Poster 230.  Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil.  Abstracts...  J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.589.

 

 

Partial Purification of a new proteolytic enzyme from Bothrops neuwiedi pauloensis snake venom. 

 

1Costa, F.L.S.; 1Rodrigues, R.S.; 1França, J.B.; 1Rodrigues, V.M.; 1Hamaguchi, A. and 1Homsi-Brandeburgo, M.I.

 

1 Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Minas Gerais, Brasil

 

Snake venoms, particularly those belonging to the genus Bothrops, are known to contain many proteolytic enzymes wich degrade a variety of natural substrates such as casein, hemoglobin, collagen, elastin, insulin, glucagon and fibrinogen. These proteases cause blood coagulation,hemorrhage, fibrinogenolysis and necrosis.The present work reports the partial purification of a proteolytic enzyme from Bothropsneuwiedi pauloensis snake venom.  The crude venom was submitted on CM-Sepharose fast flow and five fractions were obtained (CM1-CM5). CM4 fraction was submitted on Heparin-Agarose and resulted in two subfractions (H1 and H2). The H2 subfraction showed a major band with Mr 40,000 (as estimated by SDS-PAGE) and a basic character on PAGE in native conditions. The coagulant activity of H2 (10µg) on bovine plasma was assayed when 50 µL of this solution were added to 200 µL bovine plasma at 37ºC and the clotting time was recorded. Bothropsneuwiedi pauloensis venom induced bovine plasma coagulation in aproximately 10s and H2 subfracion induced in 7 seconds. Proteolytic activities upon fibrinogen and casein also were determinated. Samples of 75µL of bovine fibrinogen or casein (1mg/mL PBS) were incubated with H2 subfraction (10µg) at 37ºC for 1h, pH 8.0. These activities were analysed by 14% (w/v) SDS-PAGE. H2 subfraction degraded the Aa chain of bovine fibrinogen more rapidly than the Bb chain after 1h incubation. The proteolytic activity of H2 upon casein evaluated by SDS-PAGE showed that the casein was completely degraded after 60 min incubation resulting into a variety of shorter peptides. This preliminary study showed us that Bothropsneuwiedi pauloensis snake venom contains a prominent proteolytic enzyme which may induce relevant hemostatic alterations.

 

Suport: CNPq, UFU.

 

Correspondenceto: rsantos23@hotmail.com