Fluorescent assay for riboflavin binding to cytochrome P450 2B4.
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.
Shumyantseva
Victoria V
VV
Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya st. 10, Moscow 119992, Russia. victoria@ibmh.msk.su
Bulko
Tatiana V
TV
Petushkova
Natalia A
NA
Samenkova
Natalia F
NF
Kuznetsova
Galina P
GP
Archakov
Alexander I
AI
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002417:Cattle; D000072467:Cytochrome P450 Family 2; D012256:Riboflavin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D012256:Riboflavin