In biological systems, the one electron (incomplete) reduction of molecular O2 during respiration produces the highly reactive superoxide anion radical (O2-), which is known to be the source of most reactive oxygen species (ROS). Further reduction of the reactive O2-radical by the enzyme superoxide dismutase (SOD) results in the creation of H2O2. This is a relatively stable, (non-radical-electron-state) oxygen form which unlike its more oxidized precursor, is capable of readily penetrating cell membrane barriers and influencing intracellular metabolic activities. Many cells produce low levels of O2- and H2O2 in response to a variety of extracellular stimuli, such as cytokines or peptide growth factors. The addition of exogenous H2O2 or the intracellular production in response to receptor stimulation affects the function of various proteins, including protein kinases, protein phosphatases, transcription factors, phospholipases, ion channels, and G proteins. H2O2 and O2 may participate in the production of singlet oxygen and peroxynitrite. Generation of these species may be concurrent with reactions involving iron, and under some circumstances, they might be important contributors to H2O2 toxicity.
ICT’s Hydrogen Peroxide (H2O2) Fluorescent Detection Kit is designed to quantitatively measure H2O2 in a variety of samples. A hydrogen peroxide standard is provided to generate a standard curve. Samples are mixed with the Fluorescent Substrate and the reaction initiated by addition of horseradish peroxidase. The reaction is incubated at room temperature for 15 minutes. Any H2O2 present in the sample will oxidize the HRP which in turn oxidizes the colorless non-fluorescent substrate; converting it into a fluorescence capable product. Increasing concentration levels of H2O2 cause a corresponding increase in HRP oxidation rates which translate into a linear increase in fluorescent product.