Regeneration of bovine pancreatic ribonuclease A. 1. Steady-state distribution.

The regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol has been studied at 25 degrees C, pH 8.0, by using a variety of current experimental techniques, including quenching the regeneration reaction with 2-aminoethyl methanethiosulfonate, fractionation of intermediates by HPLC, and analysis by both UV and disulfide-specific detection systems. The disulfide-containing protein intermediates achieve a steady-state distribution after which the native protein regenerates at a rate comparable to the rates observed previously during the regeneration of RNase A with glutathione. Equilibrium constants at 25 degrees C, pH 8.0, for the interconversion of species containing different numbers of disulfide bonds are evaluated from the concentrations in the steady-state distribution. These equilibrium constants are compared with those obtained earlier when native RNase A is regenerated with glutathione. The observed equilibrium constants (with the dithiothreitol system) for the interconversions among all intermediates are very similar once statistical factors arising from the different numbers of disulfide-containing species in each grouping are taken into account. None of the disulfide-containing intermediates has any significant enzymatic activity, in agreement with earlier conclusions that these intermediates are considerably disordered. This is in sharp contrast to disulfide-containing intermediates populated during the regeneration of bovine pancreatic trypsin inhibitor, which have significant nativelike structure.