Interactions of Food Proteins - American Chemical Society

Chapter 9

Surface Activity of Bovine Whey Proteins at the Phospholipid—Water Interface

Downloaded by OHIO STATE UNIV LIBRARIES on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch009

Donald G. Cornell Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118

The interaction of the major whey proteins of cows' milk with phospholipids in a monolayer was studied with a Langmuir Film Balance. The phospholipids were mixtures of phosphatidylcholine and phosphatidylglycerol. The ultraviolet and circular dichroism spectra of transferred films were used to determine the quantity and conformation of proteins interacting with the lipids in the monolayer. The proteins were dissolved in the aqueous subphase and migrated to the phospholipid/water interface where adsorption was controlled by the solution pH and calcium ion concentration. At neutral pH, above the isoionic point of a l l proteins studied, protein-phospholipid interaction was minimal. Some binding of protein to the lipids was observed when the subphase was at the isoionic pH of the protein. Increased lipid-protein binding was observed when the subphase was below the isoionic pH of the protein; under these conditions a film of protein about one molecule thick formed beneath the lipid monolayer. Calcium in the subphase interfered with lipid-protein binding, but the effect was pH dependent. Circular dichroism spectra of transferred films showed that the secondary structure of the proteins was largely preserved in the lipid-protein complexes. An electrostatic mechanism for lipid-protein interaction is discussed. Dispersed f a t p a r t i c l e s i n a s t a b l e system such as m i l k are prevented from c o a l e s c i n g by r e p u l s i v e f o r c e s between t h e i r s u r f a c e l a y e r s . Phospholipids a s s i s t i n s t a b i l i z This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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ing t h e l i p i d phase o f milk through formation o f a b i l a y e r f a t g l o b u l e membrane (FGM) on the s u r f a c e o f t h e o i l d r o p l e t s (1). During milk p r o c e s s i n g steps such as homogenization, the f a t g l o b u l e p a r t i c l e s i z e i s deceased and t h e o i l / w a t e r i n t e r f a c i a l area i s i n c r e a s e d . The f r e s h l y exposed o i l s u r f a c e i s able t o adsorb a d d i t i o n a l s u r f a c e a c t i v e agents such as p h o s p h o l i p i d s and p r o t e i n s to g i v e a s t a b i l e emulsion. Since there i s enough phos p h o l i p i d i n m i l k t o form a f i l m on a g r e a t l y expanded o i l / w a t e r i n t e r f a c e , t h i s l i p i d undoubtedly p l a y s an important r o l e i n s t a b i l i z i n g d a i r y and other food products which u t i l i z e homogenized milk. Although much work has been done on the s u r f a c e a c t i v i t y o f p r o t e i n s a t the o i l / w a t e r i n t e r f a c e , most o f t h e e f f o r t s have centered on the i n t e r a c t i o n of p r o t e i n s with n e u t r a l o i l (hydrocarbons, t r i g l y c e r i d e s ) s u r f a c e s ; i n t e r a c t i o n s with the p o l a r l i p i d s such as the p h o s p h o l i p i d s have r e c e i v e d much l e s s a t t e n t i o n . I n t e r a c t i o n between p h o s p h o l i p i d s and p r o t e i n s depends t o a degree on s o l u t i o n pH and mineral ( p r i n c i p a l l y calcium) content and on the proper t i e s o f the l i p i d and p r o t e i n s themselves. T h i s o f course i m p l i e s t h a t milk products, when used as a food a d d i t a t i v e , may e x h i b i t d i f f e r e n t f u n c t i o n a l s u r f a c e p r o p e r t i e s i n d i f f e r e n t foods. A c c o r d i n g l y we decided t o i n v e s t i g a t e t h e i n t e r a c t i o n o f p r o t e i n s with phospho l i p i d s over a range o f pH's and calcium i o n concen t r a t i o n s which were s e l e c t e d t o i n c l u d e the c o n d i t i o n s found i n a v a r i e t y o f foods. The c a s e i n s are the major milk p r o t e i n s used t o s t a b i l i z e food emulsions, however whey i s r e c e i v i n g increased a t t e n t i o n as a food a d d i t i v e . T h i s i s e s p e c i a l l y t r u e i n a c i d foods where the whey p r o t e i n s remain s o l u b l e . In such food uses, the end-use e n v i r o n ment o f whey can vary c o n s i d e r a b l y i e . pH, s a l t , mineral content, e t c . We are studying the i n t e r a c t i o n o f t h e major whey p r o t e i n s with f i l m forming substances such as p h o s p h o l i p i d s under a range o f s o l u t i o n c o n d i t i o n s t y p i c a l o f food systems. In t h i s work we r e p o r t on the i n t e r a c t i o n o f phospholipids i n monolayers with t h e major whey p r o t e i n s , b e t a - l a c t o g l o b u l i n (β-LG), alphal a c t a l b u m i n (α-LA), and bovine serum albumin (BSA). Mixtures o f p a l m i t o y l o l e o y l p h o s p h a t i d y l c h o l i n e (POPC) and p a l m i t o y l o l e o y l p h o s p h a t i d y l g l y c e r o l (POPG) were chosen t o simulate the z w i t t e r i o n i c (POPC) and charged (POPG) p h o s p h o l i p i d s of milk. The pH of t h e s o l u t i o n s ranged from 4.4 t o 7; the calcium c o n c e n t r a t i o n was 0 t o 8mM. These c o n d i t i o n s i n c l u d e the ranges o f pH and calcium i o n ( f r e e , uncomplexed Ca ) found i n many foods t o which m i l k or d a i r y products might be added. 2+

