Bp-13 PLA₂: Purification and neuromuscular activity of a new Asp49 toxin isolated from bothrops pauloensis snake venom

A new PLA₂ (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on -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 PLA₂ myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA₂ activity required Ca²⁺; its PLA₂ activity was inhibited by Mg²⁺, Mn²⁺, Sr²⁺, and Cd²⁺ in the presence and absence of 1 mM Ca²⁺. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P<0.05). Both the replacement of Ca²⁺ by Sr²⁺ and temperature lowering (24°C) inhibited the Bp-13 PLA₂-induced twitch-tension blockade. Bp-13 PLA₂ 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 PLA₂ 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.