The Interaction of Bovine Serum Albumin with Surfactants Studied by

The interaction between bovine serum albumin (BSA) and several surfactants has been investigated by light scattering. Anionic (sodium dodecyl sulfate,...
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Langmuir 2000, 16, 922-927

The Interaction of Bovine Serum Albumin with Surfactants Studied by Light Scattering Ank Valstar,* Mats Almgren, and Wyn Brown Department of Physical Chemistry, Box 532, University of Uppsala, 751 21 Uppsala, Sweden

Marilena Vasilescu Romanian Academy, I. G. Murgulescu Institute of Physical Chemistry, Splaiul Independentei 202, 77208 Bucharest, Romania Received April 12, 1999. In Final Form: August 16, 1999 The interaction between bovine serum albumin (BSA) and several surfactants has been investigated by light scattering. Anionic (sodium dodecyl sulfate, SDS), cationic (dodecyl trimethylammonium bromide, DTAB), and nonionic (polyoxyethylene 8 lauryl ether, C12E8) surfactants, all containing a C12 alkyl chain, were used to study the effect of different headgroups on the complex formation. The hydrodynamic radii of the complexes obtained by dynamic light scattering indicate that cooperative binding of DTAB occurs at higher surfactant concentrations than in comparative solutions of SDS and C12E8. The effect of chain length is shown for the cationic surfactants DTAB and cetyl trimethylammonium bromide (CTAB, C16 alkyl chain). The higher surface activity of CTAB results in complex formation at a lower surfactant concentration compared to DTAB. The hydrodynamic radii of the BSA-SDS and BSA-DTAB complexes at saturation were determined as ∼5.9 nm and ∼4.8 nm, respectively. The hydrodynamic radius of the reduced BSA-SDS complex is somewhat smaller than the corresponding native BSA-SDS complex. Static light scattering (SLS) measurements were performed on BSA-SDS systems to determine the number of BSA molecules in the complex. Prior to SLS measurements the BSA-SDS solutions were dialyzed against a large volume of SDS solution in order to determine the refractive index increment ∂n/∂cBSA at constant chemical potential. It was observed that a very long dialysis time (several weeks) was needed to reach equilibrium. Measurements on solutions that had not reached equilibrium resulted in improbably high values of the number of BSA molecules in the complex.

Introduction Protein-surfactant interactions have been much studied.1 In these studies the globular protein bovine serum albumin (BSA) has often been used. BSA, which functions biologically as a carrier for fatty acid anions and other simple amphiphiles in the bloodstream, has a molecular weight M0 of 66 411 gmol-1 (calculated from the amino acid composition) and consists of 583 amino acids in a single polypeptide chain.2 The protein contains 17 disulfide bridges and one free SH group, which can cause it to form covalently linked dimers.3 The isoelectric point in 0.15 M NaCl is about 4.7; bound chloride ions cause it to be lower than the isoionic point (about pH 5.2).2 At the isoionic point at which essentially all of the carboxylic acids are deprotonated and the amino, guanidino, and imidazole groups are protonated, the total charge consists of about 100 each positive and negative charges.2 Guo et al.4 summarized the different models that have been proposed to describe the protein-SDS complex. Of these, the necklace model5 seems to have the strongest support. It was based originally on results from free * To whom correspondence should be addressed. E-mail: [email protected]. (1) Ananthapadmanabhan, K. P. In Interactions of Surfactants with Polymers and Proteins, Goddard, E. D., Ananthapadmanbhan, K. P., Eds.; CRC Press Inc.: London, 1993; pp 319-365. (2) Peters, T. J. All about Albumin Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (3) Foster, J. F. In Albumin Structure, Function and Uses; Rosenoer, V. M., Oratz, M., Rothschild, M. A., Eds.; Pergamon Press Inc.: Oxford, U.K., 1977; pp 53-84. (4) Guo, X.-H.; Chen, S.-H. Chem. Phys. 1990, 49, 129-139. (5) Shirahama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309319.

