人血清转铁蛋白标准物质纯化

Purification of Human Serum Transferrin Reference Material

建立了一种从Cohn血浆分离副产物组分IV中纯化高纯度转铁蛋白（Tf）标准物质的技术,对Cohn组分IV进行复溶、离心和过滤,采用Capto DEAE阴离子交换色谱进行初分离,再通过穿透式Octyl-Sepharose 4 Fast Flow疏水色谱进一步精制;对纯化产品进行了结构、纯度、活性等表征,采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、基质辅助激光解吸电离飞行时间质谱、高效液相色谱及酶联免疫吸附法对Tf标准物质进行了分析.结果表明,Tf产品纯度大于99%,收率为79%;圆二色光谱分析显示Tf的二级结构未发生改变,铁离子结合能力实验表明Tf可结合2个铁离子,保持活性.

A two-step chromatographic method of purifying transferrin （Tf） from Cohn fraction IV, a byproduct of the Cohn plasma fractionation process, was established. After diluting, centrifugation and filtration, the Cohn fraction IV was firstly purified by Capto DEAE ion-exchange chromatography, then further purified by hydrophobic interaction chromatography. The characterization of purified Tf product was analyzed by SDS-PAGE, HPLC and ELISA. The results indicated that the purity of Tf was greater than 99% and the yield was 79%. The circular dichroism spectrum proved that the secondary structure of the purified Tf product have not changed, and iron binding ability experiment proved that the purified Tf product can bind two iron ions.