r y p s i n , l y s o z y m e , p e p s i n c o n a l b u m i n , o v a l b u m i n , b o v i n e serum a l b u m i n , K - c a s e i n , . 3 - l a c t o g l o b u l i n and 3 - c a s e i n f o r m a d i v e r s e , g r o u p . . Some o f t h e p r o p e r t i e s o f t h e s e p r o t e i n s .are. l i s t e d i n T a b l e s 1, 2, and 3. The r i b o n u c l e a s e m o l e c u l e h a s t h e d i m e n s i o n s 38 x28 x 22 A (.Richards and W y c k o f f , 1 9 7 1 ) . I t c o n s i s t s o f a s i n g l e p o l y p e p t i d e c h a i n o f 124- amino a c i d r e s i d u e s . Due t o I t s . h i g h c o n t e n t o f b a s i c amino a c i d s , r i b o n u c l e a s e - A . h a s i t s i s o i o n i c p o i n t a t pH 9.6. The p r o t e i n e x i s t s , m a i n l y as a monomer b u t h a s b e e n shown.to f o r m d i m e r s . a n d h i g h e r a g g r e g a t e s . 25 Table 1. The molecular weight, f r i c t i o n a l r a t i o and number of. d i s u l p h i d e bonds i n the model p r o t e i n s P r o t e i n Molecular F r i c t i o n a l § SS weight . . . c o e f f i c i e n t bonds ribonuclease-.A (1 ). 1 2,64-0 1 .066 4 ovomucoid (2) 25,000 1 .350 16 t r y p s i n (3) 23,800 1 .154 6 lysozyme (4) 13,930 1 .21 0 4 pepsin (5) 35.,500 . 1 .54 3 conalbumin (6) 83,180 1 .288 14 ovalbumin (7) 4-5,000 1 .170 1 bovine serum 66,210 .1 .350 17 . albumin (8) K-casein (9) 23,000* 1 6-lactoglobulin.-(.T0) . 36,000 1 .250 2 3-casein (11). 23,980* 1 .835 0 * molecular weight of monomer Refe r e n c e s . f o r the.data i n t h i s t a b l e are as f o l l o w s : (1) (4) Sober,. 1 970; White et a l . 1973 (2) Ikeda ,- 1.-963 26 T a b l e 1 ( c o n t i n u e d ) (3) S o b e r , 1 9 7 0 ; I n a g a m i , 1972 (5) F r u t o n , . 1 9 7 2 ; E d e l h o c h , 1957 (6) S o b e r , 1970; A z a r i and F e e n e y , 1961 ( 7 ) T a b o r s k y , 1974 (8) P e t e r s J r . , 1975 (10) M c K e n z i e , 1971 (9) (11) S w a i s g o o d , 1973 27 Table 2. P e r c e n t a g e s of a - h e l i x . , 3-sheet and t u r n s i n the model p r o t e i n s * P r o t e i n a 3 t u r n s h e l i x sheet r i b o n u c l e a s e - A (1) 19 38 ovomucoid (2) 29 t r y p s i n 1 3.-5 31 .0 35.4 lysozyme (3) 29 16. p e p s i n 33.4 46.9 conalbum.in (4) 28 32 ovalbumin 44.1 20.5 19.5 b o v i n e serum albumi n 52.1 2.2 29.6 K - c a s e i n 20... 1 33.1 29.0 3 - l a c t o g l o b u l i n 35 .8. 30.9 17.3 B - c a s e i n 13.9 23.0 33.0 *These v a l u e s were r e p o r t e d by Pham (1981) w i t h the f o l l o w i n g e x c e p t i o n s : (•1 ) , (3) Chen e t a l , 1972 (2) Ikeda,. 1-9.68. . (4) Tan, 1971 Table 3. The- amino a c i d c o m p o s i t i o n of the model p r o t e i n s CD 1 ^ 9 •H <; 3 4 5 •H S CO o tti o •H ^2 1 0 CO 1 •H rH O rH E PH O CD CO X o o co E PH •H 3 > © O o -p PH . O 2 ,P rH > O 8 « 9 I IP! 1 0 PS1 1 •H O H •H 0 •H 0 0 E E CO o?3 CO •H cS cti > rH X o i—IO O O 0 H i Ii—I 1 co cd c ahD 00. asp 15 29 t h r 1 0 13 s e r 15 12 g l u "8 U pro k 8 g i y 5 u a l a 1 2 11 v a l 9 U eys / 2 8 16 met 2 i l e 3 3 l e u 12 t y r 6 6 phe 3 5 t r p 0 0 l y s 1 0 13 h i s a r g 6 amide 12 13 t o t a l # r e s i d u e s 1 24- 1 82 21 21 44 1 0 7 28 34 10 44 14 5 27 8 2 15 24 12 38 14 1 2 1 8 17 6 21 12 8 4 2 2 . 5 14 6 27 13 8- 28 10 . 3 1 8 3 3 14 3 6 . 6 1 2 6 1 . 3 1 1 2 11 2 26 16 36 216 ... 1 29 341 84. 32 53 39 16 32 48 35 26 80 51 75 35 14 28 66 19 16 60 34 46 52 27 35 25 8 34 12 16 4 28 24 13 55 32 61 23 9 19 29 21 26 20 3 2 71 20 56 15 7 17 37 15 22 24 33 36 779 382 566 1 2 31 9 15 16 9 13 14 16 27 30 39 20 16 34 2 7 5 14 29 5 11 19 19 2 8 0 2 4 6 1 2 20 1 0 8 44 22 9 8 4 4 8 9 1 2 1 9 30 11 3 2 6 5 3 4 21 26 27 169 291 209 29 Table 3 ( c o n t i n u e d ) ^ R e f e r e n c e s f o r the. d a t a i n t h i s t a b l e are as f o l l o w s : 1. H i l l and Schmidt, -1962 2. Oegema J r . , and J o u r d i a n , 1974-3. Inagami, 1972 4.. C a n f i e l d , 1963 5. F r u t o n , 1972 6. W i l l i a m s , 1961 7. Taborsky, 1 974-8. P e t e r s J r . , 1975 9. 11. Swaisgood, 1973 10. P i e z et a l , 1961 30 Carbohydrate accounts f o r 23% of the molecular weight of ovomucoid (Spiro., 1 973).. Each molecule c o n s i s t s of a s i n g l e p o l y p e p t i d e c h a i n bearing 2 g a l a c t o s e , 7 mannose.,.23 N-acetylglucosamine and 1 s i a l i c a c i d residue.. This p r o t e i n has i t s i s o i o n i c p o i n t at pH 4.8.. I t shows high heat s t a b i l i t y (Feeney, 1964). T r y p s i n e x i s t s predominantly i n the form of a s i n g l e polypeptide..chain although i t a l s o e x i s t s as two- and t h r e e - c h a i n s t r u c t u r e s h e l d together by d i s u l p h i d e bonds ...(Keil, 1971)* The i s o i o n i c p o i n t of t r y p s i n i s pH TO.. 8.... T r y p s i n i s most .stable around pH 3 and undergoes autolysis., above p.H 5 i n the absence of c a l c i u m . The lysozyme . molecule .is approximately o , e l l i p s o i d with dimensions 45 x 30 x 30 A (Hamaguchi and Hayashi, 1972).. . As a. r e s u l t of i t s high content of b a s i c amino acids, lysozyme has i t s i s o i o n i c point at pH 11.0. The molecule . c o n s i s t s of a s i n g l e poly-peptide c h a i n at a c i d pH,. . Between pH's 5 and 9 d i m e r i z a t i o n occurs and at higher. .pH' s polymers l a r g e r than the' dimer form. Pepsin c o n s i s t s of a s i n g l e p o l y p e p t i d e c h a i n . I t has an,extremely.high p r o p o r t i o n of 31 c a r b o x y l groups.and t h e r e f o r e has i t s i s o i o n i c p o i n t below pH 1.O.. Under the i n f l u e n c e of an e l e c t r i c f i e l d t h i s protein, moves as an anion, at t>H 1.0. At a c i d pH pepsin undergoes slow a u t o l y s i s . Conalbumin i s the only m e t a l l o p r o t e i n i n the group. Each m o l e c u l e . c o n s i s t s . o f one p o l y p e p t i d e chain which can bind, two atoms of i r o n or copper (Feeney, 196-4). The i s o i o n i c p o i n t of conalbumin i s at pH 6.6. Carbohydrate makes up 2.8$ of the weight of the .molecule, since there are 4 mannose and 8 N-acetylglucosamine resideus. per.molecule of conalbumin(Spiro, 1973). Ovalbumin .is also, a g l y c o p r o t e i n ( S p i r o , 1973). The carbohydrate moiety of ovalbumin accounts f o r 2.3$ of the molecular, weight and c o n s i s t s of 5 mannose and. 3 N-ac.e.tylglucosamine r e s i d u e s . The molecule c o n s i s t s of .'a s i n g l e p o l y p e p t i d e c h a i n i n which about h a l f the. amino, a c i d r e s i d u e s are hydrophobic (.Taborsky, 1974)-. The i s o i o n i c p o i n t of ovalbumin i s at pH, 4.6. A .variety of agents denature ovalbumin producing c o m p l e t e , i r r e v e r s i b l e u n f o l d i n g of the molecule,.,. This p r o t e i n i s p a r t i c u -l a r l y s u s c e p t i b l e t o , surface d e n a t u r a t i o n . Bovine serum.albumin c o n s i s t s of a s i n g l e 32 po l y p e p t i d e chain, arranged i n t o a s e r i e s of loops by 17 disulphid'e bonds (.Peter Jr..., 1 975). This loop s t r u c t u r e g i v e s the molecule c o n s i d e r a b l e f l e x i b i l i t y i n s p i t e of i t s high content of d i s u l p h i d e bonds.. Bovine serum albumin has i t s i s o i o n i c p o i n t at pH 4-.4-. 3 - L a c t o g l o b u l i n i s a dimer at n e u t r a l pH, d i s s o c i a t i n g below.. pH. 3.5 and above pH. 7.5 (McKenzie, 1971). X-ray a n a l y s i s r e v e a l s t h a t the dimer c o n s i s t s o of two spheres of molecular weight 18,000 and 36 A i n o diameter, which impinge by 2.3 A at. t h e i r s urface of c o n t a c t . The i s o i o n i c . p o i n t of. B - l a c t o g l o b u l i n i s ' a t pH 5.3. Both K- and 3-casein are w e l l known f o r t h e i r tendency to associate, to. produce polymers with molecular weights.as .large as.650,000 and 690,000 r e s p e c t i v e l y (Swaisgood,. 1 973).. B-Casein e x i s t s i n the monomer form at 4-°G and shows i n c r e a s e d l e v e l s of p o l y m e r i z a t i o n . a s the.temperature i n c r e a s e s . 3-Casein has a random c o i l s t r u c t u r e f r e e from c r o s s - l i n k a g e by d i s u l p h i d e bonds... I t s i s o i o n i c p o i n t occurs, at. pH. 5.3. U n l i k e the behaviour of 8-casein, the p o l y m e r i z a t i o n of. K-cas.ein i s indepen-dent of temperature, and. at l e a s t some of the u n i t s i n 33 the polymers are l i n k e d by i n t e r m o l e c u l a r d i s u l p h i d e bonds. The i s o i o n i c p o i n t of.. K - c a s e i n occurs at pH 5.3. This p r o t e i n c o n t a i n s a t r i s a c c h a r i d e , a-N-acetylnurami.dyl (2-6.)-(3-galactosyl-(1-3 or 6 ) -N - a c e t y l glucosamine... ..The number of r e s i d u e s per monomer i s thought to vary from zero to f i v e . 34 I I I . MATERIALS AND METHODS 1 . M a t e r i a l s Bovine serum albumin 4-BSA.) :to 8 8 , "ovalbumin #1319 and lysozyme #1782 were purchased from ICN N a t i o n a l Biochemicals ( C l e v e l a n d , OH). 8 - L a c t o g l o b u l i n #L-6879 from cow's milk, ovomucoid #.T-9253» t r y p s i n #T-8128 p a n c r e a t i c type I I , r i b o n u c l e a s e - A #R-5000, conalbumin #C-0755 type 1 from c h i c k e n egg white and po r c i n e pepsin #P-7012 were obtained from Sigma Chemicals (St. L o u i s , MO)... Soy p r o t e i n . i s o l a t e was obtained from General M i l l s . Inc.. (Minneapolis, MN). Defatted canola meal was obtained from Canbra Foods L t d . (Lethbridge, A l b e r t a ) * Pea p r o t e i n i s o l a t e M412-04-6 Century c u l t i v a r was s u p p l i e d by POS P i l o t P l a n t Corp. ( U n i v e r s i t y of Saskatchewan, Saskatoon, Sask.). Pro-pulse W100 was s u p p l i e d by . G r i f f i t h L a b o r a t o r i e s L t d . (Scarborough, O.nt. ) . Promine-D and sunflower concentrate were obtained from C e n t r a l Soya Co. (C h i -cago, I L ) . V i t a l wheat g l u t e n , Whetpro 15% was purchased from I n d u s t r i a l Grain Products L t d . (Thunder Bay, Ont.). c I s - P a r i n a r i c a c i d was purchased from Molecular Probe L t d . ( .Piano. TX) . U l t r a - p u r e guanidine h y d r o c h l o r i d e was obtained from Schwarz-Mann L t d .(Orangeburg, N.I- ). A l c a l a s e was obtained from Novo I n d u s t r i e s (Copenhagen, Denmark). Rexyn 35 101 and Rexyn 300 were purchased from.Fisher S c i e n t i f i c Co. ( F a i r Lawn, NJ ). 2. Methods 2.1. A c i d S o l u b i l i z e d Gluten The procedure of Wu.eJ^.al. (1976) was used f o r the p r e p a r a t i o n of a c i d s o l u b i l i z e d g l u t e n from v i t a l g l u t e n . One hundred ml . of 0..5% suspension of g l u t e n i n a 1:1 mixture of .concentrated a c e t i c a c i d and water were r e f l u x e d f o r 2 .hours... A f t e r a d j u s t i n g the pH to 4— 5 with 1M sodium, hydroxide, the suspension was c e n t r i f u g e d at 5000.. x g f o r 1,0 min and the super-natant discarded.. The r e s i d u e was suspended i n water and 6.M sodium hydroxide added to. r a i s e the pH to 7.5. The mixture was c e n t r i f uged at. 500.0 ,x. g- f o r 10 minutes and the supernatant c o l l e c t e d and d i a l y z e d f o r 2 days at 4°C. The product was f r e e z e d r i e d . 2.2. 3-Casein 8-Casein was prepared from skim milk by urea f r a c t i o n a t i o n (.Aschaff enburg.,. 1 963).. The. pH of 1 1 of milk was adjusted., to 4-.6 with 1M h y d r o c h l o r i c a c i d and the r e s u l t i n g p r e c i p i t a t e f i l t e r e d through 36 cheese c l o t h and washed with water.. By a d d i t i o n of 1M sodium hydroxide to r a i s e the pH to 7.5, the p r e c i p i t a t e was d i s p e r s e d i n 500 ml water c o n t a i n i n g 180g urea. The volume of l i q u i d was adjusted to 1 1 and the pH lowered to 4.6 with h y d r o c h l o r i c a c i d . Then, the mixture was f i l t e r e d through Whatman No. 4- paper under vacuum. H y d r o c h l o r i c a c i d was added to the f i l t r a t e u n t i l a pH of 4.9 was reached.. The f i l t r a t e was d i l u t e d to 3 1, warmed to 30°C.and l e f t standing overnight f o r p r e c i p i t a t i o n of. 8-casein... The p r e c i p i -t a t e of B - c a s e i n was c o l l e c t e d by c e n t r i f u g i n g at 650 x g f o r 10. min,.. The crude B-casein was r e d i s p e r s e d i n 400 ml 3.-3M urea by a d d i t i o n of sodium hydroxide u n t i l pH 7.5 was reached... Then., h y d r o c h l o r i c a c i d was added, to lower the pH to 4.5, and the mixture was warmed, to. 37'°C... The B-case.in p r e c i p i t a t e d was c o l l e c t e d by c e n t r i f u g i n g at 650 x g. I t was d i s p e r s e d i n c o l d water by a d j u s t i n g the pH to 7.0 with sodium hydroxide..and then f r e e z e d r i e d . The crude B-casein was p u r i f i e d by chroma-tography on a 5 x 1.4 cm .column of, DEAE c e l l u l o s e as d e s c r i b e d by Thompson and -Pepper (1 964). A 2 g sample of B - c a s e i n , . d i s s o l v e d i n 30 ml im i d a z o l e - H C l b u f f e r ( pH 7.0, y= 0....01 , 3.3M urea) was a p p l i e d to 37 the column. Chromatography was c a r r i e d out using a l i n e a r sodium c h l o r i d e g r a d i e n t of 0... 0 to 0.3M and a flow r a t e ot 80 ml/hr... The eluti.on of p r o t e i n was monitored by i t s absorbance at 280.nm.. The e l u t e d p r o t e i n was d i a l y z e d f o r 4 days at 4-°C. 2.3. K-Casein ic-Casein was. prepared from skim milk as de s c r i b e d by Z i t t l e and Custer (1963). The pH of 1 1 of milk was adjusted, to 4~..6 by a d d i t i o n of 1M h y d r o c h l o r i c a c i d . .The c a s e i n p r e c i p i t a t e d was f i l t e r e d through cheese cloth., washed with warm water, and d i s s o l v e d i n 1 1.of 6....6M urea... Two hundred ml of 3.5M sulphu r i c , a c i d and 2 1 water were.added t o lower the pH to 1.5. The mixture w a s . l e f t standing f o r 2 hours. Then, the. p r e c i p i t a t e formed was removed by f i l t r a t i o n through Whatman No... U paper and d i s -carded. K-Gasein.was p r e c i p i t a t e d from the supernatant by a d d i t i o n of 132 ,g ammonium sulphate.. The p r e c i p i t a t e was c o l l e c t e d by. c e n t r i f ugation at 600...x.g f o r 10 min and suspended .in water.. 1M sodium hydroxide was added to give a pH of. 7...5 and the mixture was l y o p h i l i z e d . The K - c a s e i n was p u r i f i e d as f o l l o w s . A 1% s o l u t i o n of K-casein. at pH 7....0 was mixed with 38 twice as much eth a n o l . Then,. 1M ammonium ace t a t e i n 75$ ethanol was added unt.il. a p r e c i p i t a t e formed. The supernatant was decanted and-the p r e c i p i t a t e d i s s o l v e d i n water, b.y a d d i t i o n of 1.M sodium hydroxide to b r i n g the pH to 7.5* The s o l u t i o n was d i a l y z e d f o r 3 days at 4-°C ag a i n s t d i s t i l l e d water and then f r e e z e d r i e d . 2.4-. Canola P r o t e i n I s o l a t e ' Canola protein, i s o l a t e , was prepared as d e s c r i b e d by N.akai et.. al...,. 1 980... Seventy gm of d e f a t t e d canola meal .were.blended with 700 ml water f o r 10 min i n a S o r v a l l Omnimixer.at ,5.,-000 rpm. The pH was maintained at 10...0 by a d d i t i o n of 1M sodium hydr oxide..... The s l u r r y was. f i l t e r e d through cheese c l o t h and the f i l t r a t e c e n t r i f uged. .at 16„000 x g f o r 15 min. A f t e r a d j u s t i n g the pH t o , 4-.2 with 1M h y d r o c h l o r i c a c i d , the p r e c i p i t a t e was c o l l e c t e d . by c e n t r i f u g a t i o n at 16,000 x g f o r 15 min and suspended in,-350 ml water. The pH of the suspension was r a i s e d , to . 8...2 b.y a d d i t i o n of 1M sodium hydroxide and b l e n d i n g . f o r 10 minutes i n the Omnimixer at 5,000 rpm. The. sample wa.s then f r e e z e dried.. 