Kinetics of Protein Sorption on Phospholipid Membranes Measured by

Chapter 14

Kinetics of Protein Sorption on Phospholipid Membranes Measured by Ellipsometry 1

Peter A. Cuypers , George M . Willems, Jos M . M . Kop, Jan W. Corsel, Marie P. Janssen, and Wim Th. Hermens

Downloaded by UNIV OF GUELPH LIBRARY on June 11, 2012 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch014

Department of Biophysics, Biomedical Centre, University of Limburg, P.O. Box 616, 6200 MD Maastricht, Netherlands

The sorption kinetics of prothrombin, fibrinogen and albumin on phospholipid bilayers were studied by el lipsometry. Using an unstirred layer model, it is possible to detect the presence of a transport limi tation in sorption kinetics and to estimate the thick ness of the unstirred layer. Prothrombin sorption is reversible and calcium-depend ent. The prothrombin association constant K is de pendent on the surface concentration of protein and on the composition of the phospholipid bilayers, indicat ing interacting binding sites. The initial rate of prothrombin adsorption is transport limited in all conditions studied. Values of the sorption rate con stants k and k are dependent on the surface con centration. The rate of adsorption decreases for high er surface concentration and the intrinsic values of k and k can be estimated as soon as the adsorp tion rate drops below the diffusional limit. Similar effects are seen for the adsorption of albumin and fibrinogen. a

on

on

off

off

Prothrombin adsorption remains reversible on pure phosphatidylserine (PS) bilayers and on a mixture of 80% PS and 20% phosphatidylcholine (PC). For PS/PC mixtures with less than 80% PS the initial reversible prothrombin adsorption is followed by a slow second surface reaction which causes irreversible adsorption. A similar slow surface reaction is seen for fibrinogen on 100% PS. Elimination of calcium after adsorption of fibrinogen gives a fast desorption of part of the adsorbed layer, possibly due to increased negative charge of the fibrinogen molecules. The

present

research

program

i n Maastricht

on

protein

adsorption

'Current address: Dutch State Mines Research BV, P.O. Box 18, 6160 MD Geleen, Netherlands 0097-6156/87/0343-0208$06.00/0 © 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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started m 1976 w i t h the m o d i f i c a t i o n o f a manual Rudolph e l l i p s o meter such t h a t t h e a d s o r p t i o n o f p r o t e i n s a t s o l i d - l i q u i d i n t e r f a c e s c o u l d be f o l l o w e d a u t o m a t i c a l l y under w e l l d e f i n e d c o n d i t i o n s (J_) · I n i t i a l s t u d i e s w i t h t h i s i n s t r u m e n t showed t h a t the r e f r a c t i v e i n d e x and t h i c k n e s s of the adsorbed p r o t e i n s d i f f e r e d c o n s i d e r a b l y depend i n g on the u n d e r l y i n g s u r f a c e s . I t was a l s o shown t h a t the s t r u c t u r e o f a f i b r i n o g e n l a y e r a f t e r a d s o r p t i o n on the s u r f a c e changed w i t h time ( 2 , 3 ) . In the f o l l o w i n g y e a r s s e v e r a l t e c h n i q u e s were d e v e l o p e d f o r the d e p o s i t i o n o f p h o s p h o l i p i d mono- and d o u b l e l a y e r s on solid s u b s t r a t e s and the s t a b i l i t y and temperature-dependent b e h a v i o r o f t h e s e membranes i n b u f f e r s o l u t i o n s was s t u d i e d (4_). I t was also shown t h a t the t h i c k n e s s and r e f r a c t i v e i n d e x o f the adsorbed p r o t e i n l a y e r s can g i v e s t r u c t u r a l i n f o r m a t i o n l i k e the water c o n t e n t , s w e l l i n g and s h r i n k i n g o f the l a y e r and p e n e t r a t i o n o f t h e p r o t e i n s i n t o the u n d e r l a y i n g p h o s p h o l i p i d l a y e r s (5-10)· In 1983 we were a b l e t o v a l i d a t e two e x a c t f o r m u l a e , b a s e d on t h e L o r e n z - L o r e n t z e q u a t i o n s , a l l o w i n g the c a l c u l a t i o n o f the mass o f the adsorbed l a y e r from the r e f r a c t i v e i n d e x and t h i c k n e s s . This e x p e r i m e n t a l v a l i d a t i o n was performed by measuring s t a c k e d m u l t i l a y e r s o f known mass o f p h o s p h a t i d y l s e n n e and by t h e a d s o r p t i o n of r a d i o l a b e l e d albumin and p r o t h r o m b i n on t h e s e m u l t i l a y e r s (9) · U s i n g t h e s e e q u a t i o n s the d i s s o c i a t i o n c o n s t a n t s and the number o f b i n d i n g s i t e s o f p r o t h r o m b i n on 14:0/14:0 PS and 18:1/18:1 PS (DOPS) monolayers c o u l d be measured ( 9 , 1 0 ) . Working i n the f i e l d o f b l o o d c o a g u l a t i o n , t h i s s t u d y was extended t o the a d s o r p t i o n on double l a y e r s of d i f f e r e n t mixtures of p h o s p h o l i p i d s . B i n d i n g s i t e s were found w i t h a t l e a s t two d i f f e r e n t d i s s o c i a t i o n constants for p r o t h r o m b i n on DOPS double l a y e r s . V a l u e s o f the d i s s o c i a t i o n con s t a n t s were s t r o n g l y i n f l u e n c e d by a d d i n g p h o s p h a t i d y l c h o l i n e (DOPC) t o the b i l a y e r s ( 7 , 1 0 ) . U n t i l t h e n , c o n c l u s i o n s were based on e q u i l i brium measurements. The prothrombin adsorption on DOPS l a y e r s i s r e v e r s i b l e and o f f e r s an e x c e p t i o n a l model f o r i n v e s t i g a t i o n o f ad s o r p t i o n as w e l l as d e s o r p t i o n k i n e t i c s . U s i n g an u n s t i r r e d l a y e r model a s i m p l e g r a p h i c a l r e p r e s e n t a t i o n o f the r e s u l t s a l l o w e d de t e c t i o n o f t r a n s p o r t l i m i t a t i o n i n s o r p t i o n k i n e t i c s ( 12 ) · The ini t i a l r a t e o f p r o t h r o m b i n a d s o r p t i o n i s t r a n s p o r t - l i m i t e d under a l l conditions studied. Adsorption of albumin and f i b r i n o g e n i s much slower and i s d e t e r m i n e d by the i n t r i n s i c r a t e o f p r o t e i n b i n d i n g

Π2). The u n s t i r r e d l a y e r a d s o r p t i o n model can be g e n e r a l i z e d by t h e i n t r o d u c t i o n o f s u r f a c e c o n c e n t r a t i o n dependent s o r p t i o n r a t e con stants k and k . T h i s s u b j e c t i s c u r r e n t l y b e i n g s t u d i e d as w e l l as t h e e x i s t e n c e or a second, i r r e v e r s i b l e , s u r f a c e r e a c t i o n f o l l o w i n g r e v e r s i b l e i n i t i a l a d s o r p t i o n f o r f i b r i n o g e n and p r o t h r o m b i n on a 60% DOPS/40% DOPC m i x t u r e . A r e c e n t m o d i f i c a t i o n o f the e l l i p s o m e t e r w i t h a r o t a t i n g r e f l e c t i n g s u r f a c e , i n s t e a d o f s t i r r i n g the b u f f e r , a l l o w s much b e t t e r c o n t r o l o f the hydrodynamics i n t h i s system ( m preparation). o n

M a t e r i a l s and

Q f

Methods

The e l l i p s o m e t e r . E l l i p s o m e t r y i s an o p t i c a l t e c h n i q u e f o r the mea surement o f changes i n the p o l a r i z a t i o n o f l i g h t caused by r e f l e c t i o n . These changes are s t r o n g l y i n f l u e n c e d by the p r e s e n c e o f v e r y t h i n (0.1 - 100 nm) f i l m s o f p h o s p h o l i p i d s and p r o t e i n s d e p o s i t e d on


