A TEST OF THE LINEAR EXTRAPOLATION OF UNFOLDING FREE-ENERGY CHANGES OVER AN EXTENDED DENATURANT CONCENTRATION RANGE

Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding DELTA-G vs [GdnHCl] in the transition zone. In order to extend unfolding DELTA-G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding DELTA-G values from the DSC measurements were corrected to 25-degrees-C using the Gibbs-Helmholtz equation and mapped onto the DELTA-G vs [GdnHCl] plot. The dependence of unfolding DELTA-G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater-than-or-equal-to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (DELTA-G-degrees N-U) in excellent agreement with the DELTA-G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding DELTA-G value of 8.0 kcal/mol at 25-degrees-C in the absence of denaturant. Within error, DELTA-G-degrees N-U values obtained from urea- and GdnHCl-induced unfolding are in agreement, suggesting that urea, GdnHCl, and thermal unfolding involve the same N reversible U equilibrium in this system.