Proteins at Interfaces - American Chemical Society

Chapter 11

Consequences of Protein Adsorption at Fluid Interfaces F.

MacRitchie

Wheat Research Unit, Commonwealth Scientific and Industrial Research Organization,

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 16, 2016 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch011

P.O. Box 7, North Ryde 2113, Australia

The consequences of protein adsorption are discussed in terms of (a) the changes in structure, properties and reactivity of adsorbed protein and (b) the role played by adsorbed protein in phenomena where interfaces exert an influence. Discussion is mainly restricted to the air/aqueous interface. The changes in configuration of proteins on adsorption and the topics of interfacial coagulation, desorption from monolayers and the applicability of the Gibbs Adsorption Equation are considered with reference to the fundamental question of reversibility of adsorption. Some of the evidence which has been used in support of irreversibility can be rationalized on the basis of a reversible process. Studies of protein monolayers reflect a flexible chain configuration where the behavior is governed by segments of the molecule, usually of 6-10 amino acid residues in size, rather than whole rigid molecules. Some of the phenomena in which protein adsorption is implicated include the fluidity of interfaces, the precipitation of proteins from solution by shaking, the formation and stability of foams and emulsions, bacterial adhesion and the reactivity of enzymes. The consequences o f p r o t e i n a d s o r p t i o n may be c o n v e n i e n t l y d i s c u s s e d i n terms of (a) t h e e f f e c t s o f a d s o r p t i o n on t h e s t r u c t u r e and p r o p e r t i e s o f t h e p r o t e i n i t s e l f , and (b) t h e i n f l u e n c e on v a r i o u s phenomena i n w h i c h interfacial b e h a v i o r p l a y s an i m p o r t a n t r o l e . T h i s review w i l l focus on s t u d i e s a t t h e air/aqueous interface, a l t h o u g h c o n c l u s i o n s w i l l i n many c a s e s be a p p l i c a b l e t o o t h e r t y p e s of i n t e r f a c e . The a i r / a q u e o u s i n t e r f a c e has c e r t a i n a d v a n t a g e s experimentally. In contrast t o i n t e r f a c e s with s o l i d s , i t i s p o s s i b l e t o m a n i p u l a t e m o l e c u l e s by u s e o f a f i l m b a l a n c e a n d measure f i l m p r o p e r t i e s and t h e k i n e t i c s o f v a r i o u s p r o c e s s e s a s a 0097-6156/87/0343-0165$06.00/0 © 1987 American Chemical Society

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

166

PROTEINS AT INTERFACES

f u n c t i o n of p r e c i s e l y determined two-dimensional concentrations. In p a r t i c u l a r , t h e s u r f a c e p r e s s u r e , which i s a measure o f changes i n s u r f a c e f r e e energy t h a t r e s u l t from adsorption, i s a fundamental parameter t h a t can be measured s i m p l y and a c c u r a t e l y .


E f f e c t s of Adsorption

on P r o t e i n S t r u c t u r e and F u n c t i o n

One o f t h e s t r i k i n g f e a t u r e s o f p r o t e i n a d s o r p t i o n i s t h a t many p r o t e i n s , a l t h o u g h h i g h l y s o l u b l e i n aqueous s o l u t i o n , f o r m f i l m s which a r e e x t r e m e l y s t a b l e and d i f f i c u l t t o desorb. This property was h i g h l i g h t e d by Langmuir and S c h a e f f e r who a p p l i e d t h e Gibbs A d s o r p t i o n E q u a t i o n i n i t s s i m p l e form dJI/d l n c = c kT b s

(1)

t o c a l c u l a t e t h e i n c r e a s e i n s o l u b i l i t y t h a t s h o u l d accompany compression of a surface f i l m of p r o t e i n . Here, Π i s t h e s u r f a c e p r e s s u r e , C t h e number o f m o l e c u l e s per u n i t area of the surface and C^ t h e c o n c e n t r a t i o n i n s o l u t i o n i n e q u i l i b r i u m w i t h t h e s u r f a c e film. F o r a p r o t e i n o f m o l e c u l a r w e i g h t 35,000 (ovalbumin), i t was c a l c u l a t e d (J_) t h a t an i n c r e a s e i n Π o f 15 mNrn" s h o u l d i n c r e a s e t h e s o l u b i l i t y o f t h e f i l m by a f a c t o r o f 1 0 . Because l i t t l e tendency f o r p r o t e i n s t o d i s s o l v e i s normally o b s e r v e d on compression of t h e i r s u r f a c e f i l m s t o surface pressures of the order o f 20 mNm" , t h i s was i n t e r p r e t e d t o mean t h a t p r o t e i n a d s o r p t i o n was n o t a r e v e r s i b l e p r o c e s s a n d t h e r e f o r e t h e G i b b s E q u a t i o n was not applicable. g

1

9 5

1

S i n c e t h e q u e s t i o n o f w h e t h e r p r o t e i n a d s o r p t i o n c a n be c o n s i d e r e d t o be a r e v e r s i b l e p r o c e s s i s b a s i c t o an u n d e r s t a n d i n g of p r o t e i n behavior a t i n t e r f a c e s , i t i s proposed t o d i s c u s s t h e p r o b l e m i n some d e t a i l . A p a r t f r o m t h e l o s s o f s o l u b i l i t y on a d s o r p t i o n , o t h e r o b s e r v a t i o n s t h a t have been i n t e r p r e t e d as evidence f o r i r r e v e r s i b i l i t y a r e : 1. P r o t e i n s , on a d s o r p t i o n a t f l u i d i n t e r f a c e s , undergo a change f r o m t h e i r g l o b u l a r c o n f i g u r a t i o n i n s o l u t i o n t o an e x t e n d e d c h a i n s t r u c t u r e . T h i s has o f t e n been r e f e r r e d t o as s u r f a c e denaturation. 2. An i n s o l u b l e coagulum i s f r e q u e n t l y formed when p r o t e i n mono l a y e r s a r e compressed t o h i g h s u r f a c e p r e s s u r e s , when p r o t e i n s a d s o r b a t q u i e s c e n t i n t e r f a c e s o r when p r o t e i n s o l u t i o n s a r e shaken. 3. In c e r t a i n cases, l o s s e s of b i o l o g i c a l a c t i v i t y (enzymatic, immunological) have been r e p o r t e d as a r e s u l t o f a d s o r p t i o n . On t h e o t h e r h a n d , w e l l - d e f i n e d a d s o r p t i o n i s o t h e r m s o f p r o t e i n s have been r e p o r t e d . F i g u r e 1 shows one example, t h a t f o r c h y m o t r y p s i n i n p u r e w a t e r a t 20°C. The a t t a i n m e n t o f s t e a d y s u r f a c e pressure v a l u e s , which i n c r e a s e with i n c r e a s i n g p r o t e i n c o n c e n t r a t i o n i n s o l u t i o n i n d i c a t i n g t r u e e q u a l i z a t i o n o f b u l k and surface chemical p o t e n t i a l s , argues i n favor of a r e v e r s i b l e adsorption process. In a d d i t i o n , d e s o r p t i o n from p r o t e i n monolayers has been measured. How t o r a t i o n a l i z e t h e s e a p p a r e n t l y c o n f l i c t i n g r e s u l t s t h e r e f o r e p r e s e n t s an i n t r i g u i n g c h a l l e n g e .


11.

