Proteins at Interfaces - American Chemical Society

Chapter 1

Proteins at Interf aces: Current Issues and Future Prospects 1

Thomas A. Horbett and John L . Brash

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Downloaded by MEMORIAL UNIV OF NEWFOUNDLAND on November 26, 2014 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch001

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Department of Chemical Engineering, BF-10, and Center for Bioengineering, University of Washington, Seattle, WA 98195 Department of Chemical Engineering, McMaster University, Hamilton, Ontario L8S 4L7, Canada 2

The ability of proteins to influence a wide v a r i e t y of processes that occur at interfaces i s well recognized. The b i o c o m p a t i b i l i t y of clinical implants, mammalian and b a c t e r i a l cell adhesion to surfaces, initiation of blood coagulation, complement a c t i v a t i o n by surfaces, s o l i d phase immunoassays, and p r o t e i n binding to cell surface receptors all involve proteins at i n t e r f a c e s . Furthermore, p r a c t i c a l problems such as contact lens f o u l i n g , foaming of p r o t e i n s o l u t i o n s , and f o u l i n g of equipment i n the food processing industry, are d i r e c t consequences of the r e l a t i v e l y high surface a c t i v i t y of p r o t e i n s . In general, any process i n v o l v i n g an i n t e r f a c e in which contact with a p r o t e i n s o l u t i o n occurs i s l i k e l y to be influenced by p r o t e i n adsorption to the i n t e r f a c e . Thus, several reviews of p r o t e i n adsorption have been published (1-5). Since previous reviews provide e x c e l l e n t coverage of the g e n e r a l l y well understood or frequently studied aspects of the i n t e r f a c i a l behavior of p r o t e i n s , t h i s chapter w i l l focus on several facets of p r o t e i n adsorption that have so far not been examined i n much detail. W h i l e t h i s a p p r o a c h i s a t y p i c a l f o r an o v e r v i e w c h a p t e r , i t i s i n k e e p i n g w i t h t h e i n t e n t o f t h i s book t o p r o v i d e i n f o r m a t i o n t o t h e r e a d e r t h a t r e f l e c t s more r e c e n t d e v e l o p m e n t s i n t h i s f i e l d . F u r t h e r m o r e , as w i l l be seen, t h e t o p i c s t o be d i s c u s s e d n e c e s s i t a t e r e e x a m i n a t i o n o f p r e v i o u s s t u d i e s and p r o v i d e some u n i f y i n g views o f t h i s r a t h e r d i v e r s e s c i e n c e . The main t o p i c s t o be p r e s e n t e d i n c l u d e t h e o r i g i n s of the surface a c t i v i t y of p r o t e i n s , m u l t i p l e s t a t e s o f a d s o r b e d p r o t e i n s , and t h e c o m p e t i t i v e adsorption b e h a v i o r o f p r o t e i n s . These t o p i c s were c h o s e n b e c a u s e i t appears that a b e t t e r understanding o f each i s necessary

0097-6156/87/0343-0001 $09.25/0 © 1987 American Chemical Society

In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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PROTEINS AT INTERFACES


t o d e s c r i b e many o f t h e i n t e r f a c i a l phenomena i n v o l v i n g p r o t e i n s , y e t t h e fundamental concepts u n d e r l y i n g each have n o t been d i s c u s s e d a s f u l l y a s we hope t o do i n t h i s c h a p t e r . F i n a l l y , we d e s c r i b e some f u t u r e a r e a s o f r e s e a r c h t h a t a r e l i k e l y t o y i e l d important advances i n our u n d e r s t a n d i n g o f p r o t e i n b e h a v i o r a t i n t e r f a c e s . On t h e O r i g i n s o f D i f f e r e n c e s Proteins

