Langmuir 1998, 14, 2167-2173
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Elution Process of Adsorbed Fibrinogen by SDS: Competition between Removal and Anchoring Maria Zembala,*,† Jean Claude Voegel,† and Pierre Schaaf‡,§ Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 1, 30-239 Cracow, Poland, INSERM U.424, Fe´ de´ ration de Recherches Odontologiques, 11 rue Humann, 67085 Strasbourg Cedex, France, Institut Charles Sadron, CNRS-ULP, 6 rue Boussingault, 67083 Strasbourg Cedex, France, and Ecole Europe´ ene de Chimie, Polyme` res et Mate´ riaux de Strasbourg, 1 rue Blaise Pascal, BP 296F, 67008 Strasbourg Cedex, France Received October 21, 1997. In Final Form: January 26, 1998 The elution of proteins from various surfaces by surfactants was widely studied in the literature in connection with practical applications but also as a tool to understand protein adsorption behavior. In this paper we present the results obtained for the following system: fibrinogen adsorbed at a silica surface eluted by sodium dodecyl sulfate (SDS) solutions of various concentrations and at various ionic strengths. The process was followed by measuring the radioactivity of labeled protein. The elution kinetics was determined and described quantitatively by an apparent rate constant k and an elutability parameter. An unexpected nonmonotonic dependence of k as a function of SDS concentration cSDS was obtained for the first time. This nonmonotonic behavior of k was explained assuming double action of SDS molecules on the fibrinogen adsorption layer: SDS with its detergent ability removes adsorbed protein but, being at the same time a strong denaturing agent, it is able to transform adsorbed molecules into an irreversibly bound nonelutable form. The observed dependence of k on cSDS is then the result of competition between these two processes. Two-step experiments were performed in which the existence of the SDS-induced fastening of fibrinogen molecules to the surface was confirmed. A simple kinetic model was proposed in which two parameters related to the removal and anchoring subprocesses were introduced. The comparison between the experimental data and the model allowed us to get access to these parameters.
Introduction The elution of adsorbed proteins by surfactants is a commonly observed process which is of great practical importance for all cleaning procedures (for example in the food industry or for removal of macromolecules from chromatographic columns). The elution process results from the interplay of several of the following individual effects: interactions of the surfactant molecules with a surface, with a protein, and with themselves; interactions of the proteins with a sorbent surface; and possible denaturation of the proteins in contact with surfaces or with surfactant molecules. From the enumeration of these individual possible contributions one realizes that no single elution mechanism exists, and at the present time it is not possible to predict the elution behavior of a given protein/surface/surfactant system. Thus, it is of primary importance to gain a better understanding of the mechanisms of the elution process. In this paper we focus our attention on the system fibrinogen/silica/sodium dodecyl sulfate (SDS). The adsorption of proteins, in particular of fibrinogen on solid surfaces, is known to be partially irreversible: if one replaces the protein solution in contact with the surface by pure buffer, only a part of the adsorbed protein population desorbs.1 A similar behavior is observed for some protein/surfactant systems. The removable amount of macromolecules (elutability) for a given surface and a given surfactant has even been used to quantify the strength of the protein binding to the surface.2 Another * To whom correspondence should be addressed. Permanent address: Polish Academy of Sciences. † Fe ´ de´ration de Recherches Odontologiques. ‡ Institut Charles Sadron. § Ecole Europe ´ ene de Chimie. (1) Chan, B. M. C.; Brash, J. L. J. Colloid Interface Sci. 1981, 82, 217.
