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Influence of conformational changes on diffusion properties of bovine serum albumin: a holographic interferometry study
Colloids and Surfaces B: Biointerfaces 25 (2002) 99 – 108 www.elsevier.com/locate/colsurfb
Influence of conformational changes on diffusion properties of bovine serum albumin: a holographic interferometry study Laurence Reyes a, Jacques Bert a,*, Jean Fornazero a, Richard Cohen b, Laurence Heinrich b a
De´partement de Physique des Mate´riaux (CNRS UMR 5586), Uni6ersite´ Claude Bernard, LYON1, 43, Bd du 11 No6embre 1918, F-69622 Lyon-Villeurbanne, France b EA 3090 Biomate´riaux et remodelages matriciels, Uni6ersite´ Claude Bernard, LYON1, ISPB, 8, A6. Rockefeller, F-69373 Lyon Cedex 08, France Received 14 May 2001; received in revised form 6 June 2001; accepted 13 August 2001
Abstract The effect of various stresses (heating, or salt excess) applied to a protein solution (BSA) have been evidenced via an efficient dynamic process: mass diffusion. The measurements were performed with a holographic interferometry technique, which is particularly interesting for the study of transport processes. The diffusion coefficient has been found to be quite independent of the BSA concentration but strongly dependent on the ionic strength of the solvent and also on heating, both processes leading to a protein conformational change as demonstrated by photon correlation spectroscopy (PCS) measurements. In all cases, the increase of the protein size leads to the decrease of the diffusion coefficient value. An application of these first experiments has been the eventual evidence of conformational changes of proteins in a solution used as a lubricating fluid for the tribological measurements of prosthetic joints. Up to 0.5 million constraint cycles performed in a hip-joint simulator, the protein solutions showed a stable or very slight increase of the diffusion coefficient. The bacteriological contamination being absent, this experiment proves that this kind of friction test has no or very slight effect on protein conformation thus showing the reliability of such trials on prosthetic elements. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Protein; Conformational changes; Holography; Hip-joint simulator; Total hip replacement; Lubricating fluid
1. Introduction
* Corresponding author. Tel.: + 33-4-72-44-8155; fax: + 33-4-72-43-2925. E-mail address: [email protected] (J. Bert).
The diffusion of macromolecules is an important subject of study in many fields of biological sciences, particularly, to understand the transport of macromolecular drugs and naturally occurring macromolecules in living tissue [1]. Proteic solu-
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Fig. 1. Holographic interferometry apparatus.
tions are also used as lubricating fluids in the simulators during the tribological tests of prosthetic joints, for the reduction of the friction between prosthetic surfaces. The tribological properties of these solutions are bound to the adsorption of proteins onto these surfaces. Nevertheless, friction tests are very constraining to proteins in solution, the stress induced eventually leading to conformational changes and consequently to a modification of the fluid rheological properties. In order to prevent dramatic changes in the protein structure, and their consequences on the frictional behavior of prosthetic materials, we have followed the evolution of the conformation of proteins when they are submitted to denaturing agents. So, the aim of this study was to detect the starting point of a protein deconformation, inducing a change of their diffusion properties, measured by holographic interferometry (Fig. 1). Various investigators have focused on protein diffusion through parameters such as pH and
ionic strength (IS) of the medium, and macromolecule concentration, which affect surface charge and mobility [2–4]. So, first, we will validate our method with the study of the influence of these last parameters on the diffusion, then we will analyze the effect of the stress induced by a friction test on the macromolecule conformation. Diffusion is the process by which matter is transported from one part of a system to another as a result of random molecular motions. Experimentally, it can be illustrated by two homogeneous solutions with different concentrations, superimposed in a vertical cell (Fig. 2). In this configuration, the movement of both cations and anions in the same direction, from high-concentration to low-concentration regions, ensures the electrical neutrality. So, a matter flux is established between these two regions with spontaneous compensation of the concentration difference that has been created. Each ion moves under the influence of two forces: the chemical potential gradient and the electric field, due to the motion of the opposite charge ions; as a consequence, an electrical potential gradient is created by the more mobile ions inducing an identical velocity for each type of ion [5,6]. Even though in most studies on diffusion process two equal volumes are superimposed in a vertical cell, we have chosen to work using a thin layer configuration, i.e. a tiny volume of the first solution with concentration C1 that diffuses into a 60 times greater volume of the second solution concentration C2. As will be seen below, this configuration permits to approach an infinite dilu-
Fig. 2. Fringe pattern evolution with time during the diffusion process.
L. Reyes et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 99–108
tion. In the first approximation, the cell containing the two solutions is considered as one-dimensional; the mean diffusion fluxes then have the direction of the main cell axis, so are perpendicular to the horizontal initial separation plane between the solutions. The mathematical theory of diffusion in isotropic solvents is based on the hypothesis that the rate of transfer of the diffusing substance through a unit area and at a given time, is proportional to the concentration gradient normal to the section (first Fick’s law). The second Fick’s law can be introduced to follow the concentration gradient evolution with time in isotropic solvents for which the structure and diffusion properties are identical at any point perpendicular to the iso-concentration horizontal planes. It can be written: #C #2C =D 2 #t #x
(1)
where C (mol l − 1) is the diffusing substance concentration, x (cm) is the space coordinate along the diffusion axis, normal to the plane section and D (cm2 s − 1) is the diffusion coefficient that can reasonably be considered as constant for a very slight C variation. This equation relates the time dependence of the solute concentration to the flux variation along the diffusion axis [7]. So, the main goal of this work was to apply a common technique, holographic interferometry, to the study of biomolecule conformation through their diffusing behavior.
2. Materials and methods
2.1. Methods of measurements The recording of refractive index variations has been widely used for many years to determine the transport properties of transparent materials [6,8 – 10]. The refractive index is related to the concentration gradient; so, mass transfer coefficients can be obtained by translating the experimentally observed refractive index profile to a concentration profile [9]. Many methods have been developed for the study of the diffusion processes in liquids (static and dynamic light scat-
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tering measurements [2,4,11], Gouy interferometry [3]…). Among optical methods, we have chosen holographic interferometry because it permits the visualization of a whole experimental cell on a single recording medium (paper or digital), instead of only getting a point information. In addition, the holographic real-time exposure technique permits to follow an evolution with time, and therefore it is very well adapted to slow processes like diffusion.
