4 mg of dry crude venom), and two specimens (T221 and T224) with a similar venom profile from Fujian province, China (4.7 mg combined weight of PLX4720 dried venom), in which high and low molecular weight PLA2s respectively formed the major components of the venom. The purification of the PLA2s was carried out using Reverse-phase HPLC on 1 mg of crude venom. All the fractions were manually

collected and a MALDI–TOF–MS analysis was performed in order to confirm the final mass of each fraction. Finally, the quantity and purity of each manually collected fraction was assessed by size exclusion chromatography. Haemorrhagic activity was assessed by exposing blood vessels serving unhatched chick embryos to filter paper discs (2 mm diameter) loaded with fixed concentrations of venom samples in 0.9% w/v NaCl (44), using Bothrops jararaca venom as a positive control and 0.9% w/v NaCl alone as a

negative control ( Sells et al., 1998). Haemorrhagic activity was measured as the time taken for a haemorrhagic corona to appear around the disc, SCH772984 datasheet and the area of the corona after continuous contact with the disc for 2hr. Myotoxic and neurotoxic activity were assessed by incubating mouse soleus muscles at room temperature in oxygenated Liley’s fluid for three hours in the presence of samples of venom or venom fractions at a fixed concentration of 10 μg ml−1. At the end of the period of incubation, muscles were lightly fixed, cryoprotected, frozen in liquid N2 and sectioned at 6 μm (TS) and 10 μm (LS). For the assessment of myotoxicity, sections were stained with H & E and evidence of frank necrosis, hyper-contraction, and oedematous separation of necrotic muscle fibres ( Harris et al., 1975) was sought. For the assessment of neurotoxicity sections were labelled with a primary antibody for synaptophysin (a protein specific to synaptic vesicles) and a primary antibody for neurofilament (a protein specific to axons) and then to a secondary antibody conjugated to a fluorescent tag. Each section was counter-labelled Depsipeptide molecular weight with alpha-bungarotoxin conjugated to a fluorescent tag to identify

the ACh receptors at the neuromuscular junction. Neurotoxicity was assessed by the absence of labelling for synaptophysin at the neuromuscular junction, or by abnormal labelling of neurofilament ( Dixon and Harris, 1999 and Prasarnpun et al., 2005). At least two muscles were used for each compound. We used SMS (http://www.bioinformatics.org/sms2/protein_gravy.html) and Protparam (EXPASY) to calculate a number of sequence-based features including pI (isoelectric point), MW (theoretical average molecular weight, without any correction made for disulphide bridges), net charge, GRAVY (GRand AVerage of hYdropathy [Kyte and Doolittle, 1982]), aliphatic index (a measure of the thermostability of globular proteins), instability index and amino acid composition (%).