Please wait a minute...
物理化学学报  2017, Vol. 33 Issue (5): 976-983    DOI: 10.3866/PKU.WHXB201702089
论文     
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 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
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 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
 全文: PDF(1096 KB)   HTML 输出: BibTeX | EndNote (RIS) |
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
收稿日期: 2017-01-13 出版日期: 2017-02-08
中图分类号:  O642  
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)
通讯作者: 白光月,王玉洁,赵扬     E-mail: baiguangyue@htu.cn;yujiewang2001@163.com;zhaoyang@htu.cn
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
白光月
刘君玲
王九霞
王玉洁
李艳娜
赵扬
姚美焕

引用本文:

白光月,刘君玲,王九霞,王玉洁,李艳娜,赵扬,姚美焕. 阳离子双子表面活性剂诱导的α-CT超活性和构象变化[J]. 物理化学学报, 2017, 33(5): 976-983.

Guang-Yue BAI,Jun-Ling LIU,Jiu-Xia WANG,Yu-Jie WANG,Yan-Na LI,Yang ZHAO,Mei-Huan YAO. Enzymatic Superactivity and Conformational Change of α-CT Induced by Cationic Gemini Surfactant. Acta Physico-Chimica Sinca, 2017, 33(5): 976-983.

链接本文:

http://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB201702089        http://www.whxb.pku.edu.cn/CN/Y2017/V33/I5/976

Fig 1  Variation of the relative activity (V12-10-12/Vb) of α-CT with concentration of 12-10-12 (C12-10-12) The symbols indicate that α-CT was incubated 10 min (●), 3 h (○) and 21 h (Δ) in 12-10-12 solution. The concentrations of the other components except 12-10-12 were constant, 0.020 g·L-1 α-CT, 0.097 mmol·L-1 2-NA, 10 mmol·L-1 PBS (pH = 7.3), respectively.
Fig 2  Variation of observed enthalpy per mole of 12-10-12 with the concentration of 12-10-12 at 298.15 K () in water; () in 10 mmol·L-1 PBS (pH 7.3); (Δ) in 10 mmol·L-1 PBS with 0.02 g·L-1 α-CT in the cell. The C12-10-12 in the syringe is 6.2 mmol·L-1.
Fig 3  ITC results for titration of 12-10-12 into α-CT solution at 298.15 K (Ⅰ) Variation of observed enthalpy with the concentration of 12-10-12, (Ⅱ) Difference in the observed enthalpy values in the presence of α-CT and in its absence. The α-CT concentration (g·L-1) in 10 mmol·L-1 PBS (pH 7.3): (●) 0; () 0.05; (▲) 0.10; () 0.20; and () 0.30, respectively. The C12-10-12 in the syringe is 2.0 mmol·L-1
Fig 4  Fluorescence spectra of α-CT at the different incubation time in PBS at 298 K The incubation time increases along the arrow direction: 0.33, 3.0, 24, 48 and 72 h, respectively, and the Cα-CT was kept at 0.02 g·L-1.
Fig 5  Fluorescence spectra of α-CT at different incubation time in different 12-10-12 concentrations at 298 K The concentration of 12-10-12: (Ⅰ) 0.025 mmol·L-1, (Ⅱ) 0.073 mmol·L-1, (Ⅲ) 0.16 mmol·L-1. The incubation times (unit in h) were denoted as a, 0.33 in buffer; b, 0.17; c, 2.0; d, 3.0; e, 23; f, 47; g, 96; h, 144; i, 190. The arrows point to the corresponding curve or the direction of increase in incubation time. Notes: before addition of 12-10-12 the buffered α-CT solution was incubated 0.33 h in PBS and the Cα-CT is kept at 0.02 g·L-1.
Fig 6  DSC curves for α-CT solutions incubated 20 min in buffer and 12-10-12/buffer (Ⅰ) The 12-10-12 concentrations (mmol·L-1) increase along the arrow direction to be: 0, 0.005, 0.01, 0.02, 0.04, 0.08, 0.12, 0.16, 0.20, 0.25, 0.28, respectively. (Ⅱ) Two representative original curves were presented in symbol (○) for C12-10-12 = 0 and (△) for C12-10-12 = 0.08 mmol·L-1. Multi-peak fitting lines of experimental curve Cp vs T for α-CT/12-10-12 were obtained through Gaussian function and the corresponding temperatures were marked with Tm, 1 and Tm, 2.
