Validation of the chloramine-T induced oxidation of human serum albumin as a model for oxidative damage in vivo
Validation of the chloramine-T induced oxidation of human serum albumin as a model for oxidative damage in vivo
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DOI：
10.1023/a:1023219420935
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发表时间：
2003-04-01
影响因子：
3.7
通讯作者：
Otagiri, M
中科院分区：
文献类型：
--
作者：
Anraku, M;Kragh-Hansen, U;Otagiri, M
目的。使用人血清白蛋白（HSA）作为模型检查了使用氯胺-T作为模拟氧化应激的模型化合物的有效性。通过用氯胺-T温和处理以及将(34)Cys突变为丝氨酸(C34S)来研究重要的氧化位点。方法。使用高效液相色谱（HPLC）结合荧光检测来确认氯胺-T作为氧化剂的有效性。通过与5,5'-二硫双(2-硝基苯甲酸)反应、用溴化氰消化、然后进行毛细管电泳来检测氧化氨基酸残基。通过光谱技术检查蛋白质构象。结果。通过对人血清的HPLC分析，证实了使用氯胺-T作为氧化剂的有效性。在低氯胺-T浓度(CT0.1-HSA、CT1-HSA)下，(34)Cys和Met残基被氧化，在中等浓度(CT10-HSA)下，色氨酸残基也似乎被氧化，在最高浓度(CT50-HSA)下，发现HSA位点II的净电荷更负。 HSA 的两个最高氧化水平（CT10-HSA、CT50-HSA）导致构象变化，疏水区域的暴露增加、华法林和酮洛芬的高亲和力结合减少以及酯酶样活性降低。后一种蛋白质还具有较短的血浆半衰期和增加的肝脏清除率。结论。我们成功地使用氯胺-T 模拟了 HSA 的氧化损伤，结果表明，当 HSA 暴露于氧化应激时，位点 II 比位点 I 和 (34)Cys 受到的影响更大。
Purpose. The validity of using chloramine- T as a model compound for mimicing oxidative stress was examined using human serum albumin ( HSA) as a model. Important sites of oxidation were studied by mild treatment with chloramine- T and by mutating (34)Cys for a serine ( C34S).Methods. High- performance liquid chromatography ( HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues were detected by reaction with 5,5 '- dithiobis( 2- nitro benzoic acid), digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques.Results. From the HPLC analysis of human serum, the validity of using chloramine- T as an oxidizing agent was confirmed. At low chloramine-T concentrations ( CT0.1- HSA, CT1- HSA), (34)Cys and Met residues were oxidized, at medium concentrations ( CT10- HSA), the tryptophan residue also appeared to be oxidized, and at the highest concentration ( CT50- HSA), the net charge of Site II of HSA was found to be more negative. The two highest levels of oxidation of HSA ( CT10- HSA, CT50- HSA) resulted in conformational changes with an increased exposure of hydrophobic regions, decreased high-affinity bindings of warfarin and ketoprofen and a reduced esterase-like activity. The latter protein also has a shorter plasma half- life and an increased liver clearance.Conclusions. We succeeded in imitating oxidative damage to HSA using chloramine- T and the findings show that Site II is more affected than Site I and (34)Cys, when HSA is exposed to oxidative stress.