RESUMO
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 percent 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 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, 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.
Assuntos
Animais , Camundongos , Coagulação Sanguínea , Bothrops , Coagulantes/isolamento & purificação , Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Antivenenos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia em Agarose , Cromatografia por Troca Iônica , Costa Rica , Coagulantes/administração & dosagem , Coagulantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Fibrinogênio/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Mordeduras de Serpentes/fisiopatologia , Trombina/químicaRESUMO
The adjuvant of several immunostimulant molecules on the murine antibody response to Bothrops asper snake venom were evaluated. Mice receiving venom together with either sodium alginate, calcium alginate, aluminum hydroxide, muramyl dipeptide, killed Brucella abortus or B. abortus smooth lipopolysaccharide developed a similar antibody response. Despite the fact that in some cases animals injected with venom and Salmonella montevideo lipopolysaccharide developed a significantly higher antibody titer when compared to other experimental groups, no statistically significant differences were observed in most of the comparisons.