Langmuir 1999, 15, 2635-2643
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Interactions of Globular Proteins with Surfactants Studied with Fluorescence Probe Methods Marilena Vasilescu* and Daniel Angelescu Romanian Academy, “I. G. Murgulescu” Institute of Physical Chemistry, Splaiul Independentei 202, 77208 Bucharest, Romania
Mats Almgren and Ank Valstar Physical Chemistry Department, Uppsala University, P.O. 532, S-751 21 Uppsala, Sweden Received October 12, 1998. In Final Form: January 25, 1999 Steady-state and dynamic fluorescence measurements were used to investigate the interactions and structures of complexes formed between bovine serum albumin (BSA) and anionic, cationic, and nonionic surfactants: sodium dodecyl sulfate (SDS), N-cetyl-N,N,N-trimethylammonium bromide (CTAB), and octaoxyethylene glycol n-dodecyl ether (C12E8), respectively. The lysozyme (Lys)-SDS complex was also studied. The measurements were carried out at different pH’s and ionic strengths. In all systems, micellelike clusters adsorbed on protein were evidenced. The average aggregate numbers are smaller than those of free micelles and are not strongly influenced by pH and ionic strength. The fluorescence lifetime of pyrene in BSA/surfactant complexes was constant at low surfactant concentrations, started to decrease at approximately the same protein-surfactant ratio (0.15 mM BSA/1 mM surfactant) regardless of the surfactant type or pH buffer, and, at high surfactant concentrations, merged to the lifetime values corresponding to free micelles. The results of the fluorescence techniques support a “necklace and bead” model of the complex for BSA/surfactant systems, with protein wrapped around the micelles. For the Lys/SDS complex, the model essentially is the same; however, some differences, due to their different sizes, appear. Lysozyme is smaller and more rigid and does not wrap up well around the micelle.
Introduction Interactions between surfactants and globular proteins have been extensively studied1-21 for various reasons: there are technical applications for drug delivery, cosmetics, and detergency; the interactions are important in several biochemical separation methods; the interactions with fatty acids and their salts are very important for a (1) Goddard, E. D. Interactions of Surfactant with Polymers and proteins. Protein-surfactant Interactions; Ananthapadmanabhan, K. P., Ed.; CRC Press: Boca Raton, New York, 1993; Chapter 8. (2) Reynolds, J. A.; Tanford, C. Proc. Natl. Acad. Sci. 1970, 66, 1002. (3) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley-Interscience: New York, 1980; Chapter 14. (4) Jones, M. N. Biochem. J. 1975, 151, 109. Jones, M. N. Chem. Soc. Rev. 1992, 88, 1003. (5) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161. (6) Rao, P. F.; Yakagi, T. J. Biochemistry 1989, 106, 365. (7) Laurie, O.; Oakes, J. J. Chem. Soc., Faraday Trans. 1 1975, 71, 1324. (8) Oakes, J. J. Chem. Soc., Faraday Trans. 1 1974, 70, 2200. (9) Smith, M. L.; Muller, W. Biochem. Biophys. Res. Commun. 1975, 62 (3), 723. (10) Tsuji, K.; Takagi, T. J. Biochem. 1975, 77, 511. (11) Turro, N. J.; Lei, X.-G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525. (12) Shirama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309. (13) Moren, A. K.; Khan, A. Langmuir 1995, 11, 3636. (14) Greener, J.; Contestable, B. A.; Bale, M. D. Macromolecules 1987, 20, 2490. (15) Chen, S. H.; Teixeira, J. Phys. Rev. Lett. 1986, 57, 2583. (16) Guo, X. H.; Chen, S. H. Chem. Phys. 1990, 149, 129. (17) Guo, X. H.; Zhao, N. M.; Chen, S. H.; Teixeira, J. Biopolymers 1990, 29, 335. (18) Tanner, R. E.; Herpigny, B.; Chen, S. H.; Rha, C. K. J. Chem. Phys. 1982, 76, 3866. (19) Shinagawa, S.; Kameyama, K.; Takagi, T. Biochim. Biophys. Acta 1993, 1161, 79. (20) Shinagawa, S.; Sato, M.; Kameyama, K.; Takagi, T. Langmuir 1994, 10, 1690. (21) Wright, A. K.; Thompson, M. R.; Miller, R. L. Biochemistry 1975, 14, 3224.
number of enzymes, etc. It is generally accepted that binding of ionic surfactant molecules to proteins disrupts the native structure of most globular proteins.1,3 For general aspects of the interactions between ionic surfactants and water-soluble proteins, sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) are often used as an archetype system;8,11,16,17,19,20 if this is a wise choice may be questioned, but they are nevertheless systems that are repeatedly studied. Serum albumins are transport proteins, with the capability to bind a variety of small molecules: fatty acids, amino acids, steroids, and calcium ions, as well as numerous pharmaceuticals. Bovine serum albumin, with a molecular mass of 66 200 Da, consists of 581 amino acids in a single polypeptide chain. The structure of the closely related human seric albumin (HSA) has been determined to a resolution of 2.8 Å.22 Helped by 17 disulfide bridges, the protein is folded into a shape that can be roughly described as an equilateral triangle with sides of about 80 Å and a thickness of about 30 Å. At neutral pH, it has a net negative charge (pI ) 5.2) and is known to undergo conformational changes at both low and high pH’s.23 Lysozyme (Lys) is another globular protein, that has been used for investigating the interaction with ionic surfactants.24-36 Compared with BSA, Lys has a compact (22) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (23) Vijai, K.; Forster, J. Biochemistry 1967, 6, 1152. (24) Mattice, W. L.; Riser, J. R.; Clark, D. Biochemistry 1976, 15, 4264. (25) Jones, M. N.; Manley, Ph. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1737. (26) Jones, M. N.; Manley, Ph. J. Chem. Soc., Faraday Trans. 1 1980, 76, 654. (27) Fukushima, K.; Murata, Y.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1982, 55, 1376. (28) Imoto, T.; Sumi, S.; Tsuru, M.; Yagishita, K. Agric. Biol. Chem. 1979, 43, 1809.
