新型氧化应激标志物AOPPs的体外制备标准化和检测  

Standardization of the preparation and determination of novel biomarker-AOPPs

作　　者：卢燕雯[1] 朱秋毓[1] 丁峰[1] 顾勇[1] 林善锬[1] 

机构地区：[1]复旦大学附属华山医院肾病科-复旦大学肾脏病研究所,上海200040

出　　处：《复旦学报（医学版）》2008年第1期21-25,共5页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金项目(300300160)

摘　　要：目的研究次氯酸氧化的人血清白蛋白(human serum albumin-advanced oxidative protein products,HSA-AOPPs)的制备标准化、次氯酸的清除以及微量HSA-AOPPs的检测。方法于搅拌条件下在HSA(60mg/mL)内滴加次氯酸至终浓度为60mmol/L即制得HSA-AOPPs。透析法或层析法清除次氯酸。碘量法测定次氯酸残留量。微量HSA-AOPPs的测定采用高效凝胶色谱法(HP-SEC),曲线下面积(AUC)用于定量计算。结果恒定的次氯酸加入量对HSA-AOPPs的产量非常关键。当次氯酸和HSA两者克分子浓度之比为1∶66左右时最为理想。过量次氯酸可导致高分子量AOPPs的破坏,大量蛋白碎片产生。层析法清除次氯酸残留优于透析法。色谱法检测AOPPs的灵敏度比分光光度法要高100倍以上。结论AOPPs的标准化制备关键在于恒定次氯酸对HSA的克分子比值。HP-SEC方法可用于微量AOPPs的测定。Objective To standardize the preparation of hypochlorous-treated human serum albumin （HSA-AOPPs）, cleanup of hypochlorous acid （HOC1） residue in HSA-AOPPs preparation as well as the determination of trace AOPPs. Methods Purified HSA （60 mg/mL） was oxidized with 60mmol/ L HOC1 （final concentration） under a stirring condition. The iodometry was used for the determination of HOC1 residue in HSA-AOPPs preparation after purification steps by column chromatography or by dialysis. High performance size-exclusion chromatography （HP-SEC） was also used for quantitative analysis of trace AOPPs based on the area under curve （AUC） summarized from all the eluting protein area. Results Accuracy addition of HOC1 was pivotal to a consistent concentration of HSA-AOPPs. It was best for the high yield of high molecular weight AOPPs when the molar ratio HSA/HOC1 of 1：66.5 was achieved and also for the elimination of the residues of HOC1 in HSA-AOPPs by column chromatography in comparison with by dialysis. Overload of oxidant resulted in massive breakage of HMW-AOPPs with a cluster of protein fragments formation. There was a more than 100-fold increment in detecting the sensitivity by HPLC, compared to spectrophotometry. Conclusions Exact and consistent HSA/HOC1 ratio is the key to standardize the HSA-AOPPs preparation. HP-SEC is a valid mean for the detection of trace AOPPs.

关 键 词：人血清白蛋白 次氯酸残留量 高级氧化蛋白产物 氧化修饰 高效凝胶色谱 

分 类 号：O657.7[理学—分析化学]