Sensitive assay of cytochrome P450scc activity by high-performance liquid chromatography.
We have developed a simple procedure for analyzing the reaction intermediates and product of the cholesterol side-chain cleavage system by high-performance liquid chromatography with uv absorption monitoring. After the cholesterol side-chain cleavage system had been incubated and the reaction then halted by heat treatment, the product was converted into 3-one-4-en steroid showing intense absorption at 240 nm upon reaction with cholesterol oxidase. The converted steroids were then analyzed by normal-phase HPLC. In consequence, the catalytic activity of the reconstituted adrenocortical cytochrome P450scc system was readily assayed with a sensitivity more than 10-fold higher by this conversion. Also, it was shown that 22R-hydroxy-cholest-4-en-3-one could serve as a good substrate for cytochrome P450scc and that the 20R,22R-dihydroxy derivative could be clearly detected as a reaction intermediate in the reconstituted system.
Sugano
S
S
Department of Biochemistry, School of Nursing, Kitasato University, Kanagawa, Japan.
Morishima
N
N
Ikeda
H
H
Horie
S
S
D000818:Animals; D002417:Cattle; D002783:Cholestenones / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013256:Steroids / Q000378:metabolism
D002783:Cholestenones; D013256:Steroids; C000475:cholest-4-en-3-one; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D002789:Cholesterol Oxidase