Both the nascent
SVMPs and the processed DC domains have been isolated from viperoid venoms (Fox and Serrano, 2005 and Fox and Serrano, 2008). In the present work, we describe the isolation procedure and partial PR-171 supplier characterization of a fibrinogenolytic metalloproteinase from B. moojeni venom, which belongs to the PIIIb subclass of SVMPs. Desiccated B. moojeni venom was purchased from Bioagents Serpentarium (Batatais-SP, Brazil). Acrylamide, ammonium bicarbonate, ammonium persulfate, benzamidine, bromophenol blue, ethylenediaminetetracetic acid (EDTA), bovine fibrinogen, β-mercaptoethanol, N,N′-methylene-bis-acrylamide, leupeptin, phenanthroline, phenylmethylsulphonyl fluoride (PMSF), sodium dodecyl sulfate (SDS) and N,N,N′,N′-tetramethylethylenediame (TEMED) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Glycine, Tris, molecular weight markers for electrophoresis and all chromatographic media were purchased from GE Healthcare (Sweden). All other reagents used were of analytical grade. Male Swiss mice (20 g ± 5 g) were obtained from Centro de Bioterismo e Experimentação Animal (CEBEA) at the Federal University of Uberlândia
(Uberlândia-MG, Brazil). The animals were housed in a temperature-controlled room (23 °C) on an automatic 12 h light/dark cycle (light phase 6 a.m. to 6 p.m.). Food and water http://www.selleckchem.com/products/DAPT-GSI-IX.html were freely available until the beginning of the experiments. The experimental protocol was approved by the Ethics Committee on Animal Experimentation of the Federal University of Uberlândia (CEUA/UFU, Protocol number 028/09). Protein isolation was carried out in two stages. Crude B. moojeni venom (400 mg) was dispersed in 2.0 mL of 0.05 M ammonium bicarbonate buffer (pH 7.8), clarified by centrifugation at 10,000 × g for 10 min and applied on a DEAE-Sephacel column (1.5 × 15 cm). Chromatography was carried out at a flow rate of 40 mL/h, with a convex concentration gradient of the same buffer (0.05–0.6 M). The seventh fraction,
named D7, was pooled, lyophilized, dissolved in 2.0 mL of 0.05 M ammonium bicarbonate (pH 7.8) and submitted to second stage separation 17-DMAG (Alvespimycin) HCl using a HiPrep Sephacryl S-300 column (2.6 × 60 cm). Samples were eluted from this column with the same buffer at a flow rate of 1 mL/min. All peaks were monitored by measuring absorbance at 280 nm. The isolated enzyme was named moojenin. To evaluate the degree of purity, the isolated moojenin was passed through a reverse-phase C2/C18 column (4.6 × 100 mm) using an FPLC Äkta Purifier UPC-10 system (GE Healthcare, Uppsala, Sweden). The column was equilibrated with solvent A (0.1% trifluoroacetic acid), and eluted with a linear concentration gradient from 0 to 100% of solvent B (70% acetonitrile, 0.1% trifluoroacetic acid), at a flow rate of 0.5 mL/min for 93 min. Absorbance was monitored at 280 nm.