高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (1): 90.doi: 10.7503/cjcu20180397

• 有机化学 • 上一篇    下一篇

基于ThT荧光寿命检测蛋清溶菌酶蛋白寡聚体

李成1, 宋吉雪1, 刘婷婷1, 李妍1, 刘冰南1, 王亮1, 肖珊1, 李琳2, 耿旭辉3(), 王际辉1,2()   

  1. 1. 大连工业大学生物工程学院, 大连 116034
    2. 东莞理工学院化学工程与能源技术学院, 东莞 523808
    3. 中国科学院分离分析化学重点实验室, 大连 116023
  • 收稿日期:2018-05-30 出版日期:2019-01-10 发布日期:2018-12-12
  • 作者简介:

    联系人简介: 王际辉, 男, 博士, 教授, 博士生导师, 主要从事天然活性物质的开发及活性方面的研究.E-mail: wangjh_dlpu@163.com;耿旭辉, 男, 博士, 副研究员, 主要从事高灵敏光学传感器方面的研究. E-mail: xuhuigeng@126.com

  • 基金资助:
    国家重点研发计划项目(批准号: 2016YFD0400203)、 辽宁省自然科学基金重点项目(批准号: 20170520043)和中国科学院分离分析化学重点实验室开放基金(批准号: KL-1704)资助.

Detection of Egg White Lysozyme Oligomers Based on Fluorescence Lifetime of ThT

LI Cheng1, SONG Jixue1, LIU Tingting1, LI Yan1, LIU Bingnan1, WANG Liang1, XIAO Shan1, LI Lin2, GENG Xuhui3,*(), WANG Jihui1,2,*()   

  1. 1. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China
    2. School of Chemical Engineering & Energy Technology, Dongguan University of Technology, Dongguan 523808, China;
    3. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian 116032, China
  • Received:2018-05-30 Online:2019-01-10 Published:2018-12-12
  • Contact: GENG Xuhui,WANG Jihui E-mail:xuhuigeng@126.com;wangjh_dlpu@163.com
  • Supported by:
    † Supported by the National Key Research Program of China(No.2016YFD0400203), the Natural Science Foundation of Liaoning Province, China(No.20170520043) and the Open Laboratory Fund of Key Laboratory of Separation and Analytical Chemistry, Chinese Academy of Sciences, China(No.KL-1704).

摘要:

采用时间分辨荧光技术, 检测了不同形态蛋白聚集体的荧光染料硫磺素T(ThT)荧光寿命. 利用蛋清溶菌酶体外制备了蛋白聚集体; 采用透射电子显微镜(TEM)及ThT稳态荧光检测了结合蛋白纤维生长的动力学曲线, 确定其形成寡聚体及纤维样聚集体的特征和时间. 通过时间相关单光子计数(TCSPC)技术测定了蛋清溶菌酶单体、 寡聚体和淀粉样纤维的ThT荧光寿命曲线, 并拟合、 计算其荧光寿命. 根据圆二色谱(CD)分析结果推测聚集体的结构不同导致其与ThT的结合状态不同, 从而影响ThT荧光寿命. 结果表明, 通过测定ThT荧光寿命可以区分蛋白单体、 寡聚体和纤维样聚集体, 并监测蛋白寡聚体的形成, 为后续病理蛋白聚集过程中形成寡聚体物质的监测提供了研究基础.

关键词: 寡聚体, 硫磺素T, 时间相关单光子计数, 荧光寿命

Abstract:

Thioflavin T(ThT) fluorescence lifetime was used to detect aggregates states by time-resolved fluorescence. The protein aggregation was made by the egg white lysozyme in vitro. The characteristics of the oligomer and fibril were determined by means of transmission electron microscopy, ThT steady-state fluorescence and the growth kinetic curve of aggregation. The time-correlated single-photon counting technique was used to measure the fluorescence lifetime of ThT incubation with the aggregations. It calculated the fluorescence lifetime by fitting to the double exponential equation. The results of circular dichroism speculated that the structure of different aggregates was different and affected the fluorescence lifetime of ThT. It demonstrated that ThT fluorescence lifetime can distinguish protein monomers, oligomers and fibrils, and monitor the formation of protein oligomers. The results provide a basis for the monitoring of oligomer substances in the proess of sub-sequent pathological protein aggregation.

Key words: Oligomer, Thioflavin T, Time dependent single photon counting, Fluorescence lifetime

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