10.1021/la981424y CCC: $18.00 © 1999 American Chemical Society Published on Web 03/25/1999
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and rigid structure and is more stable at low pH values. Lys has a molecular mass (14 350 Da) much smaller than that of BSA and consists of 129 amino acids. Its globular shape and stability have been attributed to the four disulfide bridges, hydrogen bonds, and hydrophobic interactions among the amino acids. Its isoelectric point is 11.35. It is interesting to compare the mechanism of interaction between SDS and these two different proteins. Binding studies have shown that already at a free SDS concentration as low as 0.01 mM, six or seven SDS molecules are bound per BSA.1-3,11 This strong binding is reminiscent of the binding of fatty acids, where binding constants in the range of 107-106 M-1 have been determined for three specific fatty acid binding sites; five further sites with binding constants ranging from 105 to 104 M-1 were also detected.37 With increasing SDS concentration, a general noncooperative binding sets in, presumably on hydrophobic regions of the protein. At a free concentration still far below the cmc, the cooperative binding phase starts, and the number of bound SDS molecules increases from about 20 at 0.3 mM to about 160 at 1 mM free SDS; binding of about twice that amount is reported to give saturation. The cooperative binding is supposed to be accompanied by an unfolding of the protein. Even though the disulfide bridges prevent the peptide chain from unfolding completely, an examination of the three-dimensional structure convincingly reveals that a very substantial unfolding can occur. Large amounts of adsorbed ionic surfactants can be expected both to break the intrachain hydrophobic bonding and provide an electrostatic repulsion favoring an extended structure. In the case of Lys, the shape of the binding isotherm of SDS25,26 is similar to that of BSA/SDS, but the number of bound SDS molecules is smaller and increases from about 4 at 0.25 mM to about 18 at 1 mM free SDS. At 3.16 mM, a cooperative binding starts (for 1.25 mM Lys at pH ) 3.2). The saturation of the first 18 sites, represented by cationic amino acid residues, with electrostatic and hydrophobic binding, leads to precipitation. Higher SDS concentrations redissolve the protein-surfactant complex. As to the structure of the protein-SDS complex, there are several propositions.1 Turro et al.11 recently distinguished between three principally different models and concluded that their results from fluorescence and NMR studies favored a “necklace and bead” model composed of small SDS micelles with uncoiled BSA wrapped around on the outside. This model, which is also supported by SANS results, suggests that the surfactant aggregation occurs in the same way as with other polymers and polyelectrolytes, which appears as the natural first assumption. (29) Jones, M. N.; Manley, P.; Midgley, P. J. W. J. Colloid Sci. 1981, 82, 257. (30) Jones, M. N.; Midgley, P. J. W. Biochem. J. 1984, 219, 875. (31) Jones, M. N.; Brass, A. Interaction between Small Amphipathic Molecules and Proteins. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1991; pp 65-80. (32) Fukushima, K.; Murata, Y.; Nishikido, N. Bull. Chem. Soc. Jpn. 1981, 54, 3122. (33) Murata, Y.; Okawaucki, M.; Kawamura, H.; Sugihara, G.; Tanaka, M. Interaction between ionic detergents and a protein. In Surfactant in Solution; Mittal, K. L., Bothorel, P., Eds.; Plenum Press: London, 1986; Vol. 5. (34) Gimel, J. C.; Brown, W. J. Chem. Phys. 1996, 104, 8112. (35) Ibel, K.; May, R.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311. (36) (a) Nishiyama, H.; Maeda, H. Biophys. Chem. 1992, 44, 199. (b) Tsuji, E.; Maeda, H. Colloid Polym. Sci. 1992, 270, 894. (37) Spector, A. A.; Fletcher, J. E.; Ashbrook, J. D. Biochemistry 1971, 10, 3229. Spector, A. A. Methods Enzymol. 1986, 128, 320.
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In the study of aggregative adsorption of the surfactants on polymers and polyelectrolytes, time-resolved fluorescence quenching (TRFQ) has been very useful to determine the size of the micellar-like aggregates formed by the surfactants. The method works very well if the aggregates are small and fluid, even if there are complications. It is not primarily the aggregation number that is determined but the number of aggregates, and this number is also subject to requirements on the partitioning of probes and quenchers between the aggregates and aqueous subphase and on the distribution of them among the aggregates. If the aggregates are finite, small and fluid, like micelles, then the decay curve shows this to be the case. If this is not so, it is sometimes possible to discriminate between different possibilities: thin rods, bilayers, or clusters of micelles with interchange.38 TRFQ has not been used much for the surfactant-protein complexes, to our knowledge only in one study of BSA and SDS.11 There are some complications not usually met with other polymers: the hydrophobic probe and quenchersspyrene is often used as the probe and benzophenones or pyridinium surfactants as the quencherssmay be expected to bind strongly to the protein in many cases. The present work has been performed both to test the method more extensively than what has been done before and to get some further information on protein-surfactant systems. The measurements on BSA-SDS have been partly performed under the same conditions as the earlier study to allow a comparison. Turro et al.11 used pyrene excimer formation to probe the aggregates; we used pyrene with different quenchers and also tested the simpler static method. The data regarding the interaction of cationic and nonionic surfactants with BSA are scarce.36,39-44 We also tried the application of the method to investigate BSA-CTAB (a cationic surfactant), BSA-C12E8 (a nonionic surfactant), and Lys-SDS systems. Some of the solutions were examined with the cryo-TEM method. Experimental Section Materials. Bovine serum albumin (BSA, Merck, 98% purity) and egg white lysozyme (Lys, Sigma, batch 111H7010, 95% purity) were used without further purification. Sodium dodecyl sulfate, especially pure (SDS, BDH), N-cetyl-N,N,N-trimethylammonium bromide (CTAB, Merck), and octaoxyethylene glycol n-dodecyl ether (C12E8, Nikko Chemicals) were used as the surfactants. The fluorescent probe, pyrene, was delivered by Aldrich Chemical Co. and was recrystallized from alcoholic solution. The quenchers were benzophenone (BP, Aldrich), N-cetylpyridinium chloride (CPCl, Merck), and decylpyridinium chloride (DPCl, Merck). Analytical grade reagents were used to prepare the buffers (acetate, phosphate, borate, and glycineHCl). A solution of 0.5% 2-mercaptoethanol (Fluka) was used as the reducing agent. Sample Preparation. The 0.1 M NaOAc-HOAc, pH ) 5.6 and 7.4, and 0.002 M Na2HPO4-0.064 M KH2PO4, pH ) 5.6, buffers have been used for the preparation of the SDS-BSA and CTAB-BSA solutions. The ionic strengths of the buffers were adjusted to 0.2 and 0.6 M with sodium chloride. Solutions of 0.5 and 4% surfactant in buffer were prepared and magnetically stirred 4-5 h. In the cases of Lys and Lys-SDS solutions, use has been made of 0.009 M glycine-HCl, pH ) 3.3, buffer. The (38) Almgren, M. Adv. Coll. Interface Sci. 1992, 41, 9-32. (39) Nozaki, Y.; Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1974, 249, 4452. (40) Kaneshina, S.; Tanaka, M.; Konda, T.; Mizuma, T.; Aoki, K. Bull. Chem. Soc. Jpn. 1973, 46, 2735. (41) Sen, M.; Mitra, S. P.; Chattoraj, D. K. Colloid Surf. 1981, 2, 259. (42) Koga, J.; Chen, K.; Yamazaki, Y.; Kuroki, J. Colloid Interface Sci. 1983, 91, 285. (43) Nishikida, N.; Takahara, T.; Kobayashi, H.; Tanaka, M. Bull. Chem. Soc. Jpn. 1982, 55, 3085. (44) Green, F. A. J. Colloid Interface Sci. 1971, 35, 581.
