Difference between revisions of "Miwa 2015 Free Radic Biol Med"
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{{Publication | {{Publication | ||
|title=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 H<sub>2</sub>O<sub>2</sub> release assays.. Free Radic Biol Med | |title=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 H<sub>2</sub>O<sub>2</sub> release assays.. Free Radic Biol Med 90:173-83. | ||
|info=[http://www.ncbi.nlm.nih.gov/pubmed/26577176 PMID: 26577176 Open Access] | |info=[http://www.ncbi.nlm.nih.gov/pubmed/26577176 PMID: 26577176 Open Access] | ||
|authors=Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T | |authors=Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T | ||
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|substratestates=CI, CII, ROX | |substratestates=CI, CII, ROX | ||
|instruments=Oxygraph-2k | |instruments=Oxygraph-2k | ||
}} | }} | ||
Revision as of 16:12, 13 January 2016
| 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. |
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. • Keywords: Amplex Red, Mitochondria, Liver, Reactive Oxygen Species, Measurement, Carboxylesterase
Labels: MiParea: Respiration, Pharmacology;toxicology
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Liver Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k