2.5. Enzyme Hydrolyzed P r o t e i n s The enzyme hydrolyzed food p r o t e i n s were 39 prepared by incubating. 200 ml of a 10$ d i s p e r s i o n of p r o t e i n with 2 ml A l k a l a s e a t 5 0 ° C f o r 4- hours. The pH of the mixture was a d j u s t e d to 8.0 i n i t i a l l y and was maintained at t h i s pH by a d d i t i o n of 1M sodium hydroxide.. A f t e r i n c u b a t i o n the pH of. the mixture was adju s t e d to 7.0 with 1M h y d r o c h l o r i c .acid. The mixture was then heated to. 90°C to i n a c t i v a t e the enzyme and f r e e z e d r i e d . 2.6. Foaming The foaming t e s t s were c a r r i e d out i n a 2 cm x 100 cm g l a s s column which had been c a l i b r a t e d i n ml and which had been f i t t e d with a s i n t e r e d d i s c (pore s i z e 4-0 - 60um) at the lower end . (Figure 1 ) . For each t e s t , 1$ ml of 0.1$ p r o t e i n , s o l u t i o n were poured i n t o the column. Then, a i r was bubbled i n t o the s o l u t i o n through the s i n t e r e d d i s c f o r 2 minutes, at a flow r a t e of 35 ml/min.. The volume of foam i n the. .column at t h i s time (ml foam/15 ml s.olut.ion) was taken as an index of the foaming c a p a c i t y of the p r o t e i n . The time r e q u i r e d f o r the foam to c o l l a p s e to h a l f i t s maximum volume was.measured. Using t h i s f i g u r e , the r a t e of f„oam c o l l a p s e was c a l c u l a t e d . Foam- s t a b i l i t y was. the.n,.determined as the time.-required 40 F i g u r e 1. Diagram of foaming apparatus 1) c a l i b r a t e d g l a s s column 2) s i n t e r e d d i s c 3) stopcock 4.) flowmeter 5) needle v a l v e 6) pressure gauge 7) pressure r e g u l a t o r 8) c y l i n d e r of a i r 41 f o r 50 ml of foam to breakdown.at the c a l c u l a t e d c o l l a p s e rate.. . Measurements were made, i n t r i p l i c a t e . 2.7. .Hydrophobicity P r o t e i n h y d r o p h o b i c i t y was determined using the f l u o r e s c e n c e probe, method, .of. Kat.o and Nakai -(1980) and m o d i f i c a t i o n s of t h i s method. The t e s t p r o t e i n , as a 1.5% s o l u t i o n i n 0....1M, pH 7.0 phosphate buffer,, was. exposed, t o one. of. the f o l l o w i n g treatments: 1) v a r y i n g l e v e l s of SDS. (0...002$„ 0.005$, 0.01$ and 0.05$) 2) T-M pheneth y l b i g u a n i d i n e h y d r o c h l o r i d e 3) pH 10.5 4) 5 min at.. 1 00°C . . 5) 20 min at 1 00°C i n the presence of 1..5$ SDS and 0.3$ mercaptoethanol 6) combinations of various, l e v e l s of SDS, h e a t i n g and degrees of enzymatic h y d r o l y s i s 7) 10 min at 100°C i n the . presence of. 1.5$ SDS... The sample was d i l u t e d with, 0...1 M phosphate b u f f e r pH 7.0 to make a s e r i e s of s o l u t i o n s , with c o n c e n t r a t i o n s ranging from 0.00.2$ to.0.-0.1$ protein... Then,. 10 u l of c i s -p a r i n a r i c a c i d (3.6 x 10" M i n absolute e t h a n o l c o n t a i n i n g 10 ug./ml BHA. to. prevent o x i d a t i o n ) were added to a. 2 . ml ..aliquot, of each . s o l u t i o n . An Aminco Bowman s p e c t r o f luoromet.er, No.... 4-8202 was used to measure the ..relative f l u o r e s c e n c e i n t e n s i t i e s of 42 the p a r i n a r i c a c i d - p r o t e i n conjugates. The measure-ments were made at an e x c i t a t i o n wavelength of 325 nm and an emission wavelength of 4-20 nm. The instrument was st a n d a r d i z e d so that, a mixture of 2 ml decane and 10 y l c i s - p a r i n a r i c a c i d had a f l u o r e s c e n c e i n t e n s i t y of 4-5$. P r o t e i n h y d r o p h o b i c i t y was measured as the i n i t i a l slope of the curve of % f l u o r e s c e n c e vs. % p r o t e i n . D u p l i c a t e determinations were made. 2.8. T o t a l P r o t e i n The samples were d i g e s t e d according to the m i c r o - K j e l d a h l procedure of Concon and.Soltess (1973)-A sample c o n t a i n i n g approximately 10 mg p r o t e i n was heated with 2 ml concentrated s u l p h u r i c a c i d i n the presence of 2 g of a c a t a l y s t . , '.'The c a t a l y s t c o n s i s t e d of potassium sulphate and mercuric oxide i n the r a t i o 95:2. The mixture was heated u n t i l c h a r r i n g occurred. Then, 3-4- drops of hydrogen peroxide were added. Heating was continued t i l l the mixture was c l e a r and a f t e r s l i g h t c o o l i n g the sample was d i l u t e d to 25 ml. The n i t r o g e n present was determined c o l o r i m e t r i c a l l y by r e a c t i o n with sodium s a l i c y l a t e i n the presence of sodium n i t r o p r u s s i d e and sodium h y p o c h l o r i t e , using a Technicon AutoAnalyzer I I , at a wavelength of 660 nm, acco r d i n g t o the manufacturer's i n s t r u c t i o n s . The p r o t e i n content of g l u t e n and c a s e i n were obtained by 43 m u l t i p l y i n g the determined n i t r o g e n by f a c t o r s of 5.70 and 6.38, r e s p e c t i v e l y . The f a c t o r 6.25 was used f o r a l l other samples. D u p l i c a t e determinations were made. 2.9. D I s p e r s i b i l i t y A t e n ml a l i q u o t of a 1 % d i s p e r s i o n of p r o t e i n i n 0.1M phosphate b u f f e r pH 7.0 was s t i r r e d at s e t t i n g §3 on a F i s h e r Thermomix magnetic s t i r r e r , f o r 10 min. The sample was c e n t r i f u g e d at 27,000.x g f o r 30 min and the p r o t e i n content of the supernatant determined. The r a t i o of p r o t e i n i n the supernatant to p r o t e i n d i s p e r s e d , expressed as a percentage, was used as an index of p r o t e i n s o l u b i l i t y . . Measurements were performed i n d u p l i c a t e . 2.10. V i s c o s i t y An Ostwald' viscometer was used to determine the v i s c o s i t i e s of the p r o t e i n s o l u t i o n s . The s o l u t i o n s contained 0.1$ p r o t e i n i n 0..1M phosphate b u f f e r pH 7.0. Measurements were c a r r i e d out i n d u p l i c a t e . a t 22°C according to the manufacturer's i n s t r u c t i o n s . Between readings the viscometer was cleaned with chromic a c i d , washed thoroughly with water, f l u s h e d with acetone and a i r . d r i e d . D i s t i l l e d , • d e i o n i z e d water was used f o r c a l i b r a t i o n of the viscometer. u 2.11. Non-protein N i t r o g e n Ten ml of a 1 % d i s p e r s i o n of p r o t e i n i n 0.1M phosphate b u f f e r pH 7.0 was s t i r r e d f o r 10 min at s e t t i n g #3 on a F i s h e r Thermomix magnetic s t i r r e r and then c e n t r i f u g e d at '27,000 x g f o r 30 min. The supernatant .(A) was c o l l e c t e d . . Three ml,, of 20% t r i c h l o r o a c e t i c a c i d were added to a 2 ml a l i q u o t of supernatant A to p r e c i p i t a t e the protein.. The mixture was c e n t r i f u g e d at .27,000 x g f o r 30 min and the super-natant (B) co l l e c t e d . . The. n i t r o g e n content of superna-t a n t s A and B were .determined by.the m i c r o - K j e l d a h l method. The r a t i o of the amount of n i t r o g e n i n superna-tant B to the amount of n i t r o g e n i n supernatant A expressed as. a percentage was taken as the percentage of n o n-protein n i t r o g e n i n the sample... The measurements were made i n d u p l i c a t e . 2.12. Bound SDS Two ml of a. 1..5$ p r o t e i n s o l u t i o n i n 0.1M phosphate b u f f e r , pH 7.0 c o n t a i n i n g 1.5$ SDS were heated f o r 10 min at .100 "CL The sample was cooled and the SDS-protein complex i s o l a t e d by chromatography on a column (10 mm. x 350 mm) 'of Se.phadex G-1 00 - ( A l l e n , 1974). The SDS-protein complex was e l u t e d with 0.1M 45 phosphate b u f f e r , pH 7.0 at a flow r a t e of 4- ml/hr. E l u t i o n of the complex was dete c t e d by i t s absorbance at 280 nil. The amount of SDS.present in. the eluent was determined by the methylene blue method as modified by Reynolds and Tanford (1 970).. In t h i s procedure, 0.05 ml of eluent was added to. a mixture of 20 ml chloroform and 5 ml methylene blue (24 mg/1 i n water) i n a separatory f u n n e l . A f t e r shaking f o r 2 min.,. the chloroform l a y e r was removed and i t s absorbance at 655 nm determined. The c o n c e n t r a t i o n of .SDS present was determined by comparison of the absorbance values with a standard curve, c o n s t r u c t e d using standard SDS s o l u t i o n s of 0.. 5- mg/ml - 2.0 mg/ml f o r e x t r a c t i o n . The p r o t e i n . c o n t e n t of the eluent was then determined. The bound. .SDS was c a l c u l a t e d as the r a t i o of the SDS content of the eluent i n mg/ml to the p r o t e i n content of the eluent i n mg/ml. 2.13. Simplex O p t i m i z a t i o n Simplex o p t i m i z a t i o n was c a r r i e d out to optimize the c o r r e l a t i o n between foaming c a p a c i t y and hy d r o p h o b i c i t y using Nakai's (1982) modified super-simplex o p t i m i z a t i o n program, w r i t t e n f o r a Monroe 1880-88 46 programmable ca l c u l a t o r . . This program i n c o r p o r a t e s a qu a d r a t i c r e g r e s s i o n subroutine f o r computation of the experimental c o n d i t i o n s to be used as v e r t i c e s of the simplex. The response to the treatments was evaluated using the c o r r e l a t i o n c o e f f i c i e n t of the curve of foaming c a p a c i t y v s . h y d r o p h o b i c i t y when the slope was p o s i t i v e . In cases f o r which the slope was negat i v e , the product of the c o r r e l a t i o n c o e f f i c i e n t and slope of the curve was used, since a negative c o r r e l a t i o n was unacceptable. The Bigelow. h y d r o p h o b i c i t y v a l u e s f o r the p r o t e i n s i n d i c a t e d t h at the c o r r e l a t i o n between foaming c a p a c i t y and h y d r o p h o b i c i t y should be a p o s i t i v e one. The p r i n c i p l e of the simplex o p t i m i z a t i o n method i s o u t l i n e d i n Appendix I. 2.14-. Surface t e n s i o n S o l u t i o n s of 0.1$ p r o t e i n i n 0.. 1M phosphate b u f f e r pH 7.0 were used.. The measurements were made i n t r i p l i c a t e at 22°C with a F i s h e r Surface Tensiomat Model -1 21, which had been c a l i b r a t e d . a t 49 dynes cm with a 600 mg weight according t o the manufacturer's i n s t r u c t i o n s . 2.1$. Charge Density The net proton charge on the p r o t e i n s was measured by hydrogen-ion t i t r a t i o n i n 6M guanidine h y d r o c h l o r i d e according to the procedure d e s c r i b e d by Nozaki and Tanford (1967). Two hundred and f o r t y mg 47 of p r o t e i n were d i s s o l v e d i n 6 ml water. The sample was d e i o n i z e d by passing i t through a column (10 x 120 mm) of Rexyn 300 and Rexyn 101 i n the r a t i o 3:1. The sample was c o l l e c t e d i n a v e s s e l which had been f l u s h e d with n i t r o g e n . The p r o t e i n c o n c e n t r a t i o n of the d e i o n i z e d sample was determined by comparing i t s absorbance at 280 nm with that, of a standard s o l u t i o n of the same p r o t e i n . Then, the sample was d i l u t e d to make 25 ml of s o l u t i o n 6M i n guanidine h y d r o c h l o r i d e and c o n t a i n i n g approximately 5 mg/ml of protein.. The pH of a small p o r t i o n of the d e i o n i z e d sample was measured and taken as the i s o i o n i c point of the protein.. Using 5 ml of sample and standard h y d r o c h l o r i c a c i d or potassium hydroxide (0.1M - 0.5M), the number of moles of a c i d or base r e q u i r e d to b r i n g the pH of the. p r o t e i n s o l u t i o n from i t s i s o i o n i c p o i n t t o pH 7..0 was measured. A blank t i t r a t i o n was a l s o c a r r i e d , out i n t h i s pH range using 6M guanidine hydrochloride.... The. t i t r a t i o n s were c a r r i e d out under n i t r o g e n using a s e r i e s of m i c r o l i t e r p i p e t t e s f o r a d d i t i o n of a c i d or base and a F i s h e r microprobe combination e l e c t r o d e attached.to a Beckman D i g i t a l pH meter f o r pH measurement.. A l l samples were made up with d i s t i l l e d d e i o n i z e d . water... The net proton charge was c a l c u l a t e d as the number of moles of hydrogen ions bound or l o s t per mole of p r o t e i n when the pH i s s h i f t e d 4-8 from the i s o i o n i c point to pH 7.0. This f i g u r e was d i v i d e d by the number of amino a c i d r e s i d u e s per molecule of p r o t e i n to o b t a i n i t s charge d e n s i t y . 2.16. E r r o r A n a l y s i s from R e p l i c a t e Measurements For each parameter measured, the c o e f f i c i e n t of v a r i a b i l i t y f o r the mean of t'he.replicates was c a l c u l a t e d as CV = 1 00 X = 1 00 X where CV = c o e f f i c i e n t of v a r i a b i l i t y (%) s = standard e r r o r X = mean th X^ = value of the i r e p l i c a t e N = number of r e p l i c a t e s In most cases t h i s v.alue was low .(< J+%) * However, there was a wide range i n the c o e f f i c i e n t s of v a r i a b i l i t y f o r the foaming c a p a c i t y and foam s t a b i l i t y measurements. As a r e s u l t of t h i s , t a b l e s c o n t a i n i n g foaming c a p a c i t y and foam s t a b i l i t y values a l s o i n c l u d e the standard e r r o r of the mean. A9 IV. RESULTS AND DISCUSSION 1. Choice of Model P r o t e i n s The model p r o t e i n s were chosen to provide samples with a f a i r l y wide range of average hydrophobi-c i t y values and a v a r i e t y of secondary and t e r t i a r y s t r u c t u r e s . The samples were r e s t r i c t e d to w e l l known p r o t e i n s i n order to a v. old. d i f f i c u l t i e s i n o b t a i n i n g i n f o r m a t i o n about t h e i r s t r u c t u r e . In s p i t e of the ' low c o n c e n t r a t i o n s used.,, a t o t a l , of 1 g of each p r o t e i n was r e q u i r e d f o r the foaming tests.. This f u r t h e r r e s t r i c t e d the samples to p r o t e i n s which were r e l a t i v e l y inexpensive i n a pure form. 2. Choice of c i s - P a r i n a r i c A c i d f o r Hydrophobicity Measurement s The quantum y i e l d s of a number of p o l y c y c l i c aromatic compounds and l i n e a r .polyenes, are known to i n c r e a s e In non-polar environments ( S t r y e r , 1965). As a r e s u l t of t h i s , these compounds have been used as s e n s i t i v e probes of t h e p o l a r i t y of binding s i t e s of p r o t e i n s . G i s - p a r i n a r i c a c i d was i n i t i a l l y i n t r o d u c e d f o r use as a f l u o r e s c e n t probe i n membrane s t u d i e s but 50 has r e c e n t l y been used f o r measuring the surface h y d r o p h o b i c i t y of p r o t e i n s (Kato and Nakai, 1 980; S k l a r and Hudson,. 1 976; Sklar. et. aL, 1 977). I t has a-n advantage over more commonly used f l u o r e s c e n t probes i n that i t does not bear any bulky s u b s t i t u e n t s capable of s t e r i c i n t e r f e r e n c e with b i n d i n g . In t h i s work,, c i s - p a r i n a r i c a c i d was used as a probe of the hydrophobic reg i o n s . o n p r o t e i n s i n order to permit q u a n t i t a t i v e c l a s s i f i c a t i o n of p r o t e i n s on the b a s i s of t h e i r h y d r o p h o b i c i t y and c o r r e l a t i o n of a measured h y d r o p h o b i c i t y value with foaming p r o p e r t i e s . Support f o r the use of ...cis-parinaric a c i d as a hydrophobic probe i s provided by the s i g n i f i c a n t l i n e a r c o r r e l a t i o n (r = -0.852> P<0.01 ),. which e x i s t s between the p o l a r i t y of a s e r i e s of solv.ent s, as determined by Snyder (1 978), and the quantum y i e l d . of,. c i s - p a r i n a r i c a c i d i n these s o l v e n t s ( S k l a r et al., 1977) (Figure 2). ' 3. The Importance of P r o t e i n Secondary S t r u c t u r e , Molecular S i z e , Shape and F l e x i b i l i t y f o r Foaming It has been argued that at the a i r - w a t e r i n t e r f a c e t h e . p o l a r groups of p r o t e i n s can i n t e r a c t with the aqueous phase, the non-polar groups can asso-c i a t e with, the a i r phase and hydrogen bonding can be 51 8r F i g u r e 2. R e l a t i o n s h i p between quantum y i e l d of c i s -p a r i n a r i c a c i d and solvent p o l a r i t y . * I) decane 2) ether 3) hexane 1+) cyclohexane 5) dimethylformamide 6). butanol 7) ethanol 8) chloroform 9) propanol 10) methanol II) water. """Values of p o l a r i t y and quantum y i e l d from Snyder (1978) and Sklar et a l . , (1977) re s p e c t i v e l y . 