de Roodt Adolfo Rafael, Litwin Silvana, Angel Sergio O
Area Investigación y Desarrollo/Serpentario, Instituto Nacional de Producción de Biológicos, Administración Nacional de Laboratorios e Institutos de Salud, Dr. Carlos G. Malbrán, Ministerio de Salud, Buenos Aires, Argentina.
Comp Biochem Physiol C Toxicol Pharmacol. 2003 Aug;135(4):469-79. doi: 10.1016/s1532-0456(03)00169-8.
DNA hydrolysis caused by venoms of 17 species of snakes was studied by different methodologies. Endonucleolytic activity was tested by incubation of the venoms with the plasmid pBluescript and subsequent visualization of the electrophoretic patterns in 1% agarose gels stained with ethidium bromide. DNA was sequentially degraded, from supercoiled to opened circle, to linear form, in a concentration dependent manner. The highest hydrolytic activity was observed in Bothrops (B.) neuwiedii and Naja (N.) siamensis venoms. Exonucleolytic activity was analyzed on pBluescript digested with SmaI or EcoRI. All venoms caused complete hydrolysis after 2 h of incubation. SDS-PAGE analysis in gels containing calf thymus DNA showed that the hydrolytic bands were located at approximately 30 kDa. DNA degradation was studied by radial hydrolysis in 1% agarose gels containing calf thymus DNA plus ethidium bromide and visualized by UV light. Venom of B. neuwiedii showed the highest activity whereas those of B. ammodytoides and Ovophis okinavensis (P<0.05) showed the lowest activity. Antibodies against venom of B. neuwiedii or N. siamensis neutralized the DNAse activity of both venoms. In conclusion, venom from different snakes showed endo- and exonucleolytic activity on DNA. The inhibition of DNA hydrolysis by EDTA and heterologous antibodies suggests similarities in the structure of the venom components involved.
采用不同方法研究了17种蛇毒引起的DNA水解作用。通过将蛇毒与质粒pBluescript一起孵育,随后对用溴化乙锭染色的1%琼脂糖凝胶中的电泳图谱进行可视化,来测试核酸内切酶活性。DNA依次从超螺旋形式降解为开环形式,再到线性形式,呈浓度依赖性。在巴西矛头蝮(Bothrops neuwiedii)和暹罗眼镜蛇(Naja siamensis)的毒液中观察到最高的水解活性。对用SmaI或EcoRI消化的pBluescript进行核酸外切酶活性分析。孵育2小时后,所有毒液均导致完全水解。在含有小牛胸腺DNA的凝胶中进行SDS-PAGE分析表明,水解条带位于约30 kDa处。通过在含有小牛胸腺DNA加溴化乙锭的1%琼脂糖凝胶中进行径向水解来研究DNA降解,并通过紫外光进行可视化。巴西矛头蝮的毒液显示出最高活性,而砂锯蝰(B. ammodytoides)和冲绳竹叶青(Ovophis okinavensis)的毒液活性最低(P<0.05)。针对巴西矛头蝮或暹罗眼镜蛇毒液的抗体中和了两种毒液的DNA酶活性。总之,不同蛇的毒液对DNA表现出核酸内切和核酸外切活性。EDTA和异源抗体对DNA水解的抑制作用表明所涉及的毒液成分结构具有相似性。