Biological and biochemical characterization of new basic phospholipase A 2 BmTX-I isolated from Bothrops moojeni snake venom

Abstract

BmTX-I, an Asp49 phospholipase A 2, was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14238.71 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA 2 had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and p I value 7.84, and showed a high degree of homology with basic Asp49 PLA 2 myotoxins from other Bothrops venoms. BmTX-I presented PLA 2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45 °C. Maximum PLA 2 activity required Ca 2+ and in the presence of Mg 2+, Cd 2+ and Mn 2+ it was reduced in presence or absence of Ca 2+. Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited ( P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.