Caracterização bioquimica e farmacologica de polipeptideos do veneno da aranha Phoneutria nigriventer

AUTOR(ES)
DATA DE PUBLICAÇÃO

1996

RESUMO

This thesis describes the biochemical and pharmacological characterization of three polypeptides from the venom of the spider Phoneutria nigriventer. The fractionation of Phoneutria nigriventer venom by gel filtration on Sephadex G-10-120 provided eight peaks (SI to S-VIII) of which only the first (S-I) was investigated further. lonexchange chromatography of S-I on CM-cellulose-52 yielded 16 fractions (C-I to C-XVI). Of these, Fractions C-VII + VIII and C-X +XI were further purified by reverse phase HPLC to yield two polypeptides (PNV1 and PNV2) with contractile activity. Similar purification of Fraction C-XIII by HPLC resulted in the isolation of the polypeptide with oedematogenic activity (PNV3). Ali three proteins were single chain polypeptides (as shown by SDS-PAGE under reducing conditions) with global amino acid compositions (excluding Trp) of 125, 102 and 132 residues corresponding to molecular weights of 13,900, 12,100 and 14,500 for PNV1, PNV2 and PNV3, respectively. The N-terminal amino acid sequencesof PNV1, PNV2 and PNV3 were: EAFPGQST, LAKRADICQPGKTSQRACET, and AVFAIQDQPC. The contractile activity of PNV1 and PNV2 was examined using rabbit arterial and venous vessels. The vessels were mounted in a cascade system and perfused with warm (37°C) and oxygenated (95% O2 + 5% CO2 Krebs solution. Since the spasmogenic effect of whole Phoneutria nigriventer venom in rabbit vascular smooth muscle is not affected by tetrodotoxin, it is unlikely that sodium channel activation plays a role in these tissues, as it does in both rat phrenic-diaphragm muscle-nerve preparation and guinea-pig isolated atria. Furthermore, the finding that the a-adrenoceptor antagonist phenoxybenzamine does not affect PNV-induced contractions excludes the possibility that Phoneutria nígríventer venom induces endogenous noradrenaline release from autonomic nerve endings present in the vascular walls, as occurs in guinea pig auricles. Phoneutría envenomation is mainly characterized by severe local pain, but it may be accompanied by vascular disturbances such as lung oedema and priapism. Whether these peptides with vascular smooth muscle spasmogenic activity are responsible for the mentioned permeability alterations, it remains to be further investigated. The oedema formation induced by PNV3 was investigated in rabbit skin as the local accumulation of 1251-human serum albumin. Our results showed that PNV3 significantly increased the vascular permeability in the rabbit skin. The increased microvascular permeability induced by whole Phoneutría nígríventer venom in rabbit skin involves local kinin synthesis in response to the activation of tissue (but not plasma) kallikrein-kininogen-kinin system. The biochemical identification of the peptide responsible for this activity in Phoneutría venom might provide a useful tool to further understand the role of tissue kallikrein-kinin system

ASSUNTO(S)

calicreina aranha - veneno cromatografia liquida de alta eficiencia

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