2.2. Experimental technique Measurements were made in a parallelepipedic cell the dimensions of which are 20 mm height, 9.5 mm width and 10 mm optical thickness. Before each measurement, the glass cell is plunged for one night in a diluted solution of a strong detergent (DDN 250, Franklab) and then carefully rinsed out with deionized water, in order to prevent protein adsorption on the inner walls between successive tests. The cell is inserted inside large copper blocks, which prevent temperature gradients. These blocks are mounted on a jack which allows a precise adjustment in the laser beam direction. The whole system is fixed on a vibrationless optical table with a polyethylene cover, preventing thermal instabilities due to air convection. The temperature is measured with platinum Pt 100 sensors at both ends of the cell. Experimental boundary conditions correspond to those of an infinitely long cell permitting a free diffusion as long as the diffusing species do not reach the ends of the cell. The holographic facility (Fig. 1) consists of a reference beam and an object beam originating from an argon laser (wavelength 514 nm (1)), a mirror (2) and a 50/50 beam splitter (3). Each beam energy is controlled by means of a halfwave plate (4) and a Nicol prism (5). Spatial filters (6) combined with beam expanders (7) permit the enlargement of the beam and high frequency filtering. On the reference beam, an auxiliary plate (8) is used to counterbalance the parasitic fringes coming from mechanical or thermal variations of the holographic plate (10). The object beam passes through the experimental cell (9) before interfering with the reference beam on
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the holographic plate. After the hologram processing, the cell reference state is recorded. When the diffusion starts, an interference fringe pattern appears, the variations of which are recorded by a video camera.
tion two different solutions can be obtained together with the initial, boundary and matching conditions [7,12]. In the case of thin layer diffusion, a solution of the second Fick’s law is given by:
2.3. Solution setting: the thin layer configuration
C(x,t)=
A small volume (about 50 ml) of a solution with concentration C1 is deposited with a micropipette at the bottom of the cell which is already filled with a greater volume (3000 ml) of a lower concentration solution (concentration C2) (Fig. 2). The diffusion process is assumed to be unidirectional in the direction x\ 0; the origin plane (x =0), being taken at the interface between the two solutions at time zero. According to this geometry, each plane perpendicular to the diffusion axis is an iso-concentration plane. To our knowledge, all the studies on protein diffusion were made with equal volumes of the lower solute-rich solution and of the upper solutedepleted solution (infinite layer configuration). In a preliminary study we then compared the results obtained in this configuration with those obtained with the thin layer configuration. It appears that in the infinite layer configuration the first hypothesis concerning the Fick’s law (D independent of the concentration) was not valid yet. As a matter of fact, the diffusion coefficient follows a decreasing tendency when the concentration approaches zero; consequently, for infinite dilution, the infinite layer diffusion coefficient is very close to the thin layer coefficient, those two values corresponding to the literature values. So, in this study, we have adopted a thin layer configuration. Moreover, this method permits to take advantage of its experimental simplicity because it only requires the introduction of a very small volume of the denser liquid at the bottom of the cell, which must be carefully deposited in order to avoid convective motions.
2.4. Methods of resolution The solution of the second Fick’s law Eq. (1) relates the concentration C(x,t) to the diffusion coefficient D. According to the chosen configura-
M
PDt
exp
−x2 4Dt
(2)
where M is the amount of substance per unit of surface, originally deposited in the plane x= 0. This equation applies to a semi-infinite cell extending over the region x\ 0 and with an impermeable boundary at xB 0; so, diffusion only occurs in the positive x-direction [7,12]. In the case of a thick plane diffusion, which corresponds to a substance initially confined in the region − hBxB h (thickness 2h); the solution can be written: C(x,t)=
C0 h −x h+ x erf + erf 2 2 Dt 2 Dt
(3)
where C0 is the initial concentration of the diffusing solution and erf stands for the error function [7,12]. In each case, the x= 0 plane corresponds to the maximum concentration, and the top of the cell, where no fringes are present, is taken as zero solute concentration (Fig. 2). Diffusion coefficients were first computed with both Eqs. (2) and (3). Eq. (3) gives a more rigorous result because it calls out a definite thickness whereas Eq. (2) can be considered as an approximation. However, the D values obtained with the two equations were close, providing a sound basis for the hypothesis of thin layer and, consequently, only the results computed from Eq. (2) will be given here.
2.5. Protein solutions Our aim is to study the diffusion process occurring in aqueous protein solutions usually used as lubricant fluid during tribological tests. The chosen protein is bovine serum albumin (BSA) according to the ISO/TR 9325 (November 1989) standard in application for hip and knee prosthesis friction test [13]. A few BSA properties in its native form are reported in Table 1.
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Table 1 Some characteristics of bovine serum albumin
BSA
Molecular weight (g mol−1)
Dimensions (nm×nm×nm)
Isoelectric pH (pHi)a
66 700
4×4×14
4.7
a pHi is the pH for which the global surface charge of the protein is zero. This pHi value with its neighboring represents a critical pH domain because the protein shows minimal hydration and solubility values.
At a given temperature, the ionic strength, the pH of the medium and the protein concentration are parameters which are well known to change the protein diffusion coefficient because they modulate the electrostatic interactions between protein molecules. The magnitude of the Coulomb repulsion depends on the protein surface charge, which in turn depends on the pH. In the solution, small ions screen the Coulomb interaction between the charged proteins so that for a salt concentration of 0.1 mol l − 1 the Coulomb field vanishes beyond a small fraction of the protein linear dimension. Concentration determines the probability of collisions during which the proteins come close enough to feel the Coulomb repulsion, as well as other short- and longrange mutual interactions. Thus, pH, IS and protein concentration are factors which together influence the magnitude of the diffusion coefficient [14]. Freeze dried BSA (99% purity) from Biosepra (Paris) was used without further purification in this study. BSA was diluted in different solvents such as deionized water or phosphate buffer solution. Phosphate buffer was used to maintain the pH in the physiological range whereas the addition of NaCl increases the IS. Na thimerosal was only added for antisepsis. The solvents used are described in Table 2. Photon correlation spectroscopy (PCS) measurements were also performed, because it is a helpful method for the conformational studies of proteins [15]. The PCS technique explores the temporal fluctuations in light scattered by a solution of molecules. These fluctuations are a direct function of Brownian motion of the scatterers and are related to the hydrodynamic size of the solute. PCS is a very sensitive technique for the detection of aggregates and so is adequate for the following
of the denaturation/aggregation process whatever the reason of the denaturation (heat or increasing of IS). In the present PCS study, the selected samples were characterized with a Zetasizer 3000 spectrometer (Malvern Instruments, UK).
3. Results The first part of this study is to investigate the influence on the diffusion coefficient of factors which could affect the tertiary structure of protein, and also the size and shape of molecules. BSA diffusion coefficient is first measured at room temperature, for various experimental conditions of (i) BSA concentration, (ii) IS, (iii) heat treatment. Then, the diffusion coefficient will be compared with that of a proteic solution after its use as a lubricant during a friction test.
