Hamzah Nazar Abdulameer, Raheif Eman Salman
Department of Medical Biotechnology, College of Biotechnology, University of Al-Qadisiyah, Al-Diwaniyah, Iraq.
Department of Biology, College of Science, University of Al-Qadisiyah, Al-Diwaniyah, Iraq.
Cell Mol Biol (Noisy-le-grand). 2018 May 30;64(7):19-23.
Superoxide dismutase (SOD) of the Tamarix aphylla leaves were detected at optimum conditions that collected in April, May and June. Results indicated the specific activity in the crude extract reaching to 36.76 unit/ mg protein. Crude SOD was purified by several techniques, precipitation with ammonium sulfate (50-75) %, Ion exchange chromatography using DEAE-cellulose and two steps of size exclusion chromatography on sephacryl S-200 column. The obtained specific activity (310 unit/mg protein) and purification fold 7.91. The purified enzyme revealed one band by SDS-polyacrylamide gel electrophoresis with molecular mass 85.703 kDa. while 89.125 kDa by Sephacryl S-200. The optimal pH and temperature for enzyme activity were 7.5, and 50ºC respectively. EDTA, SDS and NaN3 reduced activity, contrariwise of H2O2 and KCN, pointed to the studied SOD is MnSOD. Michalis constant Km and maximum velocity Vmax values were 0.016 mM and 55.86 mM/min, respectively by using Pyrogallol as substrate. According to the results, we conclude Tamarix aphylla produce MnSOD which can have purified by serial purification techniques for better activity and characterized for further studies.
对4月、5月和6月采集的无叶柽柳叶中的超氧化物歧化酶（SOD）在最佳条件下进行了检测。结果表明，粗提物中的比活性达到36.76单位/毫克蛋白质。通过多种技术对粗SOD进行了纯化，包括用硫酸铵（50 - 75）%沉淀、使用DEAE - 纤维素进行离子交换色谱以及在Sephacryl S - 200柱上进行两步尺寸排阻色谱。获得的比活性为（310单位/毫克蛋白质），纯化倍数为7.91。纯化后的酶在SDS - 聚丙烯酰胺凝胶电泳中显示出一条带，分子量为85.703 kDa，而在Sephacryl S - 200上为89.125 kDa。酶活性的最佳pH和温度分别为7.5和50℃。EDTA、SDS和NaN₃降低了活性，而H₂O₂和KCN则相反，表明所研究的SOD是MnSOD。以连苯三酚为底物时，米氏常数Km和最大反应速度Vmax值分别为0.016 mM和55.86 mM/分钟。根据结果，我们得出结论，无叶柽柳产生MnSOD，可通过系列纯化技术进行纯化以获得更好的活性，并进行表征以用于进一步研究。
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