A novel method for the determination of C-terminal amino acid in polypeptides by selective tritium labelling

In our previous communication (Matsuo, 1965), we reported selective ²H- and ³H-labelling reaction at the C-terminal amino acid of several N-acetylpeptides through the C-terminal racemization mechanism. The selective deuteration or tritiation is based upon the C-terminal oxazolone formation in peptides by the action of acetic anhydride or dicyclohexylcarbodiimide, followed by the base-catalyzed hydrolytic ring-opening in deuterium oxide or tritium oxide as shown in the following scheme.The above selective C-terminal tritiation has now been applied to the determination of the known C-terminal amino acids of angiotensin II (Skeggs, 1955) and beef-insulin (Sanger, 1955). The experimental results described in this communication will indicate that the C-terminal determination of polypeptides or proteins may be accomplished in a simple manipulation and in a micro-scale by this method.A modification of the above procedure for identifying C-terminal aspartic acid and proline, which were two exceptions that resisted the oxazolone formation by the above method, will also be described.