Experimental M a t e r i a l s . The phospholipids were from A v a n t i P o l a r L i p i d s , Birmingham, AL; the p r o t e i n s were from Sigma, S t .

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Louis, MO. The spreading s o l v e n t f o r the l i p i d s was ACS Reagent Grade chloroform, t r e a t e d t o remove s u r f a c e a c t i v e i m p u r i t i e s (2.). Walpole• s acetate (3) was used f o r pH 4.4 and 5.2, T r i s - H C l f o r pH 7. A low b u f f e r c o n c e n t r a t i o n (1-5 mM) was used t o minimize l i g h t s c a t t e r i n g i n the f i l m s used f o r u l t r a v i o l e t (UV) and c i r c u l a r d i c h r o i s m (CD) spectroscopy. The f o l l o w i n g are some r e l e v a n t p r o p e r t i e s of the p r o t e i n s used i n t h i s work. β-LG; molecular weight (MW), 18,300; i s o i o n i c p o i n t ( I ) , 5.1; molar a b s o r p t i v i t i e s (€ ) f o r the backbone (190 nm) band, 1.56 Χ 10 ; mean r e s i d u e a b s o r p t i v i t y f o r the 190 nm band (e ) , 9600 (7): α-LA; MW, 14,200; I , 4.4; e , 1.07 Χ 10 ; e , 8700: BSA; MW, 66,300; I, , 5.2; e , 5.06 X 10 , € , 8700. UV absorp t i v i t i e s f o r BSA and α-LA were determined i n the authors' l a b o r a t o r y (unpublished o b s e r v a t i o n s ) . A miniature T e f l o n trough (15 X 11 X 0.7 cm) with a d i p p i n g w e l l ( t o t a l depth 2 cm) was used f o r f i l m t r a n s f e r i n t h i s work. A magnetic s t i r r e r was used t o mix the p r o t e i n i n the subphase. The miniature trough was mounted i n the c r a d l e of a p r e v i o u s l y d e s c r i b e d (2) Langmuir balance from which the l a r g e trough had been removed. The b a r r i e r d r i v e , Wilhelmy p l a t e , constantpressure c o n t r o l , and f i l m t r a n s f e r were a l l provided by the l a r g e f i l m balance system. The experimental setup i s i l l u s t r a t e d s c h e m a t i c a l l y i n Figure 1. A complete d i s c u s s i o n of f i l m balance techniques has been given (4). ip

