So far, five PLA2 isoenzymes have been isolated from Lachesis spp. venoms: two acidic (LmPLA2I and LmPLA2II) from L. muta ( Fuly et al., 2003); two basic (LmTX-I and LmTX-II) from L. muta muta ( Damico et al., 2005) and one (LsPA-1) from Lachesis stenophrys ( de Assis et al., 2008). However, none have been purified from L. muta rhombeata and studied in relation to the anticoagulant activity. In this study, we report for the first time, the purification,
prediction of primary structure, anticoagulant and antithrombotic activity of the PLA2 from L. muta rhombeata venom and its relation with its enzymatic activity. Venom was collected in Serra Grande Center (IBAMA authorization number 24945-1), Bahia State Brazil, the only facility in the country totally dedicated to study and preservation of Avasimibe clinical trial the Atlantic Bushmaster, L. muta rhombeata selleckchem (www.lachesisbrasil.com.br). All chemicals and reagents were of analytical or sequencing grade. 7–8 weeks C57BL6 mice were supplied by the Animal Services Unit of the State
University of Campinas (UNICAMP). Mice were housed at room temperature on a 12 h light/dark cycle and had free access to food and water. All procedures were performed according to the general guidelines proposed by the Brazilian Council for Animal Experimentation (COBEA) and approved by the university’s Committee for Ethics in Animal Experimentation (CEEA/UNICAMP) number 1790-1. One hundred mg of crude venom of L. muta rhombeata was dissolved in 1 ml of 0.2 M Ammonium bicarbonate buffer, pH 8.0. After centrifugation at 5.000× g for 5 min, the supernatant was loaded Chlormezanone onto a Sephadex G75 column (1.5 cm × 90 cm), previously equilibrated with the same solution, under a flow rate of 12 ml/h.
Three ml fractions were collected. Five mg from selected PLA2 active fraction (FIII) was dissolved in 200 μl of 0.1% (v/v) trifluoroacetic acid (solvent A). The resulting solution was clarified by centrifugation and the supernatant was further submitted to a reversed phase chromatography on a C5 Discovery® Bio Wide Pore 10 μm (25 cm × 4.6 mm). Fractions were eluted using a linear gradient (0–100%, v/v) of acetonitrile (solvent B) at a constant flow rate of 1.0 ml/min over 50 min, and the resulting fractions were manually collected. The elution profile of both analyses was monitored at 280 nm, and the collected fractions were lyophilized and conserved at −20 °C. The homogeneity of the final material was assessed by mass spectrometry. PLA2 activity was measured using the assay described by Cho and Kezdy (1991) and Holzer and Mackessy (1996) modified for 96-well plates (Beghini et al., 2000).