The aggregation of reverse micelles

We have developed a computer simulation to model the formation of reverse micelles in two dimensions. Several of the qualitative results obtained from these calculations agree with experimental observations. Specifically, we have shown that the chain length has a large influence in determining the size and shape of the aggregate. We predict the existence of a critical tail length: Chains below this value will form an extended lamella-like structure, whereas chains longer than this value will form clusters that appear ellipsoid or circular in cross-section. Finally, we obtained a scaling law that relates the aggregation number (N) to the length of the tail (L):N∼L ^−1.14. A physical model to account for the observed exponent will be developed in a future paper.     

Stability of arginase in reverse micelles

Summary The influence of glycerol and sodium chloride on the stability of arginase entrapped in reverse micellar system of non-ionic surfactant Triton X - 100/n-hexanol/n-hexane, was studied. Entrapped enzyme shows better stability, further enhanced by the addition of these additives. However, the stability with these additives was found to be much better in reverse micelles.     

Immobilized enzymes in reverse micelles: studies with gel-entrapped trypsin and alpha-chymotrypsin in AOT reverse micelles

Trypsin and alpha-chymotrypsin were immobilized by gelentrapment in polyacrylamide cross-linked with N,N(1)-methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30-40% activity after 1 week. The enzymes obey Michaelis-Menten kinetics in the investigated concentration range with K(m) values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nalpha-benzoyl-L-arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., Z--Ala--Phe--Leu--NH(2)) using water-insoluble substrate has been performed with gelentrapped alpha-chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.     

Enhanced enzymatic activity in reverse micelles

Summary The bell shaped dependence of the superactivity of enzymes solubilized in ionic reverse micelles (RMs) on the hydration ratio (W₀) is theoretically explained. The superactivity is due to enhanced concentration of the substrate (which has the same kind of charge as that of the surfactant head groups) near the enzyme surface. The opposing effects of the increase in the absolute charge of the surface of the RM and in the water pool width with W₀ cause a maximum in activity.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

Mechanisms of protein solubilization in reverse micelles

Solubilization properties of alpha-chymotrypsin and alcohol dehydrogenase (LADH) in reverse micelles are reported for three different solubilization techniques. The solubilization properties for these two proteins depend on the method used for protein addition. The addition of a dry protein powder to a reverse-micelle-containing organic phase does not appreciably solubilize the protein until the diameter of the reverse micelle is similar to that of the protein. However, when an aqueous protein solution is injected an organic phase, protein solubilization is not strongly dependent on micelle size. For chymotrypsin, multiple protein occupancy occurs at large micelle size, with as many as 11 chymotrypsin molecules solubilized in one reverse micelle. The solubilization of chymotrypsin using a phase-transter technique with a positively charged surfactant follows the expected traned based on protein-surfactant electrostatic interactions. When a negatively charged sufactants is used for phase transfer, at low pH the solubilization data do not fit this electrostatic interaction mechanism. In this case, proteinsurfactant aggregation may be occurring at the aqueousorganic interface.     

Solubilization of ribosomes in reverse micelles.

Pig liver ribosomes have been solubilized in reverse micelles constituted by bis (2-ethyl hexyl) sodium sulfosuccinate (AOT) in isooctane and 3.6% water, v:v. The micellar ribosomal solutions are transparent, show no significant scattering and permit direct spectroscopic observation of the ribosomes to be made. Ultraviolet absorption and circular dichroic spectra have been recorded and indicate that the ribosomes maintain in the micellar environment their structural integrity. Some possible applications of these micellar systems are discussed.     

Characteristics of tyrosinase in AOT-isooctane reverse micelles

Isooctane-AOT-H(2)O is a suitable system for studying enzyme behavior in organic solvents. Tyrosinase was able to catalyze a well-known reaction in aqueous medium: oxidation of 4-methylcatechol to yield 4-methyl-o-benzoquinone. This reaction was studied using the preceding ternary system with adequate amounts of each component to make up reverse micelles. 4-Methyl-o-benzoquinone stability was demonstrated in isooctane even at alkaline pH values. Apparent K(m) and V(max) were similar to those in water, but substrate inhibition was more evident. The pH and temperature appear to be shifted toward high and low values, respectively. Characteristic parameters of reverse micelles, omega(0) (= H(2)O/AOT) and percentage of H(2)O (v/v), were investigated. The results obtained showed that the steady-state rate varies either with omega(0) or with percentage of H(2)O. The variation observed with omega(0) showed an optimal value while an increase in percentage of H(2)O can lead to decreased or increased activity depending on substrate concentration.     

