Surfactant Hydrophobic Effect on the Phase Behavior of Oppositely

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Langmuir 1998, 14, 6818-6826

Surfactant Hydrophobic Effect on the Phase Behavior of Oppositely Charged Protein and Surfactant Mixtures: Lysozyme and Sodium Alkyl Sulfates Anna Karin Moreń* and Ali Khan Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden Received April 2, 1998. In Final Form: September 9, 1998 Protein-surfactant interactions are investigated by following the phase equilibria of three lysozymesodium alkyl sulfate-water systems with surfactants of varying alkyl chain length, C10SO4, C8SO4, and C6SO4, within the concentration range 20 wt % lysozyme, 20 wt % surfactant, and 80-100 wt % water. Phase behavior similar to that for the system lysozyme-C12SO4-water is observed.1 The phase diagram of the C10SO4 system is dominated by a solution phase, a gel phase, and a multiphase precipitation region. For the C8SO4 and C6SO4 systems a solution phase, a precipitation region, and multiphase regions including gel are identified. However, no single gel phase is observed. Surfactants are sparingly soluble in aqueous solutions of lysozyme. Within the surfactant series the solubility is increased in the following order: C12SO4 < C10SO4 < C8SO4 < C6SO4. The composition of the neutral precipitate is determined to be about 8 surfactant molecules per protein molecule. The extension of the precipitation region toward higher surfactant concentrations is strongly dependent on the surfactant chain length; the shorter the chain length, the larger the precipitation region. This is explained by the maximum yield of precipitate being stable upon addition of a certain amount of excess surfactant, which is concentrated in the supernatant. The redissolution of the precipitate is induced by surfactant aggregation. The concentration of surfactant in the supernatant for the dissolution of the precipitate is less than the surfactant critical micelle concentration in water. The experimentally determined phase diagram can be understood qualitatively in terms of electrostatic and hydrophobic effects.

Introduction Proteins are formed by a mixture of amino acids in unique combinations. The different nonpolar, polar, and charged properties of the amino acids lead to complex interactions that determine the proteins’ secondary, tertiary, and quaternary structure. Due to the hydrophobic and hydrophilic properties of the amino acids, a protein exhibits a dualism that makes small amphiphilic molecules interact with proteins. Proteins interact strongly with oppositely charged surfactants in water due to a hydrophobic attraction between the surfactant tail and hydrophobic regions on the surface and in the interior of the protein as well as an electrostatic attraction between the headgroup of the surfactant and the protein. Surfactants can bind to the protein not only in the monomer form but also in an aggregated state, depending on the surfactant concentration.2,3 The interactions may result in a stabilization or a destabilization of the protein structure, depending on the surfactant concentration and the role as well as the natural environment of the protein.4,5 Interactions between proteins and surfactants are of importance not only in biological systems but also in many * To whom correspondence should be addressed. E-mail: [email protected]. Fax: 46-46-222 44 13. Work phone: 46-46-222 81 48. (1) Moreń, A. K.; Khan, A. Langmuir 1995, 11, 3636. (2) Dickinson, E. Protein in Solution and at Interfaces. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993; Chapter 7, p 295. (3) Ananthapadmanabhan, K. P. Protein-Surfactant Interactions. In Interactions of Surfactants with Polymers and Proteins, 1st ed.; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press, Inc.: London, 1993; Chapter 8, p 319. (4) Gumpen, S.; Hegg, P. O.; Martens, H. Biochim. Biophys. Acta 1979, 574, 189. (5) Waninge, R.; Paulsson, M.; Nylander, T.; Ninham, B.; Sellers, P. Int. Dairy J. 1998, 8, 141.

industrial systems. Proteins are, for example, added to washing detergents in order to improve the washing process. Another example is the sodium dodecyl sulfate (C12SO4) polyacrylamide-gel electrophoresis, where the interactions between proteins and surfactants are used to determine the molecular weight of proteins.6-8 Proteins and surfactants are also widely used to stabilize emulsions. To understand the interactions between surfactants and proteins in water different techniques have been used, for example, microcalorimetry, NMR, CD, and light scattering.9-14 Binding isotherms are also frequently determined,15-17 describing how the molar ratio of bound surfactants per protein changes upon increased surfactant concentration. In oppositely charged protein and surfactant systems, particular combinations of the electrostatic and hydrophobic interactions lead to the formation of various phases. In our previous work, the phase behavior of two oppositely charged protein and surfactant (6) Shirahama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309. (7) Takagi, T.; Tsujii, K.; Shirahama, K. J. Biochem. 1975, 77, 939. (8) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161. (9) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1980, 76, 654. (10) Chen, A.; Wu, D.; Johnson, C. S. J. Phys. Chem. 1995, 99, 828. (11) Turro, N. J.; Lei, X.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525. (12) Mattice, W. L.; Riser, J. M.; Clark, D. S. Biochemistry 1976, 15 (5), 4264. (13) Tanner, R. E.; Herpigny, B.; Chen, S.-H.; Rha, C. K. J. Chem. Phys. 1982, 16, 3866. (14) Gimel, J. C.; Brown, W. J. Chem. Phys. 1996, 104, 8112. (15) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1736. (16) Fukushima, K.; Murata, Y.; Nishikido, N.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1981, 54, 3122. (17) Fukushima, K.; Marata, Y.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1982, 55, 1376.

