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-45011β 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