Studies on the unfolding of catalase induced by urea and guanidine hydrochloride

The unfolding of bovine liver catalase induced by urea or guanidine hydrochloride (GuHCl) has been studied by "phase diagram" method of fluorescence. With increasing the concentration of urea from 0 to 0.50, 4.5 and 8.0 mol/L, the conformation of catalase is changed from the native tetramer to an expanded tetramer, a partially folded inactive dimer and the unfolded state, respectively. With increasing the concentration of GuHCl from 0 to 0.65, 2.5 and 6.0 mol/L, however, the conformation of catalase is changed from the native tetramer to a partially folded activated dimer, a partially folded monomer and the unfolded state, respectively. This indicates that the unfolding of catalase induced by urea or GuHCl follows a four-state model, but both the pathway and the mechanism for the unfolding of catalase induced by urea are different from those induced by GuHCl. The experimental results show that the "phase diagram" method of fluorescence can be used to detect unfolding intermediates of proteins. The unfolding of catalase induced by GuHCl has been studied by isothermal titration calorimetry. The intrinsic enthalpy, Gibbs free energy and entropy changes for formation of the partially folded activated dimer of the protein in the presence of low GuHCl concentrations at 25.0 degreesC were measured as -69.2 kJ(.)mol(-1), 6.43 kJ(.)mol(-1) and -254 J(.) K-1 . mol(-1) respectively. It is thus indicated that the stabilization and activation of this partially folded dimer by GuHCl originate from the entropic and the electrostatic effect of this denaturant.