[The effect of structural organization of cholesterol aggregates in aqueous-organic media on its oxidation reaction, catalyzed by cholesterol oxidase].

Cholesterol oxidation by cholesterol oxidase in homogeneous water-organic mixtures without the use of surface-active agents and the effects of reaction medium parameters on this process have been studied. It has been shown that the nature of the organic solvent, buffer salts, pH and the "age" of the cholesterol solutions determine the formation of the "substrate" properties of sterol molecules in the aggregates which is manifested as changes in the kinetic parameters of the oxidation reaction and in the spectrophotometric characteristics of the enzymatic oxidation product, 4-cholesten-3-one. Such changes are the most vividly pronounced during oxidation of "aged" cholesterol solutions prepared 24 hrs before the reaction and are concomitant with significant reductions in the maximal reaction rate, Km values of cholesterol oxidase for cholesterol and the effective molar extinction coefficient for 4-cholesten-3-one. Sonication of "aged" cholesterol solutions partly restores these indices. The observed phenomena seem to be due to the different extent of aggregation of cholesterol molecules in solutions. Other factors influencing the value of the molar extinction coefficient for 4-cholesten-3-one, such as hypochromicity and stray light, are also discussed. The enzyme has a broad pH optimum at pH 7.0. The Michaelis constant for cholesterol does not appreciably change in the pH range studied (6.0-8.0) and constitutes approximately 15 microM for freshly prepared cholesterol solutions containing 10% propanol-1. The catalytic constant measured under the same conditions is about 22 s-1 at 20 degrees C and pH 7.0. The stability of cholesterol solutions is strongly influenced by the pH of the reaction medium, nature of the organic solvent and buffer salts. Weakly alkaline solutions prove to be the most stable ones. Regardless of the nature of the buffer salt its concentration in the range of 0.02-0.2 M does not affect the reaction.