Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum.

I describe the characterization, extraction, and purification of a cholesterol:oxygen oxidoreductase (EC 1.1.3.6) from Nocardia sp. This enzyme catalyzes oxidation of cholesterol to z4-choIestenone, with production of hydrogen peroxide. It is very stable, active over a wide pH range, and has a Km of 1.4 X i0 mol/liter. It is highly specific for or 3f3-hydroxycholestanes, and may be applied to the assay of serum total cholesterol. In the procedure presented here, hydrogen peroxide is measured by reaction with quadrivalent titanium and xylenol orange. This constitutes a one-enzyme assay with stable reagents, which does not require protein precipitation and is not subject to interference from hemoglobin or bilirubin.