格列本脲蛋白结合作用的HPLC-FA法研究

HPLC-FA Study on the Protein Binding of Glibenclamide

目的:建立高效液相色谱-迎头分析法(HPLC-FA)研究格列本脲与人血清白蛋白(HSA)的结合作用。方法:采用HPLC-FA法,色谱柱为 Pinkerton GFFⅡ-S5-80(150 mm×4.6 mm,5 μm),流动相为67 mmol·L^(-1)磷酸盐等渗缓冲液(pH 7.4,I=0.17 mol·L^(-1)),流速0.2 mL·min^(-1),检测波长230 nm,进样量900μL,柱温37℃。结果:应用非线性回归参数估算求得 HSA 分子上两类结合位点结合的格列本脲分子数及相应的结合平衡常数:n_1,=1.5和 K_1=1.1(μmol·L^(-1))^(-1);n_2=3.8和 K_2=0.012(μmol·L^(-1))^(-1)。结论:HPLC-FA 法操作简便,适用于研究格列本脲与 HSA 的结合作用。本实验选定的进样体积为900μL。

Objective：To develop HPLC - FA method for study of protein binding of glibenclamide. Methods： The separation is performed using Pinkerton GFF Ⅱ - S5 - 80 internal - surface reversed - phase silica support （ 150 mm ×4. 6 mm,5 μm）at pH 7.4 in a 67 mmol · L^-1 isotonic sodium phosphate buffer at 37 ℃. Other conditions included flow rate 0.2 mL · min^ - 1, UV detection at wavelength 230 nm and injection volume 900 μL. Resuits：Nonlinear regression parameter estimation is applicable to the association constants measurement of glibenclamide to both primary and secondary sites, which are 1.1 （ μmol · L^-1 ）^ -1 and 1.5 for K1 and n1 , respectively,and 0. 012（ μ mol · L^-1 ）^ -1 and 3.8 for K2 and n2 ,respectively. Conclusions：The method is proved to be suit- able for investigation of protein binding of glibenclamide. The injection volume is 900 μL.