Elsevier

Toxicon

Volume 47, Issue 1, January 2006, Pages 47-57
Toxicon

Isolation of a new l-amino acid oxidase from Crotalus durissus cascavella venom

https://doi.org/10.1016/j.toxicon.2005.09.008Get rights and content

Abstract

A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 μM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 μg/ml.

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).

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