Purification and Partial Characterization of a Protease from Scorpaena plumieri fish venom.

Carrijo, L.C.¹, Andrich, F.¹, Cassoli, J.S.¹, Gomes, P.C.², Richardson, M.², Cordeiro, M.N.², Figueiredo, S.G.¹

1 Lab. de Toxinologia, Dept. de C. Fisiológicas, CBM-UFES; 2 Centro de Pesquisa Prof. Carlos Ribeiro Diniz, Fundação Ezequiel Dias (MG)

The scorpionfishes are the most venomous fishes in the Atlantic Ocean. The species Scorpaenaplumieri is commonly found on shallow water beaches along the Brazilian coast, where it provokes a great number of accidents. The pharmacological activities were found very unstable and this fact could be associated with proteolytic activity. In this work a gelatinolytic protease (EGe) was purified to homogeneity from a water soluble fraction of the S. plumieri venom through a combination of three chromatographic steps: gel filtration on Sephacryl S-200; ion exchange on DEAE-Cellulose and reverse phase/HPLC on a Vydac C4 column. Activity was assayed by SDS-PAGE-gelatin and the purified protease was around 2% of the whole protein in the soluble crude venom. The molecular mass of the EGe scorpionfish gelatinase estimated by SDS-PAGE was around 65 KDa under reducing conditions and 80 KDa under non-reducing conditions. Attempts to determine the N-terminal sequence by automatic Edman degradation were unsuccessful, probably due to blockage of the N-terminal group. Gelatinolytic activity was optimal at pH 8.0. Inhibitors of serine proteases such as aprotinin, PMSF and TLCK inhibited the enzymatic activity, while other inhibitors such as pepstatin, EDTA, phenanthroline and iodoacetamide did not show any effect. The results obtained confirmed that the purified protease is a proteinase with gelatinase activity belonging to the family of serine-proteases. Further studies may help to elucidate the role of this enzyme in the venom.

Supported by:CNPq, PIBIC/FACITEC-UFES, FUNED.

Correspondence to: suelygfi@cbm.ufes.br