物理化学学报 >> 2017, Vol. 33 >> Issue (5): 976-983.doi: 10.3866/PKU.WHXB201702089

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阳离子双子表面活性剂诱导的α-CT超活性和构象变化

白光月1,*(),刘君玲1,王九霞2,王玉洁2,*(),李艳娜1,赵扬1,*(),姚美焕1   

  1. 1 河南师范大学化学化工学院,精细化学品绿色制造河南省协同创新中心,绿色化学介质与反应教育部重点实验室,河南新乡453007
    2 河南科技学院化学化工学院,河南新乡453003
  • 收稿日期:2017-01-13 发布日期:2017-04-20
  • 通讯作者: 白光月,王玉洁,赵扬 E-mail:baiguangyue@htu.cn;yujiewang2001@163.com;zhaoyang@htu.cn
  • 基金资助:
    国家自然科学基金(21273061);国家自然科学基金(21573061);河南省高等学校重点科研项目(17A150032)

Enzymatic Superactivity and Conformational Change of α-CT Induced by Cationic Gemini Surfactant

Guang-Yue BAI1,*(),Jun-Ling LIU1,Jiu-Xia WANG2,Yu-Jie WANG2,*(),Yan-Na LI1,Yang ZHAO1,*(),Mei-Huan YAO1   

  1. 1 Collaborative Innovation Centre of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, Henan Province, P. R. China
    2 School of Chemistry and Chemical Engineering, Henan Institute of Science and Technology, Xinxiang 453003, Henan Province, P. R. China
  • Received:2017-01-13 Published:2017-04-20
  • Contact: Guang-Yue BAI,Yu-Jie WANG,Yang ZHAO E-mail:baiguangyue@htu.cn;yujiewang2001@163.com;zhaoyang@htu.cn
  • Supported by:
    the National Natural Science Foundation of China(21273061);the National Natural Science Foundation of China(21573061);and Scientific Research Project of Higher Education of Henan Province, China(17A150032)

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摘要：

报道了α-糜蛋白酶(α-CT)催化活性及其与阳离子双子表面活性剂(N, N'-双(十二烷基二甲基)-1, 2-二溴化癸二铵,简写为12-10-12)相互作用热力学的关系.酶活性通过紫外-可见吸收光谱法测量底物醋酸2-萘酯(2-NA)分解速率进行评估.在较短的培育时间内, 12-10-12能够激活α-CT,得到催化2-NA分解的超活性,同时也加速了酶变性的动力学过程.在低于α-CT/12-10-12体系的临界聚集浓度(cac12-10-12, CT)时,显示一个钟形的大的超活性区.酶活性随时间变化的结果表明,由12-10-12激活的α-CT有高的酶活性和低的变性稳定性.进而采用等温滴定量热(ITC)、稳态荧光光谱和差示扫描量热(DSC)技术研究了12-10-12诱导α-CT超活性的机理.结果表明酶的超活性来源于正电荷的12-10-12与α-CT相互作用对α-CT内部结构的扰动,使得酶的构象变得比处于弱相互作用平衡的天然酶的构象更加松弛,这有利于2-NA水解酸性产物释放的动力学,而同时也导致了α-CT结构的不稳定性.

关键词: 表面活性剂, α-糜蛋白酶, 超活性, 等温滴定量热, 稳态荧光, 差示扫描量热

Abstract:

This work presents the correlation of the enzymatic activity of α-chymotrypsin (α-CT) with the thermodynamics of interaction between α-CT and the cationic gemini surfactant decanediyl-α, ω-bis (dodecyldimethylammonium bromide) (12-10-12). The enzymatic activity was assessed by the rate of 2-naphthyl acetate (2-NA) hydrolysis obtained from UV-Vis absorption spectra. The superactivity of α-CT in the catalytic hydrolysis of 2-NA was obtained by activation with 12-10-12 in a short incubation time; the activated α-CT showed faster denaturation kinetics. The larger superactivities appeared in a bell shape below the critical aggregation concentration (cac12-10-12, CT) of the mixed gemini/α-CT systems in buffered aqueous solution. The results obtained from the variation of the activity with the incubation time highlight that the protein incubated in 12-10-12 has a high catalysis activity and a weakened conformational stability. The mechanism of the superactivity of α-CT in the presence of 12-10-12 has been proposed by combining the results from isothermaltitration calorimetry (ITC), steady state fluorescence, and differential scanning calorimetry (DSC). The superactivity arises from perturbation of the internal structure of α-CT by an interaction between the positively charged 12-10-12 and α-CT, which makes the conformation of α-CT looser than the native one, in the balance of a weak interaction. Such a conformation is favorable for release of the acidic product of 2-NA hydrolysis, whereas it simultaneously leads to instability of the α-CT structure.

Key words: Surfactant, α-chymotrypsin, Superactivity, Isothermal titration calorimetry, Steady state fluorescence, Differential scanning calorimetry