boundary electrophoresis and it proposed that an unfolded protein binds SDS in the form of micelle-like clusters. Results from SANS,6,7 viscometry,8 and NMR9 also agree with this model. Recently, Turro et al.10 in a study combining fluorescence, ESR, and NMR and using the necklace model, concluded that the unfolded protein wraps around the micelles. The necklace model is similar to the structures reported for complexes formed between surfactants with polymers11 and polyelectrolytes.12 Also we concluded, from a recent fluorescence study,13 that the BSA-SDS complex can be described by the necklace model, where BSA wraps around 3-4 micellelike SDS clusters at saturation. It was also found that the number of SDS molecules bound to BSA did not depend on the presence of the disulfide bridges. The present work complements this earlier study. Dynamic light scattering was performed to estimate the hydrodynamic radii of the complexes. The interaction of SDS with both native and reduced BSA was studied. As in the fluorescence study, the effect of different surfactant headgroups on complex formation was investigated. Three surfactants, all with (6) Guo, X. H.; Zhao, N. M.; Chen, S. H.; Teixeira, J. Biopolymers 1990, 29, 335-346. (7) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311-318. (8) Shinagawa, S.; Kameyama, K.; Takagi, T. BBA 1993, 1161, 7984. (9) Oakes, J. J. Chem. Soc., Faraday Trans. 1 1974, 70, 2200-2209. (10) Turro, N. J.; Lei, X.-G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525-2533. (11) Cabane, B.; Duplessix, R. J. Phys. 1982, 43, 1529-1542. (12) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 1668416693. (13) Vasilescu, M.; Angelescu, D.; Almgren, M.; Valstar, A. Langmuir 1999, 15, 2635-2643.

10.1021/la990423i CCC: $19.00 © 2000 American Chemical Society Published on Web 11/27/1999

Light Scattering of BSA/Surfactants

a C12 alkyl chain but different headgroups (i.e., SDS: negatively charged, DTAB: positively charged, and C12E8: noncharged), were used. The effect of the alkyl chain length on complex formation was studied for the positively charged surfactants. Static light scattering measurements were also made on several BSA-SDS systems, to determine the number of BSA molecules in the complex. Earlier studies by the authors14 and others,15,16 indicated (presumably wrongly) that protein-SDS complexes may comprise several protein molecules. We observed different values depending on the dialysis time. A more comprehensive study concerning the dialysis equilibrium is underway. Experimental Section Materials. BSA (Albumin fraction V, 112018) and CTAB were purchased from Merck. DTAB and C12E8 were obtained from Sigma. SDS (especially pure) was supplied by BDH. All other materials used were of analytical grade. Sample Preparation. The CH3COOH/CH3COONa buffer pH 5.6 (ionic strength 0.1 M) was prepared as described by Dawson et al.17 NaN3 (200 ppm) was added to avoid bacterial growth. The ionic strength of the buffer was adjusted to 0.2 M by adding NaCl. This buffer was used in the preparation of all solutions. The BSA monomer was separated using gel chromatography (Sephadex G-100, Pharmacia Biotech). To avoid dimerization, the free sulfhydryl group of the monomer was modified (carboxymethylated) by reaction with iodoacetic acid.18 Carboxymethylation was performed with an excess amount of iodoacetic acid for 2 h at room temperature. BSA concentrations were determined by UV-absorption measurements at 280 nm using the molar extinction coefficient 4.36 × 104 M-1 cm-1.19 An excess of β-mercaptoethanol (1%) was used to reduce monomeric BSA (10-5 M). The solutions were filtered into the light scattering cells through Anotop filters with pore size 0.1 µm; filters with a larger pore size (0.2 µm and 0.45 µm) were used for BSA-SDS solutions in the presence of β-mercaptoethanol at low SDS concentration. The critical micelle concentration (cmc) was determined by surface tension measurements (drop-weight technique). Dialysis. Samples were dialyzed prior to static light scattering experiments. BSA dissolved in buffer solution (8-10 mL) was dialyzed against a large volume of the buffer solution (i.e., 1 L). Dialysis was performed for 24 h at room temperature. Spectra Por cellulose ester membranes with a cutoff mass of 5000 g mol-1 were used. The BSA-SDS-buffer systems were treated in a similar way. The BSA-SDS buffer solution (8-10 mL) was dialyzed against the SDS-buffer solution (1 L) under the same conditions as above. The SDS concentration was well above the cmc, i.e., [SDS] ) 31.2 × 10-3 M, the BSA concentration was 1.82 × 10-4 M. The dialysis times varied from 1 day to several weeks. Dialysis was performed in order to determine the refractive index increment, ∂n/∂cBSA, at constant chemical potential of the added electrolytes. After dialysis the multicomponent BSA-SDS-buffer system can be looked upon as a two component system (i.e., the SDS-buffer solution is regarded as the solvent for BSA), and the molecular weight of BSA in the complex can be determined.20 Dilutions were made using the dialysate. Static and Dynamic Light Scattering Measurements. Static and dynamic light scattering measurements were performed using a frequency-stabilized Coherent Innova Ar ion laser (14) Valstar, A.; Brown, W.; Almgren, M. Langmuir 1999, 15, 23662374. (15) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1736-1744. (16) Gimel, J. C.; Brown, W. J. Chem. Phys. 1996, 104, 8112-8117. (17) Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. Data for Biochemical Research; Clarendon Press: Oxford, U.K., 1969. (18) Creighton, T. E. Proteins Structures and Molecular Properties, 2nd ed.; W. H. Freeman and Company: New York, 1993. (19) Wetlaufer, D. B. Adv. Prot. Chem. 1962, 17, 378. (20) Eisenberg, H. In Photon Correlation and Light Beating Spectroscopy; Cummins, H. Z., Pike, E. R., Eds.; Plenum Press: New York, 1974, pp 551-567.

