J . Phys. Chem. 1991, 95, 1467-1471
1467
Characteristics of Protein-Containing Reversed Micelles Subjected to Clathrate Hydrate Formation Conditions Huyen Nguyen,+ Vijay T.John,**+and Wayne F. Reed* Departments of Chemical Engineering and Physics, Tulane University. New Orleans, Louisiana 70118 (Received: May 23, 1990; In Final Form: July 30, 1990)
The formation qf clathrate hydrates in protein-containing reversed micellar systems, and its effects on the stability of encapsulated proteins, are discussed. The formation of methane hydrates essentially removes water from the micelles, decreasing the mean micelle size. Proteins, once encapsulated in the micelles, remain solubilized as the average micellar water content is decreased. The observation appears valid both for proteins encapsulated in relatively small single-occupancy micelles and for proteins encapsulated as large aggregates.
Introduction Reversed micelles are water-in-oil microemulsions that are capable of solubilizing a variety of proteins through encapsulation in the microaqueous phase. Because of their relevance to biomembrane mimetic phenomena,l protein extraction proce~ses,~*~ and biocatalysis in minimal water much effort has gone into the physicochemical characterizations of protein-containing reversed Knowledge of proteinsurfactant interactions and the activity of the microaqueous phase helps to understand the factors involved in protein stabilization and solubility sustenance in these systems. In this paper we attempt to further characterize reversed micellar systems containing proteins by conducting a study of clathrate hydrate formation in the microaqueous phase. Clathrate hydrates, or gas hydrates as they are often called, are crystalline inclusions of gas and water, formed when water is contacted with gas species at appropriate thermodynamic conditions of temperature and pressure. The gas molecules encaged within hydrate cavities stabilize the crystal structure through van der Waals interactions with the host water molecules.'0 Hydrate formation in bulk water has been traditionally studied due to relevance in natural gas recovery and processing;" their formation in the microaqueous phase of reversed micelles is a rather new and interesting phenomenon. In a recent paper,I2 we have shown that hydrates of methane can form in reversed micellar solution and that the thermodynamic conditions of formation are dependent on the water-to-surfactant molar ratio wo. Figure 1 summarizes the concepts with the true P-T data listed in ref 12. Each line in Figure I represents the incipient hydrate formation conditions for a solution of specific wo. At each wo, hydrate formation in reversed micelles follows univariant thermodynamic equilibria; Le., at a given temperature, hydrates form at a specific pressure. Furthermore, the smaller the wo of a reversed micellar solution, the higher the pressure for hydrate formation at a given temperature. The reasoning is rather intuitive; as wo decreases, micelle size decreases and the intramicellar water becomes more tightly bound with a lower activity.13J4 The free energy based driving force (the chemical potential gradient between water in the @-phase of empty hydrates and pure waterlo) required to reorient the water molecules to the crystalline hydrate form increases as the water activity decreases; this is manifested by the increase in the pressure for hydrate formation.I0 Examining Figure 1 from another perspective, if hydrate formation is initiated in large reversed micelles by progressively increasing the pressure at a given temperature, micelle size decreases as hydrates progressively form and the crystals nucleate and drop out of solution. The process is completely reversible; by releasing gas and decreasing the pressure, hydrates dissociate, and the water released is spontaneously back-incorporated into To whom correspondence should be addressed.
'Department of Chemical Engineering. Department of Physics.
