摘要
通过多光谱法、分子模拟技术和细胞毒性实验,研究了邻苯二甲酸二(2-乙基己基)酯(DEHP)和邻苯二甲酸二正辛酯(DNOP)与胃蛋白酶(PEP)的作用机理。荧光光谱实验表明,DEHP/DNOP对PEP产生静态猝灭,形成了稳定的复合物。其相互作用是由氢键和范德华力驱动的自发反应过程(ΔG<0),结合位点均为1;且受支链结构影响,DEHP与PEP的结合常数K_(a)(1.97×10^(4) L/mol)和分子极性指数MPI(7.65 kcal/mol)均大于DNOP(1.58×10^(4) L/mol,7.43 kcal/mol)。同步荧光光谱和分子对接结果表明,DEHP和DNOP均能与PEP的Tyr 75和Tyr 189结合,导致PEP荧光猝灭以及Tyr残基微环境的疏水性增强。圆二色谱和三维荧光光谱结果表明,DEHP与PEP结合,导致PEP二级结构中的α-Helix含量增加,β-Sheet含量下降至41.4%。DEHP和DNOP对Caco-2细胞的半数抑制浓度(IC_(50))分别为4.99×10^(-4)mol/L和3.59×10^(-2)mol/L,证实了DEHP对PEP的毒性更强。
The binding mechanisms of di(2-ethylhexyl)phthalate(DEHP)and dioctyl phthalate(DNOP)to pepsin(PEP)were investigated by multispectroscopy,molecular simulation technique and cytotoxicity assay.Fluorescence spectroscopy experiments show that DEHP/DNOP has a static quenching effect on PEP and that a stable complex is formed.The binding of PEP and DEHP/DNOP is spontaneous process driven by hydrogen bonding and van der Waals forces(ΔG<0),both of DEHP and DNOP possess one binding site with PEP.Due to the effects of side chain,the DEHP has higher binding constant K_(a)(1.97×10^(4) L/mol)and molecular polarity index MPI(7.65 kcal/mol)with PEP than DNOP.Synchronised fluorescence spectroscopy and molecular docking results showed that both DEHP and DNOP can bind to Tyr 75 and Tyr 189 of PEP,resulting in PEP fluorescence quenching and increasing hydrophobicity of the Tyr residue microenvironment.The circular dichroism and three-dimensional fluorescence results indicated that DEHP has a large effect on the secondary structure of PEP with the increase ofα-Helix.The median inhibition concentrations(IC_(50))of DEHP and DNOP on Caco-2 cells are 4.99×10^(-4)mol/L and 3.59×10^(-2)mol/L,respectively,suggesting DEHP is more toxic to gastric proteins.
作者
马兰
吴雨婷
周俊锹
张杨
吴来燕
MA Lan;WU Yuting;ZHOU Junqiao;ZHANG Yang;WU Laiyann(Key Laboratory of Resources Conversion and Pollution Control of the State Ethnic Affairs Commission,College of Resources and Environmental Science,South-Central Minzu University,Wuhan 430074,China)
出处
《分析试验室》
EI
CAS
CSCD
北大核心
2024年第10期1377-1384,共8页
Chinese Journal of Analysis Laboratory
基金
国家自然科学基金(GZQ13008)
大学生创新创业训练计划(GCX2238)
中央高校科研基本业务费(CPT22027)资助。