3.1. Influence of the protein concentration Fig. 3 shows a plot of BSA diffusivity as a function of diffusing time. For each concentration C1, the mean D values reported in Table 3, are calculated with all the experimental points. Several protein concentrations either in phosphate buffer saline solution or in deionized water are given. For each experiment, the upper solution differs from the lower, by BSA concentration only (C2 = 0). In this way, it was possible to study diffusion of BSA molecules alone. The diffusion coefficient does not change significantly with BSA concentration in the explored concentration and IS ranges. These measurements significantly confirm the choice of D independent of the concentration for the resolution of the second Fick’s law. So, the following experiments were conducted at a BSA
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Table 2 Composition of the solvents used in this study Name
Composition
Concentration (mol l−1)
pH
Ionic strengtha (mol l−1)
Saline solution Phosphate buffered solution
NaCl KH2PO4 Na2HPO4 Na thimerosal NaCl KH2PO4 Na2HPO4 Na thimerosal NaCl
0.15 9.5×10−3 0.04 4.9×10−4 0.15 9.5×10−3 0.04 4.9×10−4 3.25
6.7 7.4
0.15 0.20
7.5
3.45
High IS phosphate buffered solution
a
IS was computed according to: IS = 1/2iCi Z 2i where C (mol l−1) is the concentration and Z the valence of the ion i.
concentration of 10g l − 1 (0.15 ×10 − 6mol cm − 3), according to the concentration used in the lubrication fluid for tribological tests.
3.2. Influence of the ionic strength The results reported in Table 4 show the decrease of the diffusion coefficient of BSA with IS. Fig. 4 illustrates this tendency.
3.3. BSA submitted to stress
was used as a lubricant fluid for a friction test reproducing a classical physiological walk performed for 7 days (0.50× 106 cycles of stress). BSA diffusion coefficient was measured by holographic interferometry with sampling at 0.12× 106, 0.26×106, 0.36×106 and 0.50× 106 cycles of constraint corresponding approximately to 2, 3, 5 and 7 days of unceasing movement (Table 6). We took great care of avoiding bacteriological pollution during the entire test. Before the start of each diffusion experiment, the solution was kept for 30 min at room temperature after extraction from
In Table 5 we report the mean diffusivity obtained with a diluted phosphate buffer BSA solution, after two kinds of treatment: solution heated for 2 h at 75 °C; solution in salt excess (IS= 3.3 mol l − 1). These two treatments are known to stress the protein and can induce conformational changes. Results are reported in Fig. 5 where BSA is always the only diffusing species. They show that the diffusion coefficients of the heated and high IS solutions have the same order of magnitude. These values are clearly smaller than that of native BSA.
3.4. Comparison with the diffusion coefficient of BSA after friction test As mentioned above, the first goal of this work was to study the influence of a mechanical stress during a friction test on conformation of the BSA molecule. So, a phosphate buffered BSA solution
Fig. 3. Variation of the diffusion coefficient with diffusion time. Effect of the protein concentration for buffered and non-buffered solutions. (Concentrations given in Table 3).
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Table 3 BSA diffusion into its pure solvent Solution
Concentration C1×106 (mol cm−3) (BSA diluted in a phosphate buffered solution)
D×106 (cm2 s−1)
Solution
Concentration C1×106 (mol cm−3) (BSA diluted in deionized water)
D×106 (cm2 s−1)
1 2 3
0.15 0.30 0.60
0.60 0.59 0.66
4 5
0.15 0.30
2.54 2.04
the simulator, in order to avoid parasitic fringes due to thermal gradient in the diffusion cell. Fig. 6 shows the slight D increase with the number of cycles. This result is in opposition with the previous one which shows a smaller value of D when BSA is under stress.
4. Discussion The shape of a globular protein in solution is related to the pH and to the IS of the solvent: the pH sets the net surface charge of the protein and IS acts on the repulsive force between molecules. In an isoelectric pHi medium (4.7), the average surface charge of the BSA is zero. In deionized water or in phosphate buffered solution, where pH (7.4) is greater than the protein pHi, negative surface charges appear on the BSA surface. If salt is then added to the solution, the electrostatic repulsion between the charged macromolecules decreases because the salt ions screen the molecules from each other. So, in a solution with high IS, the macromolecules can aggregate. On the contrary, in deionized water, BSA keeps its negative surface charge and a strong electrostatic repulsion is maintained between macromolecules.
4.1. Effect of the starting concentration According to the present findings, we have concluded that in the explored concentration range, close to the concentration recommended for tribological tests, there is no effect of the initial concentration; the diffusion coefficient is then independent of the protein concentration whatever the nature of the solvent is. This result holds
if BSA is the only diffusing species. It is consistent with values found in the literature [4] and confirms the validity of the hypothesis of the independence of D with concentration.
4.2. Nature and ionic strength of the medium The influence of the ionic environment on the protein shape was studied through diffusion coefficient measurements by varying IS of the BSA solution. The explored pH range being small, the influence of the pH on the diffusivity appears negligible compared to the influence of the IS. This study then illustrates the strong influence of the ionic environment on the BSA diffusivity, which is lower in saline solution (NaCl or phosphate/buffer) than in deionized water. This finding is strengthened by the small diffusion coefficient obtained with the high IS solutions. A similar effect was observed by other authors [10] suggesting that as the double layer around the protein increases, the rate of diffusion also increases. The decreasing trend of the diffusion coefficient with IS (Table 4) indicates that the screening effect of the small ions used in our study Table 4 BSA diffusion into its pure solvent in relation with IS of the solvent Nature of the solvent IS (mol l−1)
D×106 (cm2 s−1)
Deionized water Saline solution Phosphate buffered solution High IS phosphate buffered solution
0 0.15 0.20
2.54 0.55 0.60
3.45
0.17
For solution composition see Table 2.
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Fig. 4. Variation of the diffusion coefficient with time. Effect of the solvent ionic strength.
Fig. 5. Variation of the diffusion coefficient with time. Influence of a stress on the diffusion coefficient of BSA buffered solutions.
is not complete even at high IS (3.45 mol l − 1). However, other groups [2– 4,10,15] observed that the diffusivity does not vary for IS above 0.1 mol l − 1 and chose to work with high IS solutions in order to avoid these electrostatic effects and their consequences on protein diffusion. So, electrostatic interactions seem to have a clear effect on the diffusion of macromolecules. If the formation of aggregates due to the high IS appears, the observed diffusion will be that of a ‘cluster’ of molecules, instead of that of a single molecule. For instance, Meechai et al. [4] found an onset of protein aggregation at pH 7.4 when IS increases from 1.5 to 3.3, with an average protein molecular weight 10% higher than for a single molecule.
