Proteins at Interfaces - American Chemical Society

Film, Foaming, and Emulsifying Properties of Food .... film forming properties of food proteins, i t is useful to examine .... 6.0. From Kim and Kinse...
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Chapter 39

Film, Foaming, and Emulsifying Properties of Food Proteins: Effects of Modification John E . Kinsella and Dana M . Whitehead

Proteins at Interfaces Downloaded from pubs.acs.org by UNIV OF LEEDS on 09/23/18. For personal use only.

Institute of Food Science, Cornell University, Ithaca, NY 14853

The spectrum of surface active behavior displayed by food proteins directly reflects differences in structural and physicochemical properties among the proteins originating from various sources i.e. meat, milk, legumes. Chemical or enzymatic modification of model food proteins has indicated that alteration of specific structural features e.g. net charge, disulfide bonding, size, does influence film formation, foaming and emulsifying properties. The specific sequence of amino acids in a protein determines its structure, conformation, and physicochemical properties. The structure of protein is categorized as primary, secondary, tertiary, or quaternary, depending on the progressive state of spatial arrangement of polypeptide chains of the protein (1,2). Although the primary structures of almost all major food proteins are known, the exact conformation of only a few native proteins e.g. φ-lactoglobulin has been elucidated (3-5). In an aqueous environment, the component polypeptides of a protein tend to fold in a characteristic fashion to form local­ ized secondary structures i.e. a(-helix, φ-pleated sheet, Ç-turns, or random coil (6). The integrity and stabilization of secon­ dary, tertiary, and quaternary structures of a given protein are dependent on different forces. An understanding of the various forces responsible for the native structure of proteins is fundamental in comprehending how they affect the conformation and functional properties of proteins (7.)· The non-covalent forces involved in stabilizing the second­ ary and tertiary structure and influencing the functional behavior of proteins include: hydrogen bonding, van der Waal's forces, electrostatic interactions, and hydrophobic associa­ tions. Covalent disulfide bonds are also important in maintain­ ing structural integrity of some food proteins via intramolecular and intermolecular bonds e.g. glycinin, Ç>-lactoglobulin (8_). 0097-6156/87/0343-0629$06.00/0 © 1987 American Chemical Society

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The hydrogen bond (Η-bond) i s i o n i c i n n a t u r e and s t a b i l i z e s secondary s t r u c t u r e s . Hydrogen bonding i s i n v o l v e d i n p r o t e i n -protein associations in films. Electrostatic i n t e r a c t i o n s are the major f o r c e s a f t e r hydrogen b o n d i n g . The i n v o l v e m e n t 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 i n the f u n c t i o n a l b e h a v i o r of f o o d p r o t e i n s i s i n d i c a t e d by the e f f e c t of pH on s e v e r a l p r o p e r t i e s i . e . s o l u b i l i t y , emulsion stabilization, film formation, and foaming. Modification of c a t i o n i c groups and i n t r o d u c t i o n of a n i o n i c groups s i g n i f i c a n t l y a l t e r s the p h y s i c a l p r o p e r t i e s of f o o d p r o t e i n s and improves c e r t a i n f u n c t i o n a l p r o p e r t i e s (9_). Van der Waal's f o r c e s are g e n e r a l , n o n s p e c i f i c , s h o r t - r a n g e f o r c e s which are o p e r a t i v e between c l o s e l y a p p o s e d g r o u p s i n adjacent polypeptides. The i n v o l v e m e n t of t h e s e f o r c e s i n f i l m f o r m a t i o n has not been e s t a b l i s h e d . Hydrophobic i n t e r a c t i o n s which are e n f o r c e d ( e n t r o p y driven) by the n a t u r e of water are the p r i n c i p l e f o r c e s b e h i n d p r o t e i n f o l d i n g ( 6 ) . They f a c i l i t a t e the e s t a b l i s h m e n t o f o t h e r s t a b i l i ­ z i n g i n t e r a c t i o n s (7,10). Hydrophobic i n t e r a c t i o n s , b e i n g of fundamental importance t o p r o t e i n s t r u c t u r e , are v e r y r e l e v a n t t o the f u n c t i o n a l p r o p e r t i e s of many f o o d p r o t e i n s , e s p e c i a l l y caseins. These f o r c e s a f f e c t s o l u b i l i t y , g e l a t i o n , c o a g u l a t i o n , m i c e l l e formation, f i l m formation, surfactant properties and f l a v o r b i n d i n g (7,10). Protein

