Miwa_2016_Free.Radic.Biol.Med_90_173

Reference

Title : Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays - Miwa_2016_Free.Radic.Biol.Med_90_173
Author(s) : Miwa S , Treumann A , Bell A , Vistoli G , Nelson G , Hay S , von Zglinicki T
Ref : Free Radic Biol Med , 90 :173 , 2016
Abstract :

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.

PubMedSearch : Miwa_2016_Free.Radic.Biol.Med_90_173
PubMedID: 26577176

Related information

Substrate Amplex-red    Resorufin-butyrate

Citations formats

Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T (2016)
Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays
Free Radic Biol Med 90 :173

Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T (2016)
Free Radic Biol Med 90 :173