活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用

Preparation of affinity chromatographic packings by immobi-lization of Cibacron Blue F3GA based on monodispersed non-porous hydrophilic poly (glycidylmethacry-co-ethylenedimethacrylate) particles and their application

以3.0μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观解离常数为5.26×10-5mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。

An affinity chromatographic packing was prepared by the immobilization of Cibacron Blue F3GA as dye ligands on the base of monodispersed, 3.0 μm non-porous hydrophilic poly （glycidylmethacry-co-ethylenedimethacrylate） particles. The effects of the ionic strength, organic solvent in the mobile phase and flow rate on protein retention were investigated on the chromatographic stationary phase. The apparent dissociation constant between the immobilized Cibacron Blue F3GA and lysozyme （Lys） was 5.26×10^-5 mol/L which was evaluated by frontal chromatography. Purifications of Lys from egg white and bovine serum albumin （BSA） from calf blood were performed on the dye-ligand chromatographic column, separately, and the purities of the obtained fractions were determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The purities of the purified Lys and BSA were determined to be 95% and 92%, respectively.