Behavior at

Interfaces

F i l m Formation The s u r f a c e a c t i v e p r o p e r t i e s of p r o t e i n s are r e l a t e d t o t h e i r a b i l i t y t o lower the i n t e r f a c i a l t e n s i o n between a i r / w a t e r or o i l / w a t e r i n t e r f a c e s . S u r f a c e a c t i v i t y i s a f u n c t i o n of the ease w i t h which p r o t e i n s can d i f f u s e t o , a d s o r b a t , u n f o l d , and r e a r r a n g e a t an i n t e r f a c e (11,12). Thus, s i z e , n a t i v e s t r u c t u r e and s o l u b i l i t y i n the aqueous phase are c l o s e l y c o r r e l a t e d w i t h t h e s u r f a c e a c t i v i t y of p r o t e i n s i n model systems (13-16). In model systems, i n t e r f a c i a l f i l m f o r m a t i o n i s enhanced by exposed h y d r o p h o b i c r e g i o n s on the p r o t e i n . Thus p r o t e i n s w i t h molecular ' f l e x i b i l i t y ' show s u p e r i o r surface activity, as d i s p l a y e d by the c a s e i n s . P r o t e i n c o n f o r m a t i o n a t an o i l / w a t e r i n t e r f a c e i s not f u l l y understood. Model s t r u c t u r e s have been proposed based on the p o l a r i t y of amino a c i d r e s i d u e side chains, which d e p i c t the polypeptide chain i n three segments: ' t r a i n s ' of a m i n o a c i d r e s i d u e s i n c o n t a c t w i t h the i n t e r f a c e and ' l o o p s ' (and 'tails') of r e s i d u e s p r o t r u d i n g i n t o e i t h e r b u l k phase, depending on t h e i r p o l a r i t y (13,15,17). A study with the hydrophobic s i g n a l peptide of E ^ coli lambda phage i n p h o s p h o l i p i d monolayers, showed a p r e f e r e n c e f o r p C - h e l i c a l c o n f o r m a t i o n when the p e p t i d e was i n s e r t e d i n t o the l i p i d phase ( 1 8 ) . However, i n t e r a c t i o n w i t h the l i p i d surface w i t h o u t i n s e r t i o n i n d u c e d the p e p t i d e t o adopt the φ-structure (18). These o b s e r v a t i o n s , o b t a i n e d w i t h c i r c u l a r d i c h r o i s m and F o u r i e r t r a n s f o r m - i n f r a r e d (FT-IR) d a t a , p r o v i d e t h e f i r s t d i r e c t evidence f o r i n t e r c o n v e r s i o n s between v a r i o u s conformational

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Film, Foaming, and Emulsifying Properties

