| The objectives of this work were to determine the pathways of glucagon degradation in acidic solutions, to quantitate the kinetics of each major pathway, to assess the role of primary structure on reactivity, and to propose reaction mechanisms.;Solutions of glucagon in dilute hydrochloric acid were degraded at 60°C. Degradation products were separated using RP-HPLC methods, and identified using a combination of amino acid sequencing, amino acid analysis, and mass spectrometry. Precursor-successor relationships in the degradation scheme were confirmed by initiating reactions with degradation products. Deamidation kinetics were measured using peptide fragments containing the labile amide residues. Peptide cleavage kinetics were measured using glucagon and its cleavage fragments. The role of side chain ionization was evaluated by determining the Asp side chain pKa values using NMR spectroscopy. The role of proton transfer was investigated using kinetic solvent isotope effect (KSIE) studies.;The major degradation pathways of glucagon in acidic solutions were N + 1 cleavage at Asp-9, Asp-15, and Asp-21, and deamidation at Gin-3, Gln-20, Gln-24. Minor pathways included deamidation at Asn-28, N-1 cleavage at Asp-15, peptide cleavage at Asn-28, and N + 1 peptide cleavage at Leu-26. Deamidation occurred at similar rates at the three Gln residues but was two-fold lower at Asn throughout the studied pH range. The rate constants for N + 1 cleavage at Asp-9 and Asp-15 were similar whereas rate constants for cleavage at Asp-21 was three to four fold lower. The pKa values for the side chains of Asp-9, Asp-15, and Asp-21 were found to be 3.7, 3.6, and 4.1 respectively. No kinetic solvent isotope effect was observed for the cleavage reaction whereas an inverse effect was observed for deamidation.;Based on the lack of sequence effects, pH-rate behavior, and KSIE, the deamidation mechanism involved direct hydrolysis of the amide side chain by water. Based on substrate ionization, pH-rate profiles, and KSIE, the proposed mechanism for N + 1 Asp cleavage involves nucleophilic attack of the ionized side chain carboxylate on the protonated carbonyl carbon of the peptide bond to give a cyclic anhydride intermediate. |
