Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom

The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C₁₈ reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (A D D K N P L E E A F R E A D Y E V F L E I A K N G L) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% α-helix, 19% β-sheet, 10% β-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 μg/ml), edema- (MED 4.8 μg/ml) and human platelet aggregation-inducing (ED₅₀ 33 μg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 μg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.