Purification and characterization of L-amino acid oxidase from Agkistrodon halys Pallas venom

L-amino acid oxidase (LAO, EC 1. 4. 3. 2) is widely found in snake venoms and is thought to Contribute to the toxicity in envenoming. By using of Sephadex G-150, DEAE-Sepharose CL-6B and FPLC Superose 12 chromatography, a protein with L-amino acid oxidase activity was purified and characterized from Agkistrodon haly Pallas venom. Its molecular mass was 57 kD as determined by SDS-PAGE analysis under both reducing and non-reducing conditions, and its pI was about 4.9. The protein catalysed the stereospecific oxidative deamination of L-amino acid substrate. It inhibited the platelet aggregation induced by ADP and collagen dose-dependently, even at low concentrations of 0.2 mumol/L and 0.08 mumol/L, respectively. The LAO had antibacterial effect to E. coli K12D31, and the effective concentration was as low as 0.03 g/L. Furthermore, the LAO showed cytotoxicity in crystal violet assay and apoptosis-inducing acitvity in the A549 culls. After 24 h treatment with 5 mg/L LAO, the typical DNA fragmentation pattern of apoptotic culls was observed by using of agrose gel electrophoresis.