10.1021/la980368y CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998

Surfactant Hydrophobic Effect

systems was investigated:1,18 lysozyme-sodium dodecyl sulfate (C12SO4)-water and β-lactoglobulin (BLG)-dodecyltrimethylammonium chloride (DOTAC)-water. The systems exhibit generic phase behavior. Three different phase regions are characterized, a solution phase, a gel phase, and a precipitation region. A difference between the two systems is that in the BLG-DOTAC-water system a plateau in the precipitation curve is observed, which makes the precipitation region extend toward higher surfactant concentrations. A few studies are reported on the role of the surfactant chain length in protein and surfactant interactions.9,15,19-21 It is found that long tailed surfactants interact strongly with proteins, and interactions can be observed at low surfactant and protein concentrations. Since most of the reported work is performed at very low protein concentrations, the interactions between proteins and surfactants may not be observed with short chain surfactants. To obtain more information on the interplay between the hydrophobic and electrostatic effects, we have studied the phase behavior of three protein-surfactant systems with varying surfactant chain lengths: sodium decyl sulfate (C10SO4)-, sodium octyl sulfate (C8SO4)-, and sodium hexyl sulfate (C6SO4)-lysozyme-water. The results of this study in combination with those reported for lysozyme-C12SO4-water are used to explain proteinsurfactant interactions. Experimental Section Materials. Lysozyme from chicken egg white no. L-6876, three times crystallized, dialyzed, and lyophilized, was obtained from Sigma. C10SO4 (minimum purity 99%), C8SO4 (for tenside tests), and NaCl (zur analyze) were all from Merck whereas C12SO4 (especially pure) and C6SO4 (99% pure) were from, respectively, BDH and Lancaster synthesis. Water of Millipore quality was used. Sample Preparation and Analysis. This section is divided into three parts with slightly different procedures for the sample preparation. The samples are prepared (a) to examine the general phase behavior of the lysozyme-sodium alkyl sulfates-water systems, (b) to carefully determine the transition between the solution and the precipitation region in the dilute part of the ternary system, and (c) to analyze the formation and redissolution of the precipitate as a function of the surfactant concentration at a fixed protein concentration. (a) For phase diagram determination, the samples were prepared by weighing appropriate amounts of either the components or stock solutions of protein and surfactant into glass tubes which were flame-sealed. The equilibration procedure is previously described.1 The experimentally determined phase diagrams are representative after a period of 2 weeks. Multiphase regions and single phases are stable for years, but the formation of the gel near the borderlines takes a long time and its extension may change. The single phases and multiphase regions that are identified by mixing appropriate amounts of the components are also obtained by dilution of a protein-surfactant solution containing high surfactant concentrations; the reversibility of the systems is thus ensured. (b) To detect the first appearance of the precipitate at low surfactant concentrations, samples were prepared by adding small aliquots of surfactant stock solutions to 1.5 g of 1 and 3 wt % protein solutions. The solution-precipitation transition was additionally studied at low protein concentrations by adding small amounts of protein solutions to 1.5 g of 1 wt % surfactant (18) Moreń, A. K.; Eskilsson, K.; Khan, A. Colloids Surf., B 1997, 9, 305. (19) Reynolds, J.; Herbert, S.; Steinhardt, J. Biochemistry 1968, 7, 1357. (20) Kaneshina, S.; Tanaka, M.; Kondo, T.; Mizuno, T.; Aoki, K. Bull. Chem. Soc. Jpn. 1973, 46, 2735. (21) Griffith, P. C.; Stilbs, P.; Howe, A. M.; Whitesides, T. H. Langmuir 1996, 12, 5302.

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Figure 1. Isothermal pseudoternary phase diagram for the system lysozyme-C10SO4-water at 298 K: 9, solution; b, precipitation; +, precipitate, gel, and solution; 2, gel; × in a box, blue solution. solutions. All samples were prepared in small screw-capped test tubes with total volumes up to 1.65 g. After they were mixed for about 75 min, the samples were left standing to equilibrate for at least another 48 h prior to determining the turbidity of the samples on a Perkin-Elmer UV/vis Lambda 14 spectrometer. The turbidity was measured two times or more at two different wavelengths: 400 and 500 nm. (c) All samples prepared for the purpose of analyzing the behavior of the precipitation region have a total mass of 3 g. The protein concentration was fixed at 3 wt % lysozyme, whereas the surfactant and water content were varied. The mass of the empty glass tube was determined prior to the addition of components. The contents of the sample tubes were thoroughly mixed and equilibrated for about 48 h. The equilibrated samples were centrifuged in a bench-type centrifuge (6000 rpm) for 2 h or until a clear separation was obtained between the supernatant and the precipitated solid. For the analysis of the supernatant a suitable aliquot was carefully pipetted into a preweighed glass tube which, then, was freeze-dried for at least 72 h. To obtain the total amount of precipitate in the sample, as much as possible of the supernatant was carefully removed and the wet precipitate was freeze-dried in the same way as the supernatant. The total amount of free water in the supernatant and the total amount of precipitate in the sample were obtained from standard gravimetric measurements.

