Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde
Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde
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DOI：
10.1006/abio.2000.4498
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发表时间：
2000-04-10
影响因子：
2.9
通讯作者：
Shertzer, HG
中科院分区：
文献类型：
--
作者：
Senft, AP;Dalton, TP;Shertzer, HG
由于谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）在细胞信号转导、基因调节、氧化还原调节和生化稳态中的重要性，准确测定细胞谷胱甘肽水平至关重要。已经开发了几种方法，但许多方法存在高估GSSG或细胞物质干扰或竞争GSH测定的问题。基于HPLC的测定（通过谷胱甘肽还原酶和NADPH酶促还原GSSG）似乎是有效的，但在样品通量和设备可用性方面受到限制。荧光探针邻苯二甲醛（OPA，phthalic dicarboxaldehyde）与GSH反应，具有较高的量子产率，但由于肝脏中存在未知的干扰和荧光猝灭物质，其应用受到限制。本文介绍了测定条件下，这些限制是可以避免的。通过在较低pH下使用磷酸盐缓冲测定，对非特异性反应物的干扰最小。由于OPA与NAD（P）H和其他更强的还原剂反应，酶促还原是不可能的，导致高估GSSG水平，因此使用连二亚硫酸盐还原GSSG。高样品通量结合灵敏（20 pmol检测限）和准确的测定GSH和GSSG使用OPA是可以实现的任何单色谱荧光分光光度计。描述了样品制备和储存条件，使GSH和GSSG水平至少保持4周。(C)北京：清华大学出版社.
Because of the importance of glutathione (GSH) and glutathione disulfide (GSSG) in cellular signal transduction, gene regulation, redox regulation, and biochemical homeostasis, accurate determination of cellular glutathione levels is critical. Several procedures have been developed, but many suffer from overestimating GSSG or from cellular substances interfering or competing with GSH determination. Assays based on HPLC, with enzymatic reduction of GSSG by glutathione reductase and NADPH, appear to be valid but are limited in sample throughput and availability of equipment. The fluorescence probe o-phthalaldehyde (OPA, phthalic dicarboxaldehyde) reacts with GSH and has a high quantum yield, yet its use has been limited due to unidentified interfering and fluorescence-quenching substances in liver. This paper describes assay conditions under which these limitations are avoided. By using a phosphate-buffered assay at lower pH, interference with nonspecific reactants is minimal. Since enzymatic reduction is not possible due to the reaction of OPA with NAD(P)H and other stronger reducing agents, leading to an overestimation of GSSG levels, dithionite was used to reduce GSSG. High sample throughput combined with sensitive (20-pmol limit of detection) and accurate determination of GSH and GSSG using OPA is achievable with any monochromatographic spectrofluorometer. Sample preparation and storage conditions are described that return the same levels of GSH and GSSG for at least 4 weeks. (C) 2000 Aacademic Press.