Interaction of Prothrombin with Phospholipid Monolayers at Air- and

Chapter 7

Interaction of Prothrombin with Phospholipid Monolayers at Air- and Mercury-Water Interfaces M. F. Lecompte

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 4, 2016 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch007

Laboratoire d'Electrochimie Interfaciale, Centre National de la Recherche Scientifique, 1 Place A. Briand, 92195 Meudon Principal Cedex, France

The studies on the mode of interaction of prothrombin with phospholipid monolayers, using complementary methods of surface measurement are reviewed. They were investiga ted at air-water and Hg-water interfaces respectively by radioactivity and electrochemistry. A process more com plex than a simple adsorption could be detected. Indeed, the variation of the differential capacity of a mercury electrode in direct contact with phospholipid monolayer, induced by the interaction with prothrombin could be in terpreted as a model of its penetration into the layer; this was confirmed by the study of the dynamic proper ties of the direct adsorption of this protein at the e lectrode, followed in part by the reduction of S-S brid ges at the electrode. It could be also concluded that pro thrombin resists complete unfolding at these interfaces. In o r d e r t o understand the s t r u c t u r e and s t r u c t u r a l changes o f b i o l o g i c a l components i n v o l v e d i n protein-membrane i n t e r a c t i o n s , the s u r f a c e b e h a v i o r o f p r o t e i n s must be s t u d i e d c a r e f u l l y . In s e v e r a l s t e p s o f t h e b l o o d c o a g u l a t i o n cascade, t h e a c t i v i t y of some o f the c o a g u l a t i o n f a c t o r s i s enhanced a t the s u r f a c e o f t h e p h o s p h o l i p i d membrane. P h o s p h o l i p i d s , m a i n l y those which a r e n e g a t i v e l y charged, p l a y a c r u c i a l r o l e by a c c e l e r a t i n g t h e zymogen-toenzyme c o n v e r s i o n s l e a d i n g t o c l o t f o r m a t i o n . S i n c e t h e importance of thrombin i s w e l l known i n t h i s p r o c e s s , i t was o f i n t e r e s t t o un d e r s t a n d i t s r a t h e r complex f o r m a t i o n from t h e c o r r e s p o n d i n g zymogen, p r o t h r o m b i n . Moreover, c o n v e r s i o n o f p r o t h r o m b i n i n t o thrombin i s a good example o f a t y p i c a l enzymatic a c t i v i t y t a k i n g p l a c e a t a c e l l / s o l u t i o n i n t e r f a c e . The c o n v e r s i o n r e q u i r e s a membrane-bound complex of p r o t e a s e , s u b s t r a t e and c o f a c t o r . V i t a m i n K-dependent p r o t e i n s , c o n t a i n i n g y - c a r b o x y g ^ t a m i c resi dues, l i k e p r o t h r o m b i n , a r e commonly known t o b i n d by Ca bridges t o membranes c o n t a i n i n g a c i d i c p h o s p h o l i p i d s O ) . N e v e r t h e l e s s , i t was i m p o r t a n t t o study whether i n t e r a c t i o n s o t h e r than those o f an e l e c t r o s t a t i c n a t u r e c o u l d a l s o be i n v o l v e d , such as those l e a d i n g 0097-6156/87/0343-0103$06.00/0 © 1987 American Chemical Society

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


104

PROTEINS AT INTERFACES

to penetration of proteins into membranes, since the c a t a l y t i c process i s very s p e c i f i c , during the conversion. As an example of a membrane model, phospholipid monolayers with negative charge of different density were used. It had already been found Ç2) and discussed (3) that the physical and b i o l o g i c a l behavior of phospholipid monolayers at air-water interfaces and of suspensions of liposomes are comparable i f the monolayer i s in a condensed state. Two complementary methods of surface measurements (using r a d i o a c t i v i ty and electrochemical measurements), were used to investigate the adsorption and the dynamic properties of the adsorbed prothrombin on the phospholipid monolayers. Two d i f f e r e n t interfaces, air-water and mercury-water, were examined. In this review, the behavior of prothrombin at these interfaces, in the presence of phospholipid monolayers, i s presented as compared with i t s behavior in the absence of phospholipids. An excess of l i p i d of d i f f e r e n t compositions of phosphatidyl serine (PS) and phosphatidylcholine (PC) was spread over an aqueous phase so as to form a condensed monolayer, then the proteins were injecte^ underneath the monolayer in the presence or in the absence of Ca . The adsorption occurs in s i t u and under s t a t i c conditions. The excess of l i p i d ensured a f u l l y compressed monolayer in equilibrium with the collapsed excess l i p i d layers. The contribution of this excess of l i p i d to protein adsorption was n e g l i g i b l e and there was no effect at a l l on the electrode measurements. The Air-water Interface The adsorption of prothrombin onto the l i p i d monolayer^was followed d i r e c t l y by counting the surface r a d i o a c t i v i t y of the H labelled protein using a gas-flow counter equipped with an u l t r a t h i n window as described elsewhere (4). By c a l i b r a t i n g the counter as previously described C5), i t is possible to determine the surface concentration of the radioactive protein, Γ . In Figure 1, the k i n e t i c s of adsorption of prothrombin at the i n i t i a l bulk concentration of 5 yg/ml in the presence and in the ab sence of Ca , onto a monolayer containing 100 % PS are presented as compared with the adsorption at the pure air-water interface. It i s clear that, in the presence of phospholipids, the amount of protein adsorbed i s strongly dependent on Ca concentration, while this i s not so at the pure air-wajçr interface. Nevertheless, we see that even in the absence of Ca , the prothrombin adsorption remains s i gnificant, was shown (6) that even at concentrations as low as 10 mM, Ca i s coadsorbed with prothrombin. The results showed that the surface concentration of Ca i s proportional to that of the adsorbed prothrombin, about 10 Ca being coadsorbed with one molecule of prothrombin. The surface concentrations of prothrombin, obtained at equilibrium, were plotted as a function of i t s i n i t i a l concentra||on, while the interaction occurred in the presence of d i f ferent Ca concentrations and with phospholipid monolayers of d i f f e rent compositions Ç5). The Scatchard plots obtained from the adsorption isotherms gave the binding constants, Ka. When the adsorption^ was onto pure PS monolayer, Kg turned out to be independent of Ca concentration (around 1.2 χ 10 1/mol), while Çhe maximal surface concentration, r , i s dependent. At 2 mM Ca , on the monolayers +

