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

doi:10.3724/SP.J.1123.2016.10025

色谱 ›› 2017, Vol. 35 ›› Issue (3): 332-338.DOI: 10.3724/SP.J.1123.2016.10025

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

魏强¹, 杨歌¹, 管桦², 屈锋¹

1. 北京理工大学生命学院, 北京 100081;
2. 公安部第一研究所, 北京 102200

收稿日期:2016-10-12 出版日期:2017-03-08 发布日期:2013-07-16
通讯作者: 屈锋,E-mail:qufengqu@bit.edu.cn.
基金资助:
国家自然科学基金项目（21675012，21375008，21175011）.

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

WEI Qiang¹, YANG Ge¹, GUAN Hua², QU Feng¹

1. School of Life Science, Beijing Institute of Technology, Beijing 100081, China;
2. First Research Institute of the Ministry of Public Security of People's Republic of China, Beijing 102200, China

Received:2016-10-12 Online:2017-03-08 Published:2013-07-16
Supported by:
National Natural Science Foundation of China (Nos. 21675012, 21375008, 21175011).

摘要/Abstract

摘要：

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。

关键词: 单链脱氧核糖核苷酸结合蛋白, 动力学毛细管电泳, 积分偏差, 解离常数

Abstract:

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.

Key words: dissociation constant, integral deviation, kinetic capillary electrophoresis (KCE), single-stranded deoxyribonucleic acid-binding protein (SSB)

中图分类号:

O658

魏强, 杨歌, 管桦, 屈锋. 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[J]. 色谱, 2017, 35(3): 332-338.

WEI Qiang, YANG Ge, GUAN Hua, QU Feng. Dissociation constant calculation of protein-single-stranded deoxyribonucleic acid interaction by kinetic capillary electrophoresis and its reliability analysis[J]. Chinese Journal of Chromatography, 2017, 35(3): 332-338.

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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

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

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