Distribution And Transition Of Each Conformational State During The Unfolding And Refolding Procedures Of Egg White Lysozyme Induced By Denaturant

The folded intermediates and their corresponding denaturant concentration were studied by using intrinsic fluorescence spectroscopy and phase diagram during unfolding and refolding procedures of egg white lysozyme induced by denaturant; Based on the association-disassociation interaction between the protein molecules and denaturant molecules in solution, we presented a theoretical model which described unfolding and refolding procedures of the protein molecules induced by denaturants. Through the theoretical model, two important parameters can be simultaneously obtained, they separately were the thermodynamic equilibrium constant for the transition of the protein molecules from their one conformational states to their another ones k and the number of denaturant molecules associated with one protein molecule during this procedure; By using these two characteristic parameters, we could derive the distribution and transition of each conformational state of egg white lysozyme molecules during unfolding and refolding procedures. The main results includes the following parts:1. During the unfolding and refolding procedures of egg white lysozyme induced by urea and guanidine hydrochloride, there existed a folded intermediate, when the urea concentrations in denaturation solution was about 4 mol/L or guanidine hydrochloride concentrations was about 3 mol/L; In the absence of reducing agent, there existed two folded intermediates during the refolding procedure of egg white lysozyme induced by guanidine hydrochloride, when the guanidine hydrochloride concentrations in renaturation solution was about 5.0 mol/L and 2.4 mol/L, respectively; In the presence of reducing agent, however, there was no folded intermediate during the refolding procedure of the reduced egg white lysozyme in guanidine hydrochloride solution, the refolding procedure of the reduced egg white lysozyme in urea solution existed a folded intermediate, when the urea concentrations in renaturation solution was about 4.5 mol/L.2. During the unfolding procedure of egg white lysozyme induced by urea,κ1= 1.78×10-3 L-mol-1,k2= 2.95×10-2 L-mol-1, m1= 5.14,m2= 2.58; during the unfolding procedure of egg white lysozyme induced by guanidine hydrochloride,κ1= 4.16×10-2 L-mol-1,κ2= 3.64×10-3 L-mol-1, m1= 3.41,m2= 4.86; during the refolding procedure of egg white lysozyme induced by guanidine hydrochloride, k1= 4.98×103 L-mol-1,κ2= 3.74 L-mol-1,κ3=3.26 L-mol-1, m1= 5.08,m2= 3.71,m3= 1.89; during the refolding procedure of the reduced egg white lysozyme in guanidine hydrochloride solution, k= 1.41×10-1L·mol-1,m= 3.69; during the refolding procedure of the reduced egg white lysozyme in urea solution, k1= 7.70×102 L-mol-1,κ2=6.49 L·mol-1, m1= 5.63,m2= 2.03.3. During the unfolding procedure of egg white lysozyme induced by urea solution, it was more difficult for the egg white lysozyme from the native state to the folded intermediate than it from the folded intermediate to the unfolded state; during the unfolding procedure of egg white lysozyme induced by guanidine hydrochloride solution, it was easier for the egg white lysozyme from the native state to the folded intermediate than it from the folded intermediate to the unfolded state; while during the refolding procedure of egg white lysozyme induced by guanidine hydrochloride solution, it was easier for the egg white lysozyme from the unfolded state to the first folded intermediate than it from the first folded intermediate to the second folded intermediate, which in turn easier than from the second folded intermediate to the native state; during the refolding procedure of the reduced egg white lysozyme in urea solution, it was easier for the egg white lysozyme from the unfolded state to the folded intermediate than it from the folded intermediate to the native state.4. When the parameters k and m determined, we could obtain the fractions of native egg white lysozyme molecules, the folded intermediate molecules and completely unfolded state molecules under different urea and guanidine hydrochloride concentrations. The results showed that the theoretical model not only could derive the distribution of each conformational state of egg white lysozyme molecules during unfolding and refolding procedures, but also could describe the transition of each conformational state.