I
,
,
the micelles, increasing micelle size. In this paper, we report our observations on the stability of proteins in reversed micelles subject to hydrate formation conditions and consequent removal of microaqueous-phase water. Protein solubility measurements and micelle size determinations were used to characterize the behavior of protein-containing micelles. Three proteins were studied; cytochrome c, a-chymotrypsin, and azocasein. Cytochrome c is an example of an interface-resident protein,15J6a-chymotrypsin is an example of a core-resident protein,'6J7 and casein is a fibrous protein as opposed to the other two globular proteins. Cytochrome c and azocasein were additionally chosen as model proteins since their solubilization properties could be qualitatively observed visually. Materials and Methods Cytochrome c (horse heart), a-chymotrypsin (bovine pancreatic), and azocasein (sulfanilamide-azocasein) were obtained from Sigma Chemicals and used as such. Cytochrome c and a-chymotrypsin have molecular weights of 12 389 and 25 000, respectively; the azocasein received is a mixture of a-, @-, y-, and K-azocasein with ill-defined composition. Reversed micellar constituents included sodium bis(2-ethylhexyl) sulfosuccinate (AOT; Aldrich Chemicals, 99% purity) and isooctane (Aldrich, spectrophotometric grade). Double-distilled water was used in all preparations of the microaqueous phase, and the ionic strength and pH were adjusted through addition of KCI and NaOH/HCI, respectively.
(1) Wirz, J.; Rosenbusch, J. P. In Reverse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum Press: New York, 1984. (2) Leser, M. E.; Wei, G . ; Luisi, P. L.; Maestro, M. Biochem. Biophys. Commun. 1986, 135, 629. (3) Rahaman, R. S.; Chee, J. Y.; Cabral, J. M. S.;Hatton, T. A. Biofechnol. Prog. 1988, 4 , 218. (4) Leser, M. E.; Wei, G.; Liithi, P.; Haering, G.; Hochkoeppler, A.; Blijchliger, E.; Luisi, P. L. J . Chim.Phys. 1987, 84, I 1 13. ( 5 ) Martinek, K.; Berezin, 1. V.; Khmelnitski, Yu. L.; Klyachko, N. L.; Levashov, A. V. Biocatalysis 1987, I , 9. (6) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 204. (7) Sheu, E.; Goklen, K. E.; Hatton, T. A.; Chen, S.-H. Biorechnol. frog. 1986, 2, 175. (8) Vos, K.; Laane, C.; Visser, A. J. W. G . Photochem. Photobiol. 1987, 45, 863. (9) Battistel, E.; Luisi, P. L.;Rialdi, G.J . f h y s . Chem. 1988, 92. 6680. (IO) van der Waals, J. H.; Platteeuw, J. C. Ado. Chem. Phys. 1959.2, I . ( I I ) Berecz, E.; Balla-Achs, M. Gas Hydrates; Elsevier: New York, 1983. (12) Nguyen, H.; Phillips, J. B.; John, V. T. J . Phys. Chem. 1989, 93, 8123. (13) Kubik, R.; Eicke, H.-F.; Jonsson, B. Helo. Chim. Acta 1982, 65, 170. (14) Quist, P.-0.; Halle. B. J . Chem. Soc., Faraday Trans. I 1988, 84, 1033. (IS) Brochette, P.; Petit, C.; Pileni, M. P.J . Phys. Chem. 1988, 92, 3505. (16) Petit, C.; Brochette, P.; Pileni, M. P. J . Phys. Chem. 1986, 90, 6517. (17) Pileni, M. P.; Zemb, T.; Petit, C Chem. Phys. Lett. 1985, 118, 414.
0 1991 American Chemical Society
1468 The Journal of Physical Chemistry, Vol. 95, No. 3, 1991
Nguyen et al. Supernatant Sample
P
T Figure 1. Schematic of hydrate equilibria in reversed micellar solutions. Each line represents the univariant hydrate formation characteristics for reversed micelles of a specified water/AOT molar ratio, wo.
