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The Lysozyme-Dodecyl Sulfate System. An Example of Protein-Surfactant Aggregation Anna Stenstam,* Ali Khan, and Håkan Wennerstro¨m Physical Chemistry 1, Lund University, P.O. Box 124, S-221 00 Lund, Sweden Received July 16, 2001. In Final Form: September 19, 2001 Mixtures of lysozyme and sodium dodecyl sulfate, SDS, are used to study protein-surfactant interactions. By precipitating the complex salt, CS(12), where DS- ions are the counterions to the positively charged lysozyme, a stoichiometric protein-surfactant component free from simple salts is synthesized. The solubility product of the complex salt is determined, and it provides a measure of the hydrophobic interaction in the precipitate. Aqueous systems of mixtures of the complex salt and the pure surfactant form a true threecomponent protein-surfactant-water system. The resulting phase behavior is studied and used as a fundament for theoretical discussion as well as modeling. With additional electrolyte excluded, the role of the electrostatic interactions is maximized, and the free energy balance between the different aggregates is shown to be determined by a balance between electrostatic and hydrophobic forces. We find three types of protein-surfactant aggregates: the insoluble complex salt, an electrostatically swollen gel-like state containing macroscopic aggregates, and a soluble complex containing a single protein molecule.
Introduction Protein self-association occurs under a range of circumstances in vivo. In many cases the association process is a part of a biological function as in blood clotting or the formation of muscle fibers. In other cases the aggregation leads to a perturbation of the biological function with sometimes serious physiological consequences as in the formation of cataracts in the lens of the eye or amyloid fibrils associated with Alzheimer’s and other neurological diseases. From a colloid chemistry perspective, protein selfassociation is a special case of the general problem of colloidal stability. There are two important aspects of the protein systems in this respect: first in contrast to colloids in general the system can be obtained in pure form and then represent a true single component. Second the protein has a complex molecular structure and one should expect protein-protein interactions to be highly directional. Protein self-association can be triggered by chemical transformations, it is also sensitive to physical parameters such as temperature and pressure. Moreover, it is strongly affected by changes in the properties of the medium, such as, pH, the electrolyte concentration, and the presence of a cosolvent or additive.1,2 The present paper is devoted to a study of the role of an oppositely charged surfactant.3 It is known that the interaction in an oppositely charged system is very strong, and consequently surfactants have been shown to be strong denaturing agents.2,4,5 It is also known that the strength of the interaction is rendered * To whom correspondence should be sent. Please use postal address, e-mail
[email protected], or fax +46-46-222 44 13. (1) Clark, A. H.; Lee-Tuffnell, C. D. Gelation of Globular Proteins. In Functional Properties of Food Macromolecules; Mitchell, J. R., Ledward, D. A., Eds.; Elsevier Applied Science: Amsterdam, 1986; p 203. (2) Dickinson, E. Proteins in solution and at interfaces. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; 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.: Boca Raton, FL, 1993; p 319. (4) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1736.
not only by the electrostatics of the opposite charge but also from the hydrophobic attraction between protein interface and surfactant tails.4,6-8 However, most of these earlier studies have been performed on very dilute systems. Information on the behavior at higher concentrations of both protein and surfactant has only recently begun to be reported.9,10 We have chosen to work with the protein lysozyme. It is a small globular enzyme with 18 cationic and 12 anionic residues. The isoelectric point is at pH 11.11 An aqueous solution of the protein has a pH of 6.5, and this renders the protein a positive net charge of 8.11 At this pH and the low salt conditions used in this work, lysozyme is predominantly monomeric.12 The interior of the protein globule is almost entirely hydrophobic while the interface is covered by both the charged amino acid residues and apolar patches, e.g., aromatic side groups. The considerable conformational stability is well documented13 and is primarily attributed to the four disulfide bridges. This intrinsic stability of the globular structure will turn out to be of great significance in the present study. The threedimensional structure of the crystal was determined in 1965 by Blake et al.14 and in solution; the main chain fold is very close to that of the crystal.15 The interactions of lysozyme with oppositely charged surfactants such as sodium dodecyl sulfate, SDS and (5) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1980, 76, 654. (6) Jones, M. N.; Manley, P.; Midgley, P. J. W. J. Colloid Interface Sci. 1981, 82, 257. (7) More´n, A. K.; Khan, A. Langmuir 1998, 14, 6818. (8) Griffith, P. C.; Stilbs, P.; Howe, A. M.; Whitesides, T. H. Langmuir 1996, 12, 5302. (9) More´n, A. K.; Khan, A. J. Colloid Interface Sci. 1999, 218, 397. (10) Valstar, A.; Vasilescu, M.; Vigouroux, C.; Stilbs, P.; Almgren, M. Langmuir 2001, 17, 3208. (11) Tanford, C.; Wagner, M. L. J. Am. Chem. Soc. 1954, 76, 3331. (12) Price, W. S.; Tsuchiya, F.; Arata, Y. J. Am. Chem. Soc. 1999, 121, 11503. (13) Imoto, T.; Johnsson, L. N.; North, A. C. T.; Phillips, D. C.; Rupley, J. A. Vertebrate lysozymes. In The enzymes, 3rd ed.; Boyer, P. D., Ed.; 1972; Vol. 7. (14) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206, 757. (15) Smith, L. J.; Sutcliffe, M. J.; Redfield, C.; Dobson, C. M. J. Mol. Biol. 1993, 229, 930.
