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Langmuir 1987, 3, 1128-1131
Thermal Denaturation of an Adsorbed Fibrinogen Layer Studied by Reflectometry P. Schaaf, P. DGjardin, A. Johner, and A. Schmitt* Institut Charles Sadron (CRM-EAHP),67083 Strasbourg Cedex, France Received April 29, 1987. I n Final Form: July 17, 1987
The technique of scanning angle reflectometry (SAR) allows, like ellipsometry, the determination of the characteristic parameters of an adsorbed macromolecular layer, namely its mean thickness and refractive index. We used this novel method to analyze the structural modifications induced by an interfacial thermal denaturation, below 60 "C, of a human fibrinogen layer adsorbed at the surface of silica. At low interfacial concentrations,the temperature rise results in important desorption. In contrast, denaturation of the terminal D nodules within a dense interfacial layer stabilizes it and compacts it on the surface.
Introduction There is widespread evidence that the adhesion and subsequent activation of blood platelets on artificial surfaces are mediated by an adsorbed layer of proteins, especially of fibrinogen.' It is one of the reasons why we chose, in another article,2 to study the structure of a fibrinogen layer adsorbed on hydrophilic silica. The technique of investigation was scanning angle reflectometry, around the Brewster angle, for visible light polarized parallel to the incidence plane. This method allows determination of the same parameters as in ellipsometry, namely the thickness LO(optical thickness) and the mean refractive index n, to characterize the adsorbed layer.3 The equilibrium between an interfacial layer and the bulk solution may be perturbed by replacing the solution by the pure solvent or buffer solution. Then, for most high-molecular-weight specie^^^^ and in particular for fibrinogen,6 the interfacial layer remains in a quasi-metastable state. Once such a layer is submitted to environmental pertubations like changes in temperature, pH, or ionic strength, it is possible to observe either partial desorption or interfacial structural changes, or both of these effects. Over the past decade, we have studied in our group, using a hydrodynamic technique to measure the layer thickness, L H (hydrodynamic thickness), interfacial conformational transition^.^^' It was observed that, when the adsorbed molecules are dispersed on the surface (low interfacial concentration regime), they undergo the same transitions as in solution.8 Once numerous lateral interactions exist among the adsorbed macromolecules, the laws governing the transition may change, especially with flexible polymer^.^ When solutions of fibrinogen are heated, starting at room temperature, it has been observed1°-12 that the (1)Lindon, J. N.; McManama, G.; Kushner, L.; Merrill, E. W.; Salzman, E. W. Blood 1986,68,355. (2)Schaaf, P.; Dgjardin, P.; Schmitt, A. Langmuir, in press. (3)Schaaf, P.; Dgjardin P.; Schmitt, A. Reu. Phys. Appl. 1986,21,741. (4)Schmitt, A. In Colhdes et Interfaces;Les Editions de Physique; Cazabat, A. M., VeyssiB, M., Eds.; Les Ulis; Ecole #Et6 Aussois, 1984; pp 245-288. (5)Fleer, G. J.; Lyklema, J. In Adsorption from Solution at the Solid-liquid Interface; Parfitt, C. D., Rochester, C. A., Eds.; Academic: New York, 1983;pp 153-220. (6)Voegel, J. C.;de Baillou, N.; Schmitt, A. Colloids Surf. 1985,16, 289. (7)DBjardin, P.; Toledo, C.; Pefferkorn, E.; Varoqui, R. In Ultrafiltration Membranes; Polymer Science and Technology; Cooper, A,, Ed.; Plenum: 1980;Vol. 13, pp 203-215. (8)Pefferkorn, E.;Schmitt, A.; Varoqui, R. Biopolymers 1982, 21, 1451. (9)DBjardin, P. J . Phys. (Les Ulis, Fr.) 1983,44, 537. Mihalyi, E. Proc. Natl. Acad. Sei. U.S.A. 1974, (10)Donovan, J. W.; 71,4125.
