Langmuir 2005, 21, 12115-12123
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Geometric Constraints at the Surfactant Headgroup: Effect on Lipase Activity in Cationic Reverse Micelles Rajendra Narayan Mitra, Antara Dasgupta, Debapratim Das, Sangita Roy, Sisir Debnath, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata - 700032, India Received August 16, 2005. In Final Form: October 7, 2005 The primary objective of the present article is to understand how the geometric constraints at the surfactant head affect the lipase activity in the reverse micellar interface. To resolve this issue, surfactants 1-11 were designed and synthesized, and activity was measured in 1-11/water/isooctane/n-hexanol reverse micellar systems at z ([alcohol]/[surfactant]) ) 5.6, pH 6.0 (20 mM phosphate), 25 °C across a varying range of W0 ([water]/[surfactant]) using p-nitrophenylalkanoates as the substrate. It was observed that lipase activity increases from surfactants 1 to 2 with the increment in surface area per molecule (Amin) because of the substitution by the bulky tert-butyl group at the polar head. However, the activity was found to be similar for 2-5 despite an enhancement in the hydrophilic moieties at the interface. This unchanged lipase activity is presumably due to the comparable surface area of 2 to 5 originating from the rigidity at the surfactant head. Noticeably, the enzyme activity improved from 6-8 with the simultaneous increment of both the hydroxyl group and the flexibility of the headgroup whereas that for 9-11 increased exclusively with the flexibility of the headgroup. The common parameter in both groups of surfactants 6-8 and 9-11 is the flexibility of the headgroup, which possibly enhance Amin and consequently the lipase activity. Thus, the geometric constraints at the surfactant headgroup play a crucial role in modulating the lipase activity profile probably because of the variation in interfacial area.
Introduction Reverse micelles are nanometer-scale aggregates of water and surfactant dispersed in a bulk apolar solvent1,2 and have been subjected to numerous experimental and theoretical studies because of their potential biotechnological applications.3-5 Lipases, a class of surface-active enzymes, are widely used for diversified transformations * To whom correspondence should be addressed. E-mail: bcpkd@ iacs.res.in. Fax: +(91)-33-24732805. (1) (a) Eicke, H. F.; Shepherd, T. C.; Steinmann, A. J. Colloid Interface Sci. 1976, 56, 168. (b) Zana, R.; Lang, J. Solution Behavior of Surfactants; In Mittal, K. L., Fendler, E. J., Eds.; Plenum: New York, 1982; Vol. 2. (c) Petit, C.; Bommarius, A. S.; Pileni, M. P.; Hatton, T. A. J. Phys. Chem. 1992, 96, 4653. (d) Fendler, J. H. Acc. Chem. Res. 1976, 9, 53. (2) (a) Eicke, H. F.; Kvita, P. In Reverse Micelles; Luisi, P. L., Straub, B. E.; Ed.; Plenum: New York, 1984; p 21. (b) Fendler, J. H. Membrane Mimetic Chemistry; Wiley & Sons: New York, 1982. (c) Das, D.; Roy, S.; Das, P. K. Org. Lett. 2004, 6, 4133. (d) Silber, J. J.; Biasutti, M. A.; Abuin, E. B.; Lissi, E. Adv. Colloid Interface Sci. 1999, 82, 189. (3) (a) Skargelind, P.; Jasson, M. J. Chem. Technol. Biotechnol. 1992, 54, 277. (b) Valis, T. P.; Xenakis, A.; Kolisis, F. N. Biocatalysis 1992, 6, 267. (c) Yamada, Y.; Kuboi, R.; Komasawa, I. Biotechnol. Prog. 1993, 9, 468. (d) Stark, M.; Scagerlind, P.; Holmberg, K.; Carlfors, J. Colloid Polym. Sci. 1990, 268, 384. (d) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084. (e) Abuin, E.; Lissi, E.; Duarte, R. Langmuir 2003, 19, 5374. (4) (a) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Biotechnol. Adv. 1999, 17, 293 and references therein. (b) Luisi, P. L.; Magid, L. J. CRC Crit. Rev. Biochem. 