+

m a x



7. LECOMPTE

Interaction of Prothrombin with Phospholipid Monolayers 105

Figure 1. Time dependence of adsorption of 5 yg/ml of prothrombin, at the air-water interface i n the presence of a phosphatidyl serine monolayer or i n i t s absence , i n the presence or i n the absence of 2 mM C a . + +



106 m

2


a

X

1 2

2

c o n t a i n i n g 2 5 % PS - 7 5 % P C , Γ was 1 . 7 5 x 1 0 " m o l . c m " a s c o m p a r e d w i t h 6.2x10~^ mol.cm a d s o r b e d o n p u r e PS m o n o l a y e r s ; t h e b i n d i n g c o n s t a n t was f o u n d t o b e e q u i v a l e n t t o t h e v a l u e s o b t a i n e d u s i n g p u r e PS m o n o l a y e r s . T h e s e c o n s t a n t s w e r e c o n f i r m e d , i n some c a s e s , o n m u l t i l a y e r s ( 7 ) o r o n m o n o l a y e r s (_3) b y u s i n g e l l i p s o m e t r y , b u t t h e y a r e h i g h e r t h a n t h o s e o b t a i n e d w i t h p h o s p h o l i p i d v e s i c l e s (_3). T h e s e l a s t d i f f e r e n c e s may b e a s c r i b e d t o some c a u s e s l i k e t h e d i f f e r e n t c u r v a t u r e s u s e d i n t h e t w o membrane m o d e l s a n d t h e l o w e r concentra t i o n s of p r o t e i n used w i t h the monolayers. N e v e r t h e l e s s , t h e e q u i l i b r i u m c o n d i t i o n s m i g h t depend on t h e t e c h n i q u e used and be o f impor tance i n the d i s c r e p a n c y i n t h e v a l u e s o f Ka found i n t h e l i t e r a t u r e . The m a x i m a l s u r f a c e c o n c e n t r a t i o n o f p r o t h r o m b i n a d s o r b e d o n a p u r e PS m o n o l a y e r a t 2 mM C a c o r r e s p o n d s t o a b o u t 27 n m / m o l e c u l e w h i l e a t 10 mM C a , w i t h a v a l u e o f 1 . 5 x 1 0 ~ ^ mol.cm"*2, £ç c o r r e s p o n d s t o 120 n m p e r m o l e c u l e . S i n c e t h e a r e a o c c u p i e d b y a p r o t h r o m b i n molecule a t maximal hexagonal packing o r i e n t e d w i t h i t s long a x i s p e r p e n d i c u l a r t o t h e s u r f a c e i s 18 n m , a c c o r d i n g t o t h e m o d e l g i v e n f o r p r o t h r o m b i n (J_) , t h i s c o n f i g u r a t i o n s h o u l d b e t h e o n e a p p r o a c h e d i n t h e p r e s e n c e o f C a . A t 10~3 mM C a , a p r o t h r o m b i n m o l e c u l e l i e s w i t h t h e l o n g a x i s o f i t s e l l i p s o i d a l shape p a r a l l e l t o t h e s u r f a c e . I n d e e d , i t c a n c o v e r a n a r e a o f a b o u t 50 n m a n d t h u s t h e t o t a l m a x i m a l number o f a d s o r b e d m o l e c u l e s c o v e r s a b o u t 5 0 % o f t h e a r e a . T h u s , we c a n d i s t i n g u i s h a c h a n g e i n t h e c o n f i g u r a t i o n o f t h e prothrombin molecules r e l a t i v e t o the surface o f the monolayer, i n the absence o r i n t h e presence o f C a . - 2

+ +

2

+ +

2

2

2

+ +

+ +

2

+ +

At t h e pure a i r - w a t e r i n t e r f a c e ( F i g u r e 1 ) , t h e i n i t i a l r a t e o f a d s o r p t i o n of prothrombin i s p r o p o r t i o n a l t o the square root o fthe t i m e , a s c a n be c a l c u l a t e d f r o m t h e c u r v e s , i n d i c a t i n g t h a t t h e p r o c e s s , a t t h i s i n t e r f a c e , i s d i f f u s i o n - c o n t r o l l e d , a s was o b s e r v e d f o r n a t i v e DNA (8_) . I n t h e p r e s e n c e o f p h o s p h o l i p i d m o n o l a y e r s t h e a d s o r p t i o n p r o c e s s i s s l o w e r ; a more c o m p l e x p r o c e s s m u s t t a k e p l a c e a t t h e s u r f a c e o f t h e membrane, i n a d d i t i o n t o t h e c o n f o r m a t i o n a l change o f p r o t h r o m b i n w h i c h o c c u r s i n t h e p r e s e n c e o f C a a s was f o u n d b y N e l s e s t u e n (9). I t c a n b e t h e p e n e t r a t i o n o f p r o t h r o m b i n i n t o t h e l i p i d l a y e r , as w i l l be d e s c r i b e d below. The s u r f a c e o c c u p i e d b y a p r o t e i n m o l e c u l e , a t h i g h C a concen t r a t i o n , as obtained from the p r o t e i n surface c o n c e n t r a t i o n , a t maxi mal c o v e r a g e , i s e q u i v a l e n t t o t h e s m a l l e r c r o s s - s e c t i o n a l a r e a o f a native p r o t e i n molecule i n s o l u t i o n , suggesting that the molecules are bound p e r p e n d i c u l a r l y t o t h e s u r f a c e . I t i n d i c a t e s c l e a r l y , i n t h i s c a s e , t h a t t h e p r o t e i n d o e s n o t u n f o l d a t t h i s i n t e r f a c e . The p r o t h r o m b i n m o l e c u l e c o n t a i n s a h i g h c o n c e n t r a t i o n o f S-S b o n d s , a n d i t i s known t h a t t h i s i s a v e r y i m p o r t a n t f a c t o r i n p r e s e r v i n g t h e t e r t i a r y s t r u c t u r e of proteins (10). At low calcium concentration, t h i s m i g h t a l s o b e t h e c a s e , i f we t a k e i n t o a c c o u n t t h e p o s s i b i l i t y of t h e p r o t e i n bound t o t h e s u r f a c e b e i n g a b l e t o r o t a t e i n t h e plane. + +

