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

Research output: Contribution to journalArticle

  • External authors:
  • Satomi Miwa
  • Achim Treumann
  • Amy Bell
  • Giulio Vistoli
  • Glyn Nelson
  • Thomas von Zglinicki


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.

Bibliographical metadata

Original languageEnglish
Pages (from-to)173-183
JournalFree radical biology & medicine
Early online date11 Nov 2015
StatePublished - 1 Jan 2016

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