Results and Discussion Phase Behavior. General. Knowledge of the phase equilibrium has provided important information regarding the complex interactions between protein and surfactant in water. The formation, position, and extension of single phases as well as multiphase regions reflect the importance of forces operating in the system. For a homologous series of surfactants in which the surfactant headgroup is the same and the alkyl chain lengths are different, the electrostatic effect is approximately of the same magnitude, but the hydrophobic effect is strongly dependent on the length of the alkyl chain. Thus the hydrophobic effect on interactions between the protein and surfactant can be quantified by determining the phase behavior of the protein-surfactant systems by varying the alkyl chain length of the surfactant belonging to the same homologue. The phase diagrams obtained for the three systems lysozyme-C10SO4-, -C8SO4-, and -C6SO4-water are presented in Figures 1, 2, and 3, respectively. The samples used to construct the phase diagrams are examined at

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Figure 2. Isothermal pseudoternary phase diagram for the system lysozyme-C8SO4-water at 298 K. Symbols are the same those as in Figure 1, except for 4, which signifies the coexistence of white gel and solution.

More´ n and Khan

Figure 4. Effect of surfactant chain length on the phase transition between solution (L) and precipitate (P) for the lysozyme-C12SO4-water (square), lysozyme-C10SO4-water (circle), lysozyme-C8SO4-water (triangle), and lysozymeC6SO4-water (diamond) systems at 298 K. Open symbols represent solution, and filled symbols represent precipitate. Table 1. Maximum Solubility of Surfactanta in Aqueous Lysozyme Solution (1 wt % and 3 wt %) 1 wt %

C12SO4 C10SO4 C8SO4 C6SO4

3 wt %

wt % lys

wt % surf

[surf]/ [lys]

wt % lys

wt % surf

[surf]/ [lys]

0.94 0.96 0.96 0.91

5.8 × 10-4 2.7 × 10-3 1.9 × 10-2 0.17

0.031 0.16 1.3 13

2.7 2.9 2.9 2.8

5.7 × 10-4 2.8 × 10-3 1.6 × 10-2 0.082

0.011 0.054 0.34 2.1

a Solubility is given in weight percent surfactant and in moles of surfactant per mole of protein.

Figure 3. Isothermal pseudoternary phase diagram for the system lysozyme-C6SO4-water at 298 K. Notations are as in Figure 1.

room temperature (25 °C) and standard pressure (1 atm). The phase behavior of protein-surfactant mixtures in water is presented in triangular phase diagrams. However, it should be noted that the triangular plot only represents the mixing plane of the system. The total number of independent components in the system also includes counterions of the protein and surfactant. The phase equilibria for the mixtures are examined up to 20 wt % protein and 20 wt % surfactant and between 80 and 100 wt % water. About 70-100 samples are prepared for each ternary system, and the samples are concentrated around the phase boundaries. The pH in the protein-surfactant systems is checked over a wide concentration range of protein and surfactant, and it lies in the range pH ) 6-7. The isoelectric point of the lysozyme is 11.0, and accordingly the lysozyme has a positive net charge.22 Lysozyme-C10SO4-Water System. The ternary phase diagram of the system lysozyme-C10SO4-water is shown (22) Tanford, C.; Wagner, M. L. J. Am. Chem. Soc. 1954, 76, 3331.

in Figure 1. Lysozyme is easily soluble in water within the concentrations used to construct the phase diagram. A solution of lysozyme has a very limited capability to solubilize the oppositely charged surfactant C10SO4 prior to the formation of a precipitate. The solubilization of C10SO4 in two lysozyme solutions of 1 and 3 wt % is carefully determined, and the result is given in Figure 4 and Table 1. The amount of solubilized surfactant is on the order of 10-3 wt % C10SO4 for both protein concentrations. The corresponding molar ratio of solubilized surfactant is higher for the lower protein concentration, giving 0.16 molecules of C10SO4 per protein molecule in the 1 wt % sample against [C10SO4]/[lysozyme] ) 0.054 in the 3 wt % lysozyme sample (Table 1). The multiphase precipitation region is recognized by the presence of a white precipitate at the bottom of the sample tube or by a cloudy appearance of the solution. The region extends to the very dilute water corner. Upon increasing the surfactant concentration at very low protein concentrations, the precipitate persists until a surfactant concentration of about 0.32 wt % C10SO4, at which concentration the solid is redissolved, giving rise to an isotropic solution. At higher protein concentrations, the precipitation region becomes wider with respect to surfactant concentration, and in the upper part of the precipitation region (Figure 1) the white precipitate is found to coexist with an isotropic blue solution. Previ-