way of binding strength characterization consists of the comparison of the removal kinetics. However, both aspects may depend on the type of surfactant used. A good example of surfactant-dependent behavior is provided by the study of removal of two proteins: T4 lysozyme and a mutant of this protein in which isoleucine 3 has been replaced by tryptophan (tryptophan mutant).3,4 Whereas the rate of protein removal and the removed fraction by the SDS elution are lower for T4 lysozyme than for tryptophan mutant, the opposite conclusion has been drawn for the removal of the same proteins by dodecyltrimethylammonium bromide (DTAB). The conclusions related to the elution by SDS would be expected if one takes into account the fact that the tryptophan mutant protein is less stable than T4 lysozyme. This conclusion agrees with a usual rule, that stable proteins binds less strongly to solid surfaces.5 This example shows clearly that the elution process is far from being understood. Different removal mechanisms have been proposed, and one can roughly distinguish between two cases depending on whether surfactant molecules adsorb or not at the solid surface.6 In the first case the removal process will be similar to an exchange reaction with surfactant molecules replacing gradually the links established by the protein molecules with the surface. Thus, the facility of removal strongly depends on the relative affinity of surfactant and protein molecules for the surface. Such a mechanism can (2) Bohnert, J. L.; Horbett, Th. A. J. Colloid Interface Sci. 1986, 111, 363. (3) Wahlgren, M.; Arnebrant, Th. Langmuir 1997, 13, 8. (4) McGuire, J.; Wahlgren, M. C.; Arnebrant, Th. J. Colloid Interface Sci. 1995, 170, 182. (5) Haynes, Ch. A.; Norde, W. Colloids Surf., B 1994, 2, 517. (6) Arnebrant, Th.; Wahlgren, M. In Protein at Interfaces; Horbett, Th., Brash, J. L., Eds.; ACS Symposium Series 602, American Chemical Society: Washington, DC, 1995; Chapter 17, p 239.
S0743-7463(97)01146-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/25/1998
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be found, for example, for protein removal by DTAB from negatively charged or hydrophobic surfaces. In the second case the surfactant molecules do not adsorb at the surface. This is especially the case of SDS in contact with a silica surface, both being negatively charged at the pH range usually investigated in protein adsorption studies (between 4 and 8). For such systems the elution mechanisms originate mainly in the surfactant-protein interaction causing weakening of the links between the protein molecules and the surface. The elution ability depends then on the strength of interaction between surfactant and protein molecules. It is known that the interaction of SDS with a wide variety of proteins is characterized by a high binding ratio identical for a large number of proteins (on a gram to gram basis) when the concentration of SDS monomer molecules exceeds 5 × 10-4 M. Two distinct plateaus have been found: between 5 and 8 × 10-4 M of SDS monomer, a complex with a stoichiometry of 0.4 g of SDS per 1 g of protein is formed, and for SDS monomer concentrations larger than 8 × 10-4 M, a second complex saturated with 1.4 g of SDS per 1 g of protein is detected.7 However, it has to be noticed that most of these results have been obtained for denatured proteins even if for some cases the data have also been confirmed by bringing the native proteins directly in contact with the SDS solution. The interaction of anionic surfactants, SDS in particular, with globular proteins seems to occur with the cationic sites of the protein molecule, specifically with the arginyl, histidyl and lysyl amino acid side chains.8 In addition to electrostatic interactions of the surfactant head groups with cationic proteinic sites, also the alkyl chains of the surfactant molecules may interact with the hydrophobic regions of the protein in the vicinity of the cationic sites. Generally, these interactions induce protein unfolding, exposing many hydrophobic binding sites previously buried in the core of the tertiary structure. When the protein is adsorbed at a solid surface, such a denaturation mechanism may assist more strong anchorage to the surface and thus affect the elutability of the protein by the surfactant. As will be shown, this indeed seems to be the case for the system under study. Such a competition between the elution process and the protein fixation at the surface by surfactant-induced denaturation is, to our knowledge, observed for the first time. The study was performed using a radioactive-labeling technique which allows us to follow the amount of labeled protein (fibrinogen) adsorbed at a silica surface as a function of time. This technique has proven to be adequate for the determination of kinetic parameters of adsorption or desorption processes for macromolecules at solid surfaces. The aim of the study is 2-fold: (i) quantify the elution rate by determining (to our knowledge, for the first time) an apparent elution rate constant as a function of the surfactant concentration and (ii) recognize the elution mechanism from the analysis of the rate constantsurfactant concentration dependence. Material and Methods Fibrinogen from human plasma (Mw ) 340 000), fraction I, and sodium dodecyl sulfate (SDS) (Mw ) 288.4), were purchased from Sigma. Phosphate buffer (PBS) was prepared by mixing 50 mM NaH2PO4‚H2O and 50 mM Na2HPO4‚7H2O adjusted to pH 7.5 in the presence of 0.15 M NaCl. It was used as prepared or diluted 2.5 and 5 times. These solutions correspond to ionic (7) Reynolds, J. A.; Tanford, Ch. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002. (8) Jones, M. N. Chem. Soc. Rev. 1992, 21, 127.
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Figure 1. Schematic representation of the setup. SP1 and SP2 are syringe pumps for injecting a buffer (SP1) and fibrinogen and SDS (SP2) solutions, V is a four-way valve, D is the radiation detector, T is a silica tube, C1 and C2 are containers collecting used solutions, and B is a balance for flow rate determination. strengths (I) of approximately 0.31, 0.12, and 0.06 M. Fibrinogen and SDS were dissolved in buffer of studied ionic strength. The CMC value of SDS in PBS of highest ionic strength was determined from surface tension measurements and was equal to 7.5 × 10-4 M, that is, very close to the value found for SDS in 0.3 M NaCl.9 Thus, CMC values for diluted buffers were estimated by extrapolation of CMC versus NaCl concentration data.9 They are equal to 1.3 × 10-3 M and 1.9 × 10-3 M for I ) 0.12 and I ) 0.06 M, respectively. Deionized water (Milli Q Plus, Millipore, France) was used for the preparation of all solutions. Silica tubes of diameter 0.233 and 15-cm long delivered by Striegel (Strasbourg, France) were cleaned in 5 × 10-3 M SDS in PBS, followed by careful water washing. Just before experiments the tubes were put for 5 min in contact with 10% H2SO4. Next they were rinsed with water and equilibrated with pure buffer solution during 1h under flow conditions. Fibrinogen molecules were radiolabeled with I125 using a technique derived from the method of McFarlane10 in which iodine monochloride is the iodinating agent. The NaI125 amount was chosen low enough so that only a small fraction of the molecules were labeled. The activity of labeled fibrinogen was equal on the average to 8.4 × 10 9 cpm/g of protein. Fibrinogen was stored in a concentrated form (approximately (1.5 × 10-3) % w/w) at -20 °C. The stock fibrinogen solutions were quickly thawed at 37 °C just before use and diluted with PBS to their final concentrations. Before start of the experiment, fibrinogen solutions were filtered through Millex-HV filters having 0.45µm pores (Millipore Products Div., Bedford, Great Britain). The concentrations of diluted solutions were determined from absorbance of the solutions at 280 nm, taking the value of the extinction coefficient for a 1 cm-thick layer of 1% w/w protein solution to be equal to 15.5. Experimental Setup. The experimental setup employed was similar to the one described by Boumaza et al.,11 and it is schematically presented in Figure 1. An appropriate solution was pumped by a syringe pump (SP1 for buffer or SP2 for fibrinogen and SDS solutions) via a four-way valve (V) to the silica tube (T) and then to a container (C1) placed onto a balance (B) (for flow rate determination). An additional path to the waste vessel (C2) was foreseen for initial elimination of air bubbles. The tube was connected to the valve by a short piece of elastic tubing, and a lead shielding from both sides of the tube was applied. Radiation was detected by a specially designed scintillation counter (D) (Quartz&Silica, Paris, France) and acquired by PC computer using Accuspec software (Canberra, USA). (9) Birdi, K. S. In Micellization, Solubilization and Microemulsion, Mittal, K., Ed.; 1997; Vol. 1, p 158. (10) McFarlane, A. S. Nature 1958, 182, 53. Regoeczi, E. IodineLabeled Plasma Proteins; CRC Press: Boca Raton, FL, 1984. (11) Boumaza, F.; Dejardin, Ph.; Yan, F.; Baudin, F.; Holl, Y. Biophys. Chem. 1992, 42, 87.
