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

doi:10.3724/SP.J.1123.2016.10025

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

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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).

Abstract

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)

CLC Number:

O658

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

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