1986, 20, 409. (c) Luisi, P. L. Angew. Chem., Int. Ed. Engl. 1985, 24, 439. (d) Kelley, B. D.; Wang, D. I. C.; Hatton, T. A. Biotechnol. Bioeng. 1993, 42, 1199. (e) Komives, C. F.; Osborne, D. E.; Russell, A. J. J. Phys. Chem. 1994, 98, 369. (f) Menger, F. M. Angew. Chem. Int. Ed. Engl. 1991, 30, 1086. (g) Mosler, R.; Hatton, T. A. Curr. Opin. Colloid Interface Sci. 1996, 1, 540. (h) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6411. (i) Bru, R.; Walde, P. Eur. J. Biochem. 1991, 91, 94. (j) Walde, P.; Han, D.; Luisi, P. L. Biochemistry 1993, 32, 4029. (k) Yang, F. X.; Russell, A. J. Biotechnol. Bioeng. 1995, 47, 60. (l) Bommarius, A. S.; Hatton, T. A.; Wang, D. I. C. J. Am. Chem. Soc. 1995, 117, 4515. (m) Yang, F. X.; Russell, A. J. Biotechnol. Bioeng. 1994, 43, 232. (n) Martinek, K.; Levashov, A. V.; Khmelnitsky, Y. L.; Klyachko, N. L.; Berezin, I. V. Science 1982, 218, 889. (o) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1979, 101, 6731. (5) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Kabanov, A. V.; Khmelnitsky, Y. L. Biochim. Biophys. Acta 1989, 981, 161.
such as the hydrolysis of triglycerides and esters in reverse micellar solutions.3,4a,6-8 The catalytic efficiency of such encapsulated lipase seems to be dependent on the local molar concentration of water and other ions present in the vicinity of the enzyme.9,10 To this end, Das and Chaudhuri estimated the interfacial concentration of water, [H2O]i, in cetyltrimethylammonium bromide (CTAB)/water/isooctane/nhexanol reverse micelles across the W0 (mole ratio of water to surfactant) range using a phenyl cation trapping protocol.10a The activity of Chromobacterium viscosum (CV) lipase remain essentially unchanged presumably because of the unchanged [H2O]i (28.1-31.8 M) across W0 ) 12-44.10a This scarcity of [H2O]i (almost half of the bulk water concentration) probably plays an important role in the poor efficiency of lipase in CTAB reverse micelles. Toward improving the lipase’s efficiency, in our previous study6a we found that the introduction of hydroxyethyl moieties at the polar head of surfactants dramatically enhanced the catalytic activity of CV-lipase up to 4-10fold compared to that in widely used cationic (CTAB) reverse micelles. The observed enhancement in activity (6) (a) Das, D.; Das, P. K. Langmuir 2003, 19, 9114. (b) Dasgupta, A.; Das, D.; Mitra, R. N.; Das, P. K. J. Colloid Interface Sci. In press. (7) (a) Verger, R. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1980; pp 340-392. (b) Ying, L.; Ganzuo, L.; Chengsong, M. J. Dispersion Sci. Technol. 2000, 21, 409. (8) (a) Stamatis, H.; Xenakis, A.; Menge, U.; Kolisis, F. N. Biotechnol. Bioeng. 1993, 42, 931. (b) Stamatis, H.; Kolisis, F. N.; Xenakis, A.; Bornscheuer, U.; Scheper, T.; Menge, U. Biotechnol. Lett. 1993, 15, 703. (c) Rees, G. D.; Robinson, B. H.; Stevenson, G. R. Biochim. Biophys. Acta 1995, 1259, 73. (9) (a) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239. (b) Damodaran, S. Colloids Surf., B 1998, 11, 231. (c) Das, P. K.; Srilakshmi, G. V.; Chaudhuri, A. Langmuir 1999, 15, 981. (d) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Kabanov, A. V.; Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277. (10) (a) Das, P. K.; Chaudhuri, A. Langmuir 2000, 16, 76. (b) Srilakshmi, G. V.; Chaudhuri, A. Chem.sEur. J. 2000, 6, 2847.
