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(Kd).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Kd,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Kd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Kd计算结果有影响,但在10%的偏差区间内,所求Kd值的最大相对偏差小于7%,所求Kd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.
Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is used for the determination of the complex's dissociation constant Kd.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 Kd in different conditions were determined.Moreover, the error analysis method was established based on peak's integral deviation.The results indicated that the Kd 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 Kd calculation under previous experiment conditions.But the maximum relative deviation of Kd value in 10% of the deviation interval was less than 7% and the Kd value's error could be ignored.This paper confirms that the calculation method of kinetic parameters by NECEEM is reliable.
参考文献
| [1] | Krylov SN.Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM): A novel method for biomolecular screening[J].Journal of biomolecular screening: The official journal of the Society for Biomolecular Screening,20062(2):115-122. |
| [2] | Berezovski M;Krylov SN.Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures[J].Analytical chemistry,20055(5):1526-1529. |
| [3] | Mallikaratchy P;Stahelin RV;Cao ZH;Cho WH;Tan WH.Selection of DNA ligands for protein kinase C-delta[J].Chemical communications,200630(30):3229-3231. |
| [4] | Krylov SN.;Berezovski M..Non-equilibrium capillary electrophoresis of equilibrium mixtures - appreciation of kinetics in capillary electrophoresis[J].The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry,20036(6):571-575. |
| [5] | Jaouen S;de Koning L;Gaillard C;Muselikova-Polanska E;Stros M;Strauss F.Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops[J].Journal of Molecular Biology,20054(4):822-837. |
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