NEW METHODOLOGY FOR THE ISOLATION OF AN ANTIMYOTOXIC PROTEIN FROM OPOSSUM SERUM AND A RELATIVE PROTEIN FROM HUMAN PLASMA

Chermont, S.A.(1), Rocha, S.L.G.(1), Jurgilas, P.B.(1), Neves-Ferreira, A.G.C.(1), Domont, G.B.(2), Perales, J.(1)

(1)Departamento de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro, (2)Departamento de Bioquímica, IQ, UFRJ, Rio de Janeiro.

Bothropic venom toxins are known to produce mainly local tissue damage, such as haemorrhage and myonecrosis. Natural resistance to snake venom toxic effects is observed in some animals and, in most cases, is due to the presence of soluble neutralizing proteins in their sera. From the opossum (Didelphis marsupialis) serum, we have previously isolated an acidic glycoprotein named DM64 using ion exchange, hydrophobic interaction and size exclusion chromatographies. DM64 inhibits both the myotoxicity and cytotoxicity of myotoxins I and II from Bothrops asper venom. The cDNA coding for DM64 was cloned and its deduced protein sequence showed homology to a_{1B-glycoprotein, a human plasma protein of unknown function, member of the immunoglobulin supergene family. The aim of this study was to purify DM64 from opossum serum using affinity chromatography with myotoxin. A similar protocol was used to attempt the isolation of} a_{1B-glycoprotein from human plasma. Opossum serum was submitted to an affinity column coupled with myotoxin I from Bothrops asper venom. Homogeneous DM64 was obtained after eluting the column with 0.1 M glycine/HCl pH 2.7. Human plasma was initially submitted to Blue-Sepharose chromatography. The bound fraction was eluted with 1 M NaCl and applied to the affinity column coupled with myotoxin I. A single protein was eluted with glycine/HCl. It was homogeneous by native PAGE and presented 103 kDa on Superdex200, in accordance with the molecular mass reported for the dimer of} a_{1B-glycoprotein. In conclusion, we purified DM64 from opossum serum using affinity chromatography with myotoxin. The identification of the protein isolated from human plasma using this same methodology is under way.}

CORRESPONDENCE TO:

Simone de Amorim Chermont, Rua Lucena, n 11, Rio de Janeiro, RJ, CEP: 21021320, Brasil, Email: chermont@ioc.fiocruz.br