10.1021/la011096t CCC: $20.00 © 2001 American Chemical Society Published on Web 11/03/2001
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sodium octyl sulfate, SOS have been thoroughly examined.7,16 Reports are made on several different associating features. First, the oppositely charged macromolecules are known to form a precipitate. Second, this precipitate can be resolubilized by adding more surfactant.17 The soluble complexes are reported to be of two types, of different size.16 Third, the resolubilization process is by phase behavioral studies shown to proceed via a narrow region of a gel.18 It is a major objective to establish a link between the intermolecular interactions and the macroscopic phase behavior. This is easier the fewer the components. However, globular proteins always come with inorganic salts such as sodium chloride and sodium acetate. By dissolving crystalline protein and surfactant, one obtains a four- or five-component system. To circumvent this obstacle, we have taken advantage of the fact that the protein and surfactant associate strongly. When the net positive protein lysozyme is subjected to an anionic surfactant in solution, it can be neutralized. The complex is then precipitated. If this white powder is rinsed from all common counterions, what remains is the stoichiometric salt of protein and surfactant. In this paper, it will be called a complex salt. The complex salt is a single component in a system also containing water and additional surfactant. This generates a true three-component system and implies that the Gibbs phase rule applies strictly at equilibrium. A similar approach to studies of the phase behavior of a surfactantpolyelectrolyte system was recently described by Svensson et al.19 Experimental Section (a) Materials. Lysozyme (95%), L-6876 lot no. 111H7010, from chicken egg white three times recrystallized, dialyzed, and lyophilized was obtained from Sigma. Sodium dodecyl sulfate, SDS (specially pure), was supplied by BDH and sodium octyl sulfate, SOS, from Merck. R-Deuterated SDS was synthesized by Synthelec AB (Lund, Sweden). Both surfactants form fairly monodisperse micelles above their critical micelle concentration (cmc), which is 8.3 and 133 mM, respectively.20 All chemicals were used as obtained. Millipore filtered water was used as solvent. To minimize the number of components, no buffer was used. This is possible as lysozyme is very stable and to a large extent considered self-buffering. (b) Synthesis and Characterization of the Complex Salts. The complex salts of lysozyme and anionic surfactant were prepared by precipitating the protein-surfactant complex from an aqueous solution. To obtain as much material as possible, precipitation was performed close to the point where a maximum of protein molecules are net neutralized by oppositely charged surfactants. Without any buffer present the pH of the solution is 6.5, and the lysozyme molecule has a positive net charge of 8 at this pH.11 We assume that in the precipitate the protein is neutralized by surfactant anions only. Then the stoichiometry of the complexes is 1:8, lysozyme to surfactant. Hence, the complex salt of lysozyme and SDS, abbreviated CS(12), is composed of 87 wt % lysozyme and 13 wt % dodecyl sulfate ion DS-. Correspondingly, the complex salt of lysozyme and SOS, CS(8), is composed of 90 wt % lysozyme and 10 wt % octyl sulfate ion OS-. All batches of CS(12) were prepared from mixtures of 3 wt % lysozyme and 0.37 wt % SDS in Millipore water solution. Subsequently all batches of CS(8) were prepared from mixtures of 3 wt % lysozyme and 0.48 wt % SOS. The samples were shaken (16) Valstar, A.; Brown, W.; Almgren, M. Langmuir 1999, 15, 2366. (17) Fukushima, K.; Murata, Y.; Nishikido, N.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn 1981, 54, 3122. (18) More´n, A. K.; Khan, A. Langmuir 1995, 11, 3636. (19) Svensson, A.; Piculell, L.; Cabane, B.; Ilekti, P. Accepted for publication in J. Phys. Chem. (20) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surfactant Systems; NSRDS: Washington, DC, 1971.
