Dissociation and unfolding of jack bean urease studied by fluorescence emission spectroscopy

Abstract

Abstract Thermal and chemical denaturation of urease has been studied by fluorescence spectroscopy. For thermal denaturation, a change in structure was indicated by a change in the wavelength of maximal intensity, λmax. The fluorescence peak position shifted from 330 nm to 335 nm upon heating urease from room temperature to 75° C, with a Tm corresponding to 72.4° C. The thermal denaturation was irreversible. For the chemical denaturation, the shift in λmax max was from 330 nm to 346 nm with a [GuHCl] 1 2 of 2 M, and was reversible. The denaturation of urease was found to be dependent on urease concentration, suggesting that the equilibrium involves different urease with different number of subunits before and after the transition. The free energy of unfolding was calculated at different concentrations of urease. A large ΔGu of 19.0 ± 0.3 kcal mol−1 was consistently obtained assuming a dimer to monomer dissociation. The structure stability of urease is, therefore, among the highest studied to date. Our results and other data are integrated in a dissociation model for urease where the active hexamer dissociates into folded dimers which then aggregate or dissociate into unfolded monomers. Improvement of urease stability is discussed taking into account the proposed dissociation model.