Abstract

PEREANEZ, Jaime Andrés; QUINTANA, Juan Carlos; ALARCON, Juan Carlos  and  NUNEZ, Vitelbina. ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A₂ FROM COLOMBIAN Bothrops asper VENOM. Vitae [online]. 2014, vol.21, n.1, pp.38-48. ISSN 0121-4004.

Background: Snakebites represent a relevant public health issue in many regions of the world, particularly in tropical and subtropical countries of Africa, Asia, Latin America and Oceania. Snake venoms are complex mixtures of toxic enzymes and proteins, where the most important and abundant muscle-damaging components in snake venoms are phospholipases A₂ (PLA₂s). Objective: Isolate and characterize a phospholipase A₂ from Colombian Bothrops asper venom, in order to obtain information about venom composition of this species. Materials and methods: Cation-exchange chromatography followed by reverse phase HPLC were used to purify the protein. Mass spectrometry was used to determine its molecular mass. Biochemical characterization was performed using a synthetic substrate (4-nitro-3-octanoyloxy-benzoic acid). Myotoxic and edema-inducing activity of toxin were tested in mice, by measuring the plasma creatine kinase activity and footpad diameter, respectively. Moreover, cytotoxic activity was examined to murine skeletal muscle C2C12 myoblasts and myotubes. Results: A PLA₂ of Bothrops asper venom from Colombia (BaspCol-PLA₂) was purified. Its molecular mass was 13974.6 Da. The enzyme hydrolyzed a synthetic substrate with a K_M of 3.11 mM and a V_Max of 4.47 nmol/min, showing maximum activity at 40 °C and at pH 8.0. The PLA₂ required Ca²⁺ for activity. The addition of Mg²⁺, Cd²⁺, Mn²⁺ and Zn²⁺ (10mM) in the presence of low Ca²⁺ concentration (1mM) decreased the enzyme activity. The substitution of Ca²⁺ by mentioned divalent cations also reduced the activity to levels similar to those in the absence of Ca²⁺. Three internal fragments (CCFVHDCCYGK, AAAI/ LCFRDNI/LNTYNDKK, DAAI/LCFR) identified by a mass spectrometry analysis showed similarity with previously reported B. asper PLA₂s. In mice, BaspCol-PLA₂ induced a conspicuous local myotoxic effect and moderate footpad edema. In vitro, this enzyme induced cytotoxic effect on both myoblasts and myotubes. Additionally, it was classified as weakly anticoagulant PLA₂, showing this effect at concentrations between 3 and 10 μg/mL when using human plasma. Conclusions: A PLA₂ was purified and named BaspCol-PLA₂, this enzyme displayed catalytic activity and molecular mass of 13974.6 Da. The toxin showed myotoxic, edema-forming, anticoagulant and cytotoxic activities.

Keywords : Snake venoms; snake bites; Bothrops asper; phospholipases A₂; necrosis; Colombia.

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