s t a t e s , under s p e c i f i c e n v i r o n m e n t a l c o n d i t i o n s , when t h e p e p t i d e approaches a l i p i d s u r f a c e ( 1 8 ) . During the i n i t i a l stages o f f i l m formation there i s r a p i d d i f f u s i o n (nanosecond) o f p r o t e i n s from s o l u t i o n t o t h e i n t e r face. T h i s i s t h e r m o d y n a m i c a l l y f a v o r a b l e because some o f t h e c o n f o r m a t i o n a l and h y d r a t i o n energy o f t h e p r o t e i n i s l o s t a t t h e i n t e r f a c e ( 1 2 ) . I n i t i a l l y , a t low p r o t e i n c o n c e n t r a t i o n s , there i s no b a r r i e r t o a d s o r p t i o n and f o r p r o t e i n m o l e c u l e s t h a t a r e r e a d i l y adsorbed at the i n t e r f a c e , the r a t e of a d s o r p t i o n i s diffusion controlled. But a f t e r some time, e s p e c i a l l y a t h i g h s u r f a c e p r o t e i n c o n c e n t r a t i o n s , t h e r e i s an a c t i v a t i o n e n e r g y b a r r i e r t o a d s o r p t i o n (14,19), which may i n v o l v e e l e c t r o s t a t i c , s t e r i c and o s m o t i c e f f e c t s c l o s e t o t h e i n t e r f a c i a l o r s u r f a c e layers. Under t h e l a t t e r c o n d i t i o n s , t h e a b i l i t y o f t h e p r o t e i n m o l e c u l e s t o i n t e r p e n e t r a t e and c r e a t e space i n t h e e x i s t i n g f i l m and t o r e a r r a n g e at the s u r f a c e i s rate-determining. The c a p a c i t y o f p r o t e i n s t o u n f o l d a t an i n t e r f a c e depends v e r y much on t h e c o n f o r m a t i o n a l s t a b i l i t y of f l e x i b l e segments o f t h e p r o t e i n m o l e c u l e (130. Where t h e r e i s e x t e n s i v e intramolecular a s s o c i a t i o n s and d i s u l f i d e bonding, u n f o l d i n g a t t h e i n t e r f a c e tends t o be l i m i t e d , and f o r m a t i o n o f an i n t e r f a c i a l membrane t a k e s r e l a t i v e l y l o n g e r e . g . soy p r o t e i n s compared t o c a s e i n s (10,15,20). Food P r o t e i n F i l m s i n Model Systems In o r d e r t o e l u c i d a t e r e l a t i o n s h i p s between s u r f a c e a c t i v e and f i l m f o r m i n g p r o p e r t i e s o f f o o d p r o t e i n s , i t i s u s e f u l t o examine the s u r f a c e a c t i v e p r o p e r t i e s o f p r o t e i n s whose p h y s i c a l a n d m o l e c u l a r p r o p e r t i e s a r e w e l l c h a r a c t e r i z e d e.g. Ç>-casein, b o v i n e serum albumin (BSA), lysozyme ( 1 7 ) , and Ç > - l a c t o g l o b u l i n (b-Lg) (21). These r e p r e s e n t a range o f t e r t i a r y s t r u c t u r e s f o r s o l u b l e proteins. Lysozyme i s a r i g i d and r o u g h l y e l l i p s o i d a l m o l e c u l e , whereas t h e h y d r o p h o b i c ^ - c a s e i n m o l e c u l e i s m o s t l y a random c o i l structure. The b-Lg m o l e c u l e c o n s i s t s a l m o s t e n t i r e l y o f a n t i p a r a l l e l Ç - s h e e t s t r a n d s o r g a n i z e d i n t o a f l a t t e n e d cone (5_). 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 a t an i n t e r f a c e can be measured by m o n i t o r i n g s u r f a c e c o n c e n t r a t i o n and s u r f a c e p r e s s u r e i . e . d e p r e s s i o n o f s u r f a c e t e n s i o n (V) as a f u n c t i o n o f time (17). ^ - c a s e i n i s more s u r f a c e a c t i v e t h a n serum albumin o r b - L g and much more so than lysozyme. This r e f l e c t s not only the r a t e of d i f f u s i o n o f t h e n a t i v e p r o t e i n t o t h e i n t e r f a c e , b u t a l s o i t s molecular f l e x i b i l i t y ' a n d a m p h i p a t h i c n a t u r e (15,17,22). Comparisons o f t h e s u r f a c e a d s o r p t i o n b e h a v i o r o f ^ - c a s e i n , lysozyme and BSA have been we11-documented (13-15,22). Rates o f a d s o r p t i o n a t any g i v e n s u r f a c e p r e s s u r e (2 t o 20 mN/M) and p r o t e i n c o n c e n t r a t i o n (0.01 mg/dl t o 1 mg/dl) r e f l e c t d i f f e r e n c e s i n u n f o l d i n g o f t h e p r o t e i n m o l e c u l e s as w e l l as d i f f e r e n c e s i n their i s o e l e c t r i c points, ^ - c a s e i n has a p i o f a p p r o x i m a t e l y 5.3 ( a v e r a g e o f t h e g e n e t i c v a r i a n t s ) and forms a d i l u t e monolayer o f t i g h t l y p a c k e d m o l e c u l e s (>7.7 A / r e s i d u e ) u n t i l t h e p r o t e i n c o n c e n t r a t i o n i s 1 mg/dl. F u r t h e r a d s o r p t i o n i s a v e r t e d because o f charge r e p u l s i o n and s t e r i c f a c t o r s i . e . n e t n e g a t i v e c h a r g e on t h e p r o t e i n a t pH 7 (13,23,24). Lysozyme r e t a i n s e x t e n s i v e n a t i v e s t r u c t u r e a t t h e i n t e r f a c e as i t forms a c o n c e n t r a t e d f i l m and m u l t i l a y e r s of t h e p r o t e i n a c c u m u l a t e above 1 m g / d l . Lysozyme m u l t i l a y e r f i l m s d i s p l a y g r e a t e r v i s c o s i t y , r e s i s t a n c e 1

