AFFINITY LABELING OF LYSINE-149 IN THE ANION-BINDING EXOSITE OF HUMAN ALPHA-THROMBIN WITH AN N-ALPHA-(DINITROFLUOROBENZYL)HIRUDIN C-TERMINAL PEPTIDE

In order to define structural regions in thrombin that interact with hirudin, the TV-dinitro-fluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54–64 of hirudin [GDFEEIPEEY(035SO3)L (DNFB-[35S]Hir54-64 DNFB-[35S]Hir54_64 was reacted at a 10-fold molar excess with human α-thrombin in phosphate-buffered saline at pH 7.4 and 23 °C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single 35S-labeled band of Mr ∼32 500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at Mr ∼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 α-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 of human thrombin as the major site of DNFB-Hir54-64 derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18–20 Å from the catalytic apparatus. © 1990, American Chemical Society. All rights reserved.