The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.

A major thrombin-like serine protease, acutobin, was purified from the venom of Deinagkistrdon acutus (hundred pace snake) by anionexchange-chromatography and reversed-phase HPLC. Acutobin is an Aa-fibrinogenase which may clot blood and contains about 30% carbohydrates. Its protein sequence was 80% determined by sequencing the fragments derived from CNBr-cleavage and endopeptidase-digestion of the S-alkylated proteins. Extensive screening of the venom gland cDNA after PCR amplification resulted in the identification of 4 cDNA clones encoding acutobin and 3 other serine proteases, designated as DAV-PA, DAV-KN and DAV-X. Their amino acid sequences were deduced from the cDNA sequences. Likewise, several serine proteases from the venom gland of Crotalus atrox were purified by FPLC and two related cDNAs were cloned and completely sequenced. The purified glycosylated proteases (Catroxase I and 11) were found to be either a kininase or a plasminogen activitor, but not thrombin-like (do not clot human fibrinogen). Kinetic specificities of these purified enzymes toward several synthetic chromogenic substrates were also investigated. The phylogenetic tree constructed from totally 41 complete amino acid sequences so far available for the venom proteases separate 3 major subtypes of the enzymes: the (1) procoagulating or thrombin-like enzymes, the (2) plasminogen activators and the (3) kininases. Our study and previous investigation suggest that surface loops at residues 90-100 of the enzymes are responsible for their specific functions or substrate-selectivities. In members of the same subtype, this functional site is highly similar but the sequence similarity is low between members from different subtypes.