2

631

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PROTEINS AT INTERFACES

t o shear and lower c o m p r e s s i b i l i t y ( l a r g e r d i l a t a t i o n a l modulus) than β - c a s e i n f i l m s , r e f l e c t i n g a g r e a t e r degree of c r o s s l i n k i n g ( d i s u l f i d e bonds) and 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 ( 2 3 ) . The p i of lysozyme i s c l o s e t o n e u t r a l pH, thus e l e c t r o s t a t i c r e p u l s i v e f o r c e s and s t e r i c f a c t o r s are n e g l i g i b l e a t t h i s pH. Benjamins e t a l (24) s t u d i e d the e f f e c t s of a g i n g on the e l a s t i c i t y of Ç - c a s e i n and K - c a s e i n f i l m s . The dilatational modulus of K - c a s e i n was l a r g e r than t h a t of ^ - c a s e i n and i n c r e a s ed by a f a c t o r of t h r e e w i t h f i l m age, whereas the d i l a t a t i o n a l modulus of ^ - c a s e i n f i l m s changed l i t t l e w i t h time ( 2 4 ) . K - c a s e i n u n f o l d s l e s s a t the a i r / w a t e r i n t e r f a c e s i n c e i t has l e s s random s t r u c t u r e than Ç - c a s e i n . T h i s can a l s o be i n t e r p r e t e d i n terms of K - c a s e i n h a v i n g l e s s d i r e c t c o n t a c t w i t h the f i l m s u r f a c e a t any g i v e n p r o t e i n c o n c e n t r a t i o n (25). Significant protein-protein interactions i . e . s t e r i c / e l e c t r o s t a t i c repulsion, are b e l i e v e d t o o c c u r between segments of p o l y p e p t i d e c h a i n s which extend both above and below the p l a n e of the a i r / w a t e r i n t e r f a c e i n s u r f a c e p r o t e i n f i l m s (16,26) The s u r f a c e v i s c o s i t y or r e s i s t a n c e t o shear s t r e s s of the s u r f a c e f i l m i s an index of i t s m e c h a n i c a l s t r e n g t h and i s an i m p o r t a n t parameter r e l a t e d t o the s t a b i l i t y of f i l m s and foams (23J. S u r f a c e y i e l d s t r e s s of BSA f i l m s were d e t e r m i n e d u s i n g the above parameters (23_). Maximum v a l u e s were o b t a i n e d i n the pH range of 5-6, near the i s o e l e c t r i c p o i n t of BSA, and d e c r e a s e d r a p i d l y above pH 6.0 [ T a b l e 1 ] . These o b s e r v a t i o n s a g a i n r e f l e c t the enhanced 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 between p r o t e i n compon e n t s i n the f i l m as the i s o e l e c t r i c p o i n t i s r e a c h e d and the enhanced e l e c t r o s t a t i c r e p u l s i o n between n e i g h b o r i n g m o l e c u l e s as the pH i s r a i s e d above the i s o e l e c t