表面等离子体共振技术研究肝素对高迁移率族蛋白1与晚期糖基化终产物受体亲合力的影响  

Analysis of the Impact of Heparin on the Affinity of High Mobility Group Box-1 Protein and the Receptor of Advanced Glycation End Products by Surface Plasmon Resonance Technology

作　　者：凌燕[1] 王春友[1] 杨智勇[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院胰腺外科,湖北武汉430022

出　　处：《光谱学与光谱分析》2009年第11期3084-3087,共4页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(30600593)资助

摘　　要：测定肝素与高迁移率族蛋1(HMGB1),及其与晚期糖基化终产物受体(RAGE)的亲和力,探讨肝素对HMGB1/RAGE亲和力的影响。用表面等离子体共振技术,实时分析肝素与HMGB1,HMGB1与RAGE的亲和常数,利用其binding analysis分析肝素对HMGB1/RAGE配受体结合的影响。肝素与HMGB1的动力学速率常数ka=1.78×105L.(mol.s)-1,kd=8.02×10-4.s-1,亲和常数KA=2.22×108L·mol-1,KD=4.5×10-9mol·L-1;HMGB1与RAGE的动力学常数ka=1.85×103L.(mol.s)-1,kd=1.81×10-4.s-1,亲和常数KA=1.02×107L·mol-1,KD=9.77×10-8mol·L-1。肝素与RAGE的亲和力极低。当浓度分别为50,100,1000,10000U.L-1的肝素与HMGB1混合后,后者与RAGE的亲和力下降。上述不同浓度的肝素对HMGB1/RAGE亲和力的影响没有显著差异。结果表明,肝素可与HMGB1结合并影响后者与RAGE的亲和力,该作用并不与肝素浓度呈正相关。To investigate the affinity constants of heparin with high mobility group protein 1（HMGB1） and HMGB1 with the receptor of advanced glycation end products （RAGE） and to analyze the impact of heparin on the affinity of HMGB1 and RAGE,the standard BIAcore amine coupling chemistry protocol using EDC and NHS was employed for immobilizing. Surface plasmon resonance biosensor technology was used to detect the affinity constants of heparin/HMGB1,HMGB1/RAGE and heparin/RAGE. Binding analysis was used to investigate the impact of heparin on the affinity of HMGB1 and RAGE. After the immobilization,9 000 and 5 000 RU rise of HMGB1 and RAGE respectively were obtained. These meant that the immobilized values of HMGB1 and RAGE were about 9 and 5 ng·mm^-2 respectively. The kinetic constants were ka=1.78×10^5 L·mol^-1·s^-1,kd=8.02×10^-4 s^-1,and the affinity constants were KA=2.22×10^8 L·mol^-1,the equilibrium dissociation constant KD=4.5×10^-9 mol·L^-1 for heparin and HMGB1; while the kinetic constants were ka=1.85×10^3 L·mol^-1·s^-1,kd=1.81×10^-4 s^-1,KA=1.02×10^7 L·mol^-1,KD=9.77×10^-8 mol·L^-1 for HMGB1 and RAGE; there was very low affinity of heparin with RAGE. The highest concentration of 10 000 u·L^-1 of heparin in this experiment did not reach the saturation with HMGB1. After 50 mg·L^-1 of HMGB1 was mixed with heparin of 50,100,1 000,10 000 u·L^-1,the combining amount of HMGB1 and RAGE declined from 100 to 50 RU. But there were no significant differences between different concentrations of heparin. It was concluded that heparin can combine with HMGB1 and affect the affinity of HMGB1/RAGE. In addition,this impact was not in a dose-dependent manner.

关 键 词：高迁移率族蛋白1 晚期糖基化终产物受体 肝素 表面等离子体共振技术 动力学分析 

分 类 号：O657[理学—分析化学] R965[理学—化学]