Characterization of non-covalent oligomers of proteins treated with hypochlorous acid
Characterization of non-covalent oligomers of proteins treated with hypochlorous acid
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DOI：
10.1042/bj20030685
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发表时间：
2003-10-01
影响因子：
4.1
通讯作者：
Kettle, AJ
中科院分区：
文献类型：
--
作者：
Chapman, ALP;Winterbourn, CC;Kettle, AJ
次氯酸(HOCl)是髓过氧化物酶产生的一种强有力的氧化剂，可引起许多蛋白质的聚集。当蛋白质在还原条件下用SDS/PAGE分离时，用HOC处理脱脂血红蛋白和脱脂肌红蛋白可产生一系列规则的寡聚物带。HOCI/蛋白质摩尔比为0.5：1时可检测到聚集，在10：1~20：1时聚集最大。加入HOCI后1min内形成二聚体，在接下来的30min内进一步聚集。氨基酸分析、多肽分析或电喷雾电离-质谱法均未得到HOC修饰脱脂蛋白共价交联的令人信服的证据。后者的质量增加与两个蛋氨酸残基转化为亚硫化物一致。过量5倍的Hoci会在无肌红蛋白上产生大约3个氯胺。它们经历了缓慢的腐烂。形成了蛋白质碳基，并且几乎全部位于聚合物带上。通过琥珀酸化将蛋白质上的正电基团转化为带负电的基团，导致预先形成的聚集体解离。牛磺酸氯胺处理去肌红蛋白产生蛋氨酸亚硫氧化物，但很少蛋白质羰基，并且不会导致聚集。我们得出结论，这种聚集是由于蛋白质链之间强烈的非共价相互作用造成的。我们认为，蛋白质碳基和可能的氯胺的形成，以及蛋氨酸的氧化，改变了蛋白质的折叠，使邻近分子上的疏水区域暴露出来，形成二聚体和更高分子质量的聚集体。这一过程可能导致髓过氧化物酶活性部位聚集蛋白的形成，并有助于炎症组织损伤。
Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOC] produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10: 1-20: 1. Dimers formed within 1 min of adding HOCI, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCI-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCI generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carboryls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carboryls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.