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Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.

ABSTRACT: BACKGROUND:Crotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV. METHODS:The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry. RESULTS:The first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-L's activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose. CONCLUSIONS:This research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.

SUBMITTER: Bordon KC

PROVIDER: S-EPMC4535778 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.

Bordon Karla C F KC Wiezel Gisele A GA Cabral Hamilton H Arantes Eliane C EC

The journal of venomous animals and toxins including tropical diseases 20150813

<h4>Background</h4>Crotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.<h4>Methods</h4>The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromat ...[more]

PMID: 26273287

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Project description:Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze N?-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.

Dataset Information

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.

Publications

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.

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