Hu Tao-ying, Huang Fang, Zhou Shan-shan, Liu Ying
Guang Pu Xue Yu Guang Pu Fen Xi. 2016 Dec;36(12):4141-7.
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.
全氟壬酸（PFNA）是所有全氟烷基酸（PFAAs）中在血清中第三常被检测到的，全氟烷基酸是一种有毒的新型环境污染物。PFNA对人血清白蛋白（HSA）构象乃至功能的影响仍处于研究初期。本文的尝试是通过多光谱、分子对接和等温滴定量热法（ITC）技术全面阐明PFNA与HSA的相互作用机制。消除了本文中所有荧光数据的内滤效应以获得准确的结合参数。结果表明，PFNA通过动态和静态猝灭的联合猝灭过程使HSA的荧光猝灭。通过位点标记竞争实验，已确定HSA的亚结构域IIA具有PFNA的高亲和力结合位点。此外，分子对接再次证实PFNA主要通过极性力、疏水相互作用和卤键结合在亚结构域IIA中，计算得到的自由能为-26.54 kJ·mol⁻¹，这表明PFNA分子对HSA表现出较大的结合亲和力。ITC对两类不同结合位点的热力学表征显示，第一类具有较高的亲和常数，主要由静电相互作用和卤键的焓贡献主导，而第二类具有较低的亲和常数，则以疏水相互作用为主。三维荧光显示，形成PFNA-HSA复合物后，HSA的构象发生变化，色氨酸和酪氨酸残基微环境的疏水性增加。通过圆二色性（CD）光谱定量计算了蛋白质二级结构的变化，α-螺旋含量降低约为¹4.3%，β-折叠降低5.3%，β-转角降低3.5%，无规含量从14.4%增加到37.5%。上述结果表明，PFNA与HSA的结合可改变HSA的二级结构，进一步可能影响HSA的生理功能。这些结果可为PFNA与HSA的结合机制提供见解，并为阐明PFNA在体内的转运和分布提供重要的生物物理和生化线索。
