Elsevier

Toxicon

Volume 54, Issue 3, 1 September 2009, Pages 262-271
Toxicon

Purification, characterization and biological activities of the l-amino acid oxidase from Bungarus fasciatus snake venom

https://doi.org/10.1016/j.toxicon.2009.04.017Get rights and content

Abstract

l-Amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin–Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55 kDa in SDS-PAGE and an apparent molecular weight of 70 kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against l-Tyr, l-Asp, l-Phe, l-Glu, l-Trp, l-His, l-Gln, l-Ile, l-Met, l-Leu and moderately active against l-Lys, l-Arg, l-Ala and l-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12 h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3 h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.

Introduction

LAAO (EC 1.4.3.2) is a dimeric flavoprotein which contains non-covalently bound FAD or FMN as cofactor. It catalyzes the stereospecific oxidative deamination of l-amino acid substrates to an α-keto acid along with the production of ammonia and hydrogen peroxide (H2O2) via amino acid intermediates. Venomous snake families of Viperidae, Crotalidae and Elapidae are the richest sources of LAAOs in nature (Du and Clemetson, 2002).

Snake venom LAAOs are reported to induce various toxic effects including induction of cell apoptosis (Torii et al., 1997, Ali et al., 2000) or necrosis (Braga et al., 2008), enhancement (Toyama et al., 2006, Izidoro et al., 2006, Li et al., 2008) or inhibition (Samel et al., 2008, Tõnismägi et al., 2006) of platelet aggregation, stimulation of edema formation (Stábeli et al., 2004, Izidoro et al., 2006), activation of monocyte and T cell (Wei et al., 2007), antibacterial (Lu et al., 2002, Wei et al., 2003, Toyama et al., 2006), antiviral (Zhang et al., 2003, Sant'Ana et al., 2008), antiprotozoal (França et al., 2007, Sant'Ana et al., 2008, Ciscotto et al., 2009) and antifungal (Ande et al., 2008) activities. Interestingly, LAAOs from different snake venoms not only possess differences in their primary structure, but also showed specific, sometimes even controversial actions (Du and Clemetson, 2002), indicating that pathogenesis of snake biting wound is a complicated process, and it varies between different types of snakes. We therefore investigate the toxic actions of LAAO from Bungarus fasciatus venom in the present study.

B. fasciatus snake belongs to Elapidae Family and resided in the whole of the Indo-Chinese subregion, the Malaysian peninsula and archipelago and Southern China. Many components, such as neurotoxins (Liu et al., 1989), cardiotoxins (Lu and Lo, 1981), phospholipases A2 (Liu et al., 1990, Tsai et al., 2007, Xu et al., 2007), lectins (Zha et al., 2001), cathelicidin (Wang et al., 2008), proteinases (Zhang et al., 1995) and proteinase inhibitors (Lu et al., 2008) had been purified from this snake venom. However, until recently, an LAAO from B. fasciatus venom gland has been cloned (Jin et al., 2007) with the predicted protein precursors comprised of 517 amino acid residues and shared high identity to other snake venom LAAOs except of Oh-LAAO, an LAAO purified from the snake of Ophiophagus hannah (Jin et al., 2007). Since the LAAO protein has not yet been purified before and its enzymatic and biological activities remain unknown, we purified and characterized the enzyme, and examined a number of toxic actions of the LAAO in the present study.

Section snippets

Materials

B. fasciatus venom was collected from Guangxi Province, P.R. China. Heparin–Sepharose Fast Flow (FF) and SP-Sepharose High Performance (HP) columns were from LKB Pharmacia (Uppsala, Sweden). Protein-Pak 300 SW column and reverse-phase C18 high performance liquid chromatography (HPLC) column were from Waters Corporation (Milford, MA, USA). Low molecular weight makers were from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Horseradish peroxidase, o-dianisidine,

Purification of BF-LAAO from B. fasciatus venom

SP-Sepharose HP anion exchange chromatography separated LAAO activity (in the third peak) from most protein components of crude venom of B. fasciatus (Fig. 1A). Heparin–Sepharose FF affinity chromatography isolated further LAAO activity (in the third peak of the eluent) from other proteins (Fig. 1B). The fractions with high LAAO activity were collected and designated as BF-LAAO. The yield of purified BF-LAAO from 500 mg crude venom is 4.62 mg, which accounts for a total of 0.93% protein recovery.

Discussion

A two-step chromatographic procedure was developed to purify BF-LAAO. The crude snake venom was loaded on SP-Sepharose HP column by an anion exchange mechanism in order to separate BF-LAAO from other acidic proteins and most basic proteins, such as neurotoxins (Liu et al., 1989), cytotoxins (Lu and Lo, 1981) and phospholipases A2 (Liu et al., 1989, Tsai et al., 2007, Xu et al., 2007). The Heparin–Sepharose FF affinity chromatography was able to remove non-heparin-binding proteins from BF-LAAO

Acknowledgements

We are grateful to Miss Jie-Lian Lin and Miss Qiu-Yu Chen for their assistants in the animal experiments. This project was sponsored by the Major State Basic Research Program of China (973 Program) (No. 2007CB512400) and the National Natural Science Foundation of China (No. 30801016, 30570813, 30772032).

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