i n the Surface

Activity of

Molecular Properties Influencing Surface A c t i v i t y o f Proteins. The m o l e c u l a r p r o p e r t i e s o f p r o t e i n s t h a t a r e t h o u g h t t o be r e s p o n s i b l e f o r t h e i r t e n d e n c y t o r e s i d e a t s u r f a c e s a r e summarized i n T a b l e I . The s i z e , c h a r g e , s t r u c t u r e , and other chemical p r o p e r t i e s o f p r o t e i n s t h a t presumably i n f l u e n c e s u r f a c e a c t i v i t y a r e a l l f u n d a m e n t a l l y r e l a t e d t o t h e i r amino a c i d sequence, w h i c h i s f i x e d f o r each type o f p r o t e i n b u t v a r i e s g r e a t l y among p r o t e i n s . Thus, d i f f e r e n c e s i n s u r f a c e a c t i v i t y among p r o t e i n s a r i s e f r o m v a r i a t i o n s i n t h e i r p r i m a r y structure. At t h i s point, further enquiries into the o r i g i n o f s u r f a c e a c t i v i t y d i f f e r e n c e s among p r o t e i n s become q u i t e p r o b l e m a t i c a l b e c a u s e l i t t l e d e t a i l e d information i s available that r e l a t e s v a r i a t i o n i n the p r i m a r y s t r u c t u r e o f p r o t e i n s t o changes i n t h e s u r f a c e a c t i v i t y o f t h e m o l e c u l e s . However, a d i s c u s s i o n o f s p e c i f i c f a c t o r s w i l l serve t o c l a r i f y our concepts i n t h i s regard. S i z e i e p r e s u m a b l y an i m p o r t a n t d e t e r m i n a n t o f s u r f a c e a c t i v i t y because p r o t e i n s and other m a c r o m o l e c u l e s a r e t h o u g h t t o form m u l t i p l e c o n t a c t p o i n t s when a d s o r b e d t o a s u r f a c e . The i r r e v e r s i b i l i t y t y p i c a l l y observed f o r p r o t e i n s adsorbed t o surfaces i s t h o u g h t t o be due t o t h e f a c t t h a t s i m u l t a n e o u s d i s s o c i a t i o n o f a l l t h e c o n t a c t s w i t h t h e s u r f a c e i s an u n l i k e l y e v e n t . M u l t i p l e b o n d i n g i s a l s o i n d i c a t e d by t h e r e l a t i v e l y l a r g e number o f p r o t e i n c a r b o n y l g r o u p s t h a t c o n t a c t s i l i c a s u r f a c e s upon a d s o r p t i o n ( £ ) . The bound f r a c t i o n o f p e p t i d e bond c a r b o n y l g r o u p s , a s c a l c u l a t e d f r o m s h i f t s i n i n f r a r e d f r e q u e n c i e s a f t e r a d s o r p t i o n , has been f o u n d t o be i n t h e range o f 0 . 0 5 - 0 . 2 0 (£.) . The bound f r a c t i o n s c o r r e s p o n d t o 77 c o n t a c t s p e r a d s o r b e d a l b u m i n m o l e c u l e , a n d up t o 703 c o n t a c t s p e r a d s o r b e d f i b r i n o g e n m o l e c u l e ( £ ) . On t h e o t h e r hand, s i z e i s c l e a r l y n o t on o v e r r i d i n g f a c t o r determining the surface a c t i v i t y d i f f e r e n c e s among p r o t e i n s . F o r example, h e m o g l o b i n a p p e a r s t o be f a r more s u r f a c e a c t i v e t h a n f i b r i n o g e n (2)t y e t t h e m o l e c u l a r w e i g h t o f h e m o g l o b i n (65,000) i s a p p r o x i m a t e l y 1/5 t h a t o f f i b r i n o g e n ( 3 3 0 , 0 0 0 ) . W h i l e a l b u m i n n e a r l y i s t h e same s i z e a s h e m o g l o b i n , i t i s much less surface active. F i n a l l y , s l i g h t variations i n the amino a c i d sequence o f h e m o g l o b i n make l a r g e d i f f e r e n c e s i n s u r f a c e a c t i v i t y even t h o u g h t h e s e v a r i a n t s have t h e same m o l e c u l a r w e i g h t (see b e l o w ) .



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HORBETT AND BRASH

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Current Issues and Future Prospects

Table I. Molecular P r o p e r t i e s of P r o t e i n s P o s s i b l y Influencing Their Surface A c t i v i t y

1. S i z e :

l a r g e r m o l e c u l e s may have more c o n t a c t

2. Charge : m o l e c u l e s n e a r e r a d s o r b more

their

points.

i s o e l e c t r i c pH may

easily.

3. S t r u c t u r e : a. S t a b i l i t y : b. u n f o l d i n g

l e s s s t a b l e p r o t e i n s may be more surface a c t i v e . r a t e s : more r a p i d u n f o l d i n g may f a v o r surface a c t i v i t y .

c. C r o s s - l i n k i n g :

-S-S- bonds may activity.

d. S u b u n i t s :

more s u b u n i t s activity.