+

The

Mercury-water

+

Interface

C a p a c i t a n c e , C, p r o v i d e s d i r e c t i n f o r m a t i o n o n t h e s t r u c t u r e o f t h e a d s o r b e d l a y e r (10, 11). The change i n t h e d i f f e r e n t i a l c a p a c i t y o f the e l e c t r i c a l double l a y e r between a p o l a r i z e d mercury s u r f a c e and a 0.15 M N a C l s o l u t i o n c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f p r o t e i n



7.

LECOMPTE

Interaction of Prothrombin with Phospholipid Monolayers 107

( f o r e x a m p l e ) was u s e d a s a m e a s u r e o f i t s a d s o r p t i o n o n a b a r e m e r c u r y s u r f a c e o r on a s p r e a d l i p i d m o n o l a y e r i n c o n t a c t w i t h t h e e l e c t r o d e , as w i l l be d e s c r i b e d below. A s s e e n i n F i g u r e 2, when a c o m p r e s s e d s p r e a d l a y e r o f l o n g chain l i p i d s i s brought i n t o contact w i t h a mercury e l e c t r o d e from the gaseous phase, the capacitance of the double l a y e r i s very low i n c o m p a r i s o n w i t h t h e s u p p o r t i n g e l e c t r o l y t e alone and v a r i e s b e t ween 1.5 a n d 1.9 y F . c m ~ 2 o v e r a w i d e r a n g e o f p o t e n t i a l . I t i s c h a r a c t e r i s t i c o f a h y d r o c a r b o n l a y e r , one h y d r o c a r b o n c h a i n l e n g t h t h i c k , which adheres t o the mercury s u r f a c e , w h i l e t h e p o l a r head groups o r i e n t themselves toward t h e aqueous s o l u t i o n , and i t p r e v e n t s a c c e s s o f i o n s . A t some p o s i t i v e o r n e g a t i v e p o t e n t i a l , a d e s o r p t i o n c a p a c i t a n c e p e a k c a n b e o b s e r v e d . The p e a k r e s u l t s f r o m t h e c h a r g e f l u x f o l l o w i n g the displacement of the low d i e l e c t r i c l a y e r by t h e h i g h d i e l e c t r i c a q u e o u s medium. I f t h e c o n t i n u i t y o f t h e monolayer i s p e r t u r b e d by an i n t e r a c t i n g m o l e c u l e o f h i g h e r p o l a r i t y , an i n c r e a s e i n c a p a c i t a n c e propor t i o n a l t o the degree of perturbance or p e n e t r a t i o n i s observed. I n the c a s e where t h e p e n e t r a t i n g m o l e c u l e s c o n t a i n e l e c t r o a c t i v e groups u n d e r g o i n g e l e c t r o d e r e a c t i o n , a p s e u d o c a p a c i t a n c e peak i s o b t a i n e d which i s p r o p o r t i o n a l i n s i z e t o the ease o f access of these groups (through the l i p i d l a y e r ) to the electrode surface. In the case of p r o t h r o m b i n a n d o f o t h e r p r o t e i n s , c y s t i n e may s e r v e a s s u c h a n e l e c t r o a c t i v e group. C y s t i n e i s s t r o n g l y adsorbed on t h e mercury s u r f a c e at p o s i t i v e p o t e n t i a l s of the redox p o t e n t i a l , forming a charget r a n s f e r c o m p l e x ( 1 2 ) . The s u r f a c e c o m p l e x i s t h e n r e d u c e d a t t h e r e d o x p o t e n t i a l g i v i n g r i s e t o t h e p s e u d o c a p a c i t a n c e peak. The c y s t i n e - c y s t e i n e t r a n s i t i o n o n t h e m e r c u r y e l e c t r o d e was s e e n t o b e a r e v e r s i b l e p r o c e s s ( 1 2 , 13) w h i l e t h e m e r c u r y a c t s a s a c a t a l y t i c s u r f a c e f o l l o w i n g t h e scheme: RSSR + Hg # H g ( R S )

2

or Hg (RS) 2

2

+ 2H

+

+ 2e" ^

2 RSH + Hg

A s s e e n i n F i g u r e 2, t h e a c p o l a r o g r a m r e s u l t i n g f r o m t h e i n t e r a c t i o n o f t h e m o n o l a y e r w i t h p r o t h r o m b i n shows t h a t a n e l e c t r o a c t i v e g r o u p c o n t r i b u t e s t o the capacitance curve a pseudocapacitance peak, a t a r o u n d - 0.7 V, w h i c h i s a s c r i b e d t o t h e o x y - r e d u c t i o n o f t h e d i s u l f i d e b r i d g e s a t pH 7.8. S i n c e t h e f o r m a t i o n o f c y s t e i n e r e q u i r e s hydrogen i o n s , the h a l f - w a v e p o t e n t i a l and thus a l s o the pseudocapa c i t a n c e p e a k i s s h i f t e d w i t h d e c r e a s i n g pH t o m o r e p o s i t i v e p o l a r i z a t i o n . S i n c e two c o n t r i b u t i o n s , o n e f r o m t h e p r o t e i n a n d t h e o t h e r from the p h o s p h o l i p i d monolayer, a r e i n v o l v e d i n the capacitance v a l u e s , i t was o f i m p o r t a n c e t o s t u d y p a r t i c u l a r l y t h e b e h a v i o r o f t h e p r o t e i n i n d i r e c t c o n t a c t w i t h t h e e l e c t r o d e , i n o r d e r t o be a b l e t o i n t e r p r e t b e t t e r t h e d a t a o b t a i n e d when p r o t h r o m b i n i n t e r a c t s w i t h phospholipid monolayers. D i r e c t adsorption of Prothrombin at the Mercury-water I n t e r f a c e . The a d s o r p t i o n r a t e o f p r o t h r o m b i n on a h a n g i n g m e r c u r y d r o p e l e c t r o d e (HMDE) was s t u d i e d b y m e a s u r i n g t h e d e c r e a s e o f t h e d i f f e r e n t i a l c a p a c i t y w i t h time of contact of the mercury drop w i t h the s o l u t i o n , a t a f i x e d p o t e n t i a l , - 0.5 V, i n p a r a l l e l w i t h t h e i n c r e a s e o f t h e areas o f t h e v o l t a m e t r i c peaks c o r r e s p o n d i n g t o the reduction of some S-S b o n d s o f t h e a d s o r b e d m o l e c u l e s ( 1 4 ) . T h e r e p r e s e n t a t i o n o n



108


F i g u r e 2. E f f e c t of prothrombin (5 y g / m l ) on t h e c a p a c i t a n c e o f a m o n o l a y e r o f 1 0 0 % PS as compared w i t h t h e c a p a c i t a n c e o f t h e e l e c t r o l y t e a l o n e -· ; N a C l 0.15M, T r i s 1mM, pH 7.8.


7.

interaction of Prothrombin with Phospholipid Monolayers

LECOMPTE

the same graphs of the two curves versus t and C versus t, in the presence or in the absence of ' C a , at one concentration of prothrombin for example 4.5 yg/ml, shows c l e a r l y in Figure 3 that these functions reach their saturation values simultaneously. Adsorp tion of prothrombin at the waiting potential of -0.5 V (Figure 3), near the zero charge p o t e n t i a l , causes a sharp decrease in capacitan ce. For each concentration studied (14), about the same lower l i m i t of capacitance was reached, corresponding to a saturation value in the range studied, which was therefore attributed to a completely protein-covered electrode. At about half of the maximum lowering of the capacity, an i n f l e x i o n point can be distinguished. From the k i n e t i c s of adsorption, the surface coverage could be obtained. In the case of prothrombin, the number of molecules adsor bed on the mercury surface, Γ \/2 » could be evaluated from the l i near dependence of the capacily on t ^ / Qnly at short times (< 50s), when the d i f f u s i o n layer thickness (Dt)^'^ i s s t i l l smaller than the thickness of the unstirred layer. In this region the concentration i s


+ +

2

r 1/2 t

=

(2/π1/2)

1

C p

D'

2 t

1/2

( 1 )

and thus the c o r r e l a t i o n between the lowering of capacity AC, and Γ *- obtained. By extrapolating the plot of capacitance versus t ^ / to saturation capacitance, one obtains the l i m i t i n g saturation surface concentration , r 2 · Around t= 50s, the depen dence of capacitance on t ^ starts deviating from l i n e a r i t y and above 100s, a region with linear dependence of capacitance on t i s obtained which allowed us, by extrapolation to the saturation capa citance, as described previously (14), to determine the l i m i t i n g saturation surface concentration, ^ . It was shown i n the case of polymers, below surface saturation, that the decrease of capacitance i s proportional to their surface concentration (over the whole potential region) (10, 15). It implies that the surface conformation of molecules being adsorbed i s esta blished instantaneously and then remains constant. Let us assume that this i s also the case for prothrombin. The two maximum surface concentrations T™f 2 as T ™ , equivalent to 1.5x10~^ mol.cm" , represent maximal packing for the i n i t i a l conformation of the adsor bed molecules and does not take into account a possible change i n configuration around the i n f l e x i o n point on the capacitance versus time curve. . Since the slopes of the linear v a r i a t i o n of AC = f ( t ' ) and AC = f ( t ) varied with protein concentrations at a l l concentration as according to the corresponding equations giving Γ (14), the process is d i f f u s i o n c o n t r o l l e d . It also implies immediate adsorption and n e g l i g i b l e back reaction. The saturation surface coverage values can be used for determi nation of the l i m i t i n g values of the area occupied per adsorbed pro tein. The calculated areas were equivalent to 110 nm in absence of Ca and 95 nm i n presence of C a , per molecule. The areas of the peaks obtained by c y c l i c voltametry allows the c a l c u l a t i o n of the number of the reduced cystine residues, Tg_g, or reoxidized cysteine, taking into account that the l a t t e r i s twice the former. Since the p o s i t i o n of the onset of the reduction peak i s s l i g h t l y higher than - 0.5 V, the quantity of S-S groups reduced was s

2

m a X

2

m a x

X

a x

2

2

2

+ +


2

109


1

—ι

-r


-05V

t (mm )

Figure 3. Comparison of the simultaneous variations with time of the capacitance C and the surface concentration of electroac tive d i s u l f i d e groups, calculated from the 2nd reduction sweep, for a concentration of prothrombin of 4.5 yg/ml, i n the presence or i n the absence of 2 mM C a . Waiting p o t e n t i a l : - 0.5 V + +



7. LECOMPTE

Interaction of Prothrombin with Phospholipid Monolayers

111

determined only at the second negative sweep. In Figure 3, the number of moles of reduced cystine per unit area i s plotted versus time. L i near dependence of Γ ^ on time i s observed over a r e l a t i v e l y large range of coverage ana since the slopes obtained at d i f f e r e n t concen trations were found to vary with each of them according to the cor respondent equation (14), we could calculate for each time Γ using this equation. The extrapolation of the straight lines drawn^from the i n i t i a l values, obtained from the plot of against time, at the plateau of Tg_^ versus t, allows the determination of the time necessary to complete the surface layer and therefore using the right equation allows the c a l c u l a t i o n of the actual surface concentration, at equilibrium, r , at high coverage, which i s about 3x10"^ mol. cm"*. ^ This value, together with the corresponding value of S-S (11x10"12 mol.cm ) , gives a mean value of the number of S-S groups reduced per molecule adsorbed when saturation i s reached. Only a small f r a c t i o n (3) of the t o t a l cystine residues (12) present in the whole adsorbed prothrombin molecule i s available for reduction on the electrode, i n spite of the exposure of the molecules to the mer cury electrode at p o s i t i v e p o l a r i z a t i o n s , at which cystine tends to be adsorbed on the electrode. This i s in agreement with other f i n dings that in the case of proteins only some of the S-S groups are available for electrode reaction (16, 17). The prothrombin molecule, s i m i l a r l y to other proteins, r e s i s t s complete unfolding, when adsor bed on the mercury electrode, in the range of adsorption potential studied. The degree of p a r t i a l unfolding depends on the electrode po t e n t i a l during adsorption, on the time of exposure to the surface and on the presence of C a . It was shown that at a more positive p o l a r i z a t i o n , -0.35 V, the area occupied by a molecule i s smaller than at -0.5 V and the number of S-S reduced by the molecule higher; this indicates a change i n the conformation of the molecules adsorbed at the interface depending on the electrode p o l a r i z a t i o n . The l i m i t i n g areas i n the presence of Ca are lower than in i t s absence both in the low and the high sur face concentration region, indicating smaller deviation from i t s glo bular structure in the bulk. Moreover the number of S-S reduced by a molecule was lower i n the presence of Ca than in i t s absence. Then i t could be concluded that C a causes s t a b i l i z a t i o n of the molecular structure of prothrombin at the surface. We t r i e d to answer the following question: Why i s the maximal surface concentration at adsorption equilibrium obtained from the ex trapolation of d i f f e r e n t i a l capacity against time less than half that obtained from a similar extrapolation of the voltametric peaks? The plot of as a function of the v a r i a t i o n of C from the e l e c t r o l y t e alone, AC, at d i f f e r e n t times of adsorption u n t i l sa turation i s a typical diagram which represents the dynamic picture of the growth of the adsorbed protein layer (Figure 4). It shows c l e a r l y that the contribution of a given amount of ad sorbed protein to the changes i n and C i s d i f f e r e n t i f low or high surface concentrations are considered. This plot shows essen t i a l l y two linear sections over the whole range of surface concentra tion. At low surface concentration (region A), the capacitance appears as the more sensitive probe to adsorption, while at high m a x

2

2

2

+ +

+ +

+ +



112


F i g u r e 4. Surface c o n c e n t r a t i o n o f e l e c t r o a c t i v e d i s u l f i d e bonds f r o m t h e 2 n d r e d u c t i o n · , 1 s t o x i d a t i o n sweep • , a s a f u n c t i o n o f t h e d e c r e a s e o f t h e c a p a c i t a n c e o f t h e HMDΕ i n c o n t a c t w i t h a p r o t h r o m b i n s o l u t i o n a t 4.5 u g / m l . P o t e n t i a l o f a d s o r p t i o n : - 0 . 5 V



7. LECOMPTE


113

surface concentrations the area of the voltametrie peak becomes more sensitive (region B). From this plot an answer could be given to the above question, by considering the following model for the growth of the adsorbed layer (14). While extrapolation of versus t gives informations corresponding to the "A and B" behavior of the system, extrapolation of C versus t to the equilibrium capacitance value a l lows an estimation of the hypothetical value of the number of molecu les adsorbed at maximal packing in the "A" conformation, i d e n t i c a l to the conformation of each isolated molecule at the surface. The areas obtained are larger than the cross-sectional area of a native molecu le lying in the surface (52 nm ) (JO, indicating d i f f e r e n t degrees of unfolding of the molecules which r e t a i n a considerable freedom of ro tation. In the region of the i n f l e x i o n i n the C(t) curve, the motion of molecules in the surface i s r e s t r i c t e d , because of the l a t e r a l i n teraction between the protein molecules, inducing a surface gel f o r mation (18). In region B, adsorption continues to lower the capaci tance less e f f i c i e n t l y than i n part A. In part B, each new molecule being adsorbed onto the new molecular surface network of the adlayer occupies i t s own area, which i s lower than i n part A, at the Hg sur face, causing at the same time a l a t e r a l contraction of i t s neighbour molecules. The plot of Γ g versus AC i s linear inasmuch as the sur face occupied per molecule and the number of S-S groups per adsorbed molecule are constant. From the change in the slopes i n parts A and Β (Figure 4), the adsorption process shows c l e a r l y the existence of two d i s t i n c t adsorption states for which were defined a molecular aarea (part A) and a d i f f e r e n t i a l one (part B) for the newly adsorbed molecules which might e q u i l i b r a t e with the already adsorbed ones(14). The extrapolated values of (Γ^_ )^ to A C from part A (Figure 4), divided by the value of T obtained from C versus t at saturation gives the number of S-S reduced per molecule in part A; i t i s lower 0^2) than at higher coverage 0^3) where l a t e r a l protein interactions might occur and seems to aid exposure of the s u l f i d e groups. This diagram was shown to be e s s e n t i a l l y independent of the bulk protein concentration and contains c h a r a c t e r i s t i c points which are correlated only with surface concentration. At low surface concentrations, the redox process i s nearly re v e r s i b l e , by taking in account the areas and the potentials of the peaks. At higher, the differences in the areas between reduction and oxidation peaks may be attributed to differences in the adsorption of cystine and desorption of cysteine residues at the p o s i t i v e l y or ne gatively charged mercury respectively. At the same time the protein molecule as a whole remains adsorbed by hydrophobic interactions. With other b i o l o g i c a l macromolecules (19, 20), the number of ad sorbed molecules was usually calculated from the linear dependence of the capacitance on t ^ using Equation 1, over the whole range of ad sorption. The surface concentration of hormones could also be i n f e r red d i r e c t l y from the calculated number of charges transferred bet ween the electrode and an e l e c t r o a c t i v e group, l i k e S-S, of the ad sorbed molecules, each one containing only one S-S (21, 22). This me thod could not be used for proteins, where only part of the S-S are available for the electrode reaction,as seen for prothrombin; but in this case of proteins, the method of e x p l o i t a t i o n of the data presen ted above i s very useful and quite new. Only a f r a c t i o n of the t o t a l S-S bonds i s reduced in case of 2

m a x

m a x

2



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prothrombin, i n d i c a t i n g t h a t o n l y the c y s t i n e groups i n c o n t a c t w i t h t h e m e r c u r y a r e e l e c t r o a c t i v e . I t c o u l d be c o n c l u d e d f r o m t h i s t h a t when we g e t a p e a k o f S-S r e d u c t i o n , w h i l e p r o t h r o m b i n i n t e r a c t s w i t h p h o s p h o l i p i d s , t h i s p r o t e i n had t o c r o s s t h e m o n o l a y e r i n o r d e r t h a t t h e S-S s h o u l d be r e d u c e d ; t h i s i s i n f a v o r o f t h e p e n e t r a t i o n o f p r o t h r o m b i n i n t o the l a y e r . I n t e r a c t i o n o f P r o t h r o m b i n a n d some o f i t s f r a g m e n t s w i t h P h o s p h o l i pid Monolayers. When t h e a d s o r p t i o n e q u i l i b r i u m o f t h e p r o t e i n h a s b e e n r e a c h e d , a s d e t e r m i n e d b y s u r f a c e r a d i o a c t i v i t y , a n HMDΕ was f o r m e d a n d p o s i t i o n e d i n o r d e r t o be i n c o n t a c t w i t h t h e m o n o l a y e r (23). Thus the m o n o l a y e r t r a n s f e r r e d o n t o t h e m e r c u r y e l e c t r o d e - w a t e r i n t e r f a c e s t a y e d i n e q u i l i b r i u m w i t h t h e m o n o l a y e r r e s e r v o i r on t h e a i r - w a t e r i n t e r f a c e . Then ac p o l a r o g r a m s were r e c o r d e d a f t e r e x p o s u r e o f t h e m o n o l a y e r t o t h e e l e c t r o d e a t a g i v e n p o t e n t i a l , - 0.2 V i n F i g u r e 2 a n d - 0.5 V i n t h e o t h e r s w h i c h w i l l be p r e s e n t e d . Indeed, a t -0.5V, t h e m o n o l a y e r i s a t t h e c a p a c i t a n c e minimum and more s t a b l e and t h e p o t e n t i a l i s s t i l l r e m o t e enough f r o m t h e c y s t i n e - c y s t e i n e r e d o x p s e u d o c a p a c i t a n c e p e a k p o t e n t i a l . The i n c r e a s e i n c a p a c i t a n c e , a t t h i s p o t e n t i a l , was s e l e c t e d t o r e p r e s e n t t h e e f f e c t o f t h e p r o t e i n p e n e t r a t i o n i n t o t h e l i p i d l a y e r and i s p r e s e n t e d i n F i g u r e s 5 a n d 6 a s a f u n c t i o n o f t h e p r o t e i n c o n c e n t r a t i o n i n t h e b u l k . We see c l e a r l y d i s t i n c t b e h a v i o r b e t w e e n p r o t h r o m b i n and i t s f r a g m e n t s , de p e n d i n g on t h e l i p i d c o m p o s i t i o n a n d o f t h e p r e s e n c e o f C a . + +

The p e n e t r a t i o n o f p r o t h r o m b i n i n t o a m o n o l a y e r c o n t a i n i n g 2 5 % P S - 7 5 % PC, s t a r t s o n l y a t h i g h e r p r o t h r o m b i n c o n c e n t r a t i o n s t h a n on t h e p u r e PS m o n o l a y e r , a n d a c o o p e r a t i v e d e p e n d e n c e o f t h e c a p a c i t a n c e a n d o f t h e p s e u d o c a p a c i t a n c e p e a k s on t h e p r o t h r o m b i n c o n c e n t r a t i o n i s o b s e r v e d . However, the l i m i t i n g c a p a c i t a n c e s r e a c h e d a t h i g h e r p r o t h r o m b i n c o n c e n t r a t i o n s a r e a b o u t t h e same w i t h b o t h mono l a y e r s : i t i s 7 yF.cm~2. These h i g h c a p a c i t a n c e s a r e o b t a i n e d ins t a n t a n e o u s l y upon nondamaging c o n t a c t of the m o n o l a y e r by the e l e c trode. E v e n i n t h e a b s e n c e o f Ca (Figure 6), there i s a s i g n i f i c a n t i n c r e a s e i n c a p a c i t a n c e upon a d d i t i o n of p r o t h r o m b i n . T h i s i n d i c a t e s t h a t o t h e r i n t e r a c t i o n s b e s i d e the e l e c t r o s t a t i c ones have t o take p l a c e . H o w e v e r , the i n c r e a s e w i t h c o n c e n t r a t i o n i s l e s s steep i n the l o w Ca concentration region. By t a k i n g i n t o a c c o u n t t h e r a t i o b e t w e e n t h e n u m b e r o f m o l e c u l e s a d s o r b e d on p h o s p h o l i p i d s a n d t h e n u m b e r o f S-S r e d u c e d , i t was f o u n d (23) t h a t o n l y a s m a l l f r a c t i o n of t h e t o t a l c y s t i n e r e s i d u e s o f t h e a d s o r b e d p r o t h r o m b i n m o l e c u l e s i s a v a i l a b l e f o r r e d u c t i o n on t h e e l e c t r o d e , a n d i s e q u i v a l e n t t o t h e r a t i o when p r o t h r o m b i n i s i n d i r e c t c o n t a c t w i t h t h e e l e c t r o d e . C o n s e q u e n t l y , t h e r e i s no g r o s s c o n f o r m a t i o n a l c h a n g e o f p r o t h r o m b i n , when i n t e r a c t i n g w i t h a p h o s p h o l i p i d s u r f a c e . The s i g n i f i c a n t c h a n g e i n t h i s r a t i o b e t w e e n h i g h and l o w Ca c o n c e n t r a t i o n s s u g g e s t e d a c o n f o r m a t i o n a l change b r o u g h t a b o u t by t h e l i p i d - c a l c i u m p r o t e i n b o n d s , w h i c h i s i n a g r e e m e n t w i t h the d i f f e r e n c e s o b t a i n e d f o r the a r e a s o c c u p i e d by m o l e c u l e a t t h e air-water interface. +

S i m i l a r l y t o p r o t h r o m b i n , F r a g m e n t 1 c o n t a i n i n g t h e γ-carboxyg l u t a m i c r e s i d u e s , i n c r e a s e d t h e c a p a c i t a n c e o f P S - c o n t a i n i n g mono l a y e r s , a l s o g i v i n g r i s e t o a p s e u d o c a p a c i t a n c e p e a k . The e f f e c t i n c r e a s e s w i t h Ca and w i t h F r a g m e n t 1 c o n c e n t r a t i o n and i t i s l a r -



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0.1

0.2

0.3

0.-4

Figure 5. Differential capacity of condensed monolayers containing 100% PS (a) or 25% PS-75% PC (b) at -0.5 V relative to IN Ag/AgCl electrode as a function of the protein concentrations in the bulk in the presence of Ca *. Prothrombin, o; fragment Ι , Δ ; fragment 2, · . 4

0.1

0.2

0.3

(>AM )

Figure 6. Differential capacity of condensed monolayers containing 100% PS (a) or 25% PS-75% PC (b) at -0.5 V relative to IN Ag/AgCl electrode as a function of the protein concentrations in the bulk in the absence of Ca *. Prothrombin, o; fragment 1, Δ ; fragment 2, · . 4


115

116


g e r o n a m o n o l a y e r o f p u r e PS t h a n o n a m i x e d m o n o l a y e r . T h e o b s e r v e d i n c r e a s e i n c a p a c i t a n c e i s a l s o s m a l l e r a t 10~3 mM C a . Nevertheless at h i g h Fragment 1 c o n c e n t r a t i o n , an e q u i v a l e n t s a t u r a t i o n v a l u e f o r the c a p a c i t a n c e i s reached i n each c a s e , w h i c h i s s i m i l a r t o t h e one obtained f o r prothrombin. The s i g m o i d a l c u r v e s o b t a i n e d a t 2 5 % PS a n d Ca a n d a t 1 0 0 % PS w i t h l o w C a concentration indicates a coopera t i v e e f f e c t on c a p a c i t a n c e , l i k e f o r prothrombin except that h i g h e r c o n c e n t r a t i o n s o f Fragment 1 a r e r e q u i r e d i n order t o p e n e t r a t e t h e l i p i d l a y e r . W h i l e Fragment 1 adsorbs on a monolayer c o n t a i n i n g 25% PS t i l l 0.15 yM c o r r e s p o n d i n g t o 5 y g / m l , i t d o e s n o t p e n e t r a t e i n t h i s range of c o n c e n t r a t i o n i n the bulk. I t s t a r t s p e n e t r a t i n g a f t e r a c e r t a i n s u r f a c e c o v e r a g e h a s b e e n r e a c h e d . T h i s shows d i s t i n c t l y t h a t t h e c a p a c i t a n c e measurements a s s o c i a t e d w i t h t h e r a d i o a c t i v e ones a l l o w s d i s t i n c t i o n between adsorbed and p e n e t r a t e d molecules. I n t h e c a s e o f F r a g m e n t 2, t h e d e p e n d e n c e o f c a p a c i t a n c e o n c o n c e n t r a t i o n r e m a i n s p r a c t i c a l l y t h e same w h a t e v e r t h e C a concentration. However, t h e r e i s a dependence on t h e m o n o l a y e r c o m p o s i t i o n . Fragment 2 d o e s n o t a f f e c t a t a l l t h e m i x e d m o n o l a y e r s t i l l 0.4 yM. A s was shown ( 2 3 ) , t h r o m b i n p e n e t r a t e s s i m i l a r l y b o t h l a y e r s , b u t a s i t i s n o t a n i n t a c t s t r u c t u r a l d o m a i n o f t h e w h o l e p r o t h r o m b i n ( i t comes from t h e cleavage by f a c t o r Xa o f p r e t h r o m b i n 2 ) , i t s e f f e c t on capa c i t a n c e i s not represented here. Nevertheless, there i s a l a r g e r ten d e n c y o f t h r o m b i n t o p e n e t r a t e r a t h e r t h a n F r a g m e n t 2. T h i s s u g g e s t e d t h a t t h e p r e t h r o m b i n domain i s probably r e s p o n s i b l e f o r p e n e t r a t i o n of p r o t h r o m b i n i n t o t h e l i p i d l a y e r s . + +


+ +

+ +

S i n c e S-S b r i d g e s a r e r e d u c e d i n a l l c a s e s , t h e p r o t e i n m o l e c u les have t o c r o s s t h e l a y e r i n o r d e r t o reach t h e e l e c t r o d e s u r f a c e and t o b e r e d u c e d , w h i c h i m p l i e s t h a t t h e d i f f e r e n t f r a g m e n t s p e n e t r a t e t o some e x t e n t t h e m o n o l a y e r . Conclusions The

p r o t h r o m b i n m o l e c u l e does n o t u n f o l d on t h e d i f f e r e n t i n t e r f a c e s . ++ I n t h e p r e s e n c e o f C a , some c h a n g e s o f t h e p o s i t i o n o f t h e p r o t e i n r e l a t i v e t o t h e l i p i d l a y e r c o u l d be d e t e c t e d . Ca a l s o induces sta b i l i z a t i o n o f t h e g l o b u l a r s t r u c t u r e , as measured a t a bare mercury e l e c t r o d e . Furthermore, a dynamic p i c t u r e o f t h e growth o f t h e adsor b e d p r o t h r o m b i n l a y e r o n m e r c u r y e l e c t r o d e was p r e s e n t e d a n d shows 2 d i s t i n c t adsorption states. ++ I t was c o n f i r m e d t h a t C a i n c r e a s e s t h e a d s o r p t i o n , b u t i t was found t h a t even i n t h e absence o f C a , prothrombin i n t e r a c t s w i t h phospholipids. Consequently, besides the e l e c t r o s t a t i c i n t e r a c t i o n s , some C a - i n d e p e n d e n t i n t e r a c t i o n s , w h i c h m i g h t b e h y d r o p h o b i c , a r e a l s o i n v o l v e d . The o b s e r v a t i o n t h a t p r o t h r o m b i n a n d some o f i t s f r a g ments p e n e t r a t e d t h e l a y e r i s i n a c c o r d a n c e w i t h t h i s i d e a . T h i s i m p l i e s t h a t fragments o t h e r than Fragment 1 might be i n v o l v e d i n t h e i n t e r a c t i o n . I t must be n o t i c e d , t h a t p e n e t r a t i o n c o u l d n o t be o b t a i ned b y t h e l e s s s e n s i t i v e t e c h n i q u e o f s u r f a c e p r e s s u r e m e a s u r e m e n t s (24). S i n c e i t was f o u n d t h a t a c t i v i t y o c c u r s e i t h e r o n m o n o l a y e r s o r on v e s i c l e s (_3), t h e p e n e t r a t i o n o f p r o t h r o m b i n i n t o m o n o l a y e r s w h i c h was f o u n d , a n d c o n f i r m e d o n v e s i c l e s ( 2 5 ) , m i g h t h a v e a r o l e i n t h e c a t a l y t i c t r a n s f o r m a t i o n o f prothrombin i n t o thrombin and i t s regulation. + +

+ +


7.

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Acknowledgments


The author is particularly very grateful to Professor I.R.MILLER, who provided continual encouragement and helpful advice during the suc cessive stages of this work, part of which was performed in his labo ratory in the membrane department , at the Weizmann Institute, Israel The author thanks his colleagues whose names are clear from the refe rence l i s t , and financial support from INSERM n° 845016, MRT 85C1094 and A.I.P. 9631/112 grants. Literature Cited 1. Lim, T.K.; Bloomfield, V.A.; Nelsestuen, G.L. Biochemistry 1977, 16, 4177-4181. 2. Blume, A.Biochim.Biophys.Acta 1979, 557, 32-44. 3. Kop, J.M.M.; Cuypers, P.Α.; Lindhout, T.; Hemker, H.C.;Hermens, W.T. J.Biol.Chem. 1984, 259, 13993-13998. 4. Frommer, M.A.; Miller, I.R. J.Colloid Interface Sci. 1966, 21, 245-252. 5. Lecompte, M.F.; Miller, I.R.; Elion, J.; Benarous R. Biochemis try 1980, 19, 3434-3439. 6. Lecompte, M.F.; Miller, I.R. Advances in Chemistry series 1980, 188, 117-127. 7. Cuypers, P.Α.; Corsel, J.W.; Janssen, M.P.; Kop, J.M.M., Hermens W.T.; Hemker, H.C. J.Biol.Chem. 1983, 258, 2426-2431. 8. Frommer, M.A.; Miller, I.R. J.Phys.Chem. 1968, 72, 2862-2866. 9. Nelsestuen, G.L. J.Biol.Chem. 1976, 251, 5648-5656. 10. Miller, I.R.; Bach, D. Surface and Colloid Science; Matijevic,E. Ed.; 1973, 6, 185-260. 11. Miller, I.R. Topics in Bioelectrochemistry and Bioenergetics; 1981, 4, 161-224. 12. Kolthoff, I.M.; Barnum, C. J.Am.Chem.Soc. 1941, 63, 520-526. 13. Miller, I.R.; Teva, J. J.Electroanal.Chem. 1972, 36, 157-166. 14. Lecompte, M.F.; Clavilier, J.; Dode, C.; Elion, J.; Miller, I.R. J.Electroanal.Chem. 1984, 163, 345-362. 15. Miller, I.R.; Grahame, D.C. J.Colloid Interface Sci.1961,16, 23-40. 16. Cecil, R.; Weitzmann, P.D.J. Biochem.J. 1964, 93, 1-11. 17. Pavlovic, O.; Miller, I.R. Experientia Suppl. 1971, 18,513-524. 18. Lecompte, M.F.; Rubinstein, I.; Miller, I.R. J.Colloid Interface Sci. 1983, 91, 12-19. 19. Temerk, Y.M.; Valenta, P.; Nurnberg, W. J.Electroanal.Chem. 1982, 131, 265-277. 20. Scheller, F. Bioelectrochem.Bioenerg. 1977, 4, 490-499. 21. Rishpon, J.; Miller, I.R. Bioelectrochem.Bioenerg. 1975, 2, 215- 230. 22. Rishpon, J.; Miller, I.R. J.Electroanal.Chem.1975, 65, 453-467. 23. Lecompte, M.F.; Miller, I.R. Biochemistry 1980, 19, 3439-3446. 24. Mayer, L.D.; Nelsestuen, G.L.; Brockman, H.L. Biochemistry 1983, 22, 316-321. 25. Lecompte, M.F.; Rosenberg, I.; Gitler C. Βiochem.Biophys.Res. Commun. 1984, 125, 381-386. RECEIVED January 30, 1987


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