Purification and biological effects of l-amino acid oxidase isolated from Bothrops insularis venom

doi:10.1016/j.toxicon.2007.09.003

Toxicon

Volume 51, Issue 2, February 2008, Pages 199-207

https://doi.org/10.1016/j.toxicon.2007.09.003 Get rights and content

Abstract

Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure (C₁₂₀=110.28±3.69; BiLAO₁₂₀=82.2±5.6 mmHg*); renal vascular resistance (C₁₂₀=5.48±0.53; BiLAO₁₂₀=4.12±0.42 mmHg/mL/g/min*), urinary flow (C₁₂₀=0.160±0.020; BiLAO₁₂₀=0.064±0.012 mL/g/min*), glomerular filtration rate (C₁₂₀=0.697±0.084; BiLAO₁₂₀=0.176±0.017 mL/g/min*), sodium (C₁₂₀=79.76±0.56; BiLAO₁₂₀=65.39±6.19%*), potassium (C₁₂₀=69.94±6.86; BiLAO₁₂₀=60.26±2.24%*) and chloride tubular reabsortion (C₁₂₀=78.53±2.33; BiLAO₁₂₀=64.58±6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of glomerular filtration resulted in more time for filtration and tubular reabsorption, with elevation of the total percentage of sodium and chlorine reabsorption. The maintenance of the decrease in glomerular filtration rate would determine the subsequent decreases, which were noticed in these parameters. The necrosis observed was the result of damage cell induced by l-amino acid oxidase isolated from B. insularis 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 an α-keto acid with the production of ammonia and hydrogen peroxide. l-amino acid oxidase (LAO or LAAO) is the only FAD-dependent oxidase found in snake venom and it is thought to contribute to the toxicity of the venom, possibly through generation of hydrogen peroxide formed as a result of reoxidation of the transient reduction of flavin cofactor by molecular oxygen. LAO catalyses the oxidative deamination of a number of l-amino acids, following the chemical reaction H₂NCHRCOOH+O₂+H₂O→O=CHRCOOH+NH₃+H₂O₂. LAO from snake venom has been extensively biochemically characterized as described in reviews carried out by Wellner and Meister (1961). LAO isolated from Bothrops insularis snake venom (BiLAO) was used as a chemical electrode for measuring l-amino acid (Guilbault and Hrabankova, 1970). l-amino acid oxidase is found in most snake venoms. However, the exact role of this enzyme is not yet understood. It represents approximately 30% of the whole venom of some snake species and generally displays a yellow color (Takatsuka et al., 2001). 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 Clemetson, 2002). 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 cytotoxicity, hemorrhage, hemolysis, edema, antibacterial and antiparasitic activities (Tempone et al., 2001; Ali et al., 2000; Du and Clemetson, 2002). Purified LAOs from snake venom are usually homodimeric acidic proteins with pI values between 4.4 and 8.5, FMN- or FAD-binding (∼2 mol/mol), glycol (∼3,8–4%), and with molecular masses of approximately ∼120–150 kDa for native and ∼55–66 kDa for monomeric forms (Ali et al., 2000). The snake venom LAOs have limited sequence homology 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 aim of this study was to isolate a new LAO from the whole venom of B. insularis as well as to assess the renal effects promoted by this enzyme.

Section snippets

Reagents and venom

The venom was obtained from Instituto Butantan. Chemicals and reagents used in this work were purchased from Sigma and Aldrich (Sigma Chemical Co., St. Louis, MO, USA), Applied Biosystems, Pierce and Bio Rad (USA).

l-aminoacid oxidase purification

B. insularis venom was a gift from Instituto Butantan. Purification of l-amino acid oxidase was done following the method described by Toyama et al. (2000) and Fonseca et al. (2006) and subsequently modified for the bothropic venom (Barbosa et al., 2005). Basically, an amount of 30 mg

Results of purification of l-amino acid oxidase isolated from Bothrops insularis venom

Chromatographic profile of B. insularis whole venom disclosed seven main fractions designated as I, II, III, IV, V, VI and VII and the main LAO enzymatic activity was detected in fraction IV; this fraction was then subjected to new affinity chromatography that gives two different peaks Bi II and Bi III (Fig. 1a and b). After the first HPLC molecular exclusion chromatography, fraction Bi II was submitted to new HPLC molecular exclusion for high molecular weight protein that gave one major LAO

Discussion

l-amino acid oxidases are flavo-enzymes that catalyse the stereospecific oxidative deamination of an l-amino acid substrate. LAOs are capable of activating platelets, and are also capable of cytotoxicity by inducing apoptosis, as well as of provoking hemorrhage. LAOs are the major components of many snake venoms. Some of these activities are known to date, but still poorly understood with regard to snake venoms, though they are postulated to be toxins (Du and Clemetson, 2002). As to crotalic

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Snake venom LAAOs have been reported to exhibit a wide range of pharmacological activities, including cytotoxic, edema-inducing, platelet aggregation-inducing/platelet aggregation-inhibiting, bactericidal and antiviral activities. A heat-stable form of l-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom (OH-LAAO) has been shown to exhibit very potent cytotoxicity against human tumorigenic cells but not in their non-tumorigenic counterparts, and the cytotoxicity was due to the apoptosis-inducing effect of the enzyme. In this work, the molecular mechanism of cell death induced by OH-LAAO was investigated. The enzyme exerts its apoptosis-inducing effect presumably via both intrinsic and extrinsic pathways as suggested by the increase in caspase-8 and -9 activities. Oligonucleotide microarray analysis showed that the expression of a total of 178 genes was significantly altered as a result of oxidative stress induced by the hydrogen peroxide generated by the enzyme. Of the 178 genes, at least 27 genes are involved in apoptosis and cell death. These alterations of gene expression was presumably caused by the direct cytotoxic effect of H₂O₂ generated during the enzymatic reaction, as well as the non-specific oxidative modifications of signaling molecules that eventually lead to apoptosis and cell death. The very substantial up-regulation of cytochrome P450 genes may also contribute to the potent cytotoxic action of OH-LAAO by producing excessive reactive oxygen species (ROS). In conclusion, the potent apoptosis inducing activity of OH-LAAO was likely due to the direct cytotoxic effect of H₂O₂ generated during the enzymatic reaction, as well as the non-specific oxidation of signalling molecules.

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^☆: Ethical statement: The experiments follow the methodology recommended by the international ethical standards of the scientific committee of our university (Comittê de Ètica e Pesquisa com Animais).

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Purification and biological effects of l-amino acid oxidase isolated from Bothrops insularis venom☆

Abstract

Introduction

Section snippets

Reagents and venom

l-aminoacid oxidase purification

Results of purification of l-amino acid oxidase isolated from Bothrops insularis venom

Discussion

Toxicon

Arch. Biochem. Biophys.

Toxicon

J. Biol. Chem.

Toxicon

Toxicon

Biochem. Biophys. Res. Commun.

Toxicon

Anal. Biochem.

Toxicon

Biochem. Biophys. Res. Commun.

Blood

Bioorg. Med. Chem.

Toxicon