Discriminating formation of HNO from other reactive nitrogen oxide species.
Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.
Tumor Biology Section, Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Sonia
S
Donzelli
Michael Graham
MG
Espey
Douglas D
DD
Thomas
Daniele
D
Mancardi
Carlo G
CG
Tocchetti
Lisa A
LA
Ridnour
Nazareno
N
Paolocci
S Bruce
SB
King
Katrina M
KM
Miranda
Giuseppe
G
Lazzarino
Jon M
JM
Fukuto
David A
DA
Wink
D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D019281:Dimerization; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009585:Nitrogen Dioxide / Q000378:metabolism; D009589:Nitrogen Oxides / Q000032:analysis* / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000032:analysis*
D009573:Nitrites; D009589:Nitrogen Oxides; D026361:Reactive Nitrogen Species; C510946:glutathione sulfinamide; D030421:Peroxynitrous Acid; C021229:oxyhyponitrite; D019811:Hydroxylamine; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D005978:Glutathione; C039900:nitroxyl; D009585:Nitrogen Dioxide