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 determin ation of thionitrites. The method is based on the Cu+-catalyzed homoly tic cleavage of thionitrites and electrochemical detection of the rele ased 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 reduc ing agents, The effect of Cu(NO3)(2) on the release of NO from GSNO wa s concentration-dependent. In the presence. of I mM GSH, the EC50 for Cu(NO3)(2) was 1.34 +/- 0.08 mM, Using cysteine instead of GSH, NO rel ease was quantitative at much Lower concentrations of Cu(NO3)(2) (EC50 = 8.5 +/- 2.8 mu M), NO release tvas not significantly affected by pH (7.0-9.0) and was inhibited by the Cu+-selective chelator neocuproine , whereas the Cu2+ chelator cuprizone was approximate to 16-fold less potent, Calibration of the method with GSNO, S-nitroso-N-acetyl-penici llamine, or S-nitrosated bovine serum albumin yielded linear plots of initial rates of NO release versus thionitrite concentration from 50 n M: to 5 mu M, This method may be useful for the quantitative determina tion of thionitrites in biological samples. (C) 1998 Academic Press.