Activation of J77A.1 Macrophages by Three Phospholipases A2 Isolated from Bothrops atrox Snake Venom
(a) Chromatography profile on hydrophobic interaction resin (Butyl-Sepharose HP 50 mm × 10 mm). Fractions 1, 2, and 3 obtained from CM-Sepharose chromatography were pooled and solubilized in 20 mM Ambic plus 4 M NaCl (buffer A) and applied on a Butyl-Sepharose HP column previously equilibrated with the same buffer and eluted with 20 mM Ambic (buffer B) using a step gradient of 0, 25, 50, 75, and 100%. Subsequently, a step using Milli-Q water was performed. (b) High performance chromatographic profile. Fraction 6 obtained from a Butyl-Sepharose HP column 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 of 0–70% and flow of 1 mL/min. Both elutions were monitored with absorbance at 280 nm.