The proposed research is an investigation of the mechanism of folding of the class of proteins belonging to the serine proteinase family. The role of disulfide bonds will be examined to establish whether these bonds stabilize nucleation sites or larger intermediates. Refolding of large fragments of chymotrypsinogen and trypsin will be attempted to test whether these regions of the structure fold as independent domains. Complementation of these fragments will determine if a native structure is regenerated with a regain of biological activity. Experimentally, the refolding of mixed disulfides of proteins and of protein fragments will be used. The regeneration of a native protein will be examined by 1) enzyme assays; b) stability to proteolysis; c) correct pairing of disulfide bonds; d) hydrodynamic volume; e) spectral studies with UV, CD, NMR, and tryptophan fluorescence. The mechanism of folding will be studied by identifying regions of structure that form native disulfide bonds.