Protein Adsorption at Fluid Interfaces

MACRITCHIE

S t r u c t u r e of P r o t e i n F i l m s . Spread monolayers of p r o t e i n s have pressure-area ( Π -A) curves which are generally similar, e x t r a p o l a t i n g t o an a r e a c l o s e t o 1.0 m mg~ a t Π = 0 . I t c a n be c a l c u l a t e d t h a t the t h i c k n e s s of the monolayer at t h i s area c o r r e s p o n d s t o about 1θ8. The e x a c t c o n f i g u r a t i o n h a s n o t b e e n resolved. On e n e r g e t i c g r o u n d s , i t i s e x p e c t e d t h a t the p o l y p e p t i d e backbone l i e s i n t h e p l a n e of t h e s u r f a c e w i t h p o l a r and non p o l a r s i d e c h a i n s d i r e c t e d t o w a r d s and away f r o m t h e a q u e o u s phase respectively. The s u r f a c e i n t h i s way a c t s as a g o o d s o l v e n t , i n t h i s c a s e a t w o - d i m e n s i o n a l one. When a p r o t e i n m o l e c u l e adsorbs, an a r e a of h i g h s u r f a c e f r e e energy i s r e p l a c e d by i n t e r f a c e s of low f r e e energy; i.e. p o l a r s i d e c h a i n s / w a t e r and non p o l a r s i d e c h a i n s / a i r . The a c h i e v e m e n t of t h i s f r e e energy l o w e r i n g accounts f o r the u n f o l d i n g of the m o l e c u l e at the s u r f a c e . A number of e x p e r i m e n t a l techniques have been i n t r o d u c e d to i n v e s t i g a t e t h e c o n f o r m a t i o n of p r o t e i n monolayers a f t e r removal from the s u r f a c e , e i t h e r as c o l l a p s e d o r u n c o l l a p s e d films. M a l c o l m (2) has p r e s e n t e d e v i d e n c e t o show t h a t s u r f a c e f i l m s o f a range of p o l y p e p t i d e s were i n the α - h e l i c a l c o n f o r m a t i o n , u s i n g i n f r a r e d spectroscopy, e l e c t r o n d i f f r a c t i o n and d e u t e r i u m exchange. O t h e r s t u d i e s of removed p r o t e i n f i l m s have a l s o s u p p o r t e d the p r e s e n c e of t h e α - h e l i x , using i n f r a r e d spectroscopy (_3), optical r o t a r y d i s p e r s i o n and circular dichroism (4,5). I t must be r e m e m b e r e d , h o w e v e r , t h a t w h e r e a s p o l y p e p t i d e c h a i n s may exist e x c l u s i v e l y i n the α - h e l i x form, p r o t e i n s i n t h e i r s o l u t i o n s t a t e g e n e r a l l y have t h e i r c h a i n s o n l y p a r t i a l l y i n t h i s form with r e l a t i v e l y l a r g e r o r s m a l l e r p r o p o r t i o n s i n t h e b e t a form o r random structure. F u r t h e r m o r e , because t h e a d s o r p t i o n s t e p r e p r e s e n t s a t r a n s i t i o n from a r e l a t i v e l y poor s o l v e n t (water) t o a r e l a t i v e l y g o o d s o l v e n t ( a i r / w a t e r i n t e r f a c e ) , t h e r e d o e s n o t a p p e a r t o be a s t r o n g d r i v i n g force f o r r e t e n t i o n of h e l i c a l s t r u c t u r e . A number of experimental r e s u l t s p o i n t i n d i r e c t l y toward a p r e d o m i n a n t l y random c h a i n s t r u c t u r e ; i n p a r t i c u l a r , t h e s u r f a c e f l o w and t h e c o n f i g u r a t i o n a l changes on f i l m c o m p r e s s i o n . 2


167

1

Surface V i s c o s i t y of P r o t e i n Monolayers. F i g u r e 2 shows t h e surface v i s c o s i t y (η ) of a number o f p r o t e i n s and one p o l y a m i n o a c i d as a f u n c t i o n o f Π (6). The e x t r e m e l y h i g h s u r f a c e v i s c o e l a s t i c i t y of p r o t e i n m o n o l a y e r s a p p e a r s t o be more c h a r a c t e r i s t i c o f an i n t e r a c t i n g r a n d o m c h a i n s y s t e m t h a n an a r r a y o f rigid helices. The t h e o r y of s u r f a c e v i s c o s i t y of Moore and E y r i n g {!), b a s e d on the Theory of A b s o l u t e R e a c t i o n Rates, p o s t u l a t e s t h a t t h e f l o w o f a monolayer c o n s i s t s o f movements of f l o w u n i t s , n o r m a l l y m o l e c u l e s , f r o m one e q u i l i b r i u m p o s i t i o n t o a n o t h e r , over an i n t e r mediate a c t i v a t i o n energy b a r r i e r . The e q u a t i o n d e r i v e d f o r t h e c o e f f i c i e n t of s u r f a c e v i s c o s i t y ( Γ) ) i s g

η S

= h exp / A V

AG

+

kT

ΠΔΑ

Λ /

(2)

where h i s P l a n c k ' s c o n s t a n t and Δ G i s t h e a c t i v a t i o n f r e e e n e r g y f o r f l o w a t Π = o. A G i s made up o f two t e r m s : (a) t h e w o r k r e q u i r e d to form a hole i n the s u r f a c e s u f f i c i e n t l y l a r g e f o r the m o l e c u l e t o e n t e r , and (b) t h e w o r k r e q u i r e d t o move t h e m o l e c u l e i n t o the hole; t h i s t e r m i n c l u d e s t h e work n e c e s s a r y t o break a l l



168

20


z £15

10-

/

1-0

2-0

3-0

Log c as s u r f a c e p r e s s u r e ( Π ) c o n c e n t r a t i o n (c) f o r

F i g u r e 1. A d s o r p t i o n i s o t h e r m p l o t t e d a g a i n s t the l o g a r i t h m of the bulk c h y m o t r y p s i n i n pure w a t e r a t 20°C.

l-O-

12

8 IT

16

( m Ν rrf ) 1

F i g u r e 2. L o g a r i t h m o f s u r f a c e v i s c o s i t y as a f u n c t i o n o f s u r f a c e p r e s s u r e f o r s e v e r a l p r o t e i n s and one p o l y a m i n o a c i d a t pH 5.5. o, p o l y - D L - a l a n i n e ; • , human γ - g l o b u l i n ; o, p e p s i n ; A, b o v i n e serum a l b u m i n ; Δ lysozyme. (Reproduced w i t h p e r m i s s i o n from Ref. 6. C o p y r i g h t 1970 M a r c e l Dekker). f


11.

MAC RITCHIE

169


bonds f o r m e d w i t h n e i g h b o u r i n g molecules. ΠΔΑ i s t h e n t h e a d d i t i o n a l work t h a t h a s t o be done a g a i n s t t h e s u r f a c e p r e s s u r e Π t o c r e a t e t h e h o l e o f a r e a Δ A. Equation 2 p r e d i c t s that a p l o t of log r\ v s Π s h o u l d be l i n e a r , p e r m i t t i n g t h e c a l c u l a t i o n o f àG f r o m t h e i n t e r c e p t a t Π=0 a n d Δ Α f r o m t h e s l o p e . Table I summarizes d a t a f o r t h e r e s u l t s o f F i g u r e 2. I t i s a p p a r e n t f r o m t h e d a t a t h a t Δ Α , t h e ^ a r e a o c c u p i e d by t h e f l o w u n i t i s s i m i l a r f o r a l l p r o t e i n s (100-12 ) and c o r r e s p o n d s t o s e g m e n t s o f 6-8 a m i n o a c i d r e s i d u e s . J o l y ( 8 ) , u s i n g an independent approach, a l s o c a l c u l a t e d a s i m i l a r v a l u e f o r t h e a r e a of the elementary flow u n i t s of p r o t e i n monolayers. These c a l c u l a t i o n s suggest that molecules i n the monolayer are s u f f i c i e n t l y f l e x i b l e t h a t s e g m e n t s o f t h i s s i z e , on t h e a v e r a g e , move a s u n i t s . T h i s r e s e m b l e s t h e manner i n w h i c h l o n g c h a i n h y d r o c a r b o n s a p p e a r t o d i f f u s e i n s o l u t i o n (9). As a r e s u l t , àG and η a r e p r a c t i c a l l y i n d e p e n d e n t o f m o l e c u l a r weight.


s

g

Table

I.