4 . Other chemical

reduce

may

surface

increase

surface

properties:

a. A m p h i p a t h i c i t y :

some p r o t e i n s may have more o f t h e types of side chains favored f o r bonding.

b. " O i l i n e s s " :

more " h y d r o p h o b i c " p r o t e i n s may be more s u r f a c e a c t i v e .

c. S o l u b i l i t y :

l e s s s o l u b l e p r o t e i n s may be more surface active.



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The c h a r g e and c h a r g e d i s t r i b u t i o n o f p r o t e i n s a r e l i k e l y t o i n f l u e n c e s u r f a c e a c t i v i t y b e c a u s e i t i s known t h a t most o f t h e c h a r g e d amino a c i d s r e s i d e a t t h e e x t e r i o r o f p r o t e i n m o l e c u l e s . These c h a r g e d r e s i d u e s must t h e r e f o r e come i n t o c l o s e p r o x i m i t y w i t h t h e s u r f a c e i n the process of a d s o r p t i o n . E x p e r i m e n t a l l y , proteins have f r e q u e n t l y been f o u n d t o e x h i b i t g r e a t e r a d s o r p t i o n a t o r n e a r t h e i s o e l e c t r i c pH, p e r h a p s b e c a u s e c h a r g e c h a r g e r e p u l s i o n among t h e a d s o r b e d m o l e c u l e s i s m i n i m i z e d under t h e s e c o n d i t i o n s . However, Norde has c o n c l u d e d t h a t t h e r e d u c t i o n i n a d s o r p t i o n a t pH's away from t h e i s o e l e c t r i c i s due t o s t r u c t u r a l r e a r r a n g e m e n t s i n the adsorbing molecule, r a t h e r than charge r e p u l s i o n (4.) . In t h i s c o n t e x t , i t i s p e r t i n e n t t o n o t e t h a t t h e i s o e l e c t r i c pH ( p i ) o f h e m o g l o b i n i s n e a r n e u t r a l i t y (7.2) and t h a t t h i s p r o t e i n i s much more s u r f a c e a c t i v e a t pH 7.4 t h a n e i t h e r f i b r i n o g e n ( p i = 5 . 5 ) or albumin ( p i = 4.8). I t would be o f i n t e r e s t t o compare t h e s u r f a c e a c t i v i t y o f t h e s e m o l e c u l e s a t pH's o t h e r t h a n 7.4 t o d e t e r m i n e whether t h e r a n k i n g o f s u r f a c e a c t i v i t i e s changed as t h e i s o e l e c t r i c pH o f e a c h p r o t e i n was a p p r o a c h e d . The r o l e o f p r o t e i n s u r f a c e c h a r g e i s e s p e c i a l l y i m p o r t a n t and p r o b a b l y p r e d o m i n a n t a t i n t e r f a c e s w i t h f i x e d i o n i c c h a r g e s , as shown by t h e a b i l i t y to adsorb p r o t e i n s to i o n i z e d matrices. Adsorption t o t h i s type of surface i s s t r o n g l y a f f e c t e d by t h e d e g r e e o f o p p o s i t e c h a r g e on t h e p r o t e i n and t h e d e g r e e o f c o m p e t i t i o n p r o v i d e d by l i k e c h a r g e d i o n s i n the b u f f e r . A d s o r p t i o n t o charged m a t r i c e s i s the b a s i s f o r t h e w i d e l y a p p l i e d s e p a r a t i o n o f p r o t e i n s by i o n exchange c h r o m a t o g r a p h y . S t r u c t u r a l f a c t o r s important i n 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 r e not w e l l u n d e r s t o o d . We may speculate t h a t p r o t e i n s l i k e l y t o u n f o l d t o a g r e a t e r degree or t h a t u n f o l d more r a p i d l y would be more s u r f a c e a c t i v e b e c a u s e more c o n t a c t s p e r m o l e c u l e c o u l d be formed and because the c o n f i g u r a t i o n a l entropy g a i n f a v o r s the a d s o r p t i o n . Thus, d i s u l f i d e c r o s s l i n k e d p r o t e i n s would be l e s s l i k e l y t o u n f o l d as r a p i d l y o r c o m p l e t e l y and t h e r e f o r e be l e s s s u r f a c e a c t i v e . T h i s p r e d i c t i o n i s amenable t o e x p e r i m e n t a l t e s t s i n c e r e d u c t i o n o f d i s u l f i d e bonds can be done s p e c i f i c a l l y and completely w i t h v e r y m i l d r e a g e n t s . The o n l y known t e s t o f t h i s i d e a was t h e o b s e r v a t i o n t h a t d i s u l f i d e bond r e d u c t i o n by t h i o g l y c o l l i c a c i d i n c r e a s e d t h e number o f bonds formed by a l b u m i n a d s o r b e d t o s i l i c a by about 50% (£). On t h e o t h e r hand, a d d i t i o n a l c r o s s - l i n k i n g o f a l b u m i n w i t h d i e t h y l m a l o n i m i d a t e d i d not r e d u c e t h e number o f bonds formed (£), p e r h a p s b e c a u s e n a t i v e a l b u m i n i s a l r e a d y h e a v i l y c r o s s - l i n k e d by 16 d i s u l f i d e l i n k a g e s ( £ ) . F i n a l l y , t h e e x i s t e n c e o f n o n - c o v a l e n t l y bonded s u b u n i t s i n a p r o t e i n may f a v o r s u r f a c e a c t i v i t y b e c a u s e rearrangements of the i n t e r - s u b u n i t c o n t a c t s t o a l l o w more c o n t a c t o f e a c h s u b u n i t w i t h t h e s u r f a c e can



