次氯酸对人血清白蛋白的氧化修饰影响及与高级氧化蛋白产物的关系

The oxidative modification of albumin by hydrochlorous acid and the relationship between the oxidized HSA and AOPPs

目的研究次氯酸对人血清白蛋白(HSA)的氧化修饰影响及与高级氧化蛋白产物(AOPPs)之间的关系。方法有氧条件下在恒定浓度的HSA(60 mg／ml)内加入不同浓度次氯酸(0、1、5、10、20、30、40、50、60 mmol／L,最终浓度),观察氧化剂对HSA的修饰作用。凝胶排阻色谱法检验HSA氧化修饰结果,在线光谱扫描(190 nm～400 nm)分析修饰产物的光谱特性。结果次氯酸可氧化修饰人血清白蛋白,其修饰产物主要为二聚体HSA和六聚体HSA。发现次氯酸对HSA单体和HSA二聚体的氧化修饰为一级反应:对诱导生成的AOPPs为准一级反应:而对诱导生成的HSA六聚体则为二级修饰反应。同时发现白蛋白对AOPPs的主要贡献者是六聚体形式的HSA,光谱分析表明HSA聚集体的最大吸收峰发生红移,提示HSA聚集体是由于蛋白中的酪氨酸残基通过氧化交联方式而聚集形成的。结论HSA经次氯酸处理后主要发生了蛋白聚集。而对AOPPs的主要贡献者是六聚体形式的HSA。

Objective To study the effect of oxidative modification of hydrochlorous acid （HOCI） on human serum albumin （HSA） and the relationship between the AOPPs and HOCl-treated liSA. Methods Purified HSA （60 mg/ml） was treated with HOCI （0, 1, 5, 10, 20, 30, 40, 50 and 60 mmol/L）. Size-exclusion chromatography was applied to estimate molecular weights of oxidized products of HSA by HOCl and spectrum scan from 190 nm-400 nm was performed to observe the spectrum characteristics of all variants of HSA. Results Major products of HSA after exposure to HOCl were dimer and hexmer of HSA, The first-order process could be employed to describe the oxidative dynamics of monomer and dimer of HSA oxidized by HOCl. To AOPPs formation mediated by oxidant was identified as pseudo first-order reaction. However, formation hexmer was much in accordance with second-order reaction, Hexrner was also a major contributor to AOPPs in all types of modified HSA. Spectral analysis showed that red shift of absorbance maximum of polymers of HSA occurred, suggesting that a possibility that polymers of HSA were cross linked by tyrosine residues in protein. Conclusions Protein aggregation is primary consequence of lISA after its exposure to HOCl. Hexmer of HSA is the major contributor to AOPPs.