Mechanisms of glucagon degradation at alkaline pH

Nicholas Caputo, Jessica Castle, Colin P. Bergstrom, Julie M. Carroll, Parkash A. Bakhtiani, Melanie A. Jackson, Charles Roberts, Larry David, W. Kenneth Ward

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.

Original languageEnglish (US)
Pages (from-to)40-47
Number of pages8
JournalPeptides
Volume45
DOIs
StatePublished - 2013

Fingerprint

Glucagon
Degradation
Isomerization
Bioactivity
Liquid Chromatography
Liquid chromatography
Mass Spectrometry
Mass spectrometry
Agglomeration
Peptides
Cyclic AMP-Dependent Protein Kinases
Bioassay
Biological Assay
Medical problems
Molecular Weight
Molecular weight
Pumps
Membranes
Oxidation

Keywords

  • Alkaline
  • Deamidation
  • Degradation
  • Diabetes mellitus
  • Glucagon
  • Oxidation

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Physiology
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Caputo, N., Castle, J., Bergstrom, C. P., Carroll, J. M., Bakhtiani, P. A., Jackson, M. A., ... Ward, W. K. (2013). Mechanisms of glucagon degradation at alkaline pH. Peptides, 45, 40-47. https://doi.org/10.1016/j.peptides.2013.04.005

Mechanisms of glucagon degradation at alkaline pH. / Caputo, Nicholas; Castle, Jessica; Bergstrom, Colin P.; Carroll, Julie M.; Bakhtiani, Parkash A.; Jackson, Melanie A.; Roberts, Charles; David, Larry; Ward, W. Kenneth.

In: Peptides, Vol. 45, 2013, p. 40-47.

Research output: Contribution to journalArticle

Caputo, N, Castle, J, Bergstrom, CP, Carroll, JM, Bakhtiani, PA, Jackson, MA, Roberts, C, David, L & Ward, WK 2013, 'Mechanisms of glucagon degradation at alkaline pH', Peptides, vol. 45, pp. 40-47. https://doi.org/10.1016/j.peptides.2013.04.005
Caputo N, Castle J, Bergstrom CP, Carroll JM, Bakhtiani PA, Jackson MA et al. Mechanisms of glucagon degradation at alkaline pH. Peptides. 2013;45:40-47. https://doi.org/10.1016/j.peptides.2013.04.005
Caputo, Nicholas ; Castle, Jessica ; Bergstrom, Colin P. ; Carroll, Julie M. ; Bakhtiani, Parkash A. ; Jackson, Melanie A. ; Roberts, Charles ; David, Larry ; Ward, W. Kenneth. / Mechanisms of glucagon degradation at alkaline pH. In: Peptides. 2013 ; Vol. 45. pp. 40-47.
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