r i c p o i n t , i . e . n e t charge on the p r o t e i n s u r f a c e was increased. The g e n e r a l v a l i d i t y of t h e s e r e l a t i o n s h i p s has a l s o been demonstrated w i t h ^ - l a c t o g l o b u l i n (21 ), r i b u l o s e 1,5-biphosphate c a r b o x y l a s e (25), and soy g l y c i n i n ( 2 7 ) . The a d s o r p t i o n of soy p r o t e i n a t an i n t e r f a c e i s r e l a t i v e l y s l o w c o m p a r e d to c a s e i n , and the r a t e i s a f f e c t e d by i o n i c s t r e n g t h , b e i n g h i g h e r a t 0.2 M than a t z e r o NaCl where the s u b u n i t s may be d i s s o c i a t e d . C o n c e i v a b l y the r e d u c t i o n of the z e t a p o t e n t i a l and e l e c t r o s t a t i c r e p u l s i o n (from 0 t o 0.2 M s a l t ) f a c i l i t a t e s p e n e t r a t i o n and subsequent s u r f a c e p a c k i n g ( 2 8 ) . The r a t e of p e n e t r a t i o n of a d d i t i o n a l m o l e c u l e s i n t o t h e film i n d i c a t e d t h a t the soy p r o t e i n s i n i t i a l l y adsorbed and spread e a s i l y a t the s u r f a c e (29). However, t h i s seems i n c o n s i s t e n t w i t h the h i g h l y s t a b l e d i s u l f i d e l i n k e d t e r t i a r y s t r u c t u r e of soy g l y c i n i n (30) and i t i s perhaps the c o n g l y c i n i n component t h a t forms the i n i t i a l i n t e r f a c i a l f i l m ( 3 1 ) . The b e h a v i o r o f p r o t e i n s at i n t e r f a c e s influences the f o r m a t i o n of foams and e m u l s i o n s ( 3 2 ) . S t a b i l i z a t i o n o f foams and e m u l s i o n s depends, t o a g r e a t e x t e n t , on t h e formation, r h e o l o g i c a l , and m e c h a n i c a l p r o p e r t i e s of the i n t e r f a c i a l film (22^). F a c t o r s which ensure optimum f i l m p r o p e r t i e s i n s i m p l e systems may r e t a r d f i l m f o r m a t i o n or cause d e s t a b i l i z a t i o n i n foams o r e m u l s i o n s ( 3 3 ) ; f o r example, many r h e o l o g i c a l p r o p e r t i e s o f f i l m s are maximum i n the i s o e l e c t r i c pH range of specific p r o t e i n s , y e t most p r o t e i n s have minimum s o l u b i l i t y i n t h i s pH r a n g e (34). Thus, e n v i r o n m e n t a l and processing f a c t o r s which

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633

TABLE 1 Effect

o f pH on Some F i l m and Foaming P r o p e r t i e s o f B o v i n e Serum Albumin

Foam

Film

PH

Surface Pressure

4.0 5.0 5.5 6.0 7.0 8.0

2.8 15.8 19.0 14.0 10.0 2.0

From Kim and K i n s e l l a ,

Surface Y i e l d Stress (dyne/cm)

3.0 3.8 4.0 4.3 3.0 2.2

1985 (23)