Protein

C a l c u l a t e d V a l u e s o f Δ G and Δ Α f o r P r o t e i n s and one Polyamino a c i d

(kJmole "S

3

Polyalanine γ - Globulin Pepsin Bovine Albumin Lysozyme

ΔΑ

àG

Molecular weight (χίο"· )

69 69 67 66 65

1.5 160 34 70 15

2

a) 105 110 120 100 115

The i n f l u e n c e o f a d s o r b e d p r o t e i n s on i n t e r f a c i a l v i s c o s i t y i s r e l e v a n t t o t h e f l u i d i t y o f b i o l o g i c a l membranes. An u n u s u a l e f f e c t i s o b s e r v e d when l i p i d m o l e c u l e s a r e i n c o r p o r a t e d i n t o p r o t e i n m o n o l a y e r s , f i r s t r e p o r t e d by Schulman and R i d e a l (JJHAs t h e mixed f i l m i s compressed, η i n c r e a s e s n o r m a l l y b u t t h e n goes t h r o u g h a maximum, t h e r e a f t e r d e c r e a s i n g s h a r p l y w i t h f u r t h e r i n c r e a s e o f Π (6). E v i d e n t l y , above a c e r t a i n s u r f a c e d e n s i t y , l i p i d m o l e c u l e s d i s r u p t i n t e r a c t i o n s between p r o t e i n c h a i n s . The behavior i s completely r e v e r s i b l e , η i n c r e a s i n g as t h e s u r f a c e a r e a i s expanded. A l t e r a t i o n o f t h e c o m p o s i t i o n o f t h e mixed f i l m a n d t h e s u r f a c e d e n s i t y t h u s p r o v i d e s a v e r y s e n s i t i v e means f o r v a r y i n g t h e s u r f a c e f l u i d i t y , a method t h a t may w e l l be u t i l i z e d i n b i o l o g i c a l systems. Formation of b r i t t l e monolayers t h a t g r e a t l y i n c r e a s e t h e s u r f a c e v i s c o e l a s t i c i t y a l s o may o c c u r when p r o t e i n f i l m s a r e s p r e a d on s u b s o l u t i o n s c o n t a i n i n g low c o n c e n t r a t i o n s o f d i v a l e n t m e t a l i o n s (6) o r s i l i c i c a c i d ( J J O , a g a i n suggesting mechanisms f o r t h e d e l e t e r i o u s e f f e c t s o f t h e s e s p e c i e s on membrane function. s

C o n f i g u r a t i o n a l Changes i n Compressed F i l m s . When p r o t e i n monolayers are compressed, r e l a x a t i o n processes occur which are m a n i f e s t e d by d e c r e a s e s i n s u r f a c e a r e a , A ( i f Π i s k e p t constant) o r d e c r e a s e s i n Π (A c o n s t a n t ) . P r o v i d i n g the e f f e c t s of


170


d e s o r p t i o n o r s u r f a c e c o a g u l a t i o n ( d i s c u s s e d below) a r e s e p a r a t e d , t h e d e c r e a s e s a r e t o t a l l y r e c o v e r a b l e on expansion of the monolayer. A t h i g h s u r f a c e p r e s s u r e s ( Π > 20 mNrn" ) t h e s e r e v e r s i b l e c h a n g e s a r e l a r g e and c a n o n l y be r a t i o n a l i z e d i f i t i s a s s u m e d t h a t p o r t i o n s o f t h e m o l e c u l e s l e a v e and r e - e n t e r t h e s u r f a c e . I t h a s been w e l l e s t a b l i s h e d t h a t adsorbed l i n e a r polymer m o l e c u l e s may e x i s t as t r a i n s (segments a t t a c h e d t o the s u r f a c e ) , l o o p s (segments d i s p l a c e d i n t o the a d j a c e n t phase) and t a i l s (segments a t t h e ends o f m o l e c u l e s w h i c h a l s o t e n d t o be d i s p l a c e d f r o m t h e s u r f a c e ) . T h i s m o d e l a p p e a r s t o be a p p r o p r i a t e f o r p r o t e i n m o n o l a y e r s . By u s i n g a f i l m balance to study p r o t e i n monolayers at the a i r / w a t e r i n t e r f a c e , i t i s p o s s i b l e t o q u a n t i t a t i v e l y e v a l u a t e the e q u i l i b r i u m d i s t r i b u t i o n b e t w e e n a t t a c h e d and d i s p l a c e d s e g m e n t s a t a g i v e n surface pressure. Because of t h e h i g h s u r f a c e v i s c o e l a s t i c i t y i n t h e s e monolayers, the r e l a x a t i o n p r o c e s s e s a r e s u f f i c i e n t l y s l o w t o e n a b l e them t o be f o l l o w e d e x p e r i m e n t a l l y .


1

If, at a given surface pressure Π , A i s the i n i t i a l area of t h e p r o t e i n m o n o l a y e r b e f o r e any r e l a x a t i o n h a s o c c u r r e d a n d A i s t h e a r e a a f t e r e q u i l i b r i u m has been e s t a b l i s h e d , t h e n the r a t i o of a t t a c h e d t o d i s p l a c e d segments ( r ) i s g i v e n by Q

r

=

A/A -A

(3)

Q

T h i s a s s u m e s t h a t d i s p l a c e d s e g m e n t s make no c o n t r i b u t i o n t o t h e s u r f a c e a r e a o c c u p i e d by the m o l e c u l e s . The v a r i a t i o n of A and A as Π i s i n c r e a s e d i s i l l u s t r a t e d i n F i g u r e 3 f o r a m o n o l a y e r o f b o v i n e s e r u m a l b u m i n (BSA). The v a r i a t i o n o f r w i t h Π s h o u l d be g i v e n by an e q u a t i o n of t h e form (12) Q

r

=

exp

/ AG

- ΠΔΑ

( —

(4)

λ

-) J

ν kT where A G i s t h e d i f f e r e n c e i n f r e e e n e r g y b e t w e e n a t t a c h e d and d i s p l a c e d segments a t Π = 0 and ΠΔ A i s t h e a d d i t i o n a l f r e e energy r e q u i r e d by a d i s p l a c e d s e g m e n t t o e n t e r t h e s u r f a c e a t a f i n i t e v a l u e of Π , AA b e i n g the a r e a o c c u p i e d by the segment. P l o t s of l o g r v s . Π h a v e b e e n f o u n d t o be l i n e a r f o r a number o f p r o t e i n s (13), p e r m i t t i n g A G and AA t o be e v a l u a t e d f r o m the i n t e r c e p t at Π = 0 and t h e s l o p e r e s p e c t i v e l y . ^ V a l u e s o f A G v a r i e d f r o m 4.4 t o 8.8 kT and AA r a n g e d f r o m 90A t o 160A . The s i z e o f t h e segment i s s i m i l a r t o t h a t of t h e f l o w u n i t e s t i m a t e d from s u r f a c e v i s c o s i t y d a t a and c o r r e s p o n d s t o 6-10 amino a c i d r e s i d u e s . g

g

g

g

g

2

g

g

Surface Coagulation. During the p r e p a r a t i o n of s o l u t i o n s of c e r t a i n p r o t e i n s , i t i s o b s e r v e d t h a t an i n s o l u b l e p r e c i p i t a t e may form. T h i s o c c u r s as a r e s u l t o f i n t e r f a c i a l a c t i o n a n d i s a c c e n t u a t e d by e x c e s s i v e s h a k i n g . The m e c h a n i s m a p p e a r s t o be r o u g h l y as f o l l o w s . As f r e s h i n t e r f a c e i s f o r m e d , p r o t e i n m o l e c u l e s adsorb and u n f o l d t o an extended c h a i n c o n f o r m a t i o n . At a given s u r f a c e d e n s i t y , the s o l u b i l i t y l i m i t of the p r o t e i n a t the s u r f a c e i s r e a c h e d and p r e c i p i t a t i o n o c c u r s . This i s a special case of p r e c i p i t a t i o n s i n c e i t i n v o l v e s a t r a n s i t i o n f r o m a twod i m e n s i o n a l (monolayer) to a t h r e e - d i m e n s i o n a l s t a t e (coagulated protein). The s o l u b i l i t y l i m i t i s u s u a l l y c h a r a c t e r i z e d by a


11.

