利凡诺去白蛋白人血浆再利用的实验研究Ⅱ.抗凝血酶Ⅲ和蛋白C抑制物的纯化与鉴定

Laboratory study on reproducibility of no albumin human plasma by rivanol precipitation Ⅱ.Purification and identification of antithrombin Ⅲ and protein C inhibitor

目的综合再利用利凡诺去白蛋白人血浆进行抗凝血成分抗凝血酶Ⅲ (AT Ⅲ )和蛋白C抑制物 (PCI)的分离纯化。方法以钡盐吸附上清为原料 ,采用铝盐吸附、有机沉淀、离子交换层析、分子筛层析、亲和层析和制备性等电聚焦等技术从利凡诺去白蛋白人血浆中分别纯化AT Ⅲ和PCI。结果从利凡诺去白蛋白人血浆 10L中得到了AT Ⅲ 16 0mg和PCI4.2mg ,得率分别为 12 .7%和 10 .5 % ,其相对分子量分别为 (5 7± 0 .8)和 (5 6± 0 .8)kD ;等电点分别为 4.81± 0 .0 2和 8.0 1± 0 .0 2 ;分别富含Glu、Asp、Leu和Glu、Leu、Asp ;毛细管区带电泳分析高度均一 ;与文献报道基本一致。结论该纯化路线经济、简便 。

PurposeThe aim is to isolate and purify Antithrombin Ⅲ(AT Ⅲ) and Protein C inhibitor(PCI) from no albumin human plasma with rivanol precipitation.MethodsThe isolated and purified steps included barium adsorption, adsorption onto and elution from aluminuim, PEG precipitation, ion exchange chromatography, gel exclude chromatography, affinity chromatography, preparative isoelectric focusing and so on. Results160 mg AT Ⅲ and 4.2 mg PCI were obtained from 10 L supernatant of no albumin human plasma with rivanol precipitation. Their molecular weights are (57±0.8) and (56±0.8)kD respectively. The isoelectric point, 4.81±0.02, and 8.01±0.02; yield, 12.7% and 10.5%. The purified AT Ⅲ and PCI was shown to be highly homogeneous by capillary zone electrophoresis, and rich in Glu, Asp and Leu or Glu, Leu and Asp respectively.ConclusionThese methods were simple, cheap and efficient in improving techniques. They could be used for large scale isolation and purification from no albumin human plasma with rivanol precipitation.