Bp-13 PLA_2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom

A new PLA 2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ -Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA 2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA 2 activity required Ca 2+ ; its PLA 2 activity was inhibited by Mg 2+ , Mn 2+ , Sr 2+ , and Cd 2+ in the presence and absence of 1 mM Ca 2+ . In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent ( P < 0.05 ). Both the replacement of Ca 2+ by Sr 2+ and temperature lowering (24°C) inhibited the Bp-13 PLA 2 -induced twitch-tension blockade. Bp-13 PLA 2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited ( P > 0.05 ). The main effect of this new Asp49 PLA 2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.