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Lysozyme in Catanionic Surfactant Mixtures Anna Stenstam, Ali Khan, and Håkan Wennerstro¨m* Physical Chemistry 1, Lund University, P.O. Box 124, SE-222 21 Lund, Sweden Received February 25, 2004. In Final Form: June 8, 2004 We investigate the competition between the associations of oppositely charged protein-surfactant complexes and oppositely charged surfactant complexes. In all systems examined, the most favorable complexation is the one between the two oppositely charged surfactant ions, despite the strong binding known, for example, dodecyl sulfate, DS-, to lysozyme. Thus, the phase behavior of the catanionic system is dominating the features observed also in the presence of protein. The phase behavior of the dilute protein-free dodecyltrimethylammonium chloride-sodium dodecyl sulfate-water system is presented and used as a basis for the discussion on the different solubilization mechanisms. Our results show that the mechanism for resolubilization of a protein-surfactant salt is fundamentally different when it is caused by addition of a second surfactant than when it is accomplished by an excess of the first surfactant. The competition between lysozyme and cationic amphiphiles as hosts for the anionic surfactants was studied experimentally and analyzed quantitatively. Aggregates with C12 cationic surfactants are clearly preferred by the anionic surfactants, while for C10 and particularly C8 a clear excess of cationic surfactant has to be added to completely dissolve the complex salt lysozyme-anionic surfactant.
Introduction As an ionic amphiphile is added to an aqueous solution containing an oppositely charged species, be it a polyelectrolyte, a protein, or another amphiphile, it is commonly observed that a charge neutral complex precipitates. At the point where one has added sufficient amount of the ionic amphiphile to charge neutralize the originally dissolved species, the solution phase contains nearly exclusively the electrolyte formed by the original counterions. On further addition of amphiphile the precipitated complex tends to redissolve with aggregates in the solution containing an excess of the added amphiphile.1-3 This overall trend can be analyzed in terms of the interplay between hydrophobic interactions, always promoting association, and electrostatic interactions which can be both attractive and repulsive.4 Conceptually such a balance between these two interactions is very important, and it provides the underlying theme for most association processes in biological systems. An example that has attracted much attention in recent years is the amyloid protein aggregation.5 It is, thus, of fundamental interest to quantitatively characterize such an interplay between hydrophobic and electrostatic interactions. We have previously studied a complex between the protein lysozyme and anionic sulfate surfactants and shown that this model system exhibits a rich aggregation behavior when exposed to an excess of the anionic surfactant.6 By varying the hydrocarbon chain length of the surfactant, one obtains a systematic variation in the strength of the hydrophobic interaction, while keeping other factors constant. Using this approach, it was possible to obtain * To whom correspondence should be addressed. Please use postal address, e-mail (
[email protected]), or fax (+4646-222 44 13). (1) Goddard, E. D.; Hannan, R. B. J. Am. Oil Chem. Soc. 1977, 54, 561. (2) More´n, A. K.; Khan, A. Langmuir 1995, 11, 3636. (3) Jiang, J.; Prausnitz, J. M. J. Phys. Chem. B 1999, 103, 5560. (4) Stenstam, A.; Topgaard, D.; Wennerstro¨m, H. J. Phys. Chem. B 2003, 107, 7987. (5) Koo, E. H.; Lansbury, P. T.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9989. (6) Stenstam, A.; Khan, A.; Wennerstro¨m, H. Langmuir 2001, 17, 7513.