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Methods. The p r i n c i p l e o b j e c t i v e of t h i s work was t o estimate the amount and conformation of p r o t e i n i n t e r a c t ing with phospholipids a t the l i p i d / w a t e r i n t e r f a c e . The p r o t o c o l s f o r monolayer f i l m and p r o t e i n s o l u t i o n prepa r a t i o n , f i l m and s o l u t i o n s u r f a c e pressure measurements, and t r a n s f e r of the monolayers f o r s p e c t r o s c o p i c determi n a t i o n s have a l l been given i n extenso (2, 5, 6); the f o l l o w i n g i s a summary. The s u r f a c e pressures (surface t e n s i o n lowering) of the p r o t e i n s were determined by the Wilhelmy p l a t e technique on s t i r r e d s o l u t i o n s u s i n g thoroughly cleaned glassware. Techniques i n v o l v i n g the formation and handling of l i p i d monolayers were c a r r i e d out on the miniature f i l m balance. A s e r i e s of p r e l i m i n a r y experiments were run t o determine the optimum f i l m pressure, p r o t e i n c o n c e n t r a t i o n , e t c . , f o r UV and CD spectroscopy as f o l l o w s : (1)

(2)

The s u r f a c e pressures of β-LG, α-LA, and BSA were determined as a f u n c t i o n of c o n c e n t r a t i o n (up t o about 80 mg/L) on b u f f e r e d s o l u t i o n s . T h i s estab l i s h e d the maximum surface pressure t h a t the p r o t e i n s could achieve over the range of concen trations studied. Using the miniature f i l m balance, monolayers of POPG/POPC (35/65 mol%) were formed by spreading the premixed l i p i d from chloroform s o l v e n t onto a



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F i g u r e 1. Schematic diagram of a Langmuir-Blodgett f i l m balance showing p r o t e i n s adsorbed from s o l u t i o n onto the head groups of p h o s p h o l i p i d monolayers. The i n s e t shows a l i p i d - p r o t e i n monolayer being t r a n s f e r r e d t o the s u r f a c e of a quartz p l a t e f o r u l t r a v i o l e t or c i r c u l a r dichroism spectroscopy. E i g h t p l a t e s were used which gave a t o t a l of 16 monolayers f o r spectroscopic investigations.



