title: Amino acid sequence determination of ancrod, the thrombin-like alpha-fibrinogenase from the venom of Akistrodon rhodostoma.
abstract: The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.
authors.affiliation: Department of Structural and Biophysical Chemistry, Glaxo Research Laboratories, Research Triangle Park, NC 27709.
authors.forename: W
authors.initials: W
authors.lastname: Burkhart
authors.forename: G F
authors.initials: GF
authors.lastname: Smith
authors.forename: J L
authors.initials: JL
authors.lastname: Su
authors.forename: I
authors.initials: I
authors.lastname: Parikh
authors.forename: H
authors.initials: H
authors.lastname: LeVine
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000721:Ancrod / Q000737:chemistry*; D015153:Blotting, Western; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014757:Viper Venoms / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D014757:Viper Venoms; D000721:Ancrod