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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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A Continuous Spectrophotometric Assay for NADPH-cytochrome P450 Reductase Activity Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide

  • Yim, Sung-Kun (Hormone Research Center and School of Biological Sciences and Technology, Chonnam National Univeristy) ;
  • Yun, Chul-Ho (Hormone Research Center and School of Biological Sciences and Technology, Chonnam National Univeristy) ;
  • Ahn, Tae-Ho (Department of Biochemistry, College of Veterinary Medicine) ;
  • Jung, Heung-Chae (National Research Laboratory of Microbial Display, GenoFocus, Inc.) ;
  • Pan, Jae-Gu (National Research Laboratory of Microbial Display, GenoFocus, Inc.)
  • Published : 2005.05.31
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Abstract

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 $b_5$, 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 $A_{610}$. MTT reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;20\;{\mu}M$, $k_{cat}\;=\;1,910\;min^{-1}$). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.

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References

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