A comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae
A comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae
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DOI：
10.1016/j.jphotobiol.2007.04.002
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发表时间：
2007-06-26
影响因子：
5.4
通讯作者：
Buma, Anita G. J.
中科院分区：
文献类型：
--
作者：
Janknegt, Paul J.;Rijstenbil, Jan W.;Buma, Anita G. J.
超氧化物歧化酶（SOD）等抗氧化酶在微藻和植物清除可见光和紫外线胁迫产生的活性氧中起着关键作用。然而，鲜为人知的是，酶的抗氧化应激反应在生态上重要的南极海洋微藻。SOD尤其难以分析，这可能是由于在获得可靠和可重复的酶测定所需的足够量方面存在问题。本研究旨在通过对南极硅藻短角毛藻（Chaetoceros brevis）蛋白质提取方法和SOD测定方法的比较和优化，建立一种灵敏、简便、可靠的南极微藻SOD测定方法。优化了细胞破碎（超声）和蛋白质提取程序、提取缓冲液、SOD测定方法（黄嘌呤/黄嘌呤氧化酶和NBT/核黄素光度定量法和非变性凝胶电泳定性法）和测定温度。无论使用何种类型的缓冲液，经过几次脉冲后，在低超声振幅下蛋白质提取效果最佳。提取效率在测试缓冲液之间变化很大;大多数蛋白质在1% Triton X-100存在下提取。最好使用NBT/核黄素方法结合含有磷酸钾和Triton X-100的缓冲液来量化SOD活性。此外，NBT/核黄素方法被证明是低温（5 ℃）下最可靠和最灵敏的方法。(c)2007 Elsevier B. V.保留所有权利。
Antioxidant enzymes such as superoxide dismutase (SOD) play a key role in the removal of reactive oxygen species produced during visible and ultraviolet irradiance stress in microalgae and plants. However, little is known about the enzymatic antioxidative stress responses in ecologically important Antarctic marine microalgae. SOD in particular is difficult to analyze, possibly due to problems in obtaining sufficient quantities necessary for reliable and reproducible enzymatic assays. The aim of the present work was to create a sensitive, easy-to-use and reliable method for SOD determination in Antarctic microalgal material by comparing and optimizing existing protein extraction procedures and SOD assays in the marine Antarctic diatom Chaetoceros brevis. Optimization was achieved in cell disruption (sonication) and protein extraction procedures, extraction buffers, SOD assay methods (xanthine/xanthine oxidase and NBT/ riboflavin photometric quantitative methods and native gel electrophoresis qualitative method) and the assay temperature. Protein extraction was optimal at low sonication amplitudes after a few pulses, irrespective of the type of buffer used. Extraction efficiency varied highly between the tested buffers; most protein was extracted in the presence of 1% of Triton X-100. SOD activity was best quantified using the NBT/riboflavin method in combination with a buffer containing potassium phosphate and Triton X-100. Moreover, the NBT/ riboflavin method was demonstrated to be the most reliable and sensitive method at low temperatures (5 degrees C). (c) 2007 Elsevier B.V. All rights reserved.