Langmuir 1999, 15, 6299-6303
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Elution of Adsorbed Fibrinogen from a Silica Surface by Anionic Surfactants. 2. Effect of the Hydrocarbon Chain Lengths F. Poumier,*,† P. Schaaf,‡ and J. C. Voegel† I.N.S.E.R.M. - 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, France; Ecole Europe´ enne de chimie, Polyme` res et Mate´ riaux de Strasbourg - 1, rue Blaise Pascal, 67000 Strasbourg, France Received February 26, 1999. In Final Form: May 18, 1999 The chain length effect of 8, 10, and 14 carbon homologous sodium alkyl sulfate surfactants on the elution of fibrinogen molecules adsorbed onto a silica surface has been studied. Previously, it has been shown (Zembala et al. Langmuir 1998, 14, 2167) that the release mechanism of fibrinogen by sodium dodecyl sulfate (SDS) is the result of a competition between the elution and anchoring of the adsorbed proteins. All of the removal kinetics, followed by a radioactive labeling technique, could be described by a single-exponential decay function. The apparent release rate constant k and the amount of released proteins increase with the surfactant concentration cs and attain plateau values for cs close to the critical micelle concentration (cmc). By means of two-step elution experiments (first, low-concentration surfactant injection, followed by the injection of surfactants above cmc), it was shown that all surfactants were able to anchor fibrinogen molecules onto the solid surface and that surfactants with a smaller chain length possess a higher ability to anchor proteins than surfactants with longer chains. On the contrary, for a given surfactant concentration, elution increases with the chain length.
Introduction Surfactants are known for their great elution ability for proteins adsorbed on solid surfaces, and one of the most used surfactants is sodium dodecyl sulfate (SDS). Surfactants are often used in cleaning processes and have many applications in biochemical and biological research. Another common use of surfactants is dentistry, where they constitute an important constituent of most toothpastes and prebrushing solutions.1-3 The elution can take place through two main processes: (i) solubilization of the proteins due to strong interactions of the surfactants with the protein which can weaken the protein/surface interactions, and (ii) replacement of the adsorbed proteins by surfactants that have a strong affinity for the surface. In the latter case, the proteins are in competition with the surfactants for the adsorption. The first elution mechanism seems to govern the elution of proteins by ionic surfactants and in particular by SDS on hydrophilic surfaces, whereas the second one seems to be of greater importance on hydrophobic surfaces. These elution mechanisms have been reviewed recently by Wahlgren et al.1 Due to their strong affinity for proteins, surfactants can also denaturate them. This is particularly true for SDS,4 and many studies were devoted to understanding the denaturation process of albumin and myoglobin by this surfactant.5-8 * E-mail address:
[email protected]. Telepone: (33) 03.88.24.23.96. Fax: (33) 03.88.88.24.33.99. † Fe ´ de´ration de Recherches Odontologiques. ‡ Institut Charles Sadron. (1) Wahlgren, M.; Welin-Klintstro¨m, S.; Karlson, C. A.-C. In Biopolymers at Interfaces; Malmsten, M., Ed.; Surfactant Science Series Vol. 75; Marcel Dekker: New York, 1998; Chapter14. (2) Ananthapadmanabhan, K. P. Protein-Surfactant Interactions. In Interactions of Surfactants with Polymers and Proteins; Ananthapadmanabhan, K. P., Goddard, E. D., Eds.; CRC Press: Boca Raton, FL, 1993; p 319. (3) Vassilakos, N.; Arnebrant, T.; Rundegren, J.; Glantz, P. O. Acta Odontol. Scand. 1992, 50, 179. (4) Arnebrant, T.; Simonsson, T. Acta Odontol. Scand. 1991, 49, 281.
