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.(ABSTRACT TRUNCATED AT 250 WORDS)
Santoro
M M
MM
Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale 62901.
Bolen
D W
DW
D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D016014:Linear Models; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation / Q000187:drug effects; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry*
D006146:Guanidines; D013879:Thioredoxins; D019791:Guanidine