Reductive assays for S-nitrosothiols: implications for measurements in biological systems.
Bioactive SNOs are found in many tissues. We speculated SNOs might be misidentified in conventional assays which reduce NO-3 to NO. S-Nitrosothiols were exposed to saturated VCl3 in HCl, 1% KI in acetic acid, photolysis, or CuCl and CSH in He; NO was measured by chemiluminescence. S-Nitrosothiols were readily detected in VCl3 but not in KI. Reduction in CuCl/cysteine was linear (r2 = 1.0, n = 6), sensitive to 10 pmol, and eliminated by HgCl2; it did not detect NO-2, NO-3, or 3-nitrotyrosine. S-Nitrosothiols represented approximately 2.9% of NOx assayed by VCl3 in human serum, of which <5% were low-mass species. In summary, (i) conventional assays may misidentify NO-3, but not NO-2, as SNOs; and (ii) chemiluminescence/reduction systems may be sensitive and specific as SNO assays. We suggest that assay of the SNO fraction in biological NOx may be more relevant and feasible than is now appreciated.
Fang
K
K
Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA.
Ragsdale
N V
NV
Carey
R M
RM
MacDonald
T
T
Gaston
B
B
D002851:Chromatography, High Pressure Liquid; D003300:Copper; D003545:Cysteine; D006801:Humans; D008163:Luminescent Measurements; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009603:Nitroso Compounds / Q000032:analysis*; D010084:Oxidation-Reduction
D009603:Nitroso Compounds; D003300:Copper; D019001:Nitric Oxide Synthase; D003545:Cysteine; C029892:cupric chloride