Stenstam et al. on and off for 4 h before being allowed to equilibrate at 25 °C for 24 h. The precipitates were further filtered through ordinary Millipore filter paper with a pore size of 5 µm. The filtrates were rinsed with 5 × 10 mL of Millipore filtered water. After being rinsed, the filtrates were freeze-dried for, at least, 50 h. The material was stored in an exicator. The white powder obtained was confirmed to be free from sodium ions by elementary analysis. This was performed by SGAB in Luleå, Sweden. The inductively coupled plasma atomic emission spectroscopy method did not detect any presence of Na+ ions. The method has sensitivity similar to atom absorption in open flame; i.e., a concentration of 24 mg/kg would have been detected. The name complex salt is thus found appropriate, as when the common salts are removed the surfactant and the protein truly act as each other’s counterions. As the neat protein is balanced with sodium acetate and sodium chloride buffers to the extent of 5% (according to Sigma), the precipitation procedure results in a significant reduction of the electrolyte ion content. Further, all batches of complex salt were identified as very similar by Raman spectroscopy. This was necessary as we prepared several batches in order to obtain enough material to cover the phase behavior in the high concentration range. In Raman analysis, special attention was given to the vibrational modes of the amide group in the protein backbone, i.e., the peaks at 1630-1690 cm-1. (c) Ultraviolet Spectroscopy. To measure the solubility of the complex salts in water, the concentration of protein in the top solution in equilibrium with the salt was measured. This was done using the absorbance of UV light at λ ) 280 nm. The apparatus used was a Perkin-Elmer Lambda 14 spectrometer. Samples were prepared with 1 wt % complex salt. After the samples were vortexed, they were allowed to stand for 5 days. Absorption measurements were made on the supernatant. (d) Raman Spectroscopy. Raman spectroscopy experiments were performed for two reasons. Primarily, a spectrum of the complex salt was considered as a fingerprint and all batches prepared were checked by Raman before use. In addition, the method was used in order to follow the conformational state of the protein when subjected to surfactant. For a good signal-tonoise ratio, samples with at least 10 wt % protein were used. The apparatus used was a BioRad fast-Fourier transform instrument equipped with a Nd:YAG laser. (e) Phase Diagram Determination. All samples were prepared in screw-capped vials. The complex salts were always added as the neat pulver while stock solutions of surfactant were used for the more dilute samples. After initial vortexing the samples were put in a gentle shaker for at least 16 h at 25 °C. The phase equilibria studies on the complex salt-surfactantwater system were performed by ocular observation of the samples after they had been left standing at 25 °C for at least 2 weeks. The samples were then kept for 6 months and examined from time to time to follow possible changes. Samples were prepared in the concentration regime of 80-100 wt % water. In the CS(12) system more than 70 samples were prepared only for the phase diagram determination. In the CS(8) system a limited number of samples were prepared close to the interesting phase borders.
Results and Discussion The Phase Behavior of Complex Salt-SurfactantWater Systems. One way toward understanding the complex interactions involved in a protein-surfactant system is through the phase behavior of the system. This is often illustrated by a two-dimensional phase map presenting protein, surfactant, and water as components. In this way one neglects the presence of common salts, present as counterions and buffer. As the interaction in a system of oppositely charged components, such as lysozym--SDS-water, is very much the result of the magnitude and sign of the electrostatic forces, the general screening effects of simple salts cannot be neglected. In this work, we have thus eliminated the simple salts from the system to maximize the role of the electrostatic interactions. Another simplifying consequence is that
The Lysozyme-Dodecyl Sulfate System
Figure 1. (a) Three-component phase diagram for the complex salt of lysozyme-dodecyl sulfate CS(12)-SDS-water at 25 °C: (L) isotropic solution; (G) gel phase; (S) solid CS(12). (G + L), (S + L), (S + G), and (S + G + L) are appropriate two- and three-phase regions. (b) Schematic phase diagram where the molar ratio of surfactant to protein, S/P, is noted.
CS(12)-SDS-water is a true three-component system and thus can be fully represented by a two-dimensional phase diagram where the Gibbs phase rule applies strictly at equilibrium. Figure 1 shows a phase diagram for the CS(12)-SDS-water system. It is qualitatively similar to that of the pseudoternary system lysozyme-SDS-water presented by More´n and Khan.18 We find the same analogies between the CS(8)-SOS-water phase diagram and the corresponding pseudoternary system by More´n and Khan.7 The ternary phase diagram of CS(12), SDS, and water in Figure 1 shows three main features. First, at small SDS content, a region where the complex salt is insoluble and coexisting with a solution that contains some surfactant and small amounts of protein (S + L). Second, a region where larger aggregates or networks are present throughout the sample leading to high viscosity and bluish color (G). Third, the clear nonviscous solution phase where the protein is completely solubilized by the surfactant (L). The locations of the three different regions are mainly determined by the surfactant-protein molar ratio, S/P. The solid complex salt begins to dissolve at a ratio of 9 S/P, and it is completely dissolved at a S/P ratio of 14. Within these limits the solid is gradually dissolving. The supernatant is bluish and with a viscosity that is increasing with increasing surfactant concentration. When no traces of solid can be observed, the whole sample is bluish and gelled. If the concentration of surfactant is increased above a S/P ratio of 18, the sample becomes less viscous than the gel but has retained the bluish appearance. Finally, with even more SDS to lysozyme, a large onephase area is entered at a S/P ratio of approximately 22. This isotropic solution is of low viscosity containing finite aggregates where the protein is solubilized by micellelike aggregates.16 The accuracy of the phase stability limits reported above is dominated by the sample preparation and identification of the phases. The accuracy of the first is determined by the accuracy of preparing samples gravimetrically and is obtained to 0.1 wt %. The reliability of the identification of phases is considerably harder to assess, but after 2