Denaturation of proteins can take various routes, depending on the conditions in which it takes place. In particular, when a protein adsorbs on a surface, it often denaturates, and this seems to anchor the protein more strongly to the surface.9-10 Thus, it seems possible that when surfactants are in contact with adsorbed proteins, they not only elute them, but they can also induce conformational changes that could, in principle, anchor the proteins more strongly to the surface. Despite the great number of studies that have been devoted to elucidating the elution processes of proteins by surfactants, this anchoring process, and thus the competition between elution and anchoring, has only been reported very recently.11 The system under study was fibrinogen, a plasma protein, adsorbed on a silica surface and eluted by SDS. Both the elutability, a concept introduced by Horbett and co-workers10,12,13 (and which corresponds to the proportion of removable proteins), and the elution rate were measured. It was found11 that when the adsorbed fibrinogen layer was brought directly into contact with a high SDS solution (concentration on the order of or larger than the cmc) the protein elutability was almost equal to 1. On the (5) Yamasaki, M.; Yano, H.; Aoki, K. Int. J. Biol. Macromol. 1992, 14, 305. (6) Yamasaki, M.; Yamashita, T.; Yano, H.; Tatsumi, K.; Aoki, K. Int. J. Biol. Macromol. 1996, 19, 241. (7) Moriyama, Y.; Sasaoka, H.; Ichiyanagi, T.; Takeda, K. J. Protein Chem. 1992, 11, 583. (8) Tadeka, K.; Shigeta, M.; Aoki, K. J. Colloid Interface Sci. 1987, 117, 120. (9) Arnebrant, Th.; Wahlgren, M. In Protein at Interfaces; Horbett, Th., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; p 239. (10) Bohnert, J. L.; Horbett, T. A. J. Colloid Interface Sci. 1986, 111, 363. (11) Zembala, M.; Voegel, J. C.; Schaaf, P. Langmuir 1998, 14, 2167. (12) Rapoza, R. J.; Horbett, T. A. J. Biomater. Sci. Polym. Ed. 1989, 1, 99. (13) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480.
10.1021/la990227b CCC: $18.00 © 1999 American Chemical Society Published on Web 07/16/1999
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other hand, when the adsorbed layer was first in contact with a low-concentration SDS solution that was subsequently replaced by a high-concentration SDS solution, a significant proportion of proteins remained irreversibly fixed on the surface, and the removal rate was strongly decreased. This effect was especially important at low ionic strengths. Nonmonotonic behavior of the elution rate with the surfactant solution concentration was also observed when the protein layer was brought directly in contact with the final surfactant solution.11 This also could be explained by the competition between the elution by SDS and the protein anchoring ability to the surface, this later being explained, but not proved, by the denaturation ability of proteins by SDS. In the present article, we investigate whether the competition between elution and anchoring is also observed for other surfactants or whether it is a special feature of SDS. We thus study the influence of the hydrophobic chain length of sodium alkyl sulfate surfactants on the elution mechanism of fibrinogen adsorbed on a silica surface. The ionic strength of the surfactant solution, known to be an important parameter, is fixed in this work. This study is performed, in the same manner as that by Zembala et al.,11 by means of a radioactive labeling technique. Materials and Methods Fraction I of fibrinogen from human plasma, Mw ) 340 000, (Sigma Chemical Co., St. Louis, U.S.A.), sodium octyl sulfate (C8) (Mw ) 232.3) (Sigma), sodium decyl sulfate (C10) (Mw ) 260.32) (Fluka Chemie, Buchs, Switzerland), sodium dodecyl sulfate (C12) (Mw ) 288.4) (Sigma), and sodium tetradecyl sulfate (C14) (Mw ) 316.44) (Sigma-Aldrich Chemical Co., St. Louis, U.S.A.) were used in this study without further purification. The surface tensions versus surfactant concentration increased monotonically, and no minima were detected. Phosphate buffer (PBS) was prepared by mixing 50 mM NaH2PO4‚H2O and Na2HPO4‚7H2O solutions adjusted to pH 7.5 in the presence of 0.15 M NaCl. PBS