RESUMO
A new PLA2 (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA2 (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca2+-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca2+ and had temperature and pH optima of 25 degrees C and 7.9, respectively. F16 (10 microg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA2 isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Fosfolipases A/farmacologia , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Crotalus , Diafragma/efeitos dos fármacos , Masculino , Camundongos , Dados de Sequência Molecular , Junção Neuromuscular/efeitos dos fármacos , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Fosfolipases A2 , Nervo Frênico/efeitos dos fármacos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Crotoxin, the principal neurotoxin in venom of the South American rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, contains a basic phospholipase A2 (PLA2) and an acidic protein, crotapotin. In this work, we examined the ability of rabbit anti-sera against crotoxin and its PLA2 subunit to neutralize the neurotoxicity of venom and crotoxin from C. d. cascavella in mouse phrenic nerve-diaphragm and chick biventer cervicis preparations. Immunoblotting showed that the anti-sera recognized C. d. cascavella crotoxin and PLA2. This was confirmed by ELISA, with both anti-sera having end-point dilutions of 3 x 10(-6). Anti-crotoxin serum neutralized the neuromuscular blockade in phrenic nerve-diaphragm muscle preparations at venom or crotoxin:anti-serum ratios of 1:2 and 1:3, respectively. Anti-PLA2 serum also neutralized this neuromuscular activity at a venom or crotoxin:anti-serum ratio of 1:1. In biventer cervicis preparations, the corresponding ratio for anti-crotoxin serum was 1:3 for venom and crotoxin, and 1:1 and 1:2 for anti-PLA2 serum. The neutralizing capacity of the sera in mouse preparations was comparable to that of commercial anti-serum raised against C. d. terrificus venom. These results show that anti-sera against crotoxin and PLA2 from C. d. cascavella venom neutralized the neuromuscular blockade induced by venom and crotoxin in both nerve-muscle preparations, with the anti-serum against crotoxin being slightly less potent than that against crotoxin.