毛细管电泳法评价细胞色素c与单链脱氧核糖核酸库的相互作用

以细胞色素c(Cyt c)为碱性蛋白质模型,建立了毛细管电泳评价Cyt c与3种不同链长的单链脱氧核糖核酸(ssDNA)库相互作用的评价方法,研究了离子强度对Cyt c与ssDNA库相互作用的影响。比较了Cyt c与含有20、40和60个随机碱基序列的3种不同链长的ssDNA库的作用及基于未涂层毛细管和涂层毛细管的毛细管区带电泳方法。因碱性蛋白质在未涂层毛细管管壁上存在吸附,因此利用未涂层毛细管区带电泳不能区别3种ssDNA库与其作用的差异。利用涂层毛细管电泳法,在压力辅助的反向电压下,根据游离ssDNA库的峰面积变化可比较3种ssDNA库与细胞色素c的相互作用差异。结果表明,含有20个随机寡核苷酸链长的ssDNA库与Cty c的作用最强。此外,NaCl浓度显著影响Cyt c与ssDNA60库的作用。在优化的实验条件下,0.02 mol/L NaCl有利于两者的相互作用。调节盐浓度可抑制非特异性静电作用,能提高碱性蛋白质适配体的单轮筛选效率。利用未涂层毛细管电泳分析复合物及游离ssDNA库的峰面积变化,可优化有利复合物形成的盐浓度。

Comparison of interaction between cytochrome c and single strain deoxyribonucleic acid pools based on capillary electrophoresis

An assessment method of the interaction between cytochrome c (Cyt c) selected as a representative basic protein and three lengths of single strain deoxyribonucleic acid (ssDNA) pools was presented based on capillary electrophoresis. The investigation of ionic strength effect on the interaction was made. The interactions between Cyt c and the ssDNA pools with 20 nt, 40 nt and 60 nt random sequences were compared by capillary zone electrophoresis (CZE) based on an uncoated fused capillary and a coated capillary. Using the uncoated fused capillary, the interaction difference could not be observed due to the strong adsorption of Cyt c on the inner surface of the capillary. However, under negative potential with the assistance of pressure driving, the peak area change of free ssDNA indicated the interaction difference of the three pools with Cyt c using the coated capillary, and the results showed that the ssDNA pool with 20 nt random sequence exhibited the strongest interaction. In addition, NaCl concentration had great effect on the interaction of Cyt c with ssDNA60, and 0.02 mol/L NaCl solution was favored. For the selection of basic target proteins, the adjustment of salt concentration would inhibit the non-specific electrostatic interaction, improve the selection efficiency of a selection round. The favorable salt concentration could be optimized with an uncoated fused capillary by comparing the peak area change of the complex and free ssDNA pool.