cangicum venom, previously obtained from a similar Sephadex G-50 column [46]. The neurotoxic fractions from B. granulifera and S. helianthus were submitted to reversed-phase HPLC in an ÄKTA Purifier system (Amersham Biosciences, PD-166866 purchase Uppsala, Sweden) using a semi-preparative column, CAPCELL PAK C-18, 10 mm × 250 mm

(Shiseido Corp., Kyoto, Japan). The HPLC conditions used were: 0.1% trifluoroacetic acid (TFA) in water (solvent A) and acetonitrile containing 0.1% TFA (solvent B). The chromatographic runs were performed at a flow rate of 2.5 mL/min using a 10–60% gradient of solvent B over 40 min, after an isocratic step using 10% ACN during 2.25 min. UV detection was monitored at 214 and 280 nm. Each of the individual see more sub-fractions from Fr 3-4 were manually collected and lyophilized or concentrated for further molecular mass assessments by MALDI-TOF mass spectrometry. Most intense fractions were re-purified in an analytical column (CAPCELL PAK C-18, 4.6 mm × 150 mm i.d.), using a slower gradient of 0.5%B/min to achieve better resolution. The retention

of a peptide expressed as percentage of acetonitrile (%ACN) was estimated from the formulas %ACN = 100ϕ and ϕe = ϕ0 + (Δϕ/tG)·(tR − t0 − tD) [78], therefore %ACNe = %ACN0 + (Δ%ACN/tG)·(tR − t0 − tD), being tR the retention time of compound X; t0 the elution time of a non-retained compound (6 min), tD the equipment dwell time (0.25 min), Δ%ACN/tG the gradient slope (50%/40 min = 1.25%/min), %ACNe the percentage of acetonitrile at elution of compound X, %ACN0 percentage of acetonitrile at the gradient start (10%). Then, %ACNe = 10% + 1.25%/min·(tR − 6.25 min). Considering the previous isocratic step, at 10% ACN during 2.25 min, tdelay = 2.25 min is introduced in the calculation so %ACNe = 10% + 1.25%/min·(tR − 8.50 min). The proteinaceous contents of the secretions and neurotoxic fractions were estimated by the bicinchoninic acid (BCA) method [77] following the manufacturer’s instructions (Pierce, Rockford, IL, USA). Reversed-phase chromatographic fractions were submitted to mass spectrometric

analyses, which were carried out using an AutoFlex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, USA), controlled by the FlexControl 3.0 software (Bruker Daltonics, Billerica, USA). Benzatropine The samples were mixed with two different matrixes (i) α-cyano-4-hydroxycinnamic acid matrix solution (1:2, v/v) and (ii) super-2-hydroxy-5-methoxybenzoic acid (s-DHB) (1:2, v/v) directly into a MTP AnchorChip 600/384 MALDI target plates (Bruker Daltonics, Billerica, USA) and dried at room temperature. Protein average masses (5000–20,000 Da) were obtained in linear mode with external calibration, using the Protein Calibration Standard (Bruker Daltonics, Billerica, USA). The peptide monoisotopic masses (900–5000 Da) were obtained in reflector mode with external calibration, using the Peptide Calibration Standard (Bruker Daltonics, Billerica, USA).