ously,1 we have reported that the bluish solution is a finely divided gel dispersed in solution. The gel phase which is formed at high protein concentrations is present as a narrow wedge with respect to surfactant concentration between the precipitation region and the solution region. Its width increases slightly as the protein concentration is increased. The minimum protein concentration needed to form the gel phase is about 6 wt %. The gel is highly viscous, bluish, and optically isotropic. The formation of the gel phase is preceded by that of a blue solution, and it may take about 2 weeks to form the gel. This phenomenon can be compared with that of the precipitate which is formed almost immediately after mixing surfactant and protein solutions. At surfactant concentrations just below the blue gel phase, some heterogeneous viscous samples are identified that consist of precipitate, gel, and solution in equilibrium. Upon increasing the surfactant concentration above the gel phase, a less viscous, bluish solution is formed, followed by a clear uncolored isotropic solution phase. The blue solution also extends to more dilute protein concentrations just above the precipitation region. The appearance of a blue solution is recognized in samples containing as high as 99 wt % water. To accurately define the extension of the blue solution as well as the blue solution in equilibrium with precipitate, more careful investigations are needed. The macroscopic appearance of the solution phase is clear and isotropic. It generally exhibits a viscosity close to that of water, but at high surfactant concentrations an increased viscosity is noted. Even though the solution is a homogeneous single phase, the different parts of the phase exhibit large differences on a molecular level. For example, in the narrow solution region, close to the binary protein-water axis, the negatively charged surfactant molecules are present with an excess of positively charged protein molecules. Though the structure of this part of the solution is not known, it is expected that proteinsurfactant complexes as well as surfactant monomers and free protein molecules are present in the solution. However, in the solution region above the precipitation region there is an excess of surfactant molecules. In this part of the solution phase, the soluble protein-surfactant complex has the same sign of charge as that of the surfactant molecules. Moreover, at high surfactant concentrations, the presence of surfactant aggregates both bound to protein and as free surfactant micelles has been suggested.2,3 Lysozyme-C8SO4-Water System. The ternary phase diagram determined for the system lysozyme-C8SO4water (Figure 2) forms the same three main regions as the lysozyme-C10SO4-water system: precipitation region, gel region, and solution phase. However, the two systems show different extensions and macroscopic properties of the regions. The lower borderline of the heterogeneous precipitation region lies close to the proteinwater binary axis, but the first appearance of precipitate occurs at slightly higher C8SO4 concentrations than those of the longer tailed surfactants. Both 1 wt % and 3 wt % lysozyme can solubilize C8SO4 on the order of 10-2 wt % prior to precipitation (Figure 4 and Table 1), which corresponds to a C8SO4 and lysozyme molar ratio of 1.3 and 0.34, respectively. Upon approaching the water-rich corner, the precipitation region shows a finite extension of 2.1 wt % C8SO4 along the surfactant-water axis. In addition to the investigation of the extension of the solution phase above the protein-water binary axis, we have also determined the amount of protein solubilized prior to the precipitation region at 1 wt % C8SO4 (Figure 4). It can be seen that the precipitation region extends to very dilute

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water concentrations and the surfactant solution solubilizes 4.6 × 10-3 wt % lysozyme prior to precipitation. As in the systems with longer surfactant chain lengths, the precipitation region becomes wider with increased protein concentrations. The macroscopic appearance of the gel in this system differs from that of the blue gel phase formed with the long chain surfactants C12SO4 and C10SO4. In the presence of C8SO4 the gel is white and opaque, and upon centrifugation it separates into a clear solution and a white gel. The formation of the white gel is preceded by that of a blue solution, as in the systems with surfactants having alkyl chain lengths longer than 8 carbons. The white appearance of the gel may be an effect of high ionic strength. This is due to a higher surfactant concentration in the region where the gel is formed, compared to those for the C12SO4 and C10SO4 systems. The explanation is further supported by the fact that the blue gel obtained for the lysozyme-C12SO4-water system becomes white on increasing the ionic strength by adding NaCl. The prestate of the white gel in the presence of NaCl is, as in the lysozyme-C8SO4-water system, a blue solution. The formation of turbid gels at high ionic strength is also reported in heat-set globular protein gels.23 Lysozyme-C6SO4-Water System. The phase diagram for the system lysozyme-C6SO4-water (Figure 3) is dominated by a heterogeneous precipitation region and a single solution phase. The aqueous protein solution can solubilize substantial amounts of sodium hexyl sulfate prior to the formation of a precipitate. The maximum solubilization measured at different protein concentrations (Figure 4 and Table 1) reveals that, at 1 wt % lysozyme, 13 molecules of C6SO4 are solubilized per protein molecule and the solubilization is decreased to [C6SO4]/[lys] ) 2 at 3 wt % protein. The solubilization of surfactant corresponds to about 0.1 wt % C6SO4 in the two protein solutions. The stability of the precipitation region toward higher surfactant concentrations, as the surfactant-water binary axis is approached, is about 11 wt % C6SO4. The amount of solubilized protein in 1 wt % C6SO4 is determined to be 0.1 wt % lysozyme, a slightly higher value than that in the C8SO4 system. The upper borderline between the precipitation region and solution phase at high surfactant concentrations is difficult to determine for this system. For example, a clear solution sample obtained close to the phase transition composition remains stable and clear on standing for several days at constant temperature, but the clear solution becomes cloudy upon turning the sample endover-end. In protein-surfactant systems with longer surfactant chain length, no similar behavior is observed. The phenomenon, though observed for the first time in protein-surfactant mixtures, is already reported for catanionic surfactant systems.24 The upper boundary line between the precipitation region and the solution phase was determined without disturbing the samples once the contents of the samples were mixed thoroughly and kept standing for equilibration. Lysozyme-C12SO4-Water System. The previously presented phase diagram of the lysozyme-C12SO4-water system1 is almost identical with that for the lysozymeC10SO4-water system. The same main regions are formed (precipitation region, solution phase, and blue gel phase) with minor differences in the stability of the regions. The (23) Doi, E. Trends Food Sci. Technol. 1993, 4, 1. (24) Khan, A.; Marques, E. Catanionic Surfactants. In Specialist Surfactants; Robb, I. D., Ed.; Blackie Academic & Professional. An imprint of Chapman & Hall: London, 1997; p 37.

