Refolding of bovine trypsinogen with one and two disulfide bonds reduced and carboxymethylated.

The role of specific disulfides in the refolding of bovine trypsinogen was examined with samples of the protein lacking one and two disulfide bonds. Disulfide 179 to 203 was reduced with 0.1 M sodium borohydride and the same bond and disulfide 122 to 189 was reduced with 0.002 M dithioerythritol. The newly formed sulfhydry1 groups were converted to the “‘C-labeled diand tetracarboxymethyl derivatives. The carboxymethylated proteins were fully reduced with 0.2 M mercaptoethanol in 8 M urea and treated with 0.1 M oxidized glutathione to produce the mixed disulfide of glutathione. Refolding was initiated with 3 IXIM cysteine, which catalyzed disulfide interchange and permitted further conformational changes to take place. The addition of iodoacetate stopped further folding, the regenerated protein was activated with bovine enterokinase, and the radioactively labeled trypsin derivatives were analyzed by affinity chromatography on a column of pancreatic trypsin inhibitor-Sepharose. The dicarboxymethylated trypsinogen refolded with a yield of 66% and a half-time of 119 min at 4°C. The tetracarboxymethylated trypsinogen refolded with a yield of 56% and a half-time of 217 min. Previously, we reported a yield of 70% and a half-time of 55 min for the refolding of trypsinogen (Odorzynski, T. W., and Light, A. (1979) J. Biol. Chem. 254, 4291-4295). The increase in the refolding times suggested that cysteines 122, 179, 189, and 203 normally participate in rate-limiting steps. Because the refolding yields were essentially the same for trypsinogen and the disulfide-modified derivatives, alternate folding pathways had to be followed for the carboxymethylated trypsinogens.