Quantification of Global Protein Disulfides and Thiol-Protein Mixed Disulfides to Study the Protein Dethiolation Mechanisms

Abstract

The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ↔ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman's reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 µmoles/g and after diamide exposure they were significantly augmented of about 65% -70% in liver and heart, but not in the kidney. Normal XSSP, that were 6 -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

“…Previously, researchers reported the release of GSH from protein pellets, attributing it to a nonenzymatic thiol disulfide exchange leading to the loss of PSSG [42,43]. Based on the samples processed for our isotope study, it is possible to reduce detectible GSH to below our detection limit using 10 mM NEM, and even after subsequent dissolution in 6 M urea at pH 8.0 to 9.0 no additional GSH was detected without the addition of a chemical reductant.…”

Fluorescent Probes for Imaging Protein Disulfides in Live Organisms

“…Currently, the detection of PDS generally requires three steps (Figure A–C): − (i) blocking free thiol groups, (ii) reducing the disulfide to nascent thiols, and (iii) labeling the nascent thiols with different tags for further analysis. Obviously, these in vitro assays cannot be applied to detect PDS in live organisms.…”