MACRITCHIE

171


c o a g u l a t i o n s u r f a c e pressure, Π , corresponding t o the e q u i l i b r i u m spreading pressure o f m o n o m e r i c compounds. At a quiescent i n t e r f a c e , t h e c o a g u l u m r e d u c e s t h e a v a i l a b l e i n t e r f a c e by i t s p r e s e n c e and a l s o p r e v e n t s d i f f u s i o n o f m o l e c u l e s t o i t ; t h u s , t h e coagulated f i l m tends to a l i m i t i n g thickness. In the case of a solution that i s being agitated, fresh i n t e r f a c e i s c o n t i n u a l l y b e i n g c r e a t e d so t h a t s u r f a c e c o a g u l a t i o n p r o c e e d s a t a r a t e d e p e n d e n t on t h e d e g r e e o f a g i t a t i o n . I n t h i s way, much o f t h e p r o t e i n can be r e d u c e d t o an i n s o l u b l e form. An e q u a t i o n s i m i l a r t o t h a t used t o d e s c r i b e duplex f i l m s can be u s e d t o p r e d i c t t h e c o a g u l a t i o n p r e s s u r e , Π , at a given i n t e r f a c e (14).


c

i.e.

H

=

c

Y a

- ( y

b

+

y

a b

)

(5)

where y i s t h e i n i t i a l s u r f a c e f r e e e n e r g y , γ ^ t h e i n t e r f a c i a l f r e e e n e r g y b e t w e e n non p o l a r g r o u p s o f t h e m o n o l a y e r and t h e non aqueous phase and γ i s t h e i n t e r f a c i a l f r e e energy between p o l a r groups o f t h e monolayer and t h e aqueous phase. I t can be seen f r o m t h i s equation that, other c o n d i t i o n s being equal, Jl^ i s l o w e r e d as γ , the i n i t i a l i n t e r f a c i a l f r e e energy decreases. This i s c o n s i s t e n t w i t h t h e r e s u l t t h a t c o a g u l a t e d f i l m s form more e a s i l y a t o i l / a q u e o u s t h a n a t a i r / a q u e o u s i n t e r f a c e s . A wide v a r i a t i o n i n t h e ease o f s u r f a c e c o a g u l a t i o n i s o b s e r v e d from one p r o t e i n t o a n o t h e r and, f o r a g i v e n p r o t e i n , f r o m one s e t o f c o n d i t i o n s (pH, i o n i c s t r e n g t h ) t o another. In a c o m p a r a t i v e s t u d y (VS), ovalbumin was m o s t s u s c e p t i b l e f o l l o w e d by 3 -lactoglobulin, γ-globulin h e m o g l o b i n , m y o g l o b i n and l y s o z y m e . L e a s t s u s c e p t i b l e were c y t o c h r o m e C, α - c a s e i n a n d BSA. Those p r o t e i n s t h a t are l e a s t s u s c e p t i b l e t o s u r f a c e c o a g u l a t i o n t e n d t o have h i g h e r average h y d r o p h o b i c i t y as d e f i n e d by B i g e l o w (16), a l t h o u g h 3-lactoglobulin a p p e a r s t o be an e x c e p t i o n . I t may be t h a t t h e f a i l u r e o f t h e m o n o l a y e r t o p r e s e n t a s u f f i c i e n t l y non p o l a r s u r f a c e t o t h e non aqueous phase ( s i g n i f y i n g a h i g h v a l u e f o r γ^) i s an important f a c t o r c a u s i n g s u s c e p t i b i l i t y to surface coagulation. The s e n s i t i v i t y o f s u r f a c e c o a g u l a t i o n t o p r o t e i n s t r u c t u r e i s shown by t h e r e p o r t t h a t f u l l y o x y g e n a t e d s i c k l e c e l l h e m o g l o b i n (HbS) i s much more v u l n e r a b l e t o p r e c i p i t a t i o n by s h a k i n g t h a n n o r m a l h e m o g l o b i n (HbA) d e s p i t e t h e o t h e r w i s e g r e a t s i m i l a r i t y i n s t r u c t u r e and p r o p e r t i e s (17,18). a

a

b

a

Desorption

and

the Question

of

Reversibility

A number o f i m p o r t a n t q u e s t i o n s r e v o l v e a r o u n d t h e c e n t r a l one o f r e v e r s i b i l i t y of p r o t e i n adsorption. Are p r o t e i n s , once a d s o r b e d , able to desorb? Why a r e p r o t e i n s so d i f f i c u l t t o r e m o v e f r o m a surface? What happens i n t h e s i t u a t i o n where t h e r e i s c o m p e t i t i v e a d s o r p t i o n between d i f f e r e n t p r o t e i n s o r between p r o t e i n s and o t h e r species? Do t h e d i f f e r e n t p o i n t s on an a d s o r p t i o n isotherm c o r r e s p o n d t o dynamic e q u i l i b r i a ? I f a p r o t e i n m o l e c u l e can d e s o r b , does i t r e v e r t t o i t s o r i g i n a l s o l u t i o n c o n f i g u r a t i o n and r e c o v e r its original biological activity? Can t h e G i b b s Adsorption E q u a t i o n be a p p l i e d t o p r o t e i n a d s o r p t i o n ? Some of t h e s e q u e s t i o n s may be e f f e c t i v e l y t a c k l e d by s t u d i e s w i t h t h e f i l m b a l a n c e .



172


Langmuir and Waugh (V9_) were the f i r s t t o s t u d y t h e e f f e c t s o f compression on the s t a b i l i t y o f p r o t e i n monolayers. They d i s t i n g u i s h e d between p r e s s u r e d i s p l a c e m e n t and p r e s s u r e s o l u b i l i t y . Pressure displacement r e f e r s t o the r e v e r s i b l e e x p u l s i o n o f c h a i n segments, d i s c u s s e d above, while pressure s o l u b i l i t y i s the d e s o r p t i o n of complete molecules. Pressure s o l u b i l i t y o f the pure p r o t e i n s , i n s u l i n and o v a l b u m i n , was v e r y s m a l l . However, a f t e r enzyme p r o t e o l y s i s , p r e s s u r e s o l u b i l i t y i n c r e a s e d w i t h i n c r e a s i n g d i g e s t i o n t i m e and i n c r e a s i n g s u r f a c e p r e s s u r e . Based on a t h e o r y o f p r e s s u r e s o l u b i l i t y (20), i t was e s t i m a t e d t h a t the d e g r a d a t i o n p r o d u c t s o f i n s u l i n had m o l e c u l a r w e i g h t s i n the range 1,000-2,000. As a r e s u l t o f t h e r e v e r s i b l e r e l a x a t i o n p r o c e s s e s i n p r o t e i n monolayers, d e s o r p t i o n cannot be measured as f o r monomeric compounds by t h e l o s s o f s u r f a c e a r e a , m a i n t a i n i n g Π c o n s t a n t . However, because l o s s e s of a r e a o f p r o t e i n m o n o l a y e r s a r e t o t a l l y r e c o v e r a b l e a t l o w s u r f a c e p r e s s u r e s (10 mNm"* a n d b e l o w ) , Gonzalez and M a c R i t c h i e (2J_) i n t r o d u c e d a m e t h o d f o r m e a s u r i n g p e r m a n e n t a r e a l o s s e s based on m o n i t o r i n g the a r e a a t a r e f e r e n c e p r e s s u r e o f 5 mNm" . T h i s m e t h o d h a s s u b s e q u e n t l y b e e n u s e d (22,23) t o o b t a i n d a t a on p r o t e i n d e s o r p t i o n . The r e s u l t s o f t h e s e s t u d i e s may b e summarized as f o l l o w s . 1

1

C o n f i r m a t i o n of Desorption. Permanent a r e a l o s s e s a r e o b s e r v e d f o r p r o t e i n monolayers when kept a t h i g h Π (> 15 mNm"" ). A number o f independent checks have been used t o c o n f i r m t h a t t h e s e l o s s e s a r e caused by d e s o r p t i o n . Rates o f a r e a l o s s a r e enhanced by s t i r r i n g a n d d i m i n i s h e d b y t h e p r e s e n c e o f p r o t e i n i n t h e sub p h a s e (21). U s i n g r a d i o l a b e l l e d BSA, the c o m p r e s s i b i l i t y and t h e s p e c i f i c r a d i o a c t i v i t y o f the f i l m r e m a i n e d unchanged a f t e r e x t e n s i v e l o s s e s o f a r e a and t h e r a d i o a c t i v i t y s u b s e q u e n t l y measured i n t h e sub phase a c c o u n t e d w e l l f o r t h e amount o f m a t e r i a l l o s t f r o m t h e s u r f a c e (22). 1

