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工程科学与技术:2020,52(1):175-183
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两种烟碱对映体与胃蛋白酶作用机理的研究
(1.四川大学 化学工程学院,四川 成都 610065;2.云南中烟工业有限责任公司技术中心,云南 昆明 650231)
Investigation of Interaction Between Two Enantiomers of Nicotine and Pepsin
(1.School of Chemical Eng., Sichuan Univ., Chengdu 610065, China;2.Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)
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投稿时间:2018-09-23    修订日期:2019-02-28
中文摘要: 利用分子荧光光谱法、紫外可见吸收光谱法结合分子模拟对接技术,重点围绕烟碱(NIC)两种对映体S-NIC和R-NIC对胃蛋白酶(PEP)的荧光猝灭机理、结合常数、结合模式及对酶的活性和二级结构的影响等展开了研究。Stern-Volmer方程拟合结果显示,S-NIC和R-NIC两种对映体与PEP作用时的猝灭常数(Ksv)均随温度的增大而增加,表明二者均通过动态猝灭方式使PEP的内源性荧光强度降低,R-NIC对PEP荧光的猝灭能力明显比S-NIC强,此结果得到紫外可见吸收光谱实验的进一步验证。有效结合常数(Ka)分析可知,R-NIC与PEP的结合明显优于S-NIC。由Arrhenius方程求得S-NIC和R-NIC两种对映体与PEP相互作用的活化能(Ea)分别是12.67 kJ/mol和2.65 kJ/mol,表明R-NIC与PEP碰撞结合更强烈,疏水作用力在此过程中均起主要作用。同步和3维荧光结果表明,NIC两种对映体均诱导PEP的微环境和构象发生部分变化。酶活性研究发现,PEP活性随S-NIC及R-NIC浓度的增加而逐渐降低,相同浓度下R-NIC的抑制比S-NIC更为显著。分子对接结果是:两种对映体在PEP分子中具有相同的结合位点,二者均结合在蛋白分子N端和C端之间的沟槽内。
Abstract:The fluorescence quenching mechanism, association constant, binding mode and the activity and conformation of pepsin (PEP) between two enantiomers of nicotine (NIC) and PEP were investigated through fluorescence spectroscopy, UV-vis spectroscopy and molecular docking techniques. S-NIC and R-NIC quenched the intrinsic fluorescence of PEP through dynamic quenching mode because of the increase in the Stern-Volmer quenching constant (Ksv) values with the increase in the temperature. The quenching ability of R-NIC was significantly stronger than S-NIC, as evidenced by UV-Vis spectroscopy. The effective association constant (Ka) showed that at the same temperature decreased in the order of R-NIC > S-NIC. The activation energy (Ea) was calculated to be 12.67 kJ/mol and 2.65 kJ/mol for the binding of S-NIC and R-NIC to PEP, respectively. This difference implied an unequal participation of the two enantiomers and R-NIC was more intimate to bind with PEP, and the binding of S-NIC and R-NIC with PEP were both primarily controlled by hydrophobic forces. Synchronous and three-dimensional fluorescence spectroscopy showed that the micro-environmental and conformational of PEP were changed along with the interaction between two enantiomers and PEP. In addition, the activity experiments in vitro indicated that the activity of pepsin was inhibited with increased S-NIC and R-NIC concentration, and the R-NIC was much greater than S-NIC on the activity suppression. Molecular docking study provided the same binding sites, the two enantiomers were located the groove between the N- and C-terminal domains.
文章编号:201801047     中图分类号:    文献标志码:
基金项目:中国烟草总公司科技重大专项项目110201601005(2016xx-05);云南中烟工业公司科技开发计划项目(2018XY04);云南中烟科技项目(2019XY02)
作者简介:杨继(1980-),女,博士生,工程师.研究方向:现代分析技术与生物分析.E-mail:yang ji052@163.com
引用文本:
杨继,杨柳,汤建国,刘志华,陈永宽,缪明明,唐彬,李晖.两种烟碱对映体与胃蛋白酶作用机理的研究[J].工程科学与技术,2020,52(1):175-183.
YANG Ji,YANG Liu,TANG Jianguo,LIU Zhihua,CHEN Yongkuan,MIAO Mingming,TANG Bin,LI Hui.Investigation of Interaction Between Two Enantiomers of Nicotine and Pepsin[J].Advanced Engineering Sciences,2020,52(1):175-183.