亲和色谱-氢化物发生原子荧光分光光度法纯化检测人血浆中的硒蛋白-P

摘要

开发了两步亲和色谱法:肝素-琼脂糖凝胶、Ni-琼脂糖凝胶色谱纯化人血浆中硒蛋白-P的方法,并采用氢化物发生-原子荧光分光光度法( HG - AFS)检测,成功搭建了硒蛋白-p的纯化检测平台.确定了亲和色谱纯化的最佳梯度洗脱条件,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳( SDS - PAGE)定性检测,得到了一定纯度的硒蛋白-P,其回收率达43.2％.HG - AFS方法的线性相关系数为0.9991,检出限为0.09 μg/L,日内精密度(RSD)为0.12％,日间精密度(RSD)为0.27％,加标回收率为95％～104％.该亲和色谱纯化方法简单易控、回收率高,HG - AFS检测灵敏度高,结果准确可靠.%A method was developed for the purification and detection of selenoprotein - P in human plasma. The sample was gradient eluted by two successive steps of affinity chromatography, i. e. , Heparin - Sepharose and Ni - Sepharose chromatography. The quality of the purified selenoproteinP was detected by HG - AFS. The optimum gradient elution conditions were determined and the selenoprotein - P with a certain purity was obtained by SDS - PAGE. The recovery of the two steps of af-finity chromatography reached up to 43. 2% . The correlation coefficient of HG - AFS was 0. 999 1, and the detection limit was 0. 09 μg/L. The RSDs were 0. 12% for within-day and 0. 27% for inter-day. The recoveries were in the range of 95% - 104% . The proposed method was simple and sen-sitive, and the results obtained were accurate.