was then diluted five times in order to obtain a solution with an ionic strength (I) of approximately 0.06 M. Fibrinogen and all surfactants studied were dissolved in this buffer. The cmc’s of the surfactants with I ) 0.06 M were determined from surface tension measurements and were equal to 1.2 × 10-1, 3.3 × 10-2, 2 × 10-3, and 2.75 × 10-4 M respectively for C8, C10, C12, and C14. These values are very close to the values found by Yamasaki et al.5,6 Deionized water (Millipore) was used for the preparation of the solutions. Adsorption and elution experiments were realized in a silica tube of inner diameter 1.77 mm and 150 mm long (Striegel, Strasbourg, France). It was cleaned overnight in a 10% Hellmanex solution (Hellma GMBH & Co, Mu¨llheim, Germany), followed by extensive water rinsing. Before the beginning of an experiment, the tube was brought for 5 min in contact with 10% H2SO4. It was then rinsed with water and equilibrated with buffer (I ) 0.06 M) for 1 h under flowing conditions. The surface potential of the tube has been estimated previously. It is of (-112 ( 2) mV and corresponds to a pH of 5.6 in the close vicinity of the solid surface. Contact angle measurements (θ < 10°) led to an interfacial tension of (76 ( 6) mJ m-2.14 Protein labeling was achieved with 125I using the iodine monochloride method,15 and the proteins were dissolved in PBS. After filtration of the fibrinogen solution through Millex-HV filters of 0.45 µm pores (Millipore Products Division, Bedford, Great Britain), the concentration of the labeled protein solution was determined by absorbance at 280 nm and the specific radio activity (cpm mg-1) was measured by γ counting (Minaxy γ, (14) Ball, V. Contribution a` l’e´tude de la dynamique de prote´ines adsorbe´es a` l’interface solide/solution aqueuse: adsorption, de´sorption et e´change. Doctorat d’Universite´ , Strasbourg, 1996. (15) McFarlane, A. S. Nature 1958, 182, 53. Regoeczi, E. IodineLabeled Plasma Proteins; CRC Press: Bota Raton, FL, 1984.
Poumier et al. United Technologies, Packard Instrument Co. Inc., Downers Grove, U.S.A.). Fibrinogen was stored in this concentrated form (around 0.1 wt %) at - 20 °C. Before each experiment, the frozen fibrinogen solution was rapidly thawed at 37 °C and diluted in PBS (I ) 0.06 M) to its final concentration of about 0.04 wt %.
Experimental Setup The employed experimental setup was similar to the one described by Zembala et al.11 Briefly, the cleaning and buffer solutions were injected in the silica tube by the mean of the first syringe pump. The solutions were recovered in a container and placed on a balance, in order to follow the flow rate via computer. Another pass to the waste vessel was done to eliminate bubbles. Radiation was detected in the tube by a scintillation counter (Quartz & Silice, Paris, France) and acquired by a microcomputer using Accuspec software (Canberra, Shaumburg, Illinois). The labeled protein and surfactant solutions were injected by the second syringe pump. Two kinds of elution experiments were performed. One-Step Elution Experiments. A typical one step elution experiment was divided into four different steps. Step 1: equilibration of the silica tube during 1 h with buffer (I ) 0.06 M) at a rate of 4.85 mL/h. Step 2: fibrinogen adsorption during 2 h at a rate of 2.75 mL/h using a 0.04 wt % solution dissolved in PBS (I ) 0.06 M). Step 3: 1 h of buffer washing in order to flow the fibrinogen solution out of the tube. Step 4: fibrinogen elution by surfactant solution at a rate of 9.75 mL/h. Radioactivity was detected throughout the experimental procedure. Two-Step Elution Experiments. A typical two step elution experiment was divided into five steps. Steps 1-3: identical to steps 1-3 in the one-step elution experiment. Step 4: fibrinogen elution by a surfactant solution at concentration cs(1) and a rate of 9.75 mL/h. Step 5: elution of the remaining adsorbed fibrinogen molecules by a second surfactant solution (the surfactant can be identical or of a different nature as the one used in step 4) at concentration cs(2) and a rate of 9.75 mL/h. Radioactivity again was detected throughout the experimental procedure.
