This work determines the ratio of ΔH(vH)/ΔH(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 [GdnHCl] were determined at pH 7.0 and 25 °C. The resulting plot of ΔH(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 ΔH (ΔH(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 (ΔG°(N-D)) at pH 7.0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 °C. Analysis of the resulting temperature-dependent ΔG°(N-D) data defines a van't Hoff denaturation enthalpy change (ΔH(vH)) of 26.4 ± 2.8 kcal/mol. The model-dependent van't Hoff ΔH(vH) divided by the model- independent ΔH(cal) gives a ratio of 1.1 ± 0.1 for ΔH(vH)/ΔH(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.
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