Langmuir 1996, 12, 4073-4083
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Solubilization Properties of r-Lactalbumin and β-Lactoglobulin in AOT-Isooctane Reversed Micelles Lynne E. Kawakami† and Stephanie R. Dungan*,†,‡ Department of Chemical Engineering and Materials Science and Department of Food Science and Technology, University of California, Davis, California 95616 Received July 17, 1995. In Final Form: April 25, 1996X Solubilization properties of R-lactalbumin and β-lactoglobulin in a sodium bis(ethylhexyl) sulfosuccinateisooctane reversed micellar phase were measured as a function of aqueous phase pH and ionic strength. Solubilization behavior over a wide range of pH values and ionic strengths indicates that hydrophobic, as well as electrostatic, interactions may play an important role in the solubilization of these proteins. The influence of hydrophobic interactions on the solubility behavior of R-lactalbumin and β-lactoglobulin is reflected in the effect of protein size and net charge on the degree of solubility. Salt concentration also affects the protein solubilization, most likely through its influence on the size of the reversed micellar droplets. This effect of droplet size on protein solubilization can be quantified by defining a “critical micellar radius” above which 50% or more of the protein transfers into the organic phase. The value of this parameter and its constancy over various pH values further supports the hypothesis that R-lactalbumin and β-lactoglobulin solubilize in the reversed micellar interface, stabilized by protein-surfactant hydrophobic interactions. Protein solubility was also found to be sensitive to the type of cation present in the system. Finally, the concentration of protein in the initial aqueous phase was observed to affect the solubilization of both water and protein. A strong linear relationship between water transfer and the amount of R-lactalbumin solubilized within the reversed micellar phase suggests that this protein may act as a cosurfactant, and induce expansion of the surfactant interface by its incorporation in that region.
1. Introduction Reversed micellar solutionssalso termed water-in-oil microemulsionssare nanometer scale droplets of water suspended in an organic continuum rendered thermodynamically stable by a surfactant. The surfactant molecules, oriented with their polar head groups directed toward the center of the micelle and their hydrocarbon tails protruding into the surrounding medium, stabilize an aqueous core into which hydrophilic solutes can partition. Because in some cases they allow the solubilization of proteins in organic solvents without denaturation or loss of activity,1-6 reversed micelles have potential as a liquid-liquid extraction medium for effecting protein separations.2,7-13 The ability to separate a protein from an aqueous mixture using reversed micelles depends on the solubilization behavior of that protein as a function of conditions in the aqueous and reversed micellar phase. Utilization of reversed micelles for protein separations * Author to whom all correspondence should be addressed. † Department of Chemical Engineering and Materials Science. ‡ Department of Food Science and Technology. X Abstract published in Advance ACS Abstracts, June 15, 1996. (1) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239. (2) Dekker, M.; Van’t Riet, K.; Weijers, S. R.; Baltussen, J. W. A.; Laane, C.; Bijsterbosch, B. H. Chem. Eng. J. 1986, 33, B27. (3) Fletcher, P. D. I.; Parrott, D. J. Chem. Soc., Faraday Trans. 1 1988, 84, 1131. (4) Luisi, P. L. Angew. Chem., Int. Ed. Engl. 1985, 24, 439. (5) Kinugasa, T.; Watanabe, K.; Takeuchi, H. Ind. Eng. Chem. Prod. Res. Dev. 1992, 31, 1827. (6) Marcozzi, G.; Correa, N.; Luisi, P. L.; Caselli, M. Biotechnol. Bioeng. 1991, 38, 1239. (7) Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Bioeng. 1991, 38, 1302. (8) Brandani, V.; DiGiacomo, G.; Spera, L. Process Biochem. 1993, 28, 411. (9) Go¨klen, K. E.; Hatton, T. A. Sep. Sci. Technol. 1987, 22, 831. (10) Dekker, M.; Hilhorst, R.; Laane, C. Anal. Biochem. 1989, 178, 217. (11) Giovenco, S.; Verheggen, F.; Laane, C. Enzyme Microb. Technol. 1987, 9, 470. (12) Hilhorst, R.; Fijneman, P.; Heering, D.; Wolbert, R. B. G.; Dekker, M.; Van’t Riet, K.; Bijsterbosch, B. H. Pure Appl. Chem. 1992, 64, 1765. (13) Rahaman, R. S.; Chee, J. Y.; Cabral, J. M. S.; Hatton, T. A. Biotechnol. Prog. 1988, 4, 218.
S0743-7463(95)00591-9 CCC: $12.00
thus necessitates study of the solubilization properties of a wide range of proteins. In this study we explore the equilibrium partitioning of two proteins, R-lactalbumin and β-lactoglobulin, into a reversed micellar solution of the surfactant Aerosol OT. These proteins, which are two major components of milk, have use in pharmaceutical and food applications. The major nutritional difference between human milk and infant formulas has been attributed to the different concentrations of R-lactalbumin in human and bovine milk.14 Enrichment with R-lactalbumin isolates could significantly improve the nutritional value of bovine-milkbased infant formulas which are deficient in some amino acids essential to infants. In addition, both proteins exhibit some surface activity15 and, for this reason, have application in food emulsions, foams, and gels. However, utilization of these two proteins is currently hampered by the need for a cost-effective, large scale approach for separating β-lactoglobulin and R-lactalbumin from each other and for separating both from other even higher value proteins in whey. The surface activity of β-lactoglobulin and R-lactalbumin makes these two proteins of particular fundamental interest in our study. We would like to explore whether such proteins interact significantly with the interfacial region surrounding the reversed micellar droplet and whether such interactions affect the conditions under which the proteins transfer into the microemulsion phase. The driving force leading to protein solubilization in reversed micelles has been an area of intense investigation. The majority of the data presented in the literature indicates that electrostatic forces dominate the protein solubilization process.3,8,10,12,16-22 This dominance is demonstrated by a strong dependence of solubility on the (14) Heine, W. E.; Klein, P. D.; Reeds, P. J. J. Nutr. 1991, 121, 277. (15) Suttiprasit, P.; Krisdhasima, V.; McGuire, J. J. Colloid Interface Sci. 1992, 154, 316. (16) Dungan, S. R.; Bausch, T.; Hatton, T. A.; Plucinski, P.; Nitsch, W. J. Colloid Interface Sci. 1991, 145, 33. (17) Hatton, T. A. In Ordered Media in Chemical Separations; Hinze, W. L., Armstrong, D. W., Eds.; American Chemical Society: Washington, DC, 1987; p 170.
