FPLC purification of AGA

Size fractionation of venom proteins was carried out by fast protein liquid chromatography (FPLC; ÄKTA microsystem, GE Healthcare) using a Superdex 75 column (Superdex 75 PC 3.2/30; GE Healthcare) and Ringer’s saline as a buffer, with a 50 μl/min flow. The column elution time was calibrated using native aprotinin (6.5 kDa), cytochrome C (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), and blue dextran (2,200 kDa) (Sigma Kit). For each run, 60 μl of venom extracts obtained from 50 to 75 venom apparatus were injected. Absorbance was followed at 280 nm and fractions of 50 μL were collected every minute in non-binding microplate (Greiner Bio-One). An aliquot of each collected fraction was analyzed by SDS-PAGE under reducing conditions, and the AGA-peak fractions were determined by immunoblotting and enzymatic activity assay. The fraction containing purified AGA (AGA representing more than 60% of total proteins of the fraction) was then used for estimation of kinetic constants and cross-linking experiments. Because purified AGAs were in limited quantities, we estimated AGA α- and β-subunits amounts using a silver stained gel method. Briefly, increasing volumes of the purified fractions were separated by SDS-PAGE under reducing conditions together with known amounts of BSA. Band intensities were obtained from digitalized images of the silver-stained gels using ImageJ software (http://imagej.nih.gov/ij/).