Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles

The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.     

Site-specific hydration status of an amphipathic peptide in AOT reverse micelles

Reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane (IO) and water have long been used as a means to provide a confined aqueous environment for various applications. In particular, AOT reverse micelles have often been used as a template to mimic membrane-water interfaces. While earlier studies have shown that membrane-binding peptides can indeed be incorporated into the polar cavity of AOT reverse micelles where they mostly fold into an alpha-helical structure, the underlying interactions leading to the ordered conformation are however not well understood. Herein, we have used circular dichroism (CD) and infrared (IR) spectroscopies in conjunction with a local IR marker (i.e., the CN group of a non-natural amino acid, p-cyano-phenylalanine) and a global IR reporter (i.e., the amide I' band of the peptide backbone) to probe the conformation as well as the hydration status of an antimicrobial peptide, mastoparan x (MPx), in AOT reverse micelles of different water contents. Our results show that at, w0=6, MPx adopts an alpha-helical conformation with both the backbone and hydrophobic side chains mostly dehydrated, whereas its backbone becomes partially hydrated at w0=20. In addition, our results suggest that the amphipathic alpha-helix so formed orients itself in such a manner that its positively charged, lysine-rich, hydrophilic face points toward the negatively charged AOT head groups, while its hydrophobic face is directed toward the polar interior of the water pool. This picture is in marked contrast to that observed for the binding of MPx to phospholipid bilayers wherein the hydrophobic surface of the bound alpha-helix is buried deeper into the membrane interior.     

高压CO~2对反胶束溶解蛋白质性质的影响

在308.15K下,研究了表面活性剂琥珀酸二(2-乙基己基)酯磺酸钠(Aerosol-Ot,简称AOT)的浓度和水的含量不同时,溶解的CO~2对反胶束溶解牛血清蛋白(BSA)的性质和异辛烷中AOT反胶束稳定性的影响。实验表明,在适当条件下,CO~2可以使反胶束溶液中的蛋白质全部析出。本研究对有关机理进行了初步分析。     

Water dynamics--the effects of ions and nanoconfinement

Hydrogen bond dynamics of water in highly concentrated NaBr salt solutions and reverse micelles are studied using ultrafast 2D-IR vibrational echo spectroscopy and polarization-selective IR pump-probe experiments performed on the OD hydroxyl stretch of dilute HOD in H(2)O. The vibrational echo experiments measure spectral diffusion, and the pump-probe experiments measure orientational relaxation. Both experimental observables are directly related to the structural dynamics of water's hydrogen bond network. The measurements performed on NaBr solutions as a function of concentration show that the hydrogen bond dynamics slow as the NaBr concentration increases. The most pronounced change is in the longest time scale dynamics which are related to the global rearrangement of the hydrogen bond structure. Complete hydrogen bond network randomization slows by a factor of approximately 3 in approximately 6 M NaBr solution compared to that in bulk water. The hydrogen bond dynamics of water in nanoscopically confined environments are studied by encapsulating water molecules in ionic head group (AOT) and nonionic head group (Igepal CO 520) reverse micelles. Water dynamics in the nanopools of AOT reverse micelles are studied as a function of size by observing orientational relaxation. Orientational relaxation dynamics deviate significantly from bulk water when the size of the reverse micelles is smaller than several nm and become nonexponential and slower as the size of the reverse micelles decreases. In the smallest reverse micelles, orientational relaxation (hydrogen bond structural randomization) is almost 20 times slower than that in bulk water. To determine if the changes in dynamics from bulk water are caused by the influence of the ionic head groups of AOT or the nanoconfinement, the water dynamics in 4 nm nanopools in AOT reverse micelles (ionic) and Igepal reverse micelles (nonionic) are compared. It is found that the water orientational relaxation in the 4 nm diameter nanopools of the two types of reverse micelles is almost identical, which indicates that confinement by an interface to form a nanoscopic water pool is a primary factor governing the dynamics of nanoscopic water rather than the presence of charged groups at the interface.     

