Purification and Characterization of BfPLA2: Fraction containing Acidic Asp49 Phospholipase A2 Isoforms Isolated from the Snake Bothrops fonsecai.

Abstract

Among the various proteins found in the venom of snakes belonging to the genus Bothrops, which is responsible for the highest number of snakebite accidents in Brazil, Phospholipase A2 (PLA2) is one of the most important components. The present study demonstrated the isolation of a fraction containing two Asp49 PLA2 isoforms from the endemic southeastern Brazilian snake Bothrops fonsecai, named BfPLA2. The isolation was achieved through two chromatographic techniques: RP-HPLC using a Teknokroma Europa Protein 300 C18 column, followed by anion-exchange chromatography using a HiTrap Q FF column. SDS-PAGE and two-dimensional electrophoresis revealed proteins with an approximate molecular mass of 15 kDa and an isoelectric point close to 5, indicating acidic PLA2 isoforms. Mass spectrometry analysis and bioinformatic analysis identified two isoforms with high similarity to PLA2s previously described in literature, corroborating the protein family of the isolated toxins. BfPLA2 showed maximum activity at 60 °C and pH 8.5 (Mean: 223.89 U/min/mg). Cytotoxic activity against the HeLa cell line was demonstrated (IC50: 26.21 μg/mL), as well as anticoagulant activity, prolonging coagulation time by 74.1% at the highest dose, and hemolytic activity (IC50: 8.26 μg/mL). The characterization of proteins isolated from snake venoms may contribute to a deeper understanding of snakebite envenomation, especially regarding Bothrops fonsecai, whose venom remains poorly characterized.