Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays.

Satomi Miwa, Achim Treumann, Amy Bell, Giulio Vistoli, Glyn Nelson, Sam Hay, Thomas von Zglinicki

    Research output: Contribution to journalArticlepeer-review


    Amplex Red is a fluorescent probe that is widely used to detect hydrogen peroxide (H2O2) in a reaction where it is oxidised to resorufin by horseradish peroxidase (HRP) as a catalyst. This assay is highly rated amongst other similar probes thanks to its superior sensitivity and stability. However, we report here that Amplex Red is readily converted to resorufin by a carboxylesterase without requiring H2O2, horseradish peroxidase or oxygen: this reaction is seen in various tissue samples such as liver and kidney as well as in cultured cells, causing a serious distortion of H2O2 measurements. The reaction can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at concentrations which do not disturb mitochondrial function nor the ability of the Amplex Red-HRP system to detect H2O2.In vitro experiments and in silico docking simulations indicate that carboxylesterases 1 and 2 recognise Amplex Red with the same kinetics as carboxylesterase-containing mitochondria. We propose two different approaches to correct for this problem and re-evaluate the commonly performed experimental procedure for the detection of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data.
    Original languageEnglish
    Pages (from-to)173-183
    JournalFree radical biology & medicine
    Early online date11 Nov 2015
    Publication statusPublished - 1 Jan 2016


    • Amplex Red
    • Carboxylesterase
    • Liver
    • Measurement
    • Mitochondria
    • Reactive oxygen species


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