Reversible Oxidation of the Active Site Cysteine of Peroxiredoxins to Cysteine Sulfinic Acid

Abstract

The detection of sulfinylated proteins in both of these previous studies relied on complex proteomics analysis. We now describe a simple immunoblot assay for the detection of sulfinylated Prx enzymes that is based on antibodies produced in response to a sulfonylated peptide modeled on the conserved active site sequence. These antibodies recognized both sulfinic and sulfonic forms of Prx equally well and allowed the detection of sulfinylated Prx enzymes in H 2 O 2 -treated cells with high sensitivity and specificity. With the use of these antibodies, we demonstrated that not only the cytosolic enzymes Prx I and Prx II but also the mitochondrial enzyme Prx III undergo reversible sulfinylation. The generation of antibodies specific for sulfonylated peptides should provide insight into protein function similar to that achieved with antibodies to peptides containing phosphoserine or phosphothreonine.

A chemical approach for detecting sulfenic acid-modified proteins in living cells

“…The loss of signal observed at high FCCP concentrations may result from oxidation of protein sulfenic to sulfinic and sulfonic acids, which are not targets for DAz-1. This hypothesis was confirmed using an antibody that detects the sulfinic and sulfonic acid forms of peroxiredoxin, 69 regarded as a hallmark of protein 'hyperoxidation' (ESI Fig. S5 † ).…”

Sulfiredoxin Protein Is Critical for Redox Balance and Survival of Cells Exposed to Low Steady-state Levels of H2O2*

“…To do this, we determined the hyperoxidation of 2-Cys Prxs in cells by using a specific antibody that recognizes both sulfinic and sulfonic forms of 2-Cys Prxs (19). In accordance with the increase in cellular ROS level, the hyperoxidation of Prx I, II, and III was markedly increased in the pSUPER-siSrx cells as compared with what was barely detectable in the pSUPER cells exposed to low steady-state levels of H 2 O 2 (Fig.…”

Reduction of Cysteine Sulfinic Acid by Sulfiredoxin Is Specific to 2-Cys Peroxiredoxins

“…A similar procedure was followed for the preparation of sulfinic forms of Prx V with the exception that the concentration of H 2 O 2 was increased to 3 mM for Prx V. Recombinant human Prx VI (250 g) or rabbit muscle GAPDH (250 g) was incubated for 10 min at 30°C in a 250-l reaction mixture containing 4 mM H 2 O 2 and 50 mM Tris-HCl (pH 7.5). The sulfinic state of the oxidized proteins was verified by electrospray ionization-mass spectrometry (ESI-MS) as described (2,3).…”

“…However, given that sulfinic and sulfonic acids were found not to be reduced by biological thiols under physiological conditions (1), the hyperoxidation reactions that give rise to these moieties were thought to be irreversible and to occur only under conditions of extreme oxidative stress or during protein isolation. The cysteine sulfinic acid produced during the catalytic cycle of peroxiredoxins (Prxs) was nevertheless recently found to be reducible in cells (2)(3)(4)(5)(6).…”

“…This oxidation to sulfinic acid was recently found to be a reversible step (2)(3)(4), with the back reaction being catalyzed by each of two ATP-dependent reductases designated sulfiredoxin (Srx) and sestrin (5,6). The N-Cys-SH of Prx V or Prx VI also undergoes hyperoxidation to sulfinic acid (4).…”