Skip Navigation

Publication Detail

Title: Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes.

Authors: Mishin, Vladimir; Gray, Joshua P; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

Published In Free Radic Biol Med, (2010 Jun 01)

Abstract: The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.

PubMed ID: 20188819 Exiting the NIEHS site

MeSH Terms: Animals; Cytochrome P-450 Enzyme System/metabolism*; Horseradish Peroxidase/metabolism*; Humans; Hydrogen Peroxide/analysis; Hydrogen Peroxide/metabolism*; Microsomes, Liver/enzymology*; NADPH-Ferrihemoprotein Reductase/metabolism; Oxazines*; Rats; Reactive Oxygen Species/metabolism; Recombinant Proteins/metabolism

Back
to Top