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Figure 2. Typical row signal run registered during the whole experiment: step 1, the background signal counted during tube equilibration with pure buffer; step 2, fibrinogen adsorption; step 3, displacement of fibrinogen solution by buffer; step 4, connections and valve exchange for the clean ones; step 5, additional buffer rinsing; step 6, fibrinogen elution by SDS solution. Typical Experiment. The preliminary measurements showed that fibrinogen elution by SDS is very fast. Distinct maxima on the radioactivity-time dependence caused by fibrinogen elution from preceding parts of the flow path were present on the radioactivity-time dependence registered at any part of a tube distant from the entrance. To ensure detection of fibrinogen exclusively from the chosen part of the tube, all parts of the setup used for the adsorption step (marked in Figure 1 by thinner lines) were replaced by the clean ones before starting the elution. Thus, the following experimental procedure was applied (see Figure 2): step 1s1-h equilibration of silica tube with buffer flowing at the rate 4.85 mL/h corresponding to the shear rate ) 1.08 s-1; step 2s2- or 3-h fibrinogen adsorption step at the flow rate 2.74 mL/h ) 0.61 s-1; step 3s1-h of buffer displacing the fibrinogen solution (flow rate 9.80 mL/h ) 2.19 s-1); step 4sexchanging the valve and tubing connections for solution introduction for the clean ones (Because the fibrinogen adsorption layer is sensitive to any contact with the gas phase, no air was let into the tube during the exchanging procedure.); step 5sadditional 15 min of buffer washing; step 6sfibrinogen elution by SDS solution flowing at the rate 9.75 mL/h, ) 2.18 s-1. The ionic strength of all solutions used for a defined experiment was the same. Radioactivity was detected during the whole experimental procedure.
Results and Discussion The fibrinogen adsorption preceding the elution step was realized at conditions (fibrinogen bulk concentration and adsorption time) chosen in such a way that plateau adsorbed amounts were reached for all studied ionic strengths. The experimentally determined values of the amount of fibrinogen adsorbed onto the quartz surface were equal to 0.167 ( 0.02 µg/cm2 for adsorption from the most concentrated buffer (I ∼ 0.31 M), 0.545 ( 0.045 µg/ cm2 for 2.5 times diluted PBS (I ∼ 0.12 M), and 0.424 ( 0.03 µg/cm2 for 5 times diluted buffer (I ∼ 0.06 M). The time evolution of the radioactivity for a typical elution part of the experiment (step 6) is presented in
Figure 3. Variation of the activity a(t) of a fibrinogen-coated tube eluted by 1.7 × 10-3 M SDS in PBS buffer of I ) 0.06 M. t ) 0 was adjusted to the first change of the signal caused by SDS introduction. Full line corresponds to the curve fitted to eq 1.
Figure 3. The activity a(t) is directly proportional to the amount of fibrinogen molecules remaining on the silica surface at time t. To characterize the kinetics of the elution process, we approximate experimental curves by a singleexponential decay function of the form
a(t) ) a exp(-kt) + b
(1)
The constant k represents an apparent kinetic rate constant, (a + b) is the signal measured at the starting point of the elution step (proportional to the fibrinogen adsorbed amount), and the ratio a/(a + b) corresponds to the elutability as it is usually defined, that is, to the fraction of removable molecules. This is the simplest fitting function one can introduce as a first approach, and we
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Figure 4. Exponential apparent elution rate constants k (part a) and reversible bound protein fraction (elutability) a/(a + b) (part b) as a function of SDS concentration. PBS buffer of I ) 0.31 M. All experiments were repeated at least two times.
