10006438  Myeloperoxidase Chlorination Assay Kit

MPO Chlorination Assay Kit



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Myeloperoxidase (MPO) is a member of the heme peroxidase superfamily and is stored within the azurophilic granules of leukocytes.1 MPO is found within circulating neutrophils, monocytes, and some tissue macrophages.2 A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide (H2O2) to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent.3 Recently, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory diseases.4,5 The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors.6 MPO also oxidizes a variety of substrates, including phenols and anilines, via the classic peroxidation cycle. The relative concentrations of chloride and the reducing substrate determine whether MPO uses H2O2 for chlorination or peroxidation. Assays based on measurement of chlorination activity are more specific for MPO than those based on peroxidase substrates because peroxidases generally do not produce hypochlorous acid. The only exception is eosinophil peroxidase that produces hypochlorous acid at pH below 5. The chlorination activity of MPO has a neutral pH optimum, therefore the assay conditions can be set so that only MPO activity is specifically measured. Cayman’s Myeloperoxidase Chlorination Assay provides a convenient fluorescence-based method for detecting the MPO chlorination activity in both crude cell lysates and purified enzyme preparations. The 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.7 Fluorescein fluorescence is analyzed with an excitation wavelength of 480-490 nm and an emission wavelength of 515-520 nm. The kit includes a MPO-specific inhibitor for distinguishing MPO activity from MPO-independent fluorescence.
1  Yamada, M., Kurahashi, K. Regulation of myeloperoxidase gene expression during differentiation of human myeloid leukemia HL-60 cells. J Biol Chem 259(5) 3021-3025 (1984).
2  Schultz, J., Kaminker, K. Myeloperoxidase of the leucocyte of normal human blood.1 I. Content and localization. Arch Biochem Biophys 96 465-467 (1962).
3  Harrison, J.E., Schultz, J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem 251(5) 1371-1374 (1976).
4  Podrez, E.A., Abu-Soud, H.M., Hazen, S.L. Myeloperoxidase-generated oxidants and atherosclerosis. Free Radic Biol Med 28(12) 1717-1725 (2000).
5  Zhang, R., Brennan, M., Fu, X., et al. Association between myeloperoxidase levels and risk of coronary artery disease. JAMA 286(17) 2136-2142 (2001).
6  Malle, E., Furtmüller, P.G., Sattler, W., et al. Myeloperoxidase: A target for new drug development?. Br J Pharmacol 152 838-854 (2007).
7  Setsukinai, K., Urano, Y., Kakinuma, K., et al. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 278(5) 3170-3175 (2003).

Download Kit Booklet 536 Kb PDF
Download Material Safety Data Sheet (MSDS) 126 Kb PDF

Pricing Information

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2 x 96 wells €258.00
Pricing updated 2010-09-07.
Prices are subject to change without notice.