4.3. Stress induced on the protein Several papers in the literature deal with the influence of heat on protein conformation. So, we have also chosen to evidence this influence through diffusion. A BSA phosphate buffered solution was heated for 2 h at 75 °C, and after cooling to ambient temperature, its diffusive behavior was studied by holographic interferometry. A smaller diffusion coefficient was obtained compared to that of the native protein, under the same conditions and concentrations. The value was closer to the one obtained in high IS conditions. Thus, we can conclude that thermal stress as well as salt excess lower BSA diffusivity and the hypothesis of a protein conformation change
Table 5 Influence of the stress on the diffusion coefficient of BSA buffered solutions (IS = 3.3 mol l−1)
Table 6 Influence of the number of constraint cycles on the diffusion coefficient of a BSA buffered solution
Nature of the treatment
D×106 (cm2 s−1)
Cycles number (million of cycles)
D×106 (cm2 s−1)
Native solution Heatinga Salt excessb
0.60 0.11 0.17
0.00 0.12 0.26 0.36 0.50
0.63 90.1 1.0 90.1 1.0 90.1 1.1 90.1 1.9 9 0.1
a b
Solution heated for 2 h at 75 °C. Solution in salt excess.
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because of a strong screening tendency; the protein can also be unfolded because of a stretching effect. Thus, we can then conclude that measurement of the diffusion coefficient is sensitive to the protein shape modification, whatever the nature of the change might be.
4.4. Photon correlation spectroscopy
Fig. 6. Variation of the diffusion coefficient with time. Evolution of D with the number of constraints cycles during the tribological test.
can be advanced. The hypothesis of an aggregation between molecules is not ruled out either, but it seems difficult to conclude about the exact process only with holographic interferometry. As a matter of fact, a great number of denaturing agents can break bonds that usually maintain the secondary and tertiary structure of the protein, but they can also act on the repulsion barrier between molecules. In the first case, an unfolding of the protein takes place, and molecules can stay in solution in this conformation, whereas in the second case, aggregation of some molecules appears because of the decrease of (i) the distance between molecules and (ii) the hydrophobic attractions between unfolded molecules. In the first situation, the diffusion coefficient could correspond to that of a single molecule whose size and shape are different, whereas in the second situation, the diffusion coefficient is that of a cluster. In both situations, the diffusion coefficient corresponds to different protein shape and size according to the heat effect on the protein structure; the diffusion coefficient suggests a larger size. On the other hand, the heated and the very high IS solutions give similar results. At this salt concentration, molecule aggregation probably appears
From BSA diffusivity values in high IS solutions, and in heated solutions, we have concluded on a protein conformation change and even a probable molecule aggregation. In order to confirm these hypotheses, measurements on photon correlation spectroscopy were undertaken. The PCS method permits the measurement of the size distribution of particles in a solution. The particle diameter of the native protein was found to be 7.1 nm. The Stockes–Einstein relation, and the diffusion coefficient show the coherence of the equivalent radius of the BSA particle. So, the diameter measurement with PCS strengthens our diffusivity values. Afterwards, the PCS method was applied to evaluate the particle diameters in heated and in high IS solutions. A diameter of 24.4 nm was found for BSA molecules, which is coherent with the shape modification of BSA, due to heat treatment. In high IS solutions, the particle diameter is 6.7 nm, so roughly that of the native protein. This result is absolutely not coherent with other results [4] and with our diffusion values (Table 5). A possible explanation might be that a great quantity of salts disturbs the PCS measurement. So, PCS confirms the result obtained for the heat-treated solution: heat provokes an increase in the protein size, and consequently, a decrease in the diffusion coefficient value.
4.5. Protein diffusion beha6ior after se6eral cycles of constraints due to tribological test In Fig. 6 we observe that whatever the number of cycles to which proteins have been submitted, the diffusion coefficient keeps an average constant value, with a very slight increase. If we compare this result with that obtained after the heat treatments where a decreasing D was associated with
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an unfolding of the molecule, we can think here that a weak increase of D could be due to the reducing shape of the molecule. One possible explanation could be a bacteriological contamination leading to the division of the protein molecule during an enzymatic proteolysis. A bacteriological analysis of the samples showed no evidence of contamination that could explain the increasing diffusion. So, one can conclude that the friction test had no drastic effect on the protein conformation and holographic interferometry is therefore a rather good method to follow the conformation evolution of BSA in a solution submitted to stress.
5. Conclusions Several important findings have been emphasized in this work. First, it has been evidenced that the BSA diffusion coefficient is independent of the initial concentration whatever the nature of the solvent in our experimental conditions (pH, IS). We also pointed out that the influence of the medium IS on the BSA diffusion coefficient is greater than the influence of the pH in the explored pH range and we have concluded on the decreasing tendency of the diffusion coefficient when the IS of the medium increases. Moreover, we have experimentally evidenced the sensitivity of the diffusion coefficient with the protein shape and PCS measurements have confirmed the modification of the protein shape due to the nature of the solvent or heat stress. So, holographic interferometry appears to be a technique, which is very sensitive to the conformation changes of protein molecules. However, after several cycles of constraints during tribological test, no really noticeable modification of the BSA diffusion was observed, particularly if we make a comparison between these results and the previous ones with various stresses. Thus, we can conclude that the stress applied on a simulator apparatus only creates very small changes in the protein conformation and the use of other techniques like UV
spectroscopy or circular dichroism could be used to improve this method.