have found that it describes satisfactorily the experimental data (as can be seen in Figure 3). Thus, there is no need to introduce a more complicated function containing more fitting parameters. However, this does not mean that function 1 corresponds to the correct kinetic law which describes the elution process. It just means that this unknown law is correctly approximated by the fitting function (1). The dependencies of the elution rate constant k and of the elutability a/(a + b) on the SDS concentration cSDS of the solution used for elution are presented in Figures 4a6a and 4 -6b, respectively. The experiments were performed using solutions of three ionic strengths, and results obtained are given in Figures 4 for I ) 0.3 M, in Figure 5 for I ) 0.12 M, and in Figure 6 for I ) 0.06 M. One can notice that the elutability (Figures 4b, 5b, and 6b) varies rapidly over a narrow SDS concentration range below the cmc. The elutability becomes constant and close to 1 for SDS concentrations above the CMC. This is in accordance with the observations of Reynolds and Tanford,7 who found that only the surfactant molecules which are in a monomeric form participate in the protein surfactant complex formation, the micelles playing no or only a minor role in this process. The most striking feature of these figures is the nonmonotonic dependence of the apparent elution rate constant k on the SDS concentration for the two lower ionic strengths studied (Figures 5a and 6a). The reproducibility of the data in the regions of the extrema has been checked several times. The SDS concentration corresponding to the minimum k value agrees very well with the inflection point on the elutability curve (compare Figure 5a to Figure 5b and Figure 6a to Figure 6b). Such a behavior can only be explained by the competition between two processes, one favoring the surfactant
Zembala et al.
Figure 5. Same as Figure 4, but all solutions were prepared in PBS buffer of I ) 0.12 M. Each point represents an average of at least two measurements. In the region of the minimum the measurements were repeated up to five times.
induced removal of the protein (elution) and the other causing the stronger anchoring of the same protein at the surface. An anchoring process could be a consequence, for example, of protein denaturation induced by SDS molecules. At the moment we have no proof of this hypothesis, but we will show that the anchoring process must depend on the SDS concentration and thus it may be related to the SDS presence. This implies that the process leading to the stronger fastening of protein at the surface starts not till the adsorbed fibrinogen has been brought in contact with the SDS solution. Thus, the initial slope of the time dependence of protein removal should be sensitive only to the elution process and it should no longer exhibit extrema versus the SDS concentration. A monotonic variation of the initial elution rate in the form of an S-shaped curve is expected, as was observed for the elutability-SDS concentration dependence.12 Such a behavior was indeed found experimentally, and it is shown in Figure 7. To get a better understanding of what is going on at the surface, we have introduced a simple model. This model is certainly oversimplified, and it should not be taken as a definitive description of the process under study but merely as an intermediate device to get a feeling for the possible process mechanisms. Let us assume that the adsorbed protein molecules by interacting with SDS molecules may either desorb from the surface or change their adsorption state into an irreversible one. Thus the fibrinogen molecules may be in two states: reversibly (labeled by “r”) and irreversibly (labeled by “i”) bound to the surface. The simplest model describing such a system (12) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480.
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Figure 7. Initial apparent elution rate 1/Γ0(dΓ/dt)t)0 versus SDS bulk concentration: (O) concentrated PBS (I ) 0.31 M); (4) 2.5 times diluted PBS (I ) 0.12 M); (0) 5 times diluted PBS (I ) 0.06 M). Presented values were obtained by linear regression of the initial part of the experimental elution curves.
