Abstract: The refolding or renaturation of reduced denaturated Insulin from bovine pancreas with 7.0 mol/Lguanidine hydrocloride(GuaHCl) solution on a moderate hydrophobic surface was studied with a high performance hydrophobic interaction chromatography(HPHIC). It showed that the reduced denaturated Insulin could be refolded partly with HPHICwhen the general mobile phase was used in HPHIC. However, in the presence of oxidized glutathione(GSSG) in the mobile phase employed, the disulfide exchange of reduced denaturated Insulin can be accelerated, and the most of three disulfide bridges of Insulin were formed correctly. The result indicates that the refolding yield of reduced denaturated Insulin with GuaHCl should be increased up to 66%. The result was further tested by the separation of the HPHICfractions of reduced denaturated Insulin with reversed phase high performance liquid chromatography(RPLC), the UVspectra, the flourescence spectra and MALDI TOF. In addition, compared to the refolding yields of reduced denaturated Insulin renaturated with size exclusive chromatography(SEC) and dilution, it showed that although GSSGwas used in the mobile phase, the reduced denaturated Insulin can not be refolded with SECat all. The refolding yield of it with dilution is only less than 2%. It proved further that during the refolding process of unfolded protein refolded on the hydrophobic surface, the interactions between Insulin and the HPHICstationary phase is very critical and may be the drive force for protein refolding and HPHICmay be the potential refolding tool of unfolded proteins.

Key words: Hydrophobic interaction chromatography, Protein refolding, Reduced denaturation, Insulin, Size exclusive chromatography, Dilution