title: Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO2 incorporation.
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 CO(2) 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 CO(2) incorporation was detected with formaldehyde in NaHCO(3). The observation that C-peptide forms Schiff bases with ketones/aldehydes, enhancing covalent attachment of CO(2), may have biological implications.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Melles
authors.forename: T
authors.initials: T
authors.lastname: Bergman
authors.forename: G
authors.initials: G
authors.lastname: Alvelius
authors.forename: A
authors.initials: A
authors.lastname: Jonsson
authors.forename: K
authors.initials: K
authors.lastname: Ekberg
authors.forename: J
authors.initials: J
authors.lastname: Wahren
authors.forename: H
authors.initials: H
authors.lastname: Jörnvall
mesh_terms: D002096:C-Peptide / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D012545:Schiff Bases
chemical_list: D002096:C-Peptide; D010446:Peptide Fragments; D012545:Schiff Bases; D002245:Carbon Dioxide