ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM

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-d...

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Autores principales:	Jaime Andrés Pereañez, Juan Carlos Quintana, Juan Carlos Alarcón, Vitelbina Núñez
Formato:	article
Lenguaje:	EN
Publicado:	Universidad de Antioquia 2014
Materias:	Venenos de serpiente mordeduras de serpiente Bothrops asper fosfolipasas A2 necrosis Colombia Food processing and manufacture TP368-456 Pharmaceutical industry HD9665-9675
Acceso en línea:	https://doaj.org/article/709f6f8651b649b6a0c35e4beb9e98f8
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spelling	oai:doaj.org-article:709f6f8651b649b6a0c35e4beb9e98f82021-12-02T05:24:22ZISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM0121-4004https://doaj.org/article/709f6f8651b649b6a0c35e4beb9e98f82014-04-01T00:00:00Zhttp://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0121-40042014000100005&lng=en&tlng=enhttps://doaj.org/toc/0121-4004Background: 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.Jaime Andrés PereañezJuan Carlos QuintanaJuan Carlos AlarcónVitelbina NúñezUniversidad de AntioquiaarticleVenenos de serpientemordeduras de serpienteBothrops asperfosfolipasas A2necrosisColombiaFood processing and manufactureTP368-456Pharmaceutical industryHD9665-9675ENVitae, Vol 21, Iss 1, Pp 38-48 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Venenos de serpiente mordeduras de serpiente Bothrops asper fosfolipasas A2 necrosis Colombia Food processing and manufacture TP368-456 Pharmaceutical industry HD9665-9675
spellingShingle	Venenos de serpiente mordeduras de serpiente Bothrops asper fosfolipasas A2 necrosis Colombia Food processing and manufacture TP368-456 Pharmaceutical industry HD9665-9675 Jaime Andrés Pereañez Juan Carlos Quintana Juan Carlos Alarcón Vitelbina Núñez ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM
description	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.
format	article
author	Jaime Andrés Pereañez Juan Carlos Quintana Juan Carlos Alarcón Vitelbina Núñez
author_facet	Jaime Andrés Pereañez Juan Carlos Quintana Juan Carlos Alarcón Vitelbina Núñez
author_sort	Jaime Andrés Pereañez
title	ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM
title_short	ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM
title_full	ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM
title_fullStr	ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM
title_full_unstemmed	ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM
title_sort	isolation and functional characterization of a basic phospholipase a2 from colombian bothrops asper venom
publisher	Universidad de Antioquia
publishDate	2014
url	https://doaj.org/article/709f6f8651b649b6a0c35e4beb9e98f8
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ISOLATION AND FUNCTIONAL CHARACTERIZATION OF A BASIC PHOSPHOLIPASE A2 FROM COLOMBIAN Bothrops asper VENOM

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