The absorbance was measured using an ELISA reader (Multiskan spectrophotometer EX, Labsystems, Finland) at λ 492 nm. The titre was established as the highest antiserum dilution that produced an absorbance three times greater than that produced by the negative control anti-tetanus serum. The phospholipase A2 LDK378 chemical structure activity of Tityus spp. venoms was evaluated as described by Price (2007), with some modifications. Microtitre plates were coated with venom samples

(30 μg) combined with buffer (10 mM Triton X-100, 5 mM phosphatidylcholine, 10 mM CaCl2, 0.9% NaCl, 0.03% bromothymol blue; pH 7.5) to a final volume of 200 μL. The activities were determined by measuring the OD at λ 620 nm using a spectrophotometer (Multiskan EX, Labsystems, Finland). As positive and negative controls, venom derived from Crotalus durissus terrificus (10 μg) and PBS was used, respectively. The phospholipase activity was expressed in nanomoles of HCl per minute per mg of venom (nmoles/min/mg) of three independent PR-171 purchase experiments. Hyaluronidase activity was measured as described previously by Pukrittayakamee et al. (1988), with slight modifications. Microtitre plates were coated with samples of Tityus spp. venoms (30 μg), 20 μL of the hyaluronic acid substrate (0.5 mg/mL) and acetate buffer (0.2 M

sodium acetate–acetic acid, pH 6.0, containing 0.15 M NaCl) in a final volume of 100 μL. The mixtures were incubated for 15 min at CYTH4 37 °C. After incubation, 200 μL of CTAB 2.5% in NaOH 2% was added to the samples. The absorbance was measured at λ 405 nm using a spectrophotometer (Multiskan EX, Labsystems, Finland) against a blank containing 100 μL of acetate buffer and 200 μL of CTAB. All of the assays were performed in duplicate. The turbidity-reducing activity was expressed as a percentage of the remaining hyaluronic acid, relative to the absorbance of the well in which venom was omitted. The results were expressed in

units of turbidity reduction (UTR) per mg of venom. The enzymatic activity of the Tityus spp. venoms was determined using the fluorescence resonance energy transfer (FRET) substrate peptide Abz-FLRRV-EDDnp. Venom samples (2 μg of protein) were mixed with 5 μM of FRET substrate, in cold phosphate-buffered saline (PBS). The pH studies were performed in 50 mM sodium citrate buffer (pH 3.0–5.3), 50 mM sodium phosphate buffer (pH 5.2–7.5) and 50 mM Tris–HCl buffer (pH 7.3–10) containing 20 mM NaCl ( Ribeiro-Guimarães et al., 2009). The relative inhibition was determined in parallel using 5 mM PMSF or 5 mM 1,10-phenanthroline, inhibitors of serine- or metalloproteinases, respectively. The stock solutions and the working concentrations of the synthetic inhibitors used in the characterisation of the proteolytic activities exhibited by the venom samples were assessed as described ( Beynon and Bond, 2001).