荧光光谱法研究二溴羟基卟啉与蛋白质的结合作用机理

Study on Binding Mechanism of Meso-tetra-(3,5-Dibromo-4-Hydroxyphenyl) Porphyrin with Protein by Fluorescence Method

应用荧光光谱法研究了meso-四(3,5-二溴-4-羟基苯基)卟啉[T(DBHP)P]与牛血清白蛋白(BSA)之间的结合反应,基于T(DBHP)P对BSA内源荧光的猝灭机理,测定了两者之间在不同温度下的结合常数,温度为27℃时,荧光猝灭法测得反应的结合常数为K=1.30×106L.mol-1,温度为48℃时,K=6.32×105L.mol-1,结合常数随温度升高而减小,由此判定该猝灭类型为静态猝灭。根据F rster非辐射能量转移理论,确定了T(DBHP)P与BSA之间的能量转移效率E=0.91,能量给体(BSA)与受体[T(DBHP)P]之间的结合距离r=2.39 nm<7 nm,符合非辐射能量转移条件。依据热力学参数ΔG<0,ΔH<0,ΔS>0确定了T(DBHP)P与BSA之间的作用力主要是静电引力。同时,利用同步荧光光谱,考察了T(DBHP)P对BSA构象的影响,结果发现,T(DBHP)P的加入使BSA构象发生变化,BSA内部残基所处环境的疏水性降低。

Studies on the binding mechanism between protein and small molecules could give us lots of useful information. For example, a detailed characterization of drug-protein binding properties was essential for understanding the function of delivery, hence, interest in the mechanism of the interaction between them has attracted much research using different methods. In the present paper, the interaction mechanism between meso-tetra- （3,5-dibromo-4-hydroxyphenyl） porphyrin [T（DBHP） P] and bovine serum albumin （BSA） was investigated using fluorescence method. Based on the mechanisms of fluorescence quenching of BSA caused by T（DBHP）P, the binding constants between T（DBHP）P and BSA were measured at different temperatures. The experiment showed that T（DBHP）P and BSA have strong interactions. The binding constants of the reaction at 27 and 48 ℃ were calculated by fluorescence method, respectively. The binding constants are K= 1.30 × 10^6 L ·mol^-1 at 27 ℃, and K= 6.32 × 10^5 L · mol^-1 at 48 ℃. Because the binding constants decreased with increasing the temperature, the sort of quenching between T（DBHP）P and BSA was determined as static quenching. By the theory of Fbrster non-radiation energy transfer, the binding distance and the energy transfer efficiency at 27 ℃ between T（DBHP）P （accepter of energy） and BSA （donor of energy） were obtainect to be 2.39 nm and 0. 91, respectively. The binding distance was less than 7 nm, therefore, the interaction was similar to the non-radiation energy transfer, and the static quenching was further proved. According to the thermodynamic parameters, the main sorts of binding force between T（DBHP）P and BSA could be judged as electrostatic force when △G〈0, △H 〈0 and △S〉0. Using the synchronous fluorescence spectra, the effect of T（DBHP）P on the conformation of BSA was studied. The results indicated that the conformation of BSA was changed when T（DBHP）P was added, and the hydrophobic properties of the environment of residues in BSA decreased. It was proved that fluorescence quenching of BSA was induced by static quenching and non-radiation energy transfer.