PMID: 2119800Jul 10, 1990Paper

Affinity labeling of lysine-149 in the anion-binding exosite of human alpha-thrombin with an N alpha-(dinitrofluorobenzyl)hirudin C-terminal peptide

Biochemistry
P BourdonJ M Maraganore

Abstract

In order to define structural regions in thrombin that interact with hirudin, the N alpha-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin [GDFEEIPEEY(O35SO3)L (DNFB-[35S]Hir54-64)]. DNFB-[35S]Hir54-64 was reacted at a 10-fold molar excess with human alpha-thrombin in phosphate-buffered saline at pH 7.4 and 23 degrees C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single 35S-labeled band of Mr approximately 32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at Mr approximately 31,000. Incorporation of the 35S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human alpha-thrombin-DNFB-Hir54-64 complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 ...Continue Reading

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Citations

Apr 28, 2012·Bioconjugate Chemistry·Bernadette V MarquezClaude F Meares
Feb 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·Y CadroyL A Harker
Jan 1, 1993·Thrombosis Research·M T Stubbs, W Bode
Dec 16, 1993·The American Journal of Cardiology·G Agnelli
Aug 11, 2004·Toxicon : Official Journal of the International Society on Toxinology·Anita M Tanaka-AzevedoIda S Sano-Martins
Sep 20, 1996·The Journal of Biological Chemistry·D A MehM W Mosesson

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