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 h...

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Veröffentlicht in:Analytical biochemistry 1989-11, Vol.182 (2), p.327-333
Hauptverfasser: SUGANO, S, MORISHIMA, N, IKEDA, H, HORIE, S
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Sprache:eng
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Zusammenfassung: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/0003-2697(89)90603-9