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weeks the relevant phases have emerged in the samples, which have then been observed for the additional 6 months to confirm the initial assignments of phase regions. The largest uncertainties are in the determination of the limits of the gel phase region since this structure takes a long time to equilibrate. The precipitate, the gel, and the isotropic solution are considered as three single phases. They are connected by multiphase regions. Consequently the gel is found between a blue solution (gel dispersed in isotropic solution) and samples of gel and insoluble solid. There is also a threephase triangle, complex salt-gel-isotropic solution. The phase behavior gives a thermodynamic characterization of the system on a free energy level. It is a considerable challenge to go from this macroscopic description to an understanding of the molecular interactions responsible for this macroscopic behavior. The practice of the true ternary system however significantly reduces the number of assumptions since Gibbs’ phase rule implies.21 Below we discuss the separate phases and correlate structure with the basic interactions operating in the system. The Insoluble Complex. The precipitate region (S + L) consists of the insoluble complex salt in equilibrium with a clear solution of excess surfactant. Since it forms a separate phase, the complex is by necessity electroneutral. Lysozyme has a net charge of +8, and the most likely process leading to the complex salt is
Ly8+ + 8DS- S Ly(DS)8(s)
(1)
Other conceivable alternatives are that there is an uptake or a production of a proton in the precipitation reaction:
Ly8+ + OH- + 7DS- S Ly(DS)7(s)
(2)
Ly8+ + H+ + 9DS- S Ly(DS)9(s)
(3)
Both of these reactions depend on the rather delicate balance between the binding of a surfactant present in low concentrations and the protonation/deprotonation of the protein, the energetics of which depend on pH and the pKa values of the protein molecule. However, in the system studied, buffers were not used, and if reactions 2 and 3 occurred to any extent, the pH would change substantially, which is not observed. (A combination of the two reactions simply adds up to reaction 1.) Chemical analysis showed that the purified complex salt does not contain any sodium counterions and we, thus, arrive at the conclusion that the complex salt is the electroneutral protein-surfactant complex with the stoichiometry Ly(DS)8. This conclusion is supported by earlier experimental work on lysozyme with three sodium alkyl sulfates of different chain lengths where maximum precipitation of lysozyme, for all three systems, occurred at a S/P ratio of 8.7 The complex salt has a low but measurable solubility in water. The UV spectrum of the supernatant in equilibrium with solid salt can be used for a quantitative determination of the protein concentration. The stability of the complex salt is due to a combination of the electrostatic attraction between the cationic protein and the anionic surfactant and the hydrophobic attraction among surfactant alkyl chains and between surfactant chains and hydrophobic groups of the protein. A way to specifically study the role of the hydrophobic interaction (21) Evans, D. F.; Wennerstro¨m, H. The colloidal domain: where physics, chemistry and biology meet, 2nd ed.; Wiley-VCH: New York, 1999; Chapter 10.
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is to change the length of the alkyl chain. For this purpose we have also prepared the complex salt of sodium octyl sulfate CS(8). The effects of hydrophobicity are manifested as a difference in solubility between the salt formed by SDS and that by SOS. As the hydrophobic attraction between the protein and the surfactant tails are much larger for the longer surfactant, the solubility of CS(12) is much lower than that for CS(8). In the study of CS(12) the observed concentration of lysozyme in the supernatant is only 5 µM, while it has increased by a factor of 22 to be 110 µM in the case of CS(8). The stoichiometry of the two complex salts is the same, and we can then calculate the difference in free energy of formation of the two complex salts. The additional four CH2 groups of SDS compared to SOS cause the difference between the two salts. The free energy change per methyl group could thus be computed.
∆G°(SDS) ) -kT ln[lysozyme]1 - 8kT ln[DS-] (4) ∆G°(SOS) ) -kT ln[lysozyme]2 - 8kT ln[OS-] (5) Now considering a pure stoichiometric salt, the surfactant concentration should be eight times the protein concentration since in this case the only source of the surfactant is from the dissolved complex salt so that
[DS-] ) 8[lysozyme]1; [OS-] ) 8[lysozyme]2 (6) The difference in standard free energy of formation is then
∆∆G° ) ∆G°(SOS) - ∆G°(SDS) ) -9/8 kT ln{[lysozyme]1/[lysozyme]2} ) -9/8 kT ln(22) Thus, for each additional CH2 group in the alkyl chain, we have an estimated contribution to the free energy of formation of
∆∆G°(CH2) ≈ -0.9kT or -2.2 kJ/mol This value is close to the free energy per CH2 group of approximately -1.0 kT or -2.5 kJ/mol typically found for formation of a micelle.22 Therefore we conclude that in addition to the electrostatic effect, the hydrophobic interaction provides a central contribution to the formation of the complex salt. In the calculation we have assumed that there is no association in the solution under the given circumstances. This implies that the formation of the complex salt is a highly cooperative process. This conclusion is supported by the observation that when SDS is added to a lysozyme solution, a precipitate starts to form directly without a buildup of soluble aggregates even in the presence of rather high protein concentrations. This behavior is in fact in contrast to the association between anionic surfactants and cationic polymers, where one usually observes a continuous association of surfactant and polymer and a precipitation occurs first close to the point where one has an overall charge balance between polymer and surfactant.23 This difference between the lysozyme and cationic polymers can be due to a combination of that the lysozyme (22) Evans, D. F.; Wennerstro¨m, H. The colloidal domain: where physics, chemistry and biology meet, 2nd ed.; Wiley-VCH: New York, 1999; Chapter 4. (23) Goddard, E. D. Polymer-Surfactant Interaction. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993.
