title: Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent.
abstract: Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH₂) were also detected. This suggests that GS(O)NH₂, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721.
authors.forename: Gail M
authors.initials: GM
authors.lastname: Johnson
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Chozinski
authors.forename: Debra J
authors.initials: DJ
authors.lastname: Salmon
authors.forename: Alan D
authors.initials: AD
authors.lastname: Moghaddam
authors.forename: Hsin Chih
authors.initials: HC
authors.lastname: Chen
authors.forename: Katrina M
authors.initials: KM
authors.lastname: Miranda
mesh_terms: D000588:Amines / Q000737:chemistry; D019281:Dimerization; D005456:Fluorescent Dyes; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000737:chemistry* / Q000378:metabolism; D009281:Naphthalenes / Q000737:chemistry; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis; D013194:Staining and Labeling
chemical_list: C507406:1-isopropyldiazen-1-ium-1,2-diolate; D000588:Amines; D005456:Fluorescent Dyes; D006834:Hydrazines; D009281:Naphthalenes; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; C510946:glutathione sulfinamide; C027717:2,3-naphthalenedicarboxaldehyde; D005978:Glutathione; C039900:nitroxyl
keywords: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; AS; Angeli's salt; Angeli’s salt; BODIPY; C-PTIO; DAF; DEA/NO; DHR; DMSO; DTPA; Free radicals; GS(O)NH(2); GSH; GSNHOH; GSNO; GSSG; Glutathione; HNO; IPA/NO; Isopropylamine NONOate; Mn(III)TPPS; N(2)O; N-hydroxyl sulfenamide; NDA; NO; NO(2)(−); NO(3)(−); NONOates; NaN(3); Naphthalene-2,3-dicarboxaldehyde; Nitric oxide; Nitroxyl; PBS; S-nitrosoglutathione; Sulfinamide; TCEP; boron-dipyrromethene; diaminofluorescein; diazeniumdiolates; diethylamine NONOate; diethylenetriaminepentaacetic acid; dihydrorhodamine; dimethyl sulfoxide; glutathione; glutathione sulfinamide; isopropylamine NONOate; manganese(III) meso-tetrakis(4-sulfonatophenyl) porphyrinate; metMb; metmyoglobin; naphthalene-2,3-dicarboxaldehyde; nitrate; nitric oxide; nitrite; nitrous oxide; nitroxyl; oxidized glutathione; phosphate-buffered saline; sodium azide; tris(2-carboxyethyl)phosphine)