Stability of hirudin, a thrombin-specific inhibitor. The structure of alkaline-inactivated hirudin.
Hirudin is a 65-amino acid polypeptide with three disulfide linkages. It is stable under extreme pH (1.47-12.9), high temperature (95 degrees 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 (Lys36-Asn37). 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 beta-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 intra- and intermolecularly cross-linked by cystine (20%), lanthionine (50%), and lysinoalanine (30%).
Chang
J Y
JY
Pharmaceutical Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D004220:Disulfides / Q000032:analysis; D004355:Drug Stability; D006629:Hirudins / Q000737:chemistry* / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013816:Thermodynamics; D013917:Thrombin / Q000037:antagonists & inhibitors*
D004220:Disulfides; D006629:Hirudins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002918:Chymotrypsin; D013917:Thrombin