title: Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech.
abstract: Hirudin is a specific polypeptide thrombin inhibitor consisting of 65 amino acids that is produced by the leech, Hirudo medicinalis. We describe a rapid method for the purification of hirudin from a leech extract. Crude hirudin, purchased from a commercial source, was first fractionated on a DEAE-HPLC column using a salt gradient. Hirudin activity was monitored by inhibition of the thrombin-mediated hydrolysis of a synthetic substrate H-D-Phenylalanyl-Pipecolyl-Arginine-p-Nitroanilide. The fractions containing antithrombin activity were pooled and further purified by reverse-phase HPLC. The homogeneity of purified hirudin was confirmed by a single amino-terminal sequence for 43 residues with Val-Val as the first two amino acids. Residue 33 was Asn rather than Asp as reported previously.
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006629:Hirudins / Q000032:analysis / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D006629:Hirudins; D013917:Thrombin