IDENTIFICATION AND PURIFICATION OF ENZYMES FROM THE VENOM OF THE SPIDER Phoneutria keyserlingi (CTENIDAE)

ASSIS SOUZA, I.; REZENDE, F. F.; OLIVEIRA, C. F. B.; GONÇALVES, J. M.; BORGES, M. H.; RICHARDSON, M.; CORDEIRO, M. N.

Laboratório de Bioquímica e Química de Proteínas, Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias

Spiders of the genus Phoneutria are frequently found near human habitations and are often involved in envenenomations. In this work, we investigated the enzymatic profile in the venom of the spider Phoneutria keyserlingi. SDS-PAGE zymogram experiments using hyaluronic acid and gelatin as substrates showed the presence of hyaluronidase and proteolytic enzymes in this venom. In addition, the alpha chain of fibrinogen was degraded when it was incubated with the venom during two hours at 37oC, confirming the proteolytic activity. The crude venom was partially purified by reverse phase chromatography on a preparative column of Vydac C4. The hyaluronidase and proteolytic activities were determined in each of the fractions collected towards the end of the gradient of acetonitrile. The fraction 38 which was eluted with 47% acetonitrile-TFA 0.1%, showed both proteolytic and fibrinogenolytic activity and the fractions 39-40 (49%), also showed hyaluronidase activity. The enzyme hyaluronidase found in the venom of many spiders acts as a spreading factor for the toxins because it degrades hyaluronic acid, a glycosaminoglycan in the extracellular matrix of connective tissues. So far there are no details available of the structure of this enzyme from the venom of any member of the sub-phylum Arachnida. We are currently completing the purification of hyaluronidase and the proteinases with the objective of determining their primary structure.

KEY WORDS: Phoneutria keyserlingi, enzymes, hyaluronidase, venoms, proteolytic activity.

FINANCIAL SUPPORT: FAPEMIG, CNPq, PUC-Minas, FUNED

CORRESPONDENCE TO: IVANA ASSIS, Laboratório de Bioquímica e Química de Proteínas, Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias. Belo Horizonte, MG, Brasil. Tel.: (31) 8865-0923 Email: souzaivana@gmail.com