荧光光谱法研究三种金属卟啉与人血清白蛋白的结合反应

Fluorescence Spectroscopy for Studies on the Interaction Between Three Metalloporphyrins With Human Serum Albumin

近年来,卟啉及金属卟啉在光动力疗法、抗癌药物研究方面备受关注,有些已被批准临床使用。人血清白蛋白(HSA)能结合、运输许多药物活性分子,详细研究金属卟啉与HSA的结合机制,对阐明卟啉类药物的作用机制具有重要的意义。研究合成了3种新型6-氯烟酸修饰的自由卟啉(4,5,6)及锌卟啉(4-Zn,5-Zn,6-Zn),并通过测试表征和理论计算的方法进行了结构确定。理论计算结果表明:3种锌卟啉中的侧链6-氯烟酸基团均远离卟啉环平面;4-Zn构型比连有取代基的5-Zn、6-Zn构型更稳定。在模拟生理条件下,采用荧光光谱法研究了3种锌卟啉与HSA之间的键合方式,通过Stern-Volmer方程、双对数方程和Van’t Hoff方程对测定结果进行计算。结果表明:(1)3种锌卟啉均能有效猝灭HSA的荧光,Stern-Volmer方程计算得到锌卟啉与HSA在不同温度下的K_(q)值均大于2.0×10^(10) L·mol^(-1)·s^(-1),表明猝灭类型为静态猝灭;(2)采用双对数方程得到锌卟啉与HSA的结合常数,除5-Zn在318 K时外,其他温度条件下的结合常数K_(A)均大于10^(3) L·mol^(-1),结合位点数n均接近1,说明形成了1∶1的复合物;(3)根据Van’t Hoff方程计算,3种锌卟啉与HSA结合的热力学参数均小于0,如4-Zn与HSA结合的热力学参数为ΔH=-123.9 kJ·mol^(-1),ΔS=-322.9 J·mol^(-1)·K^(-1),ΔG=-27.7 kJ·mol^(-1)(298 K),由此得出反应过程为自发进行,反应的主要作用力为范德华力和氢键。实验获得的数据可以为研究金属卟啉与生物小分子的作用机制提供一些有用的信息。

In recent years,porphyrins and metalloporphyrins have attracted much attention in photodynamic therapy and anticancer,and some have been approved for clinical use.Human serum albumin(HSA)can bind and transport some drug molecules.A detailed study of the binding mechanism of metalloporphyrins and HSA is of great significance in clarifying the action mechanism of porphyrin drugs.This study synthesised three kinds of novel free porphyrins modified with 6-chloronicotinic acid(4,5,6)and their Zn complex(4-Zn,5-Zn,6-Zn)and characterized by UV-Vis,IR,1H NMR,elemental analysis,fluorescence spectra and theoretical calculations.The theoretical calculation results showed that the 6-chloronicotinate moieties in the three zinc porphyrins were far away from the porphyrin ring plane.The 4-Zn configuration was more stable than the 5-Zn and 6-Zn configurations with substituents.Under simulated physiological conditions,the bonding modes between three zinc porphyrins and HSA were studied by fluorescence spectra,and the results were calculated according to the Stern-Volmer equation,double-logarithmic equation and Van’t Hoff equation.The experimental results indicated:(1)Three zinc porphyrins could all quench the fluorescence of HSA and the values of K_(q) calculated by the Stern-Volmer equation were much larger than 2.0×10^(10) L·mol^(-1)·s^(-1).Thus the quenching type was static quenching.(2)The binding constants were calculated by a double-logarithmic equation.Except for the 5-Zn at 318 K,other binding constants were all greater than 10^(3) L·mol^(-1),and the binding sites were close to 1,indicating the formation of a 1∶1 complex.(3)According to the Van’t Hoff equation,the thermodynamic parameters ΔH,ΔS,ΔG were all less than 0,eg.those of 4-Zn were calculated to be ΔH=-123.9 kJ·mol^(-1),ΔS=-322.9 J·mol^(-1)·K^(-1),ΔG=-27.7 kJ·mol^(-1)(298 K),indicating that the reaction process was spontaneous and the predominant forces between zinc porphyrins and HSA were vander waals force and hydrogen bond.The experimental data obtained in this experiment can provide useful information for studying the interaction mechanism between metalloporphyrins and biological small molecules.