Film Elasticity

2.2 5.0 5.2 5.4 2.3 1.8

Drainage half-life (min)

5.0 8.0 9.6 8.5 6.3 6.0

634

PROTEINS AT INTERFACES

a l t e r the c o n f o r m a t i o n and s t a b i l i t y of p r o t e i n s g r e a t l y a f f e c t s f i l m f o r m a t i o n and p r o p e r t i e s ( 3 4 ) . Because p r o t e i n - b a s e d foams d e p e n d u p o n t h e intrinsic molecular p r o p e r t i e s (extent and nature of protein-protein i n t e r a c t i o n s ) of the p r o t e i n , foaming p r o p e r t i e s ( f o r m a t i o n and s t a b i l i z a t i o n ) can v a r y immensely between d i f f e r e n t p r o t e i n s . The i n t r i n s i c p r o p e r t i e s of the p r o t e i n t o g e t h e r w i t h e x t r i n s i c f a c t o r s ( t e m p e r a t u r e , pH, s a l t s , and v i s c o s i t y of the c o n t i n u o u s phase) d e t e r m i n e the p h y s i c a l s t a b i l i t y of the f i l m . Films with enhanced m e c h a n i c a l s t r e n g t h (greater p r o t e i n - p r o t e i n i n t e r a c tions), and b e t t e r r h e o l o g i c a l and v i s c o e l a s t i c properties ( f l e x i b l e r e s i d u a l t e r t i a r y s t r u c t u r e ) a r e more s t a b l e (12,15), and t h i s i s r e f l e c t e d i n more s t a b l e foams/emulsions (14,33). Such f i l m s have b e t t e r v i s c o e l a s t i c p r o p e r t i e s (dilatational modulus) (35) and can adapt t o p h y s i c a l p e r t u r b a t i o n s without rupture. This i s i l l u s t r a t e d by $ - l a c t o g l o b u l i n which forms s t r o n g v i s c o u s f i l m s w h i l e c a s e i n f i l m s show l i m i t e d v i s c o s i t y due to diminished protein-protein (electrostatic) interactions and l a c k of b u l k y s t r u c t u r e ( s t e r i c e f f e c t s ) which apparently improves i n t e r a c t i o n s a t the i n t e r f a c e (7,13,19). In the case o f the major c y t o p l a s m i c p r o t e i n of l e a v e s , ribulose 1,5-biphosphate carboxylase (RUBISCO), the surface r h e o l o g i c a l p r o p e r t i e s and foam s t a b i l i t y were maximum a t pH 5.5, c l o s e to the i s o e l e c t r i c point (pH 4.8) and a l l parameters measured were g r e a t e r than any o t h e r p r o t e i n s t u d i e d ( 2 5 ) . This may be r e l a t e d t o the l a r g e m o l e c u l a r s i z e of RUBISCO, i . e . 560 000 d a l t o n s , and i t s d i s u l f i d e s t a b i l i z e d g l o b u l a r s t r u c t u r e . H y d r o p h o b i c i n t e r a c t i o n s between p r o t e i n s and f a t s are v e r y c r i t i c a l i n emulsion formation. The e m u l s i f y i n g a c t i v i t i e s o f v a r i o u s p r o t e i n s e.g. BSA, t r y p s i n , o v a l b u m i n , and lysozyme have been r e p o r t e d t o be c o r r e l a t e d w i t h t h e i r a v e r a g e net hydrophob i c i t i e s (36). I t has been s u g g e s t e d t h a t the conformational properties i . e . ' f l e x i b i l i t y of whey p r o t e i n s are i m p o r t a n t i n a d s o r p t i o n and p o s s i b l y a f f e c t s t h e i r e m u l s i f y i n g a b i l i t y (37). Subsequent s t u d i e s r e v e a l e d t h a t the c o n f o r m a t i o n a l s t a b i l i t y of Ç > - l a c t o g l o b u l i n v a r i e d depending on pH i . e . i t s c o n f o r m a t i o n was more r i g i d and r e s i s t a n t t o d e n a t u r a t i o n a t pH 3 than a t pH 7 (38). The low e m u l s i f y i n g and s u r f a c e a c t i v i t y of b-Lg a t a c i d i c pH was assumed t o be due t o low d e n a t u r a b i l i t y ( ' f l e x i b i l i t y ' ) of the m o l e c u l e (38) and might be an i m p o r t a n t f a c t o r i n g o v e r n ing surface a c t i v e p r o p e r t i e s (14). 1

