核黄素与细胞色素P450 2B4结合的荧光测定法。

Fluorescent assay for riboflavin binding to cytochrome P450 2B4.

作者信息

Shumyantseva Victoria V, Bulko Tatiana V, Petushkova Natalia A, Samenkova Natalia F, Kuznetsova Galina P, Archakov Alexander I

机构信息

Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya st. 10, Moscow 119992, Russia.

出版信息

J Inorg Biochem. 2004 Feb;98(2):365-70. doi: 10.1016/j.jinorgbio.2003.10.024.

DOI:10.1016/j.jinorgbio.2003.10.024

PMID:14729317

Abstract

The interactions between the hemoprotein cytochrome P450 2B4 (CYP 2B4) and riboflavin - a low molecular weight component of the flavoprotein NADPH-dependent cytochrome P450 reductase - were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by cytochrome P450 2B4 was used to probe the ligand-enzyme binding (lambda(ex)=385 nm, lambda(em)=520 nm). Fluorescence titration experiments showed formation of a complex between cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K(d)=8.8+/-1 microM. The fluorescence intensity of riboflavin was decreased with increasing the cytochrome P450 2B4 concentration, indicating the transfer of resonance excitation energy from riboflavin (energy donor) to the cytochrome P450 2B4 heme (energy acceptor). The data obtained are suggestive of the existence of riboflavin binding site(s) on the hemeprotein molecule.

摘要

通过荧光光谱法研究了血红蛋白细胞色素P450 2B4（CYP 2B4）与核黄素（黄素蛋白NADPH依赖性细胞色素P450还原酶的一种低分子量成分）之间的相互作用。利用细胞色素P450 2B4对核黄素荧光的猝灭来探测配体与酶的结合（激发波长λ(ex)=385 nm，发射波长λ(em)=520 nm）。荧光滴定实验表明细胞色素P450 2B4与核黄素形成了复合物，其表观解离常数K(d)=8.8±1 μM。随着细胞色素P450 2B4浓度的增加，核黄素的荧光强度降低，这表明共振激发能量从核黄素（能量供体）转移到了细胞色素P450 2B4血红素（能量受体）。所获得的数据表明血红蛋白分子上存在核黄素结合位点。