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first formation of precipitate is determined at the two lysozyme concentrations 1 and 3 wt % lysozyme, and the results are presented in Figure 4 and Table 1. Both protein solutions solubilize about 6 × 10-4 wt % C12SO4, which yields [C12SO4]/[lysozyme] ) 0.031 for the low and [C12SO4]/ [lysozyme] ) 0.011 for the high protein concentration. General Features of Phase Equilibria. The isothermal ternary systems of lysozyme-sodium alkyl sulfate-water with surfactant chain lengths between 6 and 12 carbons exhibit a similar phase behavior. Single phases of gel and solution are characterized. Multiphase regions are identified, and they agree with the laws of phase science. The stabilities of multiphase regions including the gel phase are, however, difficult to determine due to their extremely narrow existence region. It is also hard to visually detect the blue color, as the samples become very dilute. In the series of alkyl sulfates with different lengths of the hydrophobic tail, all surfactants are sparingly soluble in an aqueous protein solution, and at the solubility limit a white surfactant-protein precipitate is formed. The degree of solubility is shown to be dependent on the length of the surfactant tail. The system with the least hydrophobic surfactant (C6SO4) forms the largest solution region prior to precipitation, and as the surfactant chain length is increased, the extension of the solution region is decreased. The most narrow solution region, formed with the most hydrophobic surfactant (C12SO4) in the series, is explained by the strongest hydrophobic attraction exerted between the hydrophobic tail and the hydrophobic regions of the protein. The effect on the extension of the solution phase as the hydrophobic length of the surfactant is changed is shown for two protein concentrations in Figure 4 and Table 1. By comparing experimental data it is shown that the relative molar solubility of the surfactant in a 3 wt % protein solution is increased by a factor of about 100 by exchanging sodium dodecyl sulfate with sodium hexyl sulfate. The amount of solubilized surfactant is, however, less dependent on the protein concentration. One weight percent lysozyme generally solubilizes the same amount of surfactant or even slightly more for the shorter surfactants compared to that for 3 wt % protein. As discussed above, the heterogeneous precipitation region extends to very dilute protein concentrations and is stable within a limited surfactant concentration along the surfactant-water axis. The extension of the narrow solution region between the precipitation region and the surfactant-water axis is investigated by adding protein to a 1 wt % surfactant solution for the C8SO4 and C6SO4 systems. The ability to solubilize protein is markedly higher in the less hydrophobic C6SO4 system, at which about 0.1 wt % lysozyme is solubilized compared to 4.6 × 10-3 wt % lysozyme in the C8SO4 system. The investigation is not performed with the longer surfactant tails, C10SO4 and C12SO4, since the extension of the precipitation region with these systems is very narrow at dilute protein concentrations. However, these systems are expected to follow the trend of the C8SO4 and C6SO4 systems and form precipitate even at lower protein concentrations. The extension of the precipitation region toward higher surfactant concentrations is dependent on the surfactant hydrophobicity and increases in the order C12SO4 < C10SO4 < C8SO4 < C6SO4. The surfactant concentration necessary to redissolve the precipitate at dilute protein concentrations seems to follow the cmc for the surfactant in water, which also increases with decreasing length of the surfactant tail. An extrapolation of the redissolution line to zero protein concentration, that is, approaching the surfactant-water binary axis, yields the surfactant

