DIRECT ASSIGNMENT OF DISULFIDE BONDS BY EDMAN DEGRADATION OF SELECTEDPEPTIDE-FRAGMENTS

Disulfide linkages in peptides or proteins were analyzed by automated gas-phase Edman sequencing performed in minimized reducing agents. If cystine linkage was regulated at the same position in two peptides dur ing peptide preparation, the diphenylthiohydantoin derivative of cysti ne was significantly recovered by Edman reaction. In contrast, when th e crosslinked half cystines were present at different positions in the peptides, the derivative could be poorly detected. Upon direct sequen ce analysis of intact bovine insulin, the PTH derivatives of cystine f rom both Cys-A7 and Cys-B7 were significantly released after Edman cyc le 7 and gave approximately 20 % recovery of common PTH amino acids. H owever, Cys-A11 linked to Cys-A6 was poorly detectable after Edman cyc le 11. For general use of this method, proteins need to be subjected t o several sets of proteolytic or chemical cleavages in the hope that a t least one of the fragments will have cystine linkage at the same pos ition. This method was applied to several fragments of platelet-derive d growth factor B chain and brain-derived neurotrophic factor. (C) Mun ksgaard 1994.