52 s a t i s f i e d i n t e r m o l e c u l a r l y t h e r e f o r e there should be l i t t l e tendency f o r h e l i x formation (MacRitchie, 1978). However, c o n s i d e r a b l e evidence has been presented which suggests that p r o t e i n s r e t a i n much of t h e i r secondary structure., p a r t i c u l a r l y the a - h e l i x , at the i n t e r f a c e (Malcolm, 1 973).. I f p r o t e i n s r e t a i n t h e i r secondary s t r u c t u r e at the i n t e r f a c e . t h e r e may be some r e l a t i o n -ship between the secondary s t r u c t u r e of p r o t e i n s and t h e i r surface properties... This should be r e f l e c t e d i n t h e i r foaming c h a r a c t e r i s t i c s . . Therefore the foaming p r o p e r t i e s of the model., p r o t e i n s were examined i n r e l a t i o n to t h e i r secondary s t r u c t u r e (Tables 2, 4-). No r e l a t i o n s h i p was found between the foaming c a p a c i t y or foam s t a b i l i t y of the model., p r o t e i n s and the percentages of a-helix..,. 8-sheet, or..turns i n t h e i r molecules. This, suggested. ..that ,protein secondary s t r u c t u r e was not .important, f o r . d e t e r m i n i n g foaming. However ,. it.. i s . p o s s i b l e . that during . a d s o r p t i o n at the i n t e r f a c e there were some..changes i n the secondary s t r u c t u r e of p r o t e i n s . Loeb and Baier (1968) demonstrated that p r o t e i n s can adopt d i f f e r e n t secondary s t r u c t u r e s depending on t h e i r environment.. They noted that p o l y -methylglutamate monolayers removed f,rom the w a t e r - a i r i n t e r f a c e had v a r y i n g p r o p o r t i o n s of a.-helix and 8-sheet 53 s t r u c t u r e s , depending on the spreading solvent used. Graham and P h i l l i p s (1976) observed that the f l e x i b l e , random c o i l p r o t e i n 3-casein, had. good foaming c a p a c i t y . Lower foaming c a p a c i t y was e x h i b i t e d by lysozyme and BSA which have .more r i g i d molecules. From s t u d i e s on the surface a d s o r p t i o n p r o p e r t i e s of p r o t e i n s Graham and P h i l l i p s (1979)•concluded t h a t , f l e x i b l e molecules such as 3 - c a s e i n underg.o r a p i d rearrangement i n the interface... More r i g i d molecules such as BSA and lysozyme experience slow.. unf olding. and rearrangement i n the i n t e r f a c e . As a. r e s u l t of this,, f l e x i b l e molecules can r e a d i l y s t a b i l i z e f r e s h a i r bubbles and have good foaming c a p a c i t y . Since i t . seems. that molecular f l e x i b i l i t y i s important f o r determining foaming c a p a c i t y , the foaming c h a r a c t e r i s t i c s of the model p r o t e i n s were examined i n r e l a t i o n to the f l e x i b i l i t y , of t h e i r molecules. I t was not p o s s i b l e to measure molecular f l e x i b i l i t y d i r e c t l y . However, the m o s t . f l e x i b l e model p r o t e i n s were i d e n t i f i e d by c o n s i d e r i n g the number and l o c a t i o n of d i s u l p h i d e bonds per molecule i n . r e l a t i o n to molecular s i z e (Table 1 ) . The r a t i o n a l e f o r t h i s , was that, d i s u l p h i d e bonds reduce conformational, f l u c t u a t i o n s i n p r o t e i n molecules. U n l i k e the v a r i o u s no.ncovalent. bonds i n v o l v e d i n mainte-nance of p r o t e i n s t r u c t u r e , d i s u l p h i d e bonds are not 5k l i k e l y t o be d i s r u p t e d at. t h e i n t e r f a c e (Graham and P h i l l i p s , 1 9 7 9 c ) . The . d i s u l p h i d e bond c o n t e n t o f t h e m o l e c u l e s s h o u l d t h e r e f o r e be i m p o r t a n t f o r d e t e r m i n i n g t h e d e g r e e t o w h i c h a p r o t e i n . c a n u n f o l d a t t h e i n t e r f a c e . L a r g e m o l e c u l e s w i t h f e w . d i s u l p h i d e bonds s h o u l d be v e r y f l e x i b l e and s h o u l d . e x p e r i e n c e a l a r g e f a v o u r a b l e e n t r o p y c hange i n g o i n g f r o m a f o l d e d c o n f o r m a t i o n i n s o l u t i o n t o an u n c o i l e d f o r m a t t h e i n t e r f a c e . P e p s i n , ovalbumin,, B-casein.,. K - c a s e i n and B-l a c t o g l o b u l i n were . i d e n t i f i e d as h a v i n g f l e x i b l e m o l e c u l e s on t h i s b a s i s . . I n s p i t e o f t h e . l a r g e number o f d i s u l p h i d e b onds i n BSA t h e r e i s . e v i d e n c e t o . s u g g e s t t h a t t h i s p r o t e i n i s a l s o q u i t e f l e x i b l e (.Ku.z.man.,. 1 956; P e t e r s J r . , 1 9 7 5 ) . I n t h e p r e s e n c e o f u r e a a t . . c o n c e n t r a t i o n s as l o w as 2M b o v i n e serum a l b u m i n shows, a l a r g e i n c r e a s e i n o p t i c a l r o t a t i o n , , w h i c h i s . i n d e p e n d e n t o f t i m e . R e m o v a l o f u r e a r e t u r n s t h e o p t i c a l . r o t a t i o n t o t h e v a l u e f o r t h e n a t i v e p r o t e i n . In. a d d i t i o n , , t h e h y d r o g e n i o n t i t r a t i o n c u r v e f o r b o v i n e .serum a l b u m i n i s i n c o n s i s t e n t w i t h a r i g i d s t r u c t u r e . The h i g h , d e g r e e o f f l e x i b i l i t y , w h i c h t h e s e o b s e r v a t i o n s i n d i c a t e t h a t b o v i n e serum a l b u m i n h a s , i s , c o n s i s t e n t w i t h t h e s e c o n d a r y s t r u c t u r e o f i t s m o l e c u l e s . I n t h i s p r o t e i n t h e d i s u l p h i d e bonds l i n k t h e p o l y p e p t i d e c h a i n i n t o .a s e r i e s o f a d j a c e n t 55 l o o p s . A l l the f l e x i b l e p r o t e i n s showed good foaming c h a r a c t e r i s t i c s while the.more r i g i d p r o t e i n s i . e . lysozyme, ovomucoid and r i b o n u c l e a s e showed poor foaming p r o p e r t i e s (Table 4-).-. There was ..a s i g n i f i c a n t negative l i n e a r c o r r e l a t i o n (r =-0.806/, P<0.01) between foaming capacity, .and the number of d i s u l p h i d e bonds per u n i t molecular, weight (Figure 3). The r e g r e s s i o n equation was FC. = 1 28.9 - 25.05 SS/M where FC = foaming c a p a c i t y SS/M = number of d i s u l p h i d e bonds/molecular weight This s i g n i f i c a n t r e l a t i o n s h i p , between foaming c a p a c i t y and ' f l e x i b i l i t y ' supported the i d e a that p r o t e i n s must be q u i t e f l e x i b l e to. s t a b i l i z e f r e s h a i r bubbles e f f e c -t i v e l y and t h e r e f o r e show good foaming a b i l i t y . As shown i n F i g u r e 3,. the data suggested, that when the r a t i o ffSS bonds/molecular weight x 10"'^ was 3 or more p r o t e i n f l e x i b i l i t y was s e v e r l y r e s t r i c t e d and foaming c a p a c i t y was low. The f r i c t i o n a l c o e f f i c e n t s of the model p r o t e i n s are a l l c l o s e t o u n i t y .(Table 1 ) . T h i s i n d i c a t e s that hydrodynamically they a l l behave l i k e spheres (Tanford, 1961).. U n f o r t u n a t e l y , d i f f e r e n c e s i n these 56 Table 4-. Foaming c a p a c i t y and foam s t a b i l i t y of model p r o t e i n s Foam S t a b i l i t y P r o t e i n Foaming j Capacity min ml" 1 r i bonuclease 3 jf 1 0.0 + 0.0 ovomucoid 1 _+ 0 0.0 + 0.0 t r y p s i n 29 +_ 1 2.4 + 0.2 lysozyme 3 + 1 1 .2 + 0.2 pepsin 1 4-1 +_ 3 5.3 + 0.2 conalbumin 135 _+ 3 15.8 + 2.0 ovalbumin 11 2 +_ 2 2.6 + 0.2 bovine albumin 89 + 3 1 .8 + 0.2 K - c a s e i n •• 135 .2 58.0 + 3.0 8 - l a c t o g l o b u l i n 1 37 2 6.3 + 0.2 ' 8-casein 1 09 + 2 18.3 + 1.2 Each data p o i n t i s the average of three determinations 57 200r ure 3. R e l a t i o n s h i p between foaming ca p a c i t y " and molecular f l e x i b i l i t y measured as #SS bonds/ molecular weight. 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4-) lysozyme 5) pepsin 6) . conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-cas e i n 10) 6 - l a c t o g l o b u l i n 11) B-casein ""Each data p o i n t i s the average of three determinations 58 values f o r the i n d i v i d u a l p r o t e i n s are too small to be u s e f u l f o r i n t e r p r e t i n g t h e i r foaming p r o p e r t i e s i n terms of the hydrodynamic behaviour of the p r o t e i n s . 4-. The R e l a t i o n s h i p between P r o t e i n Hydrophobicity and Foaming P r o p e r t i e s It i s commonly accepted t h a t good foaming agents must have a mixture of h y d r o p h i l i c and hydro-phobic groups in. t h e i r molecules (Cherry and McWatters, 1981; Rosen, 1972).. Experiments were t h e r e f o r e c a r r i e d out to determine the nature of the r e l a t i o n s h i p between the foaming c a p a c i t y and foam s t a b i l i t y of p r o t e i n s and t h e i r h y d r o p h o b i c i t y . Table 5 shows the percentages of hydrophobic amino a c i d s i n the. model p r o t e i n s . These valu e s were c a l c u l a t e d from the amino, a c i d composition of the p r o t e i n s as l i s t e d i n Table 3- V a l i n e , p r o l i n e , l e u c i n e , i s o l e u c i n e , p h e n y l a l a n i n e and tryptophan were c l a s s e d as hydrophobic amino acids.. No simple r e l a t i o n s h i p was found between the percentage of hydrophobic amino a c i d s c a l c u l a t e d i n t h i s way and the foaming c a p a c i t y or foam s t a b i l i t y of the. p r o t e i n s . However, i t was i n t e r e s -t i n g to note that the best foaming c a p a c i t y was e x h i b i t e d by p r o t e i n s c o n t a i n i n g more than 31 % hydrophobic amino a c i d s . 59 Table 5. Percentage of hydrophobic groups i n the model p r o t e i n s * P r o t e i n % hydrophobic groups r i b o n u c l e a s e 23 ovomucoid 26 t r y p s i n 29 lysozyme 22 p e p s i n 36 conalbumin 32 ovalbumin 33 b o v i n e serum a l b u m i n 32 K - c a s e i n 37 3 - l a c t o g l o b u l i n 31 B - c a s e i n 38 v a l , pro, l e u , i l e , phe, t r p assumed to be hydrophobic 60 The importance of h y d r o p h o b i c i t y f o r d e t e r -mining foaming p r o p e r t i e s was demonstrated by:the p o s i t i v e r e l a t i o n s h i p between the foaming c a p a c i t y of the model p r o t e i n s and t h e i r average Bigelow hydropho-b i c i t i e s ( F i g u r e . i).  This r e l a t i o n s h i p was best d e s c r i b e d by the r e g r e s s i o n equation In (FC + 30) = 0.0041 H 0 a v e + 0.0393 where FC = foaming c a p a c i t y H 0 & v e = average h y d r o p h o b i c i t y as c a l c u l a t e d by Bigelow ( k c a l . r e s - ) The c o r r e l a t i o n between these two parameters was h i g h l y s i g n i f i c a n t (r = .0.823, P<0.01). Within the range of the experiment,, foaming c a p a c i t y i n c r e a s e d c o n t i n u o u s l y with average h y d r o p h o b i c i t y . This i m p l i e d that high h y d r o p h o b i c i t y was r e q u i r e d f o r p r o t e i n s to from the cohesive,. e l a s t i c l a y e r at the i n t e r f a c e , which i s necessary f o r s t a b i l i z i n g a i r bubbles. C a l c u l a t i o n of the average h y d r o p h o b i c i t y values i n v o l v e s summation of the c o n t r i b u t i o n s of a l l amino a c i d s i n the p r o t e i n (.Bigelow, 1 967). Th e r e f o r e , the c l o s e c o r r e l a t i o n between average h y d r o p h o b i c i t y and foaming c a p a c i t y i s consistent, with the hypothesis t h a t a d s o r p t i o n at. the i n t e r f a c e .required extensive u n c o i l i n g of the p r o t e i n s so that the maximum p o s s i b l e number of 61 200 r F i g u r e 4» R e l a t i o n s h i p between foaming capacity" ; ;"and Bigelow's average h y d r o p h o b i c i t y (H0 ) 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4.) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K - c a s e i n 10) 8 - l a c t o g l o b u l i n 11) 3-casein *Each data point i s the average of three determinat ions 62 i t s amino a c i d s may be s i t u a t e d i n the a i r - w a t e r i n t e r f a c e . There was a s i g n i f i c a n t (r = 0.807, P<0.01) semi l o g a r i t h m i c r e l a t i o n s h i p between foam s t a b i l i t y and average h y d r o p h o b i c i t y which was d e s c r i b e d by the r e g r e -s s i o n equation In (FS + 0.18) = 0.0102 H0 - 9.79 ave where FS = foam s t a b i l i t y (min) H 0 a v e = average h y d r o p h o b i c i t y as c a l c u l a t e d by Bigelow ( k c a l . r e s ) The i n c r e a s e i n foam s t a b i l i t y which accompanies i n c r e a s i n g h y d r o p h o b i c i t y could be explained, by inv.olvement of hydro-phobic '. i n t e r a c t ions i n the a s s o c i a t i o n . of molecules at the i n t e r f a c e . d u r i n g f i l m f o r m a t i o n . It has been, r e p o r t e d t h a t the surface hydro-p h o b i c i t y of p r o t e i n s shows.a s i g n i f i c a n t c o r r e l a t i o n with two s u r f a c e properties.: e m u l s i f y i n g a c t i v i t y and i n t e r f a c i a l t e n s i o n (Kato and Nakai,. 1 980). Since foaming c a p a c i t y and.foam s t a b i l i t y are a l s o surface p r o p e r t i e s of p r o t e i n s , . t h e i r , r e l a t i o n s h i p to surface h y d r o p h o b i c i t y was investigated,. The procedure of Kato and Nakai i s based on the enhancement of c i s - p a r i n a r i c a c i d f l u o r e s c e n c e , . w h i c h accompanies i t s a s s o c i a t i o n with hydrophobic s i t e s on p r o t e i n molecules. These s i t e s are l i m i t e d t o hydrophobic groups exposed at the s u r f a c e of the p r o t e i n molecule and i n c r e v i c e s l a r g e 63 F i g u r e 5. R e l a t i o n s h i p between foam s t a b i l i t y and Bigelow's average h y d r o p h o b i c i t y ( H 0 a v g ) 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4.). lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8.) bovine serum albumin 9) K - c a s e i n 10,). 6 - l a c t o g l o b u l i n . 11) 8-casein ""Each data, p o i n t i s the average of three determinations 64 enough to accommodate -the c i s - p a r i n a r i c a c i d molecule. The t e s t i s c a r r i e d , out i n the presence of 0.002$ SDS to promote s l i g h t u n f o l d i n g of the p r o t e i n s . SDS d e n a t u r a t i o n of p r o t e i n s probably i n v o l v e s b i n d i n g of the hydrocarbon p o r t i o n of SDS to exposed hydrophobic s i t e s on the p r o t e i n (B i r d ! , and S t e i n h a r d t , 1978). E l e c t r o s t a t i c r e p u l s i o n between the anion p o r t i o n s of the bound SDS molecules leads to u n f o l d i n g of the p r o t e i n p o l y p e p t i d e c h a i n . No s i g n i f i c a n t r e l a t i o n s h i p was found between the foaming c a p a c i t y of p r o t e i n s and t h e i r s urface h y d r o p h o b i c i t y (P>0.05).. This poor c o r r e l a t i o n provided f u r t h e r support f o r the idea, that p r o t e i n s were e x t e n s i v e l y u n c o i l e d at the .air-water, i n t e r f a c e . . In. view of the poor c o r r e l a t i o n between foaming c a p a c i t y and s u r f a c e hydro-p h o b i c i t y and the s i g n i f i c a n t c o r r e l a t i o n between foaming c a p a c i t y and average h y d r o p h o b i c i t y , i t was apparent that p r o t e i n s should be subjected to extensive u n c o i l i n g , p r i o r to h y d r o p h o b i c i t y measurement.,, i n order to o b t a i n a measured h y d r o p h o b i c i t y value ..to .substitute f o r the c a l c u l a t e d average h y d r o p h o b i c i t y value of Bigelow. Values obtained .in t h i s way should show good c o r r e l a t i o n with foaming c a p a c i t y . SDS i s a. powerful denaturant which i s e f f e c t i v e 6 5 200r 150 O g <100 LL 50 , 4 o l - — . J O 250 500 .8 750 1000 ure 6. R e l a t i o n s h i p between foaming c a p a c i t y and su r f a c e h y d r o p h o b i c i t y (S ) + 1) r i b o n u c l e a s e 2) o.vomucoid 3) t r y p s i n 4-) lysozyme 5) pe.psin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K - c a s e i n 10.) 3 - l a c t o g l o b u l i n 11) 3 - c a s e i n ""Each data point, i s the average of t r i p l i c a t e d e terminations + Each data point, i s the average of d u p l i c a t e determinations 66 at low concentrations,. Therefore the e f f e c t of exposing the p r o t e i n s to v a r i o u s low l e v e l s of.SDS,on the measured h y d r o p h o b i c i t y . v a l u e and i t s r e l a t i o n s h i p to foaming c a p a c i t y was i n v e s t i g a t e d . As shown i n F i g u r e s 7.,. 8 and 9, SDS l e v e l s of 0.005, 0.01 and 0.05$ d i d not. produce measured hydro-p h o b i c i t y v a l u e s which were s i g n i f i c a n t l y c o r r e l a t e d with foaming capacity.(P>0 J.i'5). For some of the- p r o t e i n s , the hydro-p h o b i c i t y i n c r e a s e d g r a d u a l l y with i n c r e a s i n g SDS concen-t r a t i o n . Other proteins,, notably bovine serum albumin, 8 - l a c t o g l o b u l i n and K-ca.sein showed no..such t r e n d . These changes i n measured, h y d r o p h o b i c i t y with ..SDS. c o n c e n t r a t i o n were p r o b a b l y , r e l a t e d to the dependence of the extent of SDS b i n d i n g on b o t h . p r o t e i n content and SDS l e v e l (Ray et al., 1966; Steinhardt. et. al.,. 1 977),.. Work by B i r d i and S t e i n h a r d t (1978) suggests that hydrophobic probes added to. SDS denatured^p'roteins are . s . o l u b i l i z e d by the SDS bound to the. proteins... Therefore, any change i n the SDS bound to t h e . p r o t e i n s would be manifested as a change i n the f l u o r e s c e n c e of t h e i r complexes with c i s -p a r i n a r i c a c i d and consequently i n t h e i r measured v a l u e s . At SDS l e v e l s of 0.-005$ and higher, the l y s o -zyme samples were turbid, due to p a r t i a l p r e c i p i t a t i o n and had very, high f l u o r e s c e n c e . values.. As a r e s u l t of 67 200 r 150 2 < O o z < o 100 50 .6 10 11 .8 200 400 600 »e ure 7. Relationship, betwe.en foaming c a p a c i t y and h y d r o p h o b i c i t y ( S ) measured i n the presence of 0.005$ SDS. 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n U) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-cas e i n 10) 3-lactoglobulin 11) 3-casein """Each data, point i s the average of t r i p l i c a t e d e terminations "+Each data p o i n t i s the average of d u p l i c a t e determinations 68 200r 150 _ < 100 o o g 50 J O .11 18 300 600 900 1200 F i g u r e 8. R e l a t i o n s h i p between foaming capacity' cand h y d r o p h o b i c i t y (S g) +measured i n the presence of 0.01$ SDS. 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-casein. 10) 8 - l a c t o g l o b u l i n 11) 3-casein ""Each data p o i n t , i s the average of t r i p l i c a t e d e terminations +Each data p o i n t i s the average of d u p l i c a t e determinations 69 200 r 150 >-H O & o 100 z < o 50 j o .9 .11 .8 2 4 200 400 600 800 F i g u r e 9. R e l a t i o n s h i p between foaming capacity' and h y d r o p h o b i c i t y (S g)^measured i n the presence of 0.05$ SDS. 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4-) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine.serum albumin 9) K - c a s e i n 10) 6 - l a c t o g l o b u l i n 11) 8-casein *Each data poi n t i s the average of t r i p l i c a t e d eterminations +Each data poi n t i s the average of d u p l i c a t e determinations 70 t h i s , lysozyme gave.very high measured h y d r o p h o b i c i t y v a l u e s i n s p i t e of i t s low average h y d r o p h o b i c i t y . A t u r b i d i t y c o r r e c t i o n , which i n v o l v e d s u b t r a c t i o n of the f l u o r e s c e n c e of.SDS denaturedlysozyme i n the absence of c i s - p a r i n a r i c a c i d from i t s f l u o r e s c e n c e i n the presence of the probe, d i d not a p p r e c i a b l y lower the measured hydrophobicity.. Due to the unusual behaviour of lysozyme i n the presence of high l e v e l s of SDS, the e f f e c t s of a v a r i e t y of other denaturing treatments on measured p r o t e i n h y d r o p h o b i c i t y and i t s r e l a t i o n s h i p with foaming c a p a c i t y were examined. Pheneth.ylbi.guani.dine h y d r o c h l o r i d e has been r e p o r t e d to be a more potent denaturant than guanidine h y d r o c h l o r i d e , which a c t s by d i s r u p t i n g i n t e r m o l e c u l a r hydrophobic i n t e r a t i o n s ..(Noelken., 1 980),. In the presence of t h i s reagent most. of. the p r o t e i n s , . h a d low hydropho-b i c i t y values. (Figure 10),,.. Apparently both BSA and 8 - l a c t o g l o b u l i n were p a r t i c u l a r l y s u s c e p t i b l e to d e n a t u r a t i o n by t h i s reagent as they had extremely high h y d r o p h o b i c i t y v a l u e s - I t was not p o s s i b l e to measure the h y d r o p h o b i c i t i e s of. 3..- or K - c a s e i n s i n c e n e i t h e r of these p r o t e i n s dissolved. ..in p h e n e t h y l b i g u a n i d i n e hydro-chlo r i d e . . Hydrophobicity measured a f t e r exposure to t h i s reagent showed no' r e l a t i o n s h i p to the foaming p r o p e r t i e s of the p r o t e i n s •• (P>0 . 05 ) . 71 200r 150 > H O <100 o o z _» < o 50 • 5 •6 ok .10 .8 1000 2000 3000 F i g u r e 10. R e l a t i o n s h i p between foaming c a p a c i t y and. h y d r o p h o b i c i t y ( S g ) +measured i n the presence of 1M ..phenethylbiguanidine h y d r o c h l o r i d e . 1) r i b o n u c l e a s e .2) ovomucoid 3) t r y p s i n 4-) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K - c a s e i n 1.0) 8-lactoglobulin 11) 6-casein *Each data point i s the average of t r i p l i c a t e d e terminations +Each data point i s the average of d u p l i c a t e determinations 72 No r e l a t i o n s h i p was found between the foaming p r o p e r t i e s of p r o t e i n s and t h e i r h y d r o p h o b i c i t i e s determined a f t e r b r i e f exposure of the p r o t e i n s to pH 10.5 .(P>:0. 05) (Figure 11).,. U n l i k e the other p r o t e i n s which had low h y d r o p h o b i c i t i e s BSA and ovomucoid had very high h y d r o p h o b i c i t i e s under t h e s e . c o n d i t i o n s . At a l k a l i n e pH, the net charge on the molecules i n c r e a s e s l e a d i n g to d e n a t u r a t i o n of .the p r o t e i n s through e l e c t r o -s t a t i c r e p u l s i o n . (Has.chemeyer and .Haschemeyer, 1 973). It was necessary to keep the pH of the p r o t e i n s o l u t i o n s below 11.0 because at very, high pH s e v e r a l r e a c t i o n s p a r t i c i p a t e i n the d e n a t u r a t i o n process; r e a c t i o n s which occur i n a l k a l i n e protein, s o l u t i o n s i n c l u d e d i s u l p h i d e -s u l p h y d r y l interchange,,.. h y d r o l y s i s of peptide and amide bonds, for m a t i o n of dehydroamino acids, and r a c e m i z a t i o n (Tanford, 196.8; Whitaker.,. .1980).. In s p i t e of t h i s precaution,, the. r e s u l t s of h y d r o p h o b i c i t y measurement suggested that bovine serum, albumin and ovomucoid experience extensive d i s o r g a n i z a t i o n , , probably due to r e a c t i o n s of t h e i r d i s u l p h i d e bonds. Both p r o t e i n s c o n t a i n very large, numbers of d i s u l p h i d e bonds (Table 1). Pepsin, ovalbumin, and conalbumin had low hydropho-b i c i t i e s r e l a t i v e to the values of the. other p r o t e i n s although they have r e l a t i v e l y high Bigelow. h y d r o p h o b i c i t i e s . 73 200r 150 ft •9 O 3 1001 o z < o 501 • 1 1 8 o l -1000 2000 So 3000 F i g u r e 11. R e l a t i o n s h i p between.foaming c a p a c i t y and h y d r o p h o b i c i t y ( S g ) +measured a f t e r a l k a l i treatment. 1) ribonuclea.se 2) . ovomucoid 3) t r y p s i n U) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K - c a s e i n . 1 0 ) , 3 - l a c t o g l o b u l i n 11) 8-casein """Each data p o i n t i s the average of t r i p l i c a t e d e terminations +Each data p o i n t i s the average of d u p l i c a t e determinations 74 Since the pH of the samples was lowered f o r f l u o r e s c e n c e measurements, i t seemed t h a t d e n a t u r a t i o n at pH 10.5 was r e a d i l y r e v e r s i b l e f o r these p r o t e i n s . As shown i n F i g u r e 12., there was no r e l a t i o n -ship between the foaming c a p a c i t y . o f p r o t e i n s and t h e i r h y d r o p h o b i c i t i e s determined a f t e r h eating f o r 5 min at 100°C (P>0.0-5). Compared with, the values obtained f o r the other p r o t e i n s the h y d r o p h o b i c i t i e s of pepsin, conalbumin, K - and 8-caseins were very low.. The low value f o r conalbumin was probably the r e s u l t of i t s tendency to coagulate even, on s l i g h t , heating.. Thermal c o a g u l a t i o n r e s u l t s from 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 probably through noncovalent i n t e r a c t i o n s and d i s u l p h i d e interchange (Haschemeyer and Haschemeyer.,, 1 973)-. Therefore, b i n d i n g of c i s - p a r i n a r i c a c i d to hydrophobic sites;on conalbumin must have been prevented b.y. the . involvement of those s i t e s i n 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 s , . A s i m i l a r explana-t i o n was a p p l i e d to the low h y d r o p h o b i c i t y v a l u e s of the c a s e i n s , which are known f o r t h e i r tendency to s e l f -a s s o c i a t e (Evans.and P h i l l i p s . , 1 979; Swaisgood, 1 973). In the ease of g-casein 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 i s enhanced by e l e v a t e d temperatures.. I t seems t h a t pepsin was q u i t e s t a b l e during short p e r i o d s of h e a t i n g at pH 7.0. 75 200 r 150 O <100 CD 5 < O 50 •11 6 %9 J O >8 o E ^ l 500 1000 1500 2000 F i g u r e 12. R e l a t i o n s h i p between foaming c a p a c i t y " and h y d r o p h o b i c i t y ( S g ) +measured a f t e r o heating at 100 C f o r 5 minutes. 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 1+) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-cas e i n 10). 6 - l a c t o g l o b u l i n 11) B-casein ";;"Each data p o i n t i s the average of t r i p l i c a t e d eterminations +Each data p o i n t i s the average of d u p l i c a t e determinations 76 Heating p r o t e i n s f o r prolonged p e r i o d s i n the presence of mercapt o.ethanol and high c o n c e n t r a t i o n s of SDS r e s u l t s i n the r e d u c t i o n , of dis.ulphide bonds and d i s s o c i a t i o n of p r o t e i n s i n t o t h e i r b a s i c subunits without d i s r u p t i n g peptide bonds (Deutsch, 1976). T r e a t i n g the samples i n t h i s way before measuring h y d r o p h o b i c i t y r e s u l t e d :in high hydro.phobicity values f o r a l l the samples.. There was nc' s i g n i f i c a n t r e l a -t i o n s h i p ' between h y d r o p h o b i c i t y determined a f t e r h e a t i n g the p r o t e i n s f o r 20 minutes at 100°C i n the presence of 0.3% mercaptoethanol and 1..5$ SDS and the foaming p r o p e r t i e s of the p r o t e i n s '(p>0. 05j _ (-Figure 13). A milder treatment,, which i n v o l v e d heating the samples f o r 1.0. min at 1.0Q°C i n the presence of 1.5$ SDS, gave encouraging results.. A s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n (r = 0.8 2.0.,;. P<0.01 ) was found between the average h y d r o p h o b i c i t y v a l u e s of Bigelow and hydropho-b i c i t i e s ' measured i n t h i s way (Figure 1 4-)- /^Consequently , there was a s i g n i f i c a n t . p o s i t i v e r e l a t i o n s h i p between foaming c a p a c i t y and the measured h y d r o p h o b i c i t y value (Figure 15). The r e g r e s s i o n equation was In (FC + 30) = 0.00U S g + 3.52 where FC = foaming c a p a c i t y S = measured h y d r o p h o b i c i t y 77 200 r 150 1 0 6 • 9 O 2 giool o J1 .8 < o u. 50 450 900 1350 1800 F i g u r e 13. R e l a t i o n s h i p between foaming c a p a c i t y " and h y d r o p h o b i c i t y (S g) +measured a f t e r exposure to 100°C f o r 20 min i n .the presence of mercaptoethanol and SDS 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4) lysozyme 5). pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K - c a s e i n 10) 6-lact.oglobulin 11) 6-casein •"""Each data p o i n t i s the average of t r i p l i c a t e d e terminations +Each data p o i n t i s the average of d u p l i c a t e determinations 78 ure 14-. R e l a t i o n s h i p between h y d r o p h o b i c i t y (S )';; measured a f t e r exposure to 100°C f o r 10 min i n the presence of 1..5% SDS and Bigelow h y d r o p h o b i c i t y (H0 a v g) 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 5) pepsin 6) conalbumin . 7) ovalbumin 8) bovine serum albumin 9) K - c a s e i n 10) 6 - l a c t o g l o b u l i n 11) 8-casein *Each data p o i n t i s the average of d u p l i c a t e determinations 79 200 r F i g u r e 15. R e l a t i o n s h i p between foaming c a p a c i t y and h y d r o p h o b i c i t y (S e)+measured a f t e r exposure to 100°C f o r 10 min i n the presence of 1.5% SDS. 1) r i b o n u c l e a s e 2). ovomucoid 3) t r y p s i n 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum.albumin 9) K - c a s e i n 10) 6 - l a c t o g l o b u l i n 11) B-casein ";fEach data p o i n t i s the average of t r i p l i c a t e d e terminations +Each data p o i n t Is the average of d u p l i c a t e determinations 80 The r value, was- O.82O (P<0....01 ) . As p r e v i o u s l y mentioned, lysozyme behaved.in an. unusual manner i n the presence of high c o n c e n t r a t i o n s of SDS. I t was t h e r e f o r e omitted from t h i s a n a l y s i s . Attempts to . a c h i e v e a. good p o s i t i v e c o r r e l a t i o n between foaming c a p a c i t y and h y d r o p h o b i c i t y , measured i n the'presence of SDS, with the i n c l u s i o n of lysozyme were u n s u c c e s s f u l . Simplex methods were used to optimize the c o r r e l a t i o n between foaming capacity, and h y d r o p h o b i c i t y . The samples were exposed to v a r i a t i o n s , i n he a t i n g temperature, SDS.concentration and exposure to enzymatic h y d r o l y s i s . . As. i n d i c a t e d i n . Table 6, the best c o r r e l a -t i o n was n o n - s i g n i f i c a n t and.was obtained at room temperature i n the absence of both SDS and enzyme treatment. Lysozyme i s the only one of the p r o t e i n s which has a high p o s i t i v e , charge at. p.H.7.0.. Therefore the p o s s i b i l i t y t h a t i t s behaviour was. due. to bi n d i n g of exce s s i v e q u a n t i t i e s of SDS anions through i o n i c i n t e r -a c t i o n , was investigated.. Information i n the l i t e r a t u r e as w e l l as measurements of the amount of SDS bound by the p r o t e i n s when they were heated f o r 10 min i n the presence of 1 .5% SDS r e f u t e d t h i s i d e a (.Table 7) (Reynolds and Tanford, 1 970). However..., S t e i n h a r d t .et a l . (1 977) 81 Table 6. R e s u l t s of simplex search Vertex Simplex V e r t i c e s Factor L e v e l * Response # # Retained ~A B C~~ 1 1 25 0.00 0.00 0.56 2 1 95 0.35 0.04- -0.60 3 1 42 1 .40 0.04 -0.33 A 1 42 0.35 0.18 -0.38 5 2 1 ,3,4- 25 0.82 0.10 -0.55 6 3 1,3,4- 36 . 