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has only a small net charge, which is easily neutralized, and that it also has a compact conformation allowing for local interactions with a few surfactant molecules. The very low solubility product is indeed a characteristic feature of the complex salt. From the equilibrium pure complex salt solution described above, we found a lysozyme concentration of 5 µm and, implying for a 1:8 stoichiometry, a solubility product of 3 × 10-41 M9 as:
Ks ) [lysozyme][surfactant]8
(7)
An independent test of the solubility product argument was obtained by adding SDS to a very dilute solution of lysozyme. On addition of SDS to lysozyme solutions of 0.1 and 0.35 mM, a precipitate is detected at SDS concentrations of 60.4 and 77.4 mM, respectively. This implies solubility products of 1.8 × 10-38 M9 and 4.5 × 10-37 M9, respectively. Even though the agreement is not quantitative, we find that the obtained results support the basic assumptions. Due to the difficulty in detecting small amounts of precipitate in these dilute systems, we obtain a slight overestimate of the solubility limit. Further there is some electrolyte present that stabilizes the soluble charged species relative to the virtually electrolyte-free system of the pure complex salt. The thorough work by Fukushima et al. 17 where the binding isotherms of SDS to lysozyme compared to precipitation curves can be used to obtain a solubility product in the same range, i.e., 1.2 × 10-34 M9. It is well established that ionic surfactants in general unfold proteins.3 The basic mechanism of action of general unfolding agents such as urea and guanidinium hydrochloride is to stabilize the unfolded protein conformations. On the other hand, surfactants have a more complex behavior in that they can also interact with native or compact conformations of globular proteins. The fact that the complex salt has a well-defined stoichiometry, that it is in a solid phase where conformational entropy effects are of limited importance, and that it forms in a highly cooperative way indicates that we are, even in the presence of this amount of surfactant, dealing with a compact protein structure rather than an unfolded highly disordered one. To characterize changes in the conformational state of the protein, we have used vibrational spectroscopy. Conformational changes affect the normal modes of the polymer backbone, and thus the vibrational modes of the amide group are studied using Raman spectroscopy. The vibration of the CdO bond, the amide I mode, is a useful probe of the secondary structure of proteins.24 The amide I mode at 1650-1655 cm-1 is characteristic for an R-helical conformation. For a β-conformation, the peak appears at 1666 cm-1. The disordered structures result in broadening of the lines. Consequently here is only reported whether the prominent conformation is an R or a β structure, although a disordered element might be present. Figure 2 shows a series of Raman spectra in the range 16001700 cm-1 of lysozyme in different phases. Raman spectroscopy indicates that lysozyme subjected to SDS is no longer in its native state. The amide I peak is shifted toward higher wavenumbers from 1660 cm-1 reported for aqueous lysozyme by Lord and Yu.25 Thus the secondary structure of lysozyme in the complex salt has a larger contribution of β structure than the neat protein. Further, the vibrational spectra of the solid reveals (24) Koenig, J. L. Raman Spectroscopy of Biological Molecules: A Review. In J. Polym. Sci.: Part D 1972, 59. (25) Lord, R. C.; Yu, N-T. J. Mol. Biol. 1970, 50, 509.
The Lysozyme-Dodecyl Sulfate System
Figure 2. The amide I peak from spectra of neat lysozyme, neat CS(12), the lysozyme-SDS gel and the soluble lysozyme-SDS complex, L. The concentration of protein is 14 wt % in all samples.