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent media, respectively. The influences of the molar ratio of water to surfactant omega0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show "superactivity" in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of omega0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at omega0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.     

Kinetic model for enzymatic hydrolysis in reverse micelles

A bound water model is developed for the interpretation of kinetic data of b-galactosidase in reverse micelles. Assessing the kinetic parameters of p-nitrophenyl-b-D-galactopyranoside hydrolysis in aqueous and reverse micellar system reveals that the major effect on hydrolytic rate is owing to the amount of free water in reverse micelles, not the enzyme molecules' structural change.     

Structure and activity of trypsin in reverse micelles

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

FTIR study of horseradish peroxidase in reverse micelles

Fourier transform infrared (FTIR) method was used to study the secondary structures of horseradish peroxidase (HRP) in aqueous solution and in reverse micelles for the first time. Results indicated that the structure of HRP in sodium bis(2-ethylhexy)sulfosuccinate (AOT) reverse micelles was close to that in aqueous solution. In cetyltrimethylammonium bromide (CTAB) and sodium dodecylfate (SDS) reverse micelles the position of some bands changed. Results indicated that the secondary structure had a close relationship with the surfactant species of the reverse micelles. Among the three types of reverse micelles, the system of AOT reverse micelles was probably the most beneficial reaction media to HRP.     

Chiroptical properties of lecithin reverse micelles and organogels

The ultraviolet absorbance and circular dichroism (CD) spectra of lecithin reverse micelles and gels were investigated in order to establish whether the formation of these noncovalent macromolecular aggregates, which was induced by the addition of water to solutions of lecithin in organic solvents, was accompanied by specific spectroscopic changes. Systems containing the synthetic short-chain lecithins, 1,2-hexanoyl-, 1,2-diheptanoyl-, 1,2-dioctanoyl-, and 1,2-dinonaoyl-sn-glycero-3-phosphatidylcholines were used as models for the long-chain lecithins, soybean phosphatidylcholine and palmitoyl-oleoyl-phosphatidylcholine. All the molecules studied had asymmetric centres, formed reverse micelles under appropriate conditions, and, while both the long-chain lecithins also formed gels, none of the short-chain molecules did. As well as having CD spectra that were simpler to interpret, spectroscopic observations on solutions of the short-chain lecithins could be carried out over a large water content range. The ester chromophore of these compounds was shown to be highly sensitive to variation in both the solvent environment and the temperature, and components of both direct solvent effects and conformational change upon the addition of water were detected in the spectra. The spectra of the longer chain lecithins were complicated by the presence of double bonds although, here again, it was found that significant changes occurred as the water content increased, as monitored by the ester chromophore. However, no specific effect that could be ascribed to gelation alone was detected. The overall picture that emerged was that the ester chromophore of anhydrous micelles gave rise to a specific negative band in the CD spectrum (λ_max ≈ 210 nm) whereas a positive CD signal (λ_max ≈ 233 nm) was associated with the same chromophore in filled (i.e., hydrated) micelles. The two signals correspond to two different conformational states of the lecithin molecule, the hydrated state being not only more conformationally restricted but also providing a less polar environment for the ester groups, while the addition of water to the system shifts the conformational equilibrium. These observations have been interpreted as showing that only a limited range of lecithin conformation is compatible with the formation of the micellar structure and that it is this constraint, together with those introduced by the overall geometry of the aggregated state, that gives rise to the changes observed in the CD spectrum.     

The behavior of proteases in lecithin reverse micelles

Reverse micelles, formed in isooctane/alcohol by phosphatidylcholines of variable chain length (i.e. 6, 7 or 8 C atoms in the fatty acid moiety) have been studied, mostly in relation to their capability of solubilizing trypsin and alpha-chymotrypsin. It has been found that the capability of the lecithin reverse micellar systems to solubilize water is strongly affected by the chain length of the alkyl group and by the alcohol used as co-surfactant. The C8-lecithin system, i.e. 1,2-dioctanoyl-sn-glycero-3-phosphocholine, in isooctane/hexanol is the system which affords the maximal solubilization of water (up to wo 60, where wo = [H2O]/[lecithin]) and of the enzymes. The water of the water pool of lecithin reverse micelles has been investigated by 1H-NMR; the proton chemical shift as a function of wo was found to be similar to the case of reverse micelles formed by the well known negatively charged surfactant sodium bis(2-ethylhexyl sulfosuccinate). 31P-NMR studies show that the ionization behavior of phosphate groups is similar to that in bulk water, suggesting no anomaly in the pH behavior of this water pool. The stability of trypsin and alpha-chymotrypsin in the various lecithin reverse micellar system is similar and occasionally better than that in aqueous solution. The same holds for the kinetic behavior (kcat and Km have been determined for a few systems). The bell-shaped curve of the pH/activity profile in lecithin reverse micelles is, for both enzymes, shifted towards more alkaline values with respect to water. Bell-shaped curves are also obtained when studying the influence of wo on the enzyme activity, with an optimal wo which is in the range 7-10, a surprisingly small value considering that we are dealing with hydrolases. Circular dichroic studies have been carried out in order to correlate the activity with the protein conformation: for both enzymes, generally no marked perturbations appear as a consequence of the solubilization in the lecithin reverse micelles, but conditions can be found under which significant alterations are present. Certain properties of the two enzymes, which in water solution are very similar, become sharply different in reverse micelles, showing that occasionally the micellization is able to enhance the relatively small structural differences between the two proteins.     

