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 continuous real-time measurement. Without correction for the background fluorescence slope, however, H2O2-independent formation of the fluorescent product UltroxRed (or resorufin) leads to artefacts. We analyzed (1) the medium specificity of the 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 showed a maximum under hypoxia when incubated in Dulbecco��s Phosphate Buffered Saline DPBS or KCl-medium. This hypoxic peak increased with the sequential number of normoxic-anoxic transitions. Even in the absence of yeast, the fluorescence slope increased at low O2 levels as a function of fluorescence intensity. The hypoxic peak was not observed in mitochondrial respiration medium MiR05. Therefore, the hypoxic peak was a medium-specific background effect unrelated to cell physiology. In MiR05, H2O2 production of yeast decreased linearly from hyperoxia to hypoxia, with a steep decline towards anoxia. Respiration and oxygen dependence expressed as p50 of yeast were higher in MiR05 than DPBS. Respiration was a hyperbolic function of oxygen concentration in the low-oxygen range. The flux-dependence of oxygen affinity explained the higher p50 in MiR05.

This work was partially funded by project NextGen-O2k which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement N�� 859770. Ondrej Sobotka��s secondments were founded by PROGRES Q40/02.