The thermal denaturation of ribonuclease A and its derivatives ribonuclease S and S protein was studied in phosphate at pH 6.8 by differential scanning calorimetry and a combination of optical spectroscopic techniques in order to probe the existence of intermediate states. Fourier Transform Infrared spectra of the amide I' band and far-uv circular dichroism spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv circular dichroism. Spectral bandshape changes with change in temperature were analyzed using factor analysis.
The denaturation of all studied molecules presented deviations from the two-state model. An intermediate differing in the secondary structure, but having the same tertiary structure as the native state, was observed in the thermal unfolding of both ribonuclease A and S. The secondary structure changes predominate in the -helical regions. The similarity between the pre-transitions of ribonuclease A and S as monitored by far-uv circular dichroism suggests that the intermediate state in the unfolding of ribonuclease S is a structural intermediate and that the dissociation of S peptide from S protein does not have an important contribution. However, dissociation and further unfolding occur simultaneously during the main transition. Some interactions between S protein and S peptide possibly occur in the thermally denatured state.
Additionally, the thermal denaturation of ribonuclease A was studied in a variety of aqueous solutions in the presence and absence of inhibitor. The thermodynamics of the denaturation was, as expected, influenced by interactions with the molecules present, the inhibitor having the largest influence. Also, phosphate decreased the reversibility of the transition, while the inhibitor increased it.