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.
Rothwarf
D M
DM
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Li
Y J
YJ
Scheraga
H A
HA
D000178:Acrylamides; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*
D000178:Acrylamides; D004220:Disulfides; D012259:Ribonuclease, Pancreatic; D004229:Dithiothreitol