期刊
ANALYTICAL BIOCHEMISTRY
卷 281, 期 2, 页码 223-229出版社
ACADEMIC PRESS INC
DOI: 10.1006/abio.2000.4583
关键词
nitric oxide; quantitation; chemiluminescence; spectrophotometric assay; nitrite; nitrate; cell culture
资金
- NCI NIH HHS [P01 CA66081] Funding Source: Medline
- NHLBI NIH HHS [R01 HL51469] Funding Source: Medline
- NIEHS NIH HHS [F32 ES05781] Funding Source: Medline
A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm, Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M-1 cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 mu M. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M-1 cm(-1), for the assay solutions described, The limit of detection of this assay was approximately 300 nM, Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M-1 cm(-1) and a detection limit of 0.5 mu M. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO. (C) 2000 Academic Press.
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