Isolation of a new l-amino acid oxidase from Crotalus durissus cascavella venom
Introduction
The enzyme l-amino acid oxidase (l-amino acid: O2 oxidoredutase, EC 1.4.3.2) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid substrate into a α-Keto acid with production of ammonia and hydrogen peroxidase. LAO is the only FAD-dependent oxidase found in snake venom and its toxicity possibly involve generation of hydrogen peroxide formed as a result of reoxidation of the transiently reduction of flavin cofactor by molecular oxygen. l-amino acid oxidase (LAO or LAAO) catalyzes the oxidative deamination of a number of l-amino acids, following the chemical reaction: RCHNH2COOH+O2+H2O(RCOCOOH+NH3+H2O2. LAO from snake venom has been extensively biochemically characterized as shown in reviews by Wellner and Meister (1961). LAO has been shown to be important for the purification and determination of certain amino acids and the preparation of α-Keto acid (Auricchio et al., 1971, Donlon and Fottrell, 1971). LAO from snake venom was used as a chemical electrode for measuring L-amino acid (Guilbault and Hrabankova, 1970). However, the exact role of LAO in snake venom is not yet understood. This protein represents approximately 30% of the whole venom of some snake species and LAO generally presents a yellow color (Takatsuka et al., 2001a, Takatsuka et al., 2001b).
LAOs from bacterial, fungal and plant species are involved in the utilization of nitrogen sources, but the function of snake venom LAOs is still poorly understood, although they play a role in inducing apoptosis, affect platelets, and are considered to be toxins (Du and Clementson, 2002). The purification of LAOs from various snake venoms has been reported by several groups. LAOs from different sources exhibit differences in specificity, stability, and other diverse biological activities, such as citocity, haemorrhage, hemolysis, edema, antibacterial and antiparasitic activities (Tempone et al., 2000, Ali et al., 2000 and Du et al., 2002). Purified LAOs from snake venom are usually homodimeric, acidic proteins with pI values between 4.4–8.5, FMN- or FAD-binding (∼2 mol/mol), glycol (∼3.8–4%), protein with the approximately molecular mass of ∼120–150 kDa in for native and ∼55–66 kDa for monomeric forms (Ali et al., 2000). The structures of snake venom LAOs have revealed limited amino acid sequence similarities with LAOs from bacterial or fungal enzymes, although these proteins probably share a similar topological architecture in addition to a preserved catalytic site (Ali et al., 2000). The Crotalus durissus cascavella venom present four main fraction named as convulxin, gyroxin, crotoxin and crotamine, where crotoxin is the main fraction of the venom (De Oliveira et al., 2003). But the precise constitution and biological action of venom remain unclear and it is the case of gyroxin that has been described a neurotoxic and photolytic but these action remain controversy. But recently we also found a new LAO enzymatically active protein in the gyroxin fraction, thus, as LAOs is very pharmacologically interesting and as did not has any description of this protein in the Crotalus durissus cascavella venom. The aims of this study were to isolate a new LAO from the whole venom of Crotalus durissus cascavella, responsible for the most serious snakebite in Ceará State In this article we isolated and studied some structural properties as well as some biological and pharmacological actions of this protein.
Section snippets
Reagents and Venom
The venom was obtained from the Instituto Butantan and all chemicals used were analytical, HPLC and sequence grade from Sigma and Aldrich Chemical, Waters, Applied Biosystems, Pierce and Bio Rad.
Molecular exclusion chromatography
Approximately 35 mg of whole venom from Crotalus durissus cascavella were dissolved in 400μl ammonium bicarbonate buffer (0.2 M; pH 8.0) and homogenized until complete dissolution, followed by clarification by high-speed centrifugation (4500×g for 4 min). The supernatant was recovered and injected onto a
Results
Crude venom from rattlesnake was first subjected to a Superdex 75 gel filtration HPLC column that separated it into four major fractions. LAO activity was found in the gyroxin fraction, which was collected and stored (Fig. 1a). Gyroxin (5 mg) was dissolved and applied onto an ion exchange chromatography column (0.75×8 cm), which previously equilibrated with ammonium bicarbonate 0.05 M. The samples of whole gyroxin were eluted using a discontinuous buffer concentration of ammonium bicarbonate
Purification and biochemical characterization
In the present work, we purified a novel l-amino acid oxidase (LAO) from Crotalus durissus cascavella whole venom to a high degree of molecular homogeneity after two chromatographic steps. This novel LAO is a highly stable molecule with a moderate acidic character and FAD as its enzymatic cofactor. This compound represents approximately 0.28% of dried Crotalus durissus cascavella venom, but other venoms may present higher concentrations of LAO (Torii et al., 1997). The biochemical properties,
Acknowledgements
This work was supported by FAPESP (04/00040-3, Fundação de Amaparo a Pesquisa do Estado de São Paulo), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).
References (25)
- et al.
Gyroxin, a toxin from the venom of Crotalus durissus terrificus, is a thrombin-like enzyme
Toxicon
(1988) - et al.
Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmohoni) snake venom
Arch. Biochem. Biophys.
(2000) - et al.
Assay of peptidase activities of intestinal brush border membrane with L-amino acid oxidase
Anal. Biochem.
(1971) - et al.
Gyroxin fails to modify in vitro release of labelled dopamine and acetylcholine from rat and mouse striatal tissue
Toxicon
(2001) - et al.
Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom
Toxicon
(2003) - et al.
Quantitative determination of intestinal peptide hydrolase activity using L-amino acid oxidase
Clin. Chim. Acta
(1971) - et al.
Antimicrobial action of achacin is mediated by L-amino acid oxidase activity
FEBS Lett.
(2002) - et al.
A spectrophotometric microplate assay for L-amino acid oxidase
Anal. Biochem.
(2001) - et al.
Platelet aggregation and antibacterial effects of an l-amino acid oxidase purified from Bothrops alternatus snake venom
Bioorg. Med. Chem.
(2004) - et al.
Antibacterial effect of different snake venoms: purification and characterization of antibacterial protein from Pseudochis australis (Australiam king or mulga snake) venom
Toxicon
(1991)
Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomho§i with special reference to platelet aggregation
Biochim. Biophys. Acta
Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation
Biochim. Biophys. Acta
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