Nuclear magnetic resonance studies of the unfolding of pancreatic ribonuclease. II. Unfolding by urea and guanidine hydrochloride.

The unfolding of ribonuclease by urea and guanidine hydrochloride has been studied by ¹H nuclear magnetic resonance spectroscopy, under conditions where the unfolding is fully reversible and concentration-independent. Both urea and guanidine produce marked changes in the chemical shift of the histidine C₍₂₎H resonances, together with small changes in other regions of the spectrum, at concentrations (0.1 to 1.0 m) far below those which are required for gross unfolding of the protein. The changes in area of the histidine C₍₂₎H resonances through the major unfolding transition produced by these denaturants give evidence for the existence of at least two intermediates in the unfolding process. The “order of unfolding” of the histidine residues is closely similar for both urea and guanidine hydrochloride unfolding, and also similar to that found for thermal unfolding at low pH (see Benz &; Roberts, 1975) accompanying paper.