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b u f f e r e d subphase. A f t e r the spreading s o l v e n t evaporated (15 min), the f i l m was compressed t o a pressure g r e a t e r than t h a t achieved by the 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 i n experiment (1) above. Then, with the compression b a r r i e r s stopped, p r o t e i n was i n j e c t e d i n t o the subphase beneath the l i p i d monolayer and the f i l m pressure was recorded f o r 30 min. Any pressure changes i n the f i l m u s u a l l y occurred during the f i r s t few minutes of a run with l i t t l e i f any change noted towards the end of the p e r i o d . A s e r i e s of such runs a t v a r y i n g p r o t e i n c o n c e n t r a t i o n e s t a b l i s h e d a pressure r i s e - p r o t e i n concentration p r o f i l e f o r that p a r t i c u l a r l i p i d monolayer/subphase combination. These p r o f i l e s were u s u a l l y i n the shape of a s h a r p l y r i s i n g pressure a t low p r o t e i n c o n c e n t r a t i o n followed by a p l a t e a u a t higher c o n c e n t r a t i o n . The knees of the plateaus g e n e r a l l y occurred between 2-3 mg/L p r o t e i n concentration. The pressure r i s e i n a l i p i d monolayer observed upon i n j e c t i n g p r o t e i n i n t o the subphase was recorded f o r a s e r i e s of l i p i d monolayers with d i f f e r e n t i n i t i a l pressures. S u f f i c i e n t p r o t e i n was i n j e c t e d i n t o the subphase t o g i v e a p r o t e i n c o n c e n t r a t i o n of 5 mg/L, which was w e l l i n t o the p l a t e a u r e g i o n e s t a b l i s h e d i n experiment (2) above. These experiments gave the pressure r i s e i n the l i p i d f i l m (upon i n j e c t i o n of p r o t e i n i n t o the subphase) as a f u n c t i o n of i n i t i a l l i p i d f i l m pressure ( j u s t before i n j e c t i o n of the p r o t e i n ) . T h i s allowed us t o estimate a " c r i t i c a l " i n i t i a l l i p i d pressure, above which no change i n the monolayer pressure was observed upon i n j e c t i o n of p r o t e i n i n t o the subphase. In a l l subsequent experiments, the i n i t i a l l i p i d pressure was kept above the " c r i t i c a l p r e s s u r e " t o prevent the p r o t e i n molecule from i n s e r t i n g a p o r t i o n of i t s e l f i n t o the monolayer. The e f f e c t of s o l u t i o n (subphase) pH and calcium c o n c e n t r a t i o n on l i p i d - p r o t e i n b i n d i n g i n the monolayers was s t u d i e d by spreading l i p i d onto subphases of d i f f e r e n t composition, i n j e c t i n g the p r o t e i n and then t r a n s f e r r i n g the f i l m t o quartz p l a t e s f o r UV and CD a n a l y s i s . E i g h t quartz p l a t e s were p a r t i a l l y immersed i n the subphase p r i o r t o spreading the l i p i d . A f t e r spreading the l i p i d , i n j e c t i n g the p r o t e i n , and e q u i l i b r a t i n g the f i l m f o r about 30 min., the monolayers were t r a n s f e r r e d t o the quartz p l a t e s (at constant f i l m pressure) and mounted i n the UV or CD instrument f o r s p e c t r o s c o p i c a n a l y s i s . The s p e c t r a l range was 180-350 nm f o r UV spectroscopy and 180-260 nm f o r CD s p e c t r o p o l a r i m e t r y . C o n t r o l experiments showed t h a t only p r o t e i n s c o n t r i b u t e d s i g n i f i c a n t l y to e i t h e r the UV or the CD s p e c t r a over the ranges s t u d i e d . A d d i t i o n a l d e t a i l s about equipment and methods f o r CD (5) and UV (7) spectroscopy have been given. In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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Adsorption o f the p r o t e i n s t o the quartz p l a t e s used f o r f i l m t r a n s f e r and t o the z w i t t e r i o n i c c h o l i n e was a n t i c i pated. Since we were i n t e r e s t e d i n a d s o r p t i o n only t o the charged l i p i d POPG, c o n t r o l experiments were run and c o n t r i b u t i o n s t o the UV s p e c t r a from p r o t e i n adsorbed t o the quartz p l a t e s and POPC was subtracted from the UV absorbance. For every UV experiment u s i n g POPC/POPG (65/35 mol%) there was a c o n t r o l run u s i n g i d e n t i c a l procedures (subphase pH, p r o t e i n c o n c e n t r a t i o n e t c . ) except f o r the l i p i d f i l m which was a monolayer o f POPC. In the case of calcium f r e e systems, the UV a b s o r p t i o n observed with the c o n t r o l f i l m s was about 20-30% o f t h a t observed with monolayers c o n t a i n i n g POPG; t h i s c o n t r i b u t i o n was subtracted before c a l c u l a t i n g the POPG-protein binding. L i g h t s c a t t e r i n g was n o t i c e a b l e i n f i l m s t r a n s f e r r e d from subphases c o n t a i n i n g 10 mM sodium c h l o r i d e ; we employed a l o g - l o g e x t r a p o l a t i o n procedure t o c o r r e c t f o r s c a t t e r i n g c o n t r i b u t i o n t o the 190 nm p r o t e i n absorp t i o n band ( 8 ) . C o n t r i b u t i o n s from t h i s source were e s t i mated t o be about 20-30% of the t o t a l UV a b s o r p t i o n s i g n a l observed a t 190 nm with 10 mM sodium c h l o r i d e i n the subphase. R e s u l t s and D i s c u s s i o n The r e s u l t s discussed here were f i r s t p u b l i s h e d sepa r a t e l y f o r B-LG (6) and f o r α-LA and BSA ( C o r n e l l , D. G.; Patterson, D. L.; Hoban, N.; C o l l o i d I n t e r f a c e S c i . . 1990, i n press.) Together these two r e p o r t s g i v e a p i c t u r e o f a l i p i d - p r o t e i n - c a l c i u m i n t e r a c t i o n scheme which f o l l o w s mass a c t i o n p r i n c i p l e s and i n v o l v e s an e l e c t r o s t a t i c mechanism. Of p a r t i c u l a r importance are the e l e c t r i c a l p r o p e r t i e s of the p r o t e i n s i n r e l a t i o n t o the s o l u t i o n pH as d i s c u s s e d below. The purpose of steps 1-3 above was t o e s t a b l i s h c o n d i t i o n s under which we were c o n f i d e n t t h a t the pro t e i n s were i n t e r a c t i n g with only the head groups o f the l i p i d s while a t the same time a c h i e v i n g maximum l i p i d p r o t e i n complexation. Step 1 e s t a b l i s h e d the maximum s u r f a c e pressure the v a r i o u s p r o t e i n s c o u l d support a t the air/water i n t e r f a c e over the c o n c e n t r a t i o n range s t u d i e d . These r e s u l t s are shown i n F i g u r e 2. In step 2, where p r o t e i n was i n j e c t e d beneath a l i p i d monolayer, the i n i t i a l l i p i d f i l m pressure was higher than the pressure achieved by p r o t e i n s a t the a i r / w a t e r i n t e r f a c e . T h i s e f f e c t i v e l y suppressed the formation o f patches o f pure p r o t e i n s i n a monolayer of l i p i d . Any f i l m pressure r i s e observed upon i n j e c t i n g p r o t e i n i n t o the subphase was taken as evidence of i n s e r t i o n of a p o r t i o n o f the p r o t e i n molecule i n t o the l i p i d monolayer i n an i n t i m a t e l i p i d p r o t e i n mixture. The r e s u l t s are shown i n F i g u r e 3. In t h i s step we were a l s o able t o e s t a b l i s h the


