Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone.

Deamidation of Asn residues is one of the major chemical pathways of degradation of proteins and peptides. Adrenocorticotropic hormone (ACTH), a 39-amino acid polypeptide with a single Asn residue, was shown in this study to be a useful model polypeptide for the study of the effects of pH and buffer concentration on the rate and pathway of deamidation. The disappearance of ACTH and appearance of deamidated ACTH were monitored by isoelectric focusing (IEF), and ammonia production was monitored spectrophotometrically using a coupled enzymatic assay. Using these analytical methods, the deamidation of ACTH was shown to follow pseudo-first-order kinetics and was dependent on pH and buffer concentrations. The separation of the deamidated ACTHs (Asp-ACTH and isoAsp-ACTH) from ACTH was successful, but attempts to separate Asp-ACTH from isoAsp-ACTH using cation-exchange HPLC and IEF were unsuccessful. Using bovine protein carboxymethyltransferase (PCM), which selectively methylates the carboxyl group of isoAsp residue, the isoAsp-ACTH could be detected at pH 7.0 and 9.6 but not at pH 1.9. These data support the hypothesis that under neutral and alkaline conditions, deamidation of ACTH proceeds through the formation of a cyclic imide intermediate (slow step), followed by its hydrolysis to the Asp-ACTH and isoAsp-ACTH (fast step). Under acidic conditions, the reaction appears to proceed via direct hydrolysis of the Asn residue to form Asp-ACTH without the formation of a cyclic imide intermediate.