Myeloperoxidase (MPO) is a highly cationic glycosolated hemoprotein that has a molecular weight of 144kD. The hemoprotein consists of two dimers linked via a disulfide bridge. Each dimmer is composed of a heavy (53kD) and light (15kD) subunit. Each heavy chain contains an independently acting protoporphyrin group containing a central iron (1-5). MPO is present in the azurophilic granules of polymorphonuclear leukocytes (PMNs) and is unique to neutrophils and monocytes. However, monocytes contain only one third of the MPO found in PMN’s. MPO utilizes H202 produced by the neutrophils to oxidize a varity of aromatic compounds to give substrate radicals for bactericidal activity (4 review). This enzyme is unique however in that it can oxidize chloride ions to produce a strong nonradical oxidant,HOCl. HOCl is the most powerful bactericidal produced by neutrophils (4 review). Excessive production of these radicals can cause oxidative stress leading to oxidative tissue injury.

Chlorination Reaction:

H2O2 + MPO + Cl- —-> HOCl + APF (non fluorescent) —-> Fluorescent Dye

Excitation:488nm; Emission: 515-530nm

Peroxidation Reaction:

H2O2 + Detection reagent (non-fluorescent)+ MPO —–> fluorescent analog

Excitation 530-571nm Emission 590-600nm

Figure 1. In this figure, the MPO standard curve was serially diluted in 1X Reaction Buffer. Reaction cocktail (RC) was prepared as described (without EPO inhibitor). Next 50µL of MPO standard and 50µL of RC was added to individual well of 96- well black plates. The plate was incubated at room and temperature in the dark. Data collected Ex:530nm, Em:590nm

Figure 2. Red Blood Cell AChE (RBC-AChE) was purified and protein concentration determined using the BCA Protein Assay Kit (Pierce). The RBC-AChE was titrated in 1X reaction buffer and activity determined using the Fluoro: AChE kit. Acetylcholine concentration = 1mM final. In the graph the background value has been subtracted (0 RBC-AChE) to generate standard curve.