Fractionation of Bothrops pirajai snake venom: Isolation and characterization of piratoxin-I, a new myotoxic protein

Abstract Whole desiccated venom of Bothrops pirajai was fractionated on a gel filtration (Sephadex G-75) column. Phospholipase A 2 , arginine esterase and clotting activity profiles of the six fractions (SI to SVI) obtained were determined. Fraction SIV from the gel filtration column was subjected to chromatography on SP-Sephadex C-25. It was resolved into five subfractions (SIV-SP 1 to SIV-SP 5 ). Fractions SIV-SP 1 , SIV-SP 2 and SIV-SP 3 showed phospholipase A 2 activity but, among these fractions, only SIV-SP 3 was homogeneous. Induction of myonecrosis by SIV-SP 3 , SIV-SP 4 , and SIV-SP 5 was demonstrated by their ability to release serum creatine kinase, and for SIV-SP 5 , to induce histological alterations in the injected mouse muscle. Chemical characterization by determination of mol. wts, isoelectric focusing and direct manual sequencing of the N-terminal region was performed for SIV-SP 3 , SIV-SP 4 and SIV-SP 5 . When compared with bothropstoxin-I, the myotoxin SIV-SP 5 showed the same total number of amino acid residues (121) and constant molar ratio for all but three amino acids. We have named this toxin piratoxin-I (PrTX-I).