Chemical modification of steroid-transforming monooxygenases with fluoresceine isothiocyanate

A. G. Lapko, G. Smettan, K. Ruckpaul, S.A. Usanov

Institute of Bioorganic Chemistry, Academy of Sciences of the Byelorussian SSR, Minsk; Central Institute of Molecular Biology, Academy of Sciences of the GDR, Berlin-Buch

Abstract: Chemical modifications of cytochrome P-450scc and cytochrome P-450_11β with fluosesceine-, diiodofluoresceine-, eosine-and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was-shown to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-45scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F₂, to be Lys³³⁸. Effect of modification with FITC on the interaction of cytochrome-P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.

Russian Journal of Bioorganic Chemistry 1991, 17 (7):921-932

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