Time-resolved fluorescence and circular dichroism of porphyrin cytochrome c and Zn-porphyrin cytochrome c incorporated in reversed micelles

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Zn-porphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrime-thylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces. Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (wo = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved. Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions. In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.     

Triplet-state kinetics of Zn-porphyrin cytochrome c in micellar media. Measurement of intermicellar exchange rates

The interactions of protein molecules with surfactant assemblies in aqueous and hydrocarbon media have been studied via the triplet-state kinetics of Zn-porphyrin cytochrome c in solutions containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] or a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. In aqueous solution, the observed triplet state decay is single exponential with a lifetime of 8 ms. In aqueous solutions of AOT and in AOT-reversed micellar solutions, biexponential triplet state decays were observed, indicating that interactions between the surfactant and the protein occur, resulting in a change in protein conformation near the porphyrin ring. In CTAB-reversed micellar solutions, quenching of the Zn-porphyrin cytochrome c triplet state by ferricyanide and methyl viologen was studied. Because the quenching is exchange-limited under the conditions used, the exchange rate constants for the water pools can be obtained from these experiments. The observed exchange rate constants are in the range (1-5) x 10(7) M-1 S-1, depending on the water content of the reversed micelles and on the type of quencher used. These values are three orders of magnitude lower than the calculated collision rate of the reversed micelles.     

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)     

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.     

Effect of hydration degree of aerosol OT reversed micelles and surfactant concentration in heptane on spectral and catalytic properties of catalase.

The influence of micelle hydration degree (w0) and AOT concentration on fluorescence, circular dichroism (CD), catalytic activity, and stability of catalase in Aerosol OT (AOT) reversed micelles in heptane was investigated. The quantitative parameters--differential fluorescence of catalase (DeltaI), protein molar ellipticity ([theta]lambda), initial rate of catalytic reaction, catalase efficiency (kcat/Km), and rate constant of enzyme inactivation (kin, sec-1)--decreased with increasing AOT concentration in micellar systems, reflecting the interaction of solubilized catalase with the AOT micellar aggregates in heptane. The dependences of all these parameters on increasing hydration degree of micelles (w0) were characterized by the appearance of maxima at w0 of 8, 15-18, and 26-30. These maxima are suggested to reflect three different states of catalase in the micellar system, distinguished by their conformations and catalytic activity, which is determined by the micellar microenvironment of the enzyme.     

Kinetic Studies on the Interaction of Ferricytochrome c with Anionic Surfactants

The kinetics of absorbance and fluorescence changes of cytochrome c as induced by an aqueous solution of the anionic surfactant sodium dodecyl sulfate (SDS) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) are studied. The results are compared with far-UV circular dichroism (CD) spectra. Both surfactants cause similar alterations in the secondary structure of cytochrome c, while their influence on the heme environment of cytochrome c is different. In the presence of AOT below and above critical micellar concentration a conversion of the low-spin native cytochrome c to a denatured low-spin protein not having methionine ligand takes place. In the presence of SDS micelles conversion of the native protein to a denatured mixed-spin form occurs. The changes in the heme group induced by both surfactants occur independently of the alterations in tertiary structure.     

Fluorescence of hemoproteins in reversed micelles of surfactants in octanes

The fluorescence of myoglobin, cytochromes b5 and c in the reversed aerosol OT (AOT) micelles in octane has been investigated. The fluorescence intensity of all the three hemoproteins is higher than that in aqueous solutions. The maxima and intensities of fluorescence in the AOT micelles depend on the [H2O]/[AOT] ratio and reflect the protein structure. Aliphatic alcohols and secondary amines (piperidine and morpholine) quench the cytochrome c fluorescence in the AOT micelles, whereas dipolar aprotic solvents (dimethylsulfoxide, dimethylformamide) significantly increase the intensity of cytochrome c fluorescence in the same micelles. The transformations of the proteins solubilized by the reversed micelles of a surfactant are discussed.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Reversed micellar extraction of trypsin: Effect of solvent on the protein transfer and activity recovery

By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid-liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT-reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. (c) 1995 John Wiley & Sons, Inc.     

Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

Extraction of bovine serum albumin using nanoparticulate reverse micelles

The extraction of a relatively large molecular weight protein, bovine serum albumin (BSA), using nano-sized reverse micelles of nonionic surfactant polyoxyethylene p-t-octylphenol (Triton-X-100) is attempted for the first time. Suitability of reverse micelles of anionic surfactant sodium bis (2-ethyl hexyl) sulfosuccinate (AOT) and Triton-X-100/AOT mixture in organic solvent toluene for BSA extraction is also investigated. Although, the size of the Triton-X-100 reverse micelle in toluene is large enough to host BSA molecule in the hydraulic core, the overall extraction efficiency is found to be low, which may be due to lack of strong driving force. AOT/toluene system resulted in complete forward extraction at aqueous pH 5.5 and a surfactant concentration of 160 mM. The back extraction with aqueous phase (pH 5.5) resulted in 100% extraction of BSA from the organic phase. The addition of Triton-X-100 to AOT reduced the extraction efficiency of AOT reverse micelles, which may be attributed to reduced hydrophobic interaction. The circular dichroism (CD) spectrum of BSA extracted using AOT/toluene reverse micelles indicated the structural stability of the protein extracted.     

Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.     

Fluorescence dynamics of staphylococcal nuclease in aqueous solution and reversed micelles

The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV vircular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.     

Important parameters affecting efficiency of protein refolding by reversed micelles

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.     

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.     

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.     

Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization

Experiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease-a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di-2-ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P](rm,max) was found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase-transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P](rm). In this work, it was found to be possible to dissociate the protein-surfactant complex and recover the precipitated protein. The kinetics of protein-surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head-groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein-surfactant complex formation as [P](rm) --> [P](rm,max). In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein-surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co-partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P](rm). (c) 1995 John Wiley & Sons Inc.     

Red-edge excitation fluorescence spectroscopy of proteins in reversed micelles

The dependence of fluorescence emission maxima ofl-tryptophan and single-tryptophan-containing proteins (ribonuclease T1, melittin, and parvalbumin) on excitation wavelength has been studied in reversed micelle systems of sodium bis(2-ethyl-1-oxyl) sulfosuccinate (AOT). No effect of fluorescence maximum shift for different excitation wavelengths is observed for ribonuclease T1, in which a single tryptophan residue is located in the nonrelaxating, nonpolar protein interior.l-Tryptophan and the rest of the studied proteins, which contain single tryptophan residues exposed to the solvent, exhibit the dipolar relaxational processes of partly immobilized water molecules in micelles. This effect depends on the molar H₂O/AOT ratio. Circular dichroism measurements prove that there have been no structural changes of the studied proteins in micellar systems. The results provide information about dynamic relaxational processes in proteins.     

Catalysis by enzymes entrapped into reversed micelles of surfactants in organic solvents. Peroxidase in the aerosol OT-water-octane system

Spectral and catalytic parameters of peroxidase solubilized in the aerosol OT-water-octane system have been studied. The spectrum of peroxidase solubilized in octane with AOT reversed micelles, a degree of surfactant hydration being above 12, is actually identical to that of the enzyme aqueous solution. On the other hand, significant spectral changes have been detected when transferring the enzyme from water to the reversed micelle medium at low degrees of surfactant hydration, precisely [H2O]/[AOT] less than 12. The reversed micelle-entrapped peroxidase catalyses the oxidation of pyrogallol with hydrogen peroxide much more actively (at [H2O]/[surfactant] approximately 13) than that in aqueous solution. The entrapment of peroxidase into surfactant reversed micelles increases precisely the catalytic constant of the reaction, i.e. the virtual reactivity of the enzyme increases ten and hundred times depending on degrees of surfactant hydration and concentration. The systems of reversed micelles may be considered as models of biomembranes. Our findings hence show that enzymes in vivo can be much more catalytically active then it appears possible to reveal in conventional experiments in vitro in aqueous solutions.