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F i g u r e 2. The s u r f a c e pressure (surface t e n s i o n lowering) of p r o t e i n s i n Walpole's acetate pH 4.4 as a function of concentration. 20

Protein Concentration (mg/L)

F i g u r e 3. The r i s e i n s u r f a c e pressure of a phosphol i p i d (POPC/POPG, 65/35 mol%) monolayer upon i n j e c t i o n of p r o t e i n i n t o the u n d e r l y i n g subphase. The i n i t i a l pressure o f the l i p i d f i l m s was 22 mN/m. Subphase; Walpole's acetate pH 4.4.



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" s a t u r a t i o n " c o n c e n t r a t i o n f o r the p r o t e i n s ; the concen t r a t i o n (or narrow range of concentrations) above which no f u r t h e r evidence of l i p i d - p r o t e i n i n t e r a c t i o n was observed. Despite the v a r i a b i l i t y i n the pressure r i s e shown by the three p r o t e i n s i n Figure 3, " s a t u r a t i o n " c o n c e n t r a t i o n appears t o occur between 2 t o 5 mg/L p r o t e i n c o n c e n t r a t i o n i n the subphase f o r a l l p r o t e i n s . In a l l subsequent experiments, s u f f i c i e n t p r o t e i n was i n j e c t e d i n t o the subphase t o achieve a c o n c e n t r a t i o n of 5mg/L. The magnitude of the pressure r i s e observed i n the l i p i d monolayer upon i n j e c t i o n of p r o t e i n i n t o the subphase depended upon the value of the i n i t i a l monolayer pressure. When the i n i t i a l l i p i d pressure was above a c e r t a i n " c r i t i c a l " value, l i t t l e or no change i n the pressure of the l i p i d monolayer was observed upon i n j e c t i o n of p r o t e i n i n t o the subphase. T h i s was taken t o mean t h a t i n s e r t i o n of the p r o t e i n i n t o the monolayer was e f f e c t i v e l y suppressed. The " c r i t i c a l pressure" was estimated by e x t r a p o l a t i n g a p l o t of pressure r i s e vs. i n i t i a l pressure t o zero pressure r i s e as shown i n F i g u r e 4. While the p e n e t r a t i o n of p r o t e i n i n t o the l i p i d por t i o n of the o i l / w a t e r i n t e r f a c e i s c e r t a i n l y an i n t e r e s t ing mechanism f o r the s t a b i l i z a t i o n of emulsions, we wished t o study 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 a t the o i l / w a t e r i n t e r f a c e without the complications of p r o t e i n insertion. For a l l subsequent experiments r e p o r t e d here 35 mN/m was the i n i t i a l l i p i d pressure. In a l l these cases the change i n the f i l m pressure (±) a f t e r i n j e c t i o n of the p r o t e i n was l e s s than 1/2 mN/m f o r the d u r a t i o n of each run, about 30-45 min. The amount of p r o t e i n i n t e r a c t i n g with the phospho l i p i d s i n monolayers at v a r i o u s subphase pH's was d e t e r mined from the UV s p e c t r a of the t r a n s f e r r e d monolayers u s i n g what i s e s s e n t i a l l y Beer's Law f o r f i l m s , namely: A = η€ Γ/6.02 Χ 10