210

PROTEINS AT INTERFACES


the r e f l e c t i n g s u r f a c e . The i n s t r u m e n t i s a m o d i f i e d Rudolph & Sons e l l i p s o m e t e r , t y p e 4303-200E shown i n F i g u r e 1. I t i s e q u i p p e d w i t h a He-Ne l a s e r and computer c o n t r o l l e d s t e p p i n g motors on a d j u s t a b l e o p t i c a l components, the p o l a r i z e r (Ρ) and the a n a l y z e r ( A ) . Measure ment o f the changes i n p o l a r i z a t i o n d u r i n g p r o t e i n a d s o r p t i o n g i v e s the v a l u e s o f t h e r e f r a c t i v e index and t h i c k n e s s o f the a d s o r b e d p r o t e i n l a y e r . T h i s measurement i s r e p e a t e d e v e r y 4 s e c o n d s . A com p l e t e d e s c r i p t i o n o f the i n s t r u m e n t i s g i v e n i n r e f s . ( 1 , 3 ) . F i g u r e 2 shows the t h r e e r e f l e c t i n g systems a n a l y z e d i n each e x p e r i m e n t . F i r s t t h e r e f l e c t i o n o f a chromium s l i d e i s measured i n b u f f e r s o l u t i o n ( F i g u r e 2 upper p a n e l ) g i v i n g the r e a l and complex p a r t o f t h e r e f r a c t i v e i n d e x o f the chromium s u r f a c e . Next the s l i d e i s c o v e r e d w i t h a p h o s p h o l i p i d b i l a y e r ( F i g u r e 2 m i d d l e p a n e l ) and the change i n p o l a r i z a t i o n i s measured a g a i n , g i v i n g the r e f r a c t i v e i n d e x and t h i c k n e s s o f the p h o s p h o l i p i d l a y e r . P r o t e i n a d s o r p t i o n on the p h o s p h o l i p i d s i s a n a l y z e d a c c o r d i n g t o the system p r e s e n t e d i n t h e lower p a n e l o f F i g u r e 2. The s u r f a c e c o n c e n t r a t i o n Γ , e x p r e s s e d as the mass o f t h e sub s t a n c e adsorbed on t h e s l i d e per u n i t s u r f a c e a r e a , can be c a l c u l a t e d from the r e f r a c t i v e index η and the t h i c k n e s s d o f the a d s o r b e d layer: 2

Γ = 3d

2

(n -n^)/[(n +2)(r(n^+2)-v(n^-1))]

(1)

where r and ν a r e the s p e c i f i c r e f r a c t i v i t y and the p a r t i a l s p e c i f i c volume o f the s u b s t a n c e d e p o s i t e d on the s l i d e and n^ i s the r e f r a c t i v e i n d e x o f the b u f f e r s o l u t i o n (9) . S t a c k i n g o f t h e monolayers o r m u l t i l a y e r s . S t a c k i n g was done w i t h a p r e p a r a t i v e L a n g m u i r - t r o u g h (Lauda, Type FW-1) a c c o r d i n g t o the meth od o f B l o d g e t t and Langmuir (11,12) · The r e f l e c t i n g s u r f a c e was d i p p ed i n t o t h e t r o u g h w i t h a s p e c i a l l y developed d i p p i n g machine a l l o w i n g r e g u l a r and e x a c t l y a d j u s t a b l e d i p p i n g - and w i t h d r a w i n g speeds o f about 2 mm/mm. The s u r f a c e p r e s s u r e o f the p h o s p h o l i p i d monolayer on the t r o u g h is critical and depends on the p h o s p h o l i p i d . T r a n s p o r t from t h e t r o u g h t o the e l l i p s o m e t e r c u v e t t e was done i n a s p e c i a l sample h o l der such t h a t exposure o f the b i l a y e r t o a i r was prevented. M a t e r i a l s . The f o l l o w i n g p h o s p h o l i p i d s were used: 1,2 d i m y r i s t o y l - s n - g l y c e r o - 3 - p h o s p h o s e r m e (DMPS) , 1,2 d i o l e o y l - s n - g l y c e r o - 3 - p h o s p h o s e r i n e (DOPS) and 1,2 d i o l e o y l - s n - g l y c e r o - 3 - p h o s p h o c h o l i n e (DOPC). B o v i n e p r o t h r o m b i n , human f i b r i n o g e n and b o v i n e albumin were u s e d . The p r o t e i n s were e i t h e r o b t a i n e d c o m m e r c i a l l y o r p r e p a r e d a c c o r d i n g t o e s t a b l i s h e d p r o c e d u r e s ( 12 ) · U n l e s s mentioned o t h e r w i s e a 0.05 M T r i s - H C l b u f f e r o f pH=7.5 was used, c o n t a i n i n g 0.1 M N a C l and 1.5 mM C a C l 2