On the meaning of enzymatic superactivity in reverse micelles

Summary Two types of superactivity can be defined. One, is with respect to the activity for a fixed average substrate concentration in the water pool; the pushing of the charged substrate by the likewise charged micellar surface is responsible for the superactivity and its bell-shaped dependence on the hydration ratio. The other, is with respect to the activity in a bulk aqueous solution having a substrate concentration equal to a fixed overall concentration [S^–]_ov in the entire reverse micellar solution. In this case, the pushing effect, the constraint of a fixed [S^–]_ov and the partitioning of the substrate in the surfactant layer are responsible for the bell-shaped dependence. Superactivity exists for low substrate partitioning in the surfactant layer, subactivity for high partitioning.     

Dynamic light scattering of cutinase in AOT reverse micelles

The fungal lipolytic enzyme cutinase, incorporated into sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles has been investigated using dynamic light scattering. The reversed micelles form spontaneously when water is added to a solution of sodium bis-(2ethylhexyl) sulfosuccinate in isooctane. When an enzyme is previously dissolved in the water before its addition to the organic phase, the enzyme will be incorporated into the micelles. Enzyme encapsulation in reversed micelles can be advantageous namely to the conversion of water insoluble substrates and to carry out synthesis reactions. However protein unfolding occurs in several systems as for cutinase in sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles. Dynamic light scattering measurements of sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles with and without cutinase were taken at different water to surfactant ratios. The results indicate that cutinase was attached to the micellar wall and that might cause cutinase unfolding. The interactions between cutinase and the bis-(2ethylhexyl) sulfosuccinate interface are probably the driving force for cutinase unfolding at room temperature. Twenty-four hours after encapsulation, when cutinase is unfolded, a bimodal distribution was clearly observed. The radii of reversed micelles with unfolded cutinase were determined and found to be considerable larger than the radii of the empty reversed micelles. The majority of the reversed micelles were empty (90-96% of mass) and the remainder (4-10%) containing unfolded cutinase were larger by 26-89 A.     

Characterization of patatin esterase activity in AOT-isooctane reverse micelles

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT-isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, w(o) (= H(2)O/AOT), and the percentage of H(2)O, theta, were investigated. The results obtained show that patatin esterase activity varies with w(o) but remains constant throughout the range of theta values studied. The variation with w(o) showed that the activity follows an S-shaped behavior pattern, reaching a maximum at about w(o) = 20 for 2% H(2)O. Patatin esterase activity was compared with p-nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP-caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 degrees C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated (K(m) = 10 mM, V(m) = 158 microM/min, V(m)/K(m) = 15.8 x 10(-3) min(-1)). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.     

Decontamination of surfaces by lysozyme encapsulated in reverse micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.     

Entrapment and condensation of DNA in neutral reverse micelles

DNA condensation and compaction is induced by a variety of condensing agents such as polycations. The present study analyzed the structure of plasmid DNA (DNA) in the small inner space of reverse micelles formed from nonionic surfactants (isotropic phase). Spectroscopic studies indicated that DNA was dissolved in an organic solvent in the presence of a neutral detergent. Fluorescent quenching of ethidium bromide and of rhodamine covalently attached to DNA suggested that the DNA within neutral, reverse micelles was condensed. Circular dichroism indicated that the DNA structure was C form (member of B family) and not the dehydrated A form. Concordantly, NMR experiments indicated that the reverse micelles contained a pool of free water, even at a ratio of water to surfactant (Wo) of 3.75. Electron microscopic analysis also indicated that the DNA was in a ring-like structure, probably toroids. Atomic force microscopic images also revealed small, compact particles after the condensed DNA structures were preserved using an innovative cross-linking strategy. In the lamellar phase, the DNA was configured in long strands that were 20 nm in diameter. Interestingly, such DNA structures, reminiscent of "nanowires," have apparently not been previously observed.     

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.