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

S. A. Ali, S. Stoeva, A. Abbasi, J. M. Alam, Rakez Kayed, M. Faigle, B. Neumeister, W. Voelter

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)216-226
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume384
Issue number2
DOIs
StatePublished - Dec 15 2000
Externally publishedYes

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Keywords

  • Apoptosis
  • Edema
  • Haemorrhage
  • Hemolysis
  • L-amino acid oxidase
  • Leaf-nosed viper
  • Mass spectrometry
  • Platelet aggregation
  • Snake
  • Toxin
  • Venom

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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