Phospholipase A2 (PLA2) assay

Slightly modified turbidimetric assays were conducted to assess the PLA₂ activities of venoms as described previously [14,32]. A fresh chicken egg was used to prepare the egg-yolk substrate solution in 0.9% NaCl solution, such that its absorbance at 740 nm corresponded to 1. A fixed concentration of crude venom samples (1 μg) prepared in 20 mM Tris-HCl buffer, time-dependent kinetic assays were performed in triplicate. Following the addition of 250 μl of the egg yolk solution, absorbance was measured for 60 min at 740 nm in an EPOCH 2 microplate spectrophotometer (BioTek Instruments, Inc., USA). Unit activity was calculated as the amount of crude venom required to reduce the absorbance of the substrate by 0.01 OD unit per min at the given wavelength [33].