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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-26.54kJ·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。
Perfluoro-nonanoic acid (PFNA) is the third-most toxic new pollutant of perfluoroalkanoic acids (PFAAs) detected in serum. At present, the effect of PFNA on the structure and even the function of human serum albumin (HSA) is still in its infancy. The binding mechanism of the interaction between PFNA and HSA has been studied by means of multispectral, molecular docking and isothermal titration calorimetry (ITC). All fluorescence data was internally filtered to obtain more accurate binding parameters. Fluorescence results show that PFNA can quench the endogenous fluorescence of HSA by means of dynamic and static quenching. The results of substitution experiments and molecular docking showed that PFNA was bonded to HSA sub-domain ⅡA hydrophobic cavity mainly through polar bond, hydrophobic force and halogen bond. The optimal docking free energy was -26.54kJ · mol-1, indicating that PFNA interacted with HSA Have a greater binding affinity. ITC shows that the combination of the two belongs to two types of binding site models and gives the corresponding thermodynamic parameters: the first type of binding sites have greater affinity, belonging to the enthalpy drive, electrostatic force and halogen bond as the main driving force; second The affinity of class binding sites is smaller, the main driving force is hydrophobic force. Three-dimensional fluorescence spectroscopy revealed that the complex of PFNA and HSA could change the conformation of HSA, resulting in enhanced hydrophobicity of Trp and Tyr residues in microenvironment. Circular dichroism (CD) was used to quantitatively determine the secondary structure contents of HSA and PFNA before and after the action: the content of α-helix, β-sheet and β-turn decreased by 14.3%, 5.3% and 3.5% Increase to 37.5%. The above results show that the combination of PFNA and HSA can change the secondary structure of HSA, which may affect the physiological function of HSA. The results elucidated the mechanism of interaction between PFNA and HSA and provided a reliable biophysical and biochemical basis for the transport and distribution of PFNA in vivo.