基于流动注射-化学发光法的BSA构象变化研究

Conformational Change of BSA Characterized by Flow Injection Chemiluminescence Method

功能蛋白质与药物的相互作用诱导的蛋白质构象研究对于理解蛋白质的生物学功能和药物的体内作用机制具有重要意义。本研究发现,碱性条件下,牛血清白蛋白(BSA)可显著增加鲁米诺(luminol)化学发光强度,而L-色氨酸(L-Trp)能浓度依赖性地抑制鲁米诺/BSA体系的发光强度,据此,建立了一种基于鲁米诺/BSA·L-1-色氨酸的蛋白构象表征化学发光新方法。结果表明,L-色氨酸对鲁米诺/BSA体系的发光强度的抑制作用与其浓度在100~5 000pmol·L-1范围内呈良好的线性关系,回归方程为lg[(I0-Is)/Is]=0.62lgc+4.71,相关系数为0.990 6。进一步通过该方程计算获得BSA与L-色氨酸相互作用的结合常数为5.13×104 L/M,结合位点数约为1。该研究可为功能蛋白构象表征研究提供方法学借鉴。

It was important to study the interaction of functional protein and its ligand,protein conformation changes and its control method in the life sciences,medical and pharmaceutical fields.A rapid method was developed to characterize the binding interaction of BSA and L-tryptophan by flow-injection chemiluminescence(FI-CL)method.The method relies on the inhibition of a luminol/BSA chemiluminescence by increasing amounts of L-ryptophan.This loss of CL with increasing amounts of L-tryptophan fits the equation lg[(I0-Is)/Is]=lgK +nlgc,allowing calculation of the binding constant(K)and stoichiometry(n).The results showed that the inhibition of L-tryptophan on the CL intensity of luminol/BSA reaction system was linear with the L-tryptophan concentration,with a regression equation of lg[(I0-Is)/Is]=0.62lgc+4.71.Using this equation,the association constant of L-tryptophan binding to BSA was calculated to be 5.13×104 L/M,and the stoichiometry was 1.This study will probably provide a method for the study of conformational characterization of functional proteins.