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molecules undergo a t least two distinct endothermic transitions, around 60 and 100 "C, respectively. This was established by using differential scanning calorimetry, and it provided additional support for a model composed of three compact and distinct domains: the two terminal nodules (fragment D) heat denaturated around 60 "C,and the central nodule (fragment E) denaturated around 100 "C. The more recent work of Medved et shows that this description is probably simplified. The thermal denaturation of a fibrinogen layer adsorbed on glass has recently been investigated using several techniques: radiolabeling with 1261,hydraulic flow through a porous filter, and differential scanning ~al0rimetry.l~We observed both desorption and changes in the layer thickness after denaturation around 60 "C of the terminal D nodules. In the present study, we wanted to complete and critically analyze these former results, since reflectometry may provide better insight into the structural alterations within the layer trapped at the interface.
Materials and Methods Details of the experimental system, the adsorption cell, the buffer, and fibrinogen solutions may be found in a previous publication.2 We explored a range of protein concentrations (1.5 x to 5 X (w/w)) well below those prevailing under physiologicalconditions (around 0.3% (w/w)). It should be recalled that the way of preparing the adsorbing silica surface is important. We achieved good reproducibilitywith the hydrophilic silica surface, not dried after cleaning with concentrated sulfochromic acid, followed by thorough rinsing with Super-Q-Millipore quality water. To control the temperature inside the adsorption cell, we built a thermostated envelope in contact with its upper part and connected it to a HAAKE F4C apparatus. This thermostat enables temperature programming with a PG 40 extension. In the experiments described the reference temperature was 25 "C.
We used, for each experiment,the following procedure. The clean silica surface was first equilibrated with buffer solution. The reflected intensity was recorded at different angles in the close vicinity of the Brewster angle. These data provided the reference signal, corresponding to an interface of the Fresnel type. An excess of fibrinogen solution at a given concentration was then injected into the cell to displace the buffer solution and adsorption took place during about 3 h. This protein solution was again replaced by the solvent until a constant signal was recorded as a function of time. The angular scanning was repeated to determine the mean parameters characteristic of the layer structure. A temperature cycling was then carried out; it involved a linear temperature rise between 25 and 70 "C, during 90 min, followed (11)Medved, L.V.; Gorkun, 0. V.; Privalov, P. L. FEBS Lett. 1983, 161,291.
(12)Doolitlle, R. F.Sci. Am. 1981,245,126. (13)De Baillou, N.;Dgjardin, P.; Schmitt, A.; Brash, J. L. J. Colloid Interface Sei. 1984,100, 167.
0 1987 American Chemical Society
Langmuir, Vol. 3, No. 6, 1987 1129
Fibrinogen Layer Studied by Reflectometry
"t
2 2 0 1 Lo IAl
180 I60 -
200
14012020 -
-
timelmn) I
I
I
100 -
+
4-
- 2.8
/
f-+
+
-2.4
-2.0
-1.6
-1.2
Figure 2. Variation of the optical layer thickness as a function of the equilibrium solution concentration expressed in percent (w/w) (decimal logarithmic scale), before (+) and after (0) the
thermal cycling procedure.
:
I n
1.352 P
1.350
1.0
1.3L8
1.3 L6 0.0
0.0
60 0
120.0
180.0
13L4
t ' 1 1 I i
f
it
t i m e lmn)
Figure 1. (a) Programmed variation of the temperature against
time, during a thermal cycle within the adsorption cell. (b) Variation of the reflectivity coefficient, for the "p" wave at the Brewster angle, as a function of time during a thermal cycle: lower curve, for the bare silica/solvent interface; upper curve, with an adsorbed fibrinogen layer (equilibriumconcentration, 5 X (w/w)) in the presence of solvent. by a symmetric temperature decrease (Figure la). With a clean interface containing no adsorbed layer, the recorded reflected intensity varied as shown in Figure l b (lower curve); the signal was indeed almost symmetric with respect to a vertical axis at t = 100 min. With the fibrinogen layer covering the silica surface, variations of the reflected intensity for a "p" wave were quite different, as seen in Figure Ib (upper curve). The symmetry was lost, which indicates that the process was no longer reversible. In addition, a significant vertical shift was observed during the temperature increase, around 55 'C. At the end of the cycle, when the reflected intensity was again stabilized at 25 "C,angular scanning was repeated, to analyze the layer's structure after the denaturation process.
