Kettle A J, Winterbourn C C
Department of Pathology, Christchurch School of Medicine, Christchurch Hospital, New Zealand.
Biochim Biophys Acta. 1988 Nov 23;957(2):185-91. doi: 10.1016/0167-4838(88)90271-3.
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.
一氯二甲基酮的氯化反应通常用于测定髓过氧化物酶催化过氧化氢和氯离子生成次氯酸的产量。我们发现，在pH 7.8时，髓过氧化物酶/H2O2/Cl-系统催化一氯二甲基酮的损失，先是快速爆发阶段，随后是慢得多的稳态阶段。在没有氯离子的情况下，一氯二甲基酮的损失仅为有氯离子时稳态速率的10%，这表明一氯二甲基酮的主要反应是与次氯酸发生反应。在稳态反应过程中，髓过氧化物酶以100%的化合物II形式存在，它不能直接参与次氯酸的形成。一氯二甲基酮是化合物II形成所必需的，因为在甲硫氨酸存在的情况下它不会形成。爆发阶段形成的次氯酸量以及次氯酸产生的稳态速率都随着髓过氧化物酶浓度的增加和一氯二甲基酮浓度的降低而增加。一氯二甲基酮的抑制作用与氯离子具有竞争性。根据这些结果以及髓过氧化物酶在没有氯离子时缓慢氧化一氯二甲基酮的能力，我们提出一氯二甲基酮与髓过氧化物酶/H2O2/Cl-系统的反应涉及一条主要途径，即次氯酸依赖性氯化反应途径和一条次要的过氧化途径。在每个酶循环中，只需一小部分化合物I与一氯二甲基酮而不是氯离子反应，化合物II就能迅速积累。因此，一氯二甲基酮不能被视为髓过氧化物酶产生次氯酸的惰性检测剂，而是起到限制该酶氯化活性的作用。在存在类似一氯二甲基酮的还原物质时，髓过氧化物酶的活性将取决于化合物II的周转速率。人血清成分在有过氧化氢存在的情况下促进了铁离子形式的髓过氧化物酶向化合物II的转化。因此，我们认为在体内，化合物II的周转速率可能决定受刺激的中性粒细胞中髓过氧化物酶依赖性次氯酸的产生速率。
