Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom

Abstract l -Amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus ( B. fasciatus ) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin–Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55 kDa in SDS-PAGE and an apparent molecular weight of 70 kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against l -Tyr, l -Asp, l -Phe, l -Glu, l -Trp, l -His, l -Gln, l -Ile, l -Met, l -Leu and moderately active against l -Lys, l -Arg, l -Ala and l -Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12 h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3 h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.