Electrochemical determination of S-nitrosothiols with a Clark-type nitric oxide electrode.
Low-molecular-mass thiols and nitric oxide (NO) form S-nitrosothiols (thionitrites) in the presence of oxygen. Thionitrites play an integral role in a variety of NO-dependent physiological processes. This study describes a sensitive analytical method for the quantitative determination of thionitrites. The method is based on the Cu+-catalyzed homolytic cleavage of thionitrites and electrochemical detection of the released NO with a Clark-type electrode. Cu+ was generated by addition of Cu(NO3)2 to samples containing 1 mM GSH or 4 mM L-cysteine as reducing agents. The effect of Cu(NO3)2 on the release of NO from GSNO was concentration-dependent. In the presence of 1 mM GSH, the EC50 for Cu(NO3)2 was 1.34 +/- 0.08 mM. Using cysteine instead of GSH, NO release was quantitative at much lower concentrations of Cu(NO3)2 (EC50 = 8.5 +/- 2.8 microM. NO release was not significantly affected by pH (7.0-9.0) and was inhibited by the Cu+-selective chelator neocuproine, whereas the Cu2+ chelator cuprizone was approximately 16-fold less potent. Calibration of the method with GSNO, S-nitroso-N-acetyl-penicillamine, or S-nitrosated bovine serum albumin yielded linear plots of initial rates of NO release versus thionitrite concentration from 50 nM to 5 microM. This method may be useful for the quantitative determination of thionitrites in biological samples.
Pfeiffer
S
S
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, Graz, A-8010, Austria.
Schrammel
A
A
Schmidt
K
K
Mayer
B
B
D000818:Animals; D002417:Cattle; D003300:Copper; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D005978:Glutathione / Q000031:analogs & derivatives / Q000032:analysis; D009569:Nitric Oxide / Q000032:analysis*; D009603:Nitroso Compounds / Q000032:analysis*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000032:analysis; D026423:S-Nitroso-N-Acetylpenicillamine; D026422:S-Nitrosoglutathione; D012710:Serum Albumin, Bovine / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis*
D009603:Nitroso Compounds; C091665:S-nitrosoalbumin; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D003300:Copper; D026423:S-Nitroso-N-Acetylpenicillamine; D005978:Glutathione; D010396:Penicillamine