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[21] Detection of S-nitrosothiols by fluorometric and colorimetric methods

1999; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(99)01083-6

1557-7988

David A. Wink, Sungmee Kim, Deborah Coffin, John C. Cook, Yoram Vodovotz, Danae Chistodoulou, David Jourd’heuil, Matthew B. Grisham,

Molecular Sensors and Ion Detection

This chapter discusses the detection of S-Nitrosothiols by fluorometric and colorimetric methods. The colorimetric method uses the components of the Griess reaction while the fluorometric method utilizes the conversion of 2, 3-diaminonaphthalene (DAN) to a fluorescent triazole. These methods are conducted at neutral rather than acidic pH that eliminates the interference of contaminating nitrite and allows the detection of nitrosation mediated by the presence of nitric oxide (NO). The colorimetric reaction described herein utilizes the chemistry that occurs among nitrosating species such as those in the NO/O2 reaction and sulfanilamide (SULF) to form a diazonium ion. The fluorometric assay is based on the reaction of DAN with nitrosating intermediates, such as those formed in the NO/O2 reaction, to yield a primary nitrosamine that is converted rapidly to a fluorescent triazole. The colorimetric assay has a detection range of 0.5–100/μM, while the fluorometric assay is effective in the range of 0.05–5μ/M S-nitrosothiol compounds (RSNO). The combination of the two assays provides a detection range from 50 nM to 100μM RSNO, required for most biological experiments.