Poster 229 - Partial purification and characterization of phopholipases A2 fraction from Bothrops

Poster 229. 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.588.

Partial Purification and Characterization Of Phopholipases A2 fraction From Bothrops neuwiedi pauloensis (Jararaca Pintada) Snake Venom.

¹Rodrigues, R.S.; ¹Izidoro, L.F.; ¹Hamaguchi, A.; ¹Homsi-Brandeburgo, M. I.; ²Soares, A. M. and ¹Rodrigues, V. M.

1 Instituto de Genética e Bioquímica, UFU, Laboratório de Química de Proteínas e Produtos Naturais; 2 Unidade de Biotecnologia, Universidade de Ribeirão Preto UNAERP, Bloco A.

Phospholipases A2 (PLA2) are present at high concentrations in snake venoms. Usually venom from a single species contains several isoforms of PLA2. In the present work, a PLA2 fraction was isolated from Bothropsneuwiedi pauloensis venom and some biochemical properties were characterized. The PLA2 fraction, designed P3 was isolated by a combination of anion exchange chromatography on Q-Sepharose with NH4HCO3 0,05 M plus 1 M NaCl, pH 8,0 buffer and Phenyl-Sepharose CL-4B (0,01 M Tris-HCl plus 4 M NaCl, pH 8.0 buffer, followed by a concentration gradient from 4 to 0 M NaCl at 25ºC in the same buffer) suggesting that this fraction contains acidic PLA2. P3 showed two bands with a Mr around 14,000 (as estimated by SDS-PAGE and native PAGE for acidic proteins). Phospholipase A2 activity upon egg yolk emulsion, which contains phosphatidylcholine as a substrate was assayed for the partial purified PLA2s. The specific activity of P3 was low about 70 U/mg/min when compared with the activity of basic phospholipases A2 (230 U/mg/min), isolated from the same snake venom in the same conditions. The edema-inducing activity was assayed after injection in the subplantar region with P3 (50µg/50µL). After 0.25, 0.5, 1, 3, and 6 h, the paw edema was measured with the aid of a low pressure spring caliper (Mitutoyo-Japan). The zero time values were then subtracted and the differences reported as median % S.D. P3 induced a relevant time dependent mouse paw edema. Indirect hemolytic activity was assayed with red blood cells from freshly collected rat blood. P3 showed no indirect hemolytic activity with concentrations up to 3mg of proteins. This PLA2 fraction was devoid of myotoxic, fibrinogenolytic, hemorrhagic and coagulant activities suggesting that edematogenic effect induced by P3 is due to acidic phospholipases A2 present in this fraction.