Purification and Characterization of L-amino Acid Oxidase from Agkistrodon halys Pallas Venom

LIU Jie-Wu,CHAI Min-Qiang,DU Xiao-Yan,SONG Jian-Guo,ZHOU Yuan-Cong^*
( Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China )

Abstract    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 μmol/L and 0.08 μmol/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 cells.After 24h treatment with 5 mg/L LAO,the typical DNA fragmentation pattern of apoptotic cells was observed by using of agrose gel electrophoresis.
Key words    L-amino acid oxidase(LAO); Agkistrodon halys Pallas; platelet aggregation; antibacterial effect; apoptosis

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