亚硝酸盐
硝酸盐
化学
一氧化氮
亚硝酸盐还原酶
硝酸还原酶
生物化学
色谱法
瓜氨酸
格里斯试验
一氧化氮合酶
酶
精氨酸
有机化学
氨基酸
作者
Thomas P. Misko,Roger Schilling,Daniela Salvemini,W. Michael Moore,Mark G. Currie
标识
DOI:10.1006/abio.1993.1449
摘要
The increasing importance of nitric oxide synthase has been underscored by the elucidation of its role in a growing number of normal and pathophysiological processes. Therefore, techniques for detection of nitrite/nitrate, oxidation products of the enzymatic conversion of arginine to citrulline and nitric oxide, should serve as useful tools in defining the contribution of NO synthase to these processes. We have developed a rapid and sensitive fluorometric assay for quantification of nitrite/nitrate based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the fluorescent product, 1-(H)-naphthotriazole. The assay can be used to detect 10 nM nitrite, making it 50-100 times more sensitive than the well-known Griess assay. Moreover, the assay is adaptable to a 96-well plate format, facilitating the handling of a large number of samples including conditioned media from cell culture or the nitrite generated by the purified enzyme. Nitrite/nitrate levels in blood can also be monitored using this assay when it is combined with a filtration step (to remove hemoglobin) followed by conversion of the nitrate to nitrite by nitrate reductase. Thus, this fluorometric method combines speed and sensitivity with the handling of a large number of samples for the quantification of nitrite generated from in vivo and in vitro sources.
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