Reversible oxidations of protein thiols have emerged as alternatives to free radical-mediated oxidative damage with which to consider the impacts of oxidative stress on cellular activities but the scope and pathways of such oxidations in tissues, including the brain, have yet to be fully defined. We report here a characterization of reversible oxidations of glutathione and protein thiols in extracts from rat brains, from two sources, which had been (1) frozen quickly after euthanasia to preserve in vivo redox states and (2) subjected to alkylation upon tissue disruption to trap reduced thiols. Brains were defined, relatively, as Reduced and Moderately Oxidized based on measured ratios of reduced (GSH) to oxidized (GSSG) glutathione. Levels of protein disulfides formed by the cross-linking of closely-spaced (vicinal) protein thiols, but not protein S-glutathionylation, were higher in extracts from the Moderately Oxidized brains compared to the Reduced brains. Moreover, the oxidized vicinal thiol proteome contains proteins that impact cellular energetics, signaling, neurotransmission, and cytoskeletal dynamics among others. These findings argue that kinetically-competent pathways for reversible, two-electron oxidations, of protein vicinal thiols can be activated in healthy brains in response to physiological oxidative stresses. We propose that such oxidations may link oxidative stress to adaptive, but also potentially deleterious, changes in neural cell activities in otherwise healthy brains.

Oxidative crosslinking of protein thiols via disulfide bonds, promoted by non‐radical peroxides, offers alternatives to traditional, oxygen radical‐centered, perspectives with which to consider the impacts of oxidative stress on brain functions. Protein disulfides, involving the bridging of closely‐spaced (vicinal) thiol pairs, as well as protein‐glutathione mixed disulfides occur readily in cells exposed to hydrogen peroxide but the scope, pathways, and relationships to health and disease of protein thiol oxidations in the brain remain to be fully established. We demonstrated recently that (i) brains of healthy adolescent rats, from different sources, could be distinguished by ratios of oxidized to reduced glutathione and (ii) moderate increases in these ratios were associated with marked elevations in the levels of total protein disulfides but not mixed disulfides. We report here that the more oxidized brains were characterized also by increases in the protein levels of thioredoxin (Trx)‐dependent peroxiredoxin (Prx) peroxidases as well as the extents of oxidations of the catalytic vicinal thiols of these enzymes. Moreover, among the non‐Prx proteins that were oxidized most prominently were glyceraldehyde‐3‐phosphate dehydrogenase and the catalytic subunit(s) of the Na+, K+‐ATPase, critical enzymatic determinants of glucose utilization and neuronal excitability, respectively. We hypothesize that radical‐free oxidations of protein vicinal thiol motifs, controlled by the Trx/Prx system, may facilitate short‐term adaptation to oxidative stress but that prolonged oxidations, i.e., protein disulfide stress, may derail healthy brain aging by increasing the vulnerability of neurons to excitotoxic insults.