0.58 0.07 -0.65 7 4- 1 ,3,4 4-8 0.35 0.04 -0.98 1 1 25 1 .00 0.00 -0.57 2 1 96 1.23 0.04- -0.3x10 3 . 1 A3 1 .94 0.04 -0.40 A 1 A3 1 .24- 0.1.8 -1 .77 -5 """Factor A = tem perature 25 - 1 00°C B = SDS l e v e l 0 - 2.0$ C = Alkalase l e v e l 0...0 - 0.2 ml/5 nil sample ^Response = r value i f slope p o s i t i v e r value x slope i f slope negative 82 Table 7. Quantity of SDS bound during heating f o r 10 min at 100°C i n 1.5% SDS P r o t e i n mg SDS/mg p r o t e i n K - c a s e i n 0.33 B - c a s e i n 0.83 lysozyme 0.68 b o v i n e serum albumin 0.4-7 ovalbumin 0.59 ovomucoid 0.90 B - l a c t o g l o b u l i n 0.93 r i b o n u c l e a s e 0.67 conalbumin 0.52 p e p s i n 0.82 t r y p s i n 1.76 ""Each data p o i n t Is the average of d u p l i c a t e determinations 83 demonstrated t h a t u n l i k e twelve other p r o t e i n s , i n c l u d i n g BSA, conalbumin, ovalbumin and 8 - l a c t o g l o b u l i n , the lysozyme-SDS complex i s extremely e f f e c t i v e i n s o l u b i l i z i n g dyes. It. seems l i k e l y that the b i n d i n g s i t e on the lysozyme-SDS complex i s a l s o very e f f e c t i v e f o r b i n d i n g c i s - p a r i n a r i c a c i d . I t was i n t e r e s t i n g t h a t . h y d r o p h o b i c i t y of lysozyme has a l s o be.en d i f f i c u l t t o measure by hydro-phobic chromatography-. Although the h y d r o p h o b i c i t y of lysozyme, as indic.at.ed. by i t s . amino a c i d composition and Bigelow hydrophobicity.,. .Is q u i t e low, hydrophobic chromatography methods i n d i c a t e d t h a t . i t . has a high h y d r o p h o b i c i t y (Keshavarz, 1977). Use of t h e . f l u o r e s c e n c e probe method on samples which, had. been, heated, f o r 10 min at 100°C In the presence of 1..,$$ SDS was adopted as being represen-t a t i v e of the average h y d r o p h o b i c i t y of p r o t e i n s . This method was s e l e c t e d because. (1..). the s i g n i f i c a n t c o r r e -l a t i o n between values obtained, by t h i s method and the average h y d r o p h o b i c i t y v a l u e s of Bigelow i n d i c a t e d t h a t t h i s procedure gave a.good, estimate of the t o t a l hydro-p h o b i c i t y of p r o t e i n s and (.2). among the v a r i e t y of types of p r o t e i n s examined only lysozyme showed i r r e g u l a r h y d r o p h o b i c i t y v a l u e s . 84-Using the modified, fluorescence, probe method, the h y d r o p h o b i c i t i e s . o f e i g h t . f o o d p r o t e i n s were determined. These. .proteins, were soy., r>ea, canola and sunflower, protein, i s o l a t e s . , p r o - p u l s e , Promine-D, whole c a s e i n . and . a c i d .. s o l u b i l l z e d g l u t e n . The r e l a -t i o n s h i p between, foaming, c a p a c i t y and. h y d r o p h o b i c i t y was then .examined, again . using both the. model and food proteins.. In order, to. i n v e s t i g a t e , t h i s r e l a t i o n s h i p with the i n c l u s i o n , of the"-food . p r o t e i n s , i t was necessary to take the d i s p e r . s i b i l i t y . of the s e .samples i n t o account, since most of .them were not. completely s o l u b l e i n phosphate- buffer, at pH 7 . 0 . Backwards. stepwise, r.egres.sion a n a l y s i s was used to evaluate, .the, f i t . of a. .variety of f u n c t i o n s to 2 the data..., The optimum. R . values., a s s o c i a t e d with q u a d r a t i c , cubic and quartio.. functions, were . 0 .54-9 , 0.539 and 0.508 respectively... An R 2 value of 0...772 (P.< 0.01) was ass o c i a t e d , with. the. e x p r e s s i o n which best, f i t the data points... The r e g r e s s i o n equation was FC<. = -26.0...4--In . S -.0.3012 S . + 158.6 In d e e - 2.724- d - 1 820 where FC = foaming c a p a c i t y S g = h y d r o p h o b i c i t y d = d i s p e r s i b i l i t y {%) 85 As indicated, i n Table 8, both .hydrophobicity and d i s p e r s i b i l i t y s i g n i f i c a n t l y i n f l u e n c e d foaming c a p a c i t y . The normalized c o e f f i c i e n t s i n d i c a t e d t hat h y d r o p h o b i c i t y was s l i g h t l y more important than d i s p e r s i b i l i t y f o r determining foaming c a p a c i t y . Although both, parameters played, an. important r o l e i n determining foaming, c a p a c i t y , approximately 23$ of the v a r i a t i o n i n foaming c a p a c i t y was s t i l l unexplained. P r o t e i n charge d e n s i t y .and molecular . f l e x i b i l i t y c o r r e c -t i o n s may improve.the c o r r e l a t i o n . F i g u r e s 16 and ..1,7. show,..the computer generated contour diagram and three dimensional p l o t of the r e l a t i o n s h i p between the...foaming, c a p a c i t y of p r o t e i n s and t h e i r h y d r o p h o b i c i t y a n d . d i s p e r s i b i l i t y . As shown i n these f i g u r e s , optimum, foaming .capacity was a s s o c i a t e d with d i s p e r s . i b . i l i t i e s above i.0.% and h y d r o p h o b i c i t y values above 700... This, suggested, that, h y d r o p h o b i c i t i e s of 700 or more are a s s o c i a t e d . w i t h . t h e good balance of h y d r o p h i l i c and hydrophobic, groups .necessary f o r e f f e c t i v e s t a b i l i z a t i o n of a i r bubbles.. Regardless of t h e i r degree of. d.i.spersi.bility, p r o t e i n s with low h y d r o p h o b i c i t y showed poor foaming c a p a c i t y . Good foaming capacity, was e x h i b i t e d by p r o t e i n s with poor d i s p e r s i b i l i t y ..(.approximately 20$) and hydro-86 Table 8. M u l t i p l e r e g r e s s i o n a n a l y s i s of foaming c a p a c i t y on h y d r o p h o b i c i t y and s o l u b i l i t y Std. F- F- Norm. V a r i a b l e E r r o r Ratio Prob.. Coeff. s e 0.1368 4.850 0.0463 1 .745 d 0..8703 9.797 0..0080 1 .605 In .S e 83.49 9.727 0.0081 2.444 In d 41 .60 14.53 0.0022 1 .916 d . f . = 17 R 2 = 0.772 F i g u r e 16. Contour diagram of the r e l a t i o n s h i p between foaming c a p a c i t y , h y d r o p h o b i c i t y and d i s p e r s i b i l i t y . F i g u r e 17. Three dimensional p l o t of the r e l a t i o n s h i p between foaming c a p a c i t y , h y d r o p h o b i c i t y and d i s p e r s i b i l i t y . 89 p h o b i c i t y values abov,e 500... This was probably due to s t a b i l i z a t i o n of.foams.by p a r t i c l e s of p r o t e i n present i n the medium. It i s w e l l known that some p a r t i c l e s can s t a b i l i z e . foams (Bikerman, 1.973). I t seems that p a r t i c l e s l y i n g . , i n the a i r - w a t e r .interface serve as a p h y s i c a l b a r r i e r to., bubble coa l e s c e n c e . This phenomenon has been.associated with p a r t i c l e s t h a t have medium .wettability.. P a r t i c l e s that are p e r f e c t l y wetted by water, are mixed i n with i t by the a i r c u r r e n t and never reach the . interface... . P a r t i c l e s which are p o o r ly wetted, are t r a n s p o r t e d to the i n t e r f a c e , where they are almost completely surrounded by the a i r phase and h a v e . l i t t l e e f f e c t on the r a t e of bubble coa l e s c e n c e . Regardless of t h e i r w e t t a b i l i t y , b i g p a r t i c l e s are poor foam s t a b i l i z e r s since t h e i r weight prevents them from r i s i n g to the a i r - w a t e r i n t e r f a c e . . Small p a r t i c l e s are a l s o poor foam s t a b i l i z e r s , , whatever t h e i r w e t t a b i l i t y . I t i s l i k e l y t h a t these parti.cle.s are so. t h i n t h a t minor d i s t o r t i o n of bubbles around them can b r i n g the bubbles i n t o contact making coalescence p o s s i b l e . 5. The Importance of Charge Density f o r Foaming In order to adsorb at a n . i n t e r f a c e c o n t a i n i n g charged p a r t i c l e s , an incoming molecule must do work 90 a g a i n s t the e l e c t r i c a l p o t e n t i a l set up at the i n t e r f a c e by p a r t i c l e s . i n the i n t e r f a c e (MacRitchie, 1978). T h e r e f o r e . i t . i s expected, t h a t . t h e r e should be some r e l a t i o n s h i p between, a p r o t e i n ' s. charge, d e n s i t y a n d i i t s foaming p r o p e r t i e s . Net proton.charge.measured.by t i t r a t i o n i n 6M guanidine hydrochloride, showed, good agreement with the c a l c u l a t e d . v a l u e s in. mo.s.t cases (Table 9). Two exceptions . were pepsin and t r y p s i n . . The. measured charge f o r t r y p s i n ' was -11 while the c a l c u l a t e d charge was + 7, probably, because of the presence .of i m p u r i t i e s i n the samples... In the case of. pepsin the measured charge was -27 while the. c a l c u l a t e d charge was -32. This d i s c r e p a n c y r e s u l t e d , from... the use. of the l i t e r a t u r e v a l u e , or pH 1.0, f o r the, i s o e l e c t r i c . p o i n t of pepsin i n the c a l c u l a t i o n , o f net .proton charge (Fruton, 1972). The pH of the de.ioni.zed_ pepsin s o l u t i o n , or pH 3.0, was used., as.the. i s o i o n i c . p o i n t ..of pepsin i n the measurment of net proton charge.. I t is. i n t e r e s t i n g t o . note that while the .measured i s o e l e c t r i c point .of, pepsin i s pH 1.0, the i s o i o n i c p o i n t c a l c u l a t e d from the amino a c i d composition of .pepsin, assuming a pK of 4. 0 f o r the a s i d e chain., car.boxyl groups i s pH 3.0 (Fruton, 1 972). It has been suggested that, t h i s l a r g e diff.er.ence may be due 91 Table 9. Net proton charge on the model p r o t e i n s at pH 7.0 P r o t e i n C a l c u l a t e d Measured * + Value Value r i b o n u c l e a s e + 3 + 3 ovomuc o i d -9 -8 t r y p s i n + 7 -11 lysozyme + 7 + 7 p e p s i n -32 -27 conalbumin -7 -5 ovalbumin -13 -1 2 bovine serum albumin -1 8 -16 K - c a s e i n -3 -1 8 - l a c t o g l o b u l i n -9 -8 8 - c a s e i n -4" -3 ^calcu l a t e d , as d e s c r i b e d by Nozaki and Tanford (1 967) using the pKa's of t i t r a b l e groups given i n Table I I I of the same paper and the amino a c i d composition of the p r o t e i n s l i s t e d i n Table 3 (Appendix II) teach data p o i n t i s the average of d u p l i c a t e determinations 92 to anions adsorbed on the p r o t e i n during i s o e l e c t r i c point measurement, or to the presence of abnormally a c i d i c c a r b o x y l groups. No c o r r e l a t i o n was found between the foaming c a p a c i t i e s of the model proteins, and t h e i r charge d e n s i t y (Figure -18) (?>0.0:> ).. This suggested that p r o t e i n charge d e n s i t y i s of minor importance f o r determining the foaming c a p a c i t i e s of p r o t e i n s . In order to ..determine whether p r o t e i n charge d e n s i t y has any i n f l u e n c e on the foaming c a p a c i t y of p r o t e i n s , the foaming c a p a c i t i e s of the model p r o t e i n s were measured under v a r y i n g c o n d i t i o n s of pH and i o n i c s t r e n g t h (Tables 10, 1.2.) . The l i t e r a t u r e i n d i c a t e s that an i o n i c s t r e n g t h of 0..»2 i s s u f f i c i e n t to minimize 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 betwee.n charged groups on p r o t e i n s (Haschemeyer and Haschemeyer, 1973). Therefore the maximum i o n i c s t r e n g t h ..used was 0.-2. A n a l y s i s of v a r i a n c e i n d i c a t e d that over, the range examined, i o n i c s t r e n g t h had no. s i g n i f i c a n t e f f e c t on. foaming c a p a c i t y (P>0.05) (Table 11). In order to av.oid e x t e n s i v e a c i d and a l k a l i d e n a t u r a t i o n of the p r o t e i n s which are.known to enhance foaming a b i l i t y , the .pH of the solutions.was kept between pH 5 and pH 9. A n a l y s i s of v a r i a n c e i n d i c a t e d that pH had a s i g n i f i c a n t e f f e c t (P<0....01 ) on foaming c a p a c i t y 93 200r 150 >-O 2 O 100 o z _> < o 50 .9 J1 10 .8 2 CHARGE -2 DENSITY x10 units, res'. ?ure 18. R e l a t i o n s h i p between foaming c a p a c i t y and charge d e n s i t y * 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin .8) bovine serum albumin 9) K - c a s e i n 10). 8 - l a c t o g l o b u l i n 11) 8-casein ~;;~Each data p o i n t i s the average of t r i p l i c a t e d e terminations +Each data poi n t i s the average of d u p l i c a t e determinations 94 Table 10. E f f e c t of i o n i c s t r e n g t h on foaming c a p a c i t y I o n i c Strength P r o t e i n 0. 01 0. ,05 0. 20 r i b o n u c l e a s e 2 _+ 1 2 + 1 3 + 1 ovomucoid 12 +_ 1 12 + 1 13 + 1 lysozyme 2 + 1 2 + 1 3 + 1 pepsin 144 + 3 147 3 149 3 conalbumin 1 4-1 3 142 + 2 1 41 +_ 2 ovalbumin 48 + 2 1 00 + 2 1 22 + 2 bovine serum albumin 95 +_ 4 1 03 + 4 98 2 K - c a s e i n 134 +_ 2 1 32 + 2 135 + 2 8 - l a c t o g l o b u l i n 1 38 _+ 2 135 + 3 134 + 4 8 - c a s e i n 1 04 +_ 1 1 09 +_ 2 1 08 + 2 Each data p o i n t i s the average of t r i p l i c a t e d eterminations 95 Table 11. ANOVA f o r the e f f e c t of i o n i c s t r e n g t h on foaming c a p a c i t y Source of df SS MS V a r i a t i o n p r o t e i n 9 94-994 1 0554 83.1 0^  " s t r e n g t h 2 3 9 8 1 ^ e r r o r 1 8 2281 127 t o t a l 29 97613 ' F2,18,0.005 7 , 2 1 96 T a b l e 12. E f f e c t o f pH on f o a m i n g c a p a c i t y PH P r o t e i n r i b o n u c l e a s e 2 + 1 2 1 6 1 o v o m u c o i d 97 + 1 1 2 + 1 11 + 1 l y s o z y m e 3 + 1 2 1 8 + 1 p e p s i n 1 51 + 2 147 + 3 34 + 1 c o n a l b u m i n 1 46 + 2 143 + 2 162 3 o v a l b u m i n 1 40 3 1 00 + 2 42 + 2 b o v i n e serum a l b u m i n 97 1 1 01 4 95 + 2 K - c a s e i n 0 + 0 134 + 4 144 4 3 - l a c t o g l o b u l i n 1 27 2 135 + 3 1 38 + 2 6 - c a s e i n 0 + 0 • 1 09 + 2 1 07 +_ 1 E a c h d a t a p o i n t i s t h e a v e r a g e o f t r i p l i c a t e d e t e r m i n a t i o n s 97 Table 1 3 . ANOVA f o r the e f f e c t of pH on foaming c a p a c i t y Source of V a r i a t i o n df SS MS p r o t e i n pH e r r o r t o t a l 9 2 16 27 891 08 3487 4-746 97341 9900 1743 296 33.37** 11 .75** ** F2,16,0.005 7 , 5 1 98 (Table 13). The p r o t e i n s showed a tendency toward decreased foaming c a p a c i t y as t h e i r net charge i n c r e a s e d . For r i b o n u c l e a s e and lysozyme, which have i s o e l e c t r i c p o i n t s of p.H 9.0 and 11.0 r e s p e c t i v e l y the charge d e n s i t y decreased with i n c r e a s i n g pH. For the other p r o t e i n s , with i s o e l e c t r i c p o i n t s i n the pH range 4- to 5, the charge d e n s i t y Increased with i n c r e a s i n g pH. These r e s u l t s suggested that although the charge d e n s i t y of p r o t e i n s a f f e c t e d t h e i r foaming a b i l i t y , i t s i n f l u e n c e was not very great. For t h i s reason m i n i m i z a t i o n of 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 with an I o n i c s t r e n g t h of 0,. 