More´ n and Khan

concentration necessary for the redissolution of the precipitate on the order of about 37% of the cmc in water for C10SO4 and 69% of the cmc for C8SO4. The cmc of C12SO4 is too small to extract any reasonable value by extrapolation. The redissolution of the protein-surfactant precipitate requires rather large amounts of C6SO4. An extrapolation of the redissolution line toward zero protein concentration (Figure 3) yields about 11 wt % surfactant. The aggregation behavior as well as the cmc of C6SO4 has not been reported. However, the self-association behavior of the sodium hexanesulfonate-water system has been studied, and a cmc ) 9.4 wt % surfactant in water is obtained.25 For alkyl chain lengths longer than six carbons, the cmc values for both alkyl sulfate and alkanesulfonate are almost equivalent for equal chain lengths. It is reasonable to assume that the cmc of hexyl sulfate is somewhere near 11 wt % surfactant. The findings indicate that aggregation of surfactant is a precondition for the redissolution of the precipitate and that the surfactant concentration at which the dissolution occurs is below the cmc of the surfactant in water. Binding isotherm studies in combination with the redissolution of the precipitate reported for the lysozymeC12SO4-water system,16 as well as the phase diagram study of the BLG-DOTAC system,18 relate the redissolution of the precipitate to the surfactant aggregation process. Moreover, the cmc of a surfactant is shown to be reduced in the presence of a polyelectrolyte or protein.5 It has been suggested that protein-micellar complexes are formed at surfactant concentrations below the cmc of the surfactant in water.26,27 Also in surfactant-polymer systems critical aggregation concentrations (cac’s) lower than the cmc of the surfactant in water are reported.28 Compared with the cmc, the total redissolution of the precipitate of C10SO4 (37% of cmc) is relatively earlier than that of C8SO4 (69% of cmc). Interestingly, due to counterion effects the cac in polymer-surfactant systems also occurs at a relatively lower surfactant concentration for long tailed surfactants. The formation of a single gel phase within the investigated part of the phase diagram is dependent on the chain length of the surfactant. Both C12SO4 and C10SO4 systems form a single gel phase. However, the single gel phase is not obtained with surfactant chain lengths of 8 and 6 carbon atoms. The gel is found to coexist with precipitate and solution via appropriate multiphase regions (Figure 1). The multiphase regions containing gel, solution, and precipitate as well as gel and solution extend to very dilute protein concentrations, but probably not as dilute as the region observed with precipitate and solution in equilibrium. In the C8SO4 system a viscous gel in equilibrium with solution is observed. The single gel phase of the C8SO4 system, probably, exists in more concentrated regions of the phase diagram. In the C6SO4 system we have identified several samples of multiphase regions containing bluish solutions. This is a good indication that the C6SO4-lysozyme-water system may form a stable gel at higher sodium hexyl sulfate and lysozyme concentrations. (25) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surfactant Systems; NSRDS: Washington, DC, 1971. (26) Edwards, K.; Chan, R. Y. S.; Sawyer, W. H. Biochemistry 1994, 33, 13304. (27) Gettins, J.; Gould, C.; Hall, D.; Jobling, P.; Rassing, J.; WynJones, E. J. Chem. Soc., Faraday Trans. 2 1980, 76, 1535. (28) Lindman, B.; Thalberg, K. Polymer-surfactant interactionss Recent developments. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993; Chapter 5, p 203.


Figure 5. (a) Weight percent precipitate of the total sample versus [C12SO4]/[lysozyme], the molar ratio between surfactant and protein, at a constant 3 wt % lysozyme. (b) Amount of water in the supernatant at 3 wt % lysozyme as a function of the molar ratio [C12SO4]/[lysozyme]. The dashed line along the x-axis shows the decrease in the total amount of water on exchanging water with surfactant.

Study of the Precipitation Region. One of the main regions that is further characterized in the ternary phase diagram of oppositely charged protein and surfactant mixtures in water is the precipitation region. An analysis of the precipitation region is expected to yield information, primarily, on the physicochemical properties of the precipitate and the role of the hydrophobic effect in the formation and redissolution of the protein-surfactant precipitate. A similar precipitate-supernatant study of the lysozyme-NaCl-water system is additionally presented, to obtain information on the behavior of the precipitation region as a function of salt concentration. For all systems, the protein concentration is fixed at 3 wt % lysozyme and the surfactant concentration is increased stepwise within the precipitation region until the precipitate is redissolved. All samples are analyzed with respect to both the amount of precipitate in the sample and the amount of water in the supernatant, to obtain complementary results. The experimental data from the analysis of the supernatant are, however, more accurate. This is due to the fact that an aliquot of supernatant, free from precipitate, can be transferred from the sample tube for further analysis, whereas the separation of precipitate from supernatant always leaves small amounts of supernatant, which upon freeze-drying contribute an additional weight to the mass of the precipitate. The results obtained are shown in Figures 5-9 for the lysozyme-C12SO4-water, lysozyme-C10SO4-water, lysozyme-C8SO4-water, lysozyme-C6SO4-water, and lysozyme-NaCl-water systems. Two plots are presented for each system: part a) shows the wt % of precipitate in the total sample versus the molar ratio between surfactant and protein ([surfactant]/[lysozyme]), and part b) presents

