嘧啶衍生物与人血清白蛋白的相互作用

在模拟生理条件下,运用荧光光谱、激光闪光光解(LFP)和分子对接等技术研究了8种具有抗肿瘤活性的嘧啶衍生物(PDs,其中PDs A 5-FU为成药,PDs B-H为实验室自制)与人血清白蛋白(HSA)的相互作用.利用Stern-Volmer方程和激光闪光光解技术分析了PDs对HSA的荧光猝灭机制,PDs A和B为静态猝灭,PDs G和H为动态猝灭.用双倒数曲线法得出5种PDs与HSA的结合常数Ka和结合位点数n,在测定条件下5种PDs与载体结合位点数均为1,且均以弱结合力结合,通过热力学参数ΔH,ΔS和ΔG推测出PDs B,C和E与HSA之间的作用力为静电作用力和疏水作用力,PDs A和D与HSA之间的作用力是氢键和范德华力,分子对接结果与其一致.根据F9rster非辐射能量转移理论(FRET)分析了HSA和PDs之间的结合距离(r),其结果均小于4 nm,符合能量转移理论.进一步利用同步荧光、三维荧光和圆二色光谱考察了PDs与HSA结合过程中HSA空间构象的变化,结果显示,仅PDs A和C对HSA的芳香族氨基酸周围的疏水性略有增强作用.体外实验结果表明,HSA可以作为优良的载体来运输和储存PDs A~E,这为嘧啶衍生物的后续研究提供了可参考的实验数据.

Interaction of Pyrimidine Derivatives with Human Serum Albumin

The interaction of eight pyrimidine derivatives( PDs, 5-FU marked as A is the positive control, other seven samples have been made in our laboratory and numbered from B to H, respectively) with human serum albumin( HSA) have been investigated by means of steady state fluorescence, laser flash photolysis ( LFP ) , UV-Vis absorption spectroscopy and molecular docking techniques under simulative physiological coditions. The fluorescence quenching mechanism of HSA and different pyrimidine derivatives have been analyzed by the Stern-Volmer equation and laser flash photolysis techniques. From these results, we can know that pyrimidine derivatives numbered from A to E are static quenching, but pyrimidine derivatives numbered from G to H are dynamic quenching. Depending on double reciprocal curve, we can obtain the binding con-stants(Ka) and the number of binding sites(n) of the five pyrimidine derivatives of static quenching and HSA. These data show that the protein and pyrimidine derivatives combinate with weak binding force and the number of binding site are equal to one. Fitting the thermodynamic parameters ΔH, ΔS and ΔG can faciliate to understand the binding force types for pyrimidine derivatives and HSA, and pyrimidine derivative B, C and E are the electrostatic and hydrophobic interactions, while for pyrimidine derivatives A and D are hydrogen bond and van der Waals force, and these experimental results are consistent with molecular docking results. Accord-ing to non-radiative energy transfer theory( FRET) , the binding distance( r) between HSA and pyrimidine de-rivaties can be obtained, which are less than 4 nm, and these results are conform to the energy transfer theory. With synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectroscopy, the space conformation changes of HSA in the drugs combining process can be investigated. The results show that pyrimi-dine derivatives B, D and E have no influence on the secondary and tertiary structure of HSA. pyrimidine de-rivative A and C have no effect on secondary structure of HSA, but the hydrophobicity around the aromatic a-mino acids for the tertiary structure are slightly enhanced. Therefore, HSA can be used as an excellent carrier to transport and store these five pyrimidine derivatives.