C12-10-12/(mmol·L-1)Tm, 1/℃ΔH1/(J·g-1)Tm, 2/℃ΔH2/(J·g-1)
048.9 ± 0.420.1 ± 0.257.9 ± 0.41.7 ± 0.8
0.00547.8 ± 0.420.3 ± 0.258.2 ± 0.40.9 ± 0.8
0.01043.9 ± 0.419.2 ± 0.251.2 ± 0.41.3 ± 0.8
0.02041.6 ± 0.417.1 ± 0.248.5 ± 0.41.6 ± 0.8
0.04039.8 ± 0.415.0 ± 0.247.4 ± 0.41.7 ± 0.8
0.08037.2 ± 0.411.4 ± 0.245.2 ± 0.42.0 ± 0.8
0.1236.8 ± 0.47.7 ± 0.244.0 ± 0.41.8 ± 0.8
0.1636.9 ± 0.47.3 ± 0.244.2 ± 0.41.7 ± 0.8
0.2036.5 ± 0.47.0 ± 0.243.8 ± 0.41.4 ± 0.8
0.2536.4 ± 0.46.3 ± 0.243.7 ± 0.41.8 ± 0.8
0.2836.8 ± 0.45.7 ± 0.243.9 ± 0.41.3 ± 0.8
Table 1  Thermodynamic parameters Tm, 1, Tm, 2, ΔH1and ΔH2 for α-CT in PBS containing different 12-10-12 concentrations
1 Savelli G. ; Spretib N. ; Di Profioa P. Curr. Opin. ColloidInterface Sci. 2000, 5, 111.
doi: 10.1016/S1359-0294(00)00043-1
2 Kumar A. ; Venkatesu P. Chem. Rev. 2012, 112, 4283.
doi: 10.1021/cr2003773
3 Simon L. M. ; Kotormán M. ; Garab G. ; Laczkó I. Biochem.Biophys. Res. Commun. 2002, 293, 416.
doi: 10.1016/S0006-291X(02)00246-2
4 Spreti N. ; Alfani F. ; Cantarella M. ; D'Amico F. ; Germani R. ; Savelli G. J. Mol.Catal. B: Enzym. 1999, 6, 99.
doi: 10.1016/S1381-1177(98)00139-8
5 Alfani F. ; Cantarella M. ; Spreti N. ; Germani R. ; Savelli G. Appl. Biochem. Biotechnol. 2000, 88, 1.
doi: 10.1385/ABAB:88:1-3:001
6 Abuin E. ; Lissi E. ; Calderón C. J. Colloid Interface Sci. 2007, 308, 573.
doi: 10.1016/j.jcis.2007.01.007
7 Abuin E. ; Lissi E. ; Duarte R. J. Mol. Catal. B: Enzym. 2004, 31, 83.
doi: 10.1016/j.molcatb.2004.07.009
8 Viparelli P. ; Alfani F. ; Cantarella M. J. Mol. Catal. B: Enzym. 2003, 21, 175.
doi: 10.1016/S1381-1177(02)00125-X
9 Celej M. S. ; D'Andrea M. G. ; Campana P. T. ; Fidelio G. D. ; Bianconi M. L. Biochem. J. 2004, 378, 1059.
doi: 10.1042/BJ20031536
10 Banerjee D. ; Pal S. K. Langmuir 2008, 24, 8163.
doi: 10.1021/la8010184
11 Suh J. ; Lee Y. ; Han S. Bioorg. Med. Chem. Lett. 1998, 8, 1331.
doi: 10.1016/S0960-894X(98)00211-X
12 Pitt-Rivers R. ; Impiombato F. S. Biochem. J. 1968, 109, 825.
doi: 10.1042/bj1090825
13 Reynolds G. J. A. ; Tanford C. Proc. Natl. Acad. Sci. U. S. A. 1970, 66, 1002.
doi: 10.1073/pnas.66.3.1002
14 Abuin E. ; Lissi E. ; Duarte R. Langmuir 2003, 19, 5374.
doi: 10.1021/la030050s
15 Wang Y. ; Bai G. ; Marques E. F. ; Yan H. J. Phys. Chem. B 2006, 110, 5294.
doi: 10.1021/jp054323z
16 Lou P. X. ; Wang Y. J. ; Bai G. Y. ; Fan C. Y. ; Wang Y. L. ActaPhys.-Chim. Sin. 2013, 29, 1401.
doi: 10.3866/PKU.WHXB201304282
娄朋晓; 王玉洁; 白光月; 范朝英; 王毅琳. 物理化学学报, 2013, 29, 1401.
doi: 10.3866/PKU.WHXB201304282
17 Wang Y. ; Lou P. ; Bai G. ; Fan C. ; Bastos M. J. Chem.Thermodyn. 2014, 73, 255.
doi: 10.1016/j.jct.2014.01.010
18 Belyaeva E. ; Gra D. ; Eremeev N. Biochemistry (Moscow) 2002, 67, 1032.
doi: 10.1023/A:1020530220774
19 Blocher M. ; Walde P. ; Dunn I. J. Biotechnol. Bioeng. 1999, 62, 36.
doi: 10.1002/(SICI)1097-0290(19990105)62:1
20 Bai G. ; Yan H. ; Thomas R. K. Langmuir 2001, 17, 4501.
doi: 10.1021/la001472u
21 Bai G. ; Wang Y. ; Ding Y. ; Zhuo K. ; Wang J. ; Bastos M. J.Chem. Thermodyn. 2016, 94, 221.
doi: 10.1016/j.jct.2015.11.017
22 Bagger H. L. ; Hoffmann S. V. ; Fuglsang C. C. ; Westh P. Biophys. Chem. 2007, 129, 251.
doi: 10.1016/j.bpc.2007.06.005
23 Lad M. D. ; Ledger V. M. ; Briggs B. ; Green R. J. ; Frazier R.A. Langmuir 2003, 19, 5098.
doi: 10.1021/la0269560
24 Nielsen A. D. ; Borch K. ; Westh P. Biochim. Biophys. Acta 2000, 1479, 321.
doi: 10.1016/S0167-4838(00)00012-1
25 Xie H. J. ; Liu C. C. ; Sun Q. ; Gu Q. ; Lei Q. F. ; Fang W. J. Acta Phys.-Chim. Sin 2016, 32, 2951.
doi: 10.3866/PKU.WHXB201609231
谢湖均; 刘程程; 孙强; 顾青; 雷群芳; 方文军. 物理化学学报, 2016, 32, 2951.
doi: 10.3866/PKU.WHXB201609231
26 Otzen D. E. ; Sehgal P. ; Westh P. J. Colloid Interface Sci. 2009, 329, 273.
doi: 10.1016/j.jcis.2008.10.021
27 Otzen D. Biochim. Biophys. Acta 2011, 1814, 562.
doi: 10.1016/j.bbapap.2011.03.003
28 Hamill A. C. ; Wang S. C. ; Lee C. T. ; Jr. Biochemistry 2007, 46, 7694.
doi: 10.1021/bi700233k
29 Castro G. R. Enzyme Microbial Technol. 2000, 27, 143.
doi: 10.1016/S0141-0229(00)00197-6
30 Kijima T. ; Yamamoto S. ; Kise H. Enzyme Microbial Technol. 1996, 18, 2.
doi: 10.1016/0141-0229(96)00042-7
31 Creagh A. L. ; Prausnitz J. M. ; Blanch H.W. Biotechnol.Bioeng. 1993, 41, 156.
doi: 10.1002/bit.260410120
32 Mozhaev V. V. ; Khmelnitsky Y. L. ; Sergeeva M. V. ; Belova A.B. ; Klyachko N. L. ; Levashov A. V. ; Martinek K. Eur. J.Biochem. 1989, 184, 597.
doi: 10.1111/j.1432-1033.1989.tb15055.x
33 Roberge M. ; Lewis R. N. A. H. ; Shareck F. ; Morosoli R. ; Kluepfel D. ; Dupont C. ; McElhaney R. N. Proteins: Structure, Function, and Genetics 2003, 50, 341.
doi: 10.1002/prot.10262
34 Fang Y. Y. ; Hu X. G. ; Yu L. ; Li W. B. ; Lin R. S. ActaPhys.-Chim. Sin. 2007, 23, 84.
doi: 10.3866/PKU.WHXB20070117
方盈盈; 胡新根; 于丽; 李文兵; 林瑞森. 物理化学学报, 2007, 23, 84.
doi: 10.3866/PKU.WHXB20070117
[1] 张晨辉,赵欣,雷津美,马悦,杜凤沛. 非离子表面活性剂Triton X-100溶液在不同生长期小麦叶片表面的润湿行为[J]. 物理化学学报, 2017, 33(9): 1846-1854.
[2] 赵文荣,郝京诚,HeinzHoffmann. 磁性非对称双链长表面活性剂囊泡凝胶[J]. 物理化学学报, 2017, 33(8): 1655-1664.
[3] 田茂章,张帆,马骋,马德胜,蒋凌翔,薛荣荣,刘卡尔顿,黄建滨. 基于广义阴阳表面活性剂体系对不同黏度区间原油的普适性降黏作用[J]. 物理化学学报, 2017, 33(8): 1665-1671.
[4] 甘永平,林沛沛,黄辉,夏阳,梁初,张俊,王奕顺,韩健峰,周彩红,张文魁. 表面活性剂对氧化铝修饰富锂锰基正极材料的影响[J]. 物理化学学报, 2017, 33(6): 1189-1196.
[5] 孔伟伟,郭爽,张永民,刘雪锋. 含硒磺基甜菜碱表面活性剂界面性能的氧化-还原响应行为[J]. 物理化学学报, 2017, 33(6): 1205-1213.
[6] 张婷, 沈杰. 含酯基Gemini表面活性剂在有机醇-水体系中的胶束热力学及聚集行为[J]. 物理化学学报, 2017, 33(4): 795-802.
[7] 袁鸿, 张静, 魏学红, 方慧敏, 袁世芳, 吴立新. 基于铕取代多金属氧簇的手性发光液晶材料[J]. 物理化学学报, 2017, 33(2): 407-412.
[8] 周萍萍, 席皙, 宋冰蕾, 裴晓梅, 崔正刚. 三聚阴离子表面活性剂/阳离子添加剂混合体系的流变行为[J]. 物理化学学报, 2016, 32(9): 2309-2317.
[9] 王娇, 杨利军, 朱甜甜, 汪慎之, 陈忠秀. 大分子拥挤环境中脂肪酸影响磷脂囊泡相变的差示扫描量热研究[J]. 物理化学学报, 2016, 32(8): 2027-2038.
[10] 孙小祥, 陈宇, 赵剑曦. 气相二氧化硅/季铵Gemini表面活性剂稳定的泡沫体系[J]. 物理化学学报, 2016, 32(8): 2045-2051.
[11] 吴伟, 刘丹丹, 徐志成, 宫清涛, 黄建滨, 张磊, 张路. 支链化甜菜碱和阳离子表面活性剂在聚甲基丙烯酸甲酯表面的吸附及其润湿性质[J]. 物理化学学报, 2016, 32(5): 1214-1220.
[12] 刘子瑜, 廖琦, 靳志强, 张磊, 张路. 分子动力学模拟电解质对阴非离子表面活性剂界面行为的影响[J]. 物理化学学报, 2016, 32(5): 1168-1174.
[13] 韩传红, 耿培培, 郭严, 陈肖肖, 郭晓冬, 张军红, 刘杰, 魏西莲. 阳离子表面活性剂和有机酸混合水溶液的热响应特性[J]. 物理化学学报, 2016, 32(4): 863-871.
[14] 谢湖均, 刘程程, 孙强, 顾青, 雷群芳, 方文军. 季铵盐型阳离子表面活性剂与牛血清白蛋白的相互作用[J]. 物理化学学报, 2016, 32(12): 2951-2960.
[15] 卞鹏程, 张大鹏, 刚洪泽, 刘金峰, 牟伯中, 杨世忠. 以油酸为原料的新型生物基支链十七烷基苯磺酸钠的合成及性质[J]. 物理化学学报, 2016, 32(11): 2753-2760.