Langmuir, Vol. 16, No. 3, 2000 923 emitting vertically polarized light at 488 nm. Vertically polarized light was collected and the signal analyzer was an ALV-5000 digital multiple-τ correlator (Langen GmbH) with 288 exponentially spaced channels. Measurements were performed at 25 ( 0.02 °C. Toluene was used as a reference in static measurements with a value of 3.1 × 10-5 cm-1 for the Rayleigh ratio Rtol.21 Below follows a brief discussion on how to determine various physical parameters from light scattering experiments. More information is to be found in several textbooks.22,23 In dynamic light scattering (DLS), the intensity-intensity autocorrelation function G2(t) is measured and is related to the normalized electric field autocorrelation g1(t), by the Siegert relation. The parameter g1(t) can be written as the Laplace transform of the distribution of the relaxation rate Γ (Γ ) τ-1, where τ is the relaxation time). The relaxation rate is related to the mutual diffusion coefficient Dm

Dm ) Γ/q2

(q f 0)

(1)

where q ) 4πns/λ0sin(θ/2), with ns the refractive index of the solution, λ0 the wavelength of the radiation in a vacuum, and θ the scattering angle. The diffusion coefficient at infinite dilution, D0, may be expressed

Dm(c) ) D0(1 + kdc)

(c f 0)

(2)

where c is the particle concentration and kd a constant. D0 is related to the hydrodynamic radius, Rh, through the StokesEinstein relation

D0 )

kbT 6πηRh

(3)

where kb is the Boltzmann constant, T the absolute temperature, and η the viscosity of the solvent. The DLS measurements were performed on nondialyzed samples. Most DLS measurements were performed at low BSA concentration (i.e., 10-5 M). At this low concentration kdc,1 (i.e., noninteracting particles) and D0 ≈ Dm. According to their q2 dependence, the particles represent diffusional species. Because the hydrodynamic radius was shown not to depend on scattering angle, most measurements were performed at one angle (i.e., θ ) 90°). Static light scattering (SLS) measurements were performed on dialyzed samples (see above). The BSA-SDS-buffer solution was considered as a binary system (i.e., the SDS-buffer solution is regarded as the solvent for BSA). The refractive index increment ∂n/∂cBSA and the excess scattered intensity were measured relative to the SDS-buffer solution. The SLS data were analyzed using the following Zimm equation as established in an earlier work16

( )

K′

∂n 2 c ∂cBSA BSA 2A2c 1 ) + 2 cBSA Rθ MBSA R

(cBSA f 0, θ f 0)

