Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads.
The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads. Reoxidation was performed under aerobic conditions, in the presence of mercaptoethanol and dehydroascorbate or with a mixture of reduced and oxidized glutathione. In 24 hours, the yields of regenerated trypsinogen were 60 to 70% with 0.2 to 0.6 mg of protein bound/ml of gel but 30% or less if greater than 1.7 mg of protein were bound. Rapid reoxidation, with dehydroascorbate as catalyst, gave molecules which could not be converted to active trypsin. However, if the incorrectly folded structures were placed in a mixture of reduced and oxidized glutathione, the molecules underwent disulfide interchange and could continue to refold. The rapidly reoxidized molecules regained their native structure with the same rate and to the same extent as they did initially in the absence of rapid reoxidation. Therefore, the rate-limiting step in the refolding of trypsinogen was disulfide interchange. The regenerated Agarose-bound trypsinogen displayed the usual properties of the native molecule in (a) its conversion to active trypsin by a process of limited proteolysis, (b) the kinetic constants of the activated product toward typical trypsin substrates, and (c) the limited cleavage of 1 disulfide bond with sodium borohydride. Refoldind of immobilized trypsin was also observed with an overall yield of 50%. Trypsin can fold spontaneously to its native structure even though it lacks the NH2-terminal hexapeptide of its precursor.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003488:Cyanogen Bromide; D004220:Disulfides; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012685:Sepharose; D012995:Solubility; D013696:Temperature; D014357:Trypsin; D014362:Trypsinogen
D004220:Disulfides; D014362:Trypsinogen; D012685:Sepharose; D014357:Trypsin; D003488:Cyanogen Bromide