A n a l y s i s of Desorption K i n e t i c s . S e v e r a l f e a t u r e s are e v i d e n t f r o m the k i n e t i c s o f desorption. P l o t s o f t h e l o g a r i t h m of the a r e a are a l i n e a r f u n c t i o n of the square r o o t of time i n agreement w i t h a d i f f u s i o n c o n t r o l l e d p r o c e s s governed by t h e e q u a t i o n :

η

=

2C

Q

Ç

Dt

γ

(6)

w h e r e η i s t h e number o f m o l e c u l e s t h a t d e s o r b i n a t i m e t , C i s the c o n c e n t r a t i o n ( i n molecules p e r u n i t volume) o f a t h i n s u b s u r f a c e l a y e r assumed t o be i n e q u i l i b r i u m w i t h t h e monolayer, D t h e d i f f u s i o n c o e f f i c i e n t and π = 3.14. This i s i l l u s t r a t e d i n F i g u r e 4 f o r d e s o r p t i o n of Β - l a c t o g l o b u l i n at d i f f e r e n t s u r f a c e pressures. F r o m F i g u r e 4, a n i n d u c t i o n p e r i o d o f 1-2 m i n i s e v i d e n t a t the commencement. D u r i n g t h i s p e r i o d , the monolayer i s r a p i d l y approaching i t s e q u i l i b r i u m configuration with respect to d i s p l a c e m e n t o f m o l e c u l a r segments. The r a t e o f d e s o r p t i o n i s g i v e n by d i f f e r e n t i a t i o n o f E q u a t i o n 6: Q


11.

30h

\

\ \A

\

^20 Ζ Ε


173


MACRITCHIE

0

\

\ \ \

\\ \\ \\ \\

10

1 50

ι 100

^

1 150

Area / molecule ( n m ) F i g u r e 3. E q u i l i b r i u m Π - Α curve o f BSA showing a r e a s o c c u p i e d by a t t a c h e d segments (A) and d i s p l a c e d segments (A ~A). Dashed l i n e i s the instantaneous Π-Α c u r v e ; i . e . t h a t w h i c h w o u l d be o b t a i n e d i f no r e l a x a t i o n o f monolayer. (Reproduced with p e r m i s s i o n from Ref. 25. C o p y r i g h t 1977 Academic P r e s s ) . 0

0-0-

4

t

l/2

8

F i g u r e 4. P l o t s o f t h e l o g a r i t h m o f t h e r a t i o o f t h e a r e a a t t i m e t t o t h e i n i t i a l a r e a as a f u n c t i o n of s q u a r e r o o t o f t i m e f o r 3 - l a c t o g l o b u l i n monolayers; o, 20 mNm" ; Δ , 25 mNm" ; O , 30 mNm" . (Reproduced w i t h p e r m i s s i o n from Ref. 23. Copyright 1985 Academic P r e s s ) . 1

1


1


174


dn/dt = C

/ D\

°

\ Tf)

t

(7)

The o t h e r c h a r a c t e r i s t i c t h a t can be deduced from e x p e r i m e n t a l d a t a i s t h a t p l o t s o f t h e r a t e a s a f u n c t i o n o f t ~ ^ ( E q u a t i o n 7) do n o t e x t r a p o l a t e t o z e r o r a t e a t i n f i n i t e t i m e as r e q u i r e d by a p u r e l y d i f f u s i o n c o n t r o l l e d p r o c e s s (21). T h i s i n d i c a t e s the presence o f a b a r r i e r t o the desorption step a t the i n t e r f a c e . This i s c o n f i r m e d by t h e v e r y much s m a l l e r v a l u e s f o r t h e e q u i l i b r i u m sub s u r f a c e c o n c e n t r a t i o n s c a l c u l a t e d from d e s o r p t i o n k i n e t i c s than t h o s e e x p e c t e d from t h e a d s o r p t i o n i s o t h e r m a t t h e same p r e s s u r e s . I f we assume t h a t t h e r a t e o f d e s o r p t i o n (dn/dt) i s c o n t r o l l e d by two b a r r i e r s , t h e d i f f u s i o n a l r e s i s t a n c e R^ ( e q u a l t o ό/D, where 6 i s t h e t h i c k n e s s o f t h e d i f f u s i o n l a y e r near t h e s u r f a c e and D i s t h e d i f f u s i o n c o e f f i c i e n t ) and t h e i n t e r f a c i a l r e s i s t a n c e R , t h e n by s u b s t i t u t i n g t h e e q u i l i b r i u m c o n c e n t r a t i o n C , o b t a i n e d from t h e a d s o r p t i o n i s o t h e r m , we may c a l c u l a t e t h e magnitude o f R f r o m t h e equation 2

Q

2

dn/dt

=

C

(8)

Q

A v a l u e o f 2.4 χ 10 s e c cm was c a l c u l a t e d f o r R (21) f r o m d a t a f o r a BSA m o n o l a y e r d e s o r b i n g u n d e r s t e a d y s t a t e c o n d i t i o n s a t a p r e s s u r e o f 25.6 mNm" . We may c o n c l u d e t h a t , a l t h o u g h t h e k i n e t i c s o f d e s o r p t i o n show t h e i n f l u e n c e o f t h e d i f f u s i o n a l r e s i s t a n c e (1.3 χ 1 0 s e c c m " ) , t h e a b s o l u t e r a t e i s g o v e r n e d b y t h e much l a r g e r i n t e r f a c i a l r e s i s t a n c e . 2

1

4

1

E f f e c t s o f M o l e c u l a r Weight. M o l e c u l a r w e i g h t appears t o be a g o v e r n i n g f a c t o r i n t h e ease of d e s o r p t i o n of p r o t e i n s . Results f o r s e v e r a l p r o t e i n s spanning a range o f m o l e c u l a r weights are summarized i n T a b l e I I . Table I I . Rates of D e s o r p t i o n of P r o t e i n s at D i f f e r e n t Surface Pressures

Rates o f D e s o r p t i o n (min~ χ 10 ) 1

Protein

Mol. wt (x10~ )

15

20

3

Insulin 3 -Lactoglobulin Myoglobin γ -Globulin Catalase

6 17.5 17 160 230

4

25 30 35 (Π, mNm" )

40

45

1

56

530 20

Source: Reproduced w i t h p e r m i s s i o n Academic P r e s s .

50 34

90 67 9

from Ref. 23.

144 20 30

40 70

Copyright


110

1985


11.


MACRITCHIE

175

In mixed monolayers o f i n s u l i n w i t h γ-globulin o r c a t a l a s e , i n s u l i n may be c o m p l e t e l y d e s o r b e d from the monolayer by s u i t a b l e c h o i c e of t h e s u r f a c e p r e s s u r e (23). B a s e d on t h e s e r e s u l t s , some p r e d i c t i o n s may be made about s i t u a t i o n s where t h e r e i s c o m p e t i t i o n f o r t h e i n t e r f a c e by a m i x t u r e o f p r o t e i n s . A f t e r c r e a t i o n of a new i n t e r f a c e , t h e c o m p o s i t i o n o f t h e a d s o r b e d f i l m w o u l d t e n d t o f a v o r the s m a l l e r molecules because of t h e i r h i g h e r d i f f u s i o n coefficients. However, t h e f i n a l c o m p o s i t i o n would f a v o r t h e h i g h m o l e c u l a r weight p r o t e i n s . An i n t e r e s t i n g r e s u l t i s t h a t the a b s o l u t e r a t e of d e s o r p t i o n f o r t h e d e s o r b i n g p r o t e i n i s t h e same i n t h e m i x e d f i l m as i n t h e pure monolayer p r o v i d i n g t h e a r e a o c c u p i e d i s over 30% of t h e t o t a l s u r f a c e a r e a (2_3). Under t h e s e c o n d i t i o n s , t h e c o n c e n t r a t i o n of p r o t e i n i n t h e s u b - s u r f a c e l a y e r i s e v i d e n t l y d e t e r m i n e d by t h e s u r f a c e p r e s s u r e and n o t t h e s u r f a c e d e n s i t y . T h i s r e s u l t has i m p l i c a t i o n s f o r s y s t e m s where p r o t e i n s are t r a n s p o r t e d a c r o s s i n t e r f a c e s o r membranes. Theory of D e s o r p t i o n . B a s e d on m e a s u r e m e n t s o f k i n e t i c s o f a d s o r p t i o n (24.), i t h a s b e e n e s t i m a t e d t h a t p r o t e i n m o l e c u l e s r e q u i r e r e d u c t i o n of t h e i r a r e a s i n the s u r f a c e t o a c r i t i c a l v a l u e o f a b o u t 1 0 0 S f o r them t o become u n s t a b l e and d e s o r b . T h i s may o c c u r e i t h e r (a) a t c o n s t a n t p r e s s u r e by f l u c t u a t i o n s of m o l e c u l e s a b o u t t h e i r mean c o n f i g u r a t i o n ( t h e p r o b a b i l i t y o f s u c h a f l u c t u a t i o n i s n e g l i g i b l y low when p r a c t i c a l l y a l l segments a r e i n t h e f o r m o f t r a i n s a t l o w Π ), o r (b) by c o m p r e s s i o n o f t h e f i l m t o a s u f f i c i e n t l y high pressure Π , c o r r e s p o n d i n g t o the c r i t i c a l area. Assuming t h a t t h e p a t h i s t h e same f o r each case, we can use t h e e q u i l i b r i u m Π -A c u r v e o f t h e p r o t e i n t o e v a l u a t e t h e f r e e energy of a c t i v a t i o n f o r d e s o r p t i o n ( Δ G ) « At a g i v e n p r e s s u r e , Π, t h i s i s g i v e n by: 2

