Elsevier

Analytical Biochemistry

Volume 258, Issue 1, 10 April 1998, Pages 68-73
Analytical Biochemistry

Regular Article
Electrochemical Determination ofS-Nitrosothiols with a Clark-Type Nitric Oxide Electrode

https://doi.org/10.1006/abio.1998.2562Get rights and content

Abstract

Low-molecular-mass thiols and nitric oxide (NO) formS-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)2to samples containing1 mM GSH or 4 mMl-cysteine as reducing agents. The effect of Cu(NO3)2on the release of NO from GSNO was concentration-dependent. In the presence of 1 mM GSH, the EC50for Cu(NO3)2was 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 μM). 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 ≈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 μM. This method may be useful for the quantitative determination of thionitrites in biological samples.

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    Maines, M. D.

    1

    To whom correspondence should be addressed. Fax: +43-316-380-9890. E-mail:[email protected].

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