Applying Capillary Electrophoresis To Study The Interaction Between Hemin And DFO

Research on the interaction between drug and the virulence factors contributes to understanding the mechanism of drugs working in body, which provides reference for pharmacokinetics and toxicology study. This plays an important role in the process of developing new drugs. Affinity capillary electrophoresis (ACE) is widely used to determine the binding constants between different substances. In this method a series of receptor (or ligand) at different concentrations are dissolved in buffer as additive, and used to be running buffer. Ligand (or receptor) at fixed concentration is the detected sample. The complex of ligand and receptor affects the apparent mobility of ligand, and the binding constant can be calculated through the mobility change of ligand.Several studies have shown that iron chelator desferrioxamine(DFO) can combine with hemin to form DFO-hemin complex, which would affect tissue damage induced by hemin. This thesis is the first time applying affinity capillary electrophoresis to study chemical information of interaction between DFO and hemin, to provide some useful pharmacology and toxicology information of DFO widely used in clinic.In this paper, electrophoresis experiment conditions were studied and the optimal conditions were determined. The detected sample was hemin, the buffer was 20 mmol/L boric acid solution (pH = 8.6) and DFO was buffer additive. The total length of cappilary was 60.2 cm and the effective length was 50 cm. First, the influence of different concentrations of DFO (10～80μM) on the mobility of hemin (80μM) was studied at different temperatures (20℃, 25℃, 30℃, 37℃). The binding constants of DFO and hemin at different temperatures (20℃, 25℃, 30℃, 37℃) were calculated through Scatchard linear regression analysisy, 12.43×10⁴ M^-1,10.13×10⁴ M^-1,7.57×10⁴ M^-1,3.46×10⁴ M^-1 respectively. The result showed that with temperature increasing, binding constants were decreasing. According to Van't Hoff equation, the values of standard Gibbs free change(ΔG) of the binding reaction were obtained, from -26.94 to -28.58 kJ·mol^–1 at different temperatures from 20℃to 37℃. Then through linear fitting of lnK against 1/T, the thermodynamic parameters can be calculated by the slope and intercept. Enthalpy change(ΔH) was–56.61 kJ·mol^–1 and entropy change(ΔS) was–94.77 J·mol^–1·K^–1. Thermodynamic datas above showed that the binding reaction was exothermic and spontaneous.