d e s

ρΠ*

(9)

Adll

des

Π

The i n t e g r a l may be s i m p l y e v a l u a t e d from t h e e q u i l i b r i u m Π-A curve of t h e p r o t e i n (see F i g u r e 3). C a l c u l a t i o n s based on F i g u r e 3 f o r BSA showed t h a t Δ G d e c r e a s e d s t e e p l y from a v a l u e o f 106 kT a t Π = 20 mNm" t o 9 kT a t Π = 28.8 mNm" (25). This coincided with t h e a p p e a r a n c e o f m e a s u r a b l e d e s o r p t i o n o f BSA o v e r t h i s r a n g e o f surface pressure. The t h e o r y a l s o e x p l a i n s t h e e f f e c t of m o l e c u l a r w e i g h t on d e s o r p t i o n s i n c e f o r a g i v e n r a t i o o f d i s p l a c e d t o a t t a c h e d segments, t h e a r e a o c c u p i e d by t h e a t t a c h e d segments w i l l be p r o p o r t i o n a l t o m o l e c u l a r w e i g h t a n d i t i s t h i s p a r a m e t e r t h a t determines Δ ^ · d e g

1

1

6

β 8

A p p l i c a t i o n of Gibbs Adsorption Equation. By s u b s t i t u t i n g C = 1/A (where A i s t h e a r e a per m o l e c u l e i n t h e adsorbed monolayer), i n t h e s i m p l e form o f t h e Gibbs A d s o r p t i o n E q u a t i o n ( E q u a t i o n 1), we o b t a i n g

d Il/dlnC

b

=

kT/A


(10)


176


A number o f i n d e p e n d e n t s t u d i e s h a v e shown t h a t w e l l - d e f i n e d a d s o r p t i o n i s o t h e r m s c a n be o b t a i n e d f o r p r o t e i n s (21, 26-28) a n d p l o t s of Π a g a i n s t t h e l o g a r i t h m of C^ a r e l i n e a r over wide ranges. The most n o t a b l e f e a t u r e i s t h a t the s l o p e s a r e r e l a t i v e l y s i m i l a r f o r m o s t p r o t e i n s a n d v a l u e s o f A c a l c u l a t e d f r o m E q u a t i o n 10 a r e v e r y much s m a l l e r t h a n t h o s e of whole p r o t e i n m o l e c u l e s e s t i m a t e d from t h e i r Π-A c u r v e s . For example, f r o m the Π j l o g C^ p l o t f o r c h y m o t r y p s i n ( F i g u r e 1), A i s c a l c u l a t e d t o be 170A . I t appears t h a t the a d s o r p t i o n behavior r e f l e c t s the f l e x i b l e l i n e a r c h a i n s t r u c t u r e o f p r o t e i n s i n t h e s u r f a c e so t h a t we a r e n o t o b s e r v i n g the c o n t r i b u t i o n s of r i g i d whole m o l e c u l e s but of partially i n d e p e n d e n t m o l e c u l a r segments. Joos (27) has p r o p o s e d a t h e o r y o f p r o t e i n a d s o r p t i o n b a s e d on t h e F r i s c h - S i m h a m o d e l f o r f l e x i b l e polymers ( 2 9 ) . The

R o l e o f P r o t e i n s i n I n t e r f a c i a l Phenomena

Some c o n s e q u e n c e s o f t h e i n t e r a c t i o n o f p r o t e i n s w i t h i n t e r f a c e s have a l r e a d y been mentioned; f o r example, the h i g h v i s c o e l a s t i c i t y i m p a r t e d t o i n t e r f a c e s and t h e p r e c i p i t a t i o n o f s o l u b l e p r o t e i n c a u s e d by s h a k i n g s o l u t i o n s . As a r e s u l t o f t h e i r u b i q u i t o u s p r e s e n c e a t i n t e r f a c e s , a wide range of a p p a r e n t l y unrelated p h e n o m e n a a r e a f f e c t e d by p r o t e i n a d s o r p t i o n . The r o l e t h a t p r o t e i n s play at i n t e r f a c e s i n b i o m e d i c a l systems i s e x t e n s i v e l y d i s c u s s e d i n o t h e r c o n t r i b u t i o n s t o t h i s book. Here i t i s i n t e n d e d t o f o c u s more on a r e a s r e l a t e d t o f o o d a n d a g r i c u l t u r e . Some o f the r e l e v a n t t o p i c s are: 1. The f o r m a t i o n and s t a b i l i t y o f d i s p e r s e d s y s t e m s ( f o a m s , emulsions e t c . ) . 2. B a c t e r i a l adhesion. 3. R e a c t i v i t y of enzymes. Because of the g r e a t c o m p l e x i t y of these systems, i t i s p r o p o s e d o n l y t o b r i e f l y i n d i c a t e some of t h e more b a s i c c o n c e p t s t h a t might be u s e f u l l y a p p l i e d i n t h e s e a r e a s . F o r m a t i o n and S t a b i l i t y o f E m u l s i o n s and Foams. The c o n t r i b u t i o n s of p r o t e i n a d s o r p t i o n t o the p r o p e r t i e s of d i s p e r s e d systems encompass problems o f g r e a t d i v e r s i t y r a n g i n g from t h e m a n u f a c t u r e o f f o o d e m u l s i o n s (mayonnaise, b u t t e r ) and foams (meringues, whipped cream, bread) t h r o u g h t h e p r e p a r a t i o n o f p h a r m a c e u t i c a l suspensions to bloat i n cattle. I t i s u s e f u l t o s e p a r a t e the ease of f o r m a t i o n o f foams and e m u l s i o n s f r o m t h e p r o b l e m of t h e i r s t a b i l i t y . Ease o f f o r m a t i o n r e q u i r e s r a p i d a d s o r p t i o n and t h e r e f o r e r e q u i r e s r e l a t i v e l y h i g h c o n c e n t r a t i o n s o f p r o t e i n s , a low net charge so t h a t t h e e l e c t r i c a l p o t e n t i a l b a r r i e r t o a d s o r p t i o n i s m i n i m a l and t h e absence of d e s t a b i l i z i n g agents. Once a f o a m o r e m u l s i o n i s formed, the d i f f e r e n t processes of c r e a m i n g and flocculation ( e m u l s i o n s ) a n d c o a l e s c e n c e b e g i n t o b r e a k down t h e dispersed system. P r o t e i n s a l o n e a r e v e r y e f f e c t i v e s t a b i l i z e r s f o r foams and o i l / w a t e r e m u l s i o n s . C o n t r a r y t o what might be p r e d i c t e d f r o m some c o l l o i d t h e o r i e s , t h e s t a b i l i t y of p r o t e i n foams and e m u l s i o n s i s g e n e r a l l y a maximum a t o r n e a r t h e i s o e l e c t r i c p o i n t o f t h e protein. T h i s i s p r o b a b l y a r e s u l t o f t h e g r e a t e r a d s o r p t i o n under these c o n d i t i o n s . I t a l s o i n d i c a t e s that f a c t o r s other than e l e c t r i c a l r e p u l s i o n are important f o r c o n f e r r i n g s t a b i l i t y . Some



11.

