PHOSPHOLIPASE A₂ ISOLATED FROM Micrurus lemniscatus VENOM

Casais e Silva, L.L.(1), Lebrum, I.(3), Gutiérrez, J.M.(4), Lomonte, B.(4), Alape-Giron, A.(4), Teixeira, C.F.P.(2)

(1)Escola Bahiana de Medicina e Saúde Pública (EBMSP), (2)Laboratório de Farmacologia, and (3)Laboratório de Bioquímica, Instituto Butantan, São Paulo, SP, Brasil, (4)Instituto Clodomiro Picado, San Jose,1000, Costa Rica.

In Brazil, Micrurus snakes are responsable by severe and fatal envenomation. Despide of their clinical importance only few studies have been carried about their venom. Phospholipase A₂ (PLA₂) enzymes are among the main toxic components present in this venom. We report the isolation, characterization and amino acid sequence of PLA₂ from the venom of M. lemniscatus from Bahia State, Brazil. Crude venom was fractionated using RP-HPLC system coupled to a C8 wide pore octylcolumn. The amino acid sequence of the select fraction (ML6) was determined by Edman sequencing procedure derived from digests utilizing trypsin and chymotrypsin. The phospholipase consists of a single polypeptide chain with a monomeric molecular weight of ~14 Kd, estimate by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Purified ML6 consists of 118 amino acid residues and revealed a high degree of homology with other PLA₂s from several sources, mainly with other elapidic myotoxic phopholipases. ML6 has anticoagulant and PLA₂ activities determined by indirect hemolysis. It produces myonecrosisin mice upon intramuscular injection with peak at 3 hours, as observed by increase of serum CK activity and histological analysis. Futhermore, an in vitro cytolytic effect, evaluated by lactic dehydrogenase enzyme release, was observed on muscular differentiated cells (myotubes) but not on murine skeletal muscle (C2C12), endothelial (tEnd) of inflammatory cells. In addition, ML6 induces increase of vascular permeability, degranulates mast cells and induces paw edema suggesting inflammatory effects. However, ML6 was not lethal after i.v. injection but it was toxic when injected into brain.

CORRESPONDENCE TO:

Luciana Lyra Casais e Silva, Rua Cristiano Ottoni 400/301, Salvador, BA, CEP: 40155-210, Brasil, E-mail: casais@usp.br