(4)

where K′ ) 4π2ns2/Naλ04, Na is Avogadro’s number, Rθ ) Rtol (Is - Isol/Itol) sin θ; Is, Isol, and Itol are the intensities scattered by the solution, the solvent, and toluene, respectively. A2c is the second virial coefficient of the complex, MBSA is the weight averaged molar mass of BSA in the complex, R ) (∂n/∂cc)/(∂n/∂cBSA), and cc and cBSA are the concentration of complex and the concentration of BSA, respectively. As can be seen from eq 4, SLS provides information on the molecular weight of the protein in the proteinSDS complex (i.e., the number of protein molecules in the complex can be calculated). Measurements were performed at one angle (θ ) 90°), since there is no angular dependence of the scattered intensity due to the small size of the particles. (21) Moreels, E.; Ceuninck, W. D.; Finsy, R. J. Chem. Phys. 1987, 86, 618-623. (22) Kratochvil, P. Classical Light Scattering from Polymer Solutions; Elsevier: Amsterdam, 1987. (23) Berne, B. J.; Pecora, R. Dynamic Light Scattering; John Wiley & Sons: New York, 1976.

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Figure 2. Static light scattering data for the BSA-SDS buffer system with varying dialysis times. [SDS] ) 31.2 × 10-3 M () 9 gL-1).

Figure 1. (a) Kc/Rθ as a function of the BSA concentration, for monomeric BSA and BSA as supplied by the manufacturer, respectively. (b) The mutual translational diffusion coefficient as a function of the BSA concentration, symbols as in (a).

Results and Discussion Static Light Scattering; Determination of the Number of BSA Molecules in the BSA-SDS Complex. The BSA monomer was separated using gel chromatography and its free SH group was carboxymethylated. The static light scattering data for BSA as supplied by the manufacturer and monomeric BSA are compared in Figure 1a. The molecular weight Mw obtained for the former, (8.9 ( 0.3) × 104 gmol-1, corresponds to an association number N ) Mw/M0 of 1.34; Matsumoto et al.24 reported a value of 1.75 (for BSA from Sigma, A0208). The molecular weight of the monomer was obtained as (7.1 ( 0.2) × 104 gmol-1, corresponding to N ) 1.07. The corresponding dynamic

light scattering data are shown in Figure 1b. The hydrodynamic radius of monomeric BSA was determined to be 3.37 ( 0.03 nm, which is similar to literature values.25,26 Rh determined for BSA as supplied by the manufacturer was 3.87 ( 0.04 nm. As described in the Experimental Section the number of BSA molecules in the complex can be determined. SLS measurements were performed on a BSA-SDS-buffer system containing 31.2 × 10-3 M SDS (well above the cmc ) 7.2 × 10-4 M).27 When the BSA-SDS solution was routinely dialyzed for 24 h, the molecular weight of BSA in the complex was obtained as (2.1 ( 0.1) × 105 gmol-1 (Figure 2), which corresponds to ∼3.1 BSA molecules in the complex. This value seemed overly high. Since we had made sure that the BSA was monomeric from the start (Figure 1), it seemed impossible that the highly negatively charged BSA-SDS complex should form trimers. Furthermore, the result can be compared to results obtained from SDS-polyacrylamide gel electrophoresis (SDSPAGE), a technique routinely used in biochemistry to estimate the molecular weight of proteins. Chromatographically prepared monomeric BSA resulted in a single band corresponding to the monomer; dimers or higher oligomers were not observed.3 However, increasing the dialysis time resulted in lower molecular weights (Figure 2): after one week of dialysis Mw ) (1.24 ( 0.05) × 104 gmol-1 (i.e., 1.9 BSA molecules per complex) was obtained, and after seven weeks Mw ) (8.8 ( 0.4) × 104 gmol-1 (i.e., 1.3 BSA molecules per complex). From the above it may be concluded that dialysis equilibrium had not been established when the measurements were performed, probably not even after seven weeks. The decrease in Mw was due to an increase in the value of the refractive index increment ∂n/∂cBSA (Figure (24) Matsumoto, T.; Inoue, H. Chemical Physics 1993, 178, 591598. (25) Takeda, K.; Sasaoka, H.; Sasa, K.; Hirai, H.; Hachiya, K.; Moriyama, Y. J. Colloid Interface Sci. 1992, 154, 385-392. (26) Oh, Y. S.; Johnson, J. C. S. J. Chem. Phys. 1981, 74, 2717-2720. (27) Jones, M. N.; Manley, P. In Surfactants in Solution, Vol. 2; Mittal, K. L., Lindman, B., Eds.; Plenum: London, 1984; pp 1403-1415.

