Amino acid sequence determination of Ancrod, the thrombin-like α-fibrinogenase from the venom of Akistrodon rhodostoma

William Burkhart, Gardiner F.H. Smith, Jui Lan Su, Indu Parikh, Harry LeVine

Research output: Contribution to journalArticlepeer-review

82 Scopus citations

Abstract

The thrombin-like serine protease and antithrombotic agent. Ancrod, was rapidly purified from the crude venom of Akistradon 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.

Original languageEnglish
Pages (from-to)297-301
Number of pages5
JournalFEBS Letters
Volume297
Issue number3
DOIs
StatePublished - Feb 10 1992

Keywords

  • Affinity chromatography
  • Anticoagulant therapy
  • Serine protease

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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