Only adult male specimens were used in this study due to their availability in field at the time. The spiders were identified by Dr Paulo César Motta from the Laboratory of Arachnids (University of Brasília, Brasília, DF, Brazil) based on morphological characteristics. The venom of eight adult male specimens of A. paulensis spiders was monthly obtained by electrical stimulation, solubilized in deionized water containing 0.12% trifluoroacetic acid (TFA) and centrifuged at 10,000 × g for 10 min. The soluble supernatant was immediately

frozen, lyophilized and stored at −20 °C. The venom dry weight was determined in a high precision analytic balance. Aliquots of 5 mg of dried venom were solubilized in deionized water, centrifuged at 10,000 × g for 10 min and the supernatant was submitted to high small molecule library screening performance liquid Bafetinib price chromatography (HPLC), using a C18 reversed-phase semipreparative column (Jupiter 5 μm, 300 Å, 250 × 10 mm, Phenomenex) using a linear gradient from solution A (0.12% TFA) to 60% solution B (0.10% TFA in acetonitrile – ACN) run for 60 min after 10 initial minutes at 0% solution B with detection at 216 and 230 nm. The fractions eluted at a flow rate of 1.5 mL/min were individually and

manually collected, vacuum dried and stored at −20 °C until use. In order to obtain the low molecular mass fraction (LMMF) and protein fraction (PF) for the evaluation of cardiotoxic activity, the fractions eluting from 0 to 35% solution B and from 35 to 74% solution B were separately collected. After removal of solvent, LMMF and PF were quantified by dry weight in a high precision analytic balance and stored at −20 °C until use. The molecular masses of the chromatographic

fractions of A. paulensis venom were performed on an UltraFlexIII MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany). The samples were reconstituted in deionized water at variable concentrations and dissolved (1:3, v:v) in an α-cyano-4-hydroxycinnamic acid matrix solution (α-cyano-4-hydroxycinnamic acid at 5 mg/mL dissolved on acetonitrile, water, trifluoroacetic acid, 5:4:1, v:v:v) spotted in triplicate onto a sample plate and allowed to dry at room temperature. The MS spectra were acquired in both reflected and linear positive modes. Calibration of the Orotic acid system was performed using a mixture of the Peptide Calibration Standard and Protein Calibration Standard I for mass spectrometry (Bruker Daltonics, Germany). Spectra were processed with MassLynx™ 3.5 (Manchester, UK) and FlexAnalysis 3.3 (Bruker Daltonics, Germany). Animals were contained in accordance with the ethical guidelines of the Brazilian Society for Neuroscience and Behavior, which follows the guidelines for animal care prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council, U.S.A.