Light Scattering of BSA/Surfactants

Figure 3. The refractive index increment ∂n/∂cBSA for the BSASDS buffer system with varying dialysis times (∂n/∂cBSA ) 0.2241 ( 0.0006 gmL-1, 0.264 ( 0.001 gmL-1 and 0.305 ( 0.003 gmL-1 for 1 day, 1 week and 7 weeks, respectively). [SDS] ) 31.2 × 10-3 M.

Figure 4. Rθ values for the BSA-SDS buffer system with varying dialysis times. [SDS] ) 31.2 × 10-3 M.

3). The refractive index increment ∂n/∂cBSA should be determined at dialysis equilibrium i.e., at constant chemical potential of the components diffusing through the dialysis membrane (i.e., all components except BSA). Apparently, equilibrium had not been achieved since ∂n/ ∂cBSA still varied with time. On the other hand, no significant change in Rθ was observed (i.e., no change in the scattered light intensity) (Figure 4). The results indicate that the molecular weight of BSA in the complex can be determined but that equilibrium

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Figure 5. Relaxation time distributions for a series of measurements at constant BSA concentration (10-5 M) and varying SDS concentration (from 0 M in the upper left corner to 20 × 10-3 M in the lower right corner, see also Figure 6). The intensity of the scattered light ranged from 40 to 150 kHz.

times are very long. The reason for the long equilibrium time is the slow diffusion of surfactant; the micelles cannot pass the membrane. Since the difference in the molecular surfactant concentration between the outer and inner solution is small (cmc ) 7.2 × 10-4 M),27 even for appreciable differences in micellar concentration, the equilibration will be slow. Determinations of the molecular weight of proteins in protein-SDS complexes as described above have been performed earlier by the authors14 and others.15,16 Dialysis times varied from 1 day14 to several days.15,16 In the latter report,15 experiments were described where lysozyme solutions were dialyzed for 4 days against 30 mM SDS solutions. The number of lysozyme molecules in the complex was determined as 3.0. Again, this number seems high, even if one takes into account the fact that lysozyme, to a certain extent, is subject to dimerization at the pH used (pH 6). Reference 14 describes experiments performed on lysozyme-SDS systems at low pH where lysozyme is predominantly in its monomeric form. Even in this case, where extra SDS was added to the inner solution to accelerate the dialysis equilibrium, the number of protein molecules in the complex deviated from 1. A more complete study concerning the effect of the dialysis conditions is being undertaken in our laboratory. Dynamic Light Scattering for Several BSA-Surfactant Systems. Figure 5 shows relaxation time distributions from a series of measurements at constant BSA concentration (10-5 M) and varying SDS concentration. At low SDS concentrations the distributions show one peak, which represents the relaxation of the BSA-SDS complex. At higher SDS concentrations a second mode at a shorter relaxation time becomes visible with an apparent Rh of 2.00 ( 0.03 nm, corresponding to the relaxation of the free SDS micelles. Figure 6 shows the corresponding hydrodynamic radii Rh of different BSA-surfactant complexes plotted as a function of the total surfactant concentration. The total concentration includes both the bound and unbound surfactant. The surfactants all contain a C12 alkyl chain, but differ in their headgroups: negatively charged (SDS), positively charged (DTAB), and noncharged (C12E8).

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Figure 6. Hydrodynamic radii for the BSA-SDS, BSA-DTAB, and BSA-C12E8 complexes as a function of the total surfactant concentration. The inset shows Rh for the BSA-C12E8 complex at low surfactant concentration. At high C12E8 concentrations the observed Rh corresponds to the free C12E8 micelles, since the free micelles dominate the relaxation distribution (see text for details). For each concentration at least three values of Rh were averaged and the error bar represents the standard deviation.

