AOT Reverse Micellar Solution Research Articles

Photoionization processes of charged pyrene derivatives in reverse micelles. The effect of the interfacial charge

Abstract The laser flash photolysis studies of (4-(1-pyrenyl)butyl)trimethylammonium bromide (PBTMA) and 1-pyrene sulfonic acid (PSA) were carried out in BHDC and AOT reverse micellar solutions in benzene. At low laser intensities only the triplet state was observed as a transient species and the photophysical properties of the probes are very similar in both systems. Notable differences arise at high laser intensities. Under these conditions a photoionization process is observed in BHDC for both probes, as is apparent by the presence of the radical cation of the pyrenyl group. Hydrated electrons are not observed at times longer than 100 ns. On the other hand the photoionization process is not detected in AOT. However, a slight photoionization may be induced in AOT by the presence of benzyl alcohol. These results are interpreted by the different ability of AOT and BHDC to interact with the geminate radical–electron pair.

Properties of conducting films of electrophoretic concentrates of silver and gold nanoparticles in AOT surfactant

Liquid concentrates of silver and gold nanoparticles with 1–2 M metal concentrations were isolated by electrophoresis in a capacitor-type cell from AOT reverse micellar solutions in n-decane. The electrophoretic concentrates and the starting micellar solutions were characterized by nonaqueous electrophoresis, transmission electron microscopy, photon correlation spectroscopy (dynamic light scattering), and spectrophotometry. The hydrodynamic diameter of silver and gold nanoparticles was 13.2 and 8.6 nm, respectively; the ζ-potential was 70 and 13 mV. The drying of the concentrates on glass and silicon substrates and subsequent treatment with a 30% solution of water in ethanol gave mirror conducting Ag, Au, Ag-Au, and Au/Ag films containing on the average 80% metal and 20 wt % AOT. The film structure, morphology, and composition were studied by scanning electron microscopy (SEM) and energy dispersion analysis (EDX).

Determination of Structural Parameters of Protein-Containing Reverse Micellar Solution by Near-Infrared Absorption Spectroscopy

We present a method based on near-infrared absorption spectroscopy of the OH stretching vibration band of water around 3400 cm(-1) to examine whether the aqueous cavity size of a protein-unfilled reverse micelle is affected by solubilization of protein, and it has been found for AOT [= bis(2-ethylhexyl)sulfosuccinate] reverse micellar solution with myoglobin that the cavity size does not change before and after solubilization of the protein at water-to-surfactant molar ratios (w(0)) from 2 to ∼18; that is, the w(0) values of the protein-filled and unfilled reverse micelles are the same as that of the reverse micellar solution, regardless of the size relationship between the aqueous cavity of the unfilled reverse micelle and the protein. On the basis of this experimental fact, we propose a model to determine the structural parameters of protein-filled reverse micelles, such as the aqueous cavity radius and the aggregation number of surfactant molecules constituting the micelle, and the molar concentration of the unfilled reverse micelle that exists with the protein-filled reverse micelle in the reverse micellar solution, and derive their values for AOT reverse micellar solution with myoglobin in the w(0) range from 2 to 24. On the other hand, circular dichroic measurements and UV-visible absorption spectroscopy of myoglobin/AOT reverse micellar solution and myoglobin/AOT aqueous solution were carried out in order to examine the conformational state of myoglobin in the reverse micellar solution. These experimental results lead to the conclusion that myoglobin is located in the aqueous cavity of the reverse micelle, although the conformational state of the protein is to some extent distorted because of the interaction with AOT compared with that of native myoglobin. Finally, it is suggested that the proposed model is appropriate for reverse micellar solution with a hydrophilic protein molecule that is located in the aqueous cavity of the reverse micelle.