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the wt % of H2O in the supernatant against [surfactant]/ [lysozyme]. In a series of samples, the total water content decreases, since the surfactant concentration increases and the protein concentration is fixed at 3 wt %. The decrease of water is shown in the supernatant figures as a broken line. For the purpose of discussion, we have subdivided the behavior of the precipitation region into four parts: the onset of precipitation, the precipitate maximum, a plateau, and precipitate redissolution. Lysozyme-C12SO4-Water. The amount of precipitate formed as a function of increasing surfactant concentration is previously reported for this system.1 The result is reproduced in Figure 5a. An analysis of the supernatant, performed in the present study, is presented as a function of C12SO4 concentration in Figure 5b. A good agreement between the two sets of data is observed. As stated above, a lysozyme solution of about 3 wt % can solubilize about 0.01 C12SO4 molecules per lysozyme molecule prior to the onset of precipitation. An increasing amount of precipitate is obtained as the surfactant concentration is further increased (see Figure 5a). In the supernatant this induces a decrease of the protein concentration; hence, the supernatant becomes relatively more rich with water, as seen in Figure 5b. The slope that describes the change in water content of the supernatant as a function of increasing surfactant concentration equals about 8 C12SO4 molecules per lysozyme molecule. This ratio corresponds to surfactants charge-neutralizing a protein molecule, since at pH 6.5 the net charge of lysozyme is +8. This finding is also reported in other studies.1,16,29 In the calculation of the slope describing the precipitate formation, all surfactant molecules are assumed to take part in the formation of the surfactantprotein precipitate. The concentration of surfactant in the supernatant is very low (Figure 4 and Table 1), and the solubilized surfactant is considered to have a negligible effect on the composition of the precipitate. The maximum precipitate is obtained at [C12SO4]/[lysozyme] ) 8, in this study as well as in the previous study; and at this concentration the most water-rich supernatant is found. At increased surfactant concentrations the precipitate starts to redissolve. The supernatant is enriched by the solubilized complex, and the relative water content is therefore decreased. Lysozyme-C10SO4-Water. The behaviors of the precipitate and supernatant at surfactant concentrations between the onset of precipitation and redissolution are respectively shown in parts a) and b) of Figure 6. The onset of precipitation occurs at [C10SO4]/[lysozyme] ) 0.05 (Table 1). As in the C12SO4 system, the slope of the supernatant curve (Figure 6b) that describes an increased formation of precipitate as well as the maximum amount of precipitate corresponds to global charge neutrality, that is, 8 C10SO4 molecules per lysozyme molecule. The maximum yield of precipitate remains unchanged with a further increase of the surfactant concentration between 8 and 11 C10SO4 molecules per lysozyme molecule, and beyond this concentration the precipitate begins to redissolve. If the behavior of the plateau region of the precipitate curve (Figure 6a) is compared with that of the supernatant (Figure 6b), the saturation of the precipitate with surfactant, that is, [C10SO4]/[lysozyme] ) 8, gives a maximum in the amount of water in the supernatant. When the surfactant concentration within the plateau region is increased, the amount of water in the supernatant decreases. The slope of the line is parallel to the slope of (29) Hegg, P.-O. Biochim. Biophys. Acta 1979, 579, 73.

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the broken line that describes the water content of the original samples in Figure 6b. This finding is an indication that the added surfactant molecules are partitioned into the supernatant and the net amount of precipitate remains unchanged within the plateau region. As the surfactant concentration is increased beyond the plateau region, the precipitate starts to redissolve. Lysozyme-C8SO4-Water. A substantial amount of sodium octyl sulfate ([C8SO4]/[lysozyme] ) 0.3) can be solubilized in an aqueous solution of 3 wt % lysozyme prior to the formation of a precipitate. The amount of precipitate (Figure 7a) and the water content of the supernatant (Figure 7b) increase as the surfactant concentration is increased until a maximum value at [C8SO4]/[lysozyme] ) 10. As the surfactant concentration is further increased, a large plateau region in the precipitate curve and a linear decrease in the amount of water in the supernatant curve are observed for several experimental values. For the C10SO4 system the linear decrease is parallel to the broken line in Figure 7b that represents the original concentration of water in the samples. For the precipitate curve, the amount of precipitate within the plateau region is found to increase slowly at high surfactant contents, though it is expected to remain constant. This is explained by the higher surfactant concentration in the supernatant, which erroneously contributes to the precipitate samples. At a molar ratio of [C8SO4]/[lysozyme] ) 50, the precipitate begins to resolubilize. To derive the composition of the precipitate using Figure 7b, the surfactant concentration needs to be either negligible compared to that in the precipitate or known

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in the supernatant. From the known amount of solubilized C8SO4 in lysozyme solutions prior to precipitation, it is apparent that the concentration of surfactant is too high in the supernatant to be neglected in the calculation of the precipitate composition. We also do not know the exact concentration of protein and surfactant in the supernatant for samples with different compositions. However, since the precipitate is neutral and the maximum in the amount of precipitate is close to global charge neutrality, it is reasonable to assume that the composition of the precipitate will be the same also for C8SO4. Lysozyme-C6SO4-Water. A precipitate is obtained at surfactant concentrations higher than 2[C6SO4]/[lysozyme]. It can be seen in Figure 8a that the amount of precipitate increases slowly, as the surfactant concentration increases, until a molar ratio of [C6SO4]/[lysozyme] ) 30. At higher surfactant concentrations a more steep increase in the amount of precipitate is observed. The precipitate reaches a maximum value at about [C6SO4]/[lysozyme] ) 50, and the plateau region extends to [C6SO4]/[lysozyme] ≈ 200. As the redissolution of the precipitate is approached, there is an increase in the amount of precipitate, which presumably originates from the high surfactant concentration of the supernatant that erroneously contributes to the precipitate. As in the systems with longer surfactant chain length, the supernatant curve (Figure 8b) is parallel to the broken line within the plateau region. This finding supports that within this region there is a constant amount of precipitate. Another observation is that the supernatant curve describing the redissolution of the precipitate does not show the steep decrease in the amount of water upon increased surfactant concentrations shown for the



