BINDING OF FLUORESCENT AND SPIN-LABELED C-TERMINAL HIRUDIN ANALOGS TOTHROMBIN

Synthetic peptides based on the sequence of the negatively charged car boxyl tail of hirudin exhibit anticoagulant activity. Several antithro mbin agents are being developed by chemical and structural optimizatio n of these ''hirupeptides''. The present work demonstrates the design and use of novel spin-labeled and fluorescent-labeled C-terminal hirud in analogs to study the interactions of these antithrombin agents with thrombin in solution. Three labeled hirulabels were synthesized based upon the amino acid sequence of the antithrombin agent MDL 28050, X-N H-(CH2)(7)-CO-Asp-Tyr-Glu-Pro-Ile-Pro-G where X = anthraniloyl, 1,5-da nsyl, or 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrol The modifications di d not significantly alter the potency of these inhibitors which showed Ki values of 100 nM. Their interactions with human and bovine thrombi n were studied by ESR and fluorescence techniques. The spin-labeled hi rupeptide was able to discern subtle differences in binding to human v ersus: bovine thrombin. The 8-aminooctanoic acid spacer arm placed the nitroxide moieties near the active site, near regions of the autolysi s loops which differentiates between human alpha- and gamma-thrombin. It was also able to discern paramagnetic quenching and fluorescence en ergy transfer interactions, respectively, between covalently attached spin labels and fluorescent probes at the active site Ser 195 and the fluorophore on the hirupeptide.