ABSTRACT
The synthesis and scavenging of hydrogen peroxide (H2O2) is tightly regulated in all cell types. H2O2 can fluctuate from low, physiological levels to intermediate, signaling ones and can further raise to participate in toxic reactions. Cell membranes contribute to the generation of gradients between the extracellular and intracellular media, as well as between cellular compartments. Elucidating steady-state levels of H2O2 at different cell compartments and the gradients across membranes is essential to understand redox signaling and toxicity effects. The most efficient tools for specific, quantitative, spatial, and timely measurements of H2O2 are genetically encoded biosensors of two families: redox-sensitive (ro)GFP fused to peroxiredoxins and HyPer derivatives. We will briefly describe here the advantages of three members of these families (roGFP2-Tpx1.C169S, HyPer, and Hyper7) and how the application of mathematical equations to the experimental data obtained with these probes has proven very useful to determine peroxide gradients across membranes, and can be used for calculating absolute and relative steady-state levels of H2O2 in different eukaryotic models.
