Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

• Published in: Brazilian journal of medical and biological research 2008-01, Vol.41 (1), p.12-17
• Main Authors: Pérez, A V, Rucavado, A, Sanz, L, Calvete, J J, Gutiérrez, J M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antivenins - therapeutic use; BIOLOGY; Blood Coagulation - drug effects; Bothrops; Bothrops asper; Chromatography, Agarose; Chromatography, Ion Exchange; Coagulants - administration & dosage; Coagulants - isolation & purification; Coagulants - pharmacology; Costa Rica; Crotalid Venoms - enzymology; Defibrin(ogen)ation; Drug Evaluation, Preclinical; Fibrinogen - metabolism; MEDICINE, RESEARCH & EXPERIMENTAL; Mice; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - pharmacology; Serine proteinase; Snake Bites - physiopathology; Snake venom; Thrombin - chemistry; Thrombin-like serine proteinase
• ISSN: 0100-879X; 1414-431X; 1414-431X
• DOI: 10.1590/S0100-879X2006005000189

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.