20

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Μ

where A i s the absorbance of η monolayers, € i s the molar a b s o r p t i v i t y of the p r o t e i n , and Γ i s the number of p r o t e i n molecules per square centimeter i n a s i n g l e mono l a y e r . The f a c t o r ηΓ/6.02 Χ ΙΟ has the same dimension a l i t y (mass per squared length) and numerical value as the l c product (path length i n centimeters times concen t r a t i o n i n moles per l i t e r ) of the c l a s s i c a l Beer's law f o r s o l u t i o n s . A s t r a i g h t f o r w a r d c a l c u l a t i o n then gave the s u r f a c e c o n c e n t r a t i o n i n m i l l i g r a m s of p r o t e i n per square meter of monolayer as presented i n Table I. The r e s u l t s i n Table I c l e a r l y show the e f f e c t of pH on p r o t e i n b i n d i n g t o the monolayer l i p i d s and supports an e l e c t r o s t a t i c mechanism f o r l i p i d - p r o t e i n i n t e r a c t i o n . At a c i d pH's the p r o t e i n s c a r r y a s i g n i f i c a n t p o s i t i v e charge over t h e i r s u r f a c e which can bind t o the negative l y charged l i p i d . At n e u t r a l pH where both the l i p i d s M

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F i g u r e 4. The i n c r e a s e i n l i p i d monolayer pressure upon i n j e c t i n g p r o t e i n i n t o the subphase as a f u n c t i o n of t h e i n i t i a l f i l m pressure. Protein concentration i n t h e pH 4 . 4 subphase, 5 mg/L.


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Table I. Protein Concentration i n Phospholipid-Protein Monolayers Protein Subphase

Concentration Units2 2

mg/m


Tris pH 7.0

2

1

1

Walpole s Acetate pH 4.4 Walpole*s Acetate pH 4.4 10 mM NaCl 1

2

0.09

molec./cm

Walpole s Acetate pH 5.2

B-LG

α-LA

BSA

[.05]

2

8 Χ 10

2

mg/m

10

1.1 [.2] 2

molec./cm

1.0 X 10

2

mg/m

2

2.8 Χ 10

2

mg/m

12

1.9 [.4] 2

molec./cm

0

0.4 [.1]

0

1.3 Χ 10

—

12

—

12

3.1 [.3]

molec./cm

1

1.7 Χ 10

12

2.6 [.6] 1.1 X 10

13

3.0 [.2] 1.0 X 10

0.9 [.3] 3.7 Χ 10

13

-

12

Numbers i n brackets are standard errors of 3-4 replicates. Errors i n the molec./cm u n i t s have been omitted f o r c l a r i t y but are p r o p o r t i o n a l l y i d e n t i c a l to the errors for the corresponding mg/M u n i t s . 2

