Isolation and Functional Characterization of an Acidic Myotoxic Phospholipase A(2) from Colombian Bothrops asper Venom

Myotoxic phospholipases A(2) (PLA(2)) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA(2) (BaCol PLA(2)) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA(2) had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA(2) showed structural homology with other acidic PLA(2) isolated from Bothrops venoms, including a non-myotoxic PLA(2) from Costa Rican B. asper. In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA(2) had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA(2) caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm.