Abstract:

The refolding of denatured hen egg white lysozymes induced by guanidine hydrochloride and the distribution and transition of stable conformations of denatured hen egg white lysozymes during the refolding procedure were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and native-PAGE, high-performance size-exclusion chromatography, intrinsic fluorescence emission spectra and fluorescence phase diagram, and biological activity assay. The results show that during the refolding procedure of denatured hen egg white lysozymes induced by guanidine hydrochloride, two stable intermediates of hen egg white lysozymes separately existed at about 5.0 and 2.4 mol/L of guanidine hydrochloride in renaturation solution, and this refolding followed a four-state model. Based on the four-state model, an equation which described the renaturation rate of denatured protein molecules under different denaturant concentrations in renaturation solution was presented and through this equation two characteristic refolding parameters, the thermodynamic refolding equilibrium constant k of the conformational transition of denatured protein molecules from their one conformational states to their another ones and the number m of denaturant molecules associated with an protein molecule during the conformational transitions, can be simultaneously derived. And further, by means of these two characteristic refolding parameters, the distribution and transition of stable conformations of egg white lysozyme molecules under different guanidine hydrochloride concentrations in denaturation solution can be well described.

Key words: Hen egg white lysozyme, Guanidine hydrochloride, Refolding, Characteristic refolding parameter, Fluorescence spectrum