Aliquots of B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi, and B. alternatus venom were obtained from the Serpentarium at the Faculty of Medicine of Ribeirão Preto, University of São Paulo (Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo), Brazil. For each species, venom was extracted from three snakes captured in different regions of São Paulo state and pooled. The venom aliquots were stored
at −20 °C until analysis. Protein content was measured by the Lowry method, modified by Hartree (1972), using bovine serum albumin as the standard. Sheep erythrocytes (Newprov, Pinhais, Brazil) were collected in heparinized tubes (Becton Dickinson, Franklin Lakes, NJ, USA), selleck chemical centrifuged for 20 min at 300 × g at 4 °C, washed 3 times with PBS, pH 7.4, and centrifuged again. Chemicals and reagents were purchased from Sigma (Sigma Chemical
Co., St. Louis, MO, USA). A modified kinetic indirect hemolytic assay, standardized in our laboratory (Tamarozzi et al., 2006), was performed to measure PLA2 activity. We used egg yolk as a substrate to develop a turbidimetric assay based on the capacity of snake venom PLA2s to hydrolyze egg phosphatidylcholine, Vorinostat producing lysophosphatidylcholine and fatty acids. The lysophosphatidylcholine accumulates on erythrocyte membranes, promoting their disruption and initiating the hemolysis process (Bierbaum et al., 1979). This assay was conducted in triplicate in a 96-well microplate (Costar, Corning Metalloexopeptidase Incorporated, NY, USA) and samples were analyzed at 650 nm, considering that this is well outside of the hemoglobin absorption wavelength and that the absorbance is proportional to the cell concentration. The hemolysis solution consisted of
10 ml of Tris–HCl buffer, pH 7.4, containing 140 mM NaCl, 2.5 mM CaCl2, 25 μl of egg yolk diluted 1:4 (v/v) in sterile saline solution (NaCl 0.9%), and 1.5 μl of erythrocytes. The reaction mixture was prepared by adding 175 μl of hemolysis solution and 75 μl of venom samples (20 μg/ml). The microplate was incubated at 37 °C and the decrease in absorbance was recorded at intervals of 5, 10, and 15 min. A saline solution containing no venom was used as a negative control. The blank solution was prepared by adding venom to the hemolysis solution to achieve a final concentration of 80 μg/ml. After 1 h of incubation at 37 °C, this solution was used to calibrate the microplate reader (Molecular Devices, Sunnyvale, CA, USA). Statistical analyses were based on the absorbance after 90 min of reaction. Proteolytic activity was assayed by a modified caseinolytic method (Sanchez et al., 1992). The reaction mixture consisted of venom (50 and 100 μg/ml) diluted in 500 μl of 50 mM Tris–HCl, pH 7.4, 2.0 mM CaCl2, and 0.5% (w/v) denatured casein. After 3 h of incubation at 37 °C, the reaction was terminated by adding 500 μl of 10% (w/v) trichloroacetic acid (TCA).
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