Poster 130 - Deoxyribonuclease II activity of Bothrops snake venoms: Partial purification of DNase

Poster 130. 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.489.

Deoxyribonuclease II Activity of Bothrops Snake Venoms: Partial Purification Of DNase II From Bothrops alternatus Snake Venom

1 Department of Pharmacology, Faculty of Medical Sciences and 2 Department of Biochemistry, Institute of Biology, State University of Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil.

Snake venoms contain a variety of enzymes that act on nucleic acids, including deoxyribonuclease (DNase II), 5´-nucleotidase, phosphatases, phosphodiesterases, and ribonuclease. DNase II is has an acidic pH optimum and does not depend on divalent cations for activity. In this study, we examined the Dnase II activity of venoms from several Bothropsspecies, including B. alternatus, B. jararaca. B. jararacussu and B. neuwiedii, and Crotalus durussis terrificus. DNase II activity was measured by the increase in absorbance at 260 nm using salmon testes DNA as substrate (Kunitz method) and by single radial enzyme diffusion (SRED) in agarose/DNA gels containing ethidium bromide. DNA fragmentation was also assessed by electrophoresis in 1.2% agarose gels followed by staining with ethidium bromide. DNase II activity was detected in all venoms except that of B. jararacussu using both the spectrophotometric and SRED assays. Bothropsalternatus venom consistently showed the highest enzyme activity, followed by that of B. jararaca. DNA fragmentation by B. alternatus venom at pH 4.7 was confirmed by agarose gel electrophoresis. Fractionation of B. alternatus venom by gel filtration on a column (1.6 x 60 cm) of Superdex 75 equilibrated and eluted with 50 mM sodium acetate buffer, pH 5.4, yielded four major protein peaks. DNase II activity was detected in the trough between the first and second peaks. Chromatography of the active fractions on a 5 ml HiTrap SP-Sepharose column equilibrated with acetate buffer followed by a linear gradient (0-1 M) of NaCl in the same buffer showed that DNase II activity eluted as a single peak at the start of the salt gradient. SDS-PAGE of the active fractions indicated the presence of proteins with a molecular mass of 24–50 kDa. These results show that Bothrops venoms contain an acidic DNase that could contribute to DNA degradation following envenomation. The molecular mass range of the partially purified DNase is similar to that of mammalian DNase II.