The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: A calorimetric study

This work determines the ratio of Delta H-vH/Delta H-cal for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [CdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of Delta H-mix vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric Delta H (Delta H-cal) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (Delta G degrees(N-D)) at pH 7.0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent Delta G degrees(N-D) data defines a van't Hoff denaturation enthalpy change (Delta H-vH) of 26.3 +/- 2.8 kcal/mol. The model-dependent van't Hoff Delta H-vH divided by the model-independent Delta H-cal gives a ratio of 1.1 +/- 0.1 For Delta H-vH/Delta H-cal a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.