Nanomolar quantification and identification of various nitrosothiols by high performance liquid chromatography coupled with flow reactors of metals and Griess reagent.
Nanomolar quantification and identification of various nitrosothiols by high performance liquid chromatography coupled with flow reactors of metals and Griess reagent.
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通过高效液相色谱结合金属和格里斯试剂的流动反应器对各种亚硝基硫醇进行纳摩尔定量和鉴定。
DOI：
10.1093/oxfordjournals.jbchem.a021774
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发表时间：
1997
影响因子：
2.7
通讯作者：
H. Maeda
中科院分区：
文献类型：
--
作者：
T. Akaike;K. Inoue;T. Okamoto;H. Nishino;M. Otagiri;S. Fujii;H. Maeda
亚硝基硫醇（RS-NOs）似乎是关键参与各种信号转导机制。我们在这里描述了一个具体的和高灵敏度的定量方法RS-NOs通过使用高效液相色谱（HPLC）结合流动反应器系统。将RS-NO应用于C18-反相或凝胶过滤柱的HPLC系统，并用10 mM乙酸钠缓冲液（pH 5.5）加0.5 mM二亚乙基三胺五乙酸（含或不含0-7%甲醇或0.15 M NaCl）洗脱。将来自HPLC柱的洗脱液与含有1.75 mM HgCl 2或1.75 mM CuSO 4的溶液混合，用于在反应盘管中通过三通连接器进行RS-NO分解。然后通过金属诱导的RS-NO分解产生的NO2与Griess试剂反应，该试剂通过第二个三通连接器注入，产生在540 nm处检测的重氮化合物。在一个单独的实验中，铜颗粒负载柱用于RS-NO降解，而不是金属离子流反应器。在所有测试的RS-NO中，即，亚硝基谷胱甘肽（GS-NO）、亚硝基-L-半胱氨酸和亚硝基白蛋白，经Hg ~（2+）反应体系和载铜柱将亚硝基转化为NO_2 ~-，回收率接近100%。而Cu ~（2+）-溶液流反应体系中，RS-NOs的回收率仅为30%。此外，RS-NOs可以在纳摩尔浓度下鉴定：检测限，150微升等分试样中为3.0 nM。这些RS-NOs表现出良好的解析洗脱曲线，即使在NO2-和NO3-的存在下。更重要的是，GS-NO的生物生成用培养基中掺入游离GSH的培养物中的RAW 264细胞定量证明。总之，我们的新RS-NO检测将是有用的，以检查RS-NO的形成和功能的生物系统。
Nitrosothiols (RS-NOs) appear to be critically involved in various signal transduction mechanisms. We describe here a specific and highly sensitive quantification method for RS-NOs by using high performance liquid chromatography (HPLC) combined with a flow reactor system. RS-NOs were applied to an HPLC system of C18-reverse phase or a gel filtration column and eluted with 10 mM sodium acetate buffer (pH 5.5) plus 0.5 mM diethylenetriamine pentaacetic acid with or without either 0-7% methanol or 0.15 M NaCl. The eluate from the HPLC column was mixed with a solution containing 1.75 mM HgCl2 or 1.75 mM CuSO4 for RS-NO decomposition in a reaction coil via a three-way connector. NO2- generated via the metal-induced RS-NO decomposition was then reacted with Griess reagent, which was infused through a second three-way connector, yielding a diazo-compound detected at 540 nm. In a separate experiment, a copper particle-loaded column was used for RS-NO degradation instead of the metal-ion flow reactor. In all RS-NOs tested, i.e., nitrosoglutathione (GS-NO), nitroso-L-cysteine, and nitrosoalbumin, the nitroso- group was converted to NO2- by the Hg2+-reaction system as well as copper-loaded column, and the recovery was almost 100%. The Cu2+-solution flow reaction system, however, yielded only 30% recovery of RS-NOs as NO2-. Also, the RS-NOs could be identified at nanomolar concentrations: detection limit, 3.0 nM in a 150-microl aliquot. These RS-NOs showed well-resolved elution profiles even in the presence of NO2- and NO3-. More importantly, biological generation of GS-NO was quantitatively demonstrated with RAW264 cells in culture incorporating free GSH in the medium. In conclusion, our novel RS-NO assay will be useful to examine the formation and functions of RS-NOs in biological systems.