Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Miwa 2015 Free Radic Biol Med

From Bioblast
Publications in the MiPMap
Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T (2015) Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays. Free Radic Biol Med 90:173-83.

ยป PMID: 26577176 Open Access

Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T (2015) Free Radic Biol Med

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. โ€ข Keywords: Amplex Red, Mitochondria, Liver, Reactive Oxygen Species, Measurement, Carboxylesterase

Cited by

  • Komlรณdi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi:10.26124/BEC:2021-0004

Labels: MiParea: Respiration, Pharmacology;toxicology 

Stress:Oxidative stress;RONS  Organism: Mouse  Tissue;cell: Liver  Preparation: Isolated mitochondria 


Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, ROX  HRR: Oxygraph-2k 

MitoFit 2021 AmR-O2