The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturation with urea (DeltaG degrees (urea)), GdnHCl (DeltaG degrees (Gdn)), and heat (DeltaG degrees (T)). Second,by numerical integration of H+ titration curves (DeltaG degrees (Deltav)) measured potentiometrically under native (100 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of stability described by DeltaG degrees (urea), DeltaG degrees (Gdn) and DeltaG degrees (T) was comparable but significantly different from the one described by DeltaG degrees (Deltav). The decrease in DeltaG degrees (Deltav) between pH 9 and pH 4 was 4 kcal/mol greater than the decrease in DeltaG degrees (urea), DeltaG degrees (Gdn), and DeltaG degrees (T) in the same pH range. In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, DeltaG degrees (Deltav) represents the free energy difference between the native state (N) and an ensemble of unstructured, or expanded, and highly screened conformations. In contrast, the shallower pH dependence of stability described by DeltaG degrees (urea) and by DeltaG degrees (T) between pH 9 and 5 was consistent with the titration of histidines with depressed, nativelike pK(a) values in the denatured state (D), These depressed pK(a) values likely reflect long-range electrostatic interactions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in DeltaG degrees (urea) at and DeltaG degrees (T) at pH < 5 suggests that near pH 5 the structural and thermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrostatic interactions with basic residues are lost, maybe as a consequence of an expansion.
