Irreversible Degradation of Histidine-96 of Prothrombin Fragment 1 during Protein Acetylation: Another Unusually Reactive Site in the Kringle

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^-1cm^-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 e-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 C₂of 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]). Unusual¹H NMR signals from histidine residues in the analogous position of other kringle sequences have been reported as well [Hochswender, S. M., Laursen, R. A., De Marco, A., & Llinas, M. (1983) Arch. Biochem. Biophys. 223, 58-67; Llinas, M., De Marco, A., Hochschwender, S. M., & Laursen, R. A. (1983) Eur. J. Biochem. 135, 379-391; Trexler, M., Banyai, L., Patthy, L., Pluck, N. D., & Williams, R. J. P. (1983) FEBS Lett. 154, 311–318]. This region of kringle structures may constitute an unusual component determined by folding of the kringle.