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ionic strength of the buffer was adjusted to 0.1 M with sodium chloride. NaN3 (200 ppm) was added to avoid bacterial growth. Some measurements were carried out in borate buffer, pH ) 9.1. Pyrene was introduced in the solutions as follows: an established amount of stock pyrene solution in ethanol was transferred in a volumetric flask and evaporated in a nitrogen stream; the surfactant solution was added to it and magnetically stirred for 24 h. A 6 × 10-6 M concentration of pyrene resulted. Benzophenone and cetylpyridinium chloride were added to solutions in a similar way. The concentrations of pyrene and benzophenone were calculated for each sample from the UV absorption spectrum, making use of the molar extinction coefficients: ) 55 000 L mol-1 cm-1 for pyrene at 336.8 nm in SDS micellar solution and at 337.5 nm in CTAB45 micellar solution and ) 18 600 Lmol-1cm-1 for BP at 255 nm.46 To ensure equilibrium, the solutions were measured after 24 h of room-temperature storage. A concentrated surfactant solution was diluted successively with a saturated pyrene solution in water or buffer in order to obtained the dependence of I1/I3 on surfactant concentration. UV absorption spectra were recorded with a Hewlett-Packard diode array spectrophotometer or a Perkin Lamda 2S UV-vis spectrometer. Steady-State Fluorescence Measurements. The critical micellar concentration (cmc) was evaluated, in the absence and presence of protein, by steady-state fluorescence measurements. The ratio of the first over the third vibrational peak (I1/I3) of pyrene fluorescence is very sensitive to the polarity of the probe microenvironment.47 As aggregation begins, pyrene passes from water into the more hydrophobic environment and the value decreases abruptly. The aggregation numbers were determined by the fluorescence quenching method (SSFQ), based on Turro and Yekta’s theory,48 using BP as the quencher. The probe-quencher pair selected satisfies the criteria imposed by application of the method.48 At the concentrations employed, the absorption of BP at the PY excitation wavelength of 340 nm, selected to avoid protein intrinsic fluorescence, was negligible. To determine the aggregation number, N, under these circumstances, the following equation was used:
ln(I0/I) ) N[Q]/([SDS] - [cmc])
(1)
where I0 and I are the fluorescence intensity without and with quencher, respectively, [SDS] is the total surfactant concentration, [cmc] is taken to be the concentration of free surfactant molecules not incorporated in the micellar aggregates, and [Q] stands for the quencher concentration in micelles. Steady-state fluorescence spectra were recorded on a SPEX Fluorolog 16, combined with SPEX DM3000 software. The slits were 1.5 (excitation) and 0.3 mm (emission). The measurements were carried out at 25 °C. Dynamic Fluorescence Measurements. The pyrene fluorescence lifetime, τ0, in various systems without quencher, was determined by the single photon counting technique, using λex ) 325 nm for the excitation and λem ) 394 nm for the emission. Time-resolved fluorescence quenching measurements (TRFQ) were performed using the technique and experimental setup described in ref 49. The results from the TRFQ measurements were fitted to the model proposed by Infelta,50 describing the decay of fluorescence intensity F(t):
ln[F(t)/F(0)] ) -A2t + A3[exp(-A4t) - 1]
(2)
where (45) Atik, S. S.; Nam, M.; Singer, L. A. Chem. Phys. Lett. 1979, 75, 190. (46) Winnik, F. M.; Winnik, M. A. Polym. J. 1990, 22, 482. (47) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039. (48) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951. (49) Almgren, M.; Hansson, P.; Mukhtar, E.; van Stam, J. Langmuir 1992, 8, 2405. (50) Infelta, P. P.; Gratzel, M.; Thomas, J. K. J. Phys. Chem. 1974, 78, 190.
A2 ) k0 + kqk•n/(kq + k•) A3 ) nkq2/(kq + k•)2 A4 ) kq + k• Here kq is the first-order rate constant for quenching in a micelle, k• the exit rate constant of a quencher leaving a micelle, k0 the unquenched decay rate of the probe (the reciprocal of the lifetime, τ0), and n the average number of quenchers per micelle. There are some conditions that the quencher and probe should fulfill.38,51 If k• is much less than kq and k0, eq 2 reduces to52
ln[F(t)/F(0)] ) -k0t + n[exp(-kqt) - 1]
(3)
This is the ideal case for the determination of aggregation numbers. Fitting the experimental data to eq 3, the following parameters are obtained: k0, n, and kq. The n values yield the micellar concentration, [M], and the aggregation number, N:
N ) n[S]mic/[Q]mic
(4)
The “mic” subscript on S and Q indicates the surfactant and quencher concentrations in micelles, respectively. In all fluorescence measurements, samples were not degassed. Experimental errors were 5% for I1/I3 and 10% for N determinations.
Results BSA/Surfactant. I1/I3 and the Pyrene Fluorescence Lifetime to Determine the Onset of Aggregation. The ratio between the intensities of the first and the third of the major vibrational peaks in pyrene’s fluorescence spectrum is often used as a measure of the polarity in the environment of the probe. Since pyrene has a much lower solubility in water (about 10-7 M) than in hydrocarbon (0.075 M), it is very strongly distributed into micelles as soon as they form, and since the transfer is accompanied by a decrease in the I1/I3 ratio, one would expect that the onset of the micelle formation would be shown by a sudden decrease in this value, if a surfactant is added to a aqueous solution containing pyrene. A sudden decrease does occur, but it is normally found that this decrease starts well before the cmc as measured by other methods. This might be because some aggregation in reality starts well before the cmc, and the pyrene method is so sensitive to the appearance of the hydrophobic domain that a minor fraction of small micelles also has an appreciable effect. Some evidence shows, however, that pyrene associates with the surfactant molecules before the self-aggregation starts (for example, pyrene and pyridinium surfactants).53 An exact and direct determination of the cmc is not possible with the pyrene I1/I3 method, therefore, but the decrease of the I1/I3 ratio certainly shows that aggregation occurs and can be also be used to monitor the changes in the cmc. With BSA present, the I1/I3 value clearly shows that pyrene is bound in a hydrophobic site on the protein: the I1/I3 value is much lower than in water (1.3 and 1.8, respectively) but not as low as in a SDS micelle (1.2), Figure 1. When surfactant is added, the decrease starts earlier if the protein is present. In Figure 1, the x axis shows the total SDS concentration, not the free concentration. Since surfactant is also adsorbed on the protein before the critical aggregation concentration (cac), it seems (51) Almgren, M. In Kinetics and Catalysis in Microheterogeneous System; Gratzel, M., Kalyanasundaram, K., Eds.; Marcel Dekker: New York, 1991; p 63. (52) Infelta, P. P. Chem. Phys. Lett. 1979, 61, 88. (53) Almgren, M.; Wang, K.; Asakawa, T. Langmuir 1997, 13, 4535.
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Figure 1. Dependence of the I1/I3 ratio on SDS concentration in water at various BSA concentrations.
Figure 2. I1/I3 ratio versus SDS concentration in pH ) 5.6 and pH ) 7.4 buffer with 1% BSA, I ) 0.2 M.
that the aggregation starts later when the protein concentration is larger. At lower surfactant concentrations, this method cannot monitor the noncooperative binding, only the moment when cooperative binding starts and pyrene “senses” the polarity difference (by means of I1/I3 variation). The lowest BSA concentration, 0.1% corresponds to 1.5 × 10-5 M, and assuming 20 SDS molecules adsorbed per protein at the cac, the concentration of bound SDS would be about 0.3 mM at this point, i.e., a considerable uncertainty since the cac appears to occur around 1 mM total SDS in this case. In a comparison of the curves for SDS with no BSA and 0.1% BSA, the cac is lower than the cmc by a factor of about 6-10. In the higher surfactant concentration range with 0.1% BSA, one can note that the I1/I3 ratio approaches the value characteristic for the free micelles, but with 1% BSA, this ratio remains lower (practically constant in this range) because at those [surfactant]/[protein] ratios, no free micelles are found. With buffer added, either at pH ) 7.4 or 5.6, in both cases adjusted to an ionic strength of 0.2 M, the cmc of SDS is obviously strongly reduced, by about a factor of 10 with the pH ) 7.4 buffer, and is slightly more with the other (which must be due to ion-specific effects, since SDS in itself is not pH sensitive). In the presence of 1% of BSA, however, the I1/I3 decrease starts at about the same value (around 1 mM) as in water, Figures 1 and 2. Note again that the x axis gives the total SDS concentration, so there
Vasilescu et al.
Figure 3. I1/I3 ratio versus CTAB concentration in various systems.
is nothing strange about the fact that the “cac” appears at a concentration that exceeds the cmc of free SDS in buffers. Since the free concentration in the former case is much smaller than the total SDS, it is quite possible that the free concentration at the cac is strongly affected by the buffers and ionic strength. At very low surfactant concentrations, the I1/I3 is lower in the case of buffered solutions than in water and pyrene bound to the protein senses in the former case a more hydrophobic microenvironment as a effect of ionic strength upon protein. At higher surfactant concentrations, the I1/I3 values noted in the aqueous and buffered solutions (pH ) 7.4) are different, 1.10 and 1.00, owing to the effect of ionic strength upon the micellar structure. A similar phenomenon was also noted in the absence of protein where, at the same concentration of surfactant, the ratio amounts to 1.20 and 1.05, respectively. It should be underlined that at lower surfactant concentrations (Figure 2, pH ) 5.6), a progressive increase of the I1/I3 ratio occurs when the surfactant concentration increases; in other words, one has to do with a gradual modification of local polarity “sensed” by pyrene, which is a more “open” structure of hydrophobic sites of pyrene solubilization. Owing to various values of the I1/I3 ratio, in the case of the two pH’s (higher in case of pH ) 5.6), one can affirm that the “detachment” of the protein molecule is different at the two pH values. Consequently, the stronger interaction at pH ) 5.6 yields a protein structure with larger exposure to water. The jump of the I1/I3 ratio indicates the formation of a more hydrophobic “entity”. Figure 3 shows the change of the I1/I3 ratio when CTAB is added to aqueous buffer solutions, both with and without 1% BSA. The I1/I3 ratio indicates clearly the micelle formation of CTAB in the buffer solutions, but there is no clear change with CTAB concentration in the solutions with protein, which suggest that the hydrophobicity is similar for pyrene bound to the protein and solubilized in the micellar subphase. Figure 4 shows the dependence of the I1/I3 ratio on the C12E8 concentration in aqueous solutions (I ) 0.2 M), without BSA and with 1% BSA. One can note that, as in the case of CTAB/BSA, at low surfactant concentrations, pyrene prefers the hydrophobic zones of the protein. A very small increase of the ratio occurs above the cmc, suggesting the formation of micelles, but since the polarities of the hydrophobic sites of the protein and the micelles are close and pyrene migrates from the protein to the micelle, it is impossible to determine the cac value.
