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Detection of N-nitrosomelatonin and other N-nitrosotryptophan derivatives by transnitrosation of APF and DAF-2

Authors :
Michael Kirsch
Herbert de Groot
Source :
Journal of Pineal Research. 40:10-17
Publication Year :
2006
Publisher :
Wiley, 2006.

Abstract

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 N2O3 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 nm 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, N2O3 is about 50-fold more effective in nitrosating melatonin at physiological pH.

Details

ISSN :
1600079X and 07423098
Volume :
40
Database :
OpenAIRE
Journal :
Journal of Pineal Research
Accession number :
edsair.doi.dedup.....6e0c05449b5d9dc965e32e17440b27d8
Full Text :
https://doi.org/10.1111/j.1600-079x.2005.00277.x