Effects of peroxidase substrates on the Amplex red/peroxidase assay: Antioxidant properties of anthracyclines

Krzysztof J. Reszka, Brett A. Wagner, C. Patrick Burns, Bradley E. Britigan

Research output: Contribution to journalArticlepeer-review

43 Scopus citations


Oxidation of Amplex red (AR) by H2O2 in the presence of horseradish peroxidase (HRP) gives rise to an intensely colored product, resorufin. This reaction has been frequently employed for measurements of low concentrations of H2O2 in biological samples. In the current study, we show that alternative peroxidase substrates, such as p-hydroquinone, acetaminophen, anticancer mitoxantrone, and ametantrone, inhibit AR oxidation by consuming H2O2 in a competitive process. In contrast, the anthracycline agents doxorubicin, daunorubicin, and 5-iminodaunorubicin are markedly less efficient as competitors in these reactions, as is salicylic acid. When [H2O2] > [AR], the generated resorufin was oxidized by HRP and H2O2. In the presence of anthracyclines, this process was inhibited and occurred with a lag time, the duration of which depended on the concentration of anthracycline. We propose that the mechanism of this inhibition is due to the antioxidant activity of anthracyclines involving the reduction of the resorufin-derived phenoxyl radical by the drugs' hydroquinone moiety back to resorufin. In addition to HRP, lactoperoxidase, myeloperoxidase, and HL-60 cells, naturally rich in myeloperoxidase, also supported these reactions. Results of this study suggest that extra caution is needed when using AR to measure cellular H 2O2 in the presence of alternative peroxidase substrates. They also demonstrate that the anticancer anthracyclines may function as antioxidants.

Original languageEnglish (US)
Pages (from-to)327-337
Number of pages11
JournalAnalytical Biochemistry
Issue number2
StatePublished - Jul 15 2005
Externally publishedYes


  • Acetaminophen
  • Ametantrone
  • Amplex red
  • Anthracyclines
  • Daunorubicin
  • Doxorubicin
  • EPR
  • Horseradish peroxidase
  • Lactoperoxidase
  • Mitoxantrone
  • Myeloperoxidase
  • Oxidation
  • Resorufin
  • Salicylic acid
  • p-Hydroquinone

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Effects of peroxidase substrates on the Amplex red/peroxidase assay: Antioxidant properties of anthracyclines'. Together they form a unique fingerprint.

Cite this