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

Vladimir Mishin, Joshua P. Gray, Diane E. Heck, Debra Laskin, Jeffrey Laskin

Research output: Contribution to journalArticle

67 Citations (Scopus)

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=5.0. pmol/assay and LLQ=30. pmol/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 (0.09. nmol hydrogen peroxide/min/100. Units 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 (0.31. nmol of hydrogen peroxide/min/100. Units 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.

Original languageEnglish (US)
Pages (from-to)1485-1491
Number of pages7
JournalFree Radical Biology and Medicine
Volume48
Issue number11
DOIs
StatePublished - Jun 1 2010

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Horseradish Peroxidase
Hydrogen Peroxide
NADPH-Ferrihemoprotein Reductase
Assays
Cytochrome P-450 Enzyme System
Enzymes
Cytochromes
Mixed Function Oxygenases
Reactive Oxygen Species
Substrates
Oxygen
Oxidation

All Science Journal Classification (ASJC) codes

  • Physiology (medical)
  • Biochemistry

Cite this

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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=5.0. pmol/assay and LLQ=30. pmol/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 (0.09. nmol hydrogen peroxide/min/100. Units 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 (0.31. nmol of hydrogen peroxide/min/100. Units 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.",
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Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes. / Mishin, Vladimir; Gray, Joshua P.; Heck, Diane E.; Laskin, Debra; Laskin, Jeffrey.

In: Free Radical Biology and Medicine, Vol. 48, No. 11, 01.06.2010, p. 1485-1491.

Research output: Contribution to journalArticle

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