Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.     

Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride

We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (muHiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by AOT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of -11.6 kcal mol(-1) and -2.6 kcal mol(-1) for GdnHCl and AOT, respectively. Hence, the AOT denatured state (D(AOT)) is less destabilised than the GdnHCl denatured state (D(GdnHCl)), relative to the native state in water. Far-UV CD spectroscopy revealed that D(AOT) retains some secondary structure, while D(GdnHCl) is essentially unstructured. Similarly, fluorescence data suggest that D(AOT) is more compact than D(GdnHCl). Activity measurements reveal that both D(AOT) and D(GdnHCl) are practically inactive (catalytic activity <1% of that of the native enzyme). The fluorescence spectrum of D(AOT) in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of D(AOT) in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data.     

A synthetic analog of plantaricin 149 inhibiting food-borne pathogenic bacteria:evidence for alpha-helical conformation involved in bacteria-membrane interaction.

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane.Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014.The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.     

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.     

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)     

Solubilization of soybean mitochondria in AOT/isooctane water-in-oil microemulsions

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.     

Renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content (W(o)) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.     

Characterization of serotonin in protein and membrane mimetic environments: a spectroscopic study

As a first step toward using the photophysical properties of serotonin to probe its interactions with biological target sites, we have examined its interactions with human serum albumin (HSA), chosen as a surrogate for the actual receptor proteins in physiological systems, and with sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/heptane/water reverse micelles, chosen as a biomembrane mimetic environment for the transmembrane portion of the receptor protein. Although the emission maximum of serotonin is relatively insensitive to the polarity of the local environment, which is attributed to lack of solvent dipolar reorientation of the 5-hydroxyindole chromophore, the fluorescence anisotropy (r) served as a useful and sensitive parameter from which the binding constants (K) and Gibbs energy changes (deltaG) were estimated for serotonin-HSA and serotonin-AOT reverse micellar interactions. Fluorescence-decay studies of serotonin show double-exponential kinetics in homogeneous aqueous solvent due to the structural heterogeneity arising from different rotamers of serotonin. In contrast, upon binding to HSA, a single-exponential fluorescence-decay profile was observed indicating the occurrence of a single structural species of serotonin in the protein environment. Furthermore, far-UV-circular-dichroism (CD) spectroscopic data indicate that the secondary structural features of HSA remain essentially intact after binding to serotonin. This preliminary research can be expected to open the door to extensive future studies on interactions of serotonin with relevant target proteins and associated cell membranes involved in its diverse physiological functions.     

Structural parameters of the myelin transmembrane proteolipid in reverse micelles.

The Folch-Pi proteolipid is the most abundant structural protein from the central nervous system myelin. This protein-lipid complex, normally insoluble in water, requires only a small amount of water for solubilization in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The characterization of the proteolipid-free and proteolipid-containing micelles was undertaken by light scattering and fluorescence recovery after fringe pattern photobleaching (FRAPP) experiments. Quasi elastic light scattering (QELS) was carried out at a high (200 mM) AOT concentration, at low water-to-surfactant mole ratio (Wo = 7) and at increasing protein occupancy. Two apparent hydrodynamic radii, differing tenfold in size, were obtained from correlation functions. The smaller one (RaH = 5.2 nm) remains constant and corresponds to that measured for protein-free micelles. The larger one increases linearly with protein concentration. In contrast, FRAPP measurements of self-diffusion coefficients were found unaffected by the proteolipid concentration. Accordingly, they have been performed at constant protein/surfactant mole ratios. The equivalent RH, extrapolated to zero AOT concentration for protein-free reverse micelles (2.9 nm) and in the presence of the proteolipid (4.6 nm), do not reveal the mode of organization previously suggested by QELS measurements. The complex picture emerging from this work represents a first step in the characterization of an integral membrane protein in reverse micelles.     

Dynamics of a membrane-bound tryptophan analog in environments of varying hydration: a fluorescence approach

Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have employed a combination of wavelength-selective fluorescence and time-resolved fluorescence spectroscopies to monitor the effect of varying degrees of hydration on the dynamics of TOE in reverse micellar environments formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Our results show that TOE exhibits red edge excitation shift (REES) and other wavelength-selective fluorescence effects when bound to reverse micelles of AOT. Fluorescence parameters such as intensity, emission maximum, anisotropy, and lifetime of TOE in reverse micelles of AOT depend on [water]/[surfactant] molar ratio (w (o)). These results are relevant and potentially useful for analyzing dynamics of proteins or peptides bound to membranes or membrane-mimetic media under conditions of changing hydration.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).     

Preferred conformation of endomorphin-1 in aqueous and membrane-mimetic environments.

