Isolamento e purificação de uma neurotoxina crotoxina do veneno de Crotalus durissus cascavella : caracterização bioquimica e biologica / \ c Daniela Gois Beghini
DISSERTAÇÃO
Português
(Broch.)
T/UNICAMP B394i
Campinas, SP : [s.n.], 2001.
88p. : il.
Orientador: Sergio Marangoni
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Resumo: A serpente Crotalus durissus cascavel/a é encontrada no cerrado do nordeste do Brasil (BARRA VI ERA, 1990) e apesar de pouco estudada sua picada constitui um importante problema de saúde pública (MARTINS et aI., 1998). A crotoxina é um complexo heterodimérico com atividade de fosfolipase A2...
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Resumo: A serpente Crotalus durissus cascavel/a é encontrada no cerrado do nordeste do Brasil (BARRA VI ERA, 1990) e apesar de pouco estudada sua picada constitui um importante problema de saúde pública (MARTINS et aI., 1998). A crotoxina é um complexo heterodimérico com atividade de fosfolipase A2 sendo o principal componente neurotóxico do veneno da Cascavel Sul Americana Crotalus durissus terrificus. A crotoxina atua bloqueando a transmissão de impulsos nervosos na junção neuromuscular causando uma paralisia flácida nas vítimas. O complexo crotoxina consiste em uma fosfolipase A2 (PLA2), componente básico, e um componente ácido, não enzimático conhecido como crotapotina. Acrotoxina do veneno de C. d. terrificus tem sido extensivamente estudada, enquanto que a crotoxina de outros venenos de Crotalus durissus sp tem sido pouco investigada.O objetivo deste trabalho foi a caracterização bioquímica e biológica do veneno e da crotoxina de Crotaus durissus cascavel/a, permitindo também um melhor entendimento do complexo crotoxina (PLA2 e crotapotina). PLA2 e crotapotina, foram purificadas através de dois passos cromatográficos: cromatografia de exclusão molecular em Superdex G-75 e cromatografia em HPLC de fase reversa. A análise cromatográfica em HPLC de fase reversa revelou quatro isoformas de crotapotina (F2, F3, F4 e F5) e apenas uma PLA2 (F6) na crotoxina do veneno de C. d. cascavel/a. A crotoxina de C. d. terrificus, ao contrário, contém três isoformas de PLA2 (Cdt F15, F16 e F17) e somente duas isoformas de crotapotina (Cdt F5 e F7). O grau de pureza das frações foi confirmado pela eletroforese em PAGE-SDS-Tricina onde mostrou que PLA2 e crotapotina migram como uma única banda com massa molecular estimada de 15 kDa e 9 kDa, respectivamente. A composição de aminoácidos da PLA2 mostrou uma grande quantidade de resíduos de meia-cisteína e um alto conteúdo de resíduos básicos, enquanto que as crotapotinas F3 e IF4 mostraram maior quantidade de resíduos carregados negativamente. Para uma melhor caracterização bioquímica avaliou-se a cinética da enzima PLA2 de C. d. cascavel/a. A PLA2 mostrou um comportamento alostérico, com atividade máxima em pH 8,3 e 35-40°C. A PLA2 de C. d. cascavel/a requer Ca2+ para exibir sua atividade, mas foi inibida por Zn2+ e CU2+. Crotapotina e heparina inibiram a atividade catalítica da enzima atuando como inibidores alostéricos. Com o objetivo de estudar a atividade biológica do veneno e da crotoxina de C. d. cascavel/a foi verificado a neurotoxicidade em preparação nervo frênico-diafragma de camundongo e preparação biventer cervicis de pintainho. O veneno de C. d. cascavel/a aqui reportado, bem como seu componente crotoxina, apresentou potente atividade neurotóxica, principalmente em preparação biventer cervicis de pintainho. O estudo miográfico, portanto, revelou que a preparação biventer cervicis de pintainho é mais sensível ao efeito paralisante do veneno e crotoxina quando comparada à preparação nervo frênico-diafragma de camundongo. A principal conclusão deste trabalho é que o veneno de C. d. cascavel/a contém pelo menos quatro isoformas de crotoxina formadas por diferentes combinações das crotapotinas com uma única isoforma de PLA2. Essa situação difere da crotoxina do veneno de C. d. terrificus, onde a heterogeneidade resulta da presença de três ou quatro isoformas de PLA2. Desde que a toxicidade do veneno depende principalmente de seu conteúdo de crotoxina, a presença das isoformas pode contribuir para variações na potência da neurotoxicidade
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Abstract: Crotalus durissus cascavel/a is a snake usually found in scrubland of Brazilian Northeast (BARRA VI ERA, 1990) and in spite of little studied its bite constitutes an important public health problem (MARTINS et aI., 1998). The crotoxin is a heterodimeric complex with phospholipase A2...
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Abstract: Crotalus durissus cascavel/a is a snake usually found in scrubland of Brazilian Northeast (BARRA VI ERA, 1990) and in spite of little studied its bite constitutes an important public health problem (MARTINS et aI., 1998). The crotoxin is a heterodimeric complex with phospholipase A2 activity and is the main neurotoxic component in venom of the South American rattlesnake Crotalus durissus terrifícus. Crotoxin acts blocking the transmission of nervous pulses in the neuromuscular junction causing a flaccid paralysis in the victims. The crotoxin complex consists of a phospholipase A2 (PLA2), basic component, and an acid component, nonenzymatic known as crotapotin. The crotoxin of the C. d. terrifícus venom has been studied extensively, while crotoxin of others Crotalus durissus sp venoms have been poorly investigated. The objective of this work was the biochemical and biological characterization of the venom and of the crotoxin of Crotaus durissus cascavel/a, also allowing a better understanding of the crotoxin complex (PLA2 and crotapotin).