quantitative values for the interaction between the lysozyme molecule, having both hydrophobic and hydrophilic patches, and the alkyl chains of the surfactant.6 In the present paper, we extend these investigations by exposing the insoluble lysozyme anionic surfactant complex to solutions containing cationic surfactants of varying alkyl chain length and concentration. This allows for observing the competition between the pairwise interactions, net-cationic protein-anionic surfactant and anionic surfactant-cationic surfactant, and how the relative strengths vary with the alkyl chain lengths of the surfactants. As a prerequisite for a proper analysis of the experiments, we also report results for the pseudo-ternary system anionic surfactant-cationic surfactant and water. Experimental Section Materials. The anionic surfactants sodium dodecyl sulfate (SDS), specially pure, was obtained from BDH in the United Kingdom and sodium octyl sulfate (SOS), for surfactant tests, was from Merck in Germany. The cationic surfactants dodecyltrimethylammonium chloride (DoTAC), decyltrimethylammonium chloride (DeTAC), and octyltrimethylammonium bromide (OTAB) were obtained from TCI Tokyo Kasei (purity >97%), Japan. All surfactants were used as supplied. The protein lysozyme was obtained from Sigma. The complex salts of lysozyme and different alkyl sulfates, Ly(DS)8 and Ly(OS)8, were prepared in the manner described by us previously.6 The use of the complex salts as the protein source only affects the range of the electrostatic interaction. The systems feature the same phases as presented and discussed in ref 6 as well as in the pseudoternary lysozyme-SDS-water system described in ref 2. The stoichiometry of 1:8 was further supported by the work of Mathis and Zana in which it is concluded that the molar composition reflects the net charge of the protein at the present pH.7 D2O from Dr. Glaser AG, Switzerland (99.8% pure), was employed as the solvent in the phase diagram determination of SDS-DoTACwater. Sample Preparation and Phase Determination. For the determination of the phase diagram of DoTAC-SDS-water, stock solutions of the two surfactants were mixed with water in various compositions. The phase behavior at 40 °C was determined after 24 h of temperature equilibration. The appearance (7) Mathis, A.; Zana, R. Colloid Polym. Sci. 2002, 280, 968.
10.1021/la049508w CCC: $27.50 © 2004 American Chemical Society Published on Web 07/29/2004
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Figure 1. Phase behavior of DoTAC-SDS-D2O. (a) Determined at 22 °C: L1, micellar solution; S + L1, solid and micellar solution; V, blue solution of vesicles; S + V solid with blue (vesicle) supernatant. The solid line indicates equimolarity, and the dashed line inidicates the stability limit of the solid DoTADS. (b) Determined at 40 °C: L1, micellar solution; S + L1, solid and micellar solution; L1 + LR, micellar solution and birefringent supernatant; V + LR, blue (vesicle) solution and birefringent supernatant; V, blue solution of vesicles. The solid line indicates equimolarity. at 22 °C was re-observed at room temperature with a slight hysteresis in time. For the solubilization of the various complex salts with a second surfactant, series have been prepared with a constant concentration of the protein-surfactant salt to which the second surfactant and water has been added in various amounts. Hence, in the Ly(DS)8 series all samples have a constant concentration of 2 mM lysozyme and 16 mM DS- while the DoTA+Clconcentration and, thus, the ratio of cationic/anionic surfactants (+/-) is varied. For the Ly(OS)8 series, the concentrations are 620 µM lysozyme and 5 mM OS- with varying concentration of the cationic surfactant, DoTAC, DeTAC, or OTAB The different phases [solid precipitate, bluish (vesicular) solution, and isotropic micellar solution] were determined by visual observation of the samples only. The presence of vesicles in the blue solutions is deduced on the basis of precedent investigations.8-10 The anisotropic (lamellar) structures were identified by examination between crossed polarizers. UV Absorbance Spectroscopy. To quantitatively follow the resolubilization of the solid lysozyme-surfactant complexes with increasing concentration of second surfactant (DoTAC, DeTAC or OTAB), the concentration of lysozyme in the supernatant in equilibrium with solid precipitate was measured by the absorption of UV light at λ ) 280 nm. The apparatus used was a PerkinElmer Lambda 14 spectrometer.
