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
LING Yan, WANG Chun-you, YANG Zhi-yong*
Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Abstract：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=1.78×105 L·mol-1·s-1, kd=8.02×10-4 s-1, and the affinity constants were KA=2.22×108 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=1.85×103 L·mol-1·s-1, kd=1.81×10-4 s-1, KA=1.02×107 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.
Key words：High mobility group protein 1 (HMGB1);Receptor of advanced glycation end products (RAGE);Heparin;Surface plasmon resonance technology (SPR);Affinity analysis
凌 燕，王春友，杨智勇*. 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[J]. 光谱学与光谱分析, 2009, 29(11): 3084-3087.
LING Yan, WANG Chun-you, YANG Zhi-yong*. 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 . SPECTROSCOPY AND SPECTRAL ANALYSIS, 2009, 29(11): 3084-3087.
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