2 had no. s i g n i f i c a n t e f f e c t on the foaming c a p a c i t i e s of the proteins.. However, at the high charge d e n s i t i e s brought about by high pH, foaming c a p a c i t y was a f f e c t e d . Pepsin provided a s t r i k i n g example of t h i s s i n c e i t foamed w e l l even at pH 7.0 at which i t s net. charge i s -27., During the foaming process, the time of contact between the a i r bubbles and the. p r o t e i n s o l u t i o n was l e s s than one second. This contact time may have been too short f o r a d s o r p t i o n t o have progressed beyond the i n i t i a l d i f f u s i o n c o n t r o l l e d , stage to the second stage. In t h i s second stage a s i g n i f i c a n t energy b a r r i e r to a d s o r p t i o n develops as a r e s u l t of the charges on 99 molecules i n the interfaces.. The e f f e c t i v e charge f o r a d s o r p t i o n may a l s o have been c o n s i d e r a b l y l e s s than the net charge on the molecule,. According'to the g e n e r a l l y accepted theory of adsorption., only a small segment of a molecule needs to adhere to the i n t e r f a c e before a d s o r p t i o n occurs spontaneously (MacRitchie, 1978). I f t h i s i s so, i t may be that the e f f e c t i v e charge f o r a d s o r p t i o n i s the charge on the part of the molecule near the i n t e r f a c e during the a c t i v a t i o n step. E i t h e r of these p o s s i b i l i t i e s may have l e d to the l a c k of c o r r e l a t i o n between the foaming c a p a c i t y of p r o t e i n s and t h e i r charge d e n s i t y and the i n s e n s i t i v i t y of the foaming c a p a c i t y of p r o t e i n s to charges i n the i o n i c s t r e n g t h of the s o l v e n t . There was a s i g n i f i c a n t r e l a t i o n s h i p (r = 0.725/, P<0.01) between the foam s t a b i l i t y of the model p r o t e i n s and t h e i r charge d e n s i t y . The r e g r e s s i o n equation was FS = 19.72 c _ 1 - - 2.1U _ -i where FS = foam s t a b i l i t y (min.ml - ) c = charge d e n s i t y ( u n i t s . r e s - ) As shown In F i g u r e 19, foam s t a b i l i t y i n c r e a s e d markedly at low charge d e n s i t y . This i n d i c a t e d that i n t e r m o l e c u l a r r e p u l s i o n between p r o t e i n molecules at the i n t e r f a c e had an important d e s t a b i l i z i n g i n f l u e n c e on foams. 1 00 F i g u r e 19. R e l a t i o n s h i p between foam s t a b i l i t y and charge density.* 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-casein. 10). 8 - l a c t o g l o b u l i n 11) 8-casein #Each data p o i n t i s the average of t r i p l i c a t e d e terminations +Each data p o i n t i s the average of d u p l i c a t e determinations 1 01 6. R e l a t i o n s h i p between S o l u t i o n . V i s c o s i t y and Foaming P r o p e r t i e s P r o p e r t i e s such as molecular f l e x i b i l i t y , s i z e , degree of h y d r a t i o n and the extent of 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 should i n f l u e n c e the foaming p r o p e r t i e s of p r o t e i n s . However., i t i s d i f f i c u l t to. o b t a i n a q u a n t i t a t i v e measure, of each of these parameters i n order to study i t s i n f l u e n c e on foaming.. The v i s c o s i t y of a p r o t e i n s o l u t i o n i s a f u n c t i o n of molecular s i z e , shape, f l e x i b i l i t y , degree of h y d r a t i o n and the extent of i n t e r m o l e c u l a r i n t e r a c t i o n s (Tanford, 1961; Yang, 1961). Studies r e l a t i n g p r o t e i n s t r u c t u r e to v i s c o s i t y have concentrated on .. i n t r i n s i c v i s c o s i t y r a t h e r than bulk s o l u t i o n viscosity... However., i n t h i s work, bulk v i s c o s i t y was used as an index of molecular s t r u c t u r e and a s s o c i a t i o n . The reason f o r t h i s was t h a t food p r o t e i n s are made up of a v a r i e t y of p r o t e i n and non-p r o t e i n components.,, which c o n t r i b u t e to the v i s c o s i t i e s of t h e i r s o l u t i o n s and make i t impo s s i b l e t o i n t e r p r e t i n t r i n s i c v i s c o s i t y measurements i n terms of p r o t e i n s t r u c t u r e . Even f o r pure p r o t e i n s i i n t e r p r e t a t i o n s of i n t r i n s i c v i s c o s i t y data i n terms of molecular s t r u c t u r e must be accepted with caution., s i n c e i t i s assumed t h a t the molecules do not e x h i b i t c o n c e n t r a t i o n dependent s e l f - a s s o c i a t i o n and that the p a r t i a l s p e c i f i c volume i s a t r u e measure of the volume of the p r o t e i n i n s o l u t i o n (Yang, 1961). 1 02 There was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n (r = 0.'84.9> P<0.01) between the foaming c a p a c i t y of the p r o t e i n s and t h e i r v i s c o s i t i e s (Figure 20). The r e g r e s s i o n equation was FC = 2388 n - 2622 where FC = foaming c a p a c i t y n •= v i s c o s i t y (Pa.s) Three of the food p r o t e i n s , namely sunflower i s o l a t e , canola i s o l a t e and pro-pulse had low v i s c o s i t i e s of approximately 1.090 Pa.s but high foaming c a p a c i t i e s . The reason f o r t h i s may have been that these samples contained i n s o l u b l e , p a r t i c l e s , , which had n e g l i g i b l e e f f e c t s on bulk v i s c o s i t y but which were capable of s t a b i l i z i n g foams. In an attempt to i n c r e a s e the c o e f f i c i e n t of d e t e r m i n a t i o n of the r e g r e s s i o n equation f o r foaming c a p a c i t y , r e g r e s s i o n a n a l y s i s was repeated using hydro-p h o b i c i t y , d i s p e r s i b i l i t y and v i s c o s i t y as the independent v a r i a b l e s . Ridge r e g r e s s i o n a n a l y s i s was used because there was a high degree of c o l l i n e a r i t y . between v i s c o s i t y and both h y d r o p h o b i c i t y and d i s p e r s i b i l i t y , as i n d i c a t e d by the c o r r e l a t i o n c o e f f i c i e n t s of 0...612 and 0.565 re s p e c -t i v e l y , which were s i g n i f i c a n t at the 0.05 l e v e l (Newell and Lee, 1981).. In the presence of the v i s c o s i t y parameter, the c o n t r i b u t i o n of d i s p e r s i b i l i t y to the r e g r e s s i o n equa-t i o n became n o n - s i g n i f i c a n t t h e r e f o r e , d i s p e r s i b i l i t y was d e l e t e d . Table 14- shows the r e s u l t s of r i d g e r e g r e s s i o n 1 03 F i g u r e 20. R e l a t i o n s h i p between foaming c a p a c i t y and bulk v i s c o s i t y + . 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-casein 10) 8 - l a c t o g l o b u l i n 11) (3-casein 12) whole c a s e i n 13) g l u t e n 14) pea i s o l a t e 15) soy i s o l a t e 16) Promine-D """Each data p o i n t i s the average of t r i p l i c a t e d e t erminations Each data p o i n t i s the average of d u p l i c a t e determinations Table 14. M u l t i p l e r e g r e s s i o n a n a l y s i s of foaming c a p a c i t y on h y d r o p h o b i c i t y and v i s c o s i t y V a r i a b l e Std. E r r o r t -value 3 -Coeff. n In' S 33.91 1 0.70 5.300^ 2.311^ 0.5696 0.2484 d.f. = 14 R 2 = 0.779 " n 0 . 0 5 ( 2 ) 1 2 = 2 - 1 7 9 1 05 a n a l y s i s f o r the optimal k value of 0.3. The r e g r e s s i o n equation which best d e s c r i b e d the r e l a t i o n s h i p between h y d r o p h o b i c i t y , v i s c o s i t y and .foaming c a p a c i t y was FC = 14-93 n + 25 .93 In S g - 1775 where FC = foaming c a p a c i t y n = bulk v i s c o s i t y (Pa.s) S g = h y d r o p h o b i c i t y This equation was s i g n i f i c a n t ..at the. 0.01 l e v e l with an R 2 of 0.779. F i g u r e s 21 and 22 show the computer generated three dimensional p l o t of, foaming, c a p a c i t y a g a i n s t hydro-p h o b i c i t y and v i s c o s i t y and the corresponding contour diagram. In g e n e r a l foaming c a p a c i t y i n c r e a s e d g r a d u a l l y with h y d r o p h o b i c i t y and v i s c o s i t y . The small r e g i o n of high foaming c a p a c i t y at low v i s c o s i t y and high hydropho-b i c i t y was probably produced by the food, p r o t e i n s which contained i n s o l u b l e ..particles capable of s t a b i l i z i n g the foam. Information i n the l i t e r a t u r e i n d i c a t e s that s o l u t i o n v i s c o s i t y i s r e l a t e d to foam s t a b i l i t y , determined by drainage methods (Bikerman., 1 973). A s i g n i f i c a n t r e l a -t i o n s h i p was a l s o found between, bulk v i s c o s i t y and foam s t a b i l i t y determined, by monitoring the r a t e of decrease of foam volume.(Figure 23).. The r e l a t i o n s h i p (r = 0.855, P<0.01) was In (FS + 0.03) = T30 n - 148 F i g u r e 21. Three dimensional p l o t of foaming c a p a c i t y a g a i n s t h y d r o p h o b i c i t y and bulk v i s c o s i t y ,1 07 gure 22. Contour diagram of the r e l a t i o n s h i p between foaming capacity, h y d r o p h o b i c i t y and d i s p e r s i b i l i t y 1 08 .9 VISCOSITY pascal, s x 10 R e l a t i o n s h i p between foam s t a b i l i t y and bulk v i s c o s i t y . + 1) r i b o n u c l e a s e 2) ovomucoid 3) t r y p s i n 4) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-casein .1,0) B - l a c t o g l o b u l i n 11) B-casein Each data p o i n t i s the average of t r i p l i c a t e d eterminations +Each data p o i n t i s the average of d u p l i c a t e determinations 1 09 where FS = foam s t a b i l i t y (min ml ') n = v i s c o s i t y (Pa.s.) M u l t i p l e r e g r e s s i o n a n a l y s i s of foam s t a b i l i t y on hydrophobicity,.. charge d e n s i t y and v i s c o s i t y was c a r r i e d out. In the presence of the charge parameter the c o n t r i b u t i o n of h y d r o p h o b i c i t y and v i s c o s i t y became n o n s i g n i f i c a n t . T h e . r e s u l t i n g r e g r e s s i o n equation was FS = 19.72 c" 1 - 2.11 where FS = foam s t a b i l i t y c = charge d e n s i t y 7. The R e l a t i o n s h i p between Surface Tension and Foaming . P r o p e r t i e s In order f o r a l i q u i d t o foam i t must c o n t a i n a d i s s o l v e d substance.capable of lowering, the s u r f a c e t e n s i o n (Bikerman, 1973). . Since work must be done against the surface t e n s i o n to c r e a t e the bubbles of a foam there may be some r e l a t i o n s h i p between su r f a c e t e n s i o n and foaming a b i l i t y . However, no s i g n i f i c a n t r e l a t i o n s h i p was found between surface t e n s i o n and e i t h e r foaming c a p a c i t y or foam s t a b i l i t y , i n t h i s work (F i g u r e s 24, 2 5 ) . I t has been demonstrated that the surface t e n s i o n of p r o t e i n s o l u t i o n s decreases with time (Gosh and B u l l , 2963). I n i t i a l l y , the decrease i s r a p i d but i t slows a f t e r a. few minutes... As a r e s u l t of t h i s , the t e n s i o n of a newly formed s u r f a c e , such as a bubble at 110 200r 150 >-$ < 100| o o z 9 11 10 _8 < o 50I 3 1 40 50 2 60 -1 70 SURFACE TENSION dyne.cm gure 2 4. R e l a t i o n s h i p between foaming c a p a c i t y and surface t e n s i o n . + 1) ribonuclease. 2) ovomucoid 3) t r y p s i n 4-) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-case i n 10) 8 - l a c t o g l o b u l i n 11) 8-casein *Each data p o i n t i s the average of t r i p l i c a t e d eterminations +Each data p o i n t i s the average of d u p l i c a t e determinations n r 60r .9 E |45| > • 30| 03 I15" LL 10 • 3_ 7 1_-8 "•"2 J40" 50 60 A 70 SURFACE TENSION dyne.cm gure 25. R e l a t i o n s h i p between foam s t a b i l i t y and surface t e n s i o n . 1) r i b o n u c l e a s e .2) ovomucoid 3) t r y p s i n U) lysozyme 5) pepsin 6) conalbumin 7) ovalbumin 8) bovine serum albumin 9) K-"casein 10) 8-lactog.lobulin 11) B-casein """Each data p o i n t i s the average of t r i p l i c a t e d eterminations 112 the i n s t a n t of formation, i s higher than the t e n s i o n of a s i m i l a r surface which has been aged. I t was not p o s s i b l e to measure the surface t e n s i o n of the p r o t e i n s o l u t i o n s at the i n s t a n t of c r e a t i o n of the s u r f a c e , using the F i s h e r Surface Tensiomat.. Therefore the absence of a c o r r e l a t i o n between surface t e n s i o n and foaming p r o p e r t i e s may have been because the s u r f a c e t e n s i o n measured d i f f e r e d from that, of a f r e s h l y formed bubble. However, i t i s a l s o p o s s i b l e that no c o r r e l a -t i o n was found because i t i s the r a t e of lowering of surface t e n s i o n and not the. magnitude of the surface t e n s i o n which i s important f o r foaming (Graham and P h i l l i p s , 1976). 8. The Importance of Molecular S i z e f o r Foaming Six food p r o t e i n s were subjected to extensive h y d r o l y s i s , which i n c r e a s e d the l e v e l s of non-protein n i t r o g e n from below 5% to l e v e l s between 27% and i0% (Table 15). H y d r o l y s i s was accompanied by a decrease i n h y d r o p h o b i c i t y f o r some of the samples and an i n c r e a s e i n d i s p e r s i b i l i t y (Table 16)... The decreased h y d r o p h o b i c i t y a s s o c i a t e d with h y d r o l y s i s , probably r e s u l t e d from i n h i b i -t i o n of the b i n d i n g of c i s - p a r i n a r i c a c i d due to the higher charge d e n s i t y on the peptides formed compared to the p r o t e i n . 11 3 Table 15. E f f e c t of enzyme h y d r o l y s i s on the l e v e l of non-protein n i t r o g e n % non-protein n i t r o g e n Sample untreated hydrolyzed pea i s o l a t e <5 4-0 sunflower " 27 canola " 27 c a s e i n " 37 ;Promine-D " 38 soy i s o l a t e - " 34 Each data p o i n t i s the average of d u p l i c a t e determinations 11 4 Table 16. E f f e c t of enzyme h y d r o l y s i s on foaming c a p a c i t y (FC), h y d r o p h o b i c i t y (S ') and d i s p e r s i b i l i t y (d) untreated h y d r o l y z e d Sample % j. j . — 5 j. T-FC S d FC S d e e pea 61 + U 277 36 0 + 0 280 49 sunflower 150 + 3 597 31 2 + 1 582 63 canola 157 +_ 3 950 kk 51 +_ 4 500 70 c a s e i n 163 4 725 53 2 + 1 302 64 Promine-D 33 + 3 927 13 5 1 432 55 soy i s o l a t e 41 + 2 822 1 2 U + 2 .485 57 *Each -data p o i n t i s the average of t r i p l i c a t e determinations +Each data p o i n t i s the average of d u p l i c a t e determinations / 115 Since hydrophobic and charged groups of a p r o t e i n are d i s t r i b u t e d non-uniformly along the poly-peptide c h a i n , h y d r o l y s i s should produce a heterogeneous mixture of p e p t i d e s , v a r y i n g i n s i z e , average hydropho-b i c i t i y and charge density... The low foaming c a p a c i t i e s of the enzyme hydrolyzed samples i n d i c a t e s t hat these peptides cannot a s s o c i a t e e f f e c t i v e l y i n t o the coherent f i l m r e q u i r e d at the i n t e r f a c e to s t a b i l i z e a i r bubbles. 