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

Author(s)

Bordon, Karla C. F. ^[1] ; Wiezel, Gisele A. ^[1] ; Cabral, Hamilton ^[2] ; Arantes, Eliane C. ^[1]

Affiliation

^[1] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Phys & Chem, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Pharmaceut Sci, BR-14040903 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: Journal of Venomous Animals and Toxins including Tropical Diseases; v. 21, AUG 13 2015.
• Web of Science Citations: 13

Abstract

BackgroundCrotalus 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. (AU)

• FAPESP's process: 14/06170-8 - Biochemical and in vitro evaluation of fibroblast activation and leishmanicidal potential of an L-amino acid oxidase (LAAO) from Crotalus durissus terrificus venom
• Grantee: Gisele Adriano Wiezel
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 11/23236-4 - Native and recombinant animal toxins: functional, structural and molecular analysis
• Grantee: Suely Vilela
• Support Opportunities: Research Projects - Thematic Grants