Figure 6. Same as Figure 5, but all solutions were prepared in PBS buffer of I ) 0.06 M.
leads to the following kinetic equations:
ΓT(t) ) Γr(t) + Γi(t) dΓr ) -(kd + ki)Γr dt dΓi ) kiΓr dt
Figure 8. Values of kb/(a + b) ) ki as a function of cSDS: full line, I ) 0.3 M (points marked by O); broken line, I ) 0.12 M (points marked by 4); and dotted line (‚‚‚) I ) 0.06 M (points marked by 0). These lines do not correspond to any fitted function but they are given to guide the eye.
with the initial conditions
Γr(t)0) ) Γ0 and Γi(t)0) ) 0 ΓT represents the total amount of protein adsorbed at the silica surface, Γr and Γi represent the amounts of protein adsorbed in the states “r” and “i”, respectively. kd corresponds to the removal rate constant and ki to the rate constant of the protein transformation from a state “r” to a state “i”. This system of equations is easy to solve, and one finds:
{ (
ΓT(t) ) Γ0
)
}
ki ki + 1exp(-Kt) K K
( )
ki ki ) k d + ki K
(2)
with K ) ki + kd. By comparing expressions 1 and 2, one sees that this simple model leads to the same type of function for the time dependence of adsorbed amount ΓT as the fitting function with the apparent rate constant k being equal to K. The initial removal rate given by
1 dΓT Γ0 dt
change. The evolution of the initial removal rate determined experimentally (see Figure 7) implies that kd increases monotonically with cSDS. Taking into account that K corresponds to an apparent rate constant k, to get the observed resultant behavior of k, the ki ) f(cSDS) dependence must behave nonmonotonously. It should increase at low SDS concentrations and then pass through a maximum before decreasing. According to expressions 1 and 2 b(a + b) corresponds to
) -kd
t)0
depends only on the elution step and not on the anchoring
Thus, the product
kb Kb ) a+b a+b should be equal to ki. Figure 8 represents the dependence of kb/(a + b) on cSDS. One can see that this quantity indeed exhibits a maximum before decreasing almost to zero at higher SDS concentrations for the lower ionic strengths investigated. It seems that our simple model contains
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Figure 9. Variation of activity a(t) for a two-step elution experiment. The first elution step was realized with 7.5 × 10-4 M SDS, and the second one was realized by SDS of concentration equal to 1.7 × 10-3 M. Arrows indicate the starting point of the action of SDS solutions. The experiments were performed in PBS of I ) 0.12 M. The curve marked by the thick line was calculated taking the kinetic parameters obtained from singlestep elution by SDS solution of higher concentration (1.7 × 10-3 M).
the essential features of the process under study. It first implies that the anchoring process must be SDS concentration-dependent. It means that the fibrinogen molecules undergo an SDS-induced state transformation into an irreversibly adsorbed form at some range of intermediate SDS concentrations whereas, at high SDS concentrations, even if another conformation change occurs, it leads to a form which is removable by SDS solution. One can speculate if the irreversible adsorbed state is not related to the first binding stoichiometry plateau found by Reynolds at Tanford,7 where the proteins form a complex with surfactant with a lower ratio of SDS to protein molecules. It is probable that the SDS-protein complex corresponding to the second binding plateau (with a higher ratio of SDS to protein) may have high enough net negative charge ensure complete elutability by SDS. To verify that within a defined region of SDS concentrations the surfactant indeed fixes the protein molecules at a surface, we have performed two-step experiments. The surface covered with adsorbed fibrinogen was first brought in contact with a SDS solution of concentration within the “anchoring” region for 1 h. After that the first SDS solution was replaced by a solution of high SDS concentration. Two second-step SDS concentrations were used: 1.7 and 2.5 × 10-3 M. Figure 9 shows the radioactivity measured during this two-step experiment. The simulated run of the second-step signal calculated assuming that the elution rate corresponding to the second elution step (high SDS concentration) is identical to the rate measured in the single-step experiment using the same high SDS concentration, is presented in the same figure. The distinct stabilizing effect of the SDS solution of low concentration is clearly visible. The apparent elution rate constants and the elutability corresponding to the last step of the two-step experiments were also determined. These quantities were put together in Figure 10 with the values found when the fibrinogen adsorption layer was eluted by the single SDS solutions (determined
Figure 10. Exponential rate constant (part a) and elutability (part b) obtained from two-step experiments as a function of SDS concentration in PBS buffer of I ) 0.12 M. Full points marked by (b, 9, and [) correspond to the second step of experiments preceded by a first-step elution marked by empty points (O, 0, ]). The other points (4) concern experiments for which only a single SDS concentration was applied and were taken from Figure 3b.
