The degradation pathways of glucagon in acidic solutions

Abstract

Objective: Glucagon is a 29 amino acid peptide hormone that exhibits degradation via both chemical and physical pathways. The objective of the studies reported herein was to identify the degradation products and scheme for glucagon hydrolysis in acidic solutions. Methods: Solutions of glucagon in 0.01 N HCl (pH 2.5) were degraded at 60°C for 70 h. One isocratic and two gradient RP-HPLC methods were developed to separate the degradation products. Structure elucidation of the separated peaks was achieved using amino acid sequencing, amino acid analysis, and mass spectrometry. Degradation was carried out in the pH range 1.5–5 to check for changes in degradation scheme with pH. Authentic samples of degradation products were degraded under similar acidic conditions to confirm precursor successor relationships in the degradation scheme. Results: Sixteen major degradation products were isolated and identified. The major pathways of degradation were found to be aspartic acid cleavage at positions 9, 15, and 21 and glutaminyl deamidation at positions 3, 20, and 24. Cleavage occurred on both sides of Asp-15 but only on the C-terminal side of Asp-9 and Asp-21. Deamidation of the Asn residue at position 28 was not detected.