additional changes in the environment of the polypeptide’s side groups. We conclude that in the complex salt the lysozyme molecule is still in a compact conformation but with a changed secondary structure compared to its soluble and its crystal, native conformation. The Gel. The most intriguing and interesting feature of the ternary system is the appearance of a gel-like structure in a well-defined region of the phase diagram. The pure gel is found in a narrow range of surfactantprotein ratios between 14 and 18. For S/P e 14 the gel coexists with the complex salt while for S/P g 18 there is a coexistence with the isotropic low viscous phase. However, in this latter case we do not observe a macroscopic phase separation between gel and isotropic solution. These systems rather appear as bluish dispersions of gellike aggregates with a viscosity that varies with the concentration. Following More´n and Khan18 we interpret this as two-phase systems containing dispersions of a gel in the isotropic solution. Several indications support the gel as a thermodynamically stable state. It is a significant observation that the gel can be formed via different routes. Moreover, it is possible to start with a cut-piece of gel and prepare a sample in the isotropic solution region. The transparent appearance of the gel is a sign of homogeneity. Furthermore, since the system is truly ternary the gel must be mapped together with the appropriate phases according to Gibbs’ phase rule, and it is as shown in Figure 1. There are literature reports on physical protein gels that have developed intermolecular covalent bonds, sulfur bridges, over time.10 In the present system the gel structure is broken simply by the addition of surfactant solution, and we conclude that the gel is purely physical also after storage for 1 year. Previous studies by More´n and Khan18 have revealed that the gel is optically isotropic, if not put under stress, and it only gives a broad featureless signal in small-angle X-ray scattering (unpublished observation). NMR relaxation studies of deuterated SDS show that in the gel the relaxation is so rapid that we fail to measure the relaxation rate. More´n and Khan measured the relaxation rate for the dispersed gel in the pseudoternary system lysozymeSDS-water,26 and their results are qualitatively reproduced with the salt-free system. This observation demonstrates that the DS- ions are present in large aggregates, and it also indicates that the local motion of the chains (26) More´n, A. K.; Nyde´n, M.; So¨derman, O.; Khan, A. Langmuir 1999, 15, 5480.
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is more restricted than that in typical hexagonal and lamellar amphiphilic aggregates. The high viscosity (no flow under gravity) and the broad NMR signals clearly demonstrate that the protein and surfactant are present in a highly aggregated form where aggregates probably are of macroscopic size. A Raman spectrum of lysozyme in the gel is shown in Figure 2. A comparison with the spectra of the native structure and the protein in the complex salt reveals that conformational differences exist with respect to these other two states. However, the indication is that the protein is not highly unfolded either. Rather, the protein exhibits more β-sheet structure. The pleated sheet structure of corresponding gel in the pseudoternary system lysozymeSDS-water has been revealed in a cryo-transmission electron microscopy study reported previously.27 β-Sheet formation has been observed for heat-set protein gels.28 Lin and Koenig even proposed the sheet formation as the cross linking mechanism in gels of bovine serum albumin.29 So what interactions lead to the formation of the gel structure? Many protein solutions can be transformed into a gel-like state by treatments that destabilize the native conformation, the boiling of eggs being an everyday manifestation of the general phenomenon. In most of these cases the elastic properties of the system are due to a random association of largely unfolded protein molecules. Our experimental observations in combination with results published previously16,27 do not support this model for the lysozyme-SDS gel. In cases where one is able to gently disturb the native conformation, one can observe a more specific association process that often results in predominantly linear aggregates.30 A specific class of such aggregation of considerable current interest is the formation of amyloid fibers under many pathological conditions.31-33 A stringent requirement on a native globular protein is that it should not self-associate. A mutation or two can lead to such an association with very harmful consequences for the individual.34 From our perspective the protein aggregation is just an example of colloidal association and is basically controlled by the same type of forces as other colloidal entities. An intriguing question then is what factors lead to a linear association rather than association in two or three dimensions. In the present study of the lysozyme-surfactant system, we modulate the association pattern by varying the surfactant-toprotein ratio. The formation of the complex salt is due to a combination of a hydrophobic and an electrostatic attraction between protein and surfactant. The stoichiometry of the complex is determined by the requirement of electroneutrality, and there is no reason to assume that the hydrophobic proteinsurfactant interaction is optimal. Thus as we add more surfactant, the tails will have a strong tendency to enter into the complex salt but this cannot occur on a macroscopic scale since charge will buildup and bringing in the sodium counterions into the solid is very costly in free energy. We have thus a situation with a conflict between the hydro(27) More´n, A. K.; Regev, O.; Khan, A. J. Colloid Interface Sci. 1999, 222, 170. (28) Clark, A. H.; Judge, F. J.; Richards, J. B.; Stubbs, J. M.; Suggett, A. Int. J. Peptide Protein Res. 1981, 17, 380. (29) Lin, V. J. C.; Koenig, J. L. Biopolymers 1976, 15, 203. (30) Doi, E. Trends Food Sci. Technol. 1993, 4, 1. (31) Chiti, F.; Webster, P.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi, G.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3590. (32) Barbu, E.; Joly, M. Faraday Soc. Discuss. 1953, 13, 77. (33) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. J. Mol. Biol. 1997, 273, 729. (34) Kelly, J. W. Current Opin. Struct. Biol. 1998, 8, 101.