10.1021/la052226r CCC: $30.25 © 2005 American Chemical Society Published on Web 11/11/2005
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Chart 1. Cationic Surfactants with Varying Headgroup Architecture
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studied using 8-anilino-1-naphthalenesulfonic acid (ANS) in 1-11/water/isooctane/n-hexanol reverse micelles to establish a correlation, if any, between lipase activity and the micropolarity and microviscosity at the micellar interface12 induced by the headgroup architecture and hydrophilicity. Experimental Section
was possibly due to the increase in [H2O]i through hydrogen bonding with the hydroxyl group.11 However, the introduction of hydroxyethyl groups not only increased the hydrophilicity at the interface but also, owing to its flexible nature, may have enhanced the headgroup size and consequently the interfacial area. Thus, the observed increase in lipase activity with hydroxyethyl groupss whether due to the increased hydrophilicity or interfacial areasdeserves further investigation. The purpose of the present study is to obtain insight into how the headgroup structure, in particular, the geometric constraints/flexibility, at the polar head influences the lipase activity in cationic reverse micelles. To this end, we have estimated the catalytic activity of CV-lipase in the reverse micelles of synthesized surfactants 1-11 (Chart 1) with varying headgroup size, hydrophilicity, and flexibility/rigidity. The lipase activity distinctly increases from 1 to 2 with an increase in headgroup area and also improves from 6 to 8 with a concomitant increment in hydroxyl moieties and headgroup area presumably due to enhanced flexibility at the surfactant head. However, the activity is almost unchanged from 3 to 5 though the hydrophilicity is expected to improve to similar extent as from 6 to 8. The observed activity in 3-5 is even comparable to that found in 2, which noticeably does not contain any hydroxyl moiety at the headgroup. The reason for unchanged lipase activity in these reverse micelles is probably due to the rigid polar head leading to almost no change in their headgroup area. The activity also increases from 9 to 11 with increasing flexibility as well as headgroup area even in absence of any hydroxyl moiety at the surfactant head. The generality of the effect of headgroup architecture on the lipase activity is also verified by estimating the enzyme efficiency with varying substrate chain length (C4-C16) and pH (2-10) of the dispersed water. Steady-state fluorescence was
Materials. Chromobacterium viscosum lipase (EC 3.1.1.3, type XII) was purchased from Sigma and was used as received. Analytical-grade CTAB from Spectrochem (India) was recrystallized three times from methanol/ether, and the material was without minima in the surface tension plot. Amberlyst A-26 chloride ion-exchange resin was purchased from B. D. H., U.K. HPLC-grade isooctane, n-hexanol, water, and all other reagents used in syntheses were purchased from SRL (India) and were of the highest analytical grade. Fluorescence probes pyrene and 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Fluka and Aldrich Chemical Co., respectively. The UV-visible absorption spectra were recorded on a Shimadzu UV-1700 spectrophotometer. 1H NMR spectra were recorded on a Bruker Avance DPX-300 spectrometer. Mass spectrometry (MS) data were acquired by electron spray ionization (ESI) techniques using 25-70 eV in a Q-tof Micro-Quadruple mass spectrophotometer, Micromass, U.K. The fluorescence spectra were recorded with a Perkin-Elmer LS55 luminescence spectrometer. Syntheses of p-nitrophenylalkanoates (Chart 1) and surfactants are given below. 