116 V. GENERAL DISCUSSION D i s u l p h i d e bonds and noncovalent f o r c e s , such as hydrogen bonds and van der Waals i n t e r a c t i o n s , act to maintain the p r o t e i n i n i t s n a t i v e conformation at the i n t e r f a c e . Since the energy of formation of these noncovalent bonds i s r e l a t i v e l y low,, they should be r e a d i l y d i s r u p t e d at the a i r - w a t e r i n t e r f a c e and should not l i m i t p r o t e i n . f l e x i b i l i t y and ease of u n f o l d i n g at the i n t e r f a c e as much as d i s u l p h i d e bonds. Therefore, the number of d i s u l p h i d e bonds per r e s i d u e was used as an index of molecular f l e x i b i l i t y . . A s i g n i f i c a n t c o r r e l a t i o n was found between p r o t e i n f l e x i b i l i t y and foaming c a p a c i t y , which i n d i c a t e d that the a b i l i t y to u n f o l d r e a d i l y at the i n t e r f a c e was important f o r good foaming c a p a c i t y . By using c i s - p a r i n a r i c a c i d as a probe of the hydrophobic regions., a f t e r the samples were u n c o i l e d by h e a t i n g i n the presence of SDS, i t was p o s s i b l e to measure the average h y d r o p h o b i c i t y of p r o t e i n s . Evidence f o r t h i s was provided by the s i g n i f i c a n t c o r r e l a t i o n between the average h y d r o p h o b i c i t y as c a l c u l a t e d by Bigelow and the measured h y d r o p h o b i c i t y v a l u e . Foaming c a p a c i t y was s i g n i f i c a n t l y c o r r e l a t e d 117 with average h y d r o p h o b i c i t y but not with s u r f a c e h y d r o p h o b i c i t y . This suggested that p r o t e i n s are e x t e n s i v e l y u n c o i l e d at the a i r - w a t e r i n t e r f a c e during foam formation.. However,, the foaming t e s t s were a l l c a r r i e d out at low p r o t e i n . c o n c e n t r a t i o n s . I t i s p o s s i b l e t h a t these low c o n c e n t r a t i o n s l e d to the formation of d i l u t e films.,, i n which the p r o t e i n s e x i s t i n f u l l y unfolded conformations.. Higher p r o t e i n concen-t r a t i o n s may give r i s e to more concentrated f i l m s , i n which the l a r g e number of p r o t e i n molecules per u n i t area of i n t e r f a c e r e s t r i c t s the p r o t e i n s to a compact c o n f i g u -r a t i o n . As a r e s u l t of t h i s there may be some r e l a t i o n s h i p between foaming c a p a c i t y at high p r o t e i n c o n c e n t r a t i o n s and s u r f a c e h y d r o p h o b i c i t y . A . . s i g n i f i c a n t r e l a t i o n s h i p was obtained between the foaming c a p a c i t y of p r o t e i n s and their, h y d r o p h o b i c i t y and d i s p e r s i b i l i t y . . . H y d r o p hobicity made a s l i g h t l y g r e a t e r c o n t r i b u t i o n than d i s p e r s i b i l i t y t.o the r e g r e s s i o n equation f o r foaming c a p a c i t y . The r e l a t i o n s h i p between h y d r o p h o b i c i t y and foaming c a p a c i t y was a t t r i b u t e d to the importance of h y d r o p h o b i c i t y f o r determining the a d s o r p t i o n behaviour of p r o t e i n s . The i n f l u e n c e of h y d r o p h o b i c i t y on s u r f a c e a d s o r p t i o n a r i s e s from the lowering of surface f r e e 118 energy which accompanies u n f o l d i n g of p r o t e i n s at the i n t e r f a c e to expose non-polar groups to the a i r (Mac-R i t c h i e , 1978). This f r e e energy change provides the d r i v i n g f o r c e f o r a d s o r p t i o n at the i n t e r f a c e . S o l u b i l i t y probably i n f l u e n c e d foaming capa-c i t y through i t s e f f e c t on the adsorbed f i l m . P r o t e i n s with poor s o l u b i l i t y had few. molecules d i s s o l v e d i n the aqueous phase a v a i l a b l e f o r a d s o r p t i o n at the I n t e r f a c e . With only a few molecules at. the interface., these p r o t e i n s were unable to provide a f i l m which completely covered the i n t e r f a c e and were unable to s t a b i l i z e the bubbles of a foam. Charge d e n s i t y i n f l u e n c e d the foaming c a p a c i t y pf p r o t e i n s but i t s r o l e i n determining t h i s property seemed to be a minor one.. This was i n d i c a t e d by the absence of a s i g n i f i c a n t c o r r e l a t i o n between charge d e n s i t y and foaming c a p a c i t y and by the need f.or l a r g e changes i n charge d e n s i t y i n order to s i g n i f i c a n t l y a l t e r the foaming capa-c i t i e s of the proteins.. However., a r o l e f o r charge d e n s i t y i n determining foaming c a p a c i t y cannot be discounted, since p r o t e i n s o l u b i l i t y , which was i n c l u d e d i n the r e g r e s s i o n equation f o r foaming capacity., i s dependent on charge d e n s i t y . There was a s i g n i f i c a n t r e l a t i o n s h i p between the foaming c a p a c i t y of p r o t e i n s and t h e i r h y d r o p h o b i c i t y and bulk v i s c o s i t y . V i s c o s i t y made a s l i g h t l y g r e a t e r c o n t r i b u t i o n than h y d r o p h o b i c i t y i n t h e : r e g r e s s i o n equation 119 d e s c r i b i n g t h i s r e l a t i o n s h i p . Although theory suggest that h y d r o p h o b i c i t y , v i s c o s i t y and d i s p e r s i b i l i t y should a l l be r e l a t e d to foaming c a p a c i t y , stepwise r e g r e s s i o n a n a l y s i s d i d not y i e l d a model f o r foaming c a p a c i t y , which i n c l u d e d s i g n i f i c a n t c o n t r i b u t i o n s from a l l three parameters ( K i n s e l l a , 1976). In s p i t e of the f a c t that stepwise i r e g r e s s i o n procedures are commonly used because- they o f f e r o b j e c t i v e methods f o r s e l e c t i n g one of the p o s s i b l e r e g r e s s i o n equations as the "best"., they may overlook an e x c e l l e n t model.(Hocking, 1976). U n f o r t u n a t e l y , there i s no u n i v e r s a l agreement among s t a t i s t i c i a n s as to the best method f o r . c a r r y i n g out r e g r e s s i o n a n a l y s i s (Zar, 1 974-) . Approximately 77$ of the v a r i a b i l i t y i n foaming c a p a c i t y was accounted f o r by two r e g r e s s i o n models; one i n v o l v i n g h y d r o p h o b i c i t y a n d . d i s p e r s i b i l i t y and the other i n v o l v i n g h y d r o p h o b i c i t y and v i s c o s i t y . . On the b a s i s of s t a t i s t i c s , i t was not p o s s i b l e to p i c k one of these as the best r e g r e s s i o n equation f o r foaming c a p a c i t y because they had approximately equal c o e f f i c i e n t s of de t e r m i n a t i o n and r e s i d u a l mean squares. In a d d i t i o n to h y d r o p h o b i c i t y , d i s p e r s i b i l i t y and v i s c o s i t y other f a c t o r s which may c o n t r i b u t e to the v a r i a t i o n i n foaming c a p a c i t y of the samples i n c l u d e 120 (1) the presence of p r o s t h e t i c groups on some of the p r o t e i n s (2) the charge d e n s i t y of the p r o t e i n s (3) the i n f l u e n c e of non-cov.alent. bonding on p r o t e i n s t r u c t u r e and a d s o r p t i o n at the i n t e r f a c e . There was a s i g n i f i c a n t negative r e l a t i o n s h i p between foam s t a b i l i t y , and .the.inverse of charge d e n s i t y . This i n d i c a t e d .that e l e c t r o . s t a t i c r e p u l s i o n between molecules at the i n t e r f a c e . i s an important d e s t a b i l i z i n g f o r c e f o r foams.. There were a l s o s i g n i f i c a n t semi-l o g a r i t h m i c r e l a t i o n s h i p s between foam s t a b i l i t y and h y d r o p h o b i c i t y and between f o a m . s t a b i l i t y and v i s c o s i t y . However, h y d r o p h o b i c i t y and ..viscosity made...nonsignif i c a n t c o n t r i b u t i o n s t o the f i n a l r e g r e s s i o n equation f o r foam s t a b i l i t y , which suggests t h a t . t h e y may play minor r o l e s i n determining foam s t a b i l i t y . Work by Mac.Ritchie (197.0) has demonstrated that i n t e r m o l e c u l a r hydrogen bonding, at the ai r - w a t e r i n t e r f a c e i s important, f o r the. development of surface v i s c o s i t y . Perhaps hydrogen.bonding i s a l s o a major f o r c e i n v o l v e d .in.the 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 of p r o t e i n s at the i n t e r f a c e and t h e r e f o r e f o r determining foam s t a b i l i t y . 121 VI. CONCLUSIONS Four c o n c l u s i o n s may be drawn from t h i s work. These are (1 ) I t i s p o s s i b l e to measure the average h y d r o p h o b i c i t y of p r o t e i n s using c i s - p a r i n a r i c a c i d as a probe of the hydrophobic r e g i o n s , a f t e r samples have been u n c o i l e d by h e a t i n g f o r 10 min at 100°C i n the presence of 1.5$ SDS. (2) Molecular shape, f l e x i b i l i t y and h y d r a t i o n as i n d i c a t e d by bulk viscosity.,- average h y d r o p h o b i c i t y , and d i s p e r s i b i l i t y ' are important.parameters f o r determining the foaming c a p a c i t y of p r o t e i n s . (3) The foaming c a p a c i t y o f . p r o t e i n s can be d e s c r i b e d by two r e g r e s s i o n equations which account f o r 77$ of the v a r i a t i o n i n t h i s f u n c t i o n a l p r o p e r t y . These equations are FC = 260.4 In S. - 0.3012 S + 158.6 In d . e e - 2.724 d - 1820 and FC = 24.74 In S + 1 797n -2115 e where FC = foaming c a p a c i t y S e = h y d r o p h o b i c i t y d = d i s p e r s i b i l i t y ($) n = v i s c o s i t y (Pa.s) (4) E l e c t r o s t a t i c r e p u l s i o n between molecules at the 12 2 i n t e r f a c e has an important d e s t a b i l i z i n g e f f e c t on foams. (5) Foam s t a b i l i t y can be r e l a t e d to the charge d e n s i t y of p r o t e i n s by the r e g r e s s i o n equation FS = 19.72 c _ 1 - 2 . 1 U where FS = f o a m . s t a b i l i t y (min. ml -') c = charge d e n s i t y ( u n i t s . r e s ) The r' value f o r t h i s equation i s 0.725 : which i s s i g n i f i c a n t , at the 0.01. l e v e l of p r o b a b i l i t y . These r e s u l t s i n d i c a t e d the importance of h y d r o p h o b i c i t y f o r determining the.foaming c a p a c i t y of p r o t e i n s o l u t i o n s . Foaming was dependent on the hydro-p h o b i c i t y of the u n c o i l e d protein, molecule r a t h e r than on surface h y d r o p h o b i c i t y , which i s important f o r e m u l s i f i c a t i o n . 1 23 V I I . LITERATURE CITED A l l e n , G. 1 974-• The bi n d i n g of sodium dodecyl sulphate to bovine serum albumin at high b i n d i n g r a t i o s . Biochem. J . 137: 575 - 578. Aschaffenburg, R. 1963. P r e p a r a t i o n of 8-casein by a modified urea f r a c t i o n a t i o n method... J . Dairy Res. 30: 259 - 260. A z a r i , P. R. and Feene.y,. R. E. 1961 . The r e s i s t a n c e s of conalbumin and i t s i r o n complex t o p h y s i c a l and chemical treatments. Arch.. Biochem... Biophys. 92: 44 - 52. Bigelow, C. C. 1967. On the average h y d r o p h o b i c i t y of p r o t e i n s and the r e l a t i o n between i t and p r o t e i n s t r u c t u r e . J . Theoret.. B i o l . 16: 1 87 - 211. Bikerman, J . J . 1973. "Foams". S p r i n g e r - V e r l a g . New York, NY. B i r d i , K. S. and S t e i n h a r d t , J . 1978. The e f f e c t s of d i v e r s e p r o t e i n s on the s o l u b i l i z a t i o n of v a r i o u s hydrophobic probes by p r o t e i n - d e t e r g e n t complexes. Biochim. Biophys. Acta 534: 219 - 227. B u l l , H. B. and Breese, K.» 1 974- Surface t e n s i o n of amino a c i d s o l u t i o n s : a h y d r o p h o b i c i t y s c a l e of amino a c i d r e s i d u e s . Arch.. Biochem. Biophys. 161 : 665 - 670. C a n e l l a , M. 1 978.. Whipping p r o p e r t i e s of sunflower p r o t e i n d i s p e r s i o n . Lebensm.-Wiss. u. -Technol. 11: 259 - 263. C a n f i e l d , R. E. 1963. The amino a c i d sequence of egg white lysozyme. J . B i o l . Chem. 238: 2698 - 2707. Chen, Y.-H., Yang, J . T. and Martinez, H. M. 1972. Determination of the secondary s t r u c t u r e s of p r o t e i n s by c i r c u l a r d i c h r o i s m and 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 . Biochem. 11 : 4-120 - 4131 . Cherry, J . P. and McWatters.,. K. H. 1981. W h i p p a b i l i t y and a e r a t i o n . i n " P r o t e i n F u n c t i o n a l i t y i n Foods". A.C.S. Symposium S e r i e s 147. Cherry, J . P., ed. American Chemical S o c i e t y , Washington, DC. Chrambach, A. and Rodbard, D.. 1971. Polyacrylamide g e l e l e c t r o p h o r e s i s . Science 172: 440 - 451. 124 C o l l i n s , G. L., Motarjemi, M. and Jameson, G. J . 1978. A method of measuring the charge on small gas bubbles. J . C o l l o i d I n t e r f a c e S c i . 63: 69 - 75. Concon, J . M. and S o l t e s s , D.. 1 973. Rapid m i c r o - K j e l d a h l d i g e s t i o n of c e r e a l g r a i n s and other b i o l o g i c a l m a t e r i a l s . Anal. Biochem. 53: 35 - 4-1 . Cooney, C. M. 1 974-- A study of foam formation by whey p r o t e i n s . Ph.D.. Thesis.. U n i v e r s i t y of Minnesota. A b s t r a c t i n D i s s . Abst... I n t . B 36: 1 123. Deutsch, D. G. 1976. E f f e c t of prolonged 100°C heat treatment i n sodium dodecyl s u l f a t e upon peptide bond cleavage. . An a l . Biochem. 71: 300 - 303. Edelhoch, H. 1957. The d e n a t u r a t i o n of peps i n . I. Macromolecular changes.. J... Am. Chem. Soc. 79: 61 00 -61 09. Evans, M. T. A. and P h i l l i p s . , M. C. 1 979. The conforma-t i o n and aggregation of bovine B-casein A. I I . Thermo-dynamics of thermal a s s o c i a t i o n and the e f f e c t s of changes i n p o l a r and apolar i n t e r a c t i o n s m i c e l l i z a t i o n . Biopolymers 18i 1123 - 114-0. Feeney, R..E. 1964. Egg P r o t e i n s i n "Symposium on Foods: P r o t e i n s and Their Reactions". ..Schultz, H. ¥. and Anglemier, A.. F., eds. AVI Pub. Co.. Westport, CT. Franzen, K. L. and K i n s e l l a , J . E. 1976a. F u n c t i o n a l p r o p e r t i e s of s u c c i n y l a t e d and a c e t y l a t e d soy p r o t e i n s . J . Agr. Food Chem.. 24: 788 - 795. Franzen, K. L. and K i n s e l l a , J... E. 1 976b. F u n c t i o n a l p r o p e r t i e s of s u c c i n y l a t e d . and a c e t y l a t e d l e a f p r o t e i n s . J . Agr. Food Chem. 24: 914 - 919. Fruton, J . S. 1972. Pepsin i n "The Enzymes. I I I . " 3rd e d i t i o n . Boyer, P.,. ed. Academic Press. New York, NY. Gosh, S. and B u l l , H. B. 1963. Adsorbed f i l m s of bovine serum albumin: t e n s i o n s at ai r - w a t e r surfaces and p a r a f f i n -water i n t e r f a c e s . Biochim. Biophys. Acta 66: 150 - 157. Graham, D. E. and P h i l l i p s , M. C. 1976. The conformation of p r o t e i n s at the ai r - w a t e r i n t e r f a c e , and t h e i r r o l e i n s t a b i l i z i n g foams.., i n "Foams". . Akers, R. J . , ed. Academic P r e s s . New York, NY. 1 25 Graham, D. E. and P h i l l i p s , M. C. 1979a. P r o t e i n s at l i q u i d i n t e r f a c e s . I. K i n e t i c s of a d s o r p t i o n and surface d e n a t u r a t i o n . J . C o l l o i d I n t e r f a c e S c i . 70: 403 - 4 U . Graham, D. E. and P h i l l i p s , M. C. 1 979b.. P r o t e i n s at l i q u i d i n t e r f a c e s . . II.. Ads o r p t i o n isotherms. J . C o l l o i d I n t e r f a c e S c i . 70: 4-15 - 426. Graham, D. E. and P h i l l i p s , M...C... 1 9 7 9 c P r o t e i n s at l i q u i d i n t e r f a c e s . . I I I . Molecular s t r u c t u r e s of adsorbed f i l m s . J.. C o l l o i d I n t e r f a c e S c i . 70: 427 - 439. Hamaguchi, K. and.Hayashl, K. 1972.. Lysozyme i n " P r o t e i n S t r u c t u r e and Function... I . " Funatsu, M.., Hiromi, K. , Imahori, K. , Murachi, T.. and Narita., K., eds. J . Wiley and Sons. New York, NY. Haschemeyer, R. H. and Haschemeyer, A. E. 1973. " P r o t e i n s : A Guide to Study by P h y s i c a l and Chemical Methods". J:. Wiley and Sons.. New york, NY. Hermansson, A.-M., Olsson, D. and Holmberg, B. 1974. F u n c t i o n a l p r o p e r t i e s of p r o t e i n s f o r food - m o d i f i c a t i o n s t u d i e s on rapeseed p r o t e i n . c o n c e n t r a t e . Lebens.-Wiss. u.-Technol. 7: 176 - 1 81. . Hermansson, A.-M., S i v i k , B. and Skjoldebrand, C. 1971. F u n c t i o n a l p r o p e r t i e s of p r o t e i n s f o r food - f a c t o r s a f f e c t i n g s o l u b i l i t y , foaming and s w e l l i n g of f i s h p r o t e i n concentrate... Lebensm..-Wiss, u.-Technol. 4: 201 - 204. H i l l , R. L. and Schmidt., .W.. R.. 1 962.. The complete h y d r o l y s i s of proteins.. J . B i o l . Chem. 237: 389 - 396. Hocking, R. R. 1 976.. The a n a l y s i s and s e l e c t i o n of v a r i a b l e s i n l i n e a r regression.. B i o m e t r i c s 32: 1 - 49. H o r i u c h i , T. , Fukushima, D.. , Sugimoto, H... and H a t t o r i , T. 1 978. Studies on enzyme-modified p r o t e i n s as' foaming agents: e f f e c t s of s t r u c t u r e on foam s t a b i l i t y . Food Chem. 3: 35 - 42. Ikeda, K. 1968. C i r c u l a r d i c h r o i s m and 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 of t r y p s i n i n h i b i t o r s . J . Biochem. 