Protein encapsulation into reversed micelles was carried out through thc so-called injection method,I8 wherein the required amount of protein is first dissolved in water and the aqueous phase injected into a solution containing isooctane and AOT. For example, a 5.2 mM cytochrome c in water solution is used to make up a reversed micellar solution containing 55.5 mM AOT and 78 pM cytochrome c at a w o of 15. Hydrate Formation. Experiments to form hydrates in reversed micelles were carried out using a 100-mL glass-windowed highpressure cell suspended in a temperature-controlled water bath. The apparatus is similar to that described by Holder and Hand19 for thermodynamic studies of hydrates in bulk water systems. About 60 mL of a protein-containing reversed micellar solution of wo 15 is introduced into the high-pressure cell, which is then pressurized with methane. At a temperature of 273.65 K, hydrates form in the microaqueous phase at a pressure of about 2.9 MPa12 (compared to 2.6 MPa in pure waterlo). Nucleation of hydrate crystals leads to their precipitation from solution (hydrates being more dense than the organic solvent). A schematic of the situation in the hydrate cell is shown in Figure 2. A sampling arrangement built into the system allows sampling of about 10 mL of the supernatant solution into a container kept at a pressure approximately 0.1-0.2 MPa below the system pressure. The sample container is then slowly depressurized, and the sample is removed and analyzed for water content through Karl Fischer titration (Mettler DL-18). The surfactant being highly soluble in the organic phase remains in the supernatant, and the water content of the supernatant can then be interpreted in terms of the wovalue of the supernatant. In depressurizing the sample, a little of the solvent is lost; this implies that the measured water concentration in the sampled supernatant is a little higher than that in the hydrate cell, and thus the experimentally measured wo of the supernatant is a little above the true value. Protein contents of the supernatant samples from the hydrate cell were measured through UV spectrophotometry (absorbance at 280 nm). With cytochrome c, the results from the absorbance measurements were corroborated by using a modified Lowry method involving precipitation of the protein with sodium deoxycholate and trichloroacetic acid.20 Unless the high-pressure sampling is done correctly, shear-induced protein denaturation leads to incorrect absorbance readings; the titration-based Lowry method on the other hand is insensitive to protein denaturation. Agreement between the two methods was found to be necessary to refine the technique of supernatant sampling from the hydrate cell so that protein denaturation was minimal. Size Analyses. Size characteristics of reversed micelles were done using quasi-elastic light scattering techniques (QELS). The dynamic and static light scattering apparatus used consisted of a Coherent Innova 90-5 argon ion laser operating at 488 nm, a Thorn EM1 head-on phototube for single photon counting, and a Brookhaven BI 2030 autocorrelator. The sample cell compartment was held at 298 K for all the scattering experiments. (18) Luisi, P. L. Angew. Chem., Int. Ed. Engl. 1985, 24, 439. (19) Holder, G. D.; Hand, J. H. AIChE J. 1982, 28, 440. (20) Sigma Chemical Company. Protein assay kit P5656, 1985.
4 Protein
Hydrate
Figure 2. Schematic of the contents of the hydrate cell.
Scattering glassware was soaked in chromic acid before thorough rinsing and drying. Full details of the system and its calibration are given elsewhere.21 Since there was no angular dependence to the light scattered by the microemulsions, as expected for particles much smaller than the wavelength of the incident light, all dynamic light scattering measurements were carried out at an observation angle 8 of 90'. Since the microemulsions are electrically neutral and the solutions studied were dilute (ca. 0.1 vol 76 microemulsion), the assumption of statistical independence of the scattered fields from subregions within the scattering volume was satisfied.22 This assumption allows the scattered electric field autocorrelation function to be simply related to the measured scattered intensity autocorrelation function so that the exponential decay time, 7, of the latter experimental quantity is related to the particle diffusion coefficient D = (27q2)-', where q = (47rn/X) sin (8/2) is the scattering vector. The index of refraction, n, was taken as 1.39 for isooctane. Autocorrelation decay curves were analyzed chiefly by the standard cumulant method,23whose first cumulant yields a zaveraged diffusion coefficient of the particle mass distribution. Because no assumptions about the distribution are needed in this method, all particle sizing