MACRITCHIE


177

f a c t o r s t h a t may be r e l e v a n t t o s t a b i l i t y a r e s u g g e s t e d by t h e p r e v i o u s d i s c u s s i o n o f f u n d a m e n t a l aspects of p r o t e i n i n t e r f a c i a l behavior. The c o a l e s c e n c e o f two e m u l s i o n drops p r o c e e d s w i t h a r e d u c t i o n of i n t e r f a c i a l area. This s i g n i f i e s that, during coalescence, there i s a c o m p r e s s i o n o f t h e adsorbed s t a b i l i z i n g f i l m s s u r r o u n d i n g t h e d r o p l e t s i f the f i l m m a t e r i a l c a n n o t d e s o r b s u f f i c i e n t l y q u i c k l y . The r e s i s t a n c e t o c o m p r e s s i o n by t h e f i l m s l e a d s t o an e l a s t i c r e s t o r i n g mechanism, t h u s c r e a t i n g an energy b a r r i e r which t e n d s t o oppose c o a l e s c e n c e (30,31). T h i s energy b a r r i e r , o f t h e f o r m J^Ad Π probably operates only i n the e a r l y stage of coalescence. Once a s u f f i c i e n t l y l a r g e p u n c t u r e i s made i n t h e c o l l i d i n g d r o p l e t s , coalescence would then p r o c e e d s p o n t a n e o u s l y . The tensiol a m i n i n o m e t e r i s an i n s t r u m e n t t h a t h a s b e e n u s e d t o m e a s u r e t h e energy o p p o s i n g r e d u c t i o n o f l i q u i d l a m e l l a e (32) and t h i s q u a n t i t y h a s b e e n c o r r e l a t e d w i t h f o a m s t a b i l i z i n g p r o p e r t i e s of d i f f e r e n t surfactants, including proteins. A h i g h energy b a r r i e r i s a c h i e v e d by a low c o m p r e s s i b i l i t y a n d a l o w t e n d e n c y t o d e s o r b . For p r o t e i n s , s h o r t c i r c u i t i n g o f t h e c o m p r e s s i o n a l e n e r g y b a r r i e r by d e s o r p t i o n does not o c c u r t o a s i g n i f i c a n t e x t e n t . The compress i b i l i t y o f p r o t e i n f i l m s depends on t h e speed o f c o m p r e s s i o n because o f t h e time-dependent r e l a x a t i o n p r o c e s s e s . F o r c o l l i d i n g d r o p l e t s , t h e s p e e d o f c o m p r e s s i o n i s e x p e c t e d t o be h i g h a n d t h u s t h e f i l m c o m p r e s s i b i l i t y c o r r e s p o n d i n g l y low. P r o t e i n s conform t o the g e n e r a l r u l e ( B a n c r o f t r u l e ) t h a t t h e phase i n which t h e s t a b i l i z e r i s s o l u b l e becomes t h e c o n t i n u o u s phase; i.e. they i n v a r i a b l y form o i l / w a t e r emulsions. A p o s s i b l e mechanism f o r d e t e r m i n i n g e m u l s i o n t y p e may be as f o l l o w s . When two d r o p l e t s approach, t h e c o n t i n u o u s medium n e a r t h e p o i n t o f c o n t a c t i s d i s p l a c e d s o t h a t i f t h e s t a b i l i z e r i s s o l u b l e i n t h e c o n t i n u o u s phase, i t i s p r e v e n t e d from d e s o r b i n g (or m o l e c u l a r segments a r e p r e v e n t e d f r o m b e i n g e x p e l l e d i n the case of p r o t e i n s ) , thus c o n t r i b u t i n g t o a sharp i n c r e a s e i n Π , producing a h i g h c o m p r e s s i o n a l energy b a r r i e r . On t h e o t h e r hand, i f m o l e c u l e s (or segments) of t h e s t a b i l i z e r a r e s o l u b l e i n t h e d i s p e r s e d p h a s e , t h e y a r e f r e e t o be d i s p l a c e d f r o m the i n t e r f a c e , t h e r e b y s h o r t - c i r c u i t i n g t h e energy b a r r i e r . The p u s h i n g out o f segments i n t o t h e aqueous phase ( f o r m a t i o n o f l o o p s a n d t a i l s ) c o u l d c o n c e i v a b l y h a v e two m a i n e f f e c t s on stability. The i n t e r a c t i o n between p r o t e i n segments, as d r o p l e t s a p p r o a c h , p r o d u c e s a s t e r i c b a r r i e r (33^/ a r e s u l t o f t h e f r e e energy i n c r e a s e accompanying a r i s e i n c o n c e n t r a t i o n o f p r o t e i n i n the i n t e r s t i t i a l l i q u i d . I t i s a l s o p o s s i b l e t h a t l o o p s and t a i l s can form b r i d g e s between d r o p l e t s (34^) r t h u s p r o m o t i n g f l o c c u l a t i o n . The tendency f o r p r o t e i n s t o f o r m t h i c k membranes, as a r e s u l t o f i n t e r f a c i a l c o a g u l a t i o n , n e e d s t o be t a k e n i n t o account, e s p e c i a l l y i n e m u l s i o n s s i n c e i t o c c u r s more e a s i l y a t o i l / w a t e r than at a i r / w a t e r i n t e r f a c e s . These t h i c k f i l m s can p r e s e n t a s t e r i c b a r r i e r s i m p l y by p r e v e n t i n g t h e d i s p e r s e d p h a s e i n t h e d r o p l e t s from coming i n t o c o n t a c t . On t h e o t h e r hand, because o f t h e i r g e l a t i n o u s and c o h e s i v e n a t u r e , t h e y a r e l i k e l y t o p r o d u c e flocculation. B a c t e r i a l Adhesion. B a c t e r i a o f t e n adhere t o i n t e r f a c e s and t h e r e s u l t i n g b i o f i l m s c a n c a u s e a n u i s a n c e on s h i p h u l l s , t o w a t e r r e t i c u l a t i o n and h y d r o - e l e c t r i c p i p e l i n e s and i n h e a t e x c h a n g e r s



