The Relative Rates of Glutamine and Asparagine Deamidation in Glucagon Fragment 22–29 under Acidic Conditions

Abstract

Our objective was to compare the relative rates of asparaginyl and glutaminyl deamidation in fragment 22–29 of the polypeptide hormone glucagon in acidic aqueous solutions. Reaction mixtures containing 22–29 (FVQWLMNT) or its degradation products were degraded at 60°C in dilute hydrochloric acid or phosphate buffer in the pH range 1–3. Degradation products were separated by high‐performance liquid chromatography and identified by amino acid sequencing, amino acid analysis, liquid chromatography‐mass spectrometry (LC‐MS), and matrix‐assisted laser desorption and ionization (MALDI). Nine major degradation products were identified, including asparaginyl and glutaminyl deamidated forms, aspartyl peptide cleavage of the asparaginyl deamidated products, and a cyclic imide intermediate. The pH dependences of rate constants for individual pathways were consistent with acid catalysis. Previous investigators have reported a greater susceptibility of asparagine residues to deamidation in neutral and alkaline solutions due to the formation of a more stable five‐membered succinimide intermediate. It has been suggested that asparagine may be more labile under acidic conditions also. We have observed a more facile deamidation for the glutamine residue under the acidic condition studied. It is proposed that the lower reactivity of the asparagine residue may be due to a decreased electrophilicity of its side chain carbonyl carbon imparted by a parallel cleavage pathway at this residue. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2332–2345, 2002