Queval Guillaume, Noctor Graham
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris XI, 91405 Orsay cedex, France.
Anal Biochem. 2007 Apr 1;363(1):58-69. doi: 10.1016/j.ab.2007.01.005. Epub 2007 Jan 10.
Glutathione, NAD, and NADP are key nonprotein redox couples in the aqueous phase of virtually all cells, whereas in plant cells ascorbate also plays an important role in redox homeostasis. This work presents the development and validation of plate reader assays that allow rapid analysis of these four redox couples in extracts of Arabidopsis leaves. Analytical methods were adapted and validated for specific measurement of oxidized and reduced forms. Oxidized and reduced forms of glutathione and ascorbate, as well as NAD(+) and NADP(+), were measured in HCl extracts, NADH, and NADPH in parallel alkaline extracts. Both standards and extracts gave linear assay responses, and recovery quotients of added metabolites through the extraction procedure were generally high. The plate reader method was validated against more conventional spectrophotometric assays and also, for glutathione, by HPLC analysis. The method was shown to yield quantitative data for six independent extracts with a total sample preparation and analysis time of 4h. Analysis of the four redox couples throughout Arabidopsis rosette development showed that redox states were relatively constant but that total pools of NAD, glutathione, and ascorbate were significantly modified by day length and developmental stage.
谷胱甘肽、NAD和NADP是几乎所有细胞水相中关键的非蛋白质氧化还原对，而在植物细胞中，抗坏血酸在氧化还原稳态中也起着重要作用。这项工作展示了酶标仪检测方法的开发与验证，该方法可快速分析拟南芥叶片提取物中的这四种氧化还原对。对分析方法进行了调整和验证，以用于氧化型和还原型的特异性测量。谷胱甘肽和抗坏血酸的氧化型和还原型，以及NAD(+)和NADP(+)在盐酸提取物中进行测量，NADH和NADPH在平行的碱性提取物中进行测量。标准品和提取物均给出线性检测响应，并且添加的代谢物在提取过程中的回收率通常较高。酶标仪方法相对于更传统的分光光度法进行了验证，对于谷胱甘肽，还通过高效液相色谱分析进行了验证。结果表明，该方法能为六个独立提取物提供定量数据，总样品制备和分析时间为4小时。对拟南芥莲座叶发育过程中四种氧化还原对的分析表明，氧化还原状态相对恒定，但NAD、谷胱甘肽和抗坏血酸的总量会因日照长度和发育阶段而发生显著变化。
