华法林与人血清蛋白结合的动力学过程和光谱性能研究

Study on the Kinetic Process and Spectral Properties of the Binding of Warfarin to Human Serum Protein

华法林作为口服维生素K拮抗剂几十年来广泛应用于治疗血栓类疾病。因此针对华法林的物理化学性质的研究成为了人们的重点。通过含时密度泛函理论(TD-DFT),模拟了华法林分子在水溶液状态下与人血清蛋白结合过程中几种状态的紫外-可见吸收光谱(UV-Vis)与荧光光谱,研究不同状态在激发过程中的跃迁方式和电荷转移过程,以及光谱的差异,探究整个动力学过程的激发态变化机理。结果表明,华法林在水溶液中紫外-可见吸收光谱表现出双吸收现象,双吸收现象主要由不同的激发态跃迁导致。未去质子化前,主要吸收峰波长为291 nm,去质子化后,吸收强度降低,波长红移,当华法林与血清蛋白结合后发生电荷转移导致吸收增益,波长307 nm吸收峰强度最高。通过计算第一激发态(S_(1))的结构和激发能模拟华法林不同状态的荧光光谱,最初状态下荧光峰为360 nm,去质子化后,荧光强度降低,波长红移,结合后结构变化导致荧光增益。根据华法林的荧光光谱变化情况,说明其在整个动力学过程中存在不同的荧光发射过程。通过分子前线轨道分析和电子-空穴分析研究整个动力学过程的电荷转移情况,表明华法林单体的荧光发射过程为局域激发,与蛋白质结合后荧光发射过程为电荷转移激发,结合后的荧光增益特征使华法林可以作为荧光探针。该研究揭示了华法林与蛋白结合过程中光谱变化机理,为以后探究分子结合动力学过程提供新型研究手段和理论支撑。

As an oral vitamin K antagonist,warfarin has been widely used in treating thrombotic diseases for decades.Therefore,the research on the kinetic process of warfarin has become people’s focus.Time-dependent density functional theory(TD-DFT)simulated the excitation and emission spectra and charge transfer processes of several states during the warfarin molecules binding to human serum proteins in aqueous solution.Study the transition mode and charge transfer during the excitation process,the differences in the spectra.Explore the exciting state change mechanism of the entire kinetic process.The results show that the UV-Vis absorption spectrum of warfarin in aqueous solution exhibits double absorption,mainly caused by different excited state transitions.Before deprotonation,the main absorption peak wavelength is 291 nm.After deprotonation,the absorption intensity decreases and the wavelength is red-shifted.When warfarin binds to serum protein,charge transfer occurs,resulting in an absorption gain of 307 nm and the absorption peak intensity is the highest.By calculating the structure and excitation energy of the first excited state(S_(1))to simulate the fluorescence spectra of different states of warfarin,the fluorescence peak in the initial state is 360 nm.After deprotonation,the fluorescence intensity decreases and the wavelength red shifts.The structural changes after the combination result in fluorescence gain.According to the changes in the fluorescence spectrum before and after the combination of warfarin and protein,warfarin has different fluorescence emission processes in the whole dynamic process.The charge transfer of the entire dynamic process of warfarin molecules is analyzed by molecular frontier orbital and electron-hole methods.The results show that the fluorescence emission process of warfarin monomer is local excitation,and the fluorescence emission process after binding with protein is charge transfer excitation.The combined fluorescence gain feature makes warfarin a fluorescent probe.This paper reveals the mechanism of spectral changes in the warfarin and protein binding process,and provides new research methods and theoretical support for future exploration of molecular binding dynamics.