固体表面特征对脲变α-糜蛋白酶折叠的贡献

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

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3.0mol·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.

The contribution of the surfaces of seven kinds of solids which are the stationary phase of hydrophobic interaction chromatography （HIC） to the folding of ureα-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 ureα-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 ureα-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 ureα-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.