Results and Discussion Determination, before and after the temperature cycle, of the parameters Lo and n, was repeated for different concentrations of the equilibrium solution, that is, of the solution used to prepare the adsorbed layer. Let us first consider the variations of the optical thickness Lo and the refractive index n as a function of the bulk equilibrium concentration (Figures 2 and 3). Before denaturation, the data may be compared with those published in another paper.2 There is however a difference between the two sets of measurements: In the former study, the layer was characterized in the presence of the fibrinogen solution, that is, at equilibrium. In the present work, measurements correspond to a layer in its metastable state, in pseudoequilibrium with the buffer solution. Other differences might be due to the fact that, between the two
I
I
- 2.8
I
-2.L
I
-2.0
I
-1.6
I
L
-1.2
Figure 3. Variation of the mean refractive index within the adsorbed layer as a function of the equilibrium concentration expressed in percent (w/w) (decimal logarithmic scale), before (+) and after (0) the thermal cycling procedure.
studies, the interface was frequently in contact with sulfochromic acid, leading perhaps to a slow evolution of its properties. We have already emphasized2 the importance of the surface preparation, which greatly influences its affinity for dissolved macromolecules. Despite this difference, there is good correlation between the two sets of data. A concentration of (w/w) separates clearly two adsorption domains. At lower concentrations, the thickness of the layer remains constant and the refractive index increases gradually. The value of the thickness is 20% lower than in ref 2; it is possible that washing with the buffer solution eliminates molecules which are loosely attached to the surface and not oriented parallel to it, in the so-called "side-on" state. The progressive increase of the mean refractive index indicates that the concentration within the layer of molecules attached "side-on" increases with the equilibrium bulk concentration, until some saturation state is reached, a t a bulk concentration c*. Above the concentration crossover c* and before denaturation, the thickness Lo increases steadily with the external concentration while the mean refractive index decreases. As suggested elsewhere,2 the model of a homogeneous layer provides only a first-order description of the layer's structure in this case. We proposed, in a recent
1130 Langmuir, Vol. 3, No. 6, 1987
Schaaf et al.
Figure 5. Model of an adsorbed layer with elongated fibrinogen molecules in the "side-on" (parallel to the interface) and "end-on" (orthogonal to the interface) adsorbed states. The characteristic parameters of this bilayer are the two thicknesses dl and d2 and the two refractive indexes nland 4; the length L of the fibrinogen molecule is not equal to the mean optical layer thickness.
-2.8
-2.1
-2.0
-1.6
-1.2
Figure 4. Variation of the product between the optical layer thickness and the refractive index difference between the layer and the solvent, as a function of the equilibrium solution concentration expressed in percent (w/w)(decimal logarithmic scale), the thermal cycling procedure. before (+) and after (0) p~blication,'~ the model of a bilayer to explain these observations. After the temperature cycle, the behavior in the low and high concentration regimes is still contrasted. Below c*, the thickness of the layer containing molecules dispersed on the surface increases during the thermal cycling procedure; the denaturation of the terminal D nodules probably changes the shape of the elongated macromolecule, which becomes less compact and more bulky and increases the fmt moment of the monomer concentration away from the surface. The mean refractive index decreases simultaneously, which means that the concentration within the layer decreases. At higher equilibrium concentrations, the opposite observations hold; the thickness of the layer decreases during the thermal cycle, while the mean refractive index increases significantly. To provide better insight into these structural alterations, it is important a t this stage to consider the product (n - nl)Lo (where nl is the refractive index of the solvent), which is to a first approximation proportional to the amount of protein adsorbed per unit area. We observe in Figure 4 that before denaturation this parameter increases slowly with the bulk concentration in the range explored (logarithmic scale). Dispersion in the data is however important. During thermal cycling, the interfacial concentration varies quite differently in the low and high concentration domains. At low concentration, it drops significantly upon denaturation; in other words, we observe an important thermal desorption of the isolated molecules adsorbed on the surface. During denaturation, some of the links established by the adsorbed molecules with the surface are probably weakened, leading to partial desorption. Above c*, if we postulate a constant refractive index increment, we must conclude that the excess superficial concentration has increased. This cannot be the case, since the layer is only in contact with buffer solution during the temperature cycling procedure. Therefore, the only reasonable explanations are as follows: (i) When the molecules are denatured at the surface and close to each other, their desorption is probably negligible compared to the situation below c*. Adjacent molecules may form intermolecular links, leading to some interfacial reticulation which stabilizes the layer. Similar results have indeed been observed for fibrinogen adsorbed at the sur(14) Schaaf, P.; D6jardin, P. Colloids Surf., special issue, in press.
