Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Automated summary

Intracellular reduced glutathione (GSH) is the most abundant intracellular thiol with concentrations ranging from 1-10 mM, playing a crucial role as a potent cellular antioxidant and denitrosylating agent against redox stress. Our study demonstrates GSH mediated denitrosylation of protein nitrosothiols in HepG2 cells using a unique spin-trapping mechanism and identifies two previously unknown substrates of GSH mediated S-denitrosylation.