Abstract
Generation of superoxide radicals (0.01-0.1 microm s(-1)) by radiolysis of aqueous solutions containing S-nitrosoglutathione (45-160 microm, pH 3.8-7.3) resulted in loss of this solute at rates varying with solute concentration, radical generation rate, and pH. The results were quantitatively consistent with the loss being attributed to competition between reaction of superoxide with S-nitrosoglutathione (rate constant 300 +/- 100 m(-1) s(-1)) and the pH-dependent disproportionation of superoxide/hydroperoxyl. This rate constant is much lower than previous estimates and seven orders of magnitude lower than the rate constants between superoxide and superoxide dismutase or superoxide and nitric oxide. This indicates that interaction between superoxide and S-nitrosoglutathione is unlikely to be biologically important, contrary to previous suggestions that reaction could serve to prevent the rapid reaction between superoxide and nitric oxide. Reductive homolysis of S-nitrosoglutathione by the carbon dioxide radical anion, a model for biological reductants such as disulfide radical anions, occurred with a rate constant of 7.4 x 10(8) m(-1) s(-1) and produced nitric oxide stoichiometrically. Thiyl radicals were not produced, indicating the alternative homolysis route to generate nitroxyl did not occur.
Highlights
Current interest in the biological chemistry of S-nitrosothiols [1,2,3,4] stems from their potential importance as transport intermediates or storage forms of nitric oxide [5, 6]
Generation of superoxide radicals (0.01– 0.1 M s؊1) by radiolysis of aqueous solutions containing S-nitrosoglutathione (45–160 M, pH 3.8 –7.3) resulted in loss of this solute at rates varying with solute concentration, radical generation rate, and pH
We have examined by direct observation whether reductive cleavage of GSNO by the carbon dioxide radical anion (CO2.) produces nitric oxide (Reaction 3) or thiyl radicals and nitroxyl (Reaction 4), GSNO ϩ CO2. ͑ϩ Hϩ) 3 GSH ϩ NO1⁄7 ϩ CO2
Summary
Chemicals—All chemicals were of the highest grade available. GSH and HCl were purchased from Aldrich; sodium formate, EDTA, and ABTS2Ϫ (diammonium salt) were from Sigma; sodium nitrite, formic acid, and buffers were from Merck. Initial Rates of Loss of GSNO by Reaction with Superoxide Radicals—Superoxide radicals were generated by ␥-radiolysis in solutions containing GSNO, and the loss of GSNO was determined from the decrease in absorbance at 335 nm. This assumes no products were formed that absorb significantly at this wavelength, as suggested by the uniform decrease in absorbance between ϳ300 and 400 nm. The shallow curves from the initial portion of the absorbance/time plots were fitted to a second-order polynomial, and the initial slope was obtained from the linear coefficient to obtain the radiolytic yield of GSNO decomposition, G(ϪGSNO) These were compblaerRaecadhtewiniCgthoanttsht3ea3np5trnofdmorucwRtiaeosancmtoifeoanOs2.uo(rfGeCd(OOa2.2f.)teϭwrit0ph.r6o8GduScNminOogl—JCTϪOh1)2.e. exponential (ϳ11 M) by pulse radiolysis (0.5 s) in solutions containing 100 –300 M GSNO. The yields of radicals (eaϪq, 1⁄7OH, and H1⁄7) were taken as 0.3, 0.3, and 0.06 mol JϪ1 (M GyϪ1) respectively
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