Facts and artefacts on the oxygen dependence of hydrogen peroxide flux using Amplex UltraRed

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 continuous real-time measurement. Without correction for the background fluorescence slope, however, H₂O₂-independent formation of the fluorescent product UltroxRed (or resorufin from Amplex™ Red) leads to artefacts.

We analysed (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.

Cite:

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.org/10.26124/bec:2021-0004