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Difference between revisions of "Komlodi 2021 MitoFit AmR-O2"

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Revision as of 11:43, 23 September 2021

Publications in the MiPMap
Sobotka O, Komlódi T, Doerrier C, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells. MitoFit Preprints 2021 (in prep).



Abstract: The fluorometric Amplex UltraRed AmR assay is frequently used for quantitative assessment of hydrogen peroxide production. It is specific to H2O2, can be calibrated accurately, and allows real-time and continuous measurements. The disadvantage is that H2O2-independent formation of the fluorescent product UltroxRed (or resorufin) is possible, which requires correction for background fluorescence slope. In the present study, we analysed (1) the medium-specificity of background fluorescence slope of the AmR assay, and (2) the oxygen dependence of H2O2 flux in baker´s yeast Saccharomyces cerevisiae. Apparent H2O2 flux, O2 concentration and O2 flux were measured simultaneously by high-resolution respirometry equipped with the fluorescence module. The apparent H2O2 flux of yeast incubated in Dulbecco´s Phosphate Buffered Saline DPBS and KCl-medium showed a maximum under hypoxia, which increased during consecutive aerobic-anoxic transitions. In these media, background fluorescence slope increased as a function of fluorescence intensity and at low O2 levels in the absence of sample, explaining the hypoxic peak as a medium-specific artefact. The artefact of the hypoxic peak was avoided in mitochondrial respiration medium MiR05 with stable background fluorescence slope. In MiR05, H2O2 production of yeast declined linearly from hyperoxia to hypoxia, with a steep decline towards anoxia. Respiration of yeast was higher in MiR05 than DPBS. Respiration was a hyperbolic function of oxygen concentration in the low-oxygen range, and the flux-dependence of oxygen affinity explained the higher p50 in MiR05. Keywords: Amplex UltraRed, AmR, hydrogen peroxide production, H2O2 flux, respiration media, mitochondrial respiration medium, MiR05, oxygen dependence, yeast, reductive stress, anoxia, hypoxia, O2 kinetics, respiration, reoxygenation Bioblast editor: Komlodi T


Labels: MiParea: Respiration 

Stress:Oxidative stress;RONS, Hypoxia  Organism: Saccharomyces cerevisiae  Tissue;cell: Other cell lines  Preparation: Intact cells 

Regulation: Oxygen kinetics  Coupling state: ROUTINE 

HRR: Oxygraph-2k, O2k-Fluorometer 


References