Mechanisms of Cytochrome C Extraction by Reverse Micelles

Ｔｈｅｒｅｖｅｒｓｅｍｉｃｅｌｌｅｓａｒｅｗａｔｅｒ ｉｎ ｏｉｌｍｉｃｒｏｅｍｕｌｓｉｏｎｄｒｏｐｌｅｔｓｓｔａｂｉｌｉｚｅｄｂｙａｓｕｒｆａｃｔａｎｔｉｎａｎｏｒｇａｎｉｃｓｏｌｖｅｎｔ .Ｔｈｅｐｏｌａｒｓｕｒｆａｃｔａｎｔｈｅａｄｇｒｏｕｐｓｓｕｒｒｏｕｎｄｓｍａｌｌｗａｔｅｒｐｏｏｌｓｗｉｔｈｉｎｗｈｉｃｈｈｙｄｒｏｐｈｉｌｉｃｍｏｌｅｃｕｌｅｓ,ｓｕｃｈａｓａｍｉｎｏａｃｉｄｓａｎｄｐｒｏｔｅｉｎｓ ,ｃａｎｂｅｓｏｌｕｂｉｌｉｚｅｄ .Ｔｈｅａｐｏｌａｒｓｕｒｆａｃ ｔａｎｔｈｙｄｒｏｃａｒ…     

Cyclic voltammetry and viscosity measurements of aggregated assemblies of anionic surfactants with nonionic surfactants and triblock copolymers

Cyclic voltammetry (CV) and viscosity measurements have been employed to study the aggregation behavior of mixed micellar systems of anionic surfactant (dioctyl sulfosuccinate sodium salt, AOT) with conventional nonionic surfactants such as Brij 35/TritonX-100/Tween 20/Tween 80/Myrj 45 and two triblock copolymers (L64 and F68). Critical micelle concentration (cmc) values have been determined for various micellar systems from CV measurements using 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) as an electroactive probe at 25 °C. Diffusion coefficient (D) has been evaluated from Randles–Sevcik equation which showed an overall decrease for most of the binary systems. The negative values of interaction parameters (β) obtained from regular solution theory suggest the synergistic behavior in all the binary systems except AOT + Tween 80 mixtures. The mixed systems of AOT with triblock copolymers showed stronger synergistic interactions than that of mixed systems of AOT with nonionic surfactants. A comparative evaluation of mixed systems of anionic surfactants AOT and sodium dodecyl sulfate with Myrj 45 and AOT + L64 and F68 has been made on the basis of different micellar parameters and structural properties of surfactants. Viscosity measurements also show similar type of interactions in the mixed micelles.     

Complexation of Al(III) by 8-Hydroxyquinoline and Drastic Fluorescence Enhancement in Reverse Micelles

Complexation of Al^IIIby 8-hydroxyquinoline and fluorescence behavior of the quinolinate(s) were studied in reverse micellar systems at low water content, and compared to aqueous media. Two surfactants were used: one was cationic (CTAC: cetyltrimethylammonium chloride) and the other was anionic (AOT: sodium bis(2-ethylhexyl)sulfosuccinate). The results obtained in the CTAC/dichloromethane system (W= [H₂O]/[surfactant] = 0.9) showed that complexation occurred very likely in the oil phase and no micellar effect was observed. On the contrary, in the presence of AOT, specific micellar effects were observed due to the presence of the anionic polar heads: stabilization of the positively charged 1:1 and 1:2 chelates, at the expense of the neutral water-insoluble 1:3 chelate which is formed in aqueous solutions under similar conditions;drastic fluorescence enhancement factorsof 120 and 100 in AOT/heptane (W= 1.5) and AOT/dichloromethane (W= 1.6), respectively. Such factors have never been reported so far in either hydroorganic or direct micellar systems. In return, the length of time for the production of the complex(es) is increased because of the microheterogeneity of the medium and the small sizes of the water pools.     

