The novel red-fluorescent probe DAR-4M measures reactive nitrogen species rather than NO.
BACKGROUND
Several fluorescent probes were designed for the measurement of nitric oxide (NO), however, questions arose regarding their specificity and sensitivity in biological samples. In the present study we tested the reaction of a novel rhodamine-based chromophore diaminorhodamine-4M (DAR-4M) with NO and other reactive nitrogen and oxygen species.


METHODS
We performed fluorometry in 96-well plates in a cell-free buffer with similar ion concentrations as the cytoplasm. Dose-response curves were generated using various NO donors and reactive nitrogen and oxygen species. The effects were compared between the red-fluorescent DAR-4M and its green-fluorescent counterpart 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).


RESULTS
DAR-4M had a markedly higher fluorescence yield to NO donors than DAF-FM, while both probes had a comparable threshold of sensitivity (in the range of 0.1 mM nitroprusside). Both dyes reacted with various NO donors in a dose-dependent manner, while superoxide, hydrogen peroxide, peroxynitrite, or nitroxyl failed to change the fluorescence intensity of the probes. DAR-4M was potentiated in the presence of peroxynitrite to react with low levels of NO donors in a similar manner to DAF-FM.


CONCLUSIONS
We conclude that DAR-4M is a suitable red-fluorescent probe for the qualitative assessment of reactive nitrogen species production, but not specific for NO. Quantitative comparisons among samples is inappropriate since the fluorescent yield is affected by the presence of other oxidants in the sample.
Lacza
Zsombor
Z
Department of Physiology/Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. zlacza@mac.com
Horváth
Eszter M
EM
Pankotai
Eszter
E
Csordás
Attila
A
Kollai
Márk
M
Szabó
Csaba
C
Busija
David W
DW
D016477:Artifacts; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry; D020030:Nitric Oxide Donors / Q000737:chemistry; D009599:Nitroprusside / Q000737:chemistry; D026361:Reactive Nitrogen Species / Q000032:analysis* / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry*
C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012235:Rhodamines; C508638:diaminorhodamine-4M; D009599:Nitroprusside; D009569:Nitric Oxide