Poster 242 - Effect of crotoxin B on plasmidial DNA extraction from E. coli (DH5a strain)

Poster 242. 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.601.

Effect Of Crotoxin B On Plasmidial DNA Extraction From E. coli (DH5strain)

^1,2Duarte, G.C., ¹Lino, G.R., ¹Rodrigues, V.M., ²Oliveira, F., ²Ferro, E.A.V., ¹Goulart, L.R., ¹Homsi-Brandeburgo, M.I., ¹Hamaguchi, A.

1 Instituto de Genética e Bioquímica, 2 Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais, Brasil

Crotoxin B (crotactine) and lysozyme are basic proteins recognized to express activity against a variety of gram-positive and gram-negative bacterial cells. Crotoxin B is phospholipase A2 isolated from Crotalusdurissus terrificus venom, and hen egg white lysozyme is a bacterial peptidoglycan hydrolase. In order to test the crotoxin B as a biotechnological tool, we studied its effect on plasmidial DNA extraction from Escherichia coli clone (DH5 strain). Crotoxin purified from crude venom of C. durissus terrificus, by gel filtration on Sephadex G-75 column, was submitted to urea treatment and then this sample was applied to DEAE-Sephadex column to isolate the crotoxin B. Its phospholipasic activity by using hen egg yolk as substrate source represented 62 U/min/mg. Lysozyme was isolated from hen egg white on Q-Sepharose column and it had bactericidal activity of 321.6U/min/mg, on Micrococuslysodeikticus by using turbidimetric assay (pH 6.0). When tested on bacterial cell culture in BHI media containing EDTA 0.001M, each purified protein demonstrated weak antibacterial activity on wild S. aureus. Crotoxin B showed strong activity against wild E. coli. Lysozyme did not exhibit action on the same E. coli culture, and when associated to Crotoxin B did not inhibit the lytic activity of this protein. Plasmidial DNA extraction was tested on E. coli (DH5 strain), by using boiling method, in the presence or absence of these enzymes. In the presence of lysozyme, it was possible to extract plasmidial DNA, RNA and proteins. The same assay was not efficient when the lysozyme was replaced by crotoxin B. Cloned E. coli incubated with crotoxin B in 10mM Tris-HCl, 100mM NaCl, 5% Triton X-100, EDTA 1mM, pH 8.0 ( the same condition as the first stage of the extraction method) and submitted to electron microscopy showed some electron-dense precipitates on the intact cell surface, suggesting that in this condition, crotoxin B formed “cages” able to obstruct cellular lysis of E. coli.