Interactions of Globular Proteins with Surfactants
Figure 4. I1/I3 ratio versus C12E8 concentration in water, I ) 0.2 M, with and without BSA.
The value of the I1/I3 ratio for BSA/C12E8, at high surfactant concentrations, is lower than that in C12E8 micelles. The fluorescence lifetime of pyrene is also affected by binding to the protein, Figure 5. Pyrene fluorescence is more long-lived in micelles than in aqueous solution and even more long-lived when pyrene is bound to the protein. The long lifetime is partly due to protection from water and possibly partly to protection from oxygen dissolved in water, as has been observed with several polymers, e.g., PEO or poly(acrylic acid).54-56 It is interesting that the τ0 value for the fluorescence of pyrene bound to the protein is much higher than in the case of pyrene solubilized in free micelles; however, the I1/I3 is higher or at most equal to that determined in the case of free micelles. This high value could account for the existence of some specific interactions of the probe with the hydrophobic zones of the protein. With increasing SDS concentration, the lifetime in the presence of the protein is reduced, while that from the aqueous solution is increased; both seem to approach a common value representative of pyrene in SDS micelles. The behavior, both with and without protein, is the same in both buffers (Figure 5a). In the case of CTAB in buffer solution, even though the buffer contains anions such as acetate and chloride (which do not quench the pyrene fluorescence) at concentrations adding up to 0.2 M, the bromide counterion from CTAB, which quenches the pyrene fluorescence, is not replaced quantitatively from the micelle interface. This is shown by the variation of the lifetime (Figure 5b), which drops slightly in the micelles when the concentration of CTAB is increased, contrary to the behavior in SDS (where a plateau is reached), and ends with a value of about 140 ns at 40 mM CTAB, as compared to 170 ns in 40 mM SDS. In the presence of BSA, the lifetime is high, about 320 ns, up to a concentration of about 1 mM CTAB; then the lifetime decreases to the value observed for the free CTAB micelles. Compared to the corresponding results with SDS, there is a more conspicuous drop in lifetime at high surfactant concentrations, when aggregates have formed, suggesting that bromide counterions are present also near the interface of the protein-bound micelles. (54) Van Stam, J.; Almgren, M.; Lindblad, C. Prog. Colloid Polym. Sci. 1991, 84, 13. (55) Van Stam, J.; Brown, W.; Fundin, J.; Almgren, M.; Lindblad C. In Colloid-Polymer Interactions. Particulate, Amphiphilic and Biological Surfaces; Dubin, P., Ed.; Penger Tong American Chem. Soc.: Washington, DC, 1993; Chapter 16. (56) Vasilescu, M.; Anghel, D.; Almgren, M.; Hansson, P.; Saito, S. Langmuir 1997, 13, 6951.
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The dependence of the τ0 values on surfactant concentration in C12E8-water solutions, I ) 0.2 M (Figure 5c), is similar to that encountered with ionic surfactants: when BSA ) 0, τ0 increases at micellization and afterward remains constant; at 1% BSA, τ0 is approximately constant up to 1 mM and afterward decreases. When merging all these results obtained for 1% BSA/surfactant (Figure 5d), one notes that τ0 starts to decrease at approximately the same surfactant concentration of 1 mM, regardless of the surfactant type or pH buffer. The critical concentration depends on the BSA concentration (see, e.g., Figure 5c for 0.1% BSA/C12E8) and represents the concentration threshold at which a massive cooperative surfactant binding occurs to form micelle-like aggregates. In the case of SDS at this concentration (around 1-2 mM), the cooperative binding results in conformational modifications of the protein, with pyrene passing into micellar clusters, which determines the decreasing τ0 value. Regarding CTAB and C12E8, following the hydrophobic interactions with protein, the cooperative binding of surfactants entails conformational modifications that affect the value of τ0. Tanford et al. noted conformational modifications of the protein following cooperative binding of cationic surfactants as early as 1974.39 Conformational modifications are also mentioned in the literature36b in the zone of cooperative binding of nonionic surfactants. It is most likely that the electric nature of the surfactant head plays an important role regarding the extent of conformational modifications produced on the protein. Aggregation Numbers from Fluorescence Quenching. Before discussing the results of the fluorescence quenching studies, some remarks about the static and time-resolved methods are in place. The time-resolved method is more reliable; the static measurements should only be used if it has been ascertained that the restrictions on the method are satisfied in the system of interest, mainly, that the quenching in the micelles is fast compared to the unperturbed lifetime of the probe fluorescence; the condition kqτ0 . 1 has been stated.57 We shall see that kq is on the order of (1-2) × 10-7 s-1 and τ0 ≈ 150 ns or larger. The condition is not well fulfilled, therefore, and the results obtained from static measurements are probably on the low side. Both methods primarily measure the fraction of fluorescent probes that is not accessible to quenching by the quencher. In the static case, it is assumed that if a quencher and excited probe occupy the same micelle, then no fluorescence is observed from that entity. In both methods, further assumptions are that all excited probes are in micelles and that the concentrations of the micellized quencher and surfactant are known. An assumption on how the quenchers are distributed over the micelles is also required; usually it is assumed that a Poissonian distribution is appropriate at low quencher concentrations. Deviations from this distribution show up as a dependence of the apparent aggregation number on the quencher concentration. If the deviation is just caused by a broad distribution of micelle size, the apparent aggregation number should decrease with the quencher concentration and give the weighted average aggregation number on extrapolation to zero quencher concentration, with a slope proportional to σ2, the variance of the weight distribution.57 It has been recently realized, however, that equally important deviations may be caused by even rather weak interactions between quenchers or quenchers and surfactants.58 In most cases, however, extrapolation to zero quencher concentration gives a good value for the aggregation number. (57) Almgren, M.; Lofroth, J. E. Colloid Interface Sci. 1981, 81, 486. (58) Almgren, M.; Hansson, P.; Wang, K. Langmuir 1996, 12, 3855.