I t has been s u g g e s t e d t h a t protein ' f l e x i b i l i t y ' i s an important s t r u c t u r a l f a c t o r governing e m u l s i f y i n g and foaming properties (39). Chemically i n d u c e d c r o s s - l i n k i n g of BSA and lysozyme g r e a t l y r e d u c e d the foaming power and foam s t a b i l i t y o f b o t h p r o t e i n s and t h e r e was a s i m i l a r though l e s s marked change in emulsifying activity and emulsion s t a b i l i t y (40). The monomeric c r o s s - l i n k e d p r o t e i n s were r e s i s t a n t t o p r o t e o l y s i s and heat-induced conformational changes, s u g g e s t i n g that molecular f l e x i b i l i t y may p l a y a r o l e i n foaming and e m u l s i o n p r o p e r t i e s (40). Effect

of M o d i f i c a t i o n

on F i l m ,

Foam, a n d

Emulsion Properties

I n t e n t i o n a l m o d i f i c a t i o n of p r o t e i n s t r u c t u r e through a l t e r a t i o n s of p r o t e i n net c h a r g e , h y d r o p h o b i c i t y , h y d r o g e n b o n d i n g , and

39.

KINSELLA AND WHITEHEAD

Film, Foaming, and Emulsifying Properties

d i s u l f i d e b o n d i n g , p r o v i d e approaches f o r s t u d y i n g t h e i m p o r t a n c e of s p e c i f i c s t r u c t u r a l f e a t u r e s on f i l m s t r u c t u r e and s t a b i l i t y , foaming c a p a c i t y , and e m u l s i o n s t a b i l i z a t i o n ( 2 0 ) . M o d i f i c a t i o n o f Net Charge Succinylation: The n e t c h a r g e on p r o t e i n m o l e c u l e s a f f e c t s t h e s o l u b i l i t y and t h e e x t e n t o f 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 . Both are i m p o r t a n t i n a c h i e v i n g o p t i m a l f i l m and s u r f a c e p r o p e r t i e s (8,9). The s o l u b i l i t y o f a p r o t e i n r e s u l t s from an e q u i l i b r i u m between p r o t e i n - s o l v e n t and 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 ( 41 ). C o n d i t i o n s which f a v o r p r o t e i n - s o l v e n t i n t e r a c t i o n s g e n e r a l l y increases the s o l u b i l i t y of a p r o t e i n . Chemical d e r i v a t i z a t i o n of t h e £-amino groups o f l y s i n e r e s i d u e s i n proteins with s u c c i n i c a n h y d r i d e improves t h e f u n c t i o n a l i t y o f f o o d p r o t e i n s

(9). P r o g r e s s i v e s u c c i n y l a t i o n of g l y c i n i n or β - l a c t o g l o b u l i n i n c r e a s e d t h e amount o f u n o r d e r e d s t r u c t u r e , e l e c t r o n e g a t i v i t y (42), s p e c i f i c v i s c o s i t y (43), h y d r a t i o n and s o l u b i l i t y (44,)# and enhanced t h e foaming and emulsifying p r o p e r t i e s (44) o f t h e succinylated proteins. Film strength as r e f l e c t e d i n s u r f a c e y i e l d s t r e s s o f g l y c i n i n was i n c r e a s e d by moderate s u c c i n y l a t i o n (