A COLORIMETRIC ASSAY FOR MEASURING CELL-FREE AND CELL-BOUND CHOLESTEROL OXIDASE

Cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) catalyzes the conversion of sterol DELTA-5-3-beta-alcohol to the corresponding DELTA-4-3-ketone with the reduction of oxygen to hydrogen peroxide. Rhodococcus species GK 1, a soil isolated microbe, produces an extracellular and a membrane-bound cholesterol oxidase; the latter is bound to the outer surface of the microbial cell membrane. A simple and sensitive assay is described to measure the two enzyme types; no enzyme extraction is needed for measuring the membrane-bound cholesterol oxidase. In this assay, hydrogen peroxide is reduced by the chromogen 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of horseradish peroxidase, and the increased absorbance is followed continuously at 600 nm (epsilon(m) = 1.82 X 10(4) M-1 . cm-1 at pH 7.0 and 30-degrees-C). The standardized assay medium contained 46.9 mM sodium-potassium phosphate buffer pH 7.0, 0.16% Triton X-100, 312.5-mu-M ABTS, 50-mu-g peroxidase (12.5 units at 25-degrees-C), 6.25% isopropanol, 306.3-mu-M cholesterol or other sterols (kept in solution with isopropanol), and cholesterol oxidase. Oxidation of one molecule of cholesterol by cholesterol oxidase gives one molecule of hydrogen peroxide which reacts with two molecules of ABTS. The method is reproducible and the results correlate well with those obtained by measuring the absorbance of DELTA-4-cholest-3-one at 240 nm (epsilon(m) = 1.40 X 10(4) M-1 . cm-1 at pH 7.0 and 30-degrees-C) and by the method of Allain et al. (Clin. Chem. 20, 470-475, 1974). In terms of efficiency, simplicity, and time saved, this coupled assay is expected to be a useful method for monitoring microbial production of cholesterol oxidase on an industrial scale, and for determining cholesterol or other sterols in biological fluids.