Myeloperoxidase (MPO) Inhibitor Screening Assay Kit is a rapid, simple, sensitive, and reliable assay suitable for use as a high throughput MPO activity inhibitor screening. In the MPO assay protocol, myeloperoxidase produces HClO from H2O2 and Cl–. Myeloperoxidase (MPO) is a peroxidase enzyme (EC 220.127.116.11) most abundantly present in neutrophil granulocytes. It is a green hemoprotein found in neutrophils and monocytes that catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates that play a role in the oxygen-dependent killing of tumor cells and microorganisms. Its heme pigment causes the green color in secretions rich in neutrophils, such as some forms of mucus. Furthermore, it can oxidize tyrosine to a tyrosyl radical using hydrogen peroxide as an oxidizing agent. Celltechgen’s MPO Inhibitor Screening Assay provides convenient fluorescence-based methods for screening inhibitors to both the chlorination and peroxidation activities of MPO. The chlorination assay utilizes the non-fluorescent 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]-benzoic acid (APF), which is selectively cleaved by hypochlorite (-OCl) to yield the highly fluorescent compound fluorescein. Fluorescein fluorescence is analyzed with an excitation wavelength of 480-490 nm and an emission wavelength of 515-520 nm. The peroxidation assay utilizes the peroxidase component of MPO, where a single two electron oxidation of native enzyme (MPO) to compound I (MPO-I) is followed by two successive one electron reductions back to native enzyme by compound II (MPO-II). The reaction between hydrogen peroxide and ADHP (10-acetyl-3,7-dihydroxyphenoxazine) produces the highly fluorescent compound resorufin. Resorufin fluorescence is analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm. The assay schemes are outlined in Figure below.
MPO Assay Buffer 50 ml
MPO Chlorination Substrate 100 µl
MPO Peroxidation Substrate (in DMSO) 100 µl
Hydrogen Peroxide (0.88 M) 50 µl
MPO Inhibitor 50 µl
Myeloperoxidase Control 50 µl
Store kit at -20°C protected from light. Allow Assay Buffer to warm to room temperature
before use. Briefly centrifuge small vials before opening.
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