title: The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon.
abstract: Chlorination of monochlorodimedon is routinely used to measure the production of hypochlorous acid catalysed by myeloperoxidase from H2O2 and Cl-. We have found that the myeloperoxidase/H2O2/Cl- system, at pH 7.8, catalysed the loss of monochlorodimedon with a rapid burst phase followed by a much slower steady-state phase. The loss of monochlorodimedon in the absence of Cl- was only 10% of the steady-state rate in the presence of Cl-, which indicates that the major reaction of monochlorodimedon was with hypochlorous acid. During the steady-state reaction, myeloperoxidase was present as 100% compound II, which cannot participate directly in hypochlorous acid formation. Monochlorodimedon was necessary for formation of compound II, since it was not formed in the presence of methionine. Both the amount of hypochlorous acid formed during the burst phase, and the steady-state rate of hypochlorous acid production, increased with increasing concentrations of myeloperoxidase and with decreasing concentrations of monochlorodimedon. Inhibition by monochlorodimedon was competitive with Cl-. From these results, and the ability of myeloperoxidase to slowly peroxidase monochlorodimedon in the absence of Cl-, we propose that the reaction of monochlorodimedon with the myeloperoxidase/H2O2/Cl- system involves a major pathway due to hypochlorous acid-dependent chlorination and a minor peroxidative pathway. Only a small fraction of compound I needs to react with monochlorodimedon instead of Cl- at each enzyme cycle, for compound II to rapidly accumulate. Monochlorodimedon, therefore, cannot be regarded as an inert detector of hypochlorous acid production by myeloperoxidase, but acts to limit the chlorinating activity of the enzyme. In the presence of reducing species that act like monochlorodimedon, the activity of myeloperoxidase would depend on the rate of turnover of compound II. Components of human serum promoted the conversion of ferric-myeloperoxidase to compound II in the presence of H2O2. We suggest, therefore, that in vivo the rate of turnover of compound II may determine the rate of myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.
authors.affiliation: Department of Pathology, Christchurch School of Medicine, Christchurch Hospital, New Zealand.
authors.forename: A J
authors.initials: AJ
authors.lastname: Kettle
authors.forename: C C
authors.initials: CC
authors.lastname: Winterbourn
mesh_terms: D002712:Chlorides; D002713:Chlorine; D003510:Cyclohexanes; D003512:Cyclohexanones; D006801:Humans; D006861:Hydrogen Peroxide; D006997:Hypochlorous Acid; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology; D009195:Peroxidase / Q000378:metabolism*
chemical_list: D002712:Chlorides; D003510:Cyclohexanes; D003512:Cyclohexanones; D002713:Chlorine; D006997:Hypochlorous Acid; C006991:chlorodimedone; D006861:Hydrogen Peroxide; D009195:Peroxidase