63: 521 - 531 . Inagami, T. 1972. T r y p s i n i n " P r o t e i n S t r u c t u r e and Fu n c t i o n . I . " Funatsu, M., Hiromi, K., Imahori, K., Murachi, T. and N a r i t a , K., eds. J . Wiley and Sons. New York, NY. 126 Kato, A. and Nakai, S. 1 980.. Hydrophobicity determined by a f l u o r e s c e n t probe method and i t s c o r r e l a t i o n with surface p r o p e r t i e s of p r o t e i n s . Biochim. Biophys. Acta 624: 13 - 20. K e i l , B. 1 9 7 1 . T r y p s i n i n "The Enzymes.. I I I " . Boyer, P., ed. 3 r d e d i t i o n . Academic P r e s s . New York, NY. Keshavarz, E. 1977- The r e l a t i o n s h i p between hydrophobi-c i t y and s u r f a c t a n t p r o p e r t i e s of p r o t e i n s . Ph.D. t h e s i s . U n i v e r s i t y of B r i t i s h Columbia. . Keshavarz, E. and Nakai, S.. 1 979. The r e l a t i o n s h i p between h y d r o p h o b i c i t y and i n t e r f a c i a l t e n s i o n of p r o t e i n s . Biochim. Biophys. Acta 576: 269 - 279. K i n s e l l a , J . E. 1 976... F u n c t i o n a l p r o p e r t i e s of p r o t e i n s i n foods: a survey. CRC Rev. Food Sci.. Nutr. 3: 219 -280. Kuehler, C. A. and S t i n e , C. M. 1974. E f f e c t of enzymatic h y d r o l y s i s on some f u n c t i o n a l p r o p e r t i e s of whey p r o t e i n s . J . Food S c i . 39: 379 - 3 8 2 . Kuzmann, W. 1956. S t r u c t u r a l f a c t o r s i n p r o t e i n denatura-t i o n . J . C e l l Comp. Physiol.. (Supplement) 47: 113 - 1 3 1 . Lindblom, M. 1974. A l k a l i treatment of yeast p r o t e i n c o n c e n t r a t e . Lebensm ..-Wis s. u.-Technol. 7: 295 - 298. Loeb, G. I. and Baier,.R. E. . 1 968.. S p e c t r o s c o p i c a n a l y s i s of p o l y p e p t i d e conformation i n polymethylglutamate mono-l a y e r s . J . C o l l o i d I n t e r f a c e Sci... 27: 38 - 45 . MacRitchie, F. 1970. Bonding i n p r o t e i n and po l y p e p t i d e monolayers. J . Macromol. S c i . A 4: 1169 - 1176. MacRitchie, F, 1978. P r o t e i n s at I n t e r f a c e s . Adv. P r o t e i n Chem. 32: 283 - 326. MacRitchie, F.. and Alexander, A. E. 1 9 6 3 a . K i n e t i c s of ad s o r p t i o n of p r o t e i n s at i n t e r f a c e s . I. The r o l e of bulk d i f f u s i o n i n adsorption... J . C o l l o i d I n t e r f a c e S c i . 18: 453 - 457. MacRitchie, F. and Alexander, A. E. 1963b. K i n e t i c s of a d s o r p t i o n of p r o t e i n s at i n t e r f a c e s . I I . The r o l e of pressure b a r r i e r s , i n a d s o r p t i o n . J . C o l l o i d I n t e r f a c e S c i . 1 8: 458 - 4 6 3 . 1 27 MacRitchie, F. and Alexander, A. E. 1963c. K i n e t i c s of a d s o r p t i o n of p r o t e i n s at i n t e r f a c e s . I I I . The r o l e of e l e c t r i c a l b a r r i e r s i n a d s o r p t i o n . J . C o l l o i d I n t e r f a c e S c i . 18: 4.64 - 469. Malcolm, B. R. 1973. The s t r u c t u r e and p r o p e r t i e s of monolayers of s y n t h e t i c p o l y p e p t i d e s at the ai r - w a t e r i n t e r f a c e . Prog. Surface Membrane S c i . 7: 183 - 229. McKenzie, H. A. 1971. "Milk P r o t e i n s " . Academic P r e s s . New York, NY. Means, G. E. and Feeney,. R. E. 1971,. "Chemical M o d i f i c a -t i o n of P r o t e i n s " . Holden-Day Inc. San F r a n c i s c o , CA. M i l l e r , R. and Groninger, Jr...,. H. S.. 1 976. F u n c t i o n a l p r o p e r t i e s of enzyme-modified a c y l a t e d f i s h p r o t e i n d e r i v a t i v e s . ,J. Food Sci... 4-1: 268 - 272. Mita, T., Nakai, K. , Hiraoka,- T. , Matsuo, S. and Matsumoto, H. 1977. Physicochemical s t u d i e s on wheat p r o t e i n foams. J . C o l l o i d I n t e r f a c e Sci.. 59: 172 - 178. Morgan, S. L. and Deming, S. N. 1 974-. Simplex o p t i m i z a -t i o n of a n a l y t i c a l chemical methods. Anal. Chem. 4-6: 1170 - 11 81 . Nakai, S.,.Ho, L., Tung, M. A. and Quinn, J . F. 1980. S o l u b i l i z a t i o n of rapeseed,. soy and sunflower p r o t e i n i s o l a t e s by surfactant, and., p r o t e i n a s e treatments. Can. I n s t . Food S c i . Technol. J . 13: 14- - 22. Nakai, S. 1982. Comparison of o p t i m i z a t i o n techniques f o r a p p l i c a t i o n to food product and process development. J . Food S c i . i n pre s s . Newell, G. J . and Lee, B... 1981.. Ridge r e g r e s s i o n : an a l t e r n a t i v e to m u l t i p l e l i n e a r r e g r e s s i o n f o r h i g h l y c o r r e l a t e d data. J.., Food Sci... 4-6: 968 - 969. Noelken, M. E. 1 980... Aqueous N-8-phenethylbiguanidine h y d r o c h l o r i d e as a sol v e n t f o r p r o t e i n molecular weight d e t e r m i n a t i o n . Anal.. Biochem. 1 04.: 228 - 230. Nozaki, Y. and Tanford., C. 1 967. Examination of t i t r a t i o n behaviour. Methods i n Enzymol... 1 1: 715 - 734-. Nozaki, Y. and Tanford,. C. 1971. The s o l u b i l i t y of amino a c i d s and two g l y c i n e peptides i n aqueous ethanol and dioxane solutions... J . Biol.. Chem. 24-6: 2211 - 2217. Oegema, J r . , T. R. and J o u r d i a n , G. W. 1 974-• The p h y s i c a l and chemical p r o p e r t i e s of a chicken egg white g l y c o -p r o t e i n p u r i f i e d by a non-denaturing methodology. Arch. Biochem. Biophys. 160: 26 - 39. 1 2.8 Peters J r . , T. 1975. Serum albumin i n "The Plasma P r o t e i n s . I." 2nd e d i t i o n . Putnam, F.. W. , ed. Academic Press. New York, NY. Pham, A.-M. 1981. P r e d i c t i o n program of secondary s t r u c t u r e from the sequence of p r o t e i n s according to the method of Chou~: and Fasman. M.Sc. thesis.. U n i v e r s i t y of B r i t i s h Columbia. P h i l l i p s , M. C. 1977. The conformation and p r o p e r t i e s of p r o t e i n s at l i q u i d interfaces... Chem. Ind. March 5: 1 70 - 1 76. P i e z , K. A., Davie, E. ¥. , F o l k , J . E.. and Gladner, J . A. 1961. 8 - L a c t o g l o b u l i n s A and B.. J . B i o l . Chem. 236: 2912 - 2916. -Ponnuswamy, P. K., Prabhakaran, M. and Manavalan, P. 1980. Hydrophobic packing and s p a t i a l arrangement of amino a c i d r e s i d u e s i n g l o b u l a r p r o t e i n s . Biochim. Biophys. Acta 623: 301 - 31 6. Ray, A., Reynolds, J . A., Pol.et, H. and S t e i n h a r d t , J . 1966. Binding of l a r g e organic anions and n e u t r a l molecules by n a t i v e bovine serum albumin. Biochem. 5: 2606 - 2616. Reynolds, J . A. and Tanford, C. 1 970.. Binding of d o d e c y l s u l f a t e to p r o t e i n s at high b i n d i n g r a t i o s . P o s s i b l e i m p l i c a t i o n s f o r the. s t a t e of p r o t e i n s i n b i o l o g i c a l membranes. P r o c . N a t . Acad. S c i . 66: 1002 -1 007. Richards, F. M. 1963. S t r u c t u r e of p r o t e i n s . Ann. Rev. Biochem. 32: 269 - 300. Richards, F. M. and Wyckoff., H.. ¥. 1971. Bovine p a n c r e a t i c r i b o n u c l e a s e i n "The Enzymes.. IV." Boyer, P., ed. 3rd e d i t i o n . Academic Press.. New York, NY. R i c h e r t , S. H.,.Morr, C. V. and Cooney, C. M. 1974. E f f e c t of heat and other p r o p e r t i e s of whey p r o t e i n concentrates... J . Food S c i . 39: 4-2 - 4-8. Rosen, M. J . 1972. The r e l a t i o n s h i p of s t r u c t u r e to p r o p e r t i e s of surfactants... I. J.. Am. O i l Chem. Soc. 49: 293 - 297. Saraga, L. 1981. P r o t e i n d e n a t u r a t i o n on a d s o r p t i o n and water a c t i v i t y of i n t e r f a c e s : an a n a l y s i s and suggestion. J . C o l l o i d I n t e r f a c e S c i . 80: 393 - 401. 1 2f Sato, Y. and Hayakawa, M. 1979. Whipping property of the p r o t e i n i s o l a t e d from Hansenula yeast grown on methanol. Lebensm.-Wis s. u.-Technol. 12: 4-1 - 46. Sato, Y. and Nakamura, R. 1977. F u n c t i o n a l p r o p e r t i e s of a c e t y l a t e d and. s u c c i n y l a t e d egg white. Agr. B i o l . Chem. 41 : 2163 - 2168. Shaw, D. J . 1969. " E l e c t r o p h o r e s i s " . Academic Press. New York, NY. S k l a r , L. A. and Hudson, B. S. 1976. Conjugated polyene f a t t y a c i d s as f l u o r e s c e n t membrane probes: model system s t u d i e s . J . Supramol. S t r u c t . 4: 409 - 425. S k l a r , L. A., Hudson, B. S., Peterson, M., Diamond, J . 1977. Conjugated polyene f a t t y a c i d s as f l u o r e s c e n t probes: s p e c t r o s c o p i c c h a r a c t e r i z a t i o n . . Biochem. 16: 813 - 818. Snyder, L. R. 1978. C l a s s i f i c a t i o n of the solvent p r o p e r t i e s of common l i q u i d s . . J . Chromatog. S c i . 16: 223 - 234. Sober, H. A. ed. 1970. CRC Handbook of Bioch e m i s t r y . 2nd e d i t i o n . Chemical Rubber Co.. Cleveland, OH. S p i r o , R. G. 1973. G l y c o p r o t e i n s . Adv. P r o t e i n Chem. 27: 349 - 467. St e i n h a r d t , J . , S c o t t , J.. R. and B i r d i , K. S. 1 977. D i f f e r e n c e s i n the s o l u b i l i z i n g e f f e c t i v e n e s s of sodium dodecyl s u l f a t e complexes of v a r i o u s p r o t e i n s . Biochem. 16: 718 - 725. S t r y e r , L. 1965. Fluorescence, spectroscopy of p r o t e i n s . Science 162: 526 - 530. Swaisgood, H. E. 1 973. The caseins... CRC Rev. Food Technol. 3: 375 - 414. Taborsky, G. 1974. Phosphoproteins. Adv. P r o t e i n Chem. 28: 1 - 210. Tan, A. T. 1971. C i r c u l a r d i c h r o i s m p r o p e r t i e s of conalbumin and i t s i r o n and copper complexes. Can. J . Biochem. 49: 1071 - 1075. 1 30'' Tanford, C. 1961. " P h y s i c a l Chemistry of Macromolecules". J . Wiley and Sons Inc. New York, NY. Tanford, C. 1962. C o n t r i b u t i o n of hydrophobic i n t e r a c t i o n s to the s t a b i l i t y of the g l o b u l a r conformation of p r o t e i n s . J . Am. Chem. Soc. 84: 4-24-0 - 424-7. Tanford, C. 1 968.. P r o t e i n denaturation.. Adv. P r o t e i n Chem. 23: 121 - 282. Tanford, C. 1973. "The Hydrophobic E f f e c t : Formation of M i c e l l e s and B i o l o g i c a l Membranes". W i l e y - I n t e r s c i e n c e . New York, NY. Tanford, C. 1978. The hydrophobic e f f e c t and the or g a n i z a -t i o n of l i v i n g matter.. Science 200: 1 01 2 - 1 01 8. Thompson, M. P. and Pepper, L.. 1 964. Genetic polymor-phism i n ca s e i n s of cows milk.. IV. I s o l a t i o n and p r o p e r t i e s of 8-caseins A, B and C. J . Dairy S c i . 47: 633 - 637. Tornberg, E. 1978. A p p l i c a t i o n of the drop volume technique to measurements.of the a d s o r p t i o n of p r o t e i n s at i n t e r f a c e s . J.. C o l l o i d I n t e r f a c e Sci.. 64: 391 - 402. Usui, S. and Sasaki,. H.. 1 978.. Zeta p o t e n t i a l measurements of bubbles i n aqueous s u r f a c t a n t s o l u t i o n s . J . C o l l o i d I n t e r f a c e Sci.. 65: 36 - 45 . Usui , S., Sasa k i , H. and Matsukawa,. H.. 1981. The dependence of zeta p o t e n t i a l on bubble s i z e as determined by the Dom e f f e c t . J . C o l l o i d I n t e r f a c e S c i . 81: 80 - 84. Wang, J . C. and K i n s e l l a , . J.. E. 1 976. F u n c t i o n a l p r o p e r t i e s of a l f a l f a l e a f p r o t e i n : foaming. J . Food S c i . 41: 498 -501 . Waniska, R. D. and'.Kinsella., J . E. 1 979. Foaming proper-t i e s of p r o t e i n s : e v a l u a t i o n of column a e r a t i o n apparatus using ovalbumin., J . Food S c i . 44: 1398 -1411 . Waugh, D. F. 1954. P r o t e i n - p r o t e i n i n t e r a c t i o n s . Adv. P r o t e i n Chem. 9: 325 - 437. Whitaker, J . R. 1980. Changes o c c u r r i n g i n p r o t e i n s i n a l k a l i n e s o l u t i o n , i n "Chemical D e t e r i o r a t i o n of P r o t e i n s " . A.C.S.. Symposium S e r i e s 123- Whitaker, J . R. and F u j i -maki, M., eds. American Chemical S o c i e t y Press, Washington, DC. 1 31 White, A., Handler, P. and Smith, E-. 1 973. " P r i n c i p l e s of B i o c h e m i s t r y " . 5th edition... McGraw-Hill. New York, NY. Wi l l i a m s , J . 1961. A comparison of c.onalbumin and t r a n s -f e r r i n i n the domestic fowl.. Biochem. J . 83: 355 - 36/+. W i l k i n s , D. J . and Myers, P.. A. 1 970. E l e c t r o p h o r e s i s and a d s o r p t i o n s t u d i e s of p r o t e i n s and t h e i r d e r i v a t i v e s on c o l l o i d s and c e l l s . i n "Surface Chemistry of B i o l o g i c a l Systems". Blank, M., Ed. Academic Press. New York, NY. Wu, C. H., Nakai, S. and Powrie, W. D. 1976. P r e p a r a t i o n and p r o p e r t i e s of a c i d - s o l u b i l i z e d g l u t e n . J . Agr. Food Chem. 24: 504 - 510. Yang, J . T. 1961. The v i s c o s i t y of macromolecules i n r e l a t i o n to molecular conformation.. Adv.. P r o t e i n Chem. 16: 323 - 400. Zar, J . H. 1 974-. " B i o s t a t i s t i c a l A n a l y s i s " . P r e n t i c e -H a l l Inc. Englewood C l i f f s , NJ. Z i t t l e , C. A. and Custer,,, J.. H. 1 963. P u r i f i c a t i o n and some of the p r o p e r t i e s of a. - c a s e i n and K - c a s e i n . J . Dairy S c i . 46: 1183 - 1188. s 1 3-2 APPENDIX I Simplex O p t i m i z a t i o n (Morgan and Deming, 1 974; Nakai, 1982) 1 . I f k f a c t o r s are being examined t h e . s t a r t i n g simplex of u n i t edge i s given by the k by k+1 matrix ' 0 0 o . . . 0 P q q • . . q D = q p q • • • q q q q • • • p where 0 = lower boundary value p = .1 {(k-1) + /k+l7 k /2 q = 1 {/FTT - 1} k /2 2. The response, at each v e r t e x of the simplex i s measured. 3. Let ¥, B and N be, r e s p e c t i v e l y , , the worst, the best and the next to worst response,.. then P i s the c e n t r o i d of the l i n e through B and N and R i s the. r e f l e c t i o n of W i n BN.. Based on the i n i t i a l simplex r e s u l t s the r e f l e c t i o n , R, .is c a l c u l a t e d where R = P + (P - W) 133 U. Based on the response at R the.next.simplex i s c a l c u l a -t e d . An expansion may be c a r r i e d out by g e n e r a t i n g the new v e r t e x E, such that E = P + y(P - ¥) Y>1 or a c o n t r a c t i o n may .be c a r r i e d out by generating the new v e r t e x C, such that C = P.+ B(P - W) 0<3<1 Quadratic,, f a c t o r i a l r e g r e s s i o n , a n a l y s i s i s used to determine which, new. vertex, should r e p l a c e the worst vert e x of the preceding simplex. . . 5. Using t h i s new simplex.,,, the. worst v e r t e x i s r e p l a c e d by performing an expansion or., contration.,. as d e s c r i b e d above. This process is. repeated u n t i l the step s i z e becomes l e s s than some ...predetermined v a l u e . 13'4 APPENDIX II C a l c u l a t i o n of net proton charge (Nozaki and Tanford, 1967) For any t i t r a b l e group pK = pH - l o g q + 0.868 coZ a ^ 1 -a where pK = negative l o g of the e q u i l i b r i u m a constant of the group a = degree of d i s s o c i a t i o n of the group Z = t o t a l charge on the p r o t e i n co = e l e c t r o s t a t i c f a c t o r As i o n i c s t r e n g t h i n c r e a s e s , the i o n atmosphere around the p r o t e i n becomes i n c r e a s i n g l y e f f e c t i v e at n e u t r a l i z i n g the charge on the p r o t e i n and co approaches zero. Since 6M guanidine h y d r o c h l o r i d e i s a strong e l e c t r o l y t e co i s taken to be zero. Therefore pK = pH - l o g a 1 -a Using t h i s equation and the pK 's given i n Table I I I of a Nozaki and Tanford's (1967) paper the degree of d i s s o c i a -t i o n , at pH 7.0, f o r each t i t r a b l e group i n the p r o t e i n i s c a l c u l a t e d . . I t i s assumed t h a t a l l c h e m i c a l l y i d e n t i c a l groups have the same pK .. From a knowledge a of the amino a c i d composition of the p r o t e i n , the t o t a l 1 35 number of hydrogen Ions bound or l o s t per molecule, when the pH i s s h i f t e d . f r o m i t s i s o i o n i c p o i n t to pH 7.0, i s c a l c u l a t e d . . This f i g u r e r e p r e s e n t s the net proton charge on the p r o t e i n . 

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