title: Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes.
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.
authors.affiliation: Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mishin
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Gray
authors.forename: Diane E
authors.initials: DE
authors.lastname: Heck
authors.forename: Debra L
authors.initials: DL
authors.lastname: Laskin
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Laskin
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010078:Oxazines; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D010078:Oxazines; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C470430:Amplex Red; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D009251:NADPH-Ferrihemoprotein Reductase