Abstract

Hydrolysis of three primary RSNOs, S -nitrosocysteine, S -nitroso- N -acetylated-cysteine, and S -nitrosoglutathione and two tertiary RSNOs, S -nitrosopenicillamine and S -nitroso- N -acetylated-penicillamine, was investigated in {598} 4 M H 2 SO 4 to determine the effects of structure on acid-catalyzed denitrosation. Rate increases of up to 38-fo1d resulted from dimethyl substitution at the C-SNO carbon. Electron donation from the methyl groups increased the proton affinity of the NITROSO sulfur, which was shown by computational methods to be the initial step in the acid-catalyzed hydrolysis. Only minor changes were observed in k obs due to N -acetylation, and activation energies of 25.7 ± 1.7 and 23.7 ± 1.3 kcal/mole were measured for S -nitroso- N -acetylated-cysteine and S -nitroso- N -acetylated-penicillamine, respectively. Acid-catalyzed GSNO decomposition at pH 2.0 exhibited a sigmoidal decomposition curve typical of autocatalytic processes. Based on strong inhibition on removal of HNO 2 , N 2 O 3 , or oxygen from the solutions, a chain-reaction mechanism is proposed in which N 2 O 3 is the chain carrier. Reaction is initiated by HNO 2 formation on hydrolysis of the S-NO bond via nucleophilic attack at the nitroso N , and the overall reaction generates GSO 3 H, GSOSG, GSO 2 SG, in addition to HNO 2 as decomposition products. Base-catalyzed GSNO hydrolysis was found to occur mainly via nucleophilic attack at the nitroso N forming a nitrosated disulfide intermediate that produces GSSG, GS(NO)S - , and a sulfur-free glutathionyl derivative as the GSX products NO 2 - was the main nitrogen product. The percent distribution of the GSX products varied with the OH - and GSNO concentrations. N 2 O was also detected and glutathionyl sulfenic acid GSOH was trapped with dimedone. Thus, base-catalyzed GSNO hydrolysis also proceeded via initial OH - attack at the nitroso sulfur forming nitroxyl (HNO), although this appears to be a minor pathway under our experimental conditions. S -Nitrosoglutathione (GSNO) undergoes denitrosation at neutral pH in the presence of excess glutathione (GSH) even when protected from light and metal-catalyzed decomposition. The major glutathionyl product from these reactions is GSSG, and minor products include GS(O)NH 2 , GSN(OH)H, and GSOH. Detection of GS(O)NH 2 and N 2 O suggests that HNO is generated during the reaction. Retardation of GSNO decomposition on addition of dimedone suggests that the mechanism is partially autocatalytic. The in-source fragmentation of reduced glutathione (GSH), S -nitrosoglutathione (GSNO), and oxidized glutathione (GSSG) in a Z-spray ESI source was investigated. The results show that the cone voltage is the major contributor to in-source GSX fragmentation. Cone-voltage-induced fragmentation of the three compounds was pH-dependent, and occurred via loss of H 2 O and cleavage of the peptide bonds into b and y ions. Loss of NH 3 was observed only for GSH at pH 2.5. Homolytic cleavage of the S-NO bond preceded fragmentation of the glutathionyl moiety, forming a GS + radical cation that yields fragment ions unique to GSNO. The results can be used to improve the detection and interpretation of mass spectra of samples containing mixtures of GSX compounds. Direct measurement of underivatized S -nitrosoglutathione (GSNO) was achieved by high-performance liquid chromatography (HPLC) using mobile phases at neutral pH and employing UV spectrophotometric (HPLC/(UV) and electrospray mass spectrometric (HPLC/ESI-MS) detection. Using UV detection, the method is robust and exhibits good linearity in the range of 0.2-100 oM GSNO with a limit of detection (LOD) of 0.2 oM GSNO. Attempts to improve sensitivity by using ESI-MS detection in an ion trap mass spectrometer resulted in the same LOD and linearity in the range of 0.2-10 oM GSNO