Results and Discussion DoTAC-SDS-Water. The phase diagrams of the aqueous surfactant system at 22 and 40 °C are shown in Figure 1. Here, only the very dilute corner of the triangular diagram is presented, because it will be used in the discussion on the mixed surfactant systems with lysozyme. The full phase behavior will be shown in detail elsewhere.11 At room temperature, (Figure 1a) the features are as follows. At the molar ratio 1:1 of the two amphiphiles the solid DoTA+DS- is formed (S). Prepared in heavy water this solid floats on top of a clear solution. This neutral complex has a Krafft temperature at 35 °C and is reported to be of a lamellar structure.12 With excess DoTAC, the solid is eventually solubilized and the samples are isotropic micellar solutions (L1). On the other hand, with excess SDS the solid is at first in equilibrium with a bluish solution (V). With increasing concentration of SDS, the solid is completely dissolved but the solutions remain (8) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadinski, J. A. Science 1989, 245, 1371. (9) Herrington, K. L.; Kaler, E. W.; Miller, D. D.; Zasadinski, J. A.; Chiruvolu, S. J. Phys. Chem. 1993, 97, 13792. (10) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.; Zasadinski, J. A. J. Phys. Chem. 1996, 100, 5874. (11) Khan, A.; Alfredsson, V. Manuscript in preparation. (12) Jokela, P.; Jo¨nsson, B.; Khan, A. J. Phys. Chem. 1987, 12, 3291.
bluish and at lower water content also birefringent (LR). Both the isotropic but bluish solutions as well as the birefringent samples are expected in the catanionic system.9,10,12-14 The bluish appearance of the solutions is due to light-scattering vesicles. Systems with such catanionic vesicles have been carefully studied previously (Herrington et al.9 and Yatcilla et al.10). Within the context of this paper, these vesicles are seen as manifestations of an attractive association of the cationic and anionic surfactant molecules. Finally, with further additions of SDS the samples are one-phase micellar solutions also on the anionic rich side. The stability of the solid is larger with respect to the addition of DoTAC compared to the addition of SDS. When the phase diagram is determined at 40 °C (Figure 1b), no solid precipitate is formed. On the cationic side, the samples that at room temperature featured the solid DOTADS are instead mainly micellar solutions in equilibrium with an anisotropic supernatant (L1 + LR). At higher +/- ratio, the samples are dissolved into a single micellar phase (L1). On the anionic side, close to the 1:1 stoichiometry where at 22 °C the solid DOTADS is in equilibrium with a blue vesicular solution, at 40 °C the vesicular solutions are instead in equilibrium with a birefringent liquid crystalline phase (V + LR). The general phase equilibrium properties of an aqueous system containing both anionic and cationic amphiphiles were analyzed by Jokela et al.15 Specifically, these authors discuss the competition between the formation of a stoichiometric crystal and a lamellar structure, which can incorporate a varying molar ratio anion/cation. For the lamellar structure one can define a critical association concentration Ccrit(λ) ) (CanionCcation)1/2, where the value depends on the anion-to-cation ratio λ. The precipitation of a solid crystal, on the other hand, occurs as one exceeds the solubility product Kss ) CanionCcation. Filipovic-Vincekovic et al. have measured such solubility products for a series of alkyl sulfate-alkylammonium chlorides, and for the symmetric C12 system Kss ) 6 × 10-10 M2, they can be deduced from Figure 2b in ref 16. For the trimethylamine system, we expect a slightly higher solubility product as a result of the larger size of the cationic headgroup. From a precipitation study supported by the analysis of the (13) Edlund, H.; Sadaghiani, A.; Khan, A. Langmuir 1997, 13, 4953. (14) Regev, O.; Khan, A. J. Colloid Interface Sci. 1996, 182, 95. (15) Jokela, P.; Jo¨nsson, B.; Wennerstro¨m, H. Prog. Colloid Polym. Sci. 1985, 70, 17. (16) Filipovic-Vincekovic, N.; Bujan, M.; Dragcevic, D.; Nekic, N. Colloid Polym. Sci. 1995, 273, 182.