S o r p t i o n e x p e r i m e n t s . E x p e r i m e n t s were p e r f o r m e d i n a c u v e t t e f i l l e d w i t h b u f f e r . The b u f f e r was c o n t i n u o u s l y s t i r r e d by a r a p i d l y r o t a t i n g magnetic s t i r r e r and the temperature was c o n t r o l l e d by a P e l t i e r element. A f t e r a d d i n g the p r o t e i n t o t h e c u v e t t e , the a d s o r p t i o n was f o l l o w e d by m e a s u r i n g the new p o s i t i o n s o f Ρ and A e v e r y 3-5 s e c o n d s . E q u i l i b r i u m was u s u a l l y a t t a i n e d a f t e r 10-60 min, depending on t h e


CUYPERS ET AL.

Kinetics of Protein Sorption


14.


211

212

PROTEINS AT INTERFACES


p r o t e i n c o n c e n t r a t i o n , D e s o r p t i o n was measured a f t e r r a p i d l y f l u s h i n g the c u v e t t e w i t h f r e s h b u f f e r (t=0) and subsequent c o n s t a n t slow f l u s h i n g i n order to maintain zero bulk c o n c e n t r a t i o n of p r o t e i n . F i g u r e 3 p r e s e n t s an example o f the e x p e r i m e n t a l d a t a o b t a i n e d d u r i n g a d s o r p t i o n and d e s o r p t i o n o f p r o t h r o m b i n on a DOPS d o u b l e l a y e r . The p o s i t i o n s o f the p o l a r i z e r Ρ and the a n a l y z e r A as a f u n c t i o n o f t i m e , and the parameters c a l c u l a t e d from t h e s e d a t a , the r e f r a c t i v e i n d e x n, the t h i c k n e s s d and the adsorbed mass Γ a r e shown. A n a l y s i s of s o r p t i o n k i n e t i c s . During adsorption a concentration p r o f i l e C ( x , t ) o f p r o t e i n i s e s t a b l i s h e d i n an u n s t i r r e d l a y e r s e p a r a t i n g the a d s o r b i n g s u r f a c e , s i t u a t e d a t x=0, from the b u f f e r s o l u t i o n . I t i s assumed t h a t i n i t i a l l y no p r o t e i n i s p r e s e n t i n the system and t h a t a t time t=0 the b u l k 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 b u f f e r i s changed t o a f i x e d v a l u e C^. I t i s a l s o assumed t h a t the a d s o r p t i o n r a t e i s p r o p o r t i o n a l t o the number o f f r e e b i n d i n g s i t e s and t o the p r o t e i n c o n c e n t r a t i o n a t the s u r f a c e . The r a t e o f d e s o r p t i o n i s assumed t o be p r o p o r t i o n a l t o the s u r f a c e c o n c e n t r a t i o n . For t h i s b i n d i n g model one has:

~

r< )

i n t

= k(n

t

(r

1

-run

c ( o , t ) - km "*: r

Kinetics of Protein Sorption on Phospholipid Membranes Measured by

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