1H NMR, elemental analysis, and mass spectroscopy data are given in Supporting Information. Methods. Synthesis of p-Nitrophenylalkanoates. The pnitrophenylalkanoate esters were prepared following the N,Ndicyclohexylcarbodiimide (DCC) coupling of the alkanoic acids with p-nitrophenol in the presence of 4-N,N-(dimethylamino)pyridine (DMAP). In brief, the alkanoic acid (1 equiv) was added to p-nitrophenol (1 equiv) in the presence of 1.1 equiv of 4-N,N(dimethylamino)pyridine (DMAP) and 1.1 equiv of N,N- dicyclohexylcarbodiimide (DCC) in dry DCM under a nitrogen atmosphere in an ice-salt (-20 °C) bath. After 12-14 h of stirring at room temperature, the reaction mixture was filtered, and the concentrated filtrate was extracted with ether. The ether part was washed with water and concentrated in a rotary evaporator, and then the desired product was purified through a silica gel (60-120 mesh) column with an acetone/hexane solvent mixture as the elutant. Yields were in the range of 70-80%. Synthesis of N-Hexadecyl-N,N,N-trymethylammonium Chloride (1). Surfactant 1 was obtained by chloride ion exchange of CTAB through an Amberlyst A-26 chloride ion-exchange resin column. The pure white material thus obtained was crystallized three times from methanol/ether prior to use. Synthesis of N-Hexadecyl-N,N-dimethyl-N-tert-butylammonium Chloride (2), N-Hexadecyl-N,N-dimethyl-N-(2-hydroxy-1,1dimethyl-ethyl)ammonium Chloride (3), N-Hexadecyl-N,Ndimethyl-N-(2-hydroxy-1-hydroxymethyl-1-methyl-ethyl)ammonium Chloride (4), and N-Hexadecyl-N,N-dimethyl-N-(2hydroxy-1,1-bis-hydroxymethyl-ethyl)ammonium Chloride (5). To synthesize surfactants 2-5, the respective primary amines, tertbutylamine, 2-amino-2-methyl-1-propanol, 2-amino-2-methyl1,3-propanediol, and 2-amino-2 (hydroxymethyl)propane-1,3-diol were refluxed with n-hexadecyl bromide in the mole ratio 1:1.2 in 30% methanol/acetonitrile for 24 h. The reaction mixture was concentrated on a rotary evaporator and dried under vacuum. The corresponding ammonium salts thus obtained were basified with an ammonia solution and stirred for half an hour followed by extraction in chloroform, and the organic part was washed with a brine solution. The chloroform extract was then concentrated and dried under vacuum. The secondary amines thus obtained were treated with iodomethane (in excess) in the presence of dry potassium carbonate in the mole ratio 1:1.2 equiv and a catalytic amount of 18-crown-6-ether in dry DMF at room temperature for 2-3 h. The reaction mixture was extracted with chloroform and washed with a 5% aqueous solution of sodium thiosulfate. The chloroform part was concentrated on a rotary
(11) Wettig, S. D.; Nowak, P.; Verrall, R. E. Langmuir 2002, 18, 5354.
(12) Falcone, R. D.; Biasutti, M. A.; Correa, N. M.; Silber, J. J.; Lissi, E.; Abuin, E. Langmuir 2004, 20, 5732.
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Scheme 1. Schematic Representation of the Preparation of Surfactants 2-11
evaporator and dried under vacuum to get the corresponding ammonium iodide salts. The white iodide salts were converted to their corresponding chloride salts by passing through an Amberlyst A-26 chloride ion-exchange column. The white chloride
salts (2-5) were crystallized from methanol/ether. The overall yields were in the range of 80-85% (Scheme 1). Synthesis of N-Hexadecyl-N,N-dimethyl-N-(2-hydroxyethyl)ammonium Chloride (6) and N-Hexadecyl-N-methyl-N,N-bis (2-
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hydroxyethyl)ammonium Chloride (7). The bromide salts of amphiphiles 6 and 7 were prepared following the procedure mentioned in a published protocol.13 n-Hexadecylbromide and the corresponding amines (N,N-dimethylethanolamine for 6, N-methyldiethanolamine for 7) were refluxed in 30% methanol/ acetonitrile in the molar ratio 1.2:1 for 24 h. The materials were then concentrated on a rotary evaporator and dried under vacuum. The white ammonium bromides were crystallized in methanol/ether and ion exchanged through an Amberlyst A-26 chloride ion-exchange resin column to get the corresponding chlorides. The colorless solid materials (6 and 7) were crystallized from methanol/ether twice. The yields were 85 and 89% for 6 and 7, respectively (Scheme 1). Synthesis of N-Hexadecyl-N-tris-(2-hydroxyethyl)ammonium Chloride (8). An aqueous solution of NaOH (3 equiv) was added dropwise to the mixture of n-hexadecylamine (1 equiv) and 2-chloroethanol (4 equiv) under refluxing condition at 130-140°C. After 24 h, the reaction mixture