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Figure 2. Solubilization of Ly(DS)8 with DoTAC as resolved by UV absorbance spectroscopy: squares with crosses, solid + clear solution; solid squares, thermotropic solid + clear solution; open squares, clear solution. The line represents the values obtained from eq 1. The fraction of lysozyme in solution is taken as the concentration in solution divided by the total concentration (weighed) in the sample.
lysozyme-containing system (see the following), we arrive at an estimate of Kss ≈ 2.5 × 10-9 M2 for the DoTA+DSsystem. Ly(DS)8 and DoTA+DS-. On adding aqueous solutions of DoTAC to the solid Ly(DS)8, it seems by eye as nothing happens prior to a critical concentration of about 80 mM DoTAC. At this high concentration, no solid remains and the samples are isotropic clear solutions (L1) depicted by open squares in Figure 2. However, a closer examination reveals that the white solid observed in the samples prior to this solubilization is not of constant composition. The analysis of the supernatant by UV spectroscopy reveals that the protein concentration steadily increases as shown in Figure 2. Moreover, when the samples are heated to 40 °C (from the normal lab temperature of 22 °C), the precipitate in samples with a lower degree of DoTAC are not affected (squares with crosses in Figure 2), while the solid at higher DoTAC concentration is resolubilized (solid squares in Figure 2). In the latter case, a small amount of a birefringent phase is found floating on top of the solution. As can be seen in Figure 2, all of the lysozyme is present in the supernatant in these samples. Thus, the solid precipitate does not contain any protein at +/- ratios above unity. The solid must, therefore, be the catanionic surfactant salt DoTADS. Consequently, it is the formation of this catanionic surfactant salt that drives the resolubilization of the protein-surfactant salt. Equation 1 presents a simple estimate of the solution concentration of lysozyme deduced from the solubility product Ksp of Ly(DS)8 and the quantitative formation of the 1:1 surfactant salt DoTADS.
1 solution 0 Clysozyme ≈ Clysozyme + (CDOTAC - C0DOTAC) 8
(1)
The lysozyme in solution is of two origins. A small amount of lysozyme is in equilibrium with the solid Ly(DS)8 already 0 . The solubility product before DoTAC is added, Clysozyme Ksp of Ly(DS)8 in water was in ref 6 determined to be 3 × 10-41 M9, consistent with the lysozyme concentration, 0 Clysozyme ) 5 µM. As a consequence of the stoichiometry, eight times as many DS- ions are also in solution at this point. As DoTAC is added the 1:1 salt, DoTADS is formed by association with the soluble DS- ions, which drives the redissolution of Ly(DS)8. For every DoTA+ ion that is added to the sample, one DS- ion and 1/8 of a lysozyme molecule 0 refers to the solubility limit of is dissolved. CDOTAC DoTADS. Using the estimate of Kss ≈ 2.5 × 10-9 M2 and 0 CDS - ≈ 40 µM based on the solubility product Ksp, we find
Figure 3. Solubilization of Ly(DS)8 with DoTAC inserted into the phase diagram of SDS-DoTAC-D2O at 22 °C (shown also in Figure 1a). The arrow with symbols represents the solubilization of constant 3 wt % Ly(DS)8, that is, 16 mM DS- with additions of DoTAC: The appearances of the samples are O, (L1); ×, (S + L1). 3 marks the cation/anion ratio where DoTADS starts to form, that is, where Ly(DS)8 starts to be resolubilized. 0 CDOTAC ≈ 63 µM. Equation 1 provides a good representation of the experimentally obtained results shown in Figure 2. We conclude that the driving force for the resolubilization of lysozyme in this case is the formation of solid DoTADS, which has a sufficiently low solubility product relative that to the surfactant-protein salt to ensure a quantitative exchange of lysozyme with eight DoTA+ ions. One can analyze this competition between the stability of Ly(DS)8(s) and DoTADS(s) in more detail on the basis of the two solubility products Ksp (surfactant-protein salt) and Kss (surfactant-surfactant salt), respectively. A derivation considering these two equilibria and three material balance equations (on Clysozyme, CSDS, and CDoTAC) yields that, in the presence of the two precipitates, the change in free lysozyme concentration with respect to the addition of DoTAC is
solution dClysozyme
