doi:

DOI: 10.3724/SP.J.1123.2016.10025

Chinese Journal of Chromatography (色谱) 2017/35:3 PP.332-338

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


Abstract:
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.

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

ReleaseDate:2017-03-21 16:31:28



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