Different molecular conformations, i.e., native, urea-unfolded, guanidine hydrochloride(GuHCl)-unfolded, urea-reduced-unfolded, GuHCl-reduced-unfolded, of lysozyme was systematically studied by reversed-phase liquid chromatography(RPLC), and weak-cation exchange chromatography(WCX), and the stoichiometric parameters Z in stoichiometeric displacement model(SDM). The difference of molecular conformations of lysozyme with their disulfide bonds cleaved or could not be found in RPLC and WCX in terms of retention. However, they could not be used to distinguish their reduced states from non-reduced ones. Each difference of molecular conformations of lysozyme, caused by urea and GuHCl solution, can only be characterized with Z. The definition of the degree of the unfolded was introduced to characterize the change of conformation.
The addition of packing material for high performance hydrophobic interaction chromatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin (α-Chy) denatured with guanidine hydrochloride (GuHCl) solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.
Ye Hua SHEN Quan BAI Yang Jun ZHANG Yin Mao WEI Hai Bo WANG Xing Du GENG
