title: Irreversible degradation of histidine-96 of prothrombin fragment 1 during protein acetylation: another unusually reactive site in the kringle.
abstract: Acetylation of prothrombin fragment 1 in acetate-borate buffer at pH 8.5 resulted in the appearance of increased light absorbance at about 250 nm. Protease digestions resulted in isolation of a single peptide (residues 94-99) with intense absorbance at about 250 nm (estimated extinction coefficient of 5000 M-1 cm-1). Amino acid analysis showed the expected composition except for the absence of His-96. Instead, an unidentified amino acid which had a ninhydrin product with absorption properties similar to those of proline eluted near aspartate. When sequenced, this peptide (YP?KPE containing epsilon-amino-acetyllysine) lacked histidine at the third position but gave a high yield of a PTH derivative that eluted near PTH-Gly from the HPLC column. Fast atom bombardment mass spectrometry of the derivatized 94-99 peptide showed a mass that was 74 units higher than expected. The histidine degradation product was identified as a di-N-acetylated side chain with an opened imidazole ring and loss of C2 of the ring. While a similar degradation pattern has previously been reported during acylation of histidine, the high chemical reactivity exhibited by His-96 was unusual. For example, under conditions sufficient for quantitative derivatization of His-96, His-105 of fragment 1 was not derivatized to a detectable level. Furthermore, His-96 in fragment 1 was at least an order of magnitude more susceptible to degradation than His-96 in the isolated 94-99 peptide. His-96 is therefore one of several neighboring amino acids of the kringle portion of fragment 1 that displays highly unusual chemistry (see also Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946-4952] and Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325-334, Elsevier Science, New York]).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108.
authors.forename: D J
authors.initials: DJ
authors.lastname: Welsch
authors.forename: G L
authors.initials: GL
authors.lastname: Nelsestuen
mesh_terms: D000089:Acetic Anhydrides; D000107:Acetylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D010669:Phenylthiohydantoin; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism*; D011516:Prothrombin / Q000032:analysis / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
chemical_list: D000089:Acetic Anhydrides; D000596:Amino Acids; D010446:Peptide Fragments; D011498:Protein Precursors; D010669:Phenylthiohydantoin; C031800:acetic anhydride; D006639:Histidine; C029733:prothrombin fragment 1; D011516:Prothrombin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin