Department of Chemical and Environmental Engineering, Yale University, New Haven, CT 06520-8286, USA.
Biochemistry. 2013 Feb 19;52(7):1260-71. doi: 10.1021/bi301523s. Epub 2013 Feb 7.
Although protein degradation by neutrophil-derived hypochlorous acid (HOCl) and eosinophil-derived hypobromous acid (HOBr) can contribute to the inactivation of pathogens, collateral damage to host proteins can also occur and has been associated with inflammatory diseases ranging from arthritis to atherosclerosis. Though previous research suggested halotyrosines as biomarkers of protein damage and lysine as a mediator of the transfer of a halogen to tyrosine, these reactions within whole proteins are poorly understood. Herein, reactions of HOCl and HOBr with three well-characterized proteins [adenylate kinase (ADK), ribose binding protein, and bovine serum albumin] were characterized. Three assessments of oxidative modifications were evaluated for each of the proteins: (1) covalent modification of electron-rich amino acids (assessed via liquid chromatography and tandem mass spectrometry), (2) attenuation of secondary structure (via circular dichroism), and (3) fragmentation of protein backbones (via sodium dodecyl sulfate-polyacrylamide gel electrophoresis). In addition to forming halotyrosines, HOCl and HOBr converted lysine into lysine nitrile (2-amino-5-cyanopentanoic acid), a relatively stable and largely overlooked product, in yields of up to 80%. At uniform oxidant levels, fragmentation and loss of secondary structure correlated with protein size. To further examine the role of lysine, a lysine-free ADK variant was rationally designed. The absence of lysine increased yields of chlorinated tyrosines and decreased yields of brominated tyrosines following treatments with HOCl and HOBr, respectively, without influencing the susceptibility of ADK to HOX-mediated losses of secondary structure. These findings suggest that lysine serves predominantly as a sacrificial antioxidant (via formation of lysine nitrile) toward HOCl and as a halogen-transfer mediator [via reactions involving ε-N-(di)haloamines] with HOBr.
尽管中性粒细胞衍生的次氯酸 (HOCl) 和嗜酸性粒细胞衍生的次溴酸 (HOBr) 引起的蛋白质降解有助于使病原体失活，但宿主蛋白质的附带损害也可能发生，并与从关节炎到动脉粥样硬化等各种炎症性疾病有关。虽然之前的研究表明，卤代酪氨酸可以作为蛋白质损伤的生物标志物，赖氨酸可以作为将卤素转移到酪氨酸的介质，但这些在整个蛋白质内的反应知之甚少。在此，通过三种方法对 HOCl 和 HOBr 与三种特征明确的蛋白质 [腺嘌呤激酶 (ADK)、核糖结合蛋白和牛血清白蛋白] 的反应进行了表征。对每种蛋白质都评估了三种氧化修饰的评估：(1) 富电子氨基酸的共价修饰（通过液相色谱和串联质谱法评估），(2) 二级结构的衰减（通过圆二色性评估），和 (3) 蛋白质骨架的断裂（通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳评估）。HOCl 和 HOBr 除了形成卤代酪氨酸外，还将赖氨酸转化为赖氨酸腈（2-氨基-5-氰基戊酸），这是一种相对稳定且很大程度上被忽视的产物，产量高达 80%。在均匀氧化剂水平下，蛋白质片段化和二级结构的损失与蛋白质大小相关。为了进一步研究赖氨酸的作用，设计了一种不含赖氨酸的 ADK 变体。赖氨酸的缺失增加了 HOCl 处理后氯化酪氨酸的产率，降低了 HOBr 处理后溴化酪氨酸的产率，而不影响 ADK 对 HOX 介导的二级结构丧失的敏感性。这些发现表明，赖氨酸主要作为 HOCl 的牺牲抗氧化剂（通过形成赖氨酸腈），并作为 HOBr 的卤素转移介质（通过涉及 ε-N-(二)卤代胺的反应）。
