USE OF 2,3-NAPHTHALENEDICARBOXALDEHYDE DERIVATIZATION FOR SINGLE-CELL ANALYSIS OF GLUTATHIONE BY CAPILLARY ELECTROPHORESIS AND HISTOCHEMICAL-LOCALIZATION ION BY FLUORESCENCE MICROSCOPY
USE OF 2,3-NAPHTHALENEDICARBOXALDEHYDE DERIVATIZATION FOR SINGLE-CELL ANALYSIS OF GLUTATHIONE BY CAPILLARY ELECTROPHORESIS AND HISTOCHEMICAL-LOCALIZATION ION BY FLUORESCENCE MICROSCOPY
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DOI：
10.1021/ac00119a010
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发表时间：
1995-12-01
影响因子：
7.4
通讯作者：
ZARE, RN
中科院分区：
文献类型：
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作者：
ORWAR, O;FISHMAN, HA;ZARE, RN
我们报道2,3-萘二甲酸与谷胱甘肽及其前体γ -谷氨酰半胱氨酸在生理条件下迅速反应形成高荧光衍生物。与先前的2,3-萘二甲酸标记伯胺的描述相反，不需要额外的CN-离子或任何其他额外的亲核试剂。发色团的荧光光谱特性(gamma（exc max) = 472 nm, lambda(em max) = 528 nm）使这些衍生物适合于荧光素波长优化的光学仪器的激发和检测。这种选择性标记化学可以定量测定谷胱甘肽在神经生物学样品中的组织化学定位，细胞内谷胱甘肽通过培养细胞或细胞悬液在2,3-萘二甲醛补充的含有dmso的生理缓冲液（pH = 7.4）中孵育2-10分钟进行标记。应用包括使用发光和共聚焦荧光显微镜对培养的NG 108-15细胞（小鼠神经母细胞瘤x大鼠胶质瘤）和原代胶质细胞和神经元细胞共培养（大鼠海马）进行成像。采用激光诱导荧光法和毛细管电泳法对NG 108-15细胞中谷胱甘肽的含量进行定量测定。
We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (gamma(exc max) = 472 nm, lambda(em max) = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples, Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantita tive determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis.