ELLIPSOMETRIC STUDIES OF BIOLOGICAL MACROMOLECULES

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ELLIPSOMETRIC STUDIES OF BIOLOGICAL MACROMOLECULES

K. Toth, G. Vuye, H. Vainer, Gy. Ronto, D. Aslanian

To cite this version:

K. Toth, G. Vuye, H. Vainer, Gy. Ronto, D. Aslanian. ELLIPSOMETRIC STUDIES OF BIOLOG- ICAL MACROMOLECULES. Journal de Physique Colloques, 1983, 44 (C10), pp.C10-521-C10-523.

�10.1051/jphyscol:198310107�. �jpa-00223467�

JOURNAL DE PHYSIQUE

Colloque CIO, supplément au n°12, Tome Hk, décembre 1983 page C10-521

ELLIPSOMETRIC STUDIES OF BIOLOGICAL MACROMOLECULES

K. Toth, G. Vuye*, H. Vainer***, Gy. Ronto and D. Aslanian**

Institute of Biophysics, Semmelweis Medical University, P.O. Box 263, H-1444 Budapest, Hungary

Laboratoire d'Optique des Solides, et

** Laboratoire de Physique des Solides, Spectrosaopie des Solides*, Université Pierre et Marie Curie, 4, place Jussieu, 75230 Paris Cedex, France

***Institut de Recherches sur les Maladies du Sang, Hôpital St-Louis, 2, place du Vocteuv-Fourniev, 75475 Paris Cedex, France

Resume - L'ellipsometrie a ete utilisee pour etudier les proprietes d'adhesion de collagene et fibrinogene sur une surface metallique (Cr). L'epaisseur et l'indice de refraction des couches obtenues ont ete etudies en fonction du temps d'incubation et de la concentration moleculaire. La meme methode a ete appliquee pour etudier aussi 1'adhesion du bacteriophage T7.

Abstract - Adsorption properties of collagen and fibrinogen on Cr metal surface has been studied by ellipsometry. The thickness and refractive index of the thin films have been determined in function of the incubation time and solute concentration. The same method has been also applied to investi- gate the adsorption of T7 bacteriophage.

Introduction - Blood coagulation leading to thrombosis can be induced in natural or artificial blood-wessel walls (1,2). The processus is influenced by several physical or physico-chemical elements such as hydrophilic or hydrophobic characte- ristic of the surface, the concentration and time-dependence of the adhesion etc.

Fibrinogen, collagen and fibronectin are between the most important molecules taking part in this event. In order to get some insight on the adsorption properties of these molecules, a simple model (molecules - Cr surface) has been used and the gro- wing layers have been studied by ellipsometrie determination of their thickness and refractive index (3-5).

Similar model-system (T7 bacteriophage - Cr surface) was used, as a first approxima- tion to study of the virus adsorption which presents the initial step of the infection of the cells.

Material and Methods - The human fibrinogen and fibronectin as well as the collagen from calf skin have been obtained from Sigma Chem. Company and used without further purification, suspended in a buffet solution (0,01 mol/1;Tris-HCl, 0,1 mol/1 NaCl at pH = 7).

T7 bacteriophage were grown up on E-coli B host and purified as described by Gaspar et al (6). Buffer solution containing 0,05 mol/1 Tris-HcL and 0,1 mol/1 NaCl at pH = 7 has been used.

Chromium coated glass slides were used as substrate. Their planarity has been previously controlled and their optical parameters (refractive index n, and extinc- tion coefficient^, were determined before every measurements.

Layers have been formed by incubation of drops of the sample at a given concentra- tion (c) and at a given time (t^n c). The excess material has been washed off subse- quently by distilled water. The layer homogenity was microscopically controlled.

*Associé au C.N.R.S. N° 154

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:198310107

C 10-522 JOURNAL

DE

PHYSIQUE

A double-modulation p h o t o e l e c t r i c e l l i p s o m e t e r was u s e d t o d e t e r m i n e t h e e l l i p t i c i t y ( t a n Y ) and t h e p h a s e s h i f t ( A ) o f t h e l i g h t r e f l e c t e d by t h e s a m p l e s . Measurements h a v e b e e n done a t t h r e e d i f f e r e n t a n g l e s o f i n c i d e n c e . The t h i c k n e s s ( d ) and t h e r e f r a c t i v e i n d e x (n) of t h e s a m p l e s have been d e t e r m i n e d by t h e Drude e q u a t i o n , u s i n g a computer program ( 5 ) .

R e s u l t s and d i s c u s s i o n s .

I n t h e t a b l e a r e p r e s e n t e d some t y p i c a l t h i c k n e s s e s o f t h e c o l l a g e n and f i b r i n o g e n l a y e r s on Cr s u r f a c e . As i t i s shown, t h i s p a r a m e t e r depends on t h e i n c u b a t i o n time and s o l u t e c o n c e n t r a t i o n .

TABLE : TYPICAL THICKNESS DATA OF COLLAGEN

AND FIBRINOGEN LAYERS

Sample S u b s t r a t e c (mglml) tin, ( m i d d (A)

C o l l a g e n C r 1 30 108 -i- 5

091 30 40

+

5

F i b r i n o g e n

0 , 1 30 20 4 2

C o l l a g e n

1 10 49 1 5

The c o r r e s p o n d i n g r e f r a c t i v e i n d e x however d i d n o t i n d i c a t e s i m i l a r dependence, t h e mean v a l u e s f o r f i b r i n o g e n and c o l l a g e n b e i n g : n ~ ; = 1 , 6 5

+

0 , 1 , n C o l l = l , 5 5 i 0 , 1 . When a c o l l a g e n l a y e r h a s b e e n formed on t h e C r s u r f a c e , t h e f i b r i n o g e n i n t e r a c t s w i t h i t , however t h e c o l l a g e n c a n n o t be a d s o r b e d on a f i b r i n o g e n f i l m . The t h i c k - n e s s o f t h e f i b r i n o g e n l a y e r o n c o l l a g e n s u b s t r a t e i s a b o u t t h e h a l f o f t h e t h i c k m s s o f t h e f i b r i n o g e n l a y e r a d s o r b e d d i r e c t l y o n t h e Cr s u r f a c e ( T a b l e ) . At low f i b r i n o - gen c o n c e n t r a t i o n t h e r e f r a c t i v e i n d e x of t h e t o t a l l a y e r shows a s l i g h t d e c r e a s e (An = -0,04*0,05 a t C F i = 0 , l mg/ml) a n d , i t i n c r e a s e s a t h i g h f i b r i n o g e n c o n c e n t r a - t i o n (An = + 0 , 1 + 0 , 0 5 a t CFi = 1 mg/ml).

A t low c o n c e n t r a t i o n ( 0 , 1 2 mg/ml) t h e f i b r o n e c t i n can n o t form a m e a s u r a b l e l a y e r on t h e Cr s u r f a c e . When t h e f i b r o n e c t i n i s a t t a c h e d on t h e c o l l a g e n f i l m , i t produ- c e s some changes i n t h e r e f r a c t i v e i n d e x o f t h e c o l l a g e n l a y e r (An = 0 , 1 4 + 0 , 0 5 ) , w i t h o u t c h a n g i n g i t s t h i c k n e s s . The p r e s e n c e o f f i b r o n e c t i n i n c o l l a g e n a c t i v a t e s t h e a d s o r p t i o n of f i h r i n o g e n a s shown by t h e g r e a t e r t h i c k n e s s o f t h e f i b r i n o g e n l a y e r compared t o t h e same l a y e r on c o l l a g e n w i t h o u t f i b r o n e c t i n (An,i/nri%lO%,Fig.).

&- A-

I n c o n c l u s i o n : t h e s e r e s u l t s p r e s e n t t h a t t h e a d s o r p t i o n o f t h e c o l l a g e n and f i b r i - nogen on t h e h y d r o p h o b i c C r s u r f a c e p r o d u c e s t h i n f i l m s o f t h i c k n e s s o f some molecu- l a r l a y e r s . The f o r m a t i o n o f t h e s u p e r p o s e d l a y e r s o f c o l l a g e n and f i b r i n o g e n i s n o t r e c i p r o c a l . The a d h e s i o n o f t h e f i b r i n o g e n on c o l l a g e n i s a c t i v a t e d by t h e f i b r o n e c - t i n .

F i g u r e : EFFECT OF FIBRONECTIN ON THE ADSORPTION OF FIBRINOGEN ON A COLLAGEN FILM

The t h i c k n e s s o f t h e a d s o r b e d l a y e r s o f b a c t e r i o p h a g e shows a s a t u r a t i o n i n f u n c t i o n of t h e phage c o n g e n t r a t i o n and i n c u b a t i o n t i m e . The s a t u r a t i o n v a l u e of t h e t h i c k - n e s s i s d = 70'5A a t c = 0,15 mg/ml ; t i n c = 20 min. The r e f r a c t i v e i n d e x of t h e l a y e r s i s n = 1,57+0,75.

The s a t u r a t i o n t h i c k n e s s i s a b o u t t h e t e n t h of t h e phage d i a m e t e r . T h i s f a c t may b e e x p l a i n e d e i t h e r by a s p a r s e a d h e s i o n of t h e p a r t i c l e s o r by t h e a d s o r p t i o n of o n l y t h e c o a t p r o t e i n s . The l a t t e r c o u l d b e due t o a d e g r a d a t i o n o f t h e phage c a p s i d s a s consequence o f t h e high-speed washing d u r i n g t h e sample p r e p a r a t i o n . R e f e r e n c e s :

1. MORRISSEY B.W., i n "The B e h a v i o r of. Blood and i t s Components a t I n t e r f a c e s "

Ann New-York. Acad. S c i .

283

(1977) 50.

2 . ASLANI4N D. e t a l , I n t . J . Q u a n t . Chem. X I 1 (2) (1977) 149.

3. ROTHEN A . , P r o g r . i n S u r f a c e and Membrane S c i .

8

^{(1974) 81.}

4 . AZZAM R.M.A. and BASHARA N . M . , i n E l l i p s o m e t r y and P o l a r i z e d L i g h t ( N o r t h H o l l a n d , Amsterdam 1977) Ch. 4 .

5 . VUYE G. T h e s i s (1981) P . e t M. C u r i e , Univ. P a r i s VI

6. GASPAR S. e t a l . , Z e i t s c h r i f t f c r A l l g . M i c r o b i o l .

19

(1979) 163.