晚期氧化蛋白产物的分离纯化和鉴定

Isolation and identification of advanced oxidation protein products

目的分离纯化以人血清白蛋白(humanserum albumin,HSA)为原料生成的晚期氧化蛋白产物(advanced ox idation protein products,AOPP),即AOPP HSA,并对其进行鉴定,以寻求一种制备高纯度且具生物活性的AOPP HSA的方法。方法采用HSA HOCl孵育法体外制备AOPP HSA粗纯品,经凝胶层析和离子交换高压液相层析(highperformance liquidchromatography,HPLC)两步层析分离纯化其中的AOPP HSA,并经紫外和荧光光谱、SDS PAGE、单核细胞TNFα分泌实验鉴定其结构特征和生物学活性。结果分离的蛋白质纯度达99.4%,分子质量为700×103,是含有双酪氨酸的蛋白交联聚合物,能够显著刺激单核细胞TNFα的分泌。结论采用上述两步层析方法能够由粗纯品中成功分离出高纯度并具生物活性的AOPP HSA,为AOPP的进一步研究奠定了基础。

Objective To isolate and identify advanced oxidation protein products from human serum albumin （AOPP-HSA）, expecting to search for a method of preparing highly purified and bioactive AOPP-HSA. Methods AOPP-HSA crude products were prepared in vitro by exposing HSA to HOC1. AOPP-HSA was isolated by gel chromatography and ion-exchange HPLC. Its structural features and biological activities were characterized by UV and fluorescence spectrum, SDS-PAGE, and the experiment of TNF-α release from monocytes. Results The isolated protein was purified up to 99.4% and was dityrosine-containing protein cross-linking products with molecular weight of 700 × 10^3. It possessed the ability of triggering the considerable release of TNF-α from monocytes. Conclusion Highly purified and bioactive AOPP-HSA can be successfully prepared by above-mentioned two-step chromatography from AOPP-HSA crude products, which builds a basis for further study of AOPP.