Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid

Abstract

This work presents an assay for total thiols and total disulfides in biological samples via HPLC quantification of 5-thio-2-nitrobenzoic acid (TNB) derived from the reaction of thiols with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent). This method also provides simultaneous quantification of glutathione (GSH) via the measurement of the GSH DTNB adduct (GSH-TNB). By using 326 nm as the detecting wavelength, the HPLC detection limit for TNB and the GSH-TNB adduct was determined to be 15 pmol and 7.5 pmol respectively. A recovery study with OVCAR-3 cells revealed that the recovery yields for TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.4±1.2% (n = 3), 98.1±5.0% (n = 3), 95.6±0.9% (n = 3), and 96.6±2.3% (n = 3) respectively. The recovery yield for GSH-TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.0±0.3% (n = 3), 95.1±4.9% (n = 3), 96.8±0.6% (n = 3), and 95.1±2.9% (n = 3) respectively. The reproducibility, expressed as the relative standard deviation for the analyte, for TNB was determined to be 2.8% (n = 6) for non-protein thiols, 3.9% (n = 6) for protein thiols, 3.6% (n = 6) for non-protein disulfides and 4.6% (n = 6) for protein disulfides. The reproducibility for GSH-TNB was determined to be 1.6% (n= 6) for non-protein thiols and 2.6% (n = 6) for non-protein disulfides. By comparing the amount of GSH determined in a biological sample before NaBH4 reduction with that after the reduction, this method can provide information associated with thiol glutathionylation which would be useful for protein glutathionylation study. This method should be applicable to cellular, subcellular, protein, or other biomatrix samples for thiol and disulfide quantification and will be a useful analytical method in the study of thiol redox state and thiol glutathionylation.