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Detection of N ‐nitrosomelatonin and other N ‐nitrosotryptophan derivatives by transnitrosation of APF and DAF‐2
Author(s) -
Kirsch Michael,
Groot Herbert
Publication year - 2006
Publication title -
journal of pineal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.881
H-Index - 131
eISSN - 1600-079X
pISSN - 0742-3098
DOI - 10.1111/j.1600-079x.2005.00277.x
Subject(s) - peroxynitrite , chemistry , detection limit , nitration , chromatography , combinatorial chemistry , biochemistry , enzyme , organic chemistry , superoxide
S ‐nitrosothiols can be analyzed with some simple detection procedures and this fact strongly accelerated the understanding of the biological impact of S ‐nitrosothiols. Unfortunately, such simple analytic methods are presently missing for low molecular weight N ‐nitrosotryptophan derivatives like N ‐nitrosomelatonin (NOMela). Here we demonstrate that commercially available primary aromatic amines, i.e. aminophenylfluorescein (APF) and 4,5‐diaminofluorescin (DAF‐2), can be used for a quantitative determination of NOMela. Under optimized conditions (e.g. pH 11) of the assays, the lifetime of N ‐nitrosotryptophan derivatives is largely prolonged and the reactivity of S ‐nitrosothiols with aromatic amines can be safely ignored. The influence of reactive nitrogen oxide species like N 2 O 3 is additionally limited at the alkaline pH and may be further decreased by working under hypoxic conditions. As a result of these optimal conditions, the APF assay has a detection limit for NOMela of about 25 n m but this assay fails to detect protein‐bound N ‐nitrosotryptophan residues. The DAF‐2 assay, however, might be used for a qualitative analysis of such residues. Due to the high efficacy of the APF assay it is safely demonstrated that in regard to peroxynitrite, N 2 O 3 is about 50‐fold more effective in nitrosating melatonin at physiological pH.