化学学报 ›› 2007, Vol. 65 ›› Issue (21): 2411-2416. 上一篇    下一篇

研究论文

固体表面特征对脲变α-糜蛋白酶折叠的贡献

刘振岭,柯从玉,李建军,耿信笃   

  1. (西北大学现代分离科学研究所 现代分离科学陕西省重点实验室 西安 710069)
  • 投稿日期:2006-10-27 修回日期:2007-04-10 发布日期:2007-11-14
  • 通讯作者: 耿信笃

Contribution of Solid Surface to Urea-denatured α-Chymotrypsin Folding

LIU Zhen-Ling; KE Cong-Yu; LI Jian-Jun; GENG Xin-Du   

  1. (Institute of Modern Separation Science, Shaanxi Key Laboratory of Modern Separation Science, Northwest University, Xi'an 710069)
  • Received:2006-10-27 Revised:2007-04-10 Published:2007-11-14
  • Contact: GENG Xin-Du

以脲变α-糜蛋白酶(α-Chy)为模型蛋白, 用蛋白折叠液相色谱法研究了该蛋白在7种不同固体表面上的折叠及其在折叠过程中形成的中间体, 选用疏水相互作用色谱(HPHIC)固定相为吸附剂, 在动态条件下着重研究了疏水色谱固定相TSK和PEG-600表面对脲变α-Chy复性效率的贡献. 用基质辅助激光解吸附离子化飞行时间质谱对3.0 mol•L-1脲变α-Chy, 在经 HPHIC柱复性并同时分离的收集组分进行确认后, 仅有一种稳定的脲变α-Chy折叠中间体. 发现PEG-600固定相表面较TSK固定相对α-Chy复性效果好. 证实了疏水性强度及固体表面配基的结构对蛋白折叠起着关键性的作用.

关键词: 疏水色谱, 固体表面, 蛋白折叠, α-糜蛋白酶, 折叠中间体

The contribution of the surfaces of seven kinds of solids which are the stationary phase of hydrophobic interaction chromatography (HIC) to the folding of urea-denatured, α-chymotrypsin (α-Chy) was investigated under a dynamic condition and the formed intermediate was isolated. The surfaces of the HIC stationary phase of PEG-600 and TSK were selected as the typical solid surfaces to study the folding efficiency of the urea-denatured α-Chy and the isolated intermediate of α-Chy. The obtained result indicates that the character of a solid surface has a significant contribution to protein folding. Compared with the TSK column, the surface of PEG-600 stationary phase is efficient for the renaturation of the urea-denatured α-Chy and for its quality control during α-Chy folding. Matrix-assisted laser desorption ionization time of a flight mass spectrometer was employed to identify the molecular weight of each of the collections after HPHIC separation by the two kinds of stationary phases and to confirm that there is only one stable folded intermediate from the mixture of the urea-denatured α-Chy. With the comparison of specific bioactivity of the refolded α-Chy, the surface of HIC PEG-600 stationary phase was proved to be better than that of TSK again. It further demonstrates that a suitable hydrophobic surface with good hydrophobic strength and a ligand structure plays a key role in protein folding.

Key words: hydrophobic interaction chromatography, solid surface, protein folding, α-chymotrypsin, folded intermediate