DETERMINATION OF CARBOXYL TERMINI OF PROTEINS WITH AMMONIUM THIOCYANATE AND ACETIC ANHYDRIDE, WITH DIRECT IDENTIFICATION OF THIOHYDANTOINS

The method described previously for sequential degradation of peptides has been extended to the determination of the carboxyl termini of proteins. Conversion of a protein into a proteinylthiohydantoin with ammonium thiocyanate and excess acetic anhydride can be carried out in homogeneous solution by using a mixture of hexafluoroacetone and acetic acid as the solvent. After separation from excess reagents by gel filtration, the proteinylthiohydantoin is cleaved in 12 m HCl. The thiohydantoin derived from the carboxyl terminus is separated from modified protein by gel filtration and identified by thin-layer chromatography. In equivocal situations, it is possible to convert the thiohydantoin, after extraction from the thin-layer plate, into an amino acid in moderate yield by a procedure which combines alkaline hydrolysis and oxidation by H2O2. Two large peptides derived from insulin have been degraded successfully by this method for two and three cycles, respectively. The carboxyl-terminal residue has been identified unambiguously in bovine pancreatic ribonuclease A, sperm whale myoglobin, hens’ egg white lysozyme, glucagon, and the catalytic and regulatory subunits of Escherichia coli aspartate transcarbamylase, but attempts to use the method sequentially with these proteins were not successful. © 1969, American Chemical Society. All rights reserved.