dCtot DoTAC
[
)
1 1 Kss -(7/8) 1 -(9/8) 1+ C + K(1/8) Clysozyme 8 64 K(1/8) lysozyme 64 sp sp
]
-1
(2)
Equation 1 is based on the assumption that the second and third terms in parentheses of eq 2 are small relative to 1. With Ksp ) 3 × 10-41 M9, Kss ) 2.5 × 10-9 M2, and solution 0 Clysozyme ) Clysozyme ) 5 µM, the correction term is about solution increases. This supports 0.3 and decreasing as Clysozyme the use of eq 1, which is valid to a good approximation solution encountered. even at the lowest Clysozyme As shown by Figure 2 and eq 1, where it is evident that the resolubilization of Ly(DS)8 with DoTAC is a function of the simple catanionic phase behavior, it is useful to base the analysis on the redissolution on this protein-free system. Hence, the dilute part of the DoTAC-SDS-D2O phase diagram determined at 22 °C (Figure 1a) is used as a basis for the discussion. In Figure 3 the resolubilization series shown in Figure 2 is inserted into the surfactant-surfactant phase diagram (Figure 1a) with the constant 3 wt % of Ly(DS)8 recalculated to a constant 16 mM of the DS- ion. As indicated by the triangle, the first sign of resolubilization (possible by UV measurements on lysozyme) coincides well with the first precipitation of DoTADS. Likewise, the concentration of DoTAC at which no solid is present is approximately 0.08 M in both the catanionic as well as the protein system. Thus, the
Lysozyme in Catanionic Surfactant Mixtures
Figure 4. Concentrations of free lysozyme, DS- ions, and DoTA+ ions in solution along the resolubilization route of Ly(DS)8 with DoTAC: full line, DoTA+; dashed line, lysozyme; dotted line, DS-. The amount of Ly(DS)8 was at all times 3 wt % (2 mM lysozyme). The different shades denote the different transitions discussed in the text.
conclusion is that lysozyme has very little, if any, effect on the phase behavior of the mixture of DoTAC and SDS. Lysozyme, although octavalent and with hydrophobic patches on its surface, cannot compete with DoTAC for the association with SDS. It is valuable to visualize the variations of the concentrations of free components in the sample because it makes the understanding of the terms of the free energy more accessible. In Figure 4 we have presented the redissolution of Ly(DS)8 by DoTAC in such a way. The free concentrations of lysozyme+8, DS-, and DoTA+ are plotted against the increasing total concentration of DoTAC in the samples. The diagram can be divided into five regions, shadowed in the figure. In this description, the regions will be discussed in the order of increasing DoTAC concentration, that is, from one to five. In the first region, DoTAC is added to the supernatant and concentrates while the other ions remain constant on the basis of the solubility product of Ly(DS)8, Ksp. The border between the first and the second region is at the concentration of DoTAC at which the solid DoTADS starts to be formed. This is determined by the solubility product of the surfactant-surfactant salt, Kss. Within the second region, Ly(DS)8 is resolubilized while DoTADS is precipitated. When all Ly(DS)8 is resolubilized, the source of DS- ions is almost emptied. Further addition of DoTAC takes the system into region three, where mainly DoTA+ is again concentrating in the supernatant. With the addition of DoTA+, the precipitation of DoTADS will continue, but because of the low concentration of DS- free in solution at this stage, this is hardly noticeable. As the free DoTA+ reaches the critical micelle concentration (cmc ) 20 mM for pure DoTAC)17 at about 36 mM of total DoTAC, micelles start to form. Lysozyme apparently does not interact with such positively charged micelles, and the DS- concentration is so low that it has only a minute influence on the cmc. Micelle formation introduces two effects that both in a synergistic way promote the dissolution of the solid catanionic salt. For an ionic surfactant, the concentration of the free surfactant ion decreases on increasing the total concentration after the cmc. Thus, the surfactant anion has to increase in concentration to keep the solubility product, Kss, constant. Additionally, the anion can be incorporated into the micelles. Initially micelles contain only a small fraction (17) Balma, R. R.; Clunie, J. S.; Goodman, J. F. Nature 1969, 222, 1159.
