Journal of Biochemical and Biophysical Methods, 11 (1985) 177-184 177 Elsevier BBM 00485 A novel approach to study of action of water-insoluble inhibitors of enzymic reactions Boris I. Kurganov a, Larissa G. Tsetlin 1, Emma A. Malakhova a, Natalia A. Chebotareva 1, Vadim Z. Lankin 2, Galina D. Glebova 1, Vladimir M. Berezovsky ~, Andrey V. Levashov 3 and Karel Martinek 3 All-Union Research Institute of Vitamins, Moscow 117246, Nauchny proezd, 14 A, 2 U.S.S.R. Cardiology Research Center, Moscow 121552, 3 Cherepkovskaja ul., 15 A, and 3 Department of Chemistry, Moscow State University, Moscow 117234, U.S.S.R. (Received 14 January 1985) (Accepted 21 March 1985) Summary The effect of water-insoluble compounds on enzyme catalytic properties was studied using a colloidal solution of water in organic solvent as reaction medium. In this microheterogeneous medium enzyme is entrapped into hydrated reversed micelles of a surfactant, the dimensions of the internal hole of the micelles being dependent on the ratio of water to surfactant. At sufficiently low values of this ratio the molecule of entrapped enzyme has limited mobility in the micelle. Because of this the interaction of the enzyme with water-insoluble compound which is added in assay solution and intercalated in the surface layer of the miceIle may be manifested. The suggested method was used to study the inhibitory action of dihydroriboflavin esters on D-amino acid oxidase from pig kidney and soybean lipoxygenase. The reaction medium was hydrated reversed micelles of Aerosol OT in octane. The method of sedimentation in an analytical ultracentrifuge has shown the dihydroriboflavin esters to be completely included into reversed micelles. Key words: water-insoluble inhibitors; D-amino acid oxidase; lipoxygenase; reversed micelles. Introduction In the presence of micelle-forming surfactants, enzymes are dissolved in water-im- miscible organic solvents with the catalytic activity retained [1-8]. This results in optically transparent solutions and therefore normal spectrophotometric methods may be applied for registration of the time course of enzymic reaction. In such systems the enzyme and water-soluble substances are located in the polar nucleus of the miceUe. In the cited works various enzymes were entrapped in hydrated reverse micelles of Aerosol OT (AOT) in octane. The dimensions of the internal hole of the 0165-022X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)