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The Ultimate Method for Determination of S‐Nitrosoglutathione (GSNO): Enzymatic/Copper Mediated Decomposition plus DAF‐NO Reaction®
Author(s) -
Pompella Alfonso,
Vecoli Cecilia,
Paolicchi Aldo,
Franzini Maria,
Barsacchi Renata,
Baldassini Riccardo,
Neglia Danilo,
Emilia Bramanti
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.65
Subject(s) - s nitrosoglutathione , chemistry , nitric oxide , copper , detection limit , ex vivo , enzyme , reproducibility , in vitro , nuclear chemistry , chromatography , biochemistry , glutathione , organic chemistry
Nitrosoglutathione (GSNO) plays an important role in the transport and metabolism of nitric oxide (NO), and seems to be involved in many patho‐physiological processes. Research and evaluation of GSNO in human diseases has long been hindered by the lack of analytical procedures provided with adequate sensitivity, specificity and reproducibility. All three requirements are now satisfied at the highest level by our patented protocol. The method is based on the innovative, unprecedented concept of using the commercial enzyme γ‐glutamyltransferase (GGT) coupled to the fast decomposition of its product CysGlyNO (CGNO) by copper ion (ancillary reaction), which give oxidized CysGly and NO. NO then is reacted with 4,5‐diaminofluorescein (DAF‐2) giving the triazole derivate, detectable by spectrofluorimetry (λex= 480 nm, λem= 515 nm) or in black microplates (λex= 485 ± 15 nm, λem= 535 ± 25 nm). The limit of quantitation (LOQc) of GSNO in phosphate buffer solution is 20 nM, the precision (CV) 5.5 % at 300 nM concentration level, and the dynamic linear range 5–300 nmol/L, depending on the DAF‐2 concentration. The method is environmental‐safe (no mercury), and achieves detection limits two orders of magnitude lower than direct UV detection. These features make it suitable for investigating GSNO pathophysiology in vitro and in vivo (Pat. appl. No. PI/2006/A/000093) .