Citation: 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. doi: 10.3724/SP.J.1123.2016.10025 shu

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

WEI Qiang ^1, ,
YANG Ge ¹ ,
GUAN Hua ² ,
QU Feng ¹

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 Date: 12 October 2016

Fund Project: National Natural Science Foundation of China (Nos. 21675012, 21375008, 21175011).

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.
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