动力学毛细管电泳法求解蛋白质与单链脱氧核糖核苷酸结合的解离常数及可靠性分析

平衡混合物的非平衡毛细管电泳(NECEEM)可用于测定复合物的解离常数(K_d)。该文以单链脱氧核糖核苷酸结合蛋白(SSB)和单链脱氧核糖核苷酸(ssDNA)相互作用为模型,分析了相互作用强弱的电泳图谱特征,测定了不同条件下复合物的K_d,建立了基于积分偏差的误差分析方法。结果表明,SSB与ssDNA相互作用强弱所致的电泳图差异、SSB与ssDNA的浓度比、毛细管分离温度均影响K_d的计算。此外,基于25℃时SSB与5′-GGTTGGTGTGGTTGG-3′(15mer)人凝血酶适配体作用模型的数值分析结果表明,在现有实验条件下,手动积分峰面积时产生的面积偏差对K_d计算结果有影响,但在10%的偏差区间内,所求K_d值的最大相对偏差小于7%,所求K_d值的误差可忽略。该文证实了基于NECEEM求解动力学相关参数的方法可靠。

Dissociation constant calculation of protein-single-stranded deoxyribonucleic acid interaction by kinetic capillary electrophoresis and its reliability analysis

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is used for the determination of the complex's dissociation constant K_d. In this paper, single-stranded deoxyribonucleic acid-binding protein (SSB) and single-stranded deoxyribonucleic acid (ssDNA) were taken as the interaction model. The electrophoretogram features of different interaction strengths were analyzed and the complex's K_d in different conditions were determined. Moreover, the error analysis method was established based on peak's integral deviation. The results indicated that the K_d calculation were influenced by the electrophoretogram differences of varied SSB-ssDNA interactions, SSB-ssDNA concentration ratio and capillary temperature. Besides, the results based on the SSB-5'-GGTTGGTGTGGTTGG-3' (15mer) aptamer of human thrombin interaction under 20℃ showed that the deviation generated when manually integrate the peak area had an effect on K_d calculation under previous experiment conditions. But the maximum relative deviation of K_d value in 10% of the deviation interval was less than 7% and the K_d value's error could be ignored. This paper confirms that the calculation method of kinetic parameters by NECEEM is reliable.