data, unless otherwise noted, are based on the first cumulant. In some cases it was desirable, nonetheless, to investigate possible bimodality of the distribution of mixed protein-occupied and unoccupied micelles. In such cases a double-exponential fit to the data was used. Polydispersity in the cumulant analysis was assessed by defining an index Q, which is the ratio of the second moment to the first moment squared in the power series expansion of the logarithm. In practice, a value of Q less than about 0.15 indicates a fairly narrow size distribution. Spurious values of D can be obtained if the sample contains dust or if the pointwise scatter in the measured autocorrelation function is large. Centrifugation and filtration (0.2-pm filter) of the sample always eliminated the former problem, whereas running the duration of the autocorrelation experiments in increasing increments and watching for the convergence of Q on a final value eliminated the latter. The light scattering data presented are expressed in terms of the equivalent hydrodynamic diameter, DH,obtained from the Stokes-Einstein equation, DH = kT/37rqD, where k is Boltzmann's constant and q is the solution viscosity which was taken as 0.491 CP for isooctane at 298 K. Good correspondence between D and
'
(21) Reed, C. E.; Xiao, L.; Reed, W. F. Biopolymers 1989, 28, 1981. (22) Berne, B.; Pecora, R. Dynamic Light Scattering, John Wiley and Sons: New York, 1976. (23) Koppel, D. E. J. Chem. Phys. 1972, 57,4314.
Protein-Containing Reversed Micelles
Figure 3. Size distribution analysis of protein-free and protein-containing micellar solutions modified by hydrate formation.
DH calculated by the above equation can only be expected for extrapolations to zero solute concentrations of solutions containing spherical particles. Microemulsions are generally thought to be spherical, and for the AOT based microemulsions at the water concentrations (wo)studied, there was only a slight negative slope of DH vs the AOT concentration, in agreement with the observations of Zulauf and E i ~ k e . ~ ~ Results and Discussion Micelle Size Modifications through Hydrate Formation. Figure 3 illustrates the mean micellar size for protein-free reversed micellar solutions and for micellar solutions containing cytochrome c or a-chymotrypsin. In all three cases, the data at wo 15 refers to the micellar solutions introduced into the high-pressure hydrate cell with the reversed micelles consisting of just the surfactant, pure water, and, where applicable, the protein. Data at lower wo refer to the situation where hydrates were formed by pressurization at 273.65 K. Following our earlier work,I2 the incipient hydrate formation conditions at wo IO and wo 5 are 3.1 and 5.0 MPa, respectively: these were approximate pressures at which supernatant samples were removed. The samples were depressurized, and water and protein contents were analyzed. Prior to size analysis, the supernatant samples were refiltered to remove any accumulated dust from the hydrate cell. Some points of note emerge from these experiments. First, hydrate formation clearly removes water from the micelles in all three cases and thus decreases wo (assuming constant surfactant concentration due to the high solubility of the surfactant in the organic phase). The result is a decrease in micelle size, which is welldocumented in earlier studies involving micelle preparations at different wo value^.^^.^^ In all cases, pressurization to 7.5 MPa (the limit of the glass-windowed cell) did not help reduce wo below approximately 4, validating deutron spin-relaxation studiesI4 indicating strongly bound water, perhaps that of hydration, at these wo values. Second, cytochrome c and a-chymotrypsin were found to retain complete solubility in the reversed micelles, both under hydrate formation conditions and subsequent to sampling and depressurization. It is also noted that while a-chymotrypsin encapsulation does not affect the mean micelle size, cytochrome c increases the mean size. The effect of cytochrome c on micelle size appears to be somewhat controversial; Sheu et al.' indicate a size increase in protein-containing micelles using small-angle neutron scattering and the phase-transfer method for protein encapsulation,18while Pileni et aI.I5J6using small-angle X-ray scattering and the injection method for encapsulation indicate a small decrease in size. While our focus has been the effect of hydrate formation on protein solubilization, we have attempted to rationalize the relatively significant increase in mean micelle size by conducting light scattering experiments on the protein-containing aqueous (24) ZulauT, M.; Eicke, H.-F. J . Phys. Chem. 1979, 83, 480. (25) Howe. A. M.; Mcdonald, J. A.; Robinson, B. H. J . Chem. Soc., Faruduy Trans. I 1987,83, 1007.