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Stenstam et al.
phobic and electrostatic interactions. In one extreme we have the complex salt where the electrostatics is optimized but not the hydrophobic interaction. The other extreme is observed at high S/P ratios where the protein is partly unfolded and hydrophobic side chains are solubilized by surfactant aggregates. Are there any plausible compromise alternatives between these extremes? The solution is to form an aggregate that extends in one or two dimensions instead of three. In this way one creates a situation where the aggregate can carry a net charge, which is neutralized by counterions in the electrical double layer in the solution surrounding the aggregate. Forming such an aggregate of lower local dimensionality from the solid must involve a major rearrangement of the molecular organization since the protein-protein interaction becomes confined to one (or two) dimension(s) rather than the full three-dimensional interaction of the compact solid. This implies a cooperativity of the process, and we expect a two-phase area separating the complex salt and the area with charged large aggregates. Similarly the breakup of the aggregates into individual protein molecules involves a major rearrangement, and again we should expect a two-phase coexistence rather than a gradual change of properties. Before turning to a quantitative model of these effects, we should first briefly discuss the isotropic solution structure. The Soluble Complexes. The aggregation behavior in the isotropic solution occurring for S/P ratios exceeding 22 has been extensively studied previously. NMR relaxation, dynamic light scattering, and NMR self-diffusion studies indicate that the aggregates present in the system are small, with little sign of direct protein-protein interactions except for the highest protein concentrations.16 With this description of the soluble complexes, we see a qualitative difference between the aggregates in the gel and in solution. In the latter case an extended protein is seen as interacting with a self-assembled surfactant aggregate with little direct protein-protein interaction. For the gel on the other hand the protein in a compact conformation interacts directly with neighboring protein molecules and this interaction is enhanced by addition of individual surfactant molecules. Model of the Phase Equilibria. A most useful route to establish the relation between the thermodynamic properties and the molecular interactions is to establish a quantitative model that accounts for the salient features of the phase equilibria. Experimentally we have found that the protein-surfactant interaction leads to the formation of three different phases: the complex salt, the gel, and the isotropic solution. We can thus consider the free energy relations of the two equilibria
(i) Ly(DS)8 + nNaDS S Ly(DS)n+8n- + nNa+ (8) i.e., the equilibrium between complex salt and gel, which involves the swelling of a compact electroneutral structure to a swollen charged but still infinite aggregate and
(ii) {Ly(DS)m}NN(m-8)- S N{Ly(DS)m}(m-8)-
(9)
i.e., the equilibrium between the gel and the isotropic solution. This involves the breakup of a macroscopic network into soluble aggregates containing a single protein molecule. The essential free energy contributions come from electrostatic surfactant-surfactant, surfactant-protein, and protein-protein interactions. In additition there is a
contribution from entropy of mixing in the isotropic solution. We will assume that, given the aggregate structure, the surfactant-protein and protein-protein interaction are composition independent and write the free energies of the three phases as
Ggel ) Ggelel + ngHF + kT(n ln Xcmc - ncmc) + Ggel′ (10) Gsolid ) 8gHF + Gsolid′
(11)
Gsolution ) Gsolutionel + ngHF + kT{ln Xcomplex - 1} (12) where gHF is a factor representing the hydrophobic attraction of surfactant tails to the protein molecule. Its value of 0.9 kT per CH2 group is found from our solubility study. Xcmc is the volume fraction of aggregates at cmc, n the number of surfactants per complex added to the complex salt, and ncmc the number of surfactants added at cmc. Xcomplex represents the volume fraction of soluble protein-surfactant complexes. The two G′ parameters include all composition-independent terms and they are our only fitting parameters. To calculate the electrostatic free energy associated for an aggregate with a net charge, we need to invoke structural models. For the isotropic solution we consider a spherical aggregate with a net charge (S/P - 8)e. An approximate analytical solution of the Poisson-Boltzmann equation is22
Gsolutionel )
{[
2kT ln(sL + (1 + sL)1/2) +
]
1 - (1 + sL2)1/2 sL
)}
(
1 + (1 + sL2)1/2 2 ln sLκRcomplex 2
(13)
Here, sL ) σL(8kTco*ro)-1/2 and κ is the inverse Debye length, i.e., a function of the concentration of screening ions from SDS. The detailed structure of the gel is at present unknown, but a simple model of a lamellar system with only counterions captures the essential behavior of the electrostatic interactions, a planar aggregate with hexagonally packed proteins of a circular cross-sectional area that is determined by the protein radius. Another possible model is the cylindrical, illustrating the formation of single chains of globular proteins, branched or cross-linked. The two geometries would most probably show the same qualitative features, and thus we choose the less complicated planar model. For the case of the counterions only, there exists a simple analytical expression for the electrostatic free energy in the planar case.35
( ){ (
Ggelel ) 2AkT
)
-σ s2 s2 ln + K - 1 + ze K K
}
(14)
K ) -σzeL/2rokT
(15)
s tan(s) ) K
(16)
This describes the free energy cost of overcharging the aggregates which has to be compensated by the hydrophobic contribution from the association of surfactants to the complex salt. The electrostatic free energy increases (35) Parsegian, V. A. Trans. Faraday Soc. 1966, 62, 848.
The Lysozyme-Dodecyl Sulfate System
Figure 3. The lamellar model of hexagonally packed proteins. (a) The surfactant molecules are assigned the volume between the spheres, i.e. the excess volume of the hexagonal entity. (b) The electrostatic interaction is modeled as the electrostatic interaction between two charged parallel planes. The surface charge is defined by the number of surfactant molecules per protein and the distance between the planes by the total sample composition.