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of anions; as the dissolution process continues this ratio increases and at a total concentration of DoTAC of 80 mM, when the solid phase is completely solubilized, these contain 16 mM DS-, and there is, thus, a 5:1 DoTA+/DSratio in the micelles. This ratio is also consistent with the linear phase border of the phase diagram in Figure 1. When in total 80 mM DoTAC has been added to the system, the samples are one-phase micellar solutions and the fifth and final region of the diagram has been reached. Further addition of DoTAC changes the ratio of cation/anion in the mixed micelles. The mixed micelles are getting richer and richer in the cationic surfactant, and the free DSconcentration decreases. It is plausible that this induces a rod-to-spherical transition. Ly(OS)8 and DoTA+Cl-. The resolubilization of lysozyme from the complex with octyl sulfate, Ly(OS)8, with the addition of DoTAC is similar compared to the discussed solubilization of Ly(DS)8. As a result of the higher solubility product of the protein complex salt made from less hydrophobic octyl sulfate, a higher initial 0 is measured.6 The concentration of lysozyme Clysozyme same ratio of +/-, 0.2, is needed to dissolve Ly(OS)8 as in the analogue case of Ly(DS)8. In this series as well as in that previously described, a solid aggregate is observed also at surfactant charge ratios above 1. However, in the former system, this solid was soluble in 40 °C and identified as DoTADS, while in the present series with octyl sulfate, increasing the temperature does not result in dissolution of the solid. Thus, the solid is concluded to be remaining Ly(OS)8. When almost all lysozyme is in solution, the small amount of solid that remains is in equilibrium with a bluish solution. And with only a little excess of cationic surfactant, the whole sample is bluish and no solid remains. The solubilization of Ly(OS)8 with DoTAC can also be set into the context of the pure surfactant system. A priori it might seem appropriate to use the DoTAC-SOS system as a comparison. However, the two surfactants SDS and SOS are only quantitatively different from each other.4 The shorter carbon chain of SOS gives in the binary system rise to a higher cmc, while in multicomponent systems it is manifested by a higher critical aggregation concentration. The interactions (electrostatic and hydrophobic) in catanionic mixtures between the alkyl sulfates and DoTAC are also qualitatively the same. But as a result of worse packing conditions and a lower hydrophobic attraction (which in turn leads to a higher solubility and a higher charge density of the lamellar bilayer),18 the equimolar structure of DoTAC-SOS has a lower Krafft temperature than the symmetric DoTAC-SDS. Consequently, the phase behavior of DoTAC-SOS is the same as for DoTAC-SDS above the Krafft temperature of the solid DoTADS. This is corroborated by a comparison of Figure 1b with the phase diagram of CTAB-SOS-water presented by Yatcilla et al.10 Thus, as a base for the solubilization of Ly(OS)8 it is possible to use the phase diagram of DoTAC-SDS-D2O determined at 40 °C and presented in Figure 1b. By using this approach, the similarities between SDS and SOS are illustrated, because their differences are considered quantitative and not qualitative. In Figure 5, the resolubilization series is inserted into the phase diagram with the constant 1 wt % of Ly(OS)8 recalculated to a constant 5 mM of the OS- ion and set equal to 5 mM DS- at 40 °C. As before, the first sample that by UV could be determined to have solubilized (18) Khan, A.; Marques, E. Catanionic surfactants. In Specialist Surfactants; Robb, I. D., Ed.; Blackie Academic and Professional: London, 1997; p 37.