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Figure 5. Pyrene fluorescence lifetime versus surfactant concentration: (a, top left) SDS in two different buffers, I ) 0.2 M; (b, top right) CTAB in buffer, pH ) 5.6, I ) 0.2 M; (c, bottom left) C12E8 in water, I ) 0.2 M; (d, bottom right) SDS, CTAB, and C12E8 in solutions with 1% BSA, I ) 0.2 M.
For the surfactant aggregates formed on proteins, a further complicationsin addition to that of estimating the amount of free surfactant and quenchersis that the probe, quencher, and surfactant adsorb on the protein prior to the cooperative, aggregative adsorption. It is further probable, at least as far as the probe and quencher are concerned, that molecules adsorbed in specific sites on the protein are transferred into the micelle-like aggregates. Since we have no information that would allow a different approach, we calculate the aggregation numbers as if all surfactant, probe, and quencher were micellized. When it comes to the studies of CxTAB adsorption using CxPCl as the quencher, the surfactants mix close to ideality, and we can assume that the proportions of the two are the same in the free solution as in the micelles.59,60 We assume that this applies to adsorbed surfactant aggregates as well. The aggregation number calculated as if both surfactants were completely associated in the aggregates is then correct, even when in reality both are present at appreciable amounts in aqueous solution or molecularly adsorbed, if only the proportion of the two surfactants in the different states is the same. The aggregation numbers for the three surfactants from steady-state fluorescence measurements are listed in Tables 1, 2, and 4 and from time-resolved measurements in Tables 3 and 4. The quenching curves obtained by TRFQ (59) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 16694. (60) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 16684.
measurements are shown in Figure 6: for 27.44 mM CTAB, BSA ) 0% (Figure 6a) and 1% (Figure 6b). One can note that kq . k• and that kq in the absence of the protein is higher than in the case where protein was present. The aggregation numbers obtained without BSA are similar to the results from other sources. For CTAB, an increase of the aggregation number with the surfactant concentration is obtained in a buffer solution (pH ) 5.6, I ) 0.2 M, Table 2). Smaller values that change less with surfactant concentration are obtained in water or 0.20 M NaCl. This is a general ion strength effect and, especially, mirrors the partial replacement of Br- at the micelle surface by other counterions. In the presence of BSA, the SDS aggregation numbers are much smaller and remarkably independent of the buffer composition. They are, surprisingly, not affected by a treatment with 2-mercaptoethanol (2-ME) to break the S-S bridges. The results (Table 1) from buffers with 0.6 M ionic strength are also closely similar to those at low ionic strength. The adsorbed surfactant aggregates are formed in a local environment, which is electrostatically similar, independent of bulk ionic strength, and also of the protein charge distribution, which changes with pH. The comparable N values, obtained by the method of pyrene fluorescence quenching with BP and by the pyrene excimer fluorescence method11 (Table 1), support the assumption that the quencher lies exclusively in micelles and that, at the surfactant concentrations utilized in the measurements of the aggregation numbers, pyrene was
Interactions of Globular Proteins with Surfactants
Langmuir, Vol. 15, No. 8, 1999 2641
Table 1. Values of the Aggregation Number, N, for the Micellar Aggregates Present in the SDS/BSA System, Determined by SSFQ Measurements (Quencher: Benzophenone)a N pH ) 5.6, I ) 0.2 [SDS], mM
[SDS]/[BSA], %/%
17.3 17.3 26.0 34.6 51.9 69.2
d 0.50/1 0.75/1 1/1 1.5/1 2/1
pH ) 5.6, I ) 0.6
2-ME 96 39 60 78 93
41 60 66 90 100
2-ME 110 38 57 75 106
39 71 74 103 107
pH ) 7.4, I ) 0.2 b
c
33 43 75
29 43 42
2-ME 97 33 48 61 91 96
pH ) 7.4, I ) 0.6 b
c
2-ME 153
32 49 63 67 106
29 49 52 70 74
38 43 42
91 95 122
67 80 106 132
The measurements, with and without 2-mercaptoethanol (2-ME), at pH ) 5.6 and pH ) 7.4 were carried out at 35 and 25 °C, respectively; the protein concentration was 1% (0.15 mM); λex ) 340 nm, λem ) 395 nm. b Values resulted from deexcitation of pyrene excimer.11 c Values resulted from small-angle neutron scattering.17 d [BSA] ) 0. a
Table 2. Values of the Aggregation Number, N, for the Micellar Aggregates in the CTAB/BSA System, Determined from SSFQ Measurements (Quencher: Benzophenone)a [CTAB], mM
N [CTAB]/ [BSA], pH ) 5.6, pH ) 5.6, pH ) 7.4, pH ) 7.4, %/% I ) 0.2 I ) 0.6 I ) 0.2 I ) 0.6
13.7 13.7 20.6 27.4 41.1 54.8
b 0.5/1 0.75/1 1/1 1.5/1 2/1
90 27 40 54 67 71
104 31 42 55 76 76
90 25 36 58 68 81
107 31 40 59 75 93
a The measurements were taken at 25 °C; the protein concentration was 1%; λex ) 340 nm, λem ) 395 nm. b [BSA] ) 0.