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HORBETT AND BRASH


p r o b a b l y o c c u r more r e a d i l y t h a n r e a r r a n g e m e n t s w i t h i n e a c h s u b u n i t . Measurement o f t h e r e l a t i v e s u r f a c e a c t i v i t y o f t h e subunits o f hemoglobin i n comparison t o t h e t e t r a m e r i c whole m o l e c u l e might p r o v i d e an i n t e r e s t i n g test of t h i s idea. C h e m i c a l d i f f e r e n c e s among p r o t e i n s a r i s i n g from t h e p a r t i c u l a r b a l a n c e o f amino a c i d r e s i d u e s i n e a c h p r o t e i n probably are a l s o important f a c t o r s i n f l u e n c i n g 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 . The a m p h i p a t h i c n a t u r e o f p r o t e i n s , due t o t h e p r e s e n c e o f h y d r o p h o b i c , h y d r o p h i l i c and c h a r g e d amino a c i d s i d e c h a i n s , p r o v i d e s an opportunity f o r bonding t o s i t e s t h a t vary considerably i n chemical nature. Thus, f o r a p a r t i c u l a r s u r f a c e , some p r o t e i n s may have more o f t h e t y p e o f r e s i d u e t h a t f a v o r s b o n d i n g t o t h e k i n d o f a d s o r p t i o n s i t e s p r e v a l e n t on t h i s s u r f a c e , and t h e r e f o r e would be more s u r f a c e a c t i v e t h a n other p r o t e i n s . More g e n e r a l l y , t h e i d e a t h a t p r o t e i n s have a h y d r o p h o b i c o r o i l y c o r e s u g g e s t s t h a t p r o t e i n s t h a t a r e more h y d r o p h o b i c may be p r e f e r r e d on many s u r f a c e s , e s p e c i a l l y i n view o f t h e a p p a r e n t i m p o r t a n c e of hydrophobic i n t e r a c t i o n s i n p r o t e i n i n t e r a c t i o n s with some s u r f a c e s . Lastly, since the s o l u b i l i t y of a p r o t e i n i n t h e b u l k phase i s a complex f u n c t i o n o f i t s o v e r a l l c h e m i c a l c o m p o s i t i o n , and b e c a u s e a d s o r p t i o n t o an i n t e r f a c e c a n be t h o u g h t o f as i n s o l u b i l i z a t i o n o r phase s e p a r a t i o n , i t c o u l d be t h a t d i f f e r e n c e s i n s o l u b i l i t y a r e important i n d i c a t o r s o f d i f f e r e n c e s i n surface a c t i v i t y . However, t h e r a t h e r h i g h s o l u b i l i t y o f h e m o g l o b i n ( c a 300 mg/ml i n s i d e r e d c e l l s ) a r g u e s a g a i n s t t h i s i d e a because t h i s p r o t e i n i s q u i t e s u r f a c e a c t i v e (1) . S u r f a c e A c t i v i t y o f Hemoglobin G e n e t i c V a r i a n t s . The b e s t e x p e r i m e n t a l e v i d e n c e on t h e m o l e c u l a r o r i g i n s o f d i f f e r e n c e s i n t h e 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 has come from s t u d y o f t h e b e h a v i o r o f h e m o g l o b i n g e n e t i c v a r i a n t s a t t h e a i r / w a t e r i n t e r f a c e (10-14). The d i f f e r e n c e s i n s u r f a c e a c t i v i t y o f t h e s e v a r i a n t s were o r i g i n a l l y i n d i c a t e d by t h e f o r t u i t o u s o b s e r v a t i o n by A s a k u r a e t a l . t h a t h e m o g l o b i n S s o l u t i o n s t e n d t o form p r e c i p i t a t e s when shaken, u n l i k e s o l u t i o n s o f t h e n o r m a l h e m o g l o b i n A v a r i a n t ( l u ) . Hemoglobin S i s p r e d o m i n a n t i n t h e r e d c e l l s o f humans w i t h t h e s i c k l e c e l l d i s e a s e . The r a t e o f p r e c i p i t a t i o n i n d u c e d by m e c h a n i c a l s h a k i n g i s r e f e r r e d t o as " m e c h a n i c a l s t a b i l i t y " i n t h i s l i t e r a t u r e . Since shaking o f p r o t e i n s o l u t i o n s induces bubble f o r m a t i o n , and b e c a u s e a g i t a t i o n w i t h o u t b u b b l e f o r m a t i o n (by slow s t i r r i n g ) c a u s e s a much s l o w e r r a t e o f p r e c i p i t a t i o n , t h e enhanced p r e c i p i t a t i o n r a t e o f h e m o g l o b i n S s o l u t i o n s was a t t r i b u t e d t o an enhanced r a t e of surface denaturation at the a i r / w a t e r - l i q u i d interface. T h i s i d e a was c o n f i r m e d by d i r e c t measurements o f t h e p r o p e r t i e s o f h e m o g l o b i n f i l m s a t t h e a i r / w a t e r i n t e r f a c e w i t h a s u r f a c e b a l a n c e . The s u r f a c e