Effect of the Media on the Quantum Yield of Singlet Oxygen (O2(1Δg)) Production by 9H‐Fluoren‐9‐one: Microheterogeneous Systems

The quantum yield (Φ_Δ) of singlet oxygen (O₂(¹Δ_g) production by 9H‐fluoren‐9‐one (FLU) is very sensitive to the nature of the solvent (0.02 in a highly polar and protic solvent, such as MeOH, to 1.0 in apolar solvents). This high sensitivity has been used for probing the interaction of FLU with micellar media and microemulsions based on anionic (sodium dodecyl sulfate, SDS; bis‐(2‐ethylhexyl)sodium sulfosuccinate, AOT), cationic (cetyltrimethylammonium chloride, CTAC) and nonionic (Triton X‐100, TX) surfactants. Values of Φ_Δ of FLU vary in a wide range (0.05–1.0) in both microheterogeneous media and neat solvent, and provide information on the microenvironment of FLU, i.e., on its localization within organized media. In ionic and nonionic micellar media, as well as in four‐component microemulsions, FLU is, to various extents, exposed to solvation by the polar and protic components of the microheterogeneous systems (water and/or butan‐1‐ol) in the micellar interfacial region (Φ_Δ=0.05–0.30). In contrast, in AOT reverse micelles (consisting of AOT as surfactant, cyclohexane as hydrophobic component, and water), FLU is located in the hydrophobic continuous pseudophase, and is totally separated from the micellar water pools (Φ_Δ≈1.0).     

Intermicellar material exchange in reverse micelles formed by ionic AOT and nonionic Igepal surfactants studied by means of pulse radiolysis. Influence of the temperature

The recombination of thiocyanate anion radicals, (SCN) ₂ ⁻ , formed pulse radiolytically within the water pools of reverse micelles stabilized with anionic AOT and nonionic Igepal surfactants, was proved as an indicator reaction to study intermicellar exchange. It was found that the exchange process is slower inIgepal than in AOT reverse micelles with the same water to surfactant ratio. The apparent activation enthalpy and entropy of the exchange process were determined in different alkanes. For the AOT and Igepal reverse micelles the activation parameters increase with the droplet size, but for the AOT systems they do not significantly change with the increase of droplet concentration. For non-percolated systems the activation parameters for Igepal reverse micelles approach those for AOT reverse micelles. This result supports existing suggestions that the mechanism of intermicellar exchange does not differ in principle between reverse micelles stabilized with ionic and nonionic surfactants.     

Reverse Micellar Extraction of β-Galactosidase from Barley (Hordeum vulgare)

The reverse micellar system of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane was used for the extraction and primary purification of beta-galactosidase (EC 3.2.1.23) from the aqueous extract of barley (Hordeum vulgare) for the first time. The process parameters such as the concentration of the surfactant, the volume of the sample injected, and its protein concentration, pH, and ionic strength of the initial aqueous phase for forward extraction, buffer pH, and salt concentration for back extraction are varied to optimize the extraction efficiency. Studies carried out with both phase transfer and injection mode of reverse micellar extraction confirmed the injection mode to be more suitable for beta-galactosidase extraction. The extent of reverse micellar solubilization of proteins increased with an increase in protein concentration of the feed sample. However, back extraction efficiency remained almost constant (13-14.4%), which indicates the selectivity of AOT reverse micelles for a particular protein under given experimental conditions. beta-Galactosidase was extracted with an activity recovery of 98.74% and a degree of purification of 7.2-fold.     

Biomimetic oxidation of N-nitrosodibenzylamine with molecular oxygen catalysed by chemical cytochrome P-450 in AOT reverse micelles

Moderate yields of benzaldehyde, benzyl alcohol and benzylamine are obtained by the biomimetic oxidation of N-nitrosodibenzylamine with molecular oxygen catalysed by water soluble anionic manganese(III) 5,10,15,20-tetraphenylporphyrin acetate/sodium dithionite/methylene blue in aerosol-OT (AOT) reverse micelles, under phase transfer conditions with AOT concentration higher than 10⁻³M. The formation of α-hydroxy-N-nitrosodibenzylamine and its decomposition products, benzaldehyde and benzyl alcohol in reverse micellar systems are governed by the ratio of water and AOT, pH and other changes in the microenvirpnment.     