Table 3. Aggregation Numbers, N, from TRFQ Measurements on Micellar Aggregates in the CTAB/BSA System (Quencher: CPCl) N [CTAB] mM % 13.7 13.7 27.4 27.4 41.1 41.1 54.8 54.8
[BSA], %
pH ) 5.6, I ) 0.2
0 1 0 1 0 1 0 1
95 26 125 57 129 67 131 79
0.5 0.5 1.0 1.0 1.5 1.5 2.0 2.0
water/NaCl, I ) 0.2
water, I)0
96 53
84 45
98 64
88 39
Table 4. Values of the Aggregation Numbers and Intramicellar Quenching Rate Constants for the Micellar Aggregates Present in the C12E8/BSA, I ) 0.2 M (pH ) 7), System (Quencher: Benzophenone) NSSFQ
NTRFQ
kq, 107 s-1
[C12E8], Surf/BSA, molar 0% 1% 0% 1% 0% 1% mM %/% ratio BSA BSA BSA BSA BSA BSA 6.0 10.0 20.0 30.0 40.0
0.3/1 0.5/1 1/1 1.5/1 2/1
40.0 66.6 133.3 199.8 266.4
75 73 78 85 99
26 35 50 53 86
77 83 94 107 100
35 39 64 74 86
1.67 1.66 1.68 1.58 1.42
1.20 1.27 1.29 1.28 1.25
solubilized in the micellar aggregate and not bound to the protein (see also Figure 2). The SDS/BSA saturation ratio is known from the literature to be about 1.5/1 (g/g) and to be rather independent of the nature of the protein and medium;1-3 consequently, up to this ratio, the aggregation numbers reflect an increase of the size of the protein-bound micelles. Above this value of the ratio, free micelles show up and the N values represent an average of the two types of micelles. The number of micelles per BSA molecule varies
between 3, at low [SDS], and about 3.8 at saturation. At pH ) 7.4 and I ) 0.6 M, the numbers are lower, between 2 and 2.4. The number of clusters is comparable to the value reported by Shinagawa et al.20 The effect of BSA on the CTAX aggregation numbers is similar to the SDS/BSA case: the values are smaller than those of the free micelles and with different buffers coincide. There is a very gratifying agreement between the results from the static (Table 2, quencher BP) and the time-resolved (Table 3, quencher CPCl) measurements. When no salt is added to the solvent (I ) 0), the aggregation numbers are different from the rest (Table 3), in this case the aggregation numbers being substantially smaller than normal (I ) 0.2 M). In comparison with SDS, the estimated aggregation numbers are smaller for the adsorbed aggregates (whereas free CTAB micelles are larger than SDS micelles). There is no evident explanation for this; part of the reason may be connected with the distribution of surfactants between aggregates and the free and molecularly adsorbed states. The CTAX values obtained using CPCl as the quencher would be most reliable in this respect. Some authors39 consider that the level of BSA saturation with CTAB is at least 1/1 (g/g) at pH ) 8. Tables 2 and 3 show that N increases with the CTAB/BSA ratio. The increase may partly be due to the formation of free micelles in the system. It is difficult to determine the percentage of the two species, but since the variation of N is similar at the two values of pH and ionic strength, we conclude that the percentage of free micelles is small. The number of clusters would then be comparable with that in the SDS/BSA system. In the case of C12E8 nonionic surfactant, the values of the aggregation numbers (Table 4) have been determined by SSFQ and TRFQ measurements, making use of BP as the quencher. Although there are discrepancies between the values determined by the two methods (those measured by SSFQ are usually smaller), one can note that the aggregation numbers of the surfactant clusters in the presence of protein are lower than the aggregation numbers of free micelles, the effect being stronger at lower surfactant concentrations, conclusions similar to those for the ionic surfactants. The kq values (Table 4) are of the same order of magnitude as those obtained in the case of CTAB/BSA. One can also note that in this case, the kq values decrease on addition of protein and vary insignificantly with the molar ratio over the investigated domain. Taking into account the similar behavior of N and τ0 values on the surfactant concentration, in the presence of BSA, one reaches the conclusion that the interaction mechanism in the cooperative binding zone is similar, that is, hydrophobic. Likewise, we assume that the model
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Figure 6. Decay curves in 27.44 mM CTAB/0% BSA (a) and 27.44 mM CTAB/1% BSA (b) solutions, pH ) 5.6, I ) 0.2 M; λex ) 325 nm, λem ) 394 nm, [CPCl] ) 1.7 × 10-4 M, and the autocorrelation function of the residuals.
of the complex is similar: “a necklace of protein-decorated micelles”, that is, micellar clusters, smaller than free micelles and having partially adsorbed on the surface the polypeptidic chain of BSA. The protein undergoes conformational modifications as a result of the hydrophobic interactions. In the case of ionic surfactants, the electrostatic repulsion of clusters entails the protein unfolding. For further information of the BSA/surfactant system, the cryo-TEM examination has been initiated. The cryoTEM micrographs are not very revealing. Solutions of CTAB and SDS in BSA alone gave rather similar micrographs: the usual small spots seen in solutions of globular micelles. The size and contrast of the entities are too small to allow high resolution; to see anything, it is necessary to use a certain underfocus, which in effect means that anything smaller than 5 nm looks as if it was of this size. In the solutions with BSA and a surfactant, there are clearly larger structures present. In some areas, there seem to be large structures decorated with small dark spots, as would result if micelles were formed on the protein. The features are not well resolved, however, and we cannot claim anything except that appearance of the micrographs can well be consistent with the other results. Dynamic light scattering experiments on this system (BSA/different surfactants) are now being undertaken to provide information on the hydrodynamic radius of the complex and on the conformation of BSA in the complex. Lys/SDS. The precipitation of the Lys/SDS complex, resulting from the electrostatic interaction at low SDS concentrations, considerably restricts the applicability of the fluorescent probe method. Under these circumstances, it was impossible to obtain the curves of I1/I3 and τ0 variation with surfactant concentration at very low concentrations in order to determine the cac. At higher SDS concentrations, the precipitate redissolves. The phase studies of the SDS/Lys/water system show that for 1% Lys in water, the precipitate redissolves at SDS/Lys ) 19 and that the ratio increases when 0.1 M NaCl is added to the system.13 Under the conditions utilized in this contribution, pH ) 3.3 and ionic strength of 0.1 M (with NaCl addition), the solution is clear at a molar ratio of at
