Abstract
Mammalian NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 enzymes and other several microsomal enzymes. It also catalyzes the one-electron reduction of many chemicals and drugs. Reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by CPR was assessed as a method for monitoring CPR activity. The electrons released from NADPH by CPR were transferred to CTC in the reaction medium, and CTC reduction activity could be assessed spectrophotometrically and spectrofluorometrically. The reduction kinetics of CTC follows classical Michaelis–Menten kinetics (K m = 50 μM, k cat = 2,520 min−1). This method offers a continuous assay of the enzymatic activity of CPR.
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Acknowledgments
This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science & Technology, Republic of Korea, by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2006-005-J03003), and by the Second Stage BK21 Project from the Ministry of Education, Science & Technology of the Republic of Korea.
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D. H. Kim and S. K. Yim are contributed equally to this work.
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Kim, DH., Yim, SK., Kim, KH. et al. Continuous spectrofluorometric and spectrophotometric assays for NADPH-cytochrome P450 reductase activity using 5-cyano-2,3-ditolyl tetrazolium chloride. Biotechnol Lett 31, 271–275 (2009). https://doi.org/10.1007/s10529-008-9865-4
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DOI: https://doi.org/10.1007/s10529-008-9865-4