牛血清白蛋白键合的液相手性整体柱制备及其蛋白柱载量的研究  

Preparation of bovine serum albumin immobilized chiral monolithic column and its protein bonding quantity

作　　者：武苏[1] 王伟[1] 季一兵[1] 

机构地区：[1]中国药科大学分析化学教研室,南京210009

出　　处：《中国药科大学学报》2016年第2期176-181,共6页Journal of China Pharmaceutical University

摘　　要：蛋白质柱载量的测定对优化蛋白质类手性整体柱的制备条件具有重要意义。采用热引发原位聚合方法制备了聚甲基丙烯酸缩水甘油酯(GMA)-乙二醇二甲基丙烯酸酯(EDMA)液相整体柱,而后采用环氧法直接键合手性选择剂牛血清白蛋白(BSA),制得高效液相手性整体柱,并且建立考马斯亮蓝(CBB)法测定手性整体柱上的蛋白柱载量。结果显示,BSA最大的柱载量为11.90 mg/g,此时致孔剂组成为环己醇-十二烷醇(65∶35)。将制备的高效液相手性整体柱用于色氨酸和华法林的拆分,色氨酸的两个对映异构体达到基线分离。高效液相手性整体柱的蛋白柱载量与手性拆分的效果呈现正相关。Determination of exact total protein bonding quantity is often a key step in the preparation of proteinimmobilized chiral monolith. In this study,we developed and evaluated a bovine serum albumin（ BSA） modified monolith based on glycidyl methacrylate（ GMA） and ethylene dimethacrylate（ EDMA） for chiral separation. The epoxy groups of the polymer were used directly for the covalent bonding of BSA. A Coomassie brilliant blue（ CBB） protein assay（ Bradford method） was established to determine the protein bonding quantity,and the influence of some key aspects such as ionic strength,p H value and reaction time were studied. The method was validated with respect to linearity,precision,accuracy and robustness. The maximum amount of immobilized BSA was11. 90 mg / g, obtained using 65 ∶ 35 cyclohexanol / dodecanol as the porogen. The monolith was successfully applied in the chiral separation of R / S-warfarin and D / L-tryptophan in only 1-20 min. Furthermore,the chromatographic conditions like p H and organic additive of the mobile phase were optimized. The chiral separation performance of this BSA-immobilized monolith is positively correlated to the protein bonding quantity.

关 键 词：高效液相手性整体柱 牛血清白蛋白 考马斯亮蓝法 蛋白柱载量 手性拆分 

分 类 号：O652.63[理学—分析化学] TQ937[理学—化学]