© 1996 American Chemical Society
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aqueous phase pH, which alters the protein’s net charge. In a reversed micellar system containing anionic surfactant, a high degree of solubilization is often observed when the pH is below the isoelectric point (pI),6,9,18,21,23,24 where the protein has a net positive charge. This would be the predicted behavior in a situation where electrostatic attraction between the oppositely charged protein and surfactant head groups is the dominating driving force for protein solubilization. Conversely, at and above the isoelectric point, where electrostatic interactions are no longer favorable, the degree of solubilization often drops dramatically.9,21 Another indication of the importance of electrostatic contributions to the solubilization driving force is a strong dependence of protein solubilization on the aqueous phase ionic strength.6,8-10,18,20,24,25 Salts mediate electrostatic interactions through the influence of counterions localized within the double layer adjacent to a charged surface. These counterions have the ability to screen attractive or repulsive interactions between charged surfaces and thereby reduce the distance over which the attraction or repulsion is felt. The observed reduction in protein solubilization with increasing salt concentration suggests that the salt reduces the driving force for solubilization, i.e., protein-surfactant electrostatic interactions. Salts also screen repulsive electrostatic interactions between surfactant head groups in the reversed micelle, leading to reduced water transfer to the reversed micellar phase and smaller micelle size.26 Electrostatic forces have also played a key role in existing theoretical investigations into the reversed micellar incorporation of proteins. Rahaman and Hatton27 used a shell-and-core model, in which a charged spherical protein sits concentrically within an oppositely charged surfactant shell, to determine successfully the influence of electrostatic interactions on the structure of empty and R-chymotrypsin-filled reversed micelles. Others have also been successful in applying electrostatic thermodynamic models to the partitioning of cations28 and proteins29,30 in reversed micelles. By experimentally investigating the effect of pH and ionic strength on R-lactalbumin and β-lactoglobulin partitioning, we explore the influence of electrostatic interactions on the solubilization behavior of these proteins within reversed micelles. In addition, we also consider the role of counterion type on protein partitioning. Various groups have observed that protein solubilization occurs over a wider range of salt concentrations when sodium18,20,24,31,32 and calcium20,24 chlorides are used to adjust (18) Kelley, B. D.; Rahaman, R. S.; Hatton, T. A. In Organized Assemblies in Chemical Analysis; Hinze, W. L., Ed.; JAI Press: Greenwich, 1994; p 123. (19) Leser, M. E.; Wei, G.; Luisi, P. L.; Maestro, M. Biochem. Biophys. Res. Commun. 1986, 135, 629. (20) Leser, M. E.; Luisi, P. L. Chimia 1990, 44, 270. (21) Matzke, S. F.; Creagh, A. L.; Haynes, C. A.; Prausnitz, J. M.; Blanch, H. W. Biotechnol. Bioeng. 1992, 40, 91. (22) Wolbert, R. B. G.; Hilhorst, R.; Voskuilen, G.; Nachtegaal, H.; Dekker, M.; Van’t Riet, K.; Bijsterbosch, B. H. Europe. J. Biochem. 1989, 184, 627. (23) Chang, Q.; Liu, H.; Chen, J. Enzyme Microb. Technol. 1994, 16, 970. (24) Nishiki, T.; Sato, I.; Kataoka, T.; Kato, D. Biotechnol. Bioeng. 1993, 42, 596. (25) Go¨klen, K. E.; Hatton, T. A. Biotechnol. Prog. 1985, 1, 69-74. (26) Sheu, E.; Go¨klen, K. E.; Hatton, T. A.; Chen, S.-H. Biotechnol. Prog. 1986, 2, 175. (27) Rahaman, R. S.; Hatton, T. A. J. Phys. Chem. 1991, 95, 1799. (28) Leodidis, E. B.; Hatton, T. A. Langmuir 1989, 5, 741. (29) Bruno, P.; Caselli, M.; Luisi, P. L.; Maestro, M.; Traini, A. J. Phys. Chem. 1990, 94, 5908. (30) Caselli, M.; Mangone, A.; Pagone, N. Ann. Chim. 1993, 83, 191. (31) Andrews, B. A.; Pyle, D. L.; Asenjo, J. A. Biotechnol. Bioeng. 1994, 43, 1052.
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the ionic strengths as opposed to potassium or barium salts. Nishiki et al.24 also observed that the addition of calcium or barium ions to the reversed micellar system broadened the pH range over which lysozyme solubilization occurred to include higher pHs, where electrostatic forces are less favorable. Whether such effects can be understood strictly within the context of electrostatic driving forces is currently unknown. Very recently, a small group of proteins have been shown to exhibit phase transfer behavior markedly different from the majority of proteins described in literature. These proteins, which are characterized as being “hydrophobic” in nature, include cytochrome-b5,33,34 lipases,7,35,36 cutinase,37 and thaumatin.31 Under conditions where the protein net charge is zero or of the same sign as the charge on the surfactant molecules, they still exhibit appreciable solubilization. As this behavior cannot be described solely by electrostatic interactions, it is believed that in these cases, hydrophobic interactions also play an integral role in protein solubilization. The nature of these interactions is still not well understood and attempts have been made only recently to investigate the extent of their influence on the solubilization driving force.33 It has been suggested that R-lactalbumin also fits into this unique group of hydrophobic proteins,31 and our data suggest that β-lactoglobulin may as well. Unusual physicochemical properties, such as high surface activity15 and hydrophobicity,38,39 make β-lactoglobulin and R-lactalbumin interesting subjects for the investigation of the impact of molecular interactions on protein solubilization. 2. Experimental Section 2.1. Proteins. All proteins were obtained from Sigma and used without further purification. R-Lactalbumin (pI ) 4.24.5; MW 14 000 Da40), a calcium binding protein, was obtained in a calcium depleted form, containing no more than 0.3 mol of Ca2+/mol of protein. Two different lots of β-lactoglobulin (pI ) 5.2; MW 36 000 Da41) were used. We observed some variation in the results obtained with different lot numbers, with deviations in β-lactoglobulin solubility found between 10 and 20% at salt concentrations below 0.3 M NaCl. The largest deviations were observed at the lowest salt concentration. 2.2. Chemicals. Sodium bis(ethylhexyl) sulfosuccinate (Aerosol OT; AOT) of 99% purity was obtained from Sigma. Aveyard et al.42 reported that purification of AOT from Sigma was not necessary to obtain reproducible results from one lot to another, and the surfactant was therefore used as received. Isooctane (ACS Grade) and all salts were obtained from Fisher Chemical and used without further purification. Water used to prepare all aqueous phases was distilled, deionized, and passed through a Barnstead Ultrapure Ion Exchange column. 2.3. Phase Transfer Experiments. Protein solubility was determined by phase transfer experiments. Equal volumes of approximately 5 mL each of the aqueous and organic phases were contacted by magnetic agitation for 25 min, which we experimentally determined to be sufficient to reach equilibrium. (32) Andrews, B. A.; Haywood, K. J. Chromatogr. A 1994, 668, 55. (33) Pires, M. J.; Cabral, J. M. S. Biotechnol. Prog. 1993, 9, 647. (34) Pires, M. J.; Cabral, J. M. S. J. Chem. Technol. Biotechnol. 1994, 61, 219. (35) Camarinha Vicente, M. L.; Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Tech. 1990, 4, 137. (36) Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Bioeng. 1991, 38, 1302. (37) Carneiro-da-Cunha, M. G.; Cabral, J. M. S.; Aires-Barros, M. R. Bioprocess Eng. 1994, 11, 203. (38) Nakai, S. J. Agric. Food Chem. 1983, 31, 676. (39) Acharya, K. R.; Stuart, D. I.; Walker, N. P. C.; Lewis, M.; Phillips, D. C. J. Mol. Biol. 1989, 208, 99. (40) Kronman, M. J.; Andreotti, R. E.; Vitols, R. Biochemistry 1964, 3, 1152. (41) Cannan, R. K.; Palmer, A. H.; Kibrick, A. C. J. Biol. Chem. 1942, 142, 803. (42) Aveyard, R.; Binks, B. P.; Clark, S.; Mead, J. J. Chem. Soc., Faraday Trans. 1 1986, 82, 125.
