全氟十二酸(PFDoA)是8～12个碳链的全氟烷酸(PFAAs)中毒性最强的新型环境污染物。 已有大量研究表明PFAAs在环境中广泛积累, 但对PFDoA与HSA的相互作用还处于起步阶段。 本研究力争在模拟生理条件下, 采用荧光猝灭法、 分子模拟技术和圆二色谱确定HSA与PFDoA的相互作用机理。 研究结果表明, PFDoA对HSA的猝灭是动态猝灭与形成PFDoA-HSA基态复合物引起的猝灭共同作用的结果。 计算得到的结合距离(r=3.65 nm)表明, PFDoA(受体)与HSA(供体)之间的相互作用发生了非辐射能量转移。 取代反应结果表明, PFDoA键合在HSA的site Ⅰ位点上。 分子对接进一步研究了PFDoA与HSA作用的详细结合情况, 表明PFDoA通过多种作用力结合在HSA的亚域IIA内, 例如, PFDoA上的O 1原子主要通过极性键与HSA上的Arg 257和Ser 287残基结合。 计算得到的最优对接能量为-25.87 kJ·mol-1, 表明PFDoA对HSA有较大的结合亲和力。 同步荧光光谱和三维荧光光谱研究了PFDoA对HSA构象的影响, 结果显示, 与PFDoA结合后, 色氨酸的微环境疏水性增加, HSA的构象也发生改变。 PFDoA与HSA作用前后圆二色谱二级结构的定量分析结果表明, PFDoA-HSA复合物的形成使螺旋稳定性降低。 该研究结果为全氟烷酸与HSA的动力学研究提供了理论依据和可靠数据, 并揭示了生物大分子与配体相互作用的化学本质。

Perfluorododecanoic acid (PFDoA) is the most toxic emerging environmental contaminant among the 8～12 carbon chain perfluoroalkyl acids (PFAAs). A large amount of knowledge in the field of environmental PFAAs has been accumulated so far, while we are still just at the beginning of research into the interaction between PFDoA and human serum albumin (HSA). The goal of this study was to comprehensively determinate the binding mechanism of PFDoA with HSA by using fluorescence quenching technique in combination with molecular modeling and circular dichroism (CD) spectroscopy under the simulative physiological conditions. The quenching of HSA fluorescence by PFDoA was found to be a result of the combination of dynamic quenching and the formation of PFDoA-HSA complex. The calculated binding distance (r=3.65 nm) indicated that the non-radioactive energy transfer came into being in the interaction between PFDoA (acceptor) and HSA (donor). By performing displacement measurements, the specific binding of PFDoA in the vicinity of site I of HSA was clarified. Furthermore, the binding details between PFDoA and HSA were further confirmed by molecular docking studies, which revealed that PFDoA was bound at subdomain IIA by multiple interactions, such as the interaction between O1 of PFDoA with Arg 257 and Ser 287 predominately through polar force. And the best calculated docking energy is -25.87 kJ·mol-1, this high negative value indicated that the PFDoA molecule exhibited large binding affinity towards HSA. The effects of PFDoA on the conformation of HSA were analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, and the results exhibited that the hydrophobicity of the microenvironment around tryptophan residue was increased and the conformation of HSA was altered after binding PFDoA. The CD spectra quantitatively calculated the protein secondary structure, which suggested a loss of helical stability after the PFDoA-HSA complex formation. The binding research presented in this paper enriches our knowledge of the interaction dynamics of perfluoroalkyl acids to the HSA and reveals the chemical essence of the interaction between biomacromolecule and ligand.