Abstract:
Processing of human proinsulin C-peptide and its C-terminal pentapeptide in blood serum was studied using reverse-phase HPLC and electrospray mass spectrometry. The results reveal degradation of both peptides, with a longer half-life for intact C-peptide than for the C-terminal pentapeptide. Products from C-peptide degradation were not distinguishable from the peptide background, suggesting endopeptidase degradation of C-peptide. In contrast, a set of products from the C-terminal pentapeptide were identifiable and corresponded to successive losses from the N terminus, showing that the pentapeptide is degraded by aminopeptidase in serum. Consistent with this finding, a slower degradation was found for the N-acetyl-protected pentapeptide. Removal of serum proteins by acetone precipitation produced N-terminally carbamate-modified C-peptide via a Schiff base intermediate (a ketimine with acetone), to which CO2 was added and acetone removed, generating a cyclic side chain via anhydride formation. The modification was not seen with the pyroglutamate form of C-peptide, with the N-terminally acetylated C-peptide, or with a control peptide having N-terminal Phe, but was found with human C-peptide, its N-terminal tetrapeptide, and a rat C-peptide fragment (all with N-terminal Glu). Hence, the modification appears to require N-terminal Glu, but this is not the only prerequisite since the C-terminal pentapeptide and another control peptide (also starting with Glu) were not modified. A peptide aldimine Schiff base leading to CO2 incorporation was detected with formaldehyde in NaHCO3. The observation that C-peptide forms Schiff bases with ketones/aldehydes, enhancing covalent attachment of CO2, may have biological implications.
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Received 27 March 2003; accepted 27 March 2003
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Melles, E., Bergman, T., Alvelius, G. et al. Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO2 incorporation. CMLS, Cell. Mol. Life Sci. 60, 1019–1025 (2003). https://doi.org/10.1007/s00018-003-3117-x
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DOI: https://doi.org/10.1007/s00018-003-3117-x