Results and Discussion Adsorption of fibrinogen was realized with an initial protein concentration of 0.04 wt % in order to attain the plateau domain of the adsorption isotherm. The amount of fibrinogen taken up by the silica tube was 0.38 ( 0.03 µg cm-2 in our experimental conditions (I ) 0.06M). On the basis of previous results,11 the experimental curves of the dynamic elution process were approximated by a single-exponential decay function of the form
a(t) ) a exp(-kt) + b
(1)
where k represents the apparent kinetic rate constant, a is the activity of the adsorbed proteins that are elutable, and b is the activity of the irreversibly bound proteins. The sum (a + b) thus corresponds to the radioactivity measured at the beginning of the elution step and is proportional to the total amount of adsorbed fibrinogen molecules before start of the elution process. The ratio a/(a + b) represents the elutability, i.e., the fraction of removable molecules. One-Step Elution Experiments. Over the whole explored surfactant concentration ranges and for the three surfactants, C8, C10, and C14, all of the elution kinetics could satisfactorily be fitted by means of eq 1. A typical example of experimental data and the corresponding fit is given in Figure 1. The evolution of the apparent elution rate constant k (curves in parts a) and of the elutability a/(a + b) (curves in parts b) with the surfactant concentration cs are respectively given in Figures 2 (C8), 3 (C10), and 4 (C14). As for the SDS (C12) case,11 the elutability increases with cs until it reaches a plateau for concentrations slightly below the cmc’s. The elutability then becomes close to 1 (almost all of the adsorbed proteins
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Figure 1. Variation of the radioactivity a(t) of a fibrinogencoated silica tube eluted by 2 × 10-1 M C8 in PBS buffer at I ) 0.06 M. The full line corresponds to the curve fitted with the aid of eq 1.
Figure 3. Apparent rate constants k (a) and elutabilities a/(a + b) (b) as a function of C10 concentration (I ) 0.06 M). ∆: One-step experiments. 2: Two-step experiments, first C10 injection (10-3 M over 1 h) followed by a second C10 injection (5 × 10-2 M over 1 h), i.e., expt 2 in Table 1. Full line has the same meaning as in Figure 2.
Figure 2. Apparent rate constants k (a) and elutabilities a/(a + b) (b) as functions of C8 concentration (I ) 0.06 M). ∆: Onestep experiments. 2: Two-step experiments with C8 injection (3.5 × 10-2 M over 1 h) followed by a second C8 injection (2 × 10-1 M over 1 h), i.e., expt 1 in Table 1. b: Two-step experiments with C14 injection (5 × 10-6 M over 1 h) followed by a C8 injection (3 × 10-1 M over 1 h), i.e., expt 5 in Table 1. Full line has no particular meaning but has been added to guide the eyes.