in single-step experiments). One finds that the apparent elution rate constants are almost identical to the values found previously for the experiments at low SDS concentrations. They are much smaller than the values found by bringing a protein layer in contact with only one SDS solution of high concentration. The elutability reached in the second-step was also smaller than values obtained in one-step experiments performed at the same high SDS concentrations. These findings give an experimental proof of the existence of the anchoring process caused by SDS solutions of low concentrations. The presented results show that the competition between the elution and the anchoring processes is not revealed at the highest ionic strength as distinctly as it is for the two lower ones. However, Figure 4b, presenting the elutability-SDS concentration dependence for I ) 0.3 M, and Figure 8, collecting the change of kb(a + b) ) ki values with cSDS, seem to indicate that such a competition still exists but that the anchoring process is effective in the range of lowest SDS concentrations studied. In this region the protein removal is slow and it is difficult to reach an experimental accuracy enabling us to detect the possible presence of a minimum on the apparent rate constant-SDS concentration dependence. The removal process starts to become effective at higher SDS concentrations, where the conformational change of the protein leads probably to the elutable form. This could be due to the fact that at high ionic strength the adsorbed fibrinogen amount is much lower in comparison to the amount adsorbed from diluted buffers (see also data of Chan and Brash1). The distinct effects resulting from the competi-
Elution Process of Adsorbed Fibrinogen by SDS
tion between removal and anchoring processes observed at the lower ionic strengths studied come from the fact that, in a given SDS concentration range, the rate constants of both processes are of the same order of magnitude. Our goal is now to verify if other protein/ surface/surfactant systems behaving in a similar way exist. Also the effect of the surfactant chain length should be determined to clarify the SDS-protein interaction. For better understanding of the removal-anchoring competition, we also plan to apply other experimental technics (optical, IR spectroscopy). Referring to numerous papers related to the elutability of adsorbed proteins by surfactants, we still believe that this process may be a good indirect measure of proteinsurface attachment strength. However, keeping in mind obtained results, one should perform elution experiments at conditions at which the removal process is dominant. Conclusions The elution of fibrinogen adsorbed at a silica surface by SDS solutions has been studied for the range of SDS concentrations from below to above the CMC for three levels of ionic strength. The elution kinetics and fraction of removable protein (elutability) depend strongly on both these parameters. A nonmonotonic dependence of an apparent rate constant on SDS concentration and a strong decrease of elutability in the region of low SDS concen-
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trations were found for the two lower ionic strengths studied. These observations were explained assuming that SDS affects the fibrinogen adsorption layer in two different ways: (i) removing it due to detergent action and (ii) causing a stronger anchorage probably due to protein denaturation. These two competitive processes are responsible for the nonmonotonic behavior of the system in relation to SDS concentration. The hypothesis was further supported by two-step experiments where a meaningful increase of resistivity to elution was registered after preliminary contact of the adsorption layer with an SDS solution of low concentration. A simple model was proposed which describes reasonably well the observed features of the process under study. Further studies will be performed in order to find if the dual role found for SDS is exhibited also by other surfactants. Also the effect of the chain length will be determined to clarify the SDS-protein interaction. Acknowledgment. One of the authors (M.Z.) wishes to thank the Faculte´ de Chirurgie Dentaire de Strasbourg for the grant enabling the realization of this work. The authors are grateful to Mrs. Graz˘ yna Para for performing surface tension measurements and to Mr. Bernard Senger for help and fruitful discussions. LA971146N