was extracted with chloroform and washed with brine solution, and the organic part was concentrated on a rotary evaporator and dried under vacuum. The residue was crystallized in methanol/ethyl acetate. The resulting mixture possessed three spots (Rf ) 0.55, 0.4, and 0) on thin-layer chromatography (TLC) using 1:3 (v/v) methanol/ chloroform as the TLC developing solvent. The desired product (Rf ) 0.55) was obtained by column chromatography on a 230400 mesh silica gel column with methanol/chloroform and was crystallized with methanol/ethyl acetate (yield ∼40%) (Scheme 1). Synthesis of N-Hexadecyl-N,N-dimethyl-N-propylammonium Chloride (9). n-Hexadecylamine (1 equiv) and n-propyl bromide (1.2 equiv) were refluxed in 30% methanol/acetonitrile for 24 h. The mixture was then concentrated on a rotary evaporator and dried under vacuum. The ammonium bromide salt was basified with ammonia solution to form N-hexadecyl-N-propylamine. This secondary amine (1 equiv) was permethylated with iodomethane (in excess) in the presence of dry potassium carbonate (1.2 equiv) and a catalytic amount of 18-crown-6-ether in dry DMF at room temperature for 2-3 h. The reaction mixture was extracted in chloroform and washed with a 5% aqueous solution of sodium thiosulfate. The organic part was concentrated on a rotary evaporator and dried under vacuum to get the corresponding ammonium iodide salt. The white iodide salt was converted to the corresponding chloride salt (9) by passing through an Amberlyst A-26 chloride ion-exchange resin column. This material was crystallized in methanol/ether. The overall yield was ∼80% (Scheme 1). Synthesis of N-Hexadecyl-N-methyl-N,N-dipropylammonium Chloride (10). Surfactant 9 was further refluxed in 30% methanol/ acetonitrile for 24 h with n-propyl bromide in the molar ratio 1:1.2. The mixture was concentrated and dried under vacuum. The corresponding ammonium bromide salt was basified with ammonia solution as mentioned above. The reaction mixture was extracted with chloroform and washed with brine solution. N-hexadecyl-N,N-dipropylamine obtained (1 equiv) was quaternized with iodomethane (excess) in the presence of dry potassium carbonate (1.2 equiv) and a catalytic amount of 18crown-6-ether in dry DMF at room temperature for 3 h. The reaction mixture was extracted with chloroform, and the organic layer was washed with a 5% aqueous solution of sodium thiosulfate. The chloroform part was concentrated on a rotary evaporator and dried under vacuum. The iodide salt was then converted to the chloride salt (10) by passing through an Amberlyst A-26 chloride ion-exchange resin column followed by crystallization from methanol/ether (yield ) 90%) (Scheme 1). Synthesis of N-Hexadecyl-N,N,N-tripropylammonium Chloride (11). n-Hexadecyl bromide and tripropylamine were refluxed in a 30% methanol/acetonitrile solvent mixture for 48 h in the mole ratio of 1:1.5. The refluxed material was concentrated on a rotary evaporator and dried under vacuum. The quaternized Nhexadecyl-N,N,N-tripropylammonium bromide salt was converted to the chloride salt (11) by passing through the chloride ion-exchange resin column. The white material obtained was crystallized three times from ether to get pure white solid 11 in 85% yield (Scheme 1). (13) Chatterjee, A.; Maiti, S.; Sanyal, S. K.; Moulik, S. P. Langmuir 2002, 18, 2998.
Mitra et al. Table 1. Critical Micelle Concentration (cmc), Surface Area Per Molecule (Amin), and Micropolarity Values (I1/I3) of the Aqueous Micelles of Surfactants 1-11 surfactant 1 2 3 4 5 6 7 8 9 10 11
cmc (mM)a
Amin (nm2)
I1/I3b
1.05 ( 0.02 0.62 ( 0.02 0.44 ( 0.04 0.41 ( 0.02 0.33 ( 0.01 0.51 ( 0.03 0.31 ( 0.02 0.054 ( 0.02 0.39 ( 0.01 0.27 ( 0.03 0.082 ( 0.01
0.86 ( 0.02 1.22 ( 0.01 1.28 ( 0.02 1.32 ( 0.01 1.36 ( 0.03 1.09 ( 0.02 1.67 ( 0.02 2.25 ( 0.01 1.02 ( 0.02 1.62 ( 0.02 2.07 ( 0.03
1.40 1.32 1.27 1.26 1.27 1.34 1.31 1.33 1.33 1.33 1.23
a cmc values were measured tensiometrically at 25 °C. b Intensity ratio due to first and third vibronic peaks of pyrene steady-state fluorescence I1/I3 indicates the micropolarity at the micellar interface.
