An oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule called “the reductant” or “electron donor” to another molecule called “the oxidant” or the “electron acceptor”. This group of enzymes typically uses NADP or NAD+ as cofactors.

Peroxidases (EC number 1.11.1.x): when combined with H2O2 and a halide (chloride, bromide, iodide) form a potent cytotoxic system which contributes to the host defense against invading microorganisms and possibly tumor cells. Neutrophils and monocytes contain the same peroxiase (myeloperoxidase, MPO) and eosinophils a different peroxidase (eosinophil peroxidase, EPO), in cytoplasmic granules and these enzymes are dischared into the pahgosome following particle ingestion.

1. Myeloperoxidase (MPO, donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7): is a tetrameric, heavily glycosylated basic (PI 10) heme protein of about 150kDa. It is compirsed of two identical disulfide linked protomers, each of which possesses a protoporphyrin containing 59-64 kDa heavy subunit and a 14 kDa light subunit. MPO is a complex heme protein which possesses multiple intermediate states, each of which are influenced by the availability of reduced oxygen species such as O2- and H2O2 and nitric oxide (NO, nitrogen monoxide). At ground state, MPO exists in the ferri (Fe(III) form. Upon addition of H2O2, the heme group of MPO is oxidized two e- equivalents forming a reactive ferryl pi cation radical intermediate termed Compound I.

MPO amplifies the oxidizing potential of H2O2 by using it as a cosubstrate to generate a variety of reactive oxidants and diffusible radical species. Formation of phenoxyl radicals by MPO, such as during oxidation of the free amino acid L-tyrosine, can promote protein cross-linking and oxidation of LDL lips. MPO may also use H2O2 and nitrite (NO2-), a major end product of NO metabolism, to generate a microbicidal oxidant capable of nitrating phenolic compounds and proteins in vitro. NO2- is ubiquitous in biological tissues and flues. During inflammatory and infectious processes where NO production is enhance, palsma and extracellular lelvels of NO2- are markedly increased. Schmitt (Biochemistry, 1999, 38, 16904-16915).

–Where MPO is found: MPO is abundant in azurophil granules in neutrophils and is promptly dischared after activation by different agonists. The heme protein is sorted in primary azurophilic granules of leukocytes and secreted into both the extracellular milieu and the phogolysosomal compartment following phagocyte activation by a variety of agonists.

Intracellular MPO is found in granulocytes in amounts which are generally constant from cell-to-cell, and the rate adn intesity of color change can accordingly be employed to accurately reflect the level of granulocytes in a blood sample being assayed. However, becasue MPO appears as an intercellular component of a granulocyte, those granulocytes present in the sample being assayed must be lysed or disrupted to release the enzyme before the sample is treated with the peroxide and the dye to make a meaningful determination of granulocyte level (US6,046,019).

–MPO activity may be determined by any of a variety of standard methods such as a colorimetric based assay where a chromophore that serves as a substrate for the peroxidase generates a product with a characteristic wavelenght which may be followed by any various spectroscopic methods including UV visible or fluorescence detection. 

Based on MPO catalyzed oxidation of different dyes, several spectrophotometric methods to assay MPO have been reported. These methods provide satisfactory measurements of MPO activity and can be used for quantitation of the number of PMNs in pure preparations. However, problems can be encountered when applying these methods to determine MPO activity in PMN containing tissues (Xia, Analytical Biochemistry 245, 93-96 (1997)). 

Xia, Analytical Biochemistry 245, 93-96 (1997) report a method for quantitation of MPO activity in PMN containing tissues which uses a one step purification on a Sephadex G075 gell filtration chromatography to separate myoglobin from tissue MPObased on the large difference between the MW of MPO, 118 kDa and myoglobin, 16 kDa,  followed by a spectrophotometric assay of MPO. 

–MPO Mass: is readily deteremined by an immunological method (e.g., ELISA). MPO mass in a sample can also be determined indirectly by in situ peroxidase staining of the bodily sample.