surfactants with longer chain lengths. These results indicate that the efficient solubilization of the precipitate with long-tailed surfactants is related to a strong cooperativity of the surfactant self-assembly process, which is not the case for the short C6SO4 system.25 Lysozyme-NaCl-Water. A 3 wt % lysozyme solution can solubilize about 2 wt % NaCl prior to precipitation. This corresponds to a molar ratio much higher than that in the surfactant systems. The amount of precipitate (Figure 9a) is continuously increased as more salt is added. However, the amount of precipitate increases over a much wider concentration range and does not exhibit the same proportional increase as found in the surfactant systems. The slope between the last two points in the supernatant curve (Figure 9b) is parallel to the slope of the dashed line, indicating that at about 6 wt % NaCl the added salt is enriched in the supernatant and no additional precipitate is formed. General Features of the Precipitation Region. As the surfactant chain length is decreased from C12SO4 to C6SO4, it is observed that the first formation of precipitate appears at an increased surfactant concentration. The amount of formed precipitate thereafter increases over a narrow surfactant concentration in all surfactant systems except the C6SO4 system. The maximum amount of obtained precipitate for the relatively long chain surfactants lies between 8 and 10 surfactant molecules per lysozyme molecule, whereas in the C6SO4 system the maximum appears first at [C6SO4]/[lysozyme] ) 50. Beyond the maximum extends a plateau for all surfactant

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Figure 9. (a) Weight percent precipitate of the total sample versus [NaCl]/[lysozyme], the molar ratio between salt and protein, at a constant 3 wt % lysozyme. (b) Amount of water in the supernatant at 3 wt % lysozyme as a function of the molar ratio [NaCl]/[lysozyme]. The dashed line along the x-axis shows the decrease in the total amount of water on exchanging water with salt.

systems. It is to be noted that no significant plateau region is observed for the C12SO4 system, since the stepwise increase of the surfactant concentration is too high compared to the extension of the plateau region. However, the presence of a plateau is reported by Fukushima et al.16 Within the plateau region the added surfactant is concentrated in the supernatant and no more precipitate is formed. The extension of the plateau becomes larger as the length of the surfactant chain becomes shorter. The plateau of the precipitate curve can be related to the extension of the precipitation region toward higher surfactant concentrations in the ternary phase diagram. The redissolution of the precipitate occurs over a significantly wider surfactant concentration for the shorter surfactants C8SO4 and C6SO4. For the system with NaCl and lysozyme the first formation of precipitate occurs at a much higher concentration than that in the surfactant-protein systems and the formed precipitate also increases over a wider concentration range. Deviations in the formation of the precipitate for the C6SO4 system may be due to the fact that the short chain hexyl sulfate is not behaving like a long tailed surfactant. No further precipitate is formed at about [NaCl]/[lysozyme] ) 500. Unlike the case for the lysozyme-surfactant systems, the precipitate is not redissolved at high concentrations of salt. This finding in combination with the results of the precipitation analysis of the surfactant systems clearly indicates that surfactant aggregation in the supernatant is a precondition for the redissolution of the precipitate.

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Concluding Remarks The most important observations previously described in detail are summarized as follows: (i) It is shown that protein-surfactant interactions, though complex, can be studied conveniently by the phase equilibrium method over a wide concentration range. Knowledge of formation of phases, such as the gel phase that has not been reported previously, as well as extension of multiphase regions is of great importance and is a good base for further studies. (ii) Starting from the protein-water axis, the interactions between lysozyme and CnSO4 (n ) 12-6) give rise, in succession, to the formation of a solution, a precipitate, and a gel and, finally, to the redissolution of precipitategel to a large solution phase with increasing surfactant concentrations.

More´ n and Khan

(iii) For an oppositely charged protein-surfactant pair, the interactions are dominated by the electrostatic and hydrophobic forces. The hydrophobic force is always attractive, and the electrostatic force can, depending on composition, be attactive and repulsive in nature. When the alkyl chain length, that is, the hydrophobic part of the surfactant, is increased, the capability of the system of both forming and redissolving the precipitate increases. The precipitate is neutral, and its composition reveals the net charge of the protein in solution. Acknowledgment. This project is financed by the Swedish Research Council for Engineering Sciences (TFR). LA980368Y

Surfactant Hydrophobic Effect on the Phase Behavior of Oppositely

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