The newly discovered endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are potent opioid peptides with the highest affinity and selectivity for the mu receptor among all known endogenous ligands. To investigate a possible correlation between these biological properties and the conformational preferences of the small peptides, a comparative structural analysis was performed of endomorphin-1 in aqueous buffer and in membrane-mimicking SDS and AOT normal and reverse micelles by the use of CD, FT-IR, fluorescence and(1)H-NMR spectroscopy. It is well established for opioid peptides that, independently of the receptor selectivity, the Tyr1 residue plays the role of the primary pharmacophore and that the orientation of the second aromatic pharmacophore relative to the tyrosine side-chain dictates the mu or delta-receptor selectivity. By varying the environment of endomorphin-1 from water to the amphipathic SDS micelles and even more efficiently to the AOT reverse micelles, the display of the aromatic side-chains changes from an interaction of the Tyr1 and Phe4 residues to a switch of the Trp3 indole group into close contact with the phenolic moiety to prevent this type of interaction and to force an orientation of the Phe4 side-chain into the opposite direction. This conformational switch is accompanied by a stabilization of the cis -Pro2 isomer and the resulting spatial array of the pharmacophoric groups correlate well with the structural model of mu receptor-bound opioid peptides. The results indicate that AOT reverse micelles with a woof 10, where almost exclusively ordered water is secluded in the cavity, constitute with their electrostatic and hydrophobic potential an excellent mimetic of amphipathic surfaces as present on lipid bilayers and on ligand-recognition and ligand-binding sites of proteins.     

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.     

Interactions of native and modified cytochrome C with a negatively charged reverse micellar liquid interface

Native and chemically modified cytochrome C were dissolved in sodium bis(2-ethylhexyl) sulphosuccinate (AOT)-oil-buffer microemulsions. The native cytochrome C contains 19 lysine residues, these groups were modified by 1) acetic anhydride or 2) succinic anhydride. At pH 8.4 the native, acetylated and succinylated proteins carry +8, –3 and –12 elementary charges, respectively. The phase behaviour of the microemulsion systems was found to be highly dependent on the charge of the proteins. Compared to a protein free system the native protein induces a L-2 phase separation at lower temperatures. The acetylated protein has a small effect on the temperature for the phase transition, whereas in the case of succinylated cytochrome C the phase transition takes place at higher temperatures. When dissolved in AOT microemulsions, the native cytochrome C has a perturbed tertiary structure, as indicated by loss of the 695 nm absorption band, while both the modified proteins retain the same optical properties when dissolved in an AOT microemulsion as in a pure buffer solution. The pertubed structure of the native cytochrome C was further investigated by testing the stability of the reduced form of the protein dissolved in the microemulsion media. The native cytochrome is unstable at W > 10, whereas the two modified proteins were found to be stable at all W-values investigated. The average location of the three proteins was determined by pulse radiolysis. The quenching rate constant of the hydrated electron depends upon the location of the probe in the reverse micelle; the succinylated protein is localised in the aqueous core of the reverse micelles, but both the native and the acetylated forms were found to be localised close to or at the AOT interface.     

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.     

Measurements of hydrodynamic diameter of AOT reverse micelles containing lipase in supercritical ethane and its enzymatic reaction

The enzymatic reaction by aerosol-OT (AOT)reverse micelles containing lipase in supercritical ethane was examined and is the focus of this paper. The reverse micelles were formed under various conditions at which their hydrodynamic diameters were measured by using the dynamic light scattering spectrophotometer. The reverse micelles in supercritical ethane were formed in the range of Wo (water/surfactant) less than six. The hydrodynamic diameter of the reverse micelles ranged from 2 to 5 microm. The hydrolysis reaction of triolein by the lipase in reverse micelles was also examined. The observations indicate that lipase in AOT reverse micelles in supercritical ethane showed activity. The conversion of triolein increased with the increase in size of reverse micelles and Wo, and reached its maximum near the critical temperature. Moreover, although the conversion of triolein increased with pressure, it was independent of pressure near the critical temperature.     

Extraction of flexibly structured protein in AOT reverse micelles: the flexible structure of protein is the dominant factor for its incorporation into reverse micelles

The extraction of flexibly-structured protein in Aerosol-OT (AOT)/isooctane reverse micelles was investigated. A flexibly-structured lysozyme was prepared by reduction and carboxymethylation of the disulfide bonds in the lysozyme molecule. For a comparison, lysozymes whose surface hydrophobicity was modified by monoacylation of the amino groups were also used. The extraction rate of the flexibly-structured lysozyme into the micellar phase was greater than that of the native and monoacylated lysozymes, although the free energy change of the lysozyme prepared by breaking the disulfide bonds was smaller than that of the lysozymes whose surfaces were monoacylated. Viscosity measurement of the micellar organic phase containing the modified lysozymes indicated that extraction of the flexibly-structured lysozyme changed the micelle–micelle interaction, while measurement of the interfacial tension between the AOT/isooctane and protein aqueous systems showed the flexibly-structured lysozyme to be the most amphiphilic in character. These results indicated that the flexible structure of a protein was more dominant than its surface hydrophobicity for its incorporation into reverse micelles, and that it leads to greater micelle–micelle interaction.     

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.     

A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.