Antimicrobial activity of AOT-isooctane reverse micelle as a bioseparation and biocatalysis tool

The antimicrobial activity of different reverse micelles on microorganisms is been compared using the disc diffusion method. The bis (2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micelle showed a more significant inhibitory effect than do other reverse. micelles, and it had an antimicrobial activity against a broad range of microorganisms. Results from an antimicrobial activity test of isooctane and a forward extraction containing soybean protein suggest that the surfactant was chiefly responsible for inhibiting microbes in AOT/isooctane reverse micelle, while isooctane hardly inhibited the microbial growth. The properties of S. aureus, cultured in the TSB with AOT reverse micellar solution, were identified by the SEM and SDS-PAGE fingerprinting of cell-wall proteins. It is concluded that the cell-wall of the S. aureus decreased in the TSB with AOT reverse micellar solution, and some cell protein subunits of the S. aureus did not occurr, especially between 14.4 and 42.7 kDa, while one new protein subunit at near 97.4 kDa occurred

Kinetics of p-nitrophenyl Acetate Hydrolysis Catalyzed by Mucor javanicus Lipase in AOT Reverse Micellar Solutions Formulated in Different Organic Solvents

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W=([water]/[AOT])=6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.

Laser-induced formation of pyrenyloxy radical from 1-hydoxypyrene and further oxidation: Micellar effects

Laser photolysis of POH was investigated in aqueous ethanol, three organic solvents with different polarity, SDS micellar and AOT reverse micellar solutions. The analysis of the laser-induced transient absorption (TA) spectra showed that the pyrenyloxy radical (PO ) was formed via a prompt deprotonation from the cation radical (POH) + . The observed micellar effects on the formation of the pyrenyloxy radical were consistent with this mechanism of formation. Based on the shift as well as the intensity of the TA peak attributable to the pyrenyloxy radical, information on the microenvironment surrounding POH (i.e., hydrophobic or hydrophilic, contact or no contact with water phase) was obtained. The secondary photochemical reaction after laser irradiation was investigated with UV/vis absorption spectroscopy. 1,6- and 1,8-pyrenediones were found to be produced as the main photoproducts.

Compressed CO2 in AOT Reverse Micellar Solution: Effect on Stability, Percolation, and Size

The effects of compressed CO(2) in sodium bis-2-ethylhexylsulfosuccinate (AOT)/decane reversed micellar solution on the stability of the micelles, interface, and micelle/micelle interactions were studied. It was demonstrated that the compressed gas could increase the solubilization of water in this system. The formation of the stabilized one-phase microemulsion was confirmed by conductivity measurements. A shift in percolation threshold to higher temperature was observed after compressed gas was added. The gyration radius (R(g)) of the reverse micelles was determined using SAXS. R(g) increases with the addition of water, while it decreases appreciably with increasing pressure of compressed gas at fixed W(0). These results were interpreted in terms of an increase of the rigidity of the interface layer and a decrease of the interdroplet attraction. The results of this work provide useful information to get insight into the mechanism of cosurfactants to stabilize reverse micelles.

Nonaggregating refolding of ribonuclease A using reverse micellar dialysis

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.

The factors affecting the backward-transfer of Bovine Serum Albumin (BSA) from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micellar solutions

The factors affecting the back-extraction efficiency of Bovine Serum Albumin (BSA, 65kDa, pI 4.9) solubilized in an AOT reverse micellar solution, prepared by the injection method, to an excess aqueous phase were investigated. In particular, effects of pH, the type of salt and its concentration in the excess aqueous phase were examined. Furthermore, by comparing CD spectra of the back-extracted BSA with the feed BSA, the structural changes of the protein during the extraction process were determined. The addition of 1:1 salt such as KCl or NaCl to the aqueous phase resulted in a 100% recovery of the protein to the aqueous phase at a pH higher than its isoelectric point pI. This high efficiency of the back-extraction might be due to the change in the interactions between the protein and micellar aggregates driven by the added salt. For 1 : 2 salts like MgCl2 or CaCl2, BSA was back extracted with lower than 20% extraction efficiency. Maximum efficiencies were achieved at about pH=7 and pH=8 for monovalent and divalent salts, respectively. From the C D spectra of back-extracted BSA, it was observed that denaturation of BSA was not significant during the extraction process.