face of glass beads, using the technique of radioactive labeling.15 (ii) This interfacial "denaturation/polymerization" may in turn alter the refractive index increment (dnldc). With synthetic polymers, it is well-known that this parameter varies significantly with the solvent/solute couple. When denaturation occurs, different monomers are indeed exposed to the solvent. An increased value of this increment after denaturation could also explain the increase in the product AnLo, a t constant adsorbed amount, the interfacial concentration being given, before and after denaturation, by the approximate relation
r
= AnLo(dn/dc)-]
Such a hypothesis implies that the structural modifications of the fibrinogen molecules are severe, altering therefore their polarizability. It should indeed be recalled that the increment dn/dc does not vary greatly with the nature of the native protein molecules, as shown by De Feijter et al.16 (iii) An alternative explanation for the increase in the product &Lo upon heat denaturation may lie in structural changes within the layers. We proposed earlier14the model of a bilayer, with molecules distributed among two interfacial states, "side-on" and uend-onn, to describe the structure of the layer above c*, before denaturation. Such a model is represented in Figure 5. If we fit, for example, the reflectivity data a t the concentration c = 5 X (w/w) to the bilayer model, choosing thicknesses dl = 100 A and d2 = 350 A, the mean refractive indexes within each sublayer may be obtained. The sum Anldl + An2d2then represents the "effective" amount of adsorbed protein; we obtain a value of 2.34 A,which is only slightly greater than the value 2.22 A of AnLo. Therefore, it appears that the most credible explanation for our experimental observations is given by both conjectures i and ii: the refractive index increment increases significantly upon denaturation, while the layer remains stabilized a t the silica surface. This conclusion is directly corroborated by the reflectivity data at the Brewster angle, presented in Figure lb. We showed2that this reflectivity is, to a first approximation, proportional to the square of the product AnLo. If we combine the observation of an increase in reflectivity during the denaturation process (Figure l b ) with the conjecture of a constant interfacial concentration I', a t the high equilibrium solution concentration, we may conclude from eq 1 that the refractive index increment increases upon denaturation.
Conclusion The interfacial denaturatian of fibrinogen molecules has been previously studied at the surface of glass beads using several techniques: hydrodynamic differential (15)Voegel, J. C.;DGjardin, P.;Strasser, C.; de Baillou, N.; Schmitt, A. Colloids Surf. 1987, 25, 139. (16)De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978,17, 1759.
Langmuir 1987, 3, 1131-1135
scanning ~alorimetry,'~ and radi01abeling.l~ We showed that the terminal D nodules undergo a t the interface a denaturation similar to the one identified in solution around 60 OC.lO-l2 In the present work, we analyzed the structural alterations of a fibrinogen layer adsorbed at the surface of pure hydrophilic silica, using the technique of angular scanning reflectometry. It has been observed that the occurrence of a molecular thermal denaturation of the adsorbed molecules induces opposite effects a t low and high surface concentrations. Fibrinogen molecules dispersed a t the surface show important desorption upon denaturation, while those remaining adsorbed adopt a conformation leading to an increased layer thickness. For a dense adsorbed layer, which presents a more complex structure, denaturation leads to
1131
a kind of superficial polymerization, which strongly stabilizes the layer. This description correlates well with a thermal desorption study performed with radiolabeled m01ecules.l~It appears therefore that SAR is a technique well adapted to study structural changes within an adsorbed layer. It would be of great potential interest to follow continuously, as a function of time, these structural changes, but this is not yet possible with our present system. Acknowledgment. The authors are grateful for financial support from the CNRS under the joint projects "GRECO Polymbres HBmocompatibles" and "ATP MaSriaux". The technical assistance of G. Maennel and F. Woehl is also acknowledged.