Solubilization of Proteins in Reversed Micelles
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Samples were incubated at 25 °C for at least 24 h, during which time phase separation occurred. Phases were then carefully separated and analyzed. The organic phase was composed of 100 mM AOT in isooctane. The aqueous phase contained 1.0 mg/mL protein in buffer or water. In order to explore the effect of initial protein concentrations, concentrations between 0.17 and 1.0 mg/mL were tested independently, and over this range of initial protein concentrations, there was no observable effect of protein concentration on the degree of solubilization. In the pH variation experiments, the buffers were composed of 0.05 M buffer salts and sufficient chloride salt to bring the net cation concentration to 0.25 M. Over the range of 0.05-0.15 M, buffer salt concentration had no effect on protein solubilization. In the ionic strength variation experiments, nonbuffered chloride salt solutions were used. 2.4. Assays. Water transfer to the reversed micellar phase was measured by Karl Fischer titration. Measurements of the water contained in the reversed micellar phase were made in triplicate and these values were used to calculate the parameter w0, which is defined as
w0 )
[H2O] [surfactant]
(1)
Here, [H2O] and [surfactant] are the molar concentrations of water and Aerosol OT, respectively, in the reversed micellar phase. On the basis of geometrical considerations, w0 is found to be directly related to the micelle radius R by43
R)
3vw w as 0
(2)
where vw is the molecular volume of water ()30.1 Å3) and as is the area per surfactant head group. as is a weak function of the micellar radius and can be modeled as27
as )
RR β+R
Figure 1. Solubility of R-lactalbumin (b) and β-lactoglobulin (9) in 100 mM AOT in isooctane reversed micellar phase vs aqueous phase pH. Net sodium ion concentration is 0.25 M. Dotted line represents the isoelectric point of the proteins.
(3)
where R ) 62.3 Å2 and β ) 6.39 Å. Because the reversed micellar phase is optically transparent, ultraviolet spectrophotometry was used to measure the protein content of the final aqueous and reversed micellar phases. Protein concentration was determined by monitoring the absorbance at 280 and 310 nm.18,27 Separate extinction coefficients were measured for the proteins in bulk aqueous and reversed micellar phases. The extinction coefficient of the reversed micellar phase containing β-lactoglobulin appeared to have a slight dependence on w0 (less than 8% difference over the entire range). An average value of the extinction coefficient was therefore used to calculate the protein content of the reversed micellar phase for β-lactoglobulin. We determined that this approximation gave rise to less than 3% difference in our results, which is smaller than the expected experimental error. A similar variation was observed by Matzke et al.21 in the extinction coefficient of alcohol dehydrogenase. The extinction coefficient was found to be relatively constant for R-lactalbumin over all w0 values studied.
3. Results and Discussion Calculation of the percent protein solubilized is based on the concentration of protein in the reversed micellar phase, with volume adjustments to account for water uptake. Closure of the mass balance was usually within 5%. Under certain conditions, in particular at low pH (pH < 5 for R-lactalbumin and pH < 6 for β-lactoglobulin) or high salt concentration (greater than 0.4 M KCl), significant protein losses were observed along with appreciable interfacial residue. Protein loss at low pH may be due to low aqueous solubility at these pHs, which are the near the isoelectric point of the proteins. Gro¨nwall44 reports that β-lactoglobulin solubility in aqueous (43) Luisi, P. L.; Giomini, M.; Pileni, M.-P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209.
solution is lowest around the isoelectric point. Also, in the range pH 3.5-5.2, β-lactoglobulin has been reported to form octamers,45 a process which further reduces protein solubility. Kronman et al.40 observed aggregation of R-lactalbumin in aqueous solution at the pI or at pH values lower than the pI. This aggregation is due to R-lactalbumin’s denatured state at low pHs40 and is essentially absent at pHs above the pI. Other researchers have observed some protein loss in reversed micellar extraction experiments under extremes of pH or ionic strength.21,24,30,32,37,46 Matzke et al.21 observed simultaneous protein loss and decreased water transfer for R-chymotrypsin at pH 4 and 0.1 M NaCl, suggesting protein loss due to aggregation with surfactant. However, as we observed no decrease in water transfer, protein loss was probably due to aggregation of protein molecules in the aqueous phase only. Protein losses under these conditions led to low levels of protein solubilization in the reversed micellar phase. 3.1. Effect of pH and Ionic Strength. The pH of the reversed micellar phase was changed by varying the buffer composition of the aqueous phase. In reversed micellar solutions made of an anionic surfactant such as Aerosol OT, most proteins exhibit significant solubilization only at pH values below their isoelectric point.6,9,18,21,23,24 However, R-lactalbumin and β-lactoglobulin both solubilize appreciably at pHs well above their respective isoelectric points, as shown in Figure 1. Reduced reversed micellar solubility for R-lactalbumin at low pH is most likely due to loss of aqueous protein solubility, as described above. Wolbert et al.22 developed a model to predict the optimum pH shift needed for maximum protein solubilization. Their model was based on the observation that in their cationic surfactant system, each protein exhibited a positive optimum pH shift (pHmax - pI) such that the protein and surfactant were oppositely charged, indicating the involvement of electrostatic interactions in the solubilization driving force. By similar reasoning, in a system of reversed micelles consisting of anionic surfactants, one would expect a protein to have a negative optimum pH shift. Indeed, such a negative shift is demonstrated by the hydrophilic proteins ribonuclease a9 and R-chymotrypsin.6,9,21 However, the data in Figure 1 indicate that (44) Gro¨nwall, A. Compt. Rend. Trav. Lab. Carlsberg, Ser. Chim. 1941-1943, 24, 185. (45) Townend, R.; Winterbottom, R. J.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82, 3161. (46) Regalado, C.; Asenjo, J. A.; Pyle, D. L. Biotechnol. Bioeng. 1994, 44, 674.
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Figure 2. Solubility of R-lactalbumin (b) and β-lactoglobulin (9) vs aqueous phase ionic strength at pH 6.2.