PLA2 and crotapotin, were purified through two chromatography's steps: molecular exclusion chromatography in Superdex G-75 and in HPLC of reverse phase. The chromatographyc analysis in HPLC of reverse phase revealed four crotapotin isoforms (F2, F3, F4 and F5) and just a PLA2 (F6) in the crotoxin of the C. d. cascavel/a venom. Crotoxin from C. d. terrifícus contained three PLA2 isoforms (Cdt F15, F16 and F17), but only two crotapotin isoforms (Cdt F5 e F7). The degree of purity of the fractions was confirmed by the electrophoresis in Page-SDS-Tricina that showed that PLA2 and crotapotin migrated as single band with estimated molecular masses of 15 kDa and 9 kDa, respectively. The composition of amino acids of PLA2 showed a great amount of residues of half-cysteines and a high content of basic residues, while the crotapotins F3 and F4 showed larger amount of residues carried negatively. For a better biochemical characterization, the kinetics of the enzyme PLA2 of C. d cascavella was evaluated. The PLA2 showed allosteric behavior, with maximal activity at pH 8,3 and 35-40°C. The C. d. cascavella PLA2 required Ca2+ for activity, but was inhibited by Cu2+ and Zn2+. Crotapotin and Heparin inhibited the catalytic activity of the enzyme by acting as allosteric inhibitors. Aiming to study the biological activity of the venom and of the crotoxin of C. d. cascavella the neurotoxicity was verified in the mouse phrenic nerve diaphragm preparation and chick biventer cervicis preparation. The C. d. cascavella venom here reported, as well as its crotoxin component, presented potent neurotoxic activity, mainly in chick biventer cervicis preparation. The myographic study also revealed remarkable differences in the sensibility of the mouse phrenic nerve diaphragm preparation and chick biventer cervicis preparation. The preparation mouse nerve-muscle was shown less sensitive to the action neuromuscular blocking of both crotoxin and total venom, when submitted to indirect stimulus. The main conclusion of this work it is that C. d. cascavella venom contains at least four crotoxin isoforms formed by different combinations of crotapotins with the single PLA2 isoform. This situation differs from that of crotoxin in C. d. terrificus venom, where the heterogeneity results from the presence of three or four PLA2 isoforms. Since the toxicity of the venom depends mainly on its content of crotoxin, the presence of isoforms may contribute to variations in neurotoxicity Ver menos
PLA2 and crotapotin, were purified through two chromatography's steps: molecular exclusion chromatography in Superdex G-75 and in HPLC of reverse phase. The chromatographyc analysis in HPLC of reverse phase revealed four crotapotin isoforms (F2, F3, F4 and F5) and just a PLA2 (F6) in the crotoxin of the C. d. cascavel/a venom. Crotoxin from C. d. terrifícus contained three PLA2 isoforms (Cdt F15, F16 and F17), but only two crotapotin isoforms (Cdt F5 e F7). The degree of purity of the fractions was confirmed by the electrophoresis in Page-SDS-Tricina that showed that PLA2 and crotapotin migrated as single band with estimated molecular masses of 15 kDa and 9 kDa, respectively. The composition of amino acids of PLA2 showed a great amount of residues of half-cysteines and a high content of basic residues, while the crotapotins F3 and F4 showed larger amount of residues carried negatively. For a better biochemical characterization, the kinetics of the enzyme PLA2 of C. d cascavella was evaluated. The PLA2 showed allosteric behavior, with maximal activity at pH 8,3 and 35-40°C. The C. d. cascavella PLA2 required Ca2+ for activity, but was inhibited by Cu2+ and Zn2+. Crotapotin and Heparin inhibited the catalytic activity of the enzyme by acting as allosteric inhibitors. Aiming to study the biological activity of the venom and of the crotoxin of C. d. cascavella the neurotoxicity was verified in the mouse phrenic nerve diaphragm preparation and chick biventer cervicis preparation. The C. d. cascavella venom here reported, as well as its crotoxin component, presented potent neurotoxic activity, mainly in chick biventer cervicis preparation. The myographic study also revealed remarkable differences in the sensibility of the mouse phrenic nerve diaphragm preparation and chick biventer cervicis preparation. The preparation mouse nerve-muscle was shown less sensitive to the action neuromuscular blocking of both crotoxin and total venom, when submitted to indirect stimulus. The main conclusion of this work it is that C. d. cascavella venom contains at least four crotoxin isoforms formed by different combinations of crotapotins with the single PLA2 isoform. This situation differs from that of crotoxin in C. d. terrificus venom, where the heterogeneity results from the presence of three or four PLA2 isoforms. Since the toxicity of the venom depends mainly on its content of crotoxin, the presence of isoforms may contribute to variations in neurotoxicity Ver menos
Isolamento e purificação de uma neurotoxina crotoxina do veneno de Crotalus durissus cascavella : caracterização bioquimica e biologica / \ c Daniela Gois Beghini
Isolamento e purificação de uma neurotoxina crotoxina do veneno de Crotalus durissus cascavella : caracterização bioquimica e biologica / \ c Daniela Gois Beghini
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