References [1] A. Pluen, P.A. Netti, R.K. Jain, D.A. Berk, Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations, Biophysical Journal 77 (1999) 542 – 552. [2] P. Doherty, G.B. Benedek, The effect of electric charge on the diffusion of macromolecules, The Journal of Chemical Physics 161 (12) (1974) 5426 – 5434. [3] Y-C. Kim, A.S. Myerson, Diffusivity of lysozyme in undersaturated, saturated and supersaturated solutions, Journal of Crystal Growth 146 (1994) 79 – 85. [4] N. Meechai, A.M. Jamieson, J. Blackwell, Translational diffusion coefficients of bovine serum albumin in aqueous solution at high ionic strength, Journal of Colloid and Interface Science 218 (1999) 167 – 175. [5] H. Dez, The`se, Universite´ Claude Bernard Lyon I, 1992. [6] A. Aouizerat-Elarby, H. Dez, B. Prevel, J.F. Jal, J. Bert, J. Dupuy-Philon, Diffusion processes in LiCl, R H2O solutions, Journal of Molecular Liquids 84 (2000) 289 – 299. [7] J. Crank, The Mathematics of Diffusion, 2nd ed., Clarendon Press, Oxford, 1975, pp. 11 – 27. [8] L. Gabelmann-Gray, H. Fenichel, Holographic interferometry study of liquid diffusion, Applied Optics 18 (3) (1979) 343 – 345. [9] W.D. Seufert, R.N. O’Brien, Determination of diffusion coefficients from the progression of interference fringes, The Journal of Physical Chemistry 88 (5) (1984) 829 – 832. [10] T. Fettah Kosar, R.J. Phillips, Measurement of protein diffusion in dextran solutions by holographic interferometry, AIChE Journal 41 (3) (1995) 701 – 711. [11] M. Placidi, S. Cannistraro, A dynamic light scattering study on mutual diffusion coefficient of BSA in concentrated aqueous solutions, Europhysics Letters 43 (4) (1998) 476 – 481. [12] Y. Adda, J. Philibert, La diffusion dans les solides, Presses Universitaires de France, Paris, 1966, pp. 135 – 202. [13] ISO/TR 9325: Implants chirurgicaux — Prothe`ses partielles et totales de hanche —Recommandations concernant les simulateurs de marche destine´ s a` l’e´ valuation des prothe`ses de hanche, afnor, novembre 1989, 7pp. [14] A.K. Gaigalas, J.B. Hubbard, M. Mc Curley, Sam Woo, Diffusion of bovine serum albumin in aqueous solutions, The Journal of Physical Chemistry 96 (5) (1992) 2355 – 2359. [15] D.D. Kong, T. Fettah Kosar, S.R. Dungan, R.J. Phillips, Diffusion of proteins and nonionic micelles in agarose gels by holographic interferometry, AIChE Journal 43 (1) (1997) 25 – 32.
Influence of conformational changes on diffusion properties of bovine serum albumin: a holographic interferometry study Laurence Reyes a, Jacques Bert a,*, Jean Fornazero a, Richard Cohen b, Laurence Heinrich b a
De´partement de Physique des Mate´riaux (CNRS UMR 5586), Uni6ersite´ Claude Bernard, LYON1, 43, Bd du 11 No6embre 1918, F-69622 Lyon-Villeurbanne, France b EA 3090 Biomate´riaux et remodelages matriciels, Uni6ersite´ Claude Bernard, LYON1, ISPB, 8, A6. Rockefeller, F-69373 Lyon Cedex 08, France Received 14 May 2001; received in revised form 6 June 2001; accepted 13 August 2001
Abstract The effect of various stresses (heating, or salt excess) applied to a protein solution (BSA) have been evidenced via an efficient dynamic process: mass diffusion. The measurements were performed with a holographic interferometry technique, which is particularly interesting for the study of transport processes. The diffusion coefficient has been found to be quite independent of the BSA concentration but strongly dependent on the ionic strength of the solvent and also on heating, both processes leading to a protein conformational change as demonstrated by photon correlation spectroscopy (PCS) measurements. In all cases, the increase of the protein size leads to the decrease of the diffusion coefficient value. An application of these first experiments has been the eventual evidence of conformational changes of proteins in a solution used as a lubricating fluid for the tribological measurements of prosthetic joints. Up to 0.5 million constraint cycles performed in a hip-joint simulator, the protein solutions showed a stable or very slight increase of the diffusion coefficient. The bacteriological contamination being absent, this experiment proves that this kind of friction test has no or very slight effect on protein conformation thus showing the reliability of such trials on prosthetic elements. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Protein; Conformational changes; Holography; Hip-joint simulator; Total hip replacement; Lubricating fluid
1. Introduction
* Corresponding author. Tel.: + 33-4-72-44-8155; fax: + 33-4-72-43-2925. E-mail address: [email protected] (J. Bert).
The diffusion of macromolecules is an important subject of study in many fields of biological sciences, particularly, to understand the transport of macromolecular drugs and naturally occurring macromolecules in living tissue [1]. Proteic solu-
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Fig. 1. Holographic interferometry apparatus.
tions are also used as lubricating fluids in the simulators during the tribological tests of prosthetic joints, for the reduction of the friction between prosthetic surfaces. The tribological properties of these solutions are bound to the adsorption of proteins onto these surfaces. Nevertheless, friction tests are very constraining to proteins in solution, the stress induced eventually leading to conformational changes and consequently to a modification of the fluid rheological properties. In order to prevent dramatic changes in the protein structure, and their consequences on the frictional behavior of prosthetic materials, we have followed the evolution of the conformation of proteins when they are submitted to denaturing agents. So, the aim of this study was to detect the starting point of a protein deconformation, inducing a change of their diffusion properties, measured by holographic interferometry (Fig. 1). Various investigators have focused on protein diffusion through parameters such as pH and
ionic strength (IS) of the medium, and macromolecule concentration, which affect surface charge and mobility [2–4]. So, first, we will validate our method with the study of the influence of these last parameters on the diffusion, then we will analyze the effect of the stress induced by a friction test on the macromolecule conformation. Diffusion is the process by which matter is transported from one part of a system to another as a result of random molecular motions. Experimentally, it can be illustrated by two homogeneous solutions with different concentrations, superimposed in a vertical cell (Fig. 2). In this configuration, the movement of both cations and anions in the same direction, from high-concentration to low-concentration regions, ensures the electrical neutrality. So, a matter flux is established between these two regions with spontaneous compensation of the concentration difference that has been created. Each ion moves under the influence of two forces: the chemical potential gradient and the electric field, due to the motion of the opposite charge ions; as a consequence, an electrical potential gradient is created by the more mobile ions inducing an identical velocity for each type of ion [5,6]. Even though in most studies on diffusion process two equal volumes are superimposed in a vertical cell, we have chosen to work using a thin layer configuration, i.e. a tiny volume of the first solution with concentration C1 that diffuses into a 60 times greater volume of the second solution concentration C2. As will be seen below, this configuration permits to approach an infinite dilu-
Fig. 2. Fringe pattern evolution with time during the diffusion process.
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tion. In the first approximation, the cell containing the two solutions is considered as one-dimensional; the mean diffusion fluxes then have the direction of the main cell axis, so are perpendicular to the horizontal initial separation plane between the solutions. The mathematical theory of diffusion in isotropic solvents is based on the hypothesis that the rate of transfer of the diffusing substance through a unit area and at a given time, is proportional to the concentration gradient normal to the section (first Fick’s law). The second Fick’s law can be introduced to follow the concentration gradient evolution with time in isotropic solvents for which the structure and diffusion properties are identical at any point perpendicular to the iso-concentration horizontal planes. It can be written: #C #2C =D 2 #t #x
(1)
where C (mol l − 1) is the diffusing substance concentration, x (cm) is the space coordinate along the diffusion axis, normal to the plane section and D (cm2 s − 1) is the diffusion coefficient that can reasonably be considered as constant for a very slight C variation. This equation relates the time dependence of the solute concentration to the flux variation along the diffusion axis [7]. So, the main goal of this work was to apply a common technique, holographic interferometry, to the study of biomolecule conformation through their diffusing behavior.