R-lactalbumin and β-lactoglobulin both have positive optimum pH shifts, suggesting that the solubilization behavior of these proteins cannot be described solely by electrostatic interactions. Similar observations of a positive optimum pH shift were also observed by Carneiroda-Cunha et al.37 and Pires and Cabral34 for the recombinant proteins cutinase and cytochrome b5. The change in R-lactalbumin and β-lactoglobulin solubility as a function of ionic strength is also inconsistent with a model in which electrostatic interactions are the predominating driving force for protein solubilization in reversed micelles. Ionic strength variation experiments indicate that protein solubilization occurs over a wide range of salt concentrations (Figure 2). Up to 4 M NaCl is needed in the aqueous phase before R-lactalbumin solubilization ceases. This behavior is in contrast to that of proteins such as R-chymotrypsin, cytochrome c, and ribonuclease a, for which a significant decline in reversed micellar solubility occurs at NaCl concentrations above 0.3 M.18 For such systems, in which protein-surfactant charge interactions play a major role, protein solubility is sensitive to salt concentration, as salts screen the electrostatic interactions that promote solubilization. Note that the solubility behavior shown in Figure 2 is also unusual in that the experiments were carried out under conditions where protein and surfactant are both negatively charged (pH 6.2), and hence electrostatics do not favor solubilization. The contrast of the solubilization behavior of R-lactalbumin and β-lactoglobulin as compared to other hydrophilic proteins becomes more apparent when one describes the solubility in terms of the Debye screening length. As mentioned earlier, salts screen charge interactions in aqueous solution, reducing the distance over which the attraction or repulsion occurs between charged surfaces. This distance is characterized by the Debye screening length κ-1
κ-1 )
( ) kBT
2n0e2
1/2
(4)
Here, is the aqueous dielectric constant, kBT is the Boltzmann energy, and e is the electronic charge. The Debye length is inversely proportional to n01/2, where n0 is the ionic strength. Thus, at high ionic strengths, such as 1.0 M NaCl, the Debye length is sufficiently short that nearby charged surfaces experience relatively little attraction to or repulsion from each other. Under these conditions, the contribution of electrostatic interactions to protein solubility is minute. Hydrophilic proteins, whose solubilization behavior is strongly dependent on
the strength of electrostatic interactions between the protein and surfactant head groups, usually do not solubilize significantly at salt concentrations as high as 1.0 M NaCl. However, β-lactoglobulin and R-lactalbumin exhibit 15% and 50% solubilization, respectively (Figure 2), at this ionic strength. These observations suggest the influence of some driving force other than electrostatic forces on the solubilization of these proteins. For salt concentrations below 0.3 M, a decrease in the reversed micellar solubility of β-lactoglobulin is observed. Unlike the behavior at low pH, this low solubility at low salt concentrations is not due to protein loss. Instead, this reduced solubility may arise from repulsive charge interactions between the protein and surfactant at this pH. It may be that at low salt concentrations, these unfavorable protein-surfactant electrostatic interactions are not sufficiently screened such that they dominate the solubilization process and result in low protein transfer. Interestingly, this effect is not observed for R-lactalbumin for salt concentrations as low as 0.1 M, perhaps because at this pH R-lactalbumin’s net negative charge is somewhat smaller than that of β-lactoglobulin. The results shown in Figures 1 and 2 indicate that even under conditions where one does not expect the net charge on the protein to interact favorably with the Aerosol OT head groups, β-lactoglobulin and R-lactalbumin solubilize significantly within the reversed micellar droplets. Two different scenarios can be proposed to explain this unusual solubilization behavior. If the distribution of charged residues on the protein surface is asymmetric, protein solubilization in reversed micelles of anionic surfactant could still occur at pHs above the isoelectric point. Because the positive and negative charges are concentrated on different sections of the protein molecule, favorable localized electrostatic interactions may still occur even though the net charge on the protein would predict otherwise. On the other hand, solubilization may be the result of the balance between electrostatic interactions and some other driving force, such as hydrophobic interactions between the nonpolar protein residues and the hydrocarbon surfactant tails in the micellar interface. Because β-lactoglobulin and R-lactalbumin solubility in AOT reversed micelles is relatively insensitive to the salt concentration (Figure 2), which would not be the case if solubilization were due to a localized electrostatic attraction, it does not appear that charge asymmetry is the reason for the appreciable solubilization above the isoelectric point. This conclusion is consistent with similar observations made by Pires and Cabral,34 Andrew et al.,31 and Regalado et al.46 in the solubilization behavior of other proteins for which hydrophobic interactions are believed to play a role. Sensitivity to salt concentration is also used to distinguish between different types of membrane proteins. Peripheral proteins, which are attached to the cell membrane via localized ionic interactions, can be distinguished from intrinsic proteins, which are embedded in the membrane by protein-surfactant hydrophobic interactions, by the fact that the former can characteristically be detached from the membrane in solutions of high ionic strength.47 Only a limited number of attempts have been made to verify the existence of hydrophobic interactions in the protein solubilization mechanism. Pires and Cabral,34 during their investigation of cytochrome b5 solubilization behavior, observed the effect of temperature on protein solubility. Hydrophobic interactions are believed to be primarily entropic, and as such, to become weaker at lower (47) Darnell, J.; Lodish, H.; Baltimore, D. Molecular Cell Biology; Scientific American Books: New York, 1990.
Solubilization of Proteins in Reversed Micelles
Figure 3. Solubility of R-lactalbumin (b) and β-lactoglobulin (9) vs net charge on protein. Net sodium ion concentration is 0.25 M.
temperatures. Pires and Cabral34 observed that the degree of cytochrome b5 solubilization is reduced by decreasing the temperature, and they therefore concluded that near the isoelectric point, the primary driving force for the solubilization of cytochrome b5 is protein-surfactant hydrophobic interactions. Leodidis and Hatton48 used amino acids to study the effect of solute hydrophobicity on solubilization. They concluded that the more hydrophobic amino acids solubilize to a greater degree in Aerosol OT reversed micelles. Experimental data could be accurately fit by a “surface monolayer” model, in which the hydrophobic amino acids were incorporated into the reversed micellar interface with their polar groups closely associated with the surfactant head groups and their hydrophobic groups penetrating the region containing the surfactant tail groups.48 The ability of this model to predict accurately trends in the amino acid solubility suggests the importance of hydrophobic interactions in the solubilization mechanism of amino acids. Since amino acids are the subunits making up a protein molecule, such interactions could be important in explaining protein solubilization behavior as well. If hydrophobic interactions do facilitate the solubilization of R-lactalbumin and β-lactoglobulin, then one would expect some correlation between the degree of solubility and protein hydrophobicity. Leodidis and Hatton49 observed a clear relation between amino acid solubility within reversed micelles and previously published hydrophobicity scales, and similar findings are also discussed in a review by Hatton.17 However, although β-lactoglobulin has been determined to be more hydrophobic than R-lactalbumin by aqueous two-phase partitioning,50 it appears that R-lactalbumin is able to partition into the reversed micellar phase at pHs further removed from its respective isoelectric point (Figure 1). This behavior may be understood by using the protein hydrogen ion titration curves to replot solubility as a function of the protein net charge, as done in Figure 3. From these results it is evident that, because of its greater hydrophobicity, β-lactoglobulin is able to solubilize under conditions of greater net negative charge and, hence, to a first approximation, stronger electrostatic repulsion. The solubilization behavior of the two proteins as a function of ionic strength also needs to be reexamined in light of the relative hydrophobicities of the two proteins. The data in Figure 2 indicates that, at high salt concen(48) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6400. (49) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6411. (50) Shanbhag, V. P.; Johansson, G.; Ortin, A. Biochem. Int. 1991, 24, 439.