Dynamics of low temperature induced water shedding from AOT reverse micelles

The effects of low temperature and ionic strength on water encapsulated within reverse micelles were investigated by solution NMR. Reverse micelles composed of AOT and pentane and solutions with varying concentrations of NaCl were studied at temperatures ranging from 20 degrees C to -30 degrees C. One-dimensional (1)H solution NMR spectroscopy was used to monitor the quantity and structure of encapsulated water. At low temperatures, e.g., -30 degrees C, reverse micelles lose water at rates that are dependent on the ionic strength of the aqueous nanopool. The final water loading (w0 = [water]/[surfactant]) of the reverse micelles is likewise dependent on the ionic strength of the aqueous phase. Remarkably, water resonance(s) at temperatures between -20 degrees C and -30 degrees C displayed fine structure indicating the presence of multiple transient water populations. Results of this study demonstrate that reverse micelles are an excellent vehicle for studies of confined water across a broad range of conditions, including the temperature range that provides access to the supercooled state.     

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.     

Surfactant-induced aggregation patterns of thiazole orange: a photophysical study

The aggregation behavior of the DNA marker dye thiazole orange (TO), has been investigated in two types of surfactant assemblies, namely, premicelles/micelles of sodium dodecyl sulfate (SDS) and pre reverse micelles/reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT). In the case of an SDS/water system, absorption spectral changes of TO signify the formation of H-aggregates and H-dimers of the dye at premicellar concentrations, which subsequently convert to the monomeric form beyond the critical micellar concentration (cmc). Interestingly, the observed changes in the absorption and emission characteristics due to the surfactant-induced formation of H-aggregates/dimers of TO are found to be useful to estimate the surfactant concentration parameters for premicellar aggregation of SDS. In the case of an AOT/n-heptane system, similarly, H-aggregates/dimers are observed at low AOT concentrations, below the cmc. However, in this case, the H-dimers persist even beyond the cmc. This is attributed to the strong tendency of TO for self-aggregation and its favorable electrostatic interactions with the AOT head groups. With increasing water content in the AOT reverse micelles, the hydration of the dye leads to the conversion of H-dimers to the monomeric form. The steady-state fluorescence results are nicely corroborated with those from time-resolved fluorescence studies and demonstrate the interesting behavior of the surfactant-induced aggregation of TO dye.     

反胶团中漆树酶催化氧化性能与反应产物鉴定

研究了漆树酶在AOT/正辛烷/水反胶团中邻氨基苯酚的催化氧化性能。发现在40℃、pH=7.6、R=[H₂O]/[AOT]≈18的条件下,漆树酶的活力最高,在含水量较低的反胶团中较稳定。当R=6.2,于30℃保存15h活力仍保持82%;"水池"内存在的1.0×10^-3mol/L的Zn²⁺、Fe³⁺、Mg²⁺等离子对漆树酶有一定的抑制作用。漆树酶对邻氨基苯酚在反胶团和水溶液介质中,其主要氧化产物均为2-氨基-吩嗪-3-酮,但在反胶团中其反应产率比在水溶液中约高0.5倍。     

Aggregation Behaviors of Tricarbocyanine Dye in Water and in AOT Reverse Micelles

The photophysical property of the tricarbocyanine dye IR144 has been extensively studied in non‐aqueous solvents. However, as a potential near‐infrared biomedical imaging probe, the photophysical property of IR144 in water is still little known. So, the aggregation behaviors of IR144 in water with steady‐state absorption spectroscopy and integrated polarization dependent femtosecond pump‐probe spectroscopy were investigated. Through comparing the absorption spectral bandshape of IR144 in water and in water pool of AOT reverse micelles, It is found that IR144 form dimer aggregates in water even at very low concentration (<1.0×10^?7 mol·L^?1). And the absorption spectrum of the IR144 aggregates always displays a bimodal feature, which is independent of the dye concentration ranging from 1.0×10^?7 to 1.0×10^?4 mol·L^?1. For better understanding the aggregation behaviors of IR144 in water, we measured the ground state recovery kinetics and the reorientation kinetics of IR144 in water and in water pool of AOT reverse micelles (W₀=[H₂O]/[AOT], W₀=40). It is found that the fluorescence quantum yield of IR144 in water is lower than that in water pool of AOT reverse micelles, and the reorientation time of IR144 in water is slower than that in water pool of AOT reverse micelles. Those kinetic measurements also verify that IR144 exists as dimer aggregates in water.     

