Enzymatic assay for cholesterol ester hydrolase activity.
A rapid and accurate method is described for the assay of cholesterol ester hydrolase (CEH) activity. Aliquots of the enzyme-substrate incubation mixture are extracted into isopropanol. The free cholesterol concentration in each extract is determined enzymatically using a single aqueous reagent containing cholesterol oxidase and peroxidase. The free cholesterol remaining after the cholesterol ester hydrolase-catalyzed esterification is converted to delta 4-cholestenone and hydrogen peroxide (H2O2); peroxidase couples H2O2 with phenol and 4-amino-antipyrine to yield a stable rose-colored product absorbing at 500 nm. The method is highly reproducible and the values correlate well with those obtained with the chromatographic radioassay of CEH activity.
D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D002789:Cholesterol Oxidase; D007700:Kinetics; D010544:Peroxidases; D013053:Spectrophotometry / Q000379:methods; D002787:Sterol Esterase / Q000032:analysis*
D002789:Cholesterol Oxidase; D010544:Peroxidases; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase