Poster 068 - Isolation and characterization of the antibacterial effects of new crotamine isoform

Poster 68. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.427.

ISOLATION AND CHARACTERIZATION OF THE ANTIBACTERIAL EFFECTS OF NEW CROTAMINE ISOFORM FROM THE Crotalus durissus ruruima RATTLESNAKE.

Toyama, D.O.¹; Toyama, M.H.², Luis O. Beriam³, Paulo P. Joazeiro⁴ and Marangoni, S¹

1 Departamento de Bioquímica, IB, UNICAMP; 2 Campus do Litoral Paulista, Unidade de São Vicente, UNESP; 3 Instituto Biologico, Campinas and 4 Departamento de Histologia e Embriologia, IB, UNICAMP.

In this present work we isolated a novel crotamine isoform from the Crotalusdurissus ruruima rattlesnake venom and its antibacterial effect was characterized. The purification of crotamine isoforms was done by combination of two chromatographic steps: a) HPLC molecular exclusion on Superdex 75 (Pharmacia) column (1x60), eluted with ammonium bicarbonate 0.2M, pH 7.9, b)reverse phase HPLC chromatography (m-Bondapack C18, Waters). The homogeneity of the sample was evaluated by Tricine SDS-PAGE. The determination of the primary structure was done by direct digestion of the reduced and carboxymethyled protein as well as the peptides obtained by enzymatic cleavage of this protein with Protase V8 enzyme. The Anti-microbial assay was done used fresh agar plate incubated with bacteria treated and no treated with crotamine and electron microscopy. A Crotamine was obtained as single peak in the molecular exclusion chromatography that after reverse phase HPLC this peak showed the presence of three different peaks. The crotamine isoforms was purified at high molecular homogeneity with a molecular mass of 5kDa, but among then only fraction 2 was the main (Cdru Crot2). This protein showed the presence of 42 amino acid residues. This protein induces strong antibacterial effect, reducing the bacterial growth rate at 78.4% for (Gran-negative) and 87.4% for (Gran-positive).This protein showed high molecular homology with other crotamine like protein as well as some defensin and other bacteriocin peptides and also induced a strong blockage effect similar to previously characterized for other similar toxins. This new Crotamine induced a high antimicrobial effect against both kind of bacteria and its activity was modulated by the presence of positively charged amino acid residues similar to found in the defensins and this effect was carried out by destruction of the membrane as shown in the electron microscopy.