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.     

Denaturation of a Recombinant Cutinase from Fusarium solani in AOT-iso-Octane Reverse Micelles: a Steady-State Fluorescence Study

Abstract— Near UV absorbance and fluorescence spectroscopy show conformational changes of a recombinant cutinase from Fusarium solani incorporated in sodium-di-2-ethylhexyl sulfosuccinate (AOT)-iso-octane reversed micelles with W₀= [H₂O]/[AOT] = 20. Excitation spectra were used to decompose cutinase absorbance in its Trp and Tyr components, showing that the latter absorb red-shifted in the native cutinase in aqueous solution as compared to free Tyr, whereas in reverse micelles and denatured cutinase no shift is detected. Emission maxima variations (λ_max 303, 311 and 335 nm, respectively in aqueous, reverse micelles and thermally denatured cutinase) reflect progressive changes in the micropolarity of the environment and exposure of Trp residues at the protein surface. The encapsulation of cutinase in AOT-iso-octane reversed micelles induces a time-dependent denaturation measured by fluorescence intensity changes at 330 nm, which match the profile of enzyme activity loss in this media.     

Tuning the cooperativity of the helix-coil transition by aqueous reverse micelles

We show in this letter that the thermodynamic properties of helical peptides can be tuned by varying the degrees of backbone hydration. The latter was achieved by solubilizing peptides in the water pool of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles with different water contents or w0 values. Far-UV circular dichroism measurements on a series of alanine-rich peptides indicate that the helicity of shorter peptides is significantly increased in AOT reverse micelles at low w0 values, as compared to the corresponding helical content in buffer. This result therefore corroborates the previous simulation studies suggesting that desolvation of backbone CO and NH groups increases the stability of monomeric helices. In addition, it was found that the thermal unfolding transition of these peptides can either be very noncooperative or very cooperative, depending on w0 and peptide chain length. A simple model, which considers the heterogeneous distribution of the water molecules inside the polar core of AOT reverse micelles as well as the geometric confinement effect exerted on the peptide by the reverse micelles, was used to interpret these results.     

Stabilizing effect of low concentrations of urea on reverse micelles

Urea is a well-known destabilizing agent for biopolymers like proteins and molecular aggregates like micelles and reverse micelles. Several theories have been proposed to explain the destabilizing/denaturing effect of urea. In this work, we present evidence for a stabilizing effect of a low concentration (<1 M) of urea incorporated in the central pool of AOT/n-heptane/water reverse micelles. Static light-scattering experiments were performed to measure (w0)cr--the molar ratio of water to AOT beyond which the micelles become unstable--as a function of the concentration of urea in the central water pool. The stabilizing effect of urea is reflected in an increase in the value of (w0)cr at low urea concentrations over that in the absence of urea. Dynamic light-scattering experiments show that the hydrodynamic radius of the micelles is smaller at low urea concentrations (<1 M) than in the absence of urea. Size-distribution analysis shows that for w0=20 the microemulsion containing 0.5 M urea in its pool is significantly more monodisperse than that containing no urea. Temperature-dependent studies in the range 15-65 degrees C indicate that the magnitude of this stabilizing effect decreases with increasing temperature, vanishing at temperatures higher than 65 degrees C. A model is proposed to explain the above results.     

An example of how to use AOT reverse micelle interfaces to control a photoinduced intramolecular charge-transfer process