178


(35). B a c t e r i a a l s o c o l o n i z e s u r f a c e s i n s o i l s , streams, oceans a n d s e d i m e n t s a s w e l l a s p l a n t r o o t s and o t h e r p l a n t s u r f a c e s a n d s u r f a c e s of h i g h e r organisms, i n c l u d i n g the s k i n . Mixtures of p r o t e i n s and g l y c o p r o t e i n s w i t h o t h e r compounds e x i s t i n s o l u t i o n a t low c o n c e n t r a t i o n s i n aqueous h a b i t a t s . Adhesion o f b a c t e r i a t o a s u r f a c e i s u s u a l l y p r e c e d e d by a d s o r p t i o n t o form a " c o n d i t i o n i n g f i l m " (36). Adsorbed p r o t e i n s a r e found t o a f f e c t t h e e l e c t r i c a l p o t e n t i a l and h y d r o p h o b i c i t y o f t h e surface and vary i n t h e i r e f f e c t s on a d h e s i o n o f b a c t e r i a . W i t h P s e u d o m o n a s NCMB202 1, a t t a c h m e n t o n t i s s u e c u l t u r e d i s h e s was r e d u c e d b y b o t h BSA a n d b o v i n e g l y c o p r o t e i n w h e r e a s a t t a c h m e n t t o p e t r i d i s h e s was o n l y r e d u c e d by BSA (36.). B u b b l e c o n t a c t a n g l e s on t h e p e t r i d i s h e s w e r e much l o w e r f o r BSA (< 15°) t h a n f o r t h e g l y c o p r o t e i n ( 6 4 ° ) . This experiment i n d i c a t e s the d i f f e r e n t e f f e c t s that adsorbed p r o t e i n s have on the h y d r o p h o b i c i t y o f s u r f a c e s . R e a c t i v i t y o f Enzymes. Enzyme s y s t e m s o c c u r n a t u r a l l y i n p l a n t s and a n i m a l s a n d t h e i r e f f e c t s c a n become i m p o r t a n t i n f o o d p r o c e s s i n g and n u t r i t i o n . Enzyme c a t a l y s i s a t t h e i n t e r f a c e s o f d i s p e r s e d systems i s o f g r e a t i n t e r e s t . Fundamental s u r f a c e s t u d i e s h a v e b e e n m a i n l y done on l i p a s e a c t i v i t y i n r e l a t i o n t o t h e d i g e s t i o n of t r i g l y c e r i d e s . The r e s u l t s o f V e r g e r and c o - w o r k e r s (37-39) a r e p a r t i c u l a r l y i n t e r e s t i n g and g i v e new i n s i g h t s i n t o t h e behavior of proteins a t interfaces. They have used a z e r o - o r d e r t r o u g h t o study t h e k i n e t i c s o f h y d r o l y s i s o f l i p i d s i n monolayers. The t r o u g h c o n s i s t s o f two compartments c o n n e c t e d by a s m a l l c a n a l , enzyme b e i n g p r e s e n t i n o n l y one compartment. When l i p i d monolayers are h y d r o l y z e d by l i p a s e s p r e s e n t i n the sub-phase, t h e r e a c t i o n p r o d u c t s become s o l u b l e and d i f f u s e away, l e a d i n g t o a d e c r e a s e o f area a tconstant surface pressure. From the l i n e a r s l o p e o f t h e a r e a - t i m e g r a p h , t h e v e l o c i t y o f t h e enzyme r e a c t i o n i s d i r e c t l y evaluated. I t i s found t h a t p r o t e i n s i n s o l u t i o n a t r e l a t i v e l y low concentrations may i n h i b i t t h e h y d r o l y s i s o f d i - and t r i g l y c e r i d e s by lipases (3.Ζ^' Marked d i f f e r e n c e s a r e found i nt h e s u s c e p t i b i l i t y o f d i f f e r e n t l i p a s e s t o i n h i b i t i o n and a l s o between the i n h i b i t i n g e f f e c t s o f d i f f e r e n t p r o t e i n s . Experiments using m i x e d l i p i d - p r o t e i n f i l m t r a n s f e r showed t h a t t h e i n h i b i t i o n o f p a n c r e a t i c l i p a s e i s due t o the p r o t e i n a s s o c i a t e d w i t h the l i p i d a t t h e s u r f a c e and n o t caused by d i r e c t p r o t e i n - e n z y m e i n t e r a c t i o n i n the aqueous phase (38). U s i n g r a d i o l a b e l l e d enzymes and p r o t e i n s , i t was f o u n d t h a t t h e i n a c t i v a t i o n o f t h e p a n c r e a t i c l i p a s e was c o r r e l a t e d w i t h a l a c k o f l i p a s e b i n d i n g t o t h e mixed l i p i d - p r o t e i n f i l m (39.)· Since a large f r a c t i o n of the l i p i d f i l m remained p o t e n t i a l l y a c c e s s i b l e t o t h e enzyme i n t h e p r e s e n c e o f t h e i n h i b i t i n g p r o t e i n , i t was b e l i e v e d t h a t t h e r o l e o f the p r o t e i n was t o m o d i f y the p r o p e r t i e s o f t h e i n t e r f a c e i n such a way as t o e i t h e r cause d e s o r p t i o n o f t h e l i p a s e o r p r e v e n t i t f r o m a t t a c h i n g a t t h e interface. T h e c h a l l e n g e o f how t h i s i s a c h i e v e d i s b o u n d t o s t i m u l a t e work w h i c h w i l l g r e a t l y i n c r e a s e o u r u n d e r s t a n d i n g o f interfacial reactions.

Literature Cited 1. 2.

Langmuir, I . ; Schaeffer, V.J. Chem. Rev. 1939, 24, 181-202. Malcolm, B.R. Prog. Surface & Membrane Sci. 1973, 7, 183-229.



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3. 4. 5. 6. 7. 8. 9. 10. 11. Pedrero, 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.


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Loeb, G.J. J. Colloid Interface Sci. 1969, 31, 572-574. Cornell, D.G. J . Colloid Interface Sci., 1979, 70, 167-180. Cornell, D.G. J. Colloid Interface Sci., 1984, 98, 283-285. MacRitchie, F. J . Macromol. Sci.-Chem. 1970, A4, 1169-1176. Moore, W.J.; Eyring, H. J . Chem. Phys. 1938, 6, 391-394. Joly, M. Biochem. Biophys. Acta 1948, 2, 624-632. Van Geet, A.L.; Adamson, A.W. J. Phys. Chem. 1964, 68, 238-246. Schulman, J.H.; Rideal, E.K. Proc. Roy. Soc. 1937, B122, 29-45. Miñones, J.; García Fernández, S.; Iribarnegaray, S.; Sanz P. J. Colloid Interface Sci. 1973, 42, 503-515. MacRitchie, F. Adv. Protein Chem. 1978, 32, 283-326. MacRitchie, F. J . Colloid Interface Sci. 1981, 79, 461-464. MacRitchie, F.; Owens, N.F. J. Colloid Interface Sci. 1969, 29, 66-71. Henson, A.F.; Mitchell, J.R.; Mussellwhite, P.R. J. Colloid Interface Sci. 1970, 32, 162-165. Bigelow, C.C. J . Theoret. Biol. 1967, 16, 187-211. Asakura, T.; Ohnishi, T.; Friedman, S.; Schwartz, E. Proc. Nat. Acad. Sci. USA 1974, 71, 1594-1598. Asakura, T.; Minakata, K.; Adachi, K.; Russell, M.O.; Schwartz, E. J. Clin. Invest. 1977, 59, 633-640. Langmuir, I.; Waugh, D.F. J. Amer. Chem. Soc. 1940, 62, 2771-2793. Langmuir, I. J. Amer. Chem. Soc. 1917, 39, 1848-1906. Gonzalez, G.; MacRitchie, F. J . Colloid Interface Sci. 1970, 32, 55-61. MacRitchie, F.; Ter-Minassian-Saraga, L. Colloids and Surfaces 1984, 10, 53-64. MacRitchie, F. J. Colloid Interface Sci. 1985, 105, 119-123. MacRitchie, F.; Alexander, A.E. J. Colloid Sci. 1963, 18, 458463. MacRitchie, F. J. Colloid Interface Sci. 1977, 61, 223-226. Benhamou, Ν.; Guastalla, J . J . Chim. Phys. 1960, 57, 745-751. Joos, P. Proc. Int. Congr. Surf. Act., 5th, 1968, 2, 513-519. P h i l l i p s , M.C.; Evans, M.T.A.; Graham, D.E.; Oldani, D. Colloid Polym. Sci. 1975, 253, 424-427. Frisch, H.; Simha, R. J . Phys. Chem. 1957, 27, 702-706. MacRitchie, F. Nature 1967, 215, 1159-1160. MacRitchie, F. J . Colloid Interface Sci. 1976, 56, 53-56. Eydt, A.J.; Rosano, H.L. J. Amer. Oil Chem. Soc. 1968, 45, 607-610. Vincent, B. Adv. Colloid Interface Sci. 1974, 4, 193-277. Smellie, R.H.; La Mer, V.K. J . Colloid Sci. 1958, 13, 589-599. Characklis, W.G. In Fouling of Heat Transfer Equipment; Somerscales, E.F.C.; Knudsen, J.G. Eds.; Hemisphere: Washington, D.C.; p 251. Fletcher, M.; Marshall, K.C.; Appl. Environ. Microbiol. 1982, 44, 184-192. Gargouri, Y.; Julien, R.; Sugihara, Α.; Verger, R.; Sarda, L. Biochim. Biophys. Acta 1984, 795, 326-331. Gargouri, Y.; Piéroni, G.; Rivière, C.; Sugihara, Α.; Sarda, L.; Verger, R. J . Biol. Chem. 1985, 260, 2268-2273. Gargouri, Y.; Piéroni, G.; Rivière, C.; Sarda, L.; Verger, R. Biochem. 1986, 25, 1733-1738.

RECEIVED January 28, 1987 Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Proteins at Interfaces - American Chemical Society

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