The Journal of Physical Chemistry, Vol. 95, NO. 3, 1991
1469
source phase from which the micelles were prepared. Noting that the initial 5.2 mM cytochrome c aqueous solution is too concentrated to do scattering experiments with (due to too much absorption), the solution was diluted IO-fold to 0.5 mM. Size analysis indicated large aggregates with particle size easily exceeding 100 nm. Further dilution to the same protein concentration as in reversed micelles (78 pM) still indicated the existence of the aggregates with high polydispersities (>OS). On the other hand, preparation of 78 pM cytochrome c in water solutions showed small particles with a mean size of 8.5 nm. We therefore think that the initial dissolution of cytochrome c in water at such large concentrations leads to protein aggregation. Being an interfacial enzyme, hydrophobic interactions may be a significant cause of aggregation in aqueous solution. Incorporation of cytochrome c into reversed micelles yields an average micelle size to 14.6 nm, but it is possible that aggregates still exist in the micellar phase. Indeed, while the wo 15 protein-free micellar solution and the wo 15 micellar solution with a-chymotrypsin showed quite narrow size distributions (polydispersity index CO.15 maximum, and double-exponential analysis yielding negligible bimodality before and after hydrate formation), the corresponding cytochrome c solution had a relatively high polydispersity index (0.38). Also, repeated preparations of the cytochrome c solution at wo 15 led to varying particle sizes ranging from 8 nm upward; the value of 14.6 nm listed in Figure 3 is the largest obtained. Furthermore, we found that when encapsulation was done by using the extraction method where the micellar solution is contacted with solid enzyme,I8 mean particle diameters as large as 23 nm could be obtained, indicating that the preparation method does affect the size distribution. Finally, double-exponential analysis of w,, 15 micelles containing 78 pM cytochrome c showed two well-separated relaxation times corresponding to 6.8 and 89.3 nm. The smaller size presumably corresponds to empty and one protein molecule occupancy micelles, and the larger size could correspond to aggregate-filled micelles. After hydrate formation and reduction to wo 11, the sizes fell to 6.2 and 69 nm. After further hydrate formation and reduction to wo 4, the sizes fell to 4.2 and 20 nm. The evidence is thus that both aggregate-free and aggregate-containing micelles lose water subsequent to hydrate formation. The observation does not, however, necessarily prove that hydrates form from both types of micelles, although the equivalence of water chemical potentials in all micelles indicates that this should be the case. Regardless of where hydrates preferentially form, rapid equilibration and redistribution of water through micellar collisions lead to the bimodal size distributions measured through light scattering. The unfortunate characteristic of protein aggregation is the inconsistency of the phenomenon; Le., we cannot clearly state the particle size of cytochrome c in reversed micelles at wo 15. Nevertheless, the effect of hydrate formation is to decrease the mean particle size, regardless of whether or not aggregates exist. Hydrate Formation and Protein Solubility. The retention of protein solubility in the micellar phase during hydrate formation was further explored. Under macroscopic two-phase conditions (an aqueous phase in contact with a reversed micellar phase), it has been clearly shown that protein solubility in reversed micelles is a strong function of pH and ionic strengtheZ6With cytochrome c for example, pH values less than the isoelectric point of the protein (10.6) stabilize the protein in reversed micelles due to electrostatic attraction between the positively charged macromolecule and the anionic surfactant headgroups. Increasing the ionic strength to values greater than 0.3 M KCI destabilizes the encapsulated protein due to charge screening of the electrostatic protein-micelle interactions. The question arises as to whether protein solubilization stability is affected in macroscopically single-phase reversed micellar solutions by changes in the ionic strength of the microaqueous phase, either introduced through sample preparation or induced through hydrate formation. Figure 4 illustrates the solubility of cytochrome c in reversed micelles as a function of ionic strength, at various (26) Goklen, K. E.; Hatton, T. A. Sep. Sci. Techno!. 1987, 22, 831.
