ONLINE DETECTION OF NITRIC-OXIDE FORMATION IN LIQUID AQUEOUS PHASE BY ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY
ONLINE DETECTION OF NITRIC-OXIDE FORMATION IN LIQUID AQUEOUS PHASE BY ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY
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DOI：
10.1016/0003-2697(91)90282-x
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发表时间：
1991-11-15
影响因子：
2.9
通讯作者：
VANIN, A
中科院分区：
文献类型：
--
作者：
MORDVINTCEV, P;MULSCH, A;VANIN, A
本文介绍了用电子顺磁共振谱（EPR）检测含氧水溶液中一氧化氮自由基（NO）的方法。用Fe ~（2+）-二乙基二硫代氨基甲酸盐（DETC）络合物捕获3-吗啉基丙酮亚胺（SIN-1）自发分解产生的NO。所得的单亚硝基-Fe 2 +-（DETC）2络合物是稳定的，并且在37°C下在g_（max）= 2.04和g_（max）= 2.02处表现出特征EPR信号，在冷冻溶液中在g_（max）处具有未分辨的三重态超精细结构，在gav= 2.03处具有各向同性三重态信号。捕获的NO的量从通过二亚硝酰基-Fe 2 +-硫代硫酸盐标准校准的三线之一的振幅计算。由于NO背景信号较低，NO的检测下限为0.5nmol/（ml × h）。由于陷阱饱和，检测上限为约10 nmol NO/ 40 mg陷阱（DETC负载的酵母细胞）。在厌氧条件下，低浓度的SIN-1的捕集效率接近60%，但随着较高的浓度和在氧气的存在下进行性下降。亚硝酸盐（高达0.1 mm）并没有增加背景NO水平。灵敏度足以在37°C下在扁平石英比色皿中在线跟踪来自SIN-1的NO释放速率。通过EPR光谱法检测到的NO释放的时间过程与通过重氮化测量的亚硝酸盐积累的时间过程相关。总之，这种方法将允许在线检测NO形成的内源性和药理学来源的含氧水介质中。
A method for the detection of the nitric oxide radical (NO) in oxygen-containing aqueous solution by means of electron paramagnetic resonance spectroscopy (EPR) is described. NO evolving from the spontaneous decomposition of 3-morpholinosydnonimine (SIN-1) was trapped by Fe2+-diethyldithiocarbamate (DETC) complex dissolved in yeast cell membranes. The resulting mononitrosyl-Fe2+-(DETC)2complex was stable and exhibited a characteristic EPR signal at g⊥= 2.04 and g‖= 2.02 with an unresolved triplet hyperfine structure at g⊥in frozen solution and an isotropic triplet signal at gav= 2.03 at 37°C. The amount of NO trapped was calculated from the amplitude of one of the triplet lines calibrated by means of a dinitrosyl-Fe2+-thiosulfate standard. The lower detection limit of NO was 0.5 nmol/(ml × h) due to a low background NO signal. The upper detection limit was about 10 nmol NO/ 40 mg traps (DETC-loaded yeast cells), because of saturation of traps. The trapping efficiency approached 60% under anaerobic conditions and with low concentrations of SIN-1, but decreased progressively with higher concentrations and in the presence of oxygen. Nitrite (up to 0.1 mm) did not increase the background NO level. The sensitivity was sufficient to follow the rate of NO release from SIN-1 on-line at 37°C in a flat quartz cuvette. The time course of NO release detected by EPR spectrometry correlated with the time course of nitrite accumulation measured by diazotation. In conclusion, this method will permit the on-line detection of NO formation from endogenous and pharmacological sources in oxygen-containing aqueous media.