NucleotidicActivity on DNA induced by Crotalus snake Venom: Isolation and Biochemical Characterization of a Nucleotidasefrom C. durissus terrificus Venom. 

^{1.3Sant`Ana, C. D.}; ¹Oliveira, D. G.; ^1,2Marcussi, S.; ³Ticli, F. K.; ³Mazzi, M. V.; ³Da Silva, J. O.; ⁴Magalhães, M. R.; ¹Marins, M.; ³Sampaio, S. V. and ¹Soares, A. M.

1 Unidade de Biotecnologia-UNAERP, Ribeirão Preto-SP; 2 Depto Bioquímica e Imunologia, FMRP-USP, Ribeirao Preto-SP; 3 Depto. Análises Clínicas, Toxicológicas e Bromatológicas-FCFRP-USP, Ribeirão Preto-SP and 4 UCG, Goiânia-GO, Brasil.

Snake venoms are complex mixtures of proteins which include several enzymes displaying a large action range. Active nucleases, including phosphodiesterases, which hydrolyze DNA and/or RNA, have already been reported in venoms of snakes from several world regions. This work reported a screening of nucleotidic activity of different Crotalus species and the isolation of a nuclease from C. durissus terrificus. Venoms from C. atrox, C. durissus cunamensis, C. d. terrificus, C. d. collineatus and C. d. cascavella were assayed for nuclease activity in agarose gel plates containing 2.0mg DNA from sperm salmon, with different concentrations of venom (25-100µg). After incubation at 37C, the plates were photographed under a UV light at different time intervals and the nucleotidic activity was expressed in cm of the resulting halos. Activity was also assayed by electrophoresis on agarose gel containing 400ng of DNA from E. coli and 1.0µg of each venom. Isolation of a nuclease from C. d. terrificus from venom was accomplished through an affinity chromatographic column, eluted with water followed by a salt concentration gradient (0 to 1M) in 0,01M Tris-HCl +0.001M EDTA, pH 7,4. The protein was assayed for purity by 12% SDS-PAGE and stained with Coomassie Brilliant Blue and Ag⁺. The nucleotidic activity was assayed by PAGE and radial diffusion in agarose gel. Venoms from species C. d. collineatus, C. d. cumanensis, C. d. terrificus, C. d. cascavella and C. atrox showed 0.8; 0.6; 0.8; 1.1; 0.65, respectively of nucleotidic activity. The highly purified enzyme, which was isolated in a single step, showed a single polypeptide chain and extremely active even at very low concentrations (0.5µg), snake venoms are promising sources of nucleotidic enzymes for biotechnological applications.

Financial support: FAPESP, CNPq, CAPES and UNAERP.

Correspondence to: cdalaqua@fcfrp.usp.br or andreimar@unaerp.br