Stabilization of pancreatic ribonuclease against urea denaturation by anion binding.

Partial renaturation of urea-denatured bovine pancreatic ribonuclease by polyanions such as phosphate and polyphosphates, as well as by nucleotides, has been observed by Sela, Anfinsen, and Harrington (1). The finding that the velocities of denaturation and renaturation of ribonuclease may be separately and easily measured (2) prompted us to reinvestigate by kinetic methods the manner by which anions impart stability to the native form of the enzyme in the presence of denaturing concentrations of urea. As expected, a number of different anions were found to inhibit the denaturation rate. In considering an analogous stabilization by calcium or strontium ions of the native structure of chymotrypsin against urea denaturation, Chervenka (3) employed a simple expression given by Johnson et al. (4) by which not only the dissociation constant of the protein-inhibitor complex but also the number of inhibitor molecules bound per molecule of protein can be determined readily. This paper presents evidence, based upon similar kinetic studies of the inhibition of ribonuclease denaturation, measurements of competitive inhibition of ribonuclease activity, and dialysis equilibrium experiments, that stabilization of the native form of ribonuclease involves binding of a single anion molecule to the catalytic site of the enzyme. From a comparison of the effect of pH upon the binding of different anions, certain ionization properties of the cationic groups at the catalytic site of ribonuclease were shown.