2

and p r o t e i n s c a r r y a net negative charge there i s e l e c t r o s t a t i c r e p u l s i o n and a reduced tendency t o i n t e r a c t . BSA shows t h i s tendency most c l e a r l y . A t pH 7, l i t t l e i f any l i p i d - p r o t e i n complex i s formed (.09 mg/M i s o f d o u b t f u l s t a t i s t i c a l s i g n i f i c a n c e ) . A t the i s o i o n i c p o i n t of BSA, pH 5.2, i t i s c l e a r t h a t the p r o t e i n i s i n t e r a c t i n g with the l i p i d . A t a more a c i d pH, 4.4, the coverage has reached 3.1 mg/M . A c o n c e n t r a t i o n t h i s high suggests the formation of a contiguous f i l m o f p r o t e i n i n i t s n a t i v e (globular) s t a t e one molecular l a y e r t h i c k . Values of t h i s order have been reported f o r many p r o t e i n s adsorbed at the air/water i n t e r f a c e (mi g r a t i n g from bulk s o l u t i o n ) , where the measurement t e c h niques were e l l i p s o m e t r y (9) and s u r f a c e r a d i o a c t i v i t y (10). Spread f i l m s , formed by d i r e c t a p p l i c a t i o n of the p r o t e i n from s o l u t i o n onto the s u r f a c e of water, gen e r a l l y have lower s u r f a c e concentrations than adsorbed f i l m s a t e q u i v a l e n t surface pressures (11). I t i s thought t h a t some degree of u n f o l d i n g occurs i n a p r o t e i n molecule when i t i s a p p l i e d t o the s u r f a c e of water, s u b j e c t t o c o n s t r a i n t s such as i n t r a m o l e c u l a r d i s u l f i d e 2

2


132


c r o s s l i n k i n g (11). In e a r l i e r work, an i n c r e a s e i n beta sheet was observed i n β-LG spread at the air/water i n t e r face (2). In the c u r r e n t work, no such change i n the secondary s t r u c t u r e of any of the p r o t e i n s was found. The CD s p e c t r a of the p r o t e i n s i n the l i p i d - p r o t e i n mono l a y e r s (Figure 5) were s i m i l a r t o t h e i r s p e c t r a i n s o l u t i o n as determined i n the a u t h o r s l a b o r a t o r y . This suggests t h a t there was no gross changes i n any of the p r o t e i n s secondary s t r u c t u r e upon i n t e r a c t i n g with the phospholipids i n the monolayers. T h i s i s c o n s i s t e n t with the p i c t u r e of g l o b u l a r p r o t e i n s adsorbing (without un f o l d i n g ) t o the phospholipids i n a monolayer. Perhaps the most i n t e r e s t i n g f i n d i n g presented i n Table I i s the amount of complexation t h a t occurs with a l i p i d by a p r o t e i n i n i t s z w i t t e r i o n i c s t a t e . T h i s i s shown by the f i l m concentrations f o r BSA a t pH 5.2 and α-LA a t pH 4.4. At f i r s t s i g h t t h i s observation seems t o run counter t o an e l e c t r o s t a t i c mechanism f o r l i p i d protein i n t e r a c t i o n , since a protein at i t s i s o i o n i c p o i n t has a net n e u t r a l charge. I f the charge d i s t r i b u t i o n i s homogeneous over the surface of such a p r o t e i n , then the molecule w i l l appear t o be n e u t r a l even a t very c l o s e range. As the three dimensional s t r u c t u r e of more and more p r o t e i n s are determined however, i t i s becoming i n c r e a s i n g l y c l e a r t h a t the charge d i s t r i b u t i o n over the s u r f a c e of most p r o t e i n s i s anything but homogeneous. C o n s i s t e n t with t h i s are e l e c t r o s t a t i c f i e l d c a l c u l a t i o n s (12-15) which show t h a t c l u s t e r s of b a s i c and a c i d i c r e s i d u e s commonly form l a r g e p o t e n t i a l envelopes over the p r o t e i n s u r f a c e . The patches of p o s i t i v e and negative charges w i l l cause s i g n i f i c a n t i n t e r a c t i o n when they are brought i n t o c l o s e enough proximity t o a charge of oppo s i t e s i g n , Lowering the pH of the aqueous phase below the i s o i o n i c p o i n t of the p r o t e i n w i l l g i v e i t a net p o s i t i v e charge, and the t o t a l area of the p o s i t i v e patches w i l l grow. T h i s should f a c i l i t a t e docking of the g l o b u l a r p r o t e i n s t o the n e g a t i v e l y charged l i p i d s , r e s u l t i n g i n increased adsorption of the p r o t e i n s at the o i l / w a t e r i n t e r f a c e . A l l of t h i s i s c o n s i s t e n t with the r e s u l t s presented i n Table I. Calcium ions can a l s o bind t o n e g a t i v e l y charged phospholipids and thus compete with p r o t e i n s i n a l i p i d p r o t e i n - c a l c i u m i n t e r a c t i o n scheme. Since calcium i s a major component of milk and many other foods, knowledge of the r o l e i t plays i n the adsorption of p r o t e i n s a t the o i l / w a t e r i n t e r f a c e i s e s s e n t i a l t o the understanding of emulsion s t a b i l i z a t i o n i n such complex systems. We have s t u d i e d the e f f e c t of calcium on l i p i d - p r o t e i n i n t e r a c t i o n i n s e v e r a l of the systems l i s t e d i n Table I simply by adding calcium t o the subphase b u f f e r before spreading the l i p i d . Except f o r calcium i n the b u f f e r s , a l l other c o n d i t i o n s and procedures were as i n step 4 above under methods. The amount of p r o t e i n complexing with l i p i d s i n the presence of calcium was determined from the UV spec-


