Bothrops snake venoms contain a variety of phospholipases (PLA2), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA2 from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (pI 5.05 and 4.49) Asp49 PLA2, with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA2 previously isolated from this venom (PA2_BOTIN, SwissProt accession number Q8QG87) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA2 showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA2 from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA2 that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. BinTX-I (30 μg/paw) produced mouse hind paw edema that was maximal after 1 h compared to after 3 h with venom (10 and 100 μg/paw); in both cases, the edema decreased after 6 h. BinTX-1 and venom (40 μg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo. © 2006 Elsevier Masson SAS. 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