2. Materials and methods
2.1. Methods of measurements The recording of refractive index variations has been widely used for many years to determine the transport properties of transparent materials [6,8 – 10]. The refractive index is related to the concentration gradient; so, mass transfer coefficients can be obtained by translating the experimentally observed refractive index profile to a concentration profile [9]. Many methods have been developed for the study of the diffusion processes in liquids (static and dynamic light scat-
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tering measurements [2,4,11], Gouy interferometry [3]…). Among optical methods, we have chosen holographic interferometry because it permits the visualization of a whole experimental cell on a single recording medium (paper or digital), instead of only getting a point information. In addition, the holographic real-time exposure technique permits to follow an evolution with time, and therefore it is very well adapted to slow processes like diffusion.
2.2. Experimental technique Measurements were made in a parallelepipedic cell the dimensions of which are 20 mm height, 9.5 mm width and 10 mm optical thickness. Before each measurement, the glass cell is plunged for one night in a diluted solution of a strong detergent (DDN 250, Franklab) and then carefully rinsed out with deionized water, in order to prevent protein adsorption on the inner walls between successive tests. The cell is inserted inside large copper blocks, which prevent temperature gradients. These blocks are mounted on a jack which allows a precise adjustment in the laser beam direction. The whole system is fixed on a vibrationless optical table with a polyethylene cover, preventing thermal instabilities due to air convection. The temperature is measured with platinum Pt 100 sensors at both ends of the cell. Experimental boundary conditions correspond to those of an infinitely long cell permitting a free diffusion as long as the diffusing species do not reach the ends of the cell. The holographic facility (Fig. 1) consists of a reference beam and an object beam originating from an argon laser (wavelength 514 nm (1)), a mirror (2) and a 50/50 beam splitter (3). Each beam energy is controlled by means of a halfwave plate (4) and a Nicol prism (5). Spatial filters (6) combined with beam expanders (7) permit the enlargement of the beam and high frequency filtering. On the reference beam, an auxiliary plate (8) is used to counterbalance the parasitic fringes coming from mechanical or thermal variations of the holographic plate (10). The object beam passes through the experimental cell (9) before interfering with the reference beam on
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the holographic plate. After the hologram processing, the cell reference state is recorded. When the diffusion starts, an interference fringe pattern appears, the variations of which are recorded by a video camera.
tion two different solutions can be obtained together with the initial, boundary and matching conditions [7,12]. In the case of thin layer diffusion, a solution of the second Fick’s law is given by:
2.3. Solution setting: the thin layer configuration
C(x,t)=
A small volume (about 50 ml) of a solution with concentration C1 is deposited with a micropipette at the bottom of the cell which is already filled with a greater volume (3000 ml) of a lower concentration solution (concentration C2) (Fig. 2). The diffusion process is assumed to be unidirectional in the direction x\ 0; the origin plane (x =0), being taken at the interface between the two solutions at time zero. According to this geometry, each plane perpendicular to the diffusion axis is an iso-concentration plane. To our knowledge, all the studies on protein diffusion were made with equal volumes of the lower solute-rich solution and of the upper solutedepleted solution (infinite layer configuration). In a preliminary study we then compared the results obtained in this configuration with those obtained with the thin layer configuration. It appears that in the infinite layer configuration the first hypothesis concerning the Fick’s law (D independent of the concentration) was not valid yet. As a matter of fact, the diffusion coefficient follows a decreasing tendency when the concentration approaches zero; consequently, for infinite dilution, the infinite layer diffusion coefficient is very close to the thin layer coefficient, those two values corresponding to the literature values. So, in this study, we have adopted a thin layer configuration. Moreover, this method permits to take advantage of its experimental simplicity because it only requires the introduction of a very small volume of the denser liquid at the bottom of the cell, which must be carefully deposited in order to avoid convective motions.
2.4. Methods of resolution The solution of the second Fick’s law Eq. (1) relates the concentration C(x,t) to the diffusion coefficient D. According to the chosen configura-
M
PDt
exp
−x2 4Dt
(2)
where M is the amount of substance per unit of surface, originally deposited in the plane x= 0. This equation applies to a semi-infinite cell extending over the region x\ 0 and with an impermeable boundary at xB 0; so, diffusion only occurs in the positive x-direction [7,12]. In the case of a thick plane diffusion, which corresponds to a substance initially confined in the region − hBxB h (thickness 2h); the solution can be written: C(x,t)=
C0 h −x h+ x erf + erf 2 2 Dt 2 Dt
(3)
where C0 is the initial concentration of the diffusing solution and erf stands for the error function [7,12]. In each case, the x= 0 plane corresponds to the maximum concentration, and the top of the cell, where no fringes are present, is taken as zero solute concentration (Fig. 2). Diffusion coefficients were first computed with both Eqs. (2) and (3). Eq. (3) gives a more rigorous result because it calls out a definite thickness whereas Eq. (2) can be considered as an approximation. However, the D values obtained with the two equations were close, providing a sound basis for the hypothesis of thin layer and, consequently, only the results computed from Eq. (2) will be given here.
2.5. Protein solutions Our aim is to study the diffusion process occurring in aqueous protein solutions usually used as lubricant fluid during tribological tests. The chosen protein is bovine serum albumin (BSA) according to the ISO/TR 9325 (November 1989) standard in application for hip and knee prosthesis friction test [13]. A few BSA properties in its native form are reported in Table 1.
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Table 1 Some characteristics of bovine serum albumin
BSA
Molecular weight (g mol−1)
Dimensions (nm×nm×nm)
Isoelectric pH (pHi)a
66 700
4×4×14
4.7
a pHi is the pH for which the global surface charge of the protein is zero. This pHi value with its neighboring represents a critical pH domain because the protein shows minimal hydration and solubility values.