5


6


b a l a n c e e x p e r i m e n t s showed t h a t s u r f a c e p r e s s u r e k i n e t i c s (π - t ) and i s o t h e r m s (π - A) f o r h e m o g l o b i n S and o t h e r v a r i a n t s were m a r k e d l y d i f f e r e n t f r o m h e m o g l o b i n A (14.) . The d e c r e a s e i n s u r f a c e p r e s s u r e f o l l o w i n g i n j e c t i o n o f h e m o g l o b i n s o l u t i o n s i n t o t h e subphase o c c u r r e d more q u i c k l y and was g r e a t e r a t s t e a d y s t a t e f o r h e m o g l o b i n S t h a n f o r h e m o g l o b i n A. Furthermore, the π - A curves f o r t h e two v a r i a n t s became much more a l i k e when done a t l o w e r t e m p e r a t u r e s (14) , i n agreement w i t h t h e observation that d i f f e r e n c e s i n mechanical s t a b i l i t y among t h e g e n e t i c v a r i a n t s t e n d t o d i s a p p e a r a t l o w e r t e m p e r a t u r e s ( 1 0 ) . The p r e s s u r e - a r e a i s o t h e r m s f o r t h e v a r i a n t s , o b t a i n e d by c o m p r e s s i o n o f t h e p r o t e i n f i l m s , a l s o showed d i s t i n c t d i f f e r e n c e s . The s h a r p i n c r e a s e i n the r e s i s t a n c e t o f u r t h e r compression ( a t t r i b u t e d t o m o n o l a y e r f o r m a t i o n ) o c c u r r e d a t an a r e a o f 8000 Â^/ m o l e c u l e f o r h e m o g l o b i n S compared t o 5000 Â ^ / m o l e c u l e f o r h e m o g l o b i n A. The g r e a t e r a r e a p e r m o l e c u l e s u g g e s t s a g r e a t e r degree o f u n f o l d i n g o f the hemoglobin S m o l e c u l e compared t o h e m o g l o b i n A. Study o f t h e m e c h a n i c a l s t a b i l i t y o f o t h e r h e m o g l o b i n v a r i a n t s has r e s u l t e d i n t h e f o l l o w i n g ranking: HbA « HbC ^ 6 G l u - > L y s ) « HbF (γ c h a i n r e p l a c e s β c h a i n ) « HbA2 (δ c h a i n r e p l a c e s β c h a i n ) « Hb Deer Lodge (β2 H i s - * A s p ) < Hb K o r l e Bu (β73 Asn -> Asp) < HbS (ββΘΙιι -» V a l ) < Hb C (p6Glu -> V a l ; β73 Asp -» Asn) (12.) . The n o t a t i o n s i n p a r e n t h e s i s i n d i c a t e t h e amino a c i d s u b s t i t u t i o n s e.g., β β ΰ ^ —» V a l means t h e g l u t a m i c a c i d a t p o s i t i o n 6 i n t h e β s u b u n i t has been replaced with a valine residue. The m a j o r i t y o f t h e s e d i f f e r e n c e s i n mechanical s t a b i l i t y are a t t r i b u t a b l e t o d i f f e r e n c e s i n s u r f a c e a c t i v i t y i . e . , t h e π-Α o r π-t i s o t h e r m s a t t h e a i r / w a t e r i n t e r f a c e have been shown t o v a r y c o n s i d e r a b l y f o r t h e s e v a r i a n t s . However, some a p p a r e n t e x c e p t i o n s t o t h i s c o r r e l a t i o n e x i s t , e . g . no d i f f e r e n c e i n t h e s u r f a c e a c t i v i t y o f Hb K o r l e Bu and HbA was o b s e r v e d d e s p i t e t h e i r d i f f e r e n c e i n m e c h a n i c a l stability. The l a r g e d i f f e r e n c e i n t h e s u r f a c e a c t i v i t i e s o f HbA a n d HbS a p p a r e n t l y a r i s e s from a s i n g l e G l u —> V a l amino a c i d s u b s t i t u t i o n a t p o s i t i o n 6 i n t h e β c h a i n . S i m i l a r l y , t h e v a r i a n t Hb CHarlem' w h i c h has an a d d i t i o n a l Asn —» Asp s u b s t i t u t i o n a t β73, i s even more u n s t a b l e . In c o n t r a s t , Hb K o r l e Bu, h a v i n g o n l y t h e Asn —» Asp s u b s t i t u t i o n a t β73, i s much more s t a b l e t h a n HbS. These r e s u l t s c l e a r l y i n d i c a t e t h a t s e e m i n g l y m i n o r changes i n p r i m a r y s t r u c t u r e c a n i n d u c e l a r g e changes i n t h e 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 . On t h e o t h e r hand, t h e d a t a a l s o show t h a t t h e m u l t i p l e d i f f e r e n c e s r e s u l t i n g H

