Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding.
Treatment of cytochrome P-450scc with fluorescein isothiocyanate (FITC) resulted in covalent labeling with 1.0 +/- 0.1 eq of FITC. Reverse-phase high performance liquid chromatography of tryptic and chymotryptic digests of the labeled protein revealed that a single FITC-labeled peptide accounted for 75% of the label. This peptide was found to be specifically labeled at lysine 338 by amino acid sequencing. The modification of lysine 338 with FITC resulted in 85 +/- 15% inhibition of adrenodoxin binding to cytochrome P-450scc. In a complementary experiment it was found that if a complex between adrenodoxin and native cytochrome P-450scc was formed in the presence of cholesterol and then treated with FITC, there was almost no labeling of lysine 338. The modification of lysine 338 by FITC was not inhibited by 22(R)-hydroxycholesterol, the first intermediate in the side chain cleavage reaction which binds to the active site 300 times more tightly than cholesterol itself. These experiments suggest that lysine 338 is located at the binding site for adrenodoxin and electrostatically interacts with one of the carboxylate groups on adrenodoxin that has been implicated in binding. The fluorescence emission of the FITC label on cytochrome P-450scc was only 14% as large as that of an equivalent concentration of FITC-labeled bovine serum albumin, suggesting that it was quenched by Forster energy transfer to the heme group.
Tuls
J
J
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Geren
L
L
Millett
F
F
D000325:Adrenodoxin / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007700:Kinetics; D008239:Lysine; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D013861:Thiocyanates / Q000494:pharmacology*; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013861:Thiocyanates; D000325:Adrenodoxin; D002786:Cholesterol Side-Chain Cleavage Enzyme; D002918:Chymotrypsin; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine