Reversible denaturation of pancreatic ribonuclease by urea.

Studies of the denaturation of proteins indicate certain characteristics of the forces which normally constrain them in their native chain conformations. In the case of bovine pancreatic ribonuclease, the description of the noncovalent bonds involved has taken on heightened interest following the recent report by White (1) that the integrity of the 4 disulfide bonds does not appear to be prerequisite for the correct refolding of ribonuclease to its active native form. The fact that air oxidation of completely reduced ribonuclease yields a fully active enzyme in about 80% yield, even though 105 different isomeric ways of re-forming the 4 disulfide bonds are possible (2) is strong evidence that an unusual specificity is inherent in the weak forces which determine the conformation of the polypeptide chain. Because the denaturation of ribonuclease in the presence of concentrated urea solutions proceeds at a moderately slow rate and is readily reversible, it is possible to study the kinetics and thermodynamics of the folding and unfolding processes and thus measure the magnitude of the forces which maintain the native structures. Specifically, we have investigated some of the factors that affect these processes, by following alterations in the ultraviolet absorption spectra, optical rotation, and enzymatic activity as criteria of the degree of unfolding of ribonuclease. The results reported here indicate that forces other than those involving simple electrostatic interactions play an important part in restoring and maintaining the native conformation of the polypeptide chain.