Investigation on the basic hydrolysis of crystal violet in mixed reverse micelles formed with AOT and nonionic surfactants

 The kinetics and thermodynamics of the basic hydrolysis of crystal violet (CV) in mixed reverse micelles formed with anionic surfactant AOT and nonionic surfactants have been investigated. It was found that the mixed reverse micelles had inhibitory effects on CV hydrolysis compared with the normal aqueous solution, and the equilibrium constant K of the reaction in mixed reverse micellar systems is smaller than that in pure water. The influence of water content and surfactant composition in reverse micelles on the second-order rate constant k ₁ of the positive reaction, on the first-order rate constant k _-1 of the reverse reaction, as well as on the equilibrium constant K of the reaction has been studied, and the results obtained were interpreted in terms of the nature of surfactants and the properties of microenvironment where the reaction took place. Received: 24 October 1997 Accepted: 18 March 1998     

Activity and kinetics studies of yeast alcohol dehydrogenase in a reverse micelle formulated from functional surfactants

Yeast alcohol dehydrogenase (YADH) showed substantial decrease in its catalytic activity due to the strong electrostatic interaction between the head groups of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and YADH in AOT reverse micelles. However, the catalytic activity of YADH in a nonionic reverse micellar interface (GGDE/TX-100) obtained from a functional nonionic surfactant N-gluconyl glutamic acid didecyl ester (GGDE) and Triton X-100 (TX-100) was higher than that in AOT reverse micelle under the respective optimum conditions. A comparison of the kinetic parameters showed that the turnover number k_cat in GGDE/TX-100 reverse micelle was 1.4 times as large as that in AOT reverse micelle, but the Michaelis constants in AOT reverse micelle for ethanol K_m^B was twice and for coenzyme NAD⁺ K_m^A was 5 times higher than their counterparts in GGDE/TX-100 reverse micelle. For the conversion of ethanol, the smaller K_m^B and larger k_cat in GGDE/TX-100 reverse micelle resulted in higher catalytic efficiency k_cat/K_m^B. The stability of YADH in GGDE/TX-100 reverse micelle was also found to be better than that in AOT reverse micelle. They were mainly attributed to the absence of electric charge on the head groups of GGDE and TX-100 in the GGDE/TX-100 reverse micelle.      

混合反胶束的电导与微结构研究

反胶束是两亲分子在非极性溶剂中形成的一种有序组合体，在医药、化工、采油、胶束催化及酶催化等领域中有重要应用．与胶束溶液相比，人们对反胶束的形成与结构的了解至今仍不充分．特别是对于由混合表面活性剂形成的反胶束的研究几乎无人涉及．本文采用动态光散射、电导及荧光光谱等手段对阴离子表面活性剂AOT与非离子表面活性剂形成的混合反胶束进行了研究，旨在探讨利用表面活性剂的复配来调节和控制反胶束的结构和性能．亚实验部分二异辛基磺化琉璃酸钠（AOT，Sigma公司）；Brij30为含4个氧乙烯基（EO基）的十二碳醇（AcrosOrgani…     

Forward and backward extractions of cytochrome c using reverse micellar system of sucrose fatty acid ester

The effects of solvent, pH, and ionic strength on the reverse micellar extraction of cytochrome c have been examined, when sucrose fatty acid esters were employed as surfactants of reverse micelles. The transparent and stable reverse micellar organic phase was formed, when the mixture of isooctane and n-butanol (7:3 v/v) was used as the bulk organic phase. The high forward extraction ratio was obtained under mild alkaline and low ionic strength conditions, while the high backward extraction ratio was obtained for acidic pH values or at high ionic strength. The activity of cytochrome c recovered from the reverse micellar phase was sufficiently retained.     

