Thomas E L, Jefferson M M, Grisham M B
Biochemistry. 1982 Nov 23;21(24):6299-308. doi: 10.1021/bi00267a040.
Myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCl) resulted in formation of mono- and dichloramine derivatives (RNHCl and RNCl2) of primary amines. The RNCl2 derivatives could undergo a reaction that resulted in incorporation of the R moiety into proteins. The probable mechanism was attack of RNCl2 or an intermediate formed in the decomposition of RNCl2 on histidine, tyrosine, and cystine residues and on lysine residues at high pH. Incorporation of radioactivity from labeled amines into stable, high molecular weight derivatives of proteins was measured by acid or acetone precipitation and by gel chromatography and electrophoresis. Whereas formation of RNCl2 was favored at low pH, the subsequent incorporation reaction was favored at high pH. Up to several hours were required for the maximum amount of incorporation, which was less than 10% of the label in RNCl2. For the amines tested, incorporation was in the order histamine greater than 1,2-diaminoethane greater than putrescine greater than taurine greater than lysine greater than glucosamine greater than leucine greater than methylamine. Initiation of the reaction required HOCl, and oxidized forms of bromide, iodide, or thiocyanate did not substitute. Inhibitors of incorporation fell into three classes. First, ammonia or amines competed with the labeled amine for reaction with HOCl, so that larger amounts of HOCl were required. Second, readily oxidized substances such as sulfhydryl or diketo compounds or thioethers (methionine) reduced RNCl2. Third, certain compounds competed with protein as the acceptor for the incorporation reaction. The amount required to block incorporation into protein depended on protein concentration. Among these inhibitors were imidazole compounds (histidine), phenols (tyrosine), and disulfides (glutathione disulfide, GSSG). Low yields of derivatives of histidine, tyrosine, and GSSG were detected by thin-layer chromatography. Acid-precipitable derivatives were obtained by reacting RNCl2 with polyhistidine or polytyrosine, and to a lesser extent with polylysine at high pH, but not with other poly(amino acids). Precipitable derivatives were also obtained by incubating MPO-containing extracts from leukocyte granules with hydrogen peroxide, Cl-, and labeled amines. The extracts were found to have a high content of substances with primary amino groups, which competed for incorporation. The results account for oxidative incorporation of amines into proteins in leukocytes and provide evidence that HOCl and nitrogen-chlorine (N-Cl) derivatives are formed in these cells. The characteristics of the incorporation reaction suggest that it would not contribute significantly to the antimicrobial activity of myeloperoxidase (MPO). Nevertheless, the reaction may provide a sensitive method for studying MPO action in vivo.
髓过氧化物酶催化氯化物（Cl-）氧化为次氯酸（HOCl），导致伯胺形成单氯胺和二氯胺衍生物（RNHCl和RNCl2）。RNCl2衍生物可发生反应，使R部分掺入蛋白质中。可能的机制是RNCl2或其分解过程中形成的中间体攻击组氨酸、酪氨酸、胱氨酸残基以及高pH值下的赖氨酸残基。通过酸或丙酮沉淀以及凝胶色谱和电泳来测量标记胺的放射性掺入稳定的高分子量蛋白质衍生物中的情况。虽然在低pH值下有利于RNCl2的形成，但随后的掺入反应在高pH值下更有利。最大掺入量需要长达数小时才能达到，且小于RNCl2中标记物的10%。对于所测试的胺，掺入顺序为组胺＞1,2 - 二氨基乙烷＞腐胺＞牛磺酸＞赖氨酸＞葡糖胺＞亮氨酸＞甲胺。反应的引发需要HOCl，溴化物、碘化物或硫氰酸盐的氧化形式不能替代。掺入抑制剂分为三类。第一，氨或胺与标记胺竞争与HOCl反应，因此需要更多的HOCl。第二，易氧化的物质，如巯基、二酮化合物或硫醚（甲硫氨酸）可还原RNCl2。第三，某些化合物与蛋白质竞争作为掺入反应的受体。阻断掺入蛋白质所需的量取决于蛋白质浓度。这些抑制剂包括咪唑化合物（组氨酸）、酚类（酪氨酸）和二硫化物（谷胱甘肽二硫化物，GSSG）。通过薄层色谱检测到组氨酸、酪氨酸和GSSG衍生物的产率较低。通过使RNCl2与聚组氨酸或聚酪氨酸反应，在高pH值下与聚赖氨酸反应程度较小，但与其他聚氨基酸不反应，可得到酸沉淀衍生物。通过将含髓过氧化物酶的白细胞颗粒提取物与过氧化氢、Cl-和标记胺一起孵育，也可得到可沉淀衍生物。发现提取物中含有大量具有伯氨基的物质，它们竞争掺入。这些结果解释了胺在白细胞中氧化掺入蛋白质的现象，并提供了证据表明这些细胞中形成了HOCl和氮 - 氯（N - Cl）衍生物。掺入反应的特点表明它对髓过氧化物酶（MPO）的抗菌活性贡献不大。然而，该反应可能为研究MPO在体内的作用提供一种灵敏的方法。