Table 5. Values of the Pyrene Fluorescence Lifetime and I1/I3 Ratio in Various Buffered (pH ) 3.3) Lysozyme Solutions soln 10% Lys 7.9% Lys 6.1% Lys 4.4% Lys 2.7% Lys 1.6% Lys 1.0% Lys 0.5% Lys 0.2% Lys 0.2% Lys/0.23% SDS 0.23% SDS
τ0, ns
I1/I3
250 166 142 164 178
1.142 1.176 1.169 1.234 1.233 1.242 1.252 1.275 1.398 1.048 1.092
least 37.5 (e.g., 0.75% SDS/1% Lys). At pH ) 9.1 and I ) 0.014, it was possible to work at a lower molar ratio, i.e., at 25 (0.5% SDS/1% Lys). At this molar ratio, according to the data in the literature, SDS is cooperatively bound to the protein25 and the formation of micelle-like clusters is possible. Lys has a tendency to associate at high concentrations to form dimers or higher oligomers.13 In the case of reduced Lys, these aggregates are stable and do not decompose after dilution.36 The formation of Lys associates is probably the reason for the variation of I1/I3 and τ with Lys concentration (Table 5). At 10% Lys concentration, pyrene senses hydrophobicity close to that in the SDS micelles. With decreasing Lys concentration, the I1/I3 ratio increases, suggesting a less hydrophobic environment in the monomeric Lys. Table 6 lists the values of pyrene fluorescence lifetimes, of quenching rate constants, and of aggregation numbers obtained for variable SDS/Lys ratios in buffered solutions (pH ) 3.3). The aggregation numbers were determined by the SSFQ and TRFQ methods with different quenchers. One can note the same order of magnitude for kq values as in the surfactant/BSA cases. The association tendency of Lys molecules, which is greater at 1% than at 0.3%, may account for the differences between the aggregation numbers at the same molar ratio,
Interactions of Globular Proteins with Surfactants Table 6. Values of the Aggregation Number, N, Pyrene Fluorescence Lifetime, τ0, and Intramicellar Quenching Rate Constant, kq, for Various SDS/Lys Ratio Values, at pH ) 3.3 and I ) 0.1 Ma SDS/Lys, %/%
R
τ0, ns
N
kq, 107 s-1
quencher
3/1 1.5/1 1.5/1 1.5/1 1.25/1 1/1 1/1 0.75/1 0.75/1 0.75/1 1/0.75 1/0.67 1/0.67 1/0.5 1/0.3 0.9/0.3 0.9/0.3 0.75/0.75 0.75/0.3
150 75 75 75 62.5 50 50 37.5 37.5 37.5 67.7 74.7 74.7 100 165 150 150 50 112.5
162 141
97 88 75 85 87 81 74 46 65 43 58 89 112 108 89 89 86 51 73
2.51 1.02 SSFQ SSFQ 1.41 1.75 1.47 SSFQ SSFQ 1.03 1.53 1.09 0.94 1.43 2.20 1.59 SSFQ SSFQ SSFQ
CPCl CPCL DPCl BP DPCl CPCl CPCl DPCL DPCl CPCl CPCl BP DPCl CPCl CPCL DPCl DPCl DPCl DPCl
142 141 142 173 140 154 154 141 149 154
a R ) SDS/Lys molar ratio; I ) ionic strength; DPCL ) decylpyridinium chloride; CPCl ) cethylpyridinium chloride; BP ) benzophenone.
Figure 7. Dependence of aggregation number on SDS concentration in solutions with 1% Lys at various pH and ionic strength values.
as can be observed from Table 6. It is possible that more than one Lys molecule is involved in the micellar cluster. Figure 7 shows the variation of N versus [SDS] at 1% Lys. One can notice that the pH seems to play little role at pH ) 3.2 and 7. At pH ) 9.1, the aggregation numbers are smaller, especially at an ionic strength at 0.014 M. However, at this ionic strength, the SDS normal micelles are smaller than at I ) 0.1 M (around 85 and about 100110, respectively). The binding isotherm of SDS on Lys has revealed that the number of bound SDS molecules may reach 70, which may result in unfolding of the protein. Saturation of the protein occurs at about 1.5% SDS/1% Lys followed by the formation of free micelles.29 Evidently a cluster/protein is present at saturation.
Langmuir, Vol. 15, No. 8, 1999 2643
Some differences related to the model of the complex between high molecular (e.g., BSA) and low molecular (e.g., Lys) proteins with SDS have been noted earlier by electric birefringence21 and circular dicroism24 measurements. On the basis of the fact that τ0 in protein-bound micelles is higher than in free micelles, we supported for BSA/SDS the necklace and bead or protein-decorated micelle model as in ref 11. The BSA polypeptidic chain is longer and more flexible, and the protein may wrap around the micellar aggregates formed, protecting the pyrene molecule from water (oxygen from water is a good quencher for pyrene fluorescence). In the case of Lys/SDS, however, the τ0 values are smaller or equal to those in free micelles, and in pure lysozyme, they are smaller than in BSA. The Lys peptide chain, which is shorter and more rigid and with smaller hydrophobic zones than in BSA, does not cover the protein-bound micelle as effectively and does not protect the micelle from water contact equally well. Anyway, essentially, the Lys/SDS complex can be described by the same necklace and bead model. Conclusions The BSA/SDS, BSA/CTAB, BSA/C12E8, and Lys/SDS systems were studied by fluorescence method to obtain information on the protein/surfactant interaction and the structure of the micelle-like cluster. The experimental data obtained suggested the following conclusions: (1) In all systems, micelle-like clusters have formed, with aggregation numbers smaller than those of free micelles. (2) The pH value and the ionic strength do not appreciably influence the aggregation numbers (or at least much less than the size of free micelles) nor do they influence the dependence of N on the surfactant/protein molar ratio. (3) The models for the BSA/SDS and Lys/SDS complexes are the same necklace and bead. In the case of BSA, because the polypeptidic chain is longer and more flexible, the possibility arises that the protein partially wraps around the micellar aggregate formed by SDS on protein. Lysozyme is smaller and more rigid and does not wrap up well around the micelle to it protect from water contact. (4) The structures of the micelle-like clusters formed on BSA and Lys are similar, a conclusion resulting from the size of the kq values (that is, the microviscosity as well). (5) The number of clusters per polypeptidic chain varies within 2-4 for BSA/surfactant and is 1 cluster in the Lys/ SDS case. (6) Although the addition of 2-mercaptoethanol brings about a more pronounced detachment of (tertiary) protein structure and, consequently, a higher flexibility of the polypeptide chain, the aggregation numbers remain unchanged. Acknowledgment. We are indebted to Dr. Go¨ran Karlsson for help with the cryo-TEM micrographs. This contribution is the result of a cooperation between the Royal Academy of Sweden and the Romanian Academy. We are grateful to the Swedish Technical Research Council (TFR) for financial support. LA981424Y
