title: A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range.
abstract: 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)
authors.affiliation: Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale 62901.
authors.forename: M M
authors.initials: MM
authors.lastname: Santoro
authors.forename: D W
authors.initials: DW
authors.lastname: Bolen
mesh_terms: 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*
chemical_list: D006146:Guanidines; D013879:Thioredoxins; D019791:Guanidine