Simultaneous analyses of oxidized and reduced forms in photosynthetic quinones by high-performance liquid chromatography with slight modifications.
Simultaneous analyses of oxidized and reduced forms in photosynthetic quinones by high-performance liquid chromatography with slight modifications.
复制标题
通过稍作修改的高效液相色谱法同时分析光合醌中的氧化型和还原型。
DOI：
10.1007/978-1-4939-0606-2_7
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发表时间：
2014
影响因子：
--
通讯作者：
Hiroshi Shimada
中科院分区：
文献类型：
--
作者：
Masaru Shibata;Hiroshi Shimada
质体醌和叶醌是叶绿体中主要的植物醌类化合物，它们在类囊体膜上充当光合电子氧化还原介质。这些醌通过两种方法进行分析：有机溶剂萃取和高效液相色谱（HPLC）分析醌含量测定。从植物醌提取过程和储存过程中氧化还原状态稳定性的角度来看，溶剂的选择非常重要。我们引入程序和溶剂，以避免醌的氧化还原状态的变化，除了实现高提取效率。传统方法存在灵敏度低的问题，并且需要制备步骤来去除干扰物质，例如植物色素。已经开发了HPLC系统，利用醌醇（还原形式）的荧光特性来测量醌。通过反相HPLC双检测器（紫外和荧光检测）检测质体醌。然而，具有较短侧链的叶绿醌和质体醌异构体的峰通常与快速洗脱颜料的大峰重叠。为了解决这些问题，在柱后还原以将醌转化为荧光醌醇之后，应用具有荧光检测的HPLC来测量快速洗脱的醌类（低疏水性醌类和醌醇类），例如叶绿醌。使用硼氢化钠或铂黑的柱后还原方法，不仅还原形式（荧光），而且氧化形式（非荧光）可以通过具有荧光检测器的HPLC清楚地测量。
Plastoquinones and phylloquinones are the major plant quinones localized in chloroplasts, and they act as photosynthetic electron redox mediators in thylakoid membranes. These quinones are analyzed by two processes: extraction with organic solvents and quinone assay by high-performance liquid chromatography (HPLC) analysis. Solvent choice is very important from the viewpoint of stability of the redox state in the extraction processes and during storage of plant quinones. We introduce procedures and solvents to avoid changes in the redox state of quinones, in addition to achieving high extraction efficiency. Traditional methods have problems of low sensitivity and require preparation steps to remove interfering substances, such as plant pigments. HPLC systems have been developed utilizing the fluorescent properties of quinols (reduced forms) to measure quinones. Plastoquinones were detected by reversed-phase HPLC with dual detectors (ultra-violet and fluorescence detection). However, the peak of phylloquinone and plastoquinone isomers with shorter side chains often overlaps with a large peak of fast-eluting pigments. To address these issues, HPLC with fluorescence detection after post-column reduction to convert quinones to fluorescent quinol was applied for measurement of fast-eluting quinones (low hydrophobicity quinones and quinols) such as phylloquinone. Using post-column reduction methods with sodium borohydride or platinum black, not only the reduced forms (fluorescent) but also the oxidized forms (non-fluorescent) could be clearly measured by HPLC with a fluorescence detector.