The hydrodynamic radius Rh of the BSA-SDS complex increases from 3.39 ( 0.02 nm at [SDS] ) 0 to 5.87 ( 0.02 nm at [SDS] ) 7 × 10-3 M and then levels off. The curve in Figure 6 may be compared to the binding isotherm. A binding isotherm shows the average number of surfactant molecules bound per protein molecule as a function of the free surfactant concentration. In general,28 the binding isotherms display four characteristic regions (with increasing surfactant concentration): (I) specific binding, (II) noncooperative binding, (III) cooperative binding, and (IV) saturation. Oakes9 determined the binding isotherm of SDS to BSA (1.5 × 10-4 M BSA, pH ) 5.6, 0.033 M ionic strength). This binding isotherm displays region II (total [SDS] between 7.5 × 10-4 and 7.8 × 10-4 M) and region III, where the average number of SDS molecules bound per BSA molecule is determined to be ∼180 at the highest total SDS concentration used (2.8 × 10-3 M). Figure 6 on the other hand clearly shows regions III (ranging from ∼2.5 × 10-4 to ∼5 × 10-3 M) and IV. The saturation binding for anionic surfactants has been found to be pHindependent1 and the hydrodynamic radius of the saturated BSA-SDS complex (∼5.9 nm) is comparable to that found by Takeda et al.25 (i.e., 6.0 nm at pH 7.0; ionic strength 0.014). As for the BSA-SDS complex, the cooperative binding region (III) and saturation (IV) are also clearly seen for the DTAB-BSA complex. Consistent with earlier observations,1 the cooperative binding of DTAB occurs at a higher concentration than that of SDS. Saturation is reached at about the same total surfactant concentration, i.e., around 7 × 10-3 M. However, the hydrodynamic radius of the DTAB-BSA complex at saturation is smaller: ∼4.8 nm compared to ∼5.9 nm for the BSA-SDS complex. The saturation binding for cationic surfactants is pH-dependent,1 and Takeda et al.25 found a value of 5.2 nm for the DTAB-BSA complex at saturation at pH 7.0, ionic strength 0.014. (28) Jones, M. N. Biochem. J. 1975, 151, 109-114.

Valstar et al.

Figure 7. Hydrodynamic radii for the BSA-DTAB and BSACTAB complexes as function of the total surfactant concentration. At high CTAB concentrations the observed Rh corresponds to the free CTAB micelles, since the free micelles dominate the relaxation distribution (see text for details).

At low C12E8 concentrations a small increase of the hydrodynamic radius (i.e., Rh ) 3.79 ( 0.02 nm at 0.50 × 10-3 M) is observed (Figure 6). This inital increase is similar to that observed for the BSA-SDS system. However, the increase is followed by a decrease. Difficulties exist in the determination of the hydrodynamic radius of the BSA-C12E8 complex. Since the cmc of C12E8 is small (7.1 × 10-5 M in water)29 micelles are already formed at low concentration. Unfortunately, the distribution shows only one peak which represents both the BSA-C12E8 complex and the C12E8 micelles. Since the relaxation times of these two species are closely similar, it is impossible to resolve them. The relaxation time distribution starts to be dominated by the free micelles at a C12E8 concentration of ∼5 × 10-3 M and at high C12E8 concentrations the observed hydrodynamic radius corresponds to the free micelles (i.e., Rh ) 3.1 nm, as was verified by measurements on C12E8 buffer solutions; result not shown). Figure 7 shows the effect of different chain lengths for the positively charged surfactants DTAB (C12) and CTAB (C16). As for DTAB, a cooperative binding region is also observed for CTAB. However, since CTAB is more hydrophobic, the complex formation starts at a much lower surfactant concentration. Again, difficulties arise when determining the Rh of the complex. Due to the rather low cmc of CTAB (4.5 × 10-4 M) compared to DTAB (6.5 × 10-3 M), micelles are formed already at low surfactant concentration. At a CTAB concentration of ∼3 × 10-3 M, the relaxation time distribution starts to be dominated by the diffusion of the free CTAB micelles, making it impossible to determine the hydrodynamic radius of the BSA-CTAB complex. The fluorescence study13 (Figure 5d) revealed that the dependence of the pyrene fluorescence lifetime on the surfactant concentration is similar for the BSA-SDS, BSA-CTAB and BSA-C12E8 systems. It was found that the massive cooperative binding starts at approximately the same total surfactant concentration. This critical concentration depends on the BSA concentration; it was (29) Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain: Where Physics, Chemistry, Biology, and Technology Meet; VCH Publishers: New York, 1994.

Light Scattering of BSA/Surfactants

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Figure 8. Relaxation time distributions for a series of measurements at constant BSA concentration (10-5 M) and varying SDS concentration (from 0 M in the upper left corner to 20 × 10-3 M in the lower right corner, see also Figure 9), in the presence of β-mercaptoethanol.

