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Hirudin N-terminal core domain residues 1-43 (r-Hir1-43) were prepared from limited proteolysis of recombinant hirudin by V8 Staphylococcal protease followed by purification with reversed-phase HPLC. r-Hir1-43 lacks the putative reactive site of hirudin (Lys47), but binds to thrombin (with Ki of 300 nM) and blocks the catalytic activity of the protease. The structural element which accounts for the thrombin inhibitory activity of r-Hir1-43 is analyzed in this report.

Crude hirudin (12.7 U/micrograms), a complex mixture of polypeptides obtained from the leech, could be separated by microbore HPLC. A combination of amino acid analysis, N-terminal microsequencing and chemical as well as enzymatic fragmentation made the primary sequence of the new isohirudins Ia-IIIb' accessible. The biological activity determined in the thrombin inhibition test showed a comparable value for all of these compounds. The results presented address the question as to whether these isohirudins are true mutations from a family of genes or a family of leeches.

Hirudin is a thrombin-specific inhibitor of Mr 8000 (65 amino acid residues). Native hirudin contains 3 disulfide linkages within the first 39 amino-terminal residues, and a highly acidic C-terminal segment which is freely accessible to enzyme digestion by both endo- and exo-peptidases. Removal of the acidic C-terminal amino acids of native hirudin by both chemical and enzymatic methods resulted in a concomitant loss of hirudin inhibition activity. It is concluded that this acidic C-terminal segment of hirudin is essential for hirudin-thrombin interaction. The implication of the hirudin-thrombin interaction for the enzymatic specificity of thrombin is also discussed.