A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts:: Application to redox profiling during Arabidopsis rosette development
A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts:: Application to redox profiling during Arabidopsis rosette development
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DOI：
10.1016/j.ab.2007.01.005
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发表时间：
2007-04-01
影响因子：
2.9
通讯作者：
Noctor, Graham
中科院分区：
文献类型：
--
作者：
Queval, Guillaume;Noctor, Graham
谷胱甘肽、NAD和NADP是几乎所有细胞水相中关键的非蛋白氧化还原偶对，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。这项工作提出了发展和验证的平板阅读器分析，允许快速分析这四氧化还原对在拟南芥叶提取物。分析方法适应并验证了氧化和还原形式的具体测量。在盐酸提取物中测定谷胱甘肽和抗坏血酸的氧化和还原形式，以及NAD（+）和NADP+，在平行碱性提取物中测定NADH和NADPH。标准品和提取物均呈线性反应，通过提取过程添加代谢物的回收率普遍较高。该方法与传统的分光光度法和高效液相色谱法对谷胱甘肽进行了验证。结果表明，该方法可获得6个独立提取物的定量数据，总样品制备和分析时间为4小时。对拟南芥花环发育过程中4对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库随着日照长度和发育阶段而显著改变。(c) 2007爱思唯尔公司版权所有。
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 4 h. 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. (c) 2007 Elsevier Inc. All rights reserved.