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Figure 4. Solubility of R-lactalbumin (b) and β-lactoglobulin (9) vs micelle radius at pH 6.2. Table 1. Critical Micelle Radii of Various Proteins in 100 mM Aerosol OT in Isooctanea protein
molecular weight
critical micelle radius (Å)
R-lactalbumin β-lactoglobulin R-chymotrypsin ribonuclease a cytochrome c
14 000 36 000 25 136 13 683 12 300
23 29 40 55 37
a Aqueous phase contains sodium salts and buffer components. R-Chymotrypsin, ribonuclease a, and cytochrome c critical micelle radii determined from the data of Kelley et al.18
trations, R-lactalbumin can solubilize into reversed micelles to a greater extent than β-lactoglobulin. However, as mentioned above, the salt concentration not only influences protein-surfactant interactions, it affects the amount of water transferred into the reversed micellar phase and, therefore, the size of the reversed micellar droplet as well. A geometrical model (eqs 2 and 3) can be used to calculate the micelle radius from the parameter w0. The value obtained represents the size of the reversed micellar droplet in the absence of protein; that size would be expected to change upon incorporation of protein. However, comparison of that radius with the amount of protein transferred provides important insights into the physical factors limiting protein solubilization. By replotting the protein solubilization data collected at various salt concentrations as a function of micelle radius (Figure 4), it appears that R-lactalbumin’s higher solubility at higher salt concentrations may be due to a size effect. R-Lactalbumin, the smaller protein, is able to solubilize into an organic phase containing smaller micelles than can β-lactoglobulin. Quantification of this size effect may be obtained by comparison of a “critical” micelle radius for each protein. The idea of a critical micelle size for micelles without protein (“empty” micelles) has been introduced by Kelley et al.18 Here, we define this characteristic length as the radius of the reversed micellar droplets (in the absence of protein), as determined by eqs 2 and 3, under conditions for which 50% protein solubilization occurs. The critical micelle radii of β-lactoglobulin and R-lactalbumin are 29 and 23 Å, respectively (Table 1). The critical micelle radius is the result of a balance between electrostatic and entropic effects in the reversed micellar system. In a theoretical study, Rahaman and Hatton27 demonstrated that primarily electrostatic interactions determine the size of reversed micelles. The size of protein-filled and empty micelles is affected by surfactant-surfactant repulsive electrostatic interactions across the micellar water pool. The presence of a protein
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Figure 5. Dependence of the critical micelle radius of R-lactalbumin (b), β-lactoglobulin (9), and R-chymotrypsin (4) on aqueous phase pH.
Figure 6. Effect of sodium counterion (b) vs potassium counterion (O) on the solubility of R-lactalbumin as a function of aqueous phase pH. Net cation concentration is 0.25 M. Dotted line represents the isoelectric point of the proteins.
molecule in the micelle effects partial screening of these interactions, allowing the formation of smaller, proteinfilled micelles18 as compared to micelles without protein. Furthermore, protein-surfactant electrostatic interactions also play a key role in determining the final size of the protein-filled micelle, at least for hydrophilic proteins. When proteins are incorporated into the reversed micellar phase, empty micelles break-up and surfactant molecules rearrange to form protein-filled micelles of a different size. This process is thermodynamically favorable because a relatively small number of empty micelles will form a larger (and hence entropically favored) number of protein-filled micelles. Kelley et al.18 argued that this provides an entropic driving force for solubilization which is directly coupled to the electrostatic driving force, as the latter determines the size of both empty and protein-filled reversed micelles. However, as the empty micelle size decreases and approaches some critical size, this driving force diminishes and protein solubilization ceases. The balance of these effects suggests that the critical micelle radius may depend on not only the protein size but also the aqueous phase pH, protein charge distribution, and solubilization environment as well. Dependence of the critical micelle radius on aqueous phase pH is expected in the instance where electrostatic interactions greatly affect the structure of protein-filled micelles. As the pH increases such that protein-surfactant electrostatic interactions become more unfavorable, the critical micelle radius is expected to increase as more water may be needed to form the protein-filled micelles under these conditions. This trend was observed by Kelley et al.18 for the protein R-chymotrypsin (Figure 5), which is hydrophilic and known to solubilize primarily inside the micellar water pool. However, the critical micelle radius of R-lactalbumin increases only slightly between pH 6.2 and 7.8 and remains constant at higher pH values. Likewise, the critical micelle radius of β-lactoglobulin shows essentially no change over the entire pH range studied (pH 6.2-8.5). These observations support the hypothesis that different physics dominate the solubilization of R-lactalbumin and β-lactoglobulin and that they may reside in a different solubilization environment, namely, the micellar interface. It is also of interest to point out that β-lactoglobulin and R-lactalbumin have much smaller critical micelle radii than hydrophilic proteins of comparable size (Table 1). Ribonuclease a is of comparable molecular weight to R-lactalbumin, and yet its critical micelle radius is more than twice as large. Although R-chymotrypsin is a protein of smaller molecular weight than β-lactoglobulin, its critical micelle radius is approximately 30% larger.
R-Chymotrypsin51 and ribonuclease a18 are expected to solubilize entirely within the micellar water pool and, therefore, may need a larger reversed micelle to accommodate both the protein and its Debye layer. Kelley et al.18 also noted that cytochrome c has a smaller critical micelle radius than ribonuclease a despite their similar molecular weights. They attributed this observation to the difference in solubilization sites for the two proteins, as cytochrome c is known to associate with the micellar interface.51 If the protein sits within the micellar interface, less water may be needed to form the protein-filled micelle since only a fraction of the protein’s surface is exposed to the micelle water pool. This results in a smaller proteinfilled micelle size and, therefore, the entropic driving force remains significant even under conditions where the empty micelle size is small. Thus, the small critical micelle size of R-lactalbumin and β-lactoglobulin support the hypothesis that these proteins solubilize in the reversed micellar interface under the influence of hydrophobic interactions. Finally, it is interesting to note that the data of Andrews et al.31 suggest that thaumatin, a protein with high surface hydrophobicity and whose solubilization appears to be influenced by hydrophobic interactions, has a small critical micelle radius as well, compared to hydrophilic proteins of comparable size. 3.2. Effect of Counterion Type. To avoid specific ion effects in the experiments discussed above, sodium chloride was used to adjust the ionic strength, as sodium is the counterion of Aerosol OT. However, cation type has been reported to have a strong effect on solubility for many proteins.6,18,19,24 To investigate whether this effect is present in the solubility behavior of R-lactalbumin and β-lactoglobulin, potassium salts were used to adjust the ionic strength and pH and the results were compared with those for sodium salts discussed above. As shown in Figure 6, R-lactalbumin is able to solubilize over a broader and higher pH range in the presence of sodium ion than when potassium ion is present. In the presence of potassium ions the solubility of R-lactalbumin drops rapidly for pH values greater than one unit above the isoelectric point. The solubilization data obtained in systems containing potassium ion is qualitatively similar to that of Andrews et al.31 for the same protein, despite the differences between conditions under which the experiments were performed in the two different studies. The broadening effect of sodium ion on the pH solubilization range was also observed for lysozyme24 and R-chymotrypsin.6 (51) Pileni, M.-P.; Zemb, T.; Petit, C. Chem. Phys. Lett. 1985, 118, 414.
Solubilization of Proteins in Reversed Micelles
Figure 7. Effect of sodium counterion (b 9) vs potassium counterion (O 0) on the solubility of R-lactalbumin (b O) and β-lactoglobulin (9 0) as a function of water transfer at pH 6.2.