Surface Tension Measurement. The critical micelle concentration (cmc) values of the surfactants in Chart 1 were measured using a tensiometer (Jencon, India) applying the Du Nou¨y ring method at 25 ( 0.1 °C in water. The cmc values were determined (Table 1) by plotting surface tension (γ) versus concentration of surfactant. The cmc values in duplicate experiments were within (2%. Steady-State Fluorescence. Steady-state pyrene monomer fluorescence measurements were performed using a PerkinElmer LS55 luminescence spectrometer at 25 °C. The concentration of pyrene used in all of the measurements was ∼1 × 10-7 M. The excitation wavelength was 337 nm, and emission spectra were recorded from 357 to 600 nm. The ratio of intensities of the first vibrational peaks (I1, 374 nm) to those of the third vibrational peaks (I3, 384 nm) of the pyrene emission spectra is very sensitive to the micropolarity of the medium around the probe, and hence this ratio is used in monitoring medium polarity. The fluorescence study in reverse micelles was carried out using 4 × 10-6 M ANS in 50 mM surfactants 1-11/water/ isooctane/n-hexanol systems at z ([alcohol]/[surfactant]) ) 5.6, W0 ) 40, 25 °C. The excitation wavelength was 360 nm and the emission spectra were observed at 380-600 nm. Fluorescence spectra of ANS were also recorded in water, isooctane, n-hexanol, and a isooctane/n-hexanol solvent mixture. The excitation and emission slit widths for both pyrene and ANS fluorescence experiments were kept constant at 10 and 2.5 nm, respectively. Preparation of Microemulsions (Phase Behavior). The mixture of surfactants, n-hexanol, and water were titrated with isooctane to prepare the microemulsions. A constant mass ratio (1:2) of the surfactant and n-hexanol was dissolved in water, forming solutions of different concentrations taken in different screw-topped test tubes and stirred until the solutions became clear. Isooctane was then added to these solutions in measured quantities at 25 °C until they were just turbid or phase separation occurred. The pseudoternary phase diagrams of the different surfactants are presented in Figure 2. To compare the effect owing to the variation in headgroup structure of amphiphiles, the phase diagrams of different surfactants are merged in Figure 2a-c. The isotropy and turbidity of the solutions were checked by the naked eye, which means that the measured phase boundaries are of fair accuracy. Activity of Interfacially Solubilized CV-Lipase. The second-order rate constant (k2) in the lipase-catalyzed hydrolysis of p-nitrophenylalkanoates in cationic reverse micelles was determined (on a Shimadzu UV-1700 spectrophotometer) at the isosbestic points as described previously.6,10a,14 In a typical experiment, 4.5 µL of the aqueous enzyme stock solution (0.34 mg/mL) and the substrate (10 µL, from 0.45 M stock solution in isooctane) were added to 1.5 mL of reverse micelles previously prepared with the desired surfactant concentration and pH (pH (14) (a) Crooks, G. E.; Rees, G. D.; Robinson, B. H.; Svensson, M.; Stephenson, G. R. Biotechnol. Bioeng. 1995, 48, 78. (b) Carlie, K.; Rees, G. D.; Robinson, B. H.; Steer, D. T.; Svensson, M. J. Chem. Soc., Faraday Trans. 1996, 92, 4701. (c) Fletcher, P. D. I.; Robinson, B. H.; Freedman, R. B.; Oldfield, C. J. Chem. Soc., Faraday Trans. 1985, 81, 2667.
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Figure 1. Representative plots of surface tension (γ) vs concentration (mM) of surfactants 2, 5, and 9 at 25 °C. refers to the pH of the aqueous buffer solutions used for preparing the w/o microemulsions; pH within the water pool of reverse micelles does not vary significantly,
