Activation of J77A.1 Macrophages by Three Phospholipases A2 Isolated from Bothrops atrox Snake Venom
(a) Chromatographic profile of Bothrops atrox venom. The crude venom of B. atrox after being solubilized was applied on CM-Sepharose (400 mm × 10 mm) previously equilibrated with Tris 50 mM pH 7.4 and eluted under a gradient of NaCl (0–1 M) in the same buffer, in 5 column volumes. The fractions with PLA2 activity are identified with (*). (b) High performance chromatographic profile. Fraction 6 obtained from CM-Sepharose was solubilized in 0.1% TFA (solvent A) and applied on a C18 column (discovery 25 mm × 46 mm, 5 μ, 300 Å) previously equilibrated with the same buffer and eluted with 0.1% TFA in 99.9% acetonitrile (solvent B) under a gradient 0–70% and flow of 1 mL/min. Both chromatograms were monitored with absorbance at 280 nm. (c) SDS-PAGE 12. 5%: samples: 1 molecular weight marker; 2 BthTX-I; 3 fraction 8; 4 fraction 9; 5 fraction 10; 6 11 fraction (BaTX-I); 7 fraction 12. (d) SDS-PAGE 12.5% in reducing conditions: electrophoretic analysis of basic Asp49 PLA2 from B. atrox. Samples: 1 molecular weight; 2 BthTX-I; 3 BaTX-II.