Miwa 2015 Free Radic Biol Med: Difference between revisions
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|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 | |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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== Cited by == | == Cited by == | ||
{{Template:Cited by | {{Template:Cited by Komlodi 2021 MitoFit AmR-O2}} | ||
{{Labeling | {{Labeling | ||
|area=Respiration, Pharmacology;toxicology | |area=Respiration, Pharmacology;toxicology | ||
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|pathways=N, S, ROX | |pathways=N, S, ROX | ||
|instruments=Oxygraph-2k | |instruments=Oxygraph-2k | ||
|additional=MitoFit 2021 AmR-O2 | |||
}} | }} | ||
Latest revision as of 21:40, 24 September 2021
| 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
Cited by
- Komlódi 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