The effect of Linoleic Acid on pH Inside Sodium bis(2–ethylhexyl)sulfosuccinate Reverse Micelles in Isooctane and on the Enzymic Activity of Soybean Lipoxygenase

The effective pH of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles (pHrm), containing buffers of different pH (pHst) and various amounts of linoleic acid, was studied within the range of compositions used to study the activity of soybean lipoxygenase in reverse micelles. Significant shifts of pHrm versus pHst were observed for the solutions of relatively higher pHst, dependent on linoleic acid and buffer concentrations. The effect diminished as pHst became closer to 7. When low-ionic-strength buffers were added to AOT solutions in isooctane, a significant buffering effect of linoleic acid in reverse micelles was observed. Solubilization of > 3 mM linoleic acid in micellar solutions containing 25 mM buffers gave the observed pHrm values almost independent of pHst. This effect diminished with the ionic strength of the buffering solution, but did not vanish even at 200 mM buffer. The observed effects result from the balance between ionization of linoleic acid and its partition between the water pool and the micellar interface. The enzymic activity of soybean lipoxygenase in the AOT reverse micellar solutions of the determined pHrm values was also studied. A significant reduction of the kinetics of the enzymic activity was observed, for all studied reverse micellar solutions. Changes of pHrm, caused by the presence of acidic substrate (linoleic acid) do not explain the observed reduction of activity directly through the effect on the enzyme. Due to unfavourable partition of the substrate between the microphases present in the systems, enhanced by reduction of pH at higher total concentrations of linoleic acid, the saturation of the enzyme with the substrate was not observed in the system and is difficult to attain experimentally in reverse micelles. A shift of the lipoxygenase activity/pHrm profile but negligible shift of the activity/pHst profile, with respect to aqueous buffer solutions, were observed. This indicates that either the information given by pH indicators used does not reveal the true pH of the enzyme in these reverse micelles, or that the sample of the enzyme is pH-insensitive over a broader pH range than results from the relationship observed for linoleic acid in aqueous solutions. The latter conclusion is consistent with the data on lipoxygenase activity towards linoleyl sulfate in aqueous solutions [Bild, G. S., Ramadoss, C. S. & Axelrod, B. (1977) Lipids 12, 732-735]. Conditions for measuring pH-independent lipoxygenase activity in reverse micelles are discussed.

Esterification activity of lipase entrapped in reverse micelles formed in liquefied gas.

For AOT-water-liquefied gas systems, we studied formation of reverse micelles, entrapping of lipase into water-pools of the reverse micelles, and esterification activity of the entrapped lipase. The formation of reverse micelles was confirmed with visual observation using a dye soluble in water for liquefied ethane and propane, but was not done for liquefied CO2. Water was solubilized as water-pools in large quantity for the system using liquefied propane and in small quantity for that using liquefied ethane. The entrapping of lipase was confirmed for the system of liquefied propane using lipase labelled with a fluorescence probe. The entrapped lipase exhibited high catalytic activity for esterification of lipophilic substrates, though the activity was a little lower than that in AOT reverse micellar solution formed using hexane as an organic medium. The activity varied with mole ratio of water to AOT, [H2O]/[AOT], and was maximized at a certain value of [H2O]/[AOT]. The value of [H2O]/[AOT] almost agreed with that in a AOT-hexane reverse micellar solution.

Drastic change of reverse micellar structure by protein or enzyme addition

On addition of cytochrome c to a AOT reverse micellar solution, the percolation process usually observed at high temperatures and surfactant concentrations, occurs at room temperature. This is observed either at relatively high water content at a given cytochrome c concentration or at low content on increasing the cytochrome c concentration. On increasing the water content a phase transition is observed with two optically transparent phases. A similar phase transition is observed on solubilizing various enzymes. The temperature of the transition appears to be strongly dependent on the location of the macromolecule in the reserve micelle.