With the aim to differentiate between local and global conformational changes, the proteolytic susceptibility of ribonuclease A (RNase A) toward proteinase K was analyzed in different denaturants and compared with spectroscopic and activity parameters.

The proteolytic degradation of RNase A was followed by SDS-PAGE as a function of time at different temperatures, guanidine hydrochloride (GdnHCl) and trifluoroethanol (TFE) concentrations. The rate constants of proteolysis were determined by densitometric evaluation of the protein bands and corrected regarding the proteinase K activity under the respective conditions. Activity of RNase A was assayed with 2´,3´cyclic CMP. Spectroscopic transitions were determined from changes of the UV absorbance at 287 nm or CD-signals at 279 nm.

Under native conditions, proteinase K cleaves RNase A at the Ala20-Ser21 peptide bond according to a pseudo-first order reaction. The rate constants of proteolysis as a function of temperature or the concentration of GdnHCl and TFE increase saltatorily above 45°C, 2 M GdnHCl, and 20% (v/v) TFE, respectively. These abrupt increases of proteolytic susceptibility are conform with the transitions curves observed by spectroscopic methods and indicate the global unfolding of RNase A. In the pre-transition region, however, a deviating behaviour is observed for RNase A in TFE. In the range of 0 - 20% (v/v) TFE the rate constants of proteolysis decrease by two orders of magnitude, which may be attributed to a decrease of the flexibility in the Ala20 region by a stabilisation of the adjacent a-helices. The activity changes of RNase A under the influence or temperature and TFE are similar to the changes of the proteolytic susceptibility. In contrast, a strong inactivation is observed even at very low GdnHCl concentrations being attributed to inhibiting effects by this denaturant rather than to conformational changes.