Reflectometry as a Technique To Study the Adsorption of Human Fibrinogen at the Silica/Solution Interface? P. Schaaf, P. Dejardin, and A. Schmitte Znstitut Charles Sadron (CRM-EAHP), 67083 Strasbourg Cedex, France Received March 3,1987. I n Final Form: June 1, 1987 In this paper, we show that the technique of reflectometry,applied to the reflection of a "p" wave around the Brewster angle, provides information similar to ellipsometry to characterize a layer of adsorbed macromolecules. Application of this method to study adsorption from solutions of human fibrinogen at the surface of pure silica leads to the following observations: (1) When the external equilibrium concentration is raised, but remains low, a layer of almost constant optical thickness builds up, while the concentration within the layer increases. (2) At an external crossover concentration close to (w/w), this first layer appears to be saturated. (3) Above this concentration threshold, an increase in the equilibrium concentration induces structural changes within the adsorbed layer, which remain to be completely explained. These observations correlate with former results obtained by using a hydrodynamic flow technique. Introduction When dissolved synthetic or biological macromolecules are trapped in the interfacial force field of solid/liquid interfaces, they may undergo conformational transitions affecting primarily their overall size and shape. In the case of biopolymers, properties related to local interactions may in turn be altered, leading to enhancement or inhibition of various biological responses. It is, therefore, of importance to characterize the structure of a macromolecular adsorbed layer, that is, to know at least its molecular lateral (mean area occupied by one molecule) and orthogonal (mean layer thickness) dimensions a t the surface. Because of its high sensitivity, the most popular method used to determine the surface concentration r, and thus the mean area per adsarbed molecule, is /3 or y radiolabeling. Various hydrodynamic techniques have been applied to measure the thickness of interfacial mono- or multilayers.l4 Ellipsometry is also known to be a powerful and sensitive tool in surface science, because it provides information on both adsorbed amounts and layer thicknesses or, in other words, on the zeroth and first moments of the monomer distribution function, in a direction normal to the i n t e r f a ~ e . ~ ~We ' recently developed a simplified version of this optical technique, namely, reflectometry? 'This paper is based in part on a presentation to the Division of Colloid and Surface Chemistry, 192nd National Meeting of the American Chemical Society, Anaheim, CA, Sept 7-12, 1986.
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and showed that the same physical parameters, the mean layer thickness and the mean refractive index within the layer, may be obtained; therefore, the adsorbed amount may also be evaluated. In the present work, we used this new approach to investigate variations in the structure and composition of an adsorbed fibrinogen layer, when the interfacial concentration was progressively increased, a t the surface of optically polished silica. Previous work related to the adsorption behavior of fibrinogen at various model interfaces led us to identify a t least two adsorption r e g i m e ~ . ~ J ~ We postulated that a t low interfacial concentration the (1) Rowland, F. W.; Eirich, F. R. J. Polym. Sci., Polym. Chem. Ed. 1966,4, 2401. (2) Silberberg, A. In Polymer Adsorption and Dispersion Stability; Goddard,E. D., Vincent, B., Eds.; ACS Symposium Series 240; American Chemical Society: Washington DC, 1984; p 161. (3) Silberberg, A,; Klein, J. Biorheology 1981, 18, 589. (4) Pefferkorn, E.; Schmitt, A.; Varoqui, R. Biopolymers 1982, 21, 1451. ( 5 ) de Baillou, N.; DBjardin, P.; Schmitt, A.; Brash, J. L. J. Colloid Interface Sci. 1984, 100, 167. (6) Ellipsometry and Polarized Light; Azzam, R. M. A., Bashara, N., Eds.; North-Holland Amsterdam, 1977. (7) Charmet, J. C.; de Gennes, P. G. J. Opt. SOC.Am. 1983,73,1777. (8)Schaaf, P.; D€jardin,P.; Schmitt, A. Rev. Phys. Appl. 1986,21,741. (9) Schmitt, A.; Varoqui, R.; Uniyal, S.; Brash, J. L.; Pusineri, C. J. Colloid Interface Sci. 1983, 92, 25. (10) de Baillou, N.;Voegel, J. C.; Schmitt, A. Colloids Surfaces 1985, 16, 271.
0 1987 American Chemical Society