a

r

l

e

m



1.

HORBETT AND BRASH


1

from replacement o f t h e β c h a i n w i t h e i t h e r t h e γ c h a i n i n HbF o r t h e 5 c h a i n i n HbA2 do n o t i n t r o d u c e s i g n i f i c a n t changes i n s u r f a c e a c t i v i t y . Thus, t h e amino a c i d c o m p o s i t i o n o f t h e γ c h a i n v a r i e s from t h e β c h a i n a t 38 out o f t h e 146 amino a c i d p o s i t i o n s (15) w i t h o u t a p p a r e n t e f f e c t on t h e s u r f a c e a c t i v i t y o f HbF compared t o t h e n o r m a l v a r i a n t , HbA. The d i f f e r e n c e s i n s u r f a c e a c t i v i t y o f some o f t h e hemoglobin g e n e t i c v a r i a n t s a r e d i f f i c u l t t o r a t i o n a l i z e i n terms o f t h e e f f e c t s o f s i z e , charge, o r c h e m i s t r y t h a t were d i s c u s s e d p r e v i o u s l y (see T a b l e I) b e c a u s e t h e s e e f f e c t s p r e s u m a b l y o p e r a t e i n an a d d i t i v e o r c u m u l a t i v e way. Thus, f o r example, i t i s d i f f i c u l t t o see how t h e change o f a s i n g l e r e s i d u e c o u l d change t h e balance o f a m p h i p a t h i c i t y g r e a t l y because a l l t h e residues of the protein influence t h i s property. I n s t e a d , t h e l a r g e e f f e c t o f s i n g l e amino a c i d s u b s t i t u t i o n s on h e m o g l o b i n s u r f a c e a c t i v i t y p o i n t s t o a very important r o l e f o r s t r u c t u r a l s t a b i l i t y i n t h e i n t e r f a c i a l behavior of p r o t e i n s . Since s t r u c t u r a l t r a n s i t i o n s i n p r o t e i n s occur i n a cooperative f a s h i o n , e.g., p r o t e i n " m e l t i n g " o c c u r s o v e r a n a r r o w range o f t e m p e r a t u r e , s t r u c t u r a l s t a b i l i t y c o u l d be s t r o n g l y i n f l u e n c e d by s i n g l e amino a c i d s u b s t i t u t i o n s . The i m p o r t a n c e o f s t r u c t u r a l t r a n s i t i o n s on t h e s u r f a c e a c t i v i t y o f Hb v a r i a n t s i s a l s o i n d i c a t e d by t h e f a c t t h a t m e c h a n i c a l s t a b i l i t y and s u r f a c e a c t i v i t y d i f f e r e n c e s among t h e v a r i a n t s t e n d t o d i s a p p e a r a t l o w e r t e m p e r a t u r e s and i n t h e d e o x y g e n a t e d s t a t e . The t e m p e r a t u r e e f f e c t s have been a t t r i b u t e d t o s t a b i l i z a t i o n of hydrophobic i n t e r a c t i o n s a t lower temperatures (14). The much g r e a t e r m e c h a n i c a l s t a b i l i t y and l o w e r s u r f a c e a c t i v i t y o f t h e d e o x y g e n a t e d forms o f h e m o g l o b i n S and o t h e r v a r i a n t s has a l s o been i n t e r p r e t e d t o mean t h a t s t r u c t u r a l t r a n s i t i o n s a r e b e i n g i n f l u e n c e d by t h e s u b s t i t u t i o n s , b e c a u s e i t i s known t h a t t h e oxy and deoxy forms d i f f e r i n t h e i r t h r e e d i m e n s i o n a l s t r u c t u r e s . F u r t h e r m o r e , d e u t e r a t i o n o f HbS r e d u c e s i t s s u r f a c e a c t i v i t y , an e f f e c t t h o u g h t t o a r i s e f r o m t h e s t r o n g e r h y d r o g e n b o n d i n g and van d e r Waals i n t e r a c t i o n s t h a t o c c u r i n t h i s s o l v e n t ( 1 4 ) . The l o c a t i o n o f t h e β6 G l u —» V a l s u b s t i t u t i o n i n HbS a t t h e o u t s i d e o f t h e m o l e c u l e , where i t does n o t c o n t a c t o t h e r r e s i d u e s , t h e f a c t t h a t HbC w i t h a β6 g l u —» l y s s u b s t i t u t i o n r e t a i n s i t s s t a b i l i t y , and f i n a l l y t h e requirement f o r t h e oxygenated s t a t e t o reduce s t a b i l i t y , suggest t h a t t h e s u r f a c e a c t i v i t y o f t h e h e m o g l o b i n m o l e c u l e (and p e r h a p s o t h e r p r o t e i n s ) i s v e r y s e n s i t i v e t o m i n o r changes i n t h e conformational states achievable.