Hydrocarbon peroxidation: protective effect of reverse micelles

Pulse radiolysis technique was used to study radical processes in AOT/n-heptane and AOT/n-heptane/water reverse micellar systems. It was found that reverse micelles, especially socalled 'wet' micelles ([H₂O]/[AOT] > 10), significantly diminished the yield of peroxyl radicals formed when the system contained trace amounts of oxygen. The possible mechanism of such protective effect of micellar aggregates is discussed in terms of scavenging of charges, both electrons and cation radicals, by micellar aggregates where they can eventually form radicals. The latter are however separated from the traces of oxygen which remains mainly in the continuous hydrocarbon phase, where it dissolves much better than in the micellar water core.     

SOLUBILIZATION IN MIXED REVERSE MICELLAR SYSTEMS OF AOT/NONIONIC SURFACTANTS/n-HEPTANE

Solubilization of water and aqueous NaCl solutions in mixed reverse micellar systems of anionic surfactant AOT and nonionic surfactants in n-heptane was studied. It was found that the maximum solubilization capacity of water was higher in the presence of certain concentrations of NaCl electrolyte, and these concentrations increased with the increase of nonionic surfactant content and their EO chain length. Soluibilization capacity was enhanced by mixing AOT with nonionic surfactants. The observed phenomena were interpreted in terms of the stability of the interfacial film of reverse micellar microdroplet and the packing parameter of the surfactant that formed mixed reverse micelles.     

Terahertz absorption spectroscopy of protein-containing reverse micellar solution

Terahertz time-domain spectroscopy has been carried out for AOT/isooctane reverse micellar solution with myoglobin at the water-to-surfactant molar ratios (w₀) of 0.2 and 4.4. The amplitude of the absorption spectrum increases with increasing the protein concentration at w₀ = 0.2, whereas it decreases at w₀ = 4.4. The molar extinction coefficients of the protein-filled reverse micelle, and the constituents, i.e., myoglobin, water, and AOT, have been derived by use of the structural parameters of the micellar solution. The experimental results are interpreted in terms of hydration onto the protein and surfactant in the reverse micelle.     

Kinetic Studies on of Veratryl the Lignin Peroxidase Catalyzed Oxidation Alcohol by H2O2 in AOT/Isooctane/ Toluene/Water Reverse Micelles

The steady state kinetics of the lignin peroxidase （LIP） catalyzed oxidation of veratryl alcohol （VA） by H2O2 in a sodium bis（2-ethylhexyl） sulfosuccinate （AOT）/isooctane/toluene/water reverse micellar medium was studied and a comparison with the corresponding aqueous medium was made to understand the effect of the reverse micellar medium on the catalytic mechanism and kinetic parameters. Results indicated that the model reaction in the AOT reverse micelle followed the ping-pong mechanism with true kcat, Km,VA and KmH2O2 being 59.6min^-1, 13.9 mmol· L^-1 and 94.8 μmol·L^-1, respectively; inhibition of high level of H2O2 on LiP followed the reversible competitive pattern with Ki being 0.140 mmol·L^-1. The reaction mechanism and inhibition pattern in the AOT reverse micellar medium were the same as those in bulk aqueous medium, but the kinetic parameters except KmH2O2 were greatly different in the two media. The kcat and Ki values in the reverse micelle were approximately 2 and 20 times smaller than the corresponding values in the aqueous solution, but the Michaelis constant of VA was approximately 100 times greater than that in the aqueous solution. The above mentioned differences in the kinetic parameters were caused by the microheterogeneity and the interface of the AOT reverse micelle, which resulted in the partitioning of VA and H2O2, and by the changes of the conformation of LiP and the reactivity of the substrates.     

