In biological systems, incomplete reduction of O2 during respiration produces superoxide anion (O2-·), which is spontaneously or enzymatically dismutated by superoxide dismutase to H2O2. Many cells produce low levels of O2-· and H2O2 in response to a variety of extracellular stimuli, such as cytokines (TGF-ß1, TNF-α, and various interleukins), peptide growth factors (PDGF; EGF, VEGF, bFGF, and insulin), the agonists of heterotrimeric G protein–coupled receptors (GPCR) such as angiotensin II, thrombin, lysophosphatidic acid, sphingosine 1-phosphate, histamine, and bradykinin, and by shear stress. 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. In 1894, Fenton described the oxidation of tartaric acid by Fe2+ and H2O2. H2O2 and O2 may participate in the production of singlet oxygen and peroxynitrite and the generation of these species may be concurrent with reactions involving iron, and under some circumstances, they might be important contributors to H2O2 toxicity.
A substantial portion of H2O2 lethality involves DNA damage by oxidants generated from iron-mediated Fenton reactions. Damage by Fenton oxidants occurs at the DNA bases or at the sugar residues. Sugar damage is initiated by hydrogen abstraction from one of the deoxyribose carbons, and the predominant consequence is eventual strand breakage and base release.
ICT’s Hydrogen Peroxide Fluorescent Detection Kit is designed to quantitatively measure H2O2 in a variety of samples. This kit is validated for use in fresh urine, buffers, and tissue culture media samples. It is species independent. Please read the complete kit insert before performing this assay. A hydrogen peroxide standard is provided to generate a standard curve for the assay. All samples should be read off the standard curve. Samples are mixed with the Fluorescent H2O2 Detection Substrate and the reaction is initiated by addition of horseradish peroxidase. The reaction is incubated at room temperature for 15 minutes. HRP is oxidized by hydrogen peroxide present in the sample. Oxidized HRP then reacts with the substrate to convert the colorless substrate into the fluorescent form. The fluorescent product is read at 590 nm with excitation at 570 nm. Increasing levels of H2O2 cause a linear increase in fluorescent product. This kit is for research use only and is not for use in diagnostic procedures.
- Prepare Assay buffer; dilute Assay Buffer Concentrate 1:5 in diH2O.
- Prepare samples; dilute > 1:10 in Assay Buffer.
- Prepare standard curve and zero (blank) standard.
- Add 50 µL blanks, standards, and samples to plate.
- Add 25 µL Fluorescent H2O2 Detection Substrate to plate.
- Prepare HRP; dilute Horseradish Peroxidase Concentrate 1:100 in Assay Buffer.
- Add 25 µL HRP Preparation to plate.
- Incubate 15 minutes at room temperature.
- Read fluorescence with excitation at 570 nm and emission at 590 nm.