光谱法和分子对接技术研究氟罗沙星与溶菌酶的相互作用

摘要

The interaction of fleroxacin(FLE)with extracelluar protein(lysozyme,LYSO)was investigated by using multi-spectral techniques and molecular docking in vitro.Fluorescence spectra studies indicated that FLE quenched LYSO fluorescence in a static mode with binding constants(Ka)of 4.10×104and 0.74×104 L·mol-1at 298 and 310 K,respectively.The thermodynamic parameters demonstrated that hydrogen bonds and van der Waals forces played the major role in the binding process.Based on the F?rster theory of nonradi-ative energy transfer,the binding distance(r)between FLE and the inner tryptophan residues of LYSO was calculated to be 3.16 nm.The Hill's coefficient(nH)was calculated by using involved equations,implying the negative cooperativity between them.Circular dichroism spectra(CD)indicated the secondary structure of LY-SO was partially destroyed by FLE with the α-helix percentage decreasing from 21.1% to 8.8%.UV-Vis spectral,synchronous fluorescence and three-dimensional fluorescence spectra revealed the binding interaction could cause conformational and micro-environmental changes of LYSO.The results of molecular docking showed that FLE was mainly bound in the active site hinge region where ASP-52,TRP-62 and TRP-63 were located,and well supported the thermodynamic results.Besides,FIE made the activity of lysozyme decrease with the increasing concentration of fleroxacin,indicating that structural changes in lysozyme can cause the in-hibition of lysozyme activity.The work clarified the interaction mechanism of FLE with LYSO at molecular level.%在模拟人体生理条件下,应用光谱法和分子对接技术对氟罗沙星(FIE)与溶菌酶(LYSO)的相互作用进行了研究.结果表明,FIE与LYSO的猝灭方式是静态猝灭,且在298和310 K温度下的猝灭常数 Ka分别为4.10×104和0.74×104L·mol-1.根据热力学参数的计算结果可知,FIE与LYSO的结合作用力主要是氢键和范德华力,结合距离(r=3.16 nm,<8 nm)表明从LYSO到FIE发生了非辐射能量转移.希尔系数的计算结果表明,在不同温度下的 nH<1,LYSO和 FIE的相互作用属于负协同作用.圆二色谱结果表明,LYSO和FIE的结合使LYSO的α-螺旋的含量由21.1% 减少到8.8%.紫外光谱、三维荧光光谱、同步荧光光谱结果表明,LYSO和FIE的相互作用改变了LYSO的构象和微环境.分子对接进一步显示FIE通过氢键、极性键、疏水作用力等与LYSO活性部位的ASP-52,TRP-62,TRP-63等氨基酸残基相互作用.溶菌酶的活性实验表明,由于以上实验结果说明 FIE引起了LYSO的构象改变,LYSO的活性随着FIE浓度的增大而降低,抑制了LYSO的活性.该研究结果为阐明FIE在机体内与LYSO的结合机理提供了可靠的实验数据和结果,为FIE对溶菌酶的的毒性评价和毒理学研究提供了理论依据.