Several hypotheses have been proposed to explain the specific ion effect. Andrews et al.31 speculated that hydrophobic interactions may be stronger in the presence of sodium ions as compared to potassium ion and that this effect is consistent with the lyotropic series. They also proposed that smaller ions produce less screening of protein-micelle electrostatic interactions and, therefore, allow more protein transfer. Their analysis, however, was based on bare ion size rather than hydrated ion size, whereas the latter may be a more relevant description of the ions in the micelle water pool. Nishiki et al.24 who also observed significant protein solubilization over a much broader range of ionic strengths in the presence of sodium ion than in the presence of potassium or barium ion, proposed that hydration of the salt molecules weakens their screening ability. Thus ions such as sodium, which are hydrated to a greater extent, screen electrostatic interactions less efficiently, allowing more protein transfer. Leodidis and Hatton28 observed that ions of smaller hydrated sizes than sodium ions can displace the larger sodium counterion from the vicinity of the surfactant head group in the micellar water pool, because the smaller ions can form a more intimate association with the head group. As a result these smaller ions partition more effectively into the micellar droplet, leading to better screening of the surfactant-surfactant repulsive electrostatic interactions within the droplet. Such screening results in a smaller micelle size. Kelley et al.18 observed that the differentiation in R-chymotrypsin solubilization behavior was virtually lost when the data were plotted as a function of w0swhich is approximately proportional to the micelle sizesfor the different salt types. The observations of the researchers cited above suggest that much can be learned by measuring protein solubilities at different ionic strengths and using these data to obtain a plot of solubilization as a function of w0 for the different cation types. By using w0 as a measure of the size of the reversed micelles, we can explore whether the effect of the cation type can be understood in terms of its influence on the micellar radius, coupled with the effect that radius has on protein solubilization. We therefore measured the solubility of R-lactalbumin and β-lactoglobulin at varying potassium chloride concentrations, and compared these measurements with solubility data collected in the presence of sodium ions. These values are converted in Figure 7 to a plot of percent protein solubilized as a function of w0 for the two cation types, obtained by also measuring water uptake present at the various ion concentrations. The data shown in Figure 7 were taken at a pH of 6.2. It
Langmuir, Vol. 12, No. 17, 1996 4079
Figure 8. Effect of sodium counterion (b 2) vs potassium counterion (O 4) on the solubility of R-lactalbumin as a function of water transfer when pH ) pI (b O) and pH > pI (2 4). Protein solubilization data from Andrews et al.31 Water transfer measured for 50 mM AOT in isooctane reversed micellar phase at various salt concentrations.
is evident that the solubility behavior is qualitatively different when different cations are present, even when the data are compared as a function of w0. The effect of counterion type on protein solubilization appears to be more than its effect on the water transfer or micellar water pool size. This distinction may be the result of specific protein-salt interactions, which could alter the conformation of the protein and hence the way the protein interacts with surfactant. Alternatively, the specific ions may influence the nature of hydrophobic interactions more directly. We have also replotted the R-lactalbumin solubility data reported by Andrews et al.31 as a function of w0 (Figure 8). In order to obtain this plot, we measured w0 values for 50 mM Aerosol OT in isooctane at various salt concentrations in the absence of protein. Under the conditions used in the Andrews et al.31 paper (0.25 mg/mL protein), the presence of protein is not expected to change significantly the amount of water transfer to the reversed micellar phase, except at the lowest salt concentrations. Here, the change should be less than 2%. The data shown in Figure 8 still indicate some influence of counterion type beyond its effect on reversed micellar radius. However, the difference between the results of Andrews et al.31 in the presence of potassium versus sodium is not nearly as dramatic as the results shown in Figure 7. It is important to point out a significant difference between our experiments and those performed by Andrews et al.31 In our measurements, buffers were used to control the pH in our reversed micellar system, whereas Andrews et al.31 used the addition of concentrated acid or base to adjust the pH. Fujii et al.52 used 31P-NMR to show that the pH of the reversed micellar water pool could be satisfactorily controlled using buffer solutions. When buffer solutions are not used, significant pH shifts are observed between the initial and final pH of the bulk aqueous phase, the latter being the reported pH. The use of concentrated acid or base instead of buffers, however, introduces no additional salts to the system; usually, a number of buffering components must be used to cover the full range of pH values investigated. In light of the differences between the data shown in Figures 7 and 8, further study is needed in this area before definite conclusions can be made regarding the effect of counterion type on the solubilization of hydrophobic proteins. (52) Fujii, H.; Kawai, T.; Nishikawa, H.; Ebert, G. Colloid Polym. Sci. 1982, 260, 697.
4080 Langmuir, Vol. 12, No. 17, 1996
3.3. Effect of Protein Concentration: Cosurfactant Effect. Measurements of protein partitioning between a water-in-oil microemulsion phase and an excess aqueous phase provide an experimental measure of the free energy difference for the protein in the two environments. When significant solubilization occurs under conditions where charge interactions should not dominate, it suggests that forces other than electrostatic ones lower the free energy between protein and surfactant in the reversed micelle. However, this is only one step in ascertaining the nature of these forces. For example, significant charge asymmetry on the protein could be responsible for protein partitioning into reversed micelles at pH values above the isoelectric point. It is also possible that the charge on the protein within the micellar droplet is not the same as in aqueous solution, although the use of buffers should help to prevent this. One barrier to better characterization of forces that drive the reversed micellar solubilization of proteins is that hydrophobic forces are not well understood. Although the way in which pH and ionic strength moderate charge interactions is fairly clear, there is not a similarly unambiguous way to regulate hydrophobic interactions. One interesting approach to tackling this difficulty was a study by Pires and Cabral33 which probed the effect of temperature on the partitioning of recombinant cytochrome b5 into reversed micelles. Hydrophobic interactions are believed to be primarily entropic, and as such, to become stronger at higher temperatures. Pires and Cabral33 observed that near the isoelectric point, the degree of cytochrome b5 transfer to the reversed micellar phase is increased by increasing the temperature. As this effect is observed even at high salt concentrations where any electrostatic interactions are expected to be effectively screened, these results suggest that at these conditions, the primary driving force for the solubilization of cytochrome b5 is protein-surfactant hydrophobic interactions. In contrast, under conditions (away from the isoelectric point) where electrostatic interactions are believed to be the dominating driving force, no such temperature dependence is observed. Another useful approach to exploring the role of hydrophobic forces in driving protein partitioning is to examine the conformation of the protein within the microemulsion. This approach has been most often employed in the area of enzymology in reversed micelles, where the possibility of hydrophobic protein-micelle interactions has long been acknowledged.53 Fluorescence and circular dichroism spectroscopy measurements on membrane and other interfacially active proteins show significant conformation changes in proteins housed within reversed micelles.54-59 Interestingly, these conformational changes are not believed to result from actual insertion of the protein within the surfactant monolayer, but instead are linked to more localized interactions of the protein with the surfactants, analogous to those for peripheral membrane proteins.56,59 Also it is important to note that these conformational studies have been applied to proteins “injected” into reversed micelles, where the amount of water and protein is set by the amount (53) Martinek, K.; Klyachko, N. L.; Kabanov, A. V.; Khmelnitsky, Y. L.; Levashov, A. V. Biochim. Biophys. Acta 1989, 981, 161. (54) Nicot, C.; Vacher, M.; Vincent, M.; Gallay, J.; Waks, M. Biochemistry 1985, 24, 7024. (55) Brown, E. D.; Yada, R. Y.; Marnagoni, A. G. Biochim. Biophys. Acta 1993, 1161, 66. (56) Marangoni, A. G. Enzyme Microb. Technol. 1993, 15, 944. (57) Walde, P.; Han, D.; Luisi, P. L. Biochemistry 1993, 32, 4029. (58) Otero, C.; Ru´a, M. L.; Robledo, L. FEBS Lett. 1995, 360, 202. (59) Lenz, V. J.; Federwisch, M.; Gattner, H. G.; Brandenburg, D.; Ho¨cker, H.; Hassiepen, U.; Wollmer, A. Biochemistry 1995, 34, 6130.
