Abstract
Myeloperoxidase (MPO) is a neutrophil enzyme that employs hydrogen peroxide (H2O2) to catalyze the oxidation of chloride (Cl–) to hypochlorous acid (HOCl). Accepted mechanism is based on rapid reaction of native MPO with H2O2to produce Compound I (MPO-I) which oxidizes Cl– through a 2e– transition generating MPO and HOCl. MPO-I also reacts with H2O2 to generate Compound II (MPO-II) which is inactive in 2e oxidation of Cl–. Nitrite ( NO2-) inhibits the 2e oxidation of Cl– by reaction with MPO-I through 1e transition generating MPO-II and nitrite radical. H2O2 consumption during steady- state catalysis was monitored amperometrically by a carbon fiber based H2O2-biosensor at 25oC. Results demonstrated that in absence of NO2- reactions were monophasic and rapid (complete H2O2 consumption occurs in 2- increases, reactions change to biphasic (rapid step followed by a slow step) and both steps have been inhibited by NO2- . A predictive kinetic model describing the inhibittory effect of NO2- was developed and applied to experimental results The model is based on the assumption that MPO–I cannot be detected during steady-state catalysis. Calculated rate constants are in agreement with those obtained from pre-steady state kinetic methods.
Highlights
Myeloperoxidase (MPO) is a human peroxidase enzyme and a lysosomal protein stored in azurophilic granules of the neutrophil
Pre-steady-state and steady-state studies based on stopped-flow mixing and optical detection were employed for studies of MPO-hydrogen peroxide-chloride system in presence and absence of nitrite [13,14]
A monophasic plot prevails in absence of NO2 (Figure 1(a)) which is demonstrated by a rapid consumption of H2O2
Summary
Myeloperoxidase (MPO) is a human peroxidase enzyme and a lysosomal protein stored in azurophilic granules of the neutrophil. The MPO catalyzed production of HOCl from hydrogen peroxide (H2O2) and chloride ion (Cl ), together with tyrosyl radical, are aimed at killing bacteria and other pathogens. Myeloperoxidase can oxidize nitrite ions to an intermediate capable of nitrating tyrosine and tyrosyl residues in proteins [8,9,10]. Pre-steady-state and steady-state studies based on stopped-flow mixing and optical detection were employed for studies of MPO-hydrogen peroxide-chloride system in presence and absence of nitrite [13,14]. In such studies, larger than physiological plasma concentrations of MPO and/or NO2 were employed to monitor measurable changes in absorbance at selected wavelengths. K3 k2 k 3 ratios were estimated and the dependence of k1 on [Cl–] and [ NO2 ] was determined
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