can be eluted) for all three surfactants investigated here. A similar evolution is observed for the elution rate constants k with cs. Plateaus are also reached, and the values of k in the plateaus are all close to 2 × 10-3 s-1 independent of the surfactant chain length. This value is on the same order but still lower than the one found by
Zembala et al.11 in the elution plateau of fibrinogen by SDS on a similar surface. The value of k was then on the order of 4 × 10-3 s-1 at the three ionic strengths tested (I ) 0.31, 0.12, and 0.06 M).11 We thus performed control experiments of the elution of fibrinogen by SDS in the following experimental conditions: cs ) 5 × 10-3 M and I ) 0.06 M (which lies above the cmc of SDS and is thus in the elution plateau according to Zembala et al.11). The elution kinetic of fibrinogen by SDS could again be fitted by a single-exponential function and the value of k was 1.8 × 10-3 s-1, which is close to 2 × 10-3 s-1. [It thus seems that between the experiments corresponding to Zembala et al.11 and the present work (more than 1 year), the adsorption surface has slightly changed, as is often the case. This then leads to different absolute values but still on the same order of magnitude of the parameters characterizing the behavior of macromolecules at the surface.] Our present findings are in agreement with the conclusions of Rapoza and Horbett,13 who also found (i) that the reduction in the size of the surfactant’s hydrophobic moiety does not alter the maximum elutability and (ii) that the surfactant chain length moves the onset of elution in the same direction and approximately by the same amplitude as it would move the cmc of the surfactant. These results also show that micelles do not seem to play a role in the protein elution mechanism because the elution plateaus always start at concentra-
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Figure 4. Apparent rate constants k (a) and elutabilities a/(a + b) (b) as functions of C14 concentration (I ) 0.06 M). 4: Onestep experiments. 2: Two-step experiments with C14 injection (5 × 10-6 M over 1 h) followed by a second C14 injection (5 × 10-4 M over 1 h), i.e., expt 4 in Table 1. b: Two-step experiments with C8 injection (3.5 × 10-2 M over 1 h) followed by a C14 injection (5 × 10-4 M over 1 h), i.e., expt 6 in Table 1. b: Twostep experiment with C8 injection (2 × 10-2 M over 1 h) followed by a C14 injection (5 × 10-4 M over 1 h) i.e., expt 7 in Table 1. Full line has the same meaning as in Figure 2.
tions at or below the cmc’s. This could be a consequence of the minor role played by the micelles in the protein/ surfactant complex formation.16 One can also notice that surfactant molecules are known to bind proteins in two different regimes: at low surfactant concentrations, the surfactant molecules bind to the proteins at specific sites such as charged amino acid groups or hydrophobic areas, whereas for concentrations larger than a critical concentration (known as the cac), the number of molecules binding to a protein strongly increases. The surfactant molecules seem to bind in this regime in a cooperative mode likely to involve selfassociation of the surfactants.17 The cac is known, for a given type of surfactant molecules, to be independent of the protein.18 Moreover, according to Reynolds et al.,16 the values of the cac relative to the C8, C10, and C12 surfactants studied here are typically in the range of concentrations where we observe strong evolutions of both the release constant k and the elutabilities. However, even (16) Reynolds, J. A.; Callaghen, J. P.; Steinhardt. Biochemistry, 1970, 9, 1232. (17) Wahlgren, M.; Arnebrant, Th. Langmuir 1997, 13, 8. (18) Reynolds, J. A.; Tanford, Ch. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002.
Poumier et al.
Figure 5. Evolution of the apparent elution rate constants k (a) and the elutabilities a/(a + b) (b) with the hydrocarbon chain lengths in the domain above cmc where elution becomes maximum. Open symbols: one-step experiments. Full symbols: two-step experiments. 4: Experiments realized with C12 and I ) 0.12 M.
if a direct relation between both phenomena seems to be likely, we do not have any proof of it and further studies in this direction would be of great interest. Two-Step Elution Experiments. To show that the elution of fibrinogen by the different surfactants is, as for SDS, the result of the competition between two processes, one favoring the removal of the proteins and the other causing a stronger anchoring of the proteins to the surface, we performed two-step elution experiments. The surfactant concentration cs(1) of the first elution step is chosen to be smaller than the onset of the elution plateau so that during this first step, only a fraction of the adsorbed proteins are eluted. If stabilization occurs, it can thus take place during this