Nguyen et al.
1470 The Journal of Physical Chemistry, Vol. 95, No, 3, 1991 80
10
-
[ M T ] - 55.5 mY pH (1.0
.
. [MT]
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-
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0 Fmmhydmbk" IFromdiratlnjrtbn
6
401
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\
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0.0
0.4
0.2
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0.8
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Concentration (M) Figure 4. Effect of ionic strength on maximum protein solubilization. KCI
0
0
5
10
15
20
WO
Figure 5. Retention of protein solubility during wo reduction by hydrate formation. The cross-hatched bar represents the initial system where protein is encapsulated through the injection method, the unfilled bar represents the same solution after water removal to w, 4 has been effected by hydrate formation, and the filled bar represents a prepared solution of w,, 5 where encapsulation is done by injection.
wovalues, where wo and salt contents were changed through sample preparation. The experiment was done by first dissolving cytochrome c in aqueous solution at pH 6; the amount of cytochrome c in aqueous solution was adjusted so that the final concentration in the reversed micellar phase would be 78 pM (1 mg/mL) if all mation up to a wo value of 4 (>7 MPa and 273.65 K) and subthe protein was solubilized. After addition of KCI to the aqueous sequent to supernatant sampling and bringing the sample to amsolutions, the required amount of protein-containing aqueous phase bient temperature and pressure conditions. The observation inwas injected to an AOT + isooctane solution to yield reversed micelles of the desired wo. The notation w ~implies , ~the ~wo of~ ~ dicates the irreversibility of protein solubilization in single-phase reversed micellar systems. While solubilization is inhibited at the micellar solution if all the protein and water added are solhigher ionic strengths, it appears that once encapsulation is ubilized into the micelles. achieved at low ionic strength, the protein remains immune to ionic The results shown in Figure 4 are the mean of two independent strength modifications in the microaqueous phase. Evidently, once experiments. As the figure indicates, while complete solubilization protein-surfactant interactions have been established, the elecoccurs for all w ~values , at ~ zero ~ added ~ ~salt levels, the protein trolyte species becomes unable to penetrate the intervening aqueous solubilization decreases significantly at higher salt concentrations, layers to screen out the interactions. We contrast the results to which is to be expected based on charge screening of proteinobservations in two-phase systems26 where protein solubility is surfactant attractions. While there is not much difference in sensitive to ionic strength. In two-phase systems, the micelles solubilization between solutions of w ~ 15 and , ~10, the ~ solution ~ ~ solubilize a maximum of water, with wo > 20 at KCI concenof w ~5 shows , ~ a higher ~ ~ sensitivity ~ to salt concentration, with trations of 0.1 M in the aqueous phase. The relatively greater protein solubilization dropping off by 0.2 M KCI. The rationale activity of water in these larger micelles, and the fact that collisions for this observation is not immediately obvious. In addition to with the oil-water interface lead to constant breakup and recharge screening of the proteinsurfactant electrostatic attractions formation of micelles, constitute possible mechanisms to explain during encapsulation, other factors play a role. Surfactant salt screening of protein-surfactant interactions. head-group repulsions are screened out at high salt levels, resulting in smaller micelle sizes and lower water sol~bilization.~ At low Exploratory experiments with encapsulated a-chymotrypsin, ribonuclease, microbial lipase, and azoalbumin all indicated that water contents, much of the water could be bound as the water of hydration of the protein, surfactant, and the electrolyte ions; the protein stays encapsulated subsequent to hydrate formation to reduce wo to the experimental minimum of about 4. The protein encapsulation would additionally require overcoming reobservation that proteins, once encapsulated in reversed micelles, pulsive hydration forces. In cases where complete protein solustay encapsulated when wo is reduced and the intramicellar ionic bilization is not observed at higher salt levels, we have found that the woof the reversed micellar solution is less than that expected strength is increased through hydrate formation leads to the question of the maximum amount of protein that can stay solubased on the amount of water put in, implying that some of the bilized in reversed micelles or, in the case of well-defined monwater is bound to unsolubilized protein or is unsolubilized itself. odispersed micelles, the maximum fractional occupancy of the As examples, in Figure 4 at an 0.7 M KCI concentration, the w o of the w ~ ,15~ solution ~ ~ , drops to about 1 I , while the wo of the w ~ , micelles. ~ ~ ~We~have carried out the maximum solubilization experiments with azocasein, primarily due to cost considerations. 5 solution drops to 4. Figure 5 illustrates the results, where the protein concentration It should be noted that the experiment of Figure 4 relates to is reported in units of mg/mL due to the imprecision of the concurrent reversed micelle formation and protein encapsulation molecular weight caused by the mixture of azocaseins present (an but does not address the irreversibility of the phenomenon, Le., average molecular weight of 185000 is reported by Sigma whether a protein once encapsulated becomes desolubilized if the Chemicals). While 8.85 mg/mL of the protein can be solubilized intramicellar ionic strength is modified. We have considered this aspect by forming hydrates of methane in reversed micelles of w, at w,, 15, only about 1.1 mg/mL can be solubilized at wo 5 . On 15 and cytochrome c concentration 78 pM, where the aqueous the other hand, removing water from micelles of wo 15 through phase used to prepare the micelles originally contained 0.2 M KCI. hydrate formation results in a solution of wo 4 containing the same As hydrates form, and hydrate crystals nucleate to be precipitated level of protein (8.85 mg/mL). Thus, hydrate formation in this out of solution, the supernatant microaqueous phase has a reduced case increases the total solubilization of azocasein by nearly an water content. If we assume that the crystalline hydrates are free order of magnitude. of salt inclusions and are thus made up of pure water, this implies These observations appear to imply that hydrate formation in an increasc in ionic strength in the supernatant water pools. If, high protein content reversed micelles of larger wo may be a for example, hydrates are formed to reduce the water content to method to obtain high occupancy reversed micelles with a mimimal a wo of 5 , a 3-fold increase in the microaqueous-phase ionic water content. In order to carry out an analysis of particle size strength to 0.6 M KCI results, an electrolyte level at which very in these concentrated reversed micellar systems, it was necessary little protcin can be solubilized (Figure 4). In contrast, we have to dilute the samples with isooctane to a protein concentration found that cytochrome c solubility is completely retained, at both of 1.5 mg/mL (AOT, 1 I mM). The dynamic light scattering the high pressurcs and low temperatures used for hydrate forresults shown in Figure 6 indicate the interesting observation that
The Journal of Physical Chemistry, Vol. 95, No. 3, 1991
Protein-Containing Reversed Micelles
1471
clearly shown the same phenomenon occurring during the encapsulation of myelin proteins, the myelin basic protein, and the Folch-Pi proteolipid. Our primary observation in this case is the fact that water can be removed from these micelles through hydrate formation, leaving the integrity of the large aggregates intact, with protein solubility completely sustained. Protein loadings in minimal water reversed micelles at a given surfactant concentration are much higher than can be achieved through other preparation methods.
1 M
0 Wo- 15
e 01 0.0 I
0
.
.
