表面等离子体共振技术分析卵黄抗体对酪氨酸酶与底物相互作用的影响

摘要

目的:采用表面等离子体共振技术(surface plasmon resonance,SPR)测定酪氨酸酶与特异性卵黄抗体(IgY)及其与底物L-多巴的亲和力,探讨特异性IgY对酪氨酸酶与底物亲和力的影响.方法:将裸金芯片进行自组装修饰,以3-巯基丙酸作为基底膜对酪氨酸酶进行固定,SPR实时监测特异性IgY及底物L-多巴与酪氨酸酶结合后芯片表面的响应信号变化.将数据导入CLAMP分析软件,进行拟合分析,确定动力学常数.结果:酪氨酸酶与特异性IgY的结合常数(4.50×106 L/mol)明显高于其与底物L-多巴的结合常数(4.95×103 L/mol),且酪氨酸酶与特异性IgV结合后,其与底物L-多巴结合常数明显下降.结论:相比底物L-多巴,酪氨酸酶更易与特异性IgY结合,而且从结合动力学角度说明特异性IgV可以明显抑制酪氨酸酶与底物结合从而抑制酶活性.%Aim:Surface plasmon resonance (SPR) was used to investigate the impact of specific egg yolk immunoglobulin (IgY) on the interaction between tyrosinase and its substrate L-dopa.Methods:Bare gold chip was modified by selfassembly.3-Mercaptopropionic acid (MPA) was used as the basement membrane to immobilize tyrosinase,and the SPR response signals of tyrosinse binding to specific IgY and L-dopa were monitored in real time.The CLAMP software was used to fit the experimental data to determine the kinetic constants.Results:The affinity constant of tyrosinase to specific IgY (4.50 × 106 L/mol) was higher than that to L-dopa (4.95 × 103 L/mol),and after binding to specific IgY,the binding constant between tyrosinase and L-dopa was decreased significantly.Conclusion:Compared with the substrate L-dopa,tyrosinase was more likely to specifically bind to IgY,and the specific IgY could significantly inhibit the binding of tyrosinase to its substrate and consequently inhibited the enzyme activity on the basis of binding kinetics.