Stability of Hirudin, a Thrombin-specific Inhibitor

Hirudin is a 65-amino acid polypeptide with three disulfide linkages. It is stable under extreme pH (1.4712.9), high temperature (95 “C), and in the presence of denaturants (6 M guanidinium chloride or 8 M urea). The thrombin inhibitory activity of hirudin remains unaffected even after cleavage of an internal peptide bond ( L y ~ ~ ‘ A s n ~ ~ ) . One condition which effectively and irreversibly inactivates hirudin is the combination of elevated temperature and alkaline pH. Structural analysis reveals that inactivation is a consequence of base-catalyzed &elimination of the disulfide bonds. The reaction leads to the conversion of hirudin to a mixture of highly heterogeneous polymers (from monomer to heptamer) which are intraand intermolecularly cross-linked by cystine (20%), lanthionine (50%), and lysinoalanine (30%).