'
0.2
'
"
1
'
2
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-
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~
"
'
~
"
"
3 4 5 6 7 8 K I T Concentration ( mM )
"
"
'
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9
'
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Figure 6. Size analysis of azocasein reversed micelles as modified by hydrate formation. The original solution containing 8.85 mg/mL protein (AOT 55.5 mM) has to be diluted with isooctane to the concentration range shown here, to allow light scattering analysis.
the micelles are large and that reduction of wo through hydrate formation results in a reduction of the mean particle size from about 107 to 78 nm. To ascertain that the large hydrodynamic radius observed is not due to significant particle-particle interactions, the micelles were further diluted with isooctane. As Figure 6 indicates, dilution did not reduce the mean particle size which must therefore be intrinsic to the azocasein reversed micellar system. Furthermore, polydispersity indexes for both wo 15 and w,, 4 solutions were less than 0.2, indicating a reasonably narrow size distribution. In experiments where the initial azocasein concentration was varied from 0.5 to 8.85 mg/mL (at 55.5 mM AOT, w, = 15 prior to the dilution by isooctane necessary for light scattering), we found that the hydrodynamic diameters remained between 107 and 122 nm. Light scattering on azocasein in aqueous solution also indicated the presence of large particles (-200 nm), implying that encapsulation of the protein does not involve significant aggregate breakdown. Static light scattering indicated aggregates of molecular weight around l o', with radii of gyration around 150 nm, and a positive second virial coefficient. This latter fact implies that the large aggregates are stable against dilution, an observation that appears valid in light of the fact that casein molecules do themselves form micellar aggregates in aqueous solution.27 Thus, the ability of reversed micelles to encapsulate fibrous proteins in large micellar aggregates is noted, although not for the first time. Earlier work by Waks and c o - w ~ r k e r shas ~~,~~ (27) Stothart, P. H. Mol. Biol. 1989, 208, 635.
Conclusions We have shown that clathrate hydrates of methane can form in protein-containing reversed micellar systems; their effect is to reduce the water content in the micelles. Proteins, once encapsulated into the micelles at larger wo values, remain encapsulated as hydrates form and the average water content is diminished; the observation appears valid both for proteins encapsulated in relatively small micelles and for proteins that aggregate in large micelles. In cases where double-exponential light scattering analysis indicates a bimodal distribution, there appears to be a decrease in lwater content upon hydrate formation for both aggregate-free and aggregate-containing micelles. The ability to modify the water content in reversed micelles has possible implications. It is well-known that the activity of encapsulated enzymes is a strong function of w , , ~ O and we have found that lipase and a-chymotrypsin activity can be modified by changing the water content of reversed micelles through hydrate formation,3' again suggesting that protein-containing micelles share in the average reduction of wo. In addition to phenomenological understanding of the behavior of protein-containing reversed micelles, the observation that high protein loadings can be obtained under minimal water conditions may have implications to biomembrane mimetics and to drug delivery systems with hardened reversed micelles.32 Continuing work seeks to understand changes in the microaqueous phase due to hydrate formation, through proton NMR and FTIR analysis, and the resulting protein gross conformational changes, using circular dichroism spectrophotometry. Acknowledgment. The authors thank Li Xiao and Robert M. Peitzsch for their assistance with the light scattering experiments. Support from the National Science Foundation (Grants CBT8802564 and CBT-8721829) is gratefully acknowledged by V.T.J. (28) Binks, B. P.; Chatenay, D.; Nicot, C.; Urbach, W.; Waks, M. Biophys. J . 1989, 55, 949. (29) Vacher, M.; Waks, M.; Nicot, C. J . Neurochem. 1989, 52, 117. (30) Kabanov. A. V.: Levashov. A. V.: Klyachko. N . L.: Namyotkin, S. N.;'Pshezhetesky, A. V.; Martinek, K. J . Thior. Biol. 1988, 13A-327. (31) Rao, M.; Nguyen, H.; John, V. T. Biofechnol. Prog. 1990, 6 , 465. (32) Speiser, P. In Reoerse Micelles; Luisi, P. L., Straub, B. E.,Eds.; Plenum Press: New York, 1984.
