The relative rates of glutamine and asparagine deamidation in glucagon fragment 22-29 under acidic conditions

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 60degreesC 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. (C) 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.