8


Multiple

States

of Adsorbed

Proteins

Background. The i d e a t h a t a d s o r b e d p r o t e i n s may e x i s t i n more t h a n one s t a t e has been t a k e n i n t o a c c o u n t i n more r e c e n t models o f p r o t e i n a d s o r p t i o n (16-18) , a l t h o u g h many e a r l i e r i n v e s t i g a t o r s u s e d a s i m p l e Langmuir model t h a t does not c o n s i d e r s u c h a p o s s i b i l i t y . However, r e v i e w o f t h e l i t e r a t u r e on p r o t e i n a d s o r p t i o n r e v e a l s t h a t t h e r e i s a l a r g e amount o f e x p e r i m e n t a l e v i d e n c e s u p p o r t i n g the concept of m u l t i p l e s t a t e s . In a d d i t i o n , i t i s f o u n d t h a t t h e r e a r e q u i t e p l a u s i b l e mechanisms t o e x p l a i n how and why p r o t e i n s c o u l d r e s i d e a t i n t e r f a c e s i n more t h a n one way. The m o t i v a t i o n f o r t h e s e t h o u g h t s came o r i g i n a l l y from a t t e m p t s t o u n d e r s t a n d changes i n t h e d e t e r g e n t e l u t a b i l i t y o f a d s o r b e d p r o t e i n s , so t h e s e e x p e r i m e n t s and an a n a l y s i s o f them a r e b r i e f l y p r e s e n t e d by way o f a c o n c r e t e i n t r o d u c t i o n t o t h e c o n c e p t o f multiple states. The a b i l i t y t o remove f i b r i n o g e n o r a l b u m i n from a v a r i e t y o f p o l y m e r i c s u r f a c e s w i t h t h e d e t e r g e n t sodium d o d e c y l s u l f a t e (SDS) was f o u n d t o g r a d u a l l y d e c r e a s e i f t h e t i m e between a d s o r p t i o n and e l u t i o n was l e n g t h e n e d (19). S i n c e t h e e l u t i o n c o n d i t i o n s were h e l d c o n s t a n t , the data suggested t h a t the b i n d i n g s t r e n g t h of the a d s o r b e d p r o t e i n s had changed o v e r t i m e , i n d i c a t i n g t h a t t h e y c o u l d e x i s t i n more t h a n one way, o r " s t a t e " on t h e surface. The r a t e o f l o s s o f SDS e l u t a b i l i t y was s t r o n g l y enhanced a t e l e v a t e d t e m p e r a t u r e s but was very ο low i f t h e samples were s t o r e d a t 4 C r a t h e r t h a n room temperature. The t e m p e r a t u r e e f f e c t s s u g g e s t t h a t t h e p o s t - a d s o r p t i o n changes i n e l u t a b i l i t y a r e p r o b a b l y due t o c o n f o r m a t i o n a l a l t e r a t i o n s which a l l o w more c o n t a c t s p e r m o l e c u l e ("molecular s p r e a d i n g " ) . The s t u d i e s a l s o i m p l y t h a t t h e i n c o m p l e t e e l u t i o n o b s e r v e d even i f e l u t i o n was done i m m e d i a t e l y a f t e r a d s o r p t i o n i s due t o t r a n s i