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 (Eristocoph is macmahoni) snake venom was purified to homogeneity in a single step usin g high performance liquid chromatography on a Nucleosil 7C(18) reverse phas e column. The molecular mass of the purified enzyme was 58734.0 Da, as dete rmined by matrix-assisted laser desorption/ionization mass spectrometry. Th e 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 cl ose structural homology with other L-amino acid oxidases isolated from diff erent snake venoms. The secondary structural contents analysis of LAG, esta blished by means of circular dichroism, revealed ca. 49% alpha -helix, 19% beta -sheet, 10% beta -turn, and 22% random coil structure. The purified LN V-LAO not only retained its specific enzymatic activity (73.46 U/mg), deter mined against L-leucine as a substrate, but also exhibited potent haemolyti c (1-10 mug/ml), edema- (MED 4.8 mug/ml) and human platelet aggregation-ind ucing (ED50 33 mug/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 acti vity in the MM6 cell culture assay. After 18 h treatment with 25-100 mug/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was obser ved by means of fluorescent microscopy and agarose gel electrophoresis. (C) 2000 Academic Press.