Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom

Abstract

This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA2 (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS–PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by “de novo sequencing,” in tandem mass spectrometry, belonging to D49-PLA2 enzyme class and exhibiting high identity (44–90%) with other myotoxics PLA2 from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35–45°C. This activity was dependent on Ca2+, other cations (Mg2+, Mn2+, Cd2+ and Zn2+) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Ex vivo, whole venom and PhTX-I PLA2 caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA2 was able to lyse myotubes.