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Figure 5. Solubilization of Ly(OS)8 with DoTAC inserted into the phase diagram of SDS-DoTAC-D2O at 40 °C (shown also in Figure 1b). The filled line with symbols represents the solubilization of constant 1 wt % Ly(OS)8, that is, 5 mM OSwith additions of DoTAC: The appearances of the samples are O, (L1); b, (B); *, (S + B); ×, (S + L1). 3 marks the cation/anion ratio where lysozyme is first resolubilized from Ly(OS)8 according to UV spectroscopy.
lysozyme in solution coincides with the composition where a catanionic lamellar structure is first formed. This structure is on the anionic side probably in the form of vesicles because the samples are bluish and such a closed lamellar structure is known to form in especially asymmetric (C12-C8) catanionic mixtures.10 With increasing DoTAC concentration, the lamellar structure incorporates more and more anionic surfactant that is taken from the solid source, Ly(OS)8, and, thus, the samples are bluish solutions in equilibrium with a decreasing amount of solid. Close to equimolarity, all lysozyme is dissolved and the samples appear as blue solutions. At a concentration of 21 mM DoTAC, the bluish tint is gone and the samples are pure micellar solutions. This transition also occurs in the protein-free system. Thus, also when the anionic surfactant is the less hydrophobic analogue, lysozyme fails to compete with DoTAC. This conclusion is consistent with eq 2 because in the ratio Kss/K1/8 sp both factors are approximately equally affected by the decrease in length of the anionic surfactant. A diagram of free ion concentrations as in Figure 4 would display both similarities and differences compared to the case of Ly(DS)8. The limits would appear at lower total DoTAC concentrations because of the higher concentration of anionic surfactant in solution already in the beginning. However, the profiles of the variations would not be the simple straight lines as in Figure 4. The system with Ly(DS)8 is relatively easy to display because of the two stoichiometric solids with defined solubility products. In the present system, with octyl sulfate, lamellar structures are formed with a stoichiometry that can vary continuously, and consequently the description of the equilibrium constant is much more complex. Thus, we settle with the conclusion that lysozyme is solubilized also in this system in a way determined by the behavior of the catanionic mixture and that this behavior is less easy to characterize quantitatively because of the flexibility of the composition in the lamellar structure. Ly(OS)8 and Less Hydrophobic Cationic Surfactants. We have seen that an anionic surfactant, be it DSor OS-, strongly prefers to aggregate with the C12 DoTA+ cationic amphiphile relative to the lysozyme. A way to obtain a more balanced situation is to use a cationic amphiphile of shorter chain length. When the chain length of the cationic surfactant is shortened, we expect the constant Kss to increase by 1 order of magnitude for each of the two carbons. One then enters eq 2, where the second
Stenstam et al.
Figure 6. Solubilization of Ly(OS)8 with DoTAC (O), DeTAC (]), and OTAB (4), respectively: open symbols, (S + L1); gray circles, (L1 + LR), and filled black symbols, (S + V).
