Colloidal Solution of Water in Organic Solvents: a Microheterogeneous Medium for Enzymatic Reactions

Martínek, Karel; Levashov, Andrey V.; Khmelnitsky, Yu. L.; Klyachko, N. L.; Березин, И.В.

doi:10.1126/science.6753152

Cited by 200 publications

(74 citation statements)

References 25 publications

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“…H # O # and n-octane were purchased from Reakhim (Moscow, Russia). The latter was purified as described in [20]. H # O # concentration was determined spectrophotometri- cally, using a molar absorption coefficient of 72.7 M −" :cm −" at 230 nm [21].…”

Section: Experimental Materialsmentioning

confidence: 99%

Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles

Gazaryan

Klyachko

Dulkis

et al. 1997

Biochemical Journal

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Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.

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Section: Experimental Materialsmentioning

confidence: 99%

Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles

Gazaryan

Klyachko

Dulkis

et al. 1997

Biochemical Journal

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“…It is seen that the kinetic parameters, particularly the values of the Michaelis constant, observed in aqueous solution differ considerably from those determined in the micellar system. This difference results in a profound change in the observed substrate specificity of HLADH on going from aqueous solution to the system of hydrated reversed AOT micelles in octane [13,14]. The dependence of the second-order rate constant, kc,,/&,, which is the measure of the substrate specificity of the enzyme, on the hydrocarbon chain length of alcohols is shown in Fig.…”

Section: Application Of the Model For Description Ojthe Kinetic Behavmentioning

confidence: 99%

“…The length of the initial linear part of kinetic curves depended on the nature of alcohol and usually was of the order of several minutes, which was sufficient for the reliable determination of the initial slope. A more detailed description of the kinetic studies of HLADH in AOT reversed micelles can be found in previous publications [13,141.…”

Section: Kinetic Assaysmentioning

confidence: 99%

“…Oxidation of aliphatic alcohols catalyzed by HLADH is a convenient model reaction for checking the validity of the proposed pseudophase approach, because alcoholic substrates form a broad series with smoothly varying hydrophobic properties and can partition among all phases of the micellar system. HLADH has been shown to retain its catalytic properties [13,14,[16][17][18][19] and conformation [20] in microemulsion systems of various types.…”

Section: Application Of the Model For Description Ojthe Kinetic Behavmentioning

confidence: 99%

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Kinetic theory of enzymatic reactions in reversed micellar systems

Khmelnitsky

Neverova

Polyakov

et al. 1990

European Journal of Biochemistry

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A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving SUbStrdteS capable of partitioning among all pseudophases of the micellar system, i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system. Micellar enzymology, which deals with enzymes entrapped in surfactant reversed micelles in orgarlic solvents, has received increasing attention during the last decade (for recent reviews see [l -41). The kinetic description of enzymatic reactions in reversed micellar systems often represents a rather complicated problem because of the microheterogeneity of the micellar medium: micellar solution is composed of tiny surfactantstabilized aqueous microdroplets (with diameter up to 200 nm) suspended in organic solvent. Solubilized enzyme molecules are localized exclusively inside these droplets (micelles), whereas the substrate can be distributed throughout the whole volume of the system. Evidently, the latter fact should be taken into account in kinetic analysis. To allow for substrate partitioning, the pseudophase approach can be used, according to which micellar systems can be regarded as being composed of two phases, namely the phase of organic solvent and a pseudophase of micelles. The pseudophase approach was successfully used for description of the kinetics of nonenzymatic [5] as well as enzymatic [6 -91 reactions in micellar systems. However, the notion that a micellar solution can be viewed as a pseudobiphasic system is to a certain extent on ...

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“…Meier and Luisi [I 61 have studied the kinetics of acetaldehyde reduction in Aerosol-OT microemulsion. Martinek et al [17,181 investigated the equilibrium constants and substrate specificity for oxidation of alcohols. Lee and Biellman [19,201 have studied kinetics for NAD and storage stability in Triton X-100, SDS and cetyhimethyhmmonium bromide microemulsions.…”

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confidence: 99%

Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol‐OT/isooctane microemulsions

Larsson¹,

Oldfield

Freedman

1989

European Journal of Biochemistry

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Changes in the enzymatic properties of horse liver alcohol dehydrogenase (HLADH; EC 1.1.1.1) were studied as a function of incubation time in Aerosol‐OT/isooctane microemulsions. The enzyme was characterized by fluorimetric binding studies of the inhibitor isobutyramide to the binary complex, HLADH‐NADH and by determination of Km,app and Vmax,app values for cyclohexanone. The Km,app values for cyclohexanone and the Kd,app for isobutyramide stay constant throughout a 48‐h incubation, whereas the Vmax,app and the total number of inhibitor binding sites decrease. Thus the inactivation process previously described corresponds to progressive loss of functional sites, while the properties of the remaining functional sites are unchanged. If no co‐enzyme is added to the system, the enzyme loses catalytic activity within less than an hour, but if co‐enzyme is added, a fraction of the HLADH enzyme population retains enzyme activity over a long period of time. Hence the presence of bound co‐enzyme significantly inhibits the process(es) leading to inactivation of the enzyme in the microemulsions.

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Colloidal Solution of Water in Organic Solvents: a Microheterogeneous Medium for Enzymatic Reactions

Cited by 200 publications

References 25 publications

Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles

Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles

Kinetic theory of enzymatic reactions in reversed micellar systems

Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol‐OT/isooctane microemulsions

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