Analytical Sciences

A Novel Spectrofluorometric Probe for the Determination of Peroxynitrite Anion Scavenging Activity of Biothiols and Amino Acids

Burcu BEKDESER,*,** Mustafa ÖZYÜREK,* Esin AKYÜZ,* and Resat APAK*,**

*Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey
**Istanbul University-Application & Research Center for the Measurement of Food Antioxidants, Istanbul University, Avcilar 34320, Istanbul, Turkey

In this study, a novel fluorometric method for the determination of peroxynitrite anion (ONOO^–) scavenging (PAS) activity of amino acids and biothiols, which can mostly trap peroxynitrite in vivo, is described. This assay is based on the conversion of a gentisic acid probe to its non-fluorescent oxidation products with ONOO^–. The attenuation of the fluorescence intensity (FI) of the probe upon peroxynitrite attack is diminished with antioxidants, the difference in FI being related to the PAS activity of the antioxidants. The IC₅₀ (50% inhibitive concentration) values of biothiols, amino acids and tissue homogenates were estimated, in comparison with the reference Pyrogallol Red (PR) bleaching method. PR is the most suitable and frequently used dye to determine PAS activity, but is relatively insensitive. The developed fluorometric assay is highly sensitive to allow determinations of the PAS activity of amino acids.

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