A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome b5, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of A610. MTT reduction followed classical Michaelis-Menten kinetics (K(m)= 20 microM, k(cat)= 1,910 min(-1)). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.
Yim
Sung-Kun
SK
Hormone Research Center and School of Biological Sciences and Technology, Chonnam National Univeristy, Gwangju 500-757, Korea.
Yun
Chul-Ho
CH
Ahn
Taeho
T
Jung
Heung-Chae
HC
Pan
Jae-Gu
JG
D000818:Animals; D005292:Ferricyanides; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins; D013053:Spectrophotometry; D013778:Tetrazolium Salts; D013844:Thiazoles
D005292:Ferricyanides; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C007931:hexacyanoferrate III; D009251:NADPH-Ferrihemoprotein Reductase; C022616:thiazolyl blue