Abstract
Denaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.