At a given temperature, the ionic strength, the pH of the medium and the protein concentration are parameters which are well known to change the protein diffusion coefficient because they modulate the electrostatic interactions between protein molecules. The magnitude of the Coulomb repulsion depends on the protein surface charge, which in turn depends on the pH. In the solution, small ions screen the Coulomb interaction between the charged proteins so that for a salt concentration of 0.1 mol l − 1 the Coulomb field vanishes beyond a small fraction of the protein linear dimension. Concentration determines the probability of collisions during which the proteins come close enough to feel the Coulomb repulsion, as well as other short- and longrange mutual interactions. Thus, pH, IS and protein concentration are factors which together influence the magnitude of the diffusion coefficient [14]. Freeze dried BSA (99% purity) from Biosepra (Paris) was used without further purification in this study. BSA was diluted in different solvents such as deionized water or phosphate buffer solution. Phosphate buffer was used to maintain the pH in the physiological range whereas the addition of NaCl increases the IS. Na thimerosal was only added for antisepsis. The solvents used are described in Table 2. Photon correlation spectroscopy (PCS) measurements were also performed, because it is a helpful method for the conformational studies of proteins [15]. The PCS technique explores the temporal fluctuations in light scattered by a solution of molecules. These fluctuations are a direct function of Brownian motion of the scatterers and are related to the hydrodynamic size of the solute. PCS is a very sensitive technique for the detection of aggregates and so is adequate for the following
of the denaturation/aggregation process whatever the reason of the denaturation (heat or increasing of IS). In the present PCS study, the selected samples were characterized with a Zetasizer 3000 spectrometer (Malvern Instruments, UK).
3. Results The first part of this study is to investigate the influence on the diffusion coefficient of factors which could affect the tertiary structure of protein, and also the size and shape of molecules. BSA diffusion coefficient is first measured at room temperature, for various experimental conditions of (i) BSA concentration, (ii) IS, (iii) heat treatment. Then, the diffusion coefficient will be compared with that of a proteic solution after its use as a lubricant during a friction test.
3.1. Influence of the protein concentration Fig. 3 shows a plot of BSA diffusivity as a function of diffusing time. For each concentration C1, the mean D values reported in Table 3, are calculated with all the experimental points. Several protein concentrations either in phosphate buffer saline solution or in deionized water are given. For each experiment, the upper solution differs from the lower, by BSA concentration only (C2 = 0). In this way, it was possible to study diffusion of BSA molecules alone. The diffusion coefficient does not change significantly with BSA concentration in the explored concentration and IS ranges. These measurements significantly confirm the choice of D independent of the concentration for the resolution of the second Fick’s law. So, the following experiments were conducted at a BSA
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Table 2 Composition of the solvents used in this study Name
Composition
Concentration (mol l−1)
pH
Ionic strengtha (mol l−1)
Saline solution Phosphate buffered solution
NaCl KH2PO4 Na2HPO4 Na thimerosal NaCl KH2PO4 Na2HPO4 Na thimerosal NaCl
0.15 9.5×10−3 0.04 4.9×10−4 0.15 9.5×10−3 0.04 4.9×10−4 3.25
6.7 7.4
0.15 0.20
7.5
3.45
High IS phosphate buffered solution
a
IS was computed according to: IS = 1/2iCi Z 2i where C (mol l−1) is the concentration and Z the valence of the ion i.
concentration of 10g l − 1 (0.15 ×10 − 6mol cm − 3), according to the concentration used in the lubrication fluid for tribological tests.
3.2. Influence of the ionic strength The results reported in Table 4 show the decrease of the diffusion coefficient of BSA with IS. Fig. 4 illustrates this tendency.
3.3. BSA submitted to stress
was used as a lubricant fluid for a friction test reproducing a classical physiological walk performed for 7 days (0.50× 106 cycles of stress). BSA diffusion coefficient was measured by holographic interferometry with sampling at 0.12× 106, 0.26×106, 0.36×106 and 0.50× 106 cycles of constraint corresponding approximately to 2, 3, 5 and 7 days of unceasing movement (Table 6). We took great care of avoiding bacteriological pollution during the entire test. Before the start of each diffusion experiment, the solution was kept for 30 min at room temperature after extraction from
In Table 5 we report the mean diffusivity obtained with a diluted phosphate buffer BSA solution, after two kinds of treatment: solution heated for 2 h at 75 °C; solution in salt excess (IS= 3.3 mol l − 1). These two treatments are known to stress the protein and can induce conformational changes. Results are reported in Fig. 5 where BSA is always the only diffusing species. They show that the diffusion coefficients of the heated and high IS solutions have the same order of magnitude. These values are clearly smaller than that of native BSA.
3.4. Comparison with the diffusion coefficient of BSA after friction test As mentioned above, the first goal of this work was to study the influence of a mechanical stress during a friction test on conformation of the BSA molecule. So, a phosphate buffered BSA solution
Fig. 3. Variation of the diffusion coefficient with diffusion time. Effect of the protein concentration for buffered and non-buffered solutions. (Concentrations given in Table 3).
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Table 3 BSA diffusion into its pure solvent Solution
Concentration C1×106 (mol cm−3) (BSA diluted in a phosphate buffered solution)
D×106 (cm2 s−1)
Solution
Concentration C1×106 (mol cm−3) (BSA diluted in deionized water)
D×106 (cm2 s−1)
1 2 3
0.15 0.30 0.60
0.60 0.59 0.66
4 5
0.15 0.30
2.54 2.04
the simulator, in order to avoid parasitic fringes due to thermal gradient in the diffusion cell. Fig. 6 shows the slight D increase with the number of cycles. This result is in opposition with the previous one which shows a smaller value of D when BSA is under stress.
4. Discussion The shape of a globular protein in solution is related to the pH and to the IS of the solvent: the pH sets the net surface charge of the protein and IS acts on the repulsive force between molecules. In an isoelectric pHi medium (4.7), the average surface charge of the BSA is zero. In deionized water or in phosphate buffered solution, where pH (7.4) is greater than the protein pHi, negative surface charges appear on the BSA surface. If salt is then added to the solution, the electrostatic repulsion between the charged macromolecules decreases because the salt ions screen the molecules from each other. So, in a solution with high IS, the macromolecules can aggregate. On the contrary, in deionized water, BSA keeps its negative surface charge and a strong electrostatic repulsion is maintained between macromolecules.
4.1. Effect of the starting concentration According to the present findings, we have concluded that in the explored concentration range, close to the concentration recommended for tribological tests, there is no effect of the initial concentration; the diffusion coefficient is then independent of the protein concentration whatever the nature of the solvent is. This result holds
if BSA is the only diffusing species. It is consistent with values found in the literature [4] and confirms the validity of the hypothesis of the independence of D with concentration.
4.2. Nature and ionic strength of the medium The influence of the ionic environment on the protein shape was studied through diffusion coefficient measurements by varying IS of the BSA solution. The explored pH range being small, the influence of the pH on the diffusivity appears negligible compared to the influence of the IS. This study then illustrates the strong influence of the ionic environment on the BSA diffusivity, which is lower in saline solution (NaCl or phosphate/buffer) than in deionized water. This finding is strengthened by the small diffusion coefficient obtained with the high IS solutions. A similar effect was observed by other authors [10] suggesting that as the double layer around the protein increases, the rate of diffusion also increases. The decreasing trend of the diffusion coefficient with IS (Table 4) indicates that the screening effect of the small ions used in our study Table 4 BSA diffusion into its pure solvent in relation with IS of the solvent Nature of the solvent IS (mol l−1)
D×106 (cm2 s−1)
Deionized water Saline solution Phosphate buffered solution High IS phosphate buffered solution
0 0.15 0.20
2.54 0.55 0.60
3.45
0.17
For solution composition see Table 2.