term in the denominator can be of order unity for a larger concentration range in Clysozyme. This implies that there is not a quantitative replacement of the lysozyme to the cationic surfactant. Figure 6 shows how the lysozyme concentration in solution increases on addition of the cationic surfactants with C12, C10, and C8 chains, that is, with DoTAC, DeTAC, and OTAB. The concentration of OTAB needed to solubilize Ly(OS)8 is high (more than four times the cmc). At this concentration and corresponding +/- ratio, no lamellar phases are observed. Instead, all samples feature a white solid and a clear supernatant. It can be concluded that lysozyme can compete for the anionic surfactant when we use cationic amphiphiles less hydrophobic than DoTAC. The resolubilization of Ly(OS)8 with DeTAC is compared to the analyzed system with DoTAC qualitatively the same. However, the association of lysozyme with octyl sulfate is strong enough to demand higher concentrations of DeTAC compared with DoTAC. With OTAB, the concentration at which the resolubilization of Ly(OS)8 is completed is several times higher than the cmc. Another conclusion can be drawn especially from the comparison of Ly(OS)8-SOS-H2O discussed thoroughly in ref 4 and Ly(OS)8-OTAB-H2O presented here: The association of lysozyme by octyl sulfate does not seem to result in an increased exposure of the hydrophobic interior. If that would be the case, a cooperative association of OTAB onto the protein-surfactant complex by the same mechanism as for SOS would be the likely path to resolubilization instead of the here presented mixed micelles. Thus, the association of SOS to Ly(OS)8 described in ref 4 must be attributed to a cooperativity toward the first eight electrostatically associated OS- tails and not to an exposed hydrophobic backbone. Conclusion The solubilization of the solid protein-surfactant complex salts by a second surfactant is governed by a completely different mechanism compared to the solubilization in excess of surfactant number one. In the latter case, it is the self-association of the surfactant onto the solid and neutral complex at a critical concentration that renders it soluble in water.4 In the cases presented here, it is the surfactant-surfactant association that is the driving force. The aggregate preferred by the respective catanionic mixtures dominates the picture. The aggregates found in these systems are the same as the ones in the respective protein-free systems, and, thus, it can be concluded that the presence of lysozyme plays a very small role in the formation of these structures. Indeed, in all the presented systems the measured lysozyme concentrations have been referred to as being the measure of free,
Lysozyme in Catanionic Surfactant Mixtures
unassociated lysozyme molecules. This can be based upon that the liquid crystalline phases, the vesicles, and the mixed micelles are positively charged. Thus, they are of the same net charge as lysozyme, which consequently is electrostatically repelled. Moreover, as the measured concentrations of lysozyme in the clear solution phase is more or less identical at 40 and at 22 °C, the idea of free lysozyme molecules is corroborated. On the basis of the known renaturation of lysozyme, it is even possible that the protein is in its native state after resolvation. In fact, preliminary activity tests indicate that the redissolved lysozyme has an activity comparable to an equally aged stock solution of lysozyme. A consequence of the results presented here is, thus, that lysozyme can be precipitated with SDS from a mixture, for example, a nonpurified batch of hen egg white proteins, and be resolvated by DoTAC, subsequently rendering a pure and active lysozyme solution. This approach might be as valid for other protein-catanionic systems as long as the second surfactant is hydrophobic enough to associate strongly with the first surfactant. The method is analogous to the artificial chaperon-assisted refolding presented by Rozema and Gellman.19 Their method, which gives very good yields of active protein, is based on an association of the protein with a surfactant molecule that in a second step is stripped (19) Rozema, D.; Gellman, S. H. Biochemistry 1996, 35, 15760.
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from the protein by association of a third molecule. In the present paper, we present a simpler version of the same principle. Thereby, we have been able to compare systems with varying hydrophobic interactions. This has made it possible to make conclusions of the comparative strengths of the protein-surfactant and surfactant-surfactant associations. Lysozyme can as a cationic colloidal species not compete with DoTAC for the association with SDS or SOS. The association between the oppositely charged surfactants is much stronger. With the shorter surfactants DeTAC or OTAB, the system is more balanced and the presence of lysozyme increases the amount needed of cationic surfactant to form the different catanionic structures. Finally, we argue for the approach used, which puts the more complex system into the context of a system with fewer components. Also, the difference in phase behavior of DoTAC with SDS or SOS is mainly due to the different Krafft points of the catanionic surfactants. It is demonstrated how interaction forces can be used to control the aggregation pattern. Acknowledgment. A.S. was funded by the Swedish foundation for strategic research in colloid and interface technology, SSF-CIT. LA049508W
