A New Method for Testing the Functional Dependence of Unfolding Free Energy Changes on Denaturant Concentration1

Abstract

Denaturations of ribonuclease A, lysozyme, and cytochrome c by guanidine hydrochloride (GdnHCI), urea, and GdnHCl-urea mixture were studied at constant temperature and pH to assess the functional dependence of denaturational free energy change (ΔG_D) on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl-induced transition curve exhibits a linear plot of ΔG_D, versus [GdnHCl] in the transition zone. To extend ΔG_Dmeasurements beyond this narrow concentration range, GdnHCl-induced unfolding was measured in the presence of different concentrations of urea. ΔG_D values from these measurements were corrected for the effect of urea on the free energy change using the appropriate relation. The corrected ΔG_D data were mapped onto the ΔG_Dversus [GdnHCl] plot. For each protein, the dependence of free energy change on denaturation was found to be linear over the full GdnHCl concentration.