I describe the characterization, extraction, and purification of a cholesterol:oxygen oxidoreductase (EC 126.96.36.199) from Nocardia sp. This enzyme catalyzes oxidation of cholesterol to Δ4-choIestenone, with production of hydrogen peroxide. It is very stable, active over a wide pH range, and has a Km of 1.4 x 10-5 mol/ liter. It is highly specific for Δ4- or Δ5-3β-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.
- Δ4- and Δ5-3β-hydroxycholestanes
- enzyme kinetics
- one-enzyme assay
- H2O2 measurement
- thin-layer chromatography
- ion-exchange chromatography
- cellular disruption
- ultraviolet spectrophotometry
- Received for publication August 7, 1973.
- © 1973 The American Association of Clinical Chemists, Inc.