Specifics of the formation of J-aggregates of cyanine dyes in solutions of AOT/water/hexane reverse micelles

The behavior of a cyanine dye (3,3′-di-(gamma-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethylthiacarbocyanine betaine pyridinium salt) was studied in AOT/water/hexane reverse micelles over a wide range of W at various concentrations of the dye, AOT, and reverse micelles. The processes occurring during the formation of the AOT/water/hexane micellar solution were studied in detail. It has been shown that, before the formation of the stable microemulsion, the dye aggregation processes occur by virtue of the interaction of the dye with the AOT anion. The amount of J-aggregates is proportional to the logarithm of the ratio of the amount of AOT molecules to the amount of dye molecules. The time behavior of J-aggregates after the formation of a micellar structure depends on the concentration of reverse micelles, thereby indicating an important role of intermicellar exchange.     

Partial purification of glutaryl-7-ACA acylase from crude cellular lysate by reverse micelles

Glutaryl-7-ACA acylase was partially purified from the cellular lysate ofPseudomonas sp. NCIMB 40409 by means of reverse micelles-water two-phases extractions. The tetrameric enzyme can be solubilized inside the reverse micelles formed by anionic (Aerosol OT, AOT) and cationic (tetradecyltrimethylammoniumbromide, TDAB) surfactants with retention of the enzymatic activity. With TDAB reverse micelles system, the acylase was partially extracted from the aqueous phase and, after backward transfer into a second water phase, a twofold purification factor was achieved. On the other hand, with the AOT micellar system, in conditions were most of the proteins but acylase, were extracted by the organic micellar solution, a sixfold increase of the specific activity of the acylase remaining in the aqueous phase was obtained.     

Improvement in enzyme activity and stability by addition of low molecular weight polyethylene glycol to sodium bis(2-ethyl-L-hexyl)sulfosuccinate/isooctane reverse micellar system

The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-1-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining>75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (V _max)exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.     

Preparation and properties of surfactant-bacillolysin ion-pair in organic solvents

The transesterification-active enzyme bacillolysin was extracted into organic solvents such as isooctane by enzyme-AOT (bis (2-ethylhexyl) sulfosuccinate) ion-pairing preserving its natural second structure and catalytic activity. Extraction efficiency was affected by the interaction mode of the two phases, ionic strength, and pH of aqueous phase, surfactant and enzyme concentration. Magnetic stirring with phase mixing was favorable for the enzyme extraction. Optimal ionic strength and pH were 8 mM CaCl₂ and 5.0, respectively. Critical number of AOT molecule for an enzyme molecule to be extracted into isooctane was 89. Optimal initial enzyme concentration in the aqueous phase was 7 mg mL⁻¹ while the initial AOT concentration in isooctane was 3 mM. Within CMC (critical micellar concentration) of AOT in isooctane, the increase of initial AOT concentration enhanced the extraction efficiency.     

Effects of Surfactant and Salt Species in Reverse Micellar Forward Extraction Efficiency of Isoflavones with Enriched Protein from Soy Flour

Suitability of reverse micelles of anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS), cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) and nonionic surfactant polyoxyethylene p-t-octylphenol (TritonX-100) in organic solvent isooctane for extraction of soy isoflavone-enriching proteins was investigated. The results showed that the order of combined isoflavone contents was SDS>CTAB>Triton X-100>AOT, while the order of protein recovery was SDS>AOT>TritonX-100>CTAB. As compared with ACN-HCl extraction, the total amount of isoflavones was lower than reverse micellar extraction. Ion strength was one of the important conditions to control extraction of isoflavone-enriching proteins with AOT reversed micelles. For the six salt systems, KNO₃, KCl, MgCl₂, CaCl₂, NaCl, and Na₂SO₄, extracted fraction of isoflavone-enriching proteins was measured. Salt solutions greatly influenced the extraction efficiency of isoflavones in an order of KNO₃>MgCl₂>CaCl₂>KCl>NaCl>Na₂SO₄, while protein in an order of MgCl₂>CaCl₂>NaCl>KNO₃>Na₂SO₄>KCl.