title: 7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching, suggesting binding at multiple protein sites.
abstract: Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern-Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (Kd1.8-2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (Kd1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (Kd2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (Kd0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.
authors.affiliation: a Physics Department , Grove City College , Grove City , PA , 16127-2104 , USA.
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Marsch
authors.affiliation: a Physics Department , Grove City College , Grove City , PA , 16127-2104 , USA.
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Carlson
authors.affiliation: b Department of Biochemistry , Vanderbilt University School of Medicine , 638B Robinson Research Building, 2200 Pierce Avenue, Nashville , TN , 37232-0146 , USA.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D001571:Benzoflavones / Q000737:chemistry*; D001665:Binding Sites; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D001571:Benzoflavones; D008024:Ligands; C011512:alpha-naphthoflavone; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human
keywords: P450 3A4; cytochromes P450; fluorescence quenching; α-naphthoflavone