化学学报 ›› 2008, Vol. 66 ›› Issue (18): 2037-2042. 上一篇    下一篇

研究论文

荧光猝灭法和动态光散射法研究尿素-水混合溶剂中牛血清 白蛋白的构象变化

边平凤a 马 林*,b 王 旭c 许 莉a 魏志强b 林瑞森a

  

  1. (a浙江大学化学系 杭州 310027)
    (b广西大学化学化工学院 南宁 530004)
    (c湖州师范学院生命科学学院 湖州 313000)

  • 收稿日期:2007-11-23 修回日期:2008-04-13 出版日期:2008-09-28 发布日期:2008-09-28
  • 通讯作者: 马林

Conformational Study of Bovine Serum Albumin in Urea-water Mixtures by Fluorescence Quenching Technique and Dynamic Light Scattering Measurements

BIAN, Ping-Feng a MA, Lin *,b WANG, Xu c XU, Li a
WEI, Zhi-Qiang b LIN, Rui-Sen a

  

  1. (a Department of Chemistry, Zhejiang University, Hangzhou 310027)
    (b School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004)
    (c School of Life Sciences, Huzhou Teachers College, Huzhou 313000)
  • Received:2007-11-23 Revised:2008-04-13 Online:2008-09-28 Published:2008-09-28
  • Contact: MA Lin

利用荧光猝灭法和动态光散射法测定尿素-水混合溶剂中牛血清白蛋白(BSA)与荧光素的结合距离和BSA的流体动力学半径, 并通过分析BSA和荧光素在BSA-尿素-水和荧光素-尿素-水三元体系以及BSA-荧光素-尿素-水四元体系中荧光光谱的变化, 探讨尿素与蛋白质分子在水溶液中相互作用的机理及其对蛋白质构象的影响. 结果显示, BSA的3个结构域在尿素-水混合溶剂中具有不同的稳定性, 其中结构域III在尿素-水混合溶剂中是不稳定的, 而结构域I和结构域II分别在尿素浓度大于3.0和4.0 mol•L-1的混合溶剂中发生去折叠. 试验发现, BSA结构域II在低于去折叠浓度的尿素-水混合溶剂中形成更为紧密的构象, 这一现象可以归因于尿素与BSA结合引起的“蛋白质粘稠效应”

关键词: 牛血清白蛋白, 尿素, 变性, 荧光猝灭, 动态光散射

The binding distance of fluorescein to bovine serum albumin (BSA) and the hydrodynamic radius of BSA in urea-water mixtures were determined by a fluorescence quenching technique and dynamic light scattering measurements, respectively, and utilized to investigate the interaction between urea and the protein and its influence on the conformation of the protein in aqueous solution, together with the analysis of the fluorescence spectra of BSA and fluorescein in the ternary mixtures of BSA-urea-water and fluorescein-urea-water and the quaternary mixtures of BSA-flurescein-urea-water. The results showed that the three domains of BSA were of different stability in urea-water mixtures, i.e., domain III was unstable even at a low urea content and domain I and II were unfolded above urea concentration of 3.0 and 4.0 mol•L-1, respectively. Domain II of BSA was found to be of more compact conformation in the mixtures up to the subdenaturating concentration of urea, which was attributed to the effect of “protein stiffening” resulting from the binding with urea.

Key words: bovine serum albumin, urea, denaturation, fluorescence quenching, dynamic light scattering