KINETICS OF CTAB-ASSISTED LYSOZYME REFOLDING in vitro
The investigation of lysozyme refolding in vitro in the presence of CTAB was carried out with emphasis on the interaction of denatured lysozyme and CTAB characterized by the formation of various protein products.The changes in surface tension as well as the lysozyme activity were determined as a function of the initial concentration of denatured lysozyme and the molecular ratio of CTAB to denatured lysozyme.It was concluded from the experimental results presented that the denatured lysozyme firstly formed a complex with CTAB upon dilution into the refolding buffer, and then refolded at the input of the oxidative-reductive pair, leading to the dissociation of denatured lysozyme-CTAB complex.The non-reductive SDS-PAGE showed three kinds of protein products obtained in the above refolding process, i.e., native lysozyme, denatured lysozyme aggregate, and CTAB-denatured lysozyme.Similar result was obtained for native lysozyme incubated with CTAB for 24 hours at 37 ℃.The kinetics of CTAB-assisted refolding of lysozyme were investigated at 0.1～0.4 mg·ml -1 lysozyme with a molecular ratio of CTAB to protein equating to 5～20.The apparent kinetics of refolding could be described by a two-state refolding model, i.e., a reversible reaction between unfolded and refolded lysozyme.The apparent rate constants were shown to be a function of the molar ratio of CTAB to protein but where slightly dependent on protein concentration.The recovery of lysozyme activity, an index of the refolding performance, reached its optimum at a molecular ratio of CTAB to denatured lysozyme of 10 and decreased against an increase of the initial concentration of denatured lysozyme.