6-Propionyl-2-(N,N-dimethyl)aminonaphtahalene, PRODAN, is widely used as a fluorescent molecular probe due to its significant Stokes shift in polar solvents. It is an aromatic compound with intramolecular charge-transfer (ICT) states which can be particularly useful as sensors. In this work, we performed absorption, steady-state, time-resolved fluorescence (TRES), and time-resolved area normalized emission (TRANES) spectroscopies on PRODAN dissolved in nonaqueous reverse micelles. The reverse micelles are composed of polar solvents/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane. Sequestered polar solvents included ethylene glycol (EG), propylene glycol (PG), glycerol (GY), formamide (FA), dimethylformamide (DMF), and dimethylacetamide (DMA). The experiments were performed with varying surfactant concentrations at a fixed molar ratio W(S) = [polar solvent]/[AOT]. In every reverse micelle studied, the results show that PRODAN undergoes a partition process between the external solvent and the reverse micelle interface. The partition constants, K(p), are quantified from the changes in the PRODAN emission and/or absorption spectra with the surfactant concentration. The K(p) values depend strongly on the encapsulated polar solvent and correlate quite well with the AOT reverse micelle interface's zones where PRODAN can exist and emits. Thus, the partition toward the reverse micelle interface is strongly favored in DMF and DMA containing micelles where the PRODAN emission comes only from an ICT state. For GY/AOT reverse micelles, the K(p) value is the lowest and only emission from the local excited (LE) state is observed. On the other hand, for EG/AOT, PG/AOT, and water/AOT reverse micelles, the K(p) values are practically the same and emission from both states (LE and ICT) is simultaneously detected. We show here that it is possible to control the PRODAN state emission by simply changing the properties of the AOT reverse micelle interfaces by choosing the appropriate polar solvent to make the reverse micelle media. Indeed, we present experimental evidence with the answer to the long time question about from which state does PRODAN emit, a process that can be controlled using the unique reverse micelle interfaces properties.     

Dynamics of solvent and rotational relaxation of glycerol in the nanocavity of reverse micelles

The dynamics of solvent and rotational relaxation of Coumarin 480 and Coumarin 490 in glycerol containing bis-2-ethyl hexyl sulfosuccinate sodium salt (AOT) reverse micelles have been investigated with steady-state and time-resolved fluorescence spectroscopy. We observed slower solvent relaxation of glycerol confined in the nanocavity of AOT reverse micelles compared to that in pure glycerol. However, the slowing down in the solvation time on going from neat glycerol to glycerol confined reverse micelles is not comparable to that on going from pure water or acetonitrile to water or acetonitrile confined AOT reverse micellar aggregates. While solvent relaxation times were found to decrease with increasing glycerol content in the reverse micellar pool, rotational relaxation times were found to increase with increase in glycerol content.     

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…     

Effect of CO(2) and CHF(3) on the Solubilization of Protein in Reverse Micelles

The effect of dissolved CO(2) and CHF(3) on the precipitation of bovine serum albumin (BSA) from reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in 2,2,4-trimethylpentane (iso-octane) has been studied at 308.15 K. It was found that BSA can be precipitated completely by CO(2) while the AOT and water remain in the iso-octane continuous phase. This opens up a possibility for recovery of protein solids directly from reverse micellar solutions. CHF(3) is also an effective agent for precipitation of BSA in the reverse micelles when the expansion coefficient of CHF(3) exceeds 0.4. However, the condition required for complete removal of the BSA using CHF(3) results in some precipitation of AOT and water from the solution. Copyright 2000 Academic Press.     

Effect of compressed CO2 on the critical micelle concentration and aggregation number of AOT reverse micelles in isooctane

The effect of compressed CO2 on the critical micelle concentration (cmc) and aggregation number of sodium bis-2-ethylhexylsulfosuccinate (AOT) reverse micelles in isooctane solution was studied by UV/Vis and fluorescence spectroscopy methods in the temperature range of 303.2-318.2 K and at different pressures or mole fractions of CO2 (X(CO2)). The capacity of the reverse micelles to solubilize water was also determined by direct observation. The standard Gibbs free energy (DeltaGo(m)), standard enthalpy (DeltaHo(m)), and standard entropy (DeltaSo(m)) for the formation of the reverse micelles were calculated by using the cmc data determined. It was discovered that the cmc versus X(CO2) curve and the DeltaGo(m) versus X(CO2) curve for a fixed temperature have a minimum, and the aggregation number and water-solubilization capacity of the reverse micelles reach a maximum at the X(CO2) value corresponding to that minimum. These results indicate that CO2 at a suitable concentration favors the formation of and can stabilize AOT reverse micelles. A detailed thermodynamic study showed that the driving force for the formation of the reverse micelles is entropy.     

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.