The Complete Covalent Structure of Hirudin. Localization of the Disulfide Bonds

Abstract

Hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis, is a single-chain polypeptide (65 amino acid residues) linked by 3 disulfide bridges. Localization of the 3 disulfide bonds could be assigned on the basis of the structures of cystine peptides derived by high performance liquid chromatography separations of thermolysinolytic digests of native hirudin. By characterization of the 9 major fragments by amino acid analysis, N-terminal amino acid determination and sequence analysis, the following disulfide linkages were identified: Cys6-Cys14, Cys16-Cys28 and Cys22-Cys39. Due to the lack of any closer sequence homology and topological structural homology to other serine proteinase inhibitor proteins, hirudin seems to be unique in its primary structure and hence designates an unknown inhibitor family.