Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A₂ isoform (Bj-V) from Bothrops jararacussu venom

A new PLA₂ Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40°C. Full PLA₂ activity required Ca ²⁺ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca²⁺.The addition of Mn²⁺ or Mg²⁺ (10 mM) in the presence of low (1 mM) Ca²⁺ slightly increased the enzyme activity, whereas Zn²⁺ and Cu²⁺ (10 mM) diminished the activity. The substitution of Ca²⁺ for Mg²⁺ or Cu ²⁺ also reduced the enzymatic activity. Bj V had PLA² activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.