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Fig. 4. Variation of the diffusion coefficient with time. Effect of the solvent ionic strength.
Fig. 5. Variation of the diffusion coefficient with time. Influence of a stress on the diffusion coefficient of BSA buffered solutions.
is not complete even at high IS (3.45 mol l − 1). However, other groups [2– 4,10,15] observed that the diffusivity does not vary for IS above 0.1 mol l − 1 and chose to work with high IS solutions in order to avoid these electrostatic effects and their consequences on protein diffusion. So, electrostatic interactions seem to have a clear effect on the diffusion of macromolecules. If the formation of aggregates due to the high IS appears, the observed diffusion will be that of a ‘cluster’ of molecules, instead of that of a single molecule. For instance, Meechai et al. [4] found an onset of protein aggregation at pH 7.4 when IS increases from 1.5 to 3.3, with an average protein molecular weight 10% higher than for a single molecule.
4.3. Stress induced on the protein Several papers in the literature deal with the influence of heat on protein conformation. So, we have also chosen to evidence this influence through diffusion. A BSA phosphate buffered solution was heated for 2 h at 75 °C, and after cooling to ambient temperature, its diffusive behavior was studied by holographic interferometry. A smaller diffusion coefficient was obtained compared to that of the native protein, under the same conditions and concentrations. The value was closer to the one obtained in high IS conditions. Thus, we can conclude that thermal stress as well as salt excess lower BSA diffusivity and the hypothesis of a protein conformation change
Table 5 Influence of the stress on the diffusion coefficient of BSA buffered solutions (IS = 3.3 mol l−1)
Table 6 Influence of the number of constraint cycles on the diffusion coefficient of a BSA buffered solution
Nature of the treatment
D×106 (cm2 s−1)
Cycles number (million of cycles)
D×106 (cm2 s−1)
Native solution Heatinga Salt excessb
0.60 0.11 0.17
0.00 0.12 0.26 0.36 0.50
0.63 90.1 1.0 90.1 1.0 90.1 1.1 90.1 1.9 9 0.1
a b
Solution heated for 2 h at 75 °C. Solution in salt excess.
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because of a strong screening tendency; the protein can also be unfolded because of a stretching effect. Thus, we can then conclude that measurement of the diffusion coefficient is sensitive to the protein shape modification, whatever the nature of the change might be.
4.4. Photon correlation spectroscopy
Fig. 6. Variation of the diffusion coefficient with time. Evolution of D with the number of constraints cycles during the tribological test.
can be advanced. The hypothesis of an aggregation between molecules is not ruled out either, but it seems difficult to conclude about the exact process only with holographic interferometry. As a matter of fact, a great number of denaturing agents can break bonds that usually maintain the secondary and tertiary structure of the protein, but they can also act on the repulsion barrier between molecules. In the first case, an unfolding of the protein takes place, and molecules can stay in solution in this conformation, whereas in the second case, aggregation of some molecules appears because of the decrease of (i) the distance between molecules and (ii) the hydrophobic attractions between unfolded molecules. In the first situation, the diffusion coefficient could correspond to that of a single molecule whose size and shape are different, whereas in the second situation, the diffusion coefficient is that of a cluster. In both situations, the diffusion coefficient corresponds to different protein shape and size according to the heat effect on the protein structure; the diffusion coefficient suggests a larger size. On the other hand, the heated and the very high IS solutions give similar results. At this salt concentration, molecule aggregation probably appears
From BSA diffusivity values in high IS solutions, and in heated solutions, we have concluded on a protein conformation change and even a probable molecule aggregation. In order to confirm these hypotheses, measurements on photon correlation spectroscopy were undertaken. The PCS method permits the measurement of the size distribution of particles in a solution. The particle diameter of the native protein was found to be 7.1 nm. The Stockes–Einstein relation, and the diffusion coefficient show the coherence of the equivalent radius of the BSA particle. So, the diameter measurement with PCS strengthens our diffusivity values. Afterwards, the PCS method was applied to evaluate the particle diameters in heated and in high IS solutions. A diameter of 24.4 nm was found for BSA molecules, which is coherent with the shape modification of BSA, due to heat treatment. In high IS solutions, the particle diameter is 6.7 nm, so roughly that of the native protein. This result is absolutely not coherent with other results [4] and with our diffusion values (Table 5). A possible explanation might be that a great quantity of salts disturbs the PCS measurement. So, PCS confirms the result obtained for the heat-treated solution: heat provokes an increase in the protein size, and consequently, a decrease in the diffusion coefficient value.
4.5. Protein diffusion beha6ior after se6eral cycles of constraints due to tribological test In Fig. 6 we observe that whatever the number of cycles to which proteins have been submitted, the diffusion coefficient keeps an average constant value, with a very slight increase. If we compare this result with that obtained after the heat treatments where a decreasing D was associated with
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an unfolding of the molecule, we can think here that a weak increase of D could be due to the reducing shape of the molecule. One possible explanation could be a bacteriological contamination leading to the division of the protein molecule during an enzymatic proteolysis. A bacteriological analysis of the samples showed no evidence of contamination that could explain the increasing diffusion. So, one can conclude that the friction test had no drastic effect on the protein conformation and holographic interferometry is therefore a rather good method to follow the conformation evolution of BSA in a solution submitted to stress.
5. Conclusions Several important findings have been emphasized in this work. First, it has been evidenced that the BSA diffusion coefficient is independent of the initial concentration whatever the nature of the solvent in our experimental conditions (pH, IS). We also pointed out that the influence of the medium IS on the BSA diffusion coefficient is greater than the influence of the pH in the explored pH range and we have concluded on the decreasing tendency of the diffusion coefficient when the IS of the medium increases. Moreover, we have experimentally evidenced the sensitivity of the diffusion coefficient with the protein shape and PCS measurements have confirmed the modification of the protein shape due to the nature of the solvent or heat stress. So, holographic interferometry appears to be a technique, which is very sensitive to the conformation changes of protein molecules. However, after several cycles of constraints during tribological test, no really noticeable modification of the BSA diffusion was observed, particularly if we make a comparison between these results and the previous ones with various stresses. Thus, we can conclude that the stress applied on a simulator apparatus only creates very small changes in the protein conformation and the use of other techniques like UV
spectroscopy or circular dichroism could be used to improve this method.
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