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Amino acid sequence determination of Ancrod, the thrombin‐like α‐fibrinogenase from the venom of Akistrodon rhodostoma
Author(s) -
Burkhart William,
Smith Gardiner F.H.,
Su Jui-Lan,
Parikh Indu,
LeVine Harry
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80559-y
Subject(s) - thrombin , biochemistry , edman degradation , peptide sequence , affinity chromatography , chemistry , proteases , serine protease , serine , protease , microbiology and biotechnology , biology , enzyme , platelet , gene , immunology
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.