Myeloperoxidase (MPO) Activity Assay

Summary

Myeloperoxiase (MPO), the most abundant protein in neutrophils (also found in monocytes), is the focus of inflammatory pathologies. Most recent work has indicated that it is an excellent biomarker for human cardiovascular risk. Its ability to catalyze reaction between chloride and hydrogen peroxide (H₂O₂) to form hypochlorous acid is unique among mammalian enzymes and is considered to be the dominant function of MPO in vivo. Hypochlorous acid is a powerful antimicrobial agent, and extremely reactive with biological molecules causing much of the damage mediated by neutrophils in inflammatory diseases. MPO also exhibits peroxidase activity that catalyzes oxidation of a number of substrates by (H₂O₂). This activity has been widely used to assess the amount of MPO. Unfortunately, its specificity is very poor for unpurified biological samples due to the presence of other peroxidases. Peroxidases however, generally do not produce hypochlorous acid; the only exception is eosinophil peroxidase that produces hypochlorous acid at pH levels below 5. The chlorination activity of MPO has a pH optinum of near neutral pH. Therefore, assay conditions can be set so to provide for MPO enzyme specificity.

Intended Use

The NWLSS™ Myeloperoxidase Activity Assay is intended for use as a simple and easy colorimetric assay for the study of MPO activity in various biological and purified samples. The assay is not species specific and is compatible in all model systems where myeloperoxidase enzyme is thought to be present.

Test Principle

The NWLSS™ Myeloperoxidase Activity Assay method has been described by Weiss and coworkers (1982). Briefly, hypochlorous acid (HOCl) is formed from MPO catalyzed reaction between chloride and hydrogen peroxide. HOCl is rapidly trapped by �-amino acid taurine to form a stable oxidant taurine chloramine. Taurine prevents accumulation of hypochlorous acid that could deactivate MPO and does not react with MPO enzyme intermediate to interfere MPO catalysis. After incubation for specific time, the MPO catalyzed reaction is stopped by adding catalase to eliminate hydrogen peroxide. Taurine chloramine thus formed is then allowed to react with 5-thio-2-nitrobenzoic acid (TNB). TNB has a chromophore that has maximal absorbance at 412 nm while its reaction product with taurin chloramine, 5-5-dithiobis(2-nitrobenzoic acid) or DTNB is colorless. By following the decrease of absorbance at 412 nm, MPO activity is measured. One unit is the amount of MPO that can produce 1.0 nmole of taurine chloramine (hypochlorous acid) at pH 6.5 and 25C during 30 minutes in the presence of 100 mM chloride and 100 mM of hydrogen peroxide. The assay principal is summarized in the reaction scheme below.

MPO 03 Reaction

Stop the reaction (1a) after 30 minutes by adding Catalase to decompose H2O2

Since DTNB and taurine does not absorb at 412 nm, the absorbance of TNB at 412 nm is measured to calculate the MPO unit directly. In the presence of MPO, the decrease of absorbance at 412 nm is proportionally (linearly) enhanced.