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