Kawakami and Dungan
Figure 9. Effect of initial amino acid concentration on the extent of water transfer into an AOT-in-isooctane microemulsion, for the amino acids glycine (b), leucine (9), and phenylalanine (2). AOT concentration is 300 mM, the aqueous phase contained sodium phosphate buffers so that the total Na+ concentration was 0.2 M and the pH value was between 6.0 and 6.5. Data were taken from work of Leodidis and Hatton.48
added by the researcher to the organic phase. These microemulsions differ from the two phase systems discussed in the current paper, where the protein, salts, and water can equilibrate with an excess aqueous phase. Fluorescence and circular dichroism spectroscopy measurements on R-lactalbumin and β-lactoglobulin in reversed micelles should provide useful clues as to the interaction of these proteins with the AOT monolayer, and such measurements will be the subject of future research in our laboratory. Here, however, we take a different approach to exploring protein-surfactant interactions, one based on recent experiments examining amino acid solubilization in reversed micelles. Leodidis and Hatton48 used amino acids to study the effect of small solute hydrophobicity on reversed micellar solubilization. Their results indicated that hydrophobic amino acids partition to a greater extent into the micelles than do more polar amino acids. More remarkably, however, they also found that increasing the concentration of the hydrophobic amino acids in the initial aqueous phase increased the amount of water transferred to the reversed micellar phase. As shown in Figure 9, phenylalanine enhances water transfer into the microemulsion substantially, compared to water transferred in the absence of amino acid, whereas the hydrophilic amino acid glycine has no effect on water transfer. In this way, these amino acids act as “cosurfactants”si.e., they are molecules that do not form microemulsions themselves, but facilitate the formation of such a microemulsion. This behavior provided strong evidence that hydrophobic amino acids are substantially incorporated within the micelle interfacial layer. The results of Leodidis and Hatton33 shown in Figure 9 demonstrate that by investigating the effect of a solute on the properties of the microemulsion, we gain valuable insight into the way in which that solute interacts with the micellar droplet. In Figure 10a is shown the effect of various initial concentrations of R-lactalbumin, β-lactoglobulin, and R-chymotrypsin on the water solubilized within the microemulsion phase. The latter was included in this study as a well-studied example of a hydrophilic protein which is believed to position itself within the aqueous water pool within the micelle, and whose solubilization is driven primarily by electrostatic interactions.27,51 These measurements were carried out using buffered solutions at a pH of 6.2 and with a salt
Solubilization of Proteins in Reversed Micelles
Langmuir, Vol. 12, No. 17, 1996 4081
Both R-lactalbumin and R-chymotrypsin can solubilize to much greater extents in the micelles; concentrations up to 4 g/L are shown in Figure 10b. The effect of R-chymotrypsin on water incorporation levels out at concentrations above 1 g/L and may be due to reequilibration effects of salts and surfactant between proteinfilled and empty micelles. The amount of water transferred in the presence of R-lactalbumin, however, increases linearly (or even more rapidly) with protein concentration and can most likely be attributed to an increase in interfacial area due to incorporation of protein within the surfactant layer. It is possible to use simple geometric arguments to estimate the increase in interfacial area caused by the addition of R-lactalbumin to the reversed micellar droplets. Assuming for simplicity a monodisperse system of microemulsion droplets of radius R, that radius will be proportional to the volume to surface area ratio of the droplets. This allows us to write for a protein-containing microemulsion the relation
R)3
Nsvs + Nwvw + Npvp Nsas + Npap
(5)
where the subscripts s, w, and p represent the surfactant, water, and protein molecules, respectively, and N, v, and a denote the number, volume, and interfacial area of those molecules in the droplets. Setting λ ) Np/Ns, eq 5 simplifies to
R)3 Figure 10. Effect of protein concentration on the extent of water transfer into an AOT-in-isooctane microemulsion, for the proteins R-chymotrypsin (9), β-lactoglobulin (2), and R-lactalbumin (b), at a NaCl concentration of 0.1 M and pH 6.2: (a) water transfer versus initial (total) protein concentration; (b) water transfer versus the reversed micellar concentration of protein.
concentration of 0.1 M sodium chloride. These conditions were chosen as those favoring transfer of all three proteins into the reversed micellar phase. The results indicate the presence of protein within the reversed micelles enhances water incorporation within the microemulsion, and in the case of R-lactalbumin, this enhancement is dramatic: 60% more water is taken up by the reversed micelles in the presence of R-lactalbumin compared to that in its absence. By contrast, the effect of R-chymotrypsin or β-lactoglobulin on water solubilization is less than 12%. A more meaningful comparison of the three proteins can be made by replotting the data from Figure 10a as a function of the reversed micellar concentration of protein. The result, shown in Figure 10b, indicates that the weak effect of β-lactoglobulin on water solubilization may be due to the fact that relatively small amounts of that protein can be accommodated within the micelle. Although the partitioning of the protein favors the reversed micellar phase at low protein concentrations, as the protein concentration is increased the capacity of the microemulsion appears to be limited to approximately 1 g/L of β-lactoglobulin. The low capacity of the micelles for this protein may be related to the large size of β-lactoglobulin relative to the two other proteins. Because of the limited concentration range for β-lactoglobulin in the reversed micelles, it is difficult to discern from Figure 10b whether this protein is substantially affecting the structure of the micelle.
vs + w0vw + λvp as + λap
(6)
Dividing this radius by the radius R° of the droplets in the absence of protein, we obtain
vw vp +λ v vs R s ) R° vw ap 1 + w0° 1+λ vs as 1 + w0
(
)(
)
(7)
where w0° is the molar ratio of water to surfactant in the absence of protein. Since we do not as yet know how the micelle radius changes with protein incorporation, we must consider a few different scenarios to pursue these geometric arguments further. First, as the interfacial area increases, the number of micelles could increase while keeping the radius of the droplets constant. In this case eq 7 can be rearranged to yield
((
)
)
w0 ap vs vp 1+ )1+λ w0° as vww0° vww0°
(8)
Alternatively, the number of micelles may stay constant while the size of the micelle increases. In this case we may write
Nsas + Npap 2
4πR
Nsas )
4πR° 2
or
(
)
ap R ) 1+λ R° as
1/2
Combining this result with eq 7 above gives
4082 Langmuir, Vol. 12, No. 17, 1996
(
)(
)
w0 vs ap 1+λ ) 1+ w0° vww0° as
3/2
-λ
Kawakami and Dungan
vp vs (9) vww0° vww0°
Equations 8 and 9 can be fit to the data in Figure 10b by using the molecular weight of R-lactalbumin to convert the reversed micellar protein concentrations to various values of λ. The molecular volume of water vw is 30.1 Å3, while the geometric parameters for AOT can be well estimated27,33 as as ) 62.3 Å2 and vs ) 640 Å3. Finally, we will take as our volume for R-lactalbumin vp ) 28 900 Å3, obtained from the reported geometry of the molecule.60 Both eqs 8 and 9 fit the data equally well and thus are represented only by the single solid curve in the plot of normalized water transfer versus λ given in Figure 11. The fit values of ap obtained for the two cases are presented in Table 2. The values shown are quite large relative to the area taken up by an AOT head groups, consistent with the much larger size of the protein molecule. Also shown in Table 2 are values for an “effective radius” r, corresponding to a circular region taken up by the protein at the interface, so that ap ) πr2. This value for r can be compared with the ellipsoidal dimensions of R-lactalbumin in solution, estimated to be 2.2 nm × 4.4 nm × 5.7 nm.60 The results indicate that according to these simple geometric arguments, the protein may occupy a region within the interface larger than its cross section in aqueous solution. In addition to the two cases considered above, it is of course possible that both the number and size change in response to protein incorporation. As a result there is a range of values for ap which could be matched to the water transfer data above. Further there are a number of simplifying assumptions made in the geometric arguments above which limit the accuracy of the analysis, with the assumption of monodispersity perhaps the most obviously incorrect one. However, until we obtain more information on the structural characteristics of the microemulsion, in particular the size of the microemulsion droplets themselves, the simple model above suggests that expansion of the microemulsion interface due to protein incorporation is a reasonable possibility. Another assumption in the geometric arguments above is that the reversed micellar droplets remain spherical upon protein incorporation. If a large portion of the protein molecule inserts itself into the interfacial region, it would seem likely that the molecule could significantly alter the localized curvature of the interface, resulting in a shift away from a spherical droplet. We observed some evidence of this possibility, in that the microemulsion appeared somewhat opalescent at high R-lactalbumin concentrations. The behavior of the microemulsion upon protein incorporation may therefore be similar to that only recently noted with synthetic, surface-active polymers,61,62 which can shift the phase behavior of microemulsions. Such behavior for a protein molecule has also been noted in a recent paper by Huruguen and Pileni,63 in which percolation thresholds in AOT reversed micelles were shown to be substantially altered by the addition of cytochrome c. 4. Conclusion The solubility behavior in a reversed micellar system of 100 mM Aerosol OT in isooctane was presented for the milk proteins R-lactalbumin and β-lactoglobulin. The (60) Swaisgood, H. E. In Developments in Dairy Chemistrys1; Fox, P. F., Ed.; Applied Science Publishers: London, 1982; pp 1-59. (61) Kabalnov, A.; Olsson, U.; Thuresson, K.; Wennerstro¨m, H. Langmuir 1994, 10, 4509. (62) Bagger-Jo¨rgensen, H.; Olsson, U.; Iliopoulos, I. Langmuir 1995, 11, 1934. (63) Huruguen, J. P.; Pileni, M. P. Eur. Biophys. J. 1991, 19, 103.
Figure 11. Comparison of eqs 8 and 9 with data (b) for the effect of R-lactalbumin concentration on the extent of water transfer. Data were taken from Figure 10b. w0 is the moles of water per mole of surfactant in the reversed micellar phase in the presence of protein, while w0° is that quantity in the absence of protein. λ is the moles of protein per mole surfactant in the reversed micellar phase. Table 2. Values for the Effective Area and Radius Occupied by r-Lactalbumin at the Reversed Micellar Interface, Using Geometric Arguments As Described in the Text
geometric assumption micelle radius remains constant number of micelles remains constant
interfacial area effective radius r ap occupied occupied by by protein (nm2) protein (nm) 77 50
5 4
solubilization of these proteins as a function of aqueous phase pH is very different than that described for hydrophilic proteins in the literature. As electrostatics dominate the solubilization process of hydrophilic proteins, they exhibit maximum solubility at pHs below their isoelectric points, where the protein and surfactant are oppositely charged. The maximum solubility of R-lactalbumin and β-lactoglobulin, however, occurred above their respective isoelectric points, indicating that their solubilization involves some driving force other than electrostatic interactions. R-Lactalbumin and β-lactoglobulin also showed significant solubility at high ionic strengths, where the solubilization of other hydrophilic proteins is normally suppressed. Insensitivity to aqueous phase ionic strength provides further evidence that this unusual solubility behavior is due to the involvement of protein-surfactant hydrophobic interactions, rather than localized electrostatic interactions due to asymmetric charge distribution. A correlation is observed between the solubilization properties of R-lactalbumin and β-lactoglobulin and their respective hydrophobicities, as demonstrated by the effect of protein size and net charge on protein solubility. The critical micelle radius has been used to provide a quantitative measure of the effect of micelle size on the solubilization properties of individual proteins. Although the critical micelle radius of hydrophilic proteins depends on the aqueous phase pH, no such dependence is observed for R-lactalbumin and β-lactoglobulin. Also, the critical micelle radii of R-lactalbumin and β-lactoglobulin are much smaller than the values observed for other hydrophilic proteins of comparable size. These observations indicate that models based on electrostatic interactions that have been developed to explain the solubility phenomena of hydrophilic proteins do not apply to R-lactalbumin and β-lactoglobulin. The solubility behavior of these proteins appears to be primarily due to hydrophobic interactions that allow the proteins to solubilize in the reversed micellar interface. On a practical basis, measurements of the influence of pH and ionic strength on the solubilization of R-lactal-
Solubilization of Proteins in Reversed Micelles
bumin and β-lactoglobulin indicate that these proteins can be separated on the basis of pH, and even more readily on the basis of sodium chloride concentration at high ionic strengths. We believe the latter effect is really a protein size effectsthe larger β-lactoglobulin is excluded from the small micelles produced at high ionic strengths. Preliminary experiments in the laboratory have also demonstrated that mixtures of the proteins can be separated, results that will discussed in a future publication. Studies on the influence of counterion type on protein solubility were performed with R-lactalbumin. The pH range for significant protein solubilization is more narrow and shifted toward lower pHs in the presence of potassium ion. The counterion type also greatly affects solubility as a function of ionic strength. Our data suggest that the differences in R-lactalbumin solubility in the presence of potassium ion versus sodium ion is not simply due to the effect of counterion type on micelle size but further investigation is needed to confirm this conclusion. The concentration of protein in the initial aqueous phase was observed to affect both the solubilization of water
Langmuir, Vol. 12, No. 17, 1996 4083
and protein. As the R-lactalbumin concentration was increased, significantly greater amounts of water could be solubilized by the reversed micellar phase. Significantly lower effects on water transfer were observed with the proteins β-lactoglobulin and R-chymotrypsin. The strong linear relationship between water transfer and the amount of R-lactalbumin solubilized within the reversed micellar phase suggests that this protein may act as a cosurfactant and induce expansion of the surfactant interface by its incorporation in that region. Acknowledgment. The authors are grateful to the California Dairy Foods Research Center (93 DUS-01) for their financial support of this work and to the NIH Biotechnology Pre-Doctoral Training Grant (T32 GM0 8343-05) for a fellowship for L. Kawakami. We would also like to thank Ms. Cristina Cheung for her assistance in determining reversed micellar phase protein extinction coefficients. LA950591K
