全氟壬酸(PFNA)是在血清中检测到第三多的全氟烷酸类(PFAAs)新型有毒环境污染物。 目前PFNA对人血清白蛋白(HSA)结构甚至是功能的影响还处于起步阶段, 借助于多光谱、 分子对接和等温滴定微量热(ITC)技术研究了PFNA和HSA相互作用的结合机理。 所有荧光数据均进行了内滤光校正以获得更准确的结合参数。 荧光结果表明PFNA通过动静态猝灭方式可以猝灭HSA的内源荧光。 取代实验和分子对接结果表明, PFNA主要通过极性键、 疏水力和卤素键键合在HSA亚域ⅡA疏水腔中, 最佳对接自由能为-26.54 kJ·mol-1, 表明PFNA分子与HSA有较大的结合亲和力。 ITC表明两者的结合属于两类结合位点模型并给出了相应的热力学参数: 第一类结合位点有较大的亲和力, 属于焓驱动, 静电力和卤键作为主要驱动力； 第二类结合位点亲和力较小, 主要驱动力是疏水力。 三维荧光光谱揭示PFNA与HSA生成复合物后, 可以改变HSA的构象, 引起Trp和Tyr残基微环境疏水性增强。 圆二色谱(CD)定量测定了HSA与PFNA作用前后的二级结构含量: α-螺旋、 β-折叠和β-转角含量分别降低14.3%, 5.3%和3.5%, 无规卷曲含量从14.4%增加到37.5%。 以上结果表明, PFNA与HSA的结合可以改变HSA的二级结构, 进而可能影响HSA的生理功能。 结果阐述了PFNA与HSA相互作用机理, 并且为PFNA在体内的运输和分配提供了可靠的生物物理和生物化学的相关依据。

Perfluorononanoic acid (PFNA) is the third most frequently detected in serum among all perfluoroalkyl acids (PFAAs) which is a kind of toxic emerging environmental contaminant. The influence of PFNA on the conformation and even function of human serum albumin (HSA) is still just at the beginning of research. The attempt of this paper was to completely elucidate the interaction mechanism of PFNA with HSA by means of multi-spectroscopic, molecular docking and isothermal titration calorimetry (ITC) techniques. The inner filter effect of all fluorescence data in the paper was eliminated to get accurate binding parameters. The results showed that the fluorescence of HSA was quenched by PFNA through a combined quenching procedure of dynamic and static quenching. Through site marker competitive experiments, subdomain IIA of HSA had been assigned to possess the high-affinity binding site of PFNA. Furthermore, molecular docking reconfirmed that PFNA was bound in subdomain IIA mainly through polar force, hydrophobic interaction and halogen-bond, and the calculated free energy was -26.54 kJ·mol-1 which indicated that the PFNA molecule exhibited large binding affinity towards HSA. The thermodynamic characterizations of two different classes of binding sites by ITC displayed that the first class with a higher affinity constant was dominated by an enthalpic contribution due to electrostatic interactions and halogen-bond, whereas the second class with a lower affinity constant was preponderated by hydrophobic interaction. The three-dimensional fluorescence revealed that the conformation of HSA was changed and the hydrophobicity of the Trp and Tyr residues microenvironment increased after formation of PFNA-HSA complex. The alterations of the protein secondary structure were quantitatively calculated from circular dichroism (CD) spectroscopy with reduction of α-helix content about 14.3%, β-sheet 5.3%, β-turn 3.5%, and augment in random content from 14.4% to 37.5%. Above results revealed that the binding of PFNA with HSA can alter the secondary structure of HSA, further probably affecting HSA physiological function. The results can provide insights with the binding mechanism of PFNA with HSA and salient biophysical and biochemical clues on elucidating the transport and distribution of PFNA in vivo.