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.
Rothwarf
D M
DM
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D010084:Oxidation-Reduction; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
D004220:Disulfides; D012259:Ribonuclease, Pancreatic; D004229:Dithiothreitol