The cellular steady-state of H2O2: latency concepts and gradients

Hydrogen peroxide (H2O2) is able to diffuse across biomembranes but, when cells are exposed to external H2O2, the fast consumption of H2O2 inside the cells due to H2O2-removing enzymes provides the driving force for setting up a H2O2 gradient across the plasma membrane. Knowing this gradient is fundamental to standardize studies with H2O2 as for the same extracellular H2O2 concentration cells with different H2O2 gradients may be exposed to different intracellular H2O2 concentrations. Here, we present the kinetic background behind the establishment of the H2O2 gradient and show how the gradient can be determined experimentally using the principle of enzyme latency. Furthermore, we discuss some of the caveats that may arise when determining the H2O2 gradient. Finally, we describe detailed protocols for the experimental determination of the H2O2 gradient across the plasma membrane in Saccharomyces cerevisiae cells and in mammalian cell lines.