first step. The surfactant concentration cs(2) of the second elution step was always chosen above the cmc and thus lies in the elution plateau. The values of the elution rate constants measured are thus independent of the surfactant concentration cs(2). The nature of the surfactants and the concentrations of the solutions used in these experiments are given in Table 1. Both the elution rate constants and the elutabilities (Figure 5 and Table 1) were determined. The elutabilities were always equal to 1 within experimental errors so that if an anchoring process took place during the first of the two elution steps it was not sufficient to anchor irreversibly the fibrinogen molecules to the surface. On the other hand, Figure 5 shows a systematic decrease of k (full symbols) when compared with the elution rate constants determined
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Table 1. Values of the Elution Rate Constant k for Two Step Elution Experiments in Different Elution Conditions experiment number 1 nature of first surfactant cs(1) (m) (cs(1)/cmc) nature of second surfactant cs(2) (m) (cs(2)/cmc) k (s-1)
C8 3.5 × 10-2 (2.9 × 10-1) C8 2 × 10-1 (1.7) 0.48 × 10-3
2
3
4
5
6
7
C10 10-3 (0.03)
C12 5 × 10-4 (0.25)
C10 5 × 10-2 (1.5) 0.8 × 10-3
C12 3 × 10-3 (1.5) 1.6 × 10-3
C14 5 × 10-6 (1.8 × 10-2) C14 5 × 10-4 (1.8) 1.8 × 10-3
C14 5 × 10-6 (1.8 × 10-2) C8 3 × 10-1 (2.5) 1.34 × 10-3
C8 3.5 × 10-2 (2.9 × 10-1) C14 5 × 10-4 (1.8) 0.95 × 10-3
C8 2 × 10-2 (1.7 × 10-1) C14 5 × 10-4 (1.8) 1.04 × 10-3
during the one-step elution experiments. This shows the existence of the anchoring of fibrinogen on the surface by the surfactants. More information is contained in two-elution step experiments with different surfactants in each step. These experiments should allow the precise identification of the surfactant with the highest anchoring ability. We compared the anchoring abilities of C8 and C14 in experiments 1 and 5 with those of experiments 4, 6, and 7 (Table 1). The comparison of the rate constants of the experiments 1 and 5 with those of the experiments 4, 6, and 7 reveals that during the first elution step the C8 surfactant molecules anchor fibrinogen molecules more strongly to the surface than do the C14 molecules [k(1) < k(5), k(6) < k(4), and k(7) < k(4)]. On the other hand, a comparison of the rate constants of experiments 4 and 5 with those of experiments 1 and 6, i.e., k(4) > k(5) and k(6) > k(1), shows that C14 leads to a more rapid elution than does C8 despite the facts that during the second elution step the number of surfactant molecules present in the solution is much smaller for the C14 solution than for the C8 solution and that the diffusion coefficient of the C14 molecules in water is smaller than that of C8. From our experiments, it thus seems that the chain length of the surfactant molecules interferes in the elution process via a dual mechanism of protein removal and anchoring, as follows. (i) When referring to the absolute surfactant concentration, the ability of the alkyl sulfate surfactants to elute strongly anchored fibrinogen molecules is increased with the chain length. This can, according to Arnebrant and Wahlgren,9 be interpreted by assuming that longer chains permit stronger interactions with adsorbed proteins, thus forming complexes that are
more easily desorbed from the surface. Longer hydrophobic chains can also increase the affinity of the surfactant molecules for the surface. The surfactants are then in competition with the proteins for the surface, which also favors the surfactant ability for elution. (ii) In an opposite manner, a stronger anchoring of the adsorbed proteins to the surface seems to be favored by small hydrocarbon chain lengths. If one considers that a stronger anchoring is linked with protein denaturation, it would imply that smaller molecules can more easily interfere or penetrate within the adsorbed protein layer and thus more easily induce protein denaturation. Unfortunately, to the authors’ knowledge, no systematic study of the chain length of alkyl sulfate surfactants on the denaturation of proteins has been reported. The present study revealed that all sodium alkyl sulfate surfactants possess a double competing ability for elution and protein anchoring that varies with the alkyl chain length. We have, however, not clearly established the correlation between the induced anchoring of adsorbed proteins by surfactants and the induced denaturation. This will constitute one of the next goals and will be accomplicated by using infrared spectroscopy in an ATR mode. Such an approach should then give more insight into the processes observed at a molecular level in the present work. Acknowledgment. The authors are grateful to Mrs. Marzena Novoryta for performing surface tension measurements and to Mrs. Maria Zembala for fruitful discussions. LA990227B