1



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133

t r a o f t r a n s f e r r e d f i l m s and formula 1 above. The q u a n t i t y o f p r o t e i n so obtained was compared t o the v a l u e s obtained f o r the same system i n the absence o f calcium (Table I ) , and p l o t t e d as the percent POPG/ p r o t e i n complex v s . calcium c o n c e n t r a t i o n as shown i n F i g u r e 6. The s o l i d l i n e s represent the best f i t t o the d i s c r e t e data p o i n t s based on the assumption o f s a t u r a t i o n b i n d i n g over the e n t i r e range o f calcium concentra t i o n s s t u d i e d . In the case of BSA a t pH 4.4 t h i s r e s u l t e d i n a r e l a t i v e l y poor f i t a t the lowest concen trations. Due t o the v a r i a b i l i t y i n the data, t h e s i g n i f i c a n c e o f t h i s "poor f i t " i s u n c e r t a i n . Because o f t h i s we have chosen t o d i s c u s s the data only i n terms o f o v e r a l l trends (see below). When e i t h e r α-LA or BSA was i n j e c t e d i n t o a subphase near the r e s p e c t i v e p r o t e i n s i s o i o n i c p o i n t , calcium ions a t the micromolar l e v e l i n t e r f e r e d with l i p i d p r o t e i n b i n d i n g . T h i s i s shown by the steep curves near the o r d i n a t e i n Figure 6 where the subphase b u f f e r i s pH 4.4 f o r α-LA and pH 5.2 f o r BSA. These r e s u l t s imply t h a t the apparent d i s s o c i a t i o n constant o f the l i p i d calcium complex i s o f the order o f micromoles o r l e s s . When the pH o f the subphase was below the i s o i o n i c p o i n t of the p r o t e i n , m i l l i m o l a r l e v e l s o f calcium were r e q u i r e d t o suppress the l i p i d - p r o t e i n i n t e r a c t i o n . This i s shown by the upper curves i n F i g u r e 6 where the subphase b u f f e r i s pH 4.4 f o r both B-LG and BSA. These r e s u l t s i n t u r n imply t h a t when the p r o t e i n c a r r i e s a net p o s i t i v e charge, i t i s b e t t e r able t o compete with the calcium ions f o r complexation t o the l i p i d . Assuming t h a t calcium binds t o POPG i n a 1/1 complex as found by Lau e t a l . (16), we can summarize t h e b i n d i n g of p r o t e i n and calcium t o monolayer l i p i d by Equation 2, 1

P

z+

+

nCaL

+

Interactions of Food Proteins - American Chemical Society

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