Regeneration of bovine pancreatic ribonuclease A: Identification of two nativelike three-disulfide intermediates involved in separate pathways

During the regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 degrees C, pH 8.0, the disulfide containing protein intermediates achieve a steady-state distribution. By manipulating the redox conditions after the attainment of the steady-state condition, it has been possible to kinetically trap and, thereby, isolate and identify the disulfide-bonded species that follow the rate-determining step in the regeneration pathway. Two three-disulfide species have been identified by peptide mapping. Both species contain three native disulfide-bond pairings, one lacks the 65-72 disulfide bond (des-[65-72]), and the other lacks the 40-95 disulfide bond (des-[40-95]). These species are the same as those identified during the reduction of RNase A. By restarting the regeneration process from isolated des-[65-72] and des-[40-95], it is shown that both intermediates lie directly on regeneration pathways.