Figure 9. Hydrodynamic radii for the BSA-SDS complex in the abscence (open circles) and presence (closed circles) of the reducing agent β-mercaptoethanol.

found to be ∼3 × 10-4 M for [BSA] ) 1.5 × 10-5 M (ref 13, Figure 5c). The light scattering data ([BSA] ) 10-5 M) revealed that the hydrodynamic radii of the BSA-SDS and BSA-C12E8 complexes start to increase in the surfactant concentration range of 2.5 × 10-4 to 5.0 × 10-4 M, which is in agreement with the concentration of the onset of massive cooperative binding as determined in the fluorescence experiments. However, as described earlier, the massive cooperative binding of DTAB occurs at a higher surfactant concentration. This is what might be expected according to the relatively high cmc of DTAB (i.e., 6.5 × 10-3 M): the higher the tendency of the surfactant to form micelles, the stronger is its affinity to bind to proteins.1 Reducing BSA (in the absence of SDS) causes aggregation (Figure 8). These aggregates, with a hydrodynamic radius up to around 140 nm, dissolve on adding SDS and become similar in size (slightly smaller) to the native BSASDS complex at an SDS concentration of about 2 × 10-3 M. The complex formation of SDS and BSA in its native form and in its reduced form are compared in Figure 9, which shows that the hydrodynamic radius of the reduced BSA-SDS complex is somewhat smaller. It is known that the number of SDS molecules bound to BSA does not depend on the presence of the disulfide bridges.13 This indicates that the presence of the disulfide bridges causes a slightly larger complex with a somewhat more open structure. Similar results have been observed for the lysozyme-SDS system.14 Tanner et al.,30 in a dynamic light scattering study, determined the hydrodynamic radius of the BSA-SDS complex in the presence of β-mercaptoethanol to be 7.2 nm (2% BSA (3.0 × 10-4 M), pH 7.2 and ionic strength 0.020). They also concluded from their data that BSA saturates above an SDS/BSA weight ratio 2.8/1, where the total concentration (bound and unbound) of SDS was used. From Figure 9 a saturation level of 3.3 can be calculated (BSA/SDS ) 10-5 M/7 × 10-3 M ) 0.66 gL-1/2.0 gL-1 ) 3.3).

The interaction between bovine serum albumin (BSA) and several surfactants has been investigated by light scattering. SDS, DTAB, and C12E8, all containing a C12 alkyl chain, were used to study the effect of different headgroups on complex formation. The hydrodynamic radii of the complexes obtained by dynamic light scattering indicate that the cooperative binding of DTAB occurs at a higher surfactant concentration than that of SDS and C12E8. The effect of different chain length on the complex formation is shown for the cationic surfactants DTAB and CTAB. The higher surface activity of CTAB results in complex formation at a lower surfactant concentration compared to DTAB. The hydrodynamic radii of the BSASDS and BSA-DTAB complexes at saturation were determined to be ∼5.9 nm and ∼4.8 nm, respectively. The hydrodynamic radius of the reduced BSA-SDS complex is somewhat smaller than the corresponding native BSASDS complex. The low cmc values for CTAB and C12E8 made it impossible to determine the hydrodynamic radii of the BSA-CTAB and BSA-C12E8 complexes; the free micelles and the complex are of similar size and the relaxation times cannot be resolved. Other techniques are needed in this case: e.g., NMR. Static light scattering (SLS) measurements were performed on BSA-SDS systems to determine the number of BSA molecules in the complex. Prior to SLS measurements the BSA-SDS solutions were dialyzed against a large volume of the SDS solution. Dialysis was performed to be able to determine the refractive index increment ∂n/∂cBSA at constant chemical potential. We observed that the dialysis time needed to achieve equilibrium was long (several weeks). Solutions that had not achieved equilibrium resulted in improbably high values of the number of BSA molecules in the complex. Longer dialysis times resulted in a number of BSA molecule per complex close to unity.

(30) Tanner, R. E.; Herpigny, B.; Chen, S.-H.; Rha, C. K. J. Chem. Phys. 1982, 76, 3866-3872.

Conclusions

Acknowledgment. We thank the Swedish Technical Research Council (TFR) for financial support. LA990423I