t i o n s that occured during adsorption — that i s , some o f t h e p r o t e i n s i n t h e a d s o r b e d l a y e r a r e i n one s t a t e (elutable) while other molecules are i n the nonelutable state. T h i s t h o u g h t l e d t o a more g e n e r a l a n a l y s i s of p r o t e i n s i n m u l t i p l e s t a t e s of a d s o r p t i o n b e c a u s e i t was c l e a r t h a t more e v i d e n c e was needed t o support t h i s hypothesis. The p o s s i b l e mechanisms t h a t c o u l d l e a d t o m u l t i p l e s t a t e s and t h e e x p e r i m e n t a l evidence t h a t supports t h i s i d e a are p r e s e n t e d i n the remainder of t h i s s e c t i o n . Mechanisms f o r M u l t i p l e S t a t e s . P o s s i b l e mechanisms t h a t c o u l d l e a d t o m u l t i p l e s t a t e s of adsorbed p r o t e i n s are summarized i n T a b l e I I and i l l u s t r a t e d i n F i g u r e s 1-5. The a d s o r p t i o n o f a p r o t e i n m o l e c u l e a t a s i t e i s l i k e l y t o be i n f l u e n c e d by t h e e x i s t e n c e o f m o l e c u l e s a l r e a d y a d s o r b e d a t n e a r b y s i t e s , by e i t h e r g e o m e t r i c r e d u c t i o n o f t h e a r e a a v a i l a b l e f o r a d s o r p t i o n as t h e s u r f a c e s i t e s become o c c u p i e d ("occupancy" e f f e c t s , F i g u r e l a ) o r by



1.

Table

II.

9

Current Issues and Future Prospers

HORBETT AND BRASH

M u l t i p l e States of Adsorbed P r o t e i n s : Some P o s s i b l e Mechanisms

1.

" I n t r i n s i c " h e t e r o g e n e i t y due t o o c c u p a n c y and a d s o r b a t e - a d s o r b a t e i n t e r a c t i o n s .

2 .

S t r u c t u r a l l y a l t e r e d forms o f t h e p r o t e i n may on t h e s u r f a c e due t o : a. b. c.

effects

exist

P r e - e x i s t i n g d i s t r i b u t i o n of conformations i n s o l u t i o n phase; R a p i d c o n f o r m a t i o n a l changes accompanying adsorption; Slow c o n f o r m a t i o n a l changes a f t e r a d s o r p t i o n ("residence time" e f f e c t s ) .

3.

Orientational exterior.

s t a t e s due

to "patchiness" of

protein

4.

M u l t i p l e b i n d i n g modes due t o a m p h i p a t h i c i t y o f p r o t e i n and mixed s i t e n a t u r e o f s u b s t r a t e .

5.

A d i s t r i b u t i o n w i t h r e g a r d t o number o f bonds p e r m o l e c u l e may be e x p e c t e d on s t a t i s t i c a l g r o u n d s .




Site size greater than molecular size

Site size less than molecular size

b

Intermolecular distance

> effective range of repulsive forces

Intermolecular distance

Proteins at Interfaces - American Chemical Society

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