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Figure 5. Calculated loci of multiphase samples connecting the gel phase to the insoluble complex salt and the solution of soluble complexes: full line, calculated region of the two-phase samples (G + L); circles, experimental data for (G + L); dotted line, calculated region of three-phase samples (S + G + L); crosses, experimental data for (S + G + L).
and (ii) above, is zero represent multiphase samples of the aggregates involved. Indeed, the resulting loci of these compositions do fit the experimental results very well. Our main conclusion in this section is that the formation of the gel in a rather narrow range of S/P ratios is due to a compromise between a hydrophobic protein-surfactant attraction and an electrostatic effect which prohibits overcharging in a compact macroscopic phase but gives a free energy penalty for aggregates that have a microscopic size in at least one direction. In these aggregates the protein takes a conformation different from the native one but it is still compact. Since the protein is confined to an aggregate, there is only a limited conformational freedom eliminating the major driving force for forming a random coil. A linear aggregate has a lower electrostatic penalty than a two-dimensional one, and future work will focus on a cylindrical/fibrous model. Concluding Discussion
Figure 4. The contributions of free energy differences illustrating the equilibrium between the gel and the complex salt, at constant 10 wt % CS(12) and increasing amount of surfactant: full line, the difference in electrostatic interaction ∆Gel; dashed line, the difference in hydrophobic attraction (n - 8)gHF; dotted line, the difference in entropy of mixing kT(n ln Xcmc - ncmc).
not only with increasing charge density but also with decreasing aggregate concentration, which can be seen as due to the fact that in the counterion only case the mean counterion concentration decreases upon dilution. With an expression for the electrostatic contribution to the formation of a large aggregate, it is possible to calculate the positioning of such a phase in a ternary phase diagram. In Figure 4 we show, as an example, how the different free energy contributions vary with surfactant concentration. In this way one can illustrate that the stability of the gel is due to a compromise between a reasonably retained protein-protein interaction and a not too strong electrostatic repulsion. At higher surfactant contents the electrostatic effect acts to disperse the system into finite aggregates. By fitting the two parameters Ggel′ and Gsolid′, we capture the concentration dependence of the phase equilibria as shown in Figure 5 providing support to the basic relevance of the model and the underlying mechanistic argument. The compositions in the phase diagram where the difference in free energy between each pair of phases, (i)
Our objective has been twofold. The experimentally determined phase diagram has been presented and supported by theoretical calculations. The calculations were based on our knowledge of the hydrophobic and electrostatic interactions. What follows is a discussion of the phase behavior in terms of these interaction forces. To further illustrate the argument, we consider the phase equilibria reached when following a sample of constant weight percent lysozyme in water subjected to more and more alkyl sulfate Initially the electrostatic as well as the hydrophobic effect contributes to the overall very attractive interaction between the surfactant and the protein. A very small amount of surfactant can be solubilized as has been shown. Instead an associative phase separation manifests itself as a precipitation of a, by oppositely charged surfactant molecules, net-neutralized protein. As this physical complex of protein and surfactants also consists of hydrophobic domains, it will no longer be soluble in the aqueous solution. When surfactant is added to the neutral complex, charges are introduced. The electrostatic interaction is increased from its minimum. The hydrophobic attractive force is most probably increased as well when the surfactant molecules influence the protein conformation. With charges, water and counterions have to enter the structure. Consequently, the structure swells. However, the hydrophobic attraction is strong enough to keep the complexes together. The result of these two opposing
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Figure 6. A schematic illustration of the molecular interactions between the protein-surfactant complex and added surfactant molecules.
phenomena is a network structure. We thus propose that this network is hydrophobically mediated with a porosity caused by the electrostatic repulsion. The compositional stability is narrow as a result of the large entropy of mixing when breakup of the network has been made possible by an increased electrostatic repulsive force. This surfactantmediated protein network can be modeled as a structure of packed globular proteins.
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Eventually, redissolution occurs when a critical concentration of surfactant molecules aggregates on the complex. This in turn happens when a critical concentration of surfactants is reached in the supernatant that is in equilibrium with the insoluble salt. Subsequently the resolubilization is a result of hydrophobic interaction and as such varying with the chain lengths of the different surfactants examined.7 The precondition of surfactant aggregation for redissolution was proposed by More´n and Khan for the lysozyme-SDS-water system.7 At the same time as the hydrophobic interaction is crucial for the mechanism, it is the electrostatic repulsion that stabilizes the solution of soluble complexes. In this paper we have, for the first time, presented the phase equilibria in a true ternary protein-surfactantwater system. Further we have demonstrated that this chemical simplification also provides generalizations of the theoretical discussion concerning the molecular mechanisms that determine the relative stability of different aggregation states. Finally, we argue that the lysozyme molecule can retain its compact form in surfactantmediated gels. Acknowledgment. We acknowledge Anna Karin More´n and Lennart Piculell for valuable suggestions as well as discussions. The project is financed by Stiftelsen fo¨r Strategisk Forskning, SSF. LA011096T
