Protein Redox State Monitoring Studies of Thiol Reactivity

Protein cysteine thiol status is a major determinant of oxidative stress and oxidant signaling. The -SulfoBiotics- Protein Redox State Monitoring Kit provides a unique opportunity to investigate protein thiol states. This system adds a 15-kDa Protein-SHifter to reduced cysteine residues, and this molecular mass shift can be detected by gel electrophoresis. Even in biological samples, Protein-SHifter Plus allows the thiol states of specific proteins to be studied using Western blotting. Peroxiredoxin 6 (Prx6) is a unique one-cysteine peroxiredoxin that scavenges peroxides by utilizing conserved Cysteine-47. Human Prx6 also contains an additional non-conserved cysteine residue, while rat Prx6 only has the catalytic cysteine. In cultured cells, cysteine residues of Prx6 were found to be predominantly fully reduced. The treatment of human cells with hydrogen peroxide (H(2)O(2)) formed Prx6 with one cysteine reduced. Since catalytic cysteine becomes oxidized in rat cells by the same H(2)O(2) treatment and treating denatured human Prx6 with H(2)O(2) results in the oxidation of both cysteines, non-conserved cysteine may not be accessible to H(2)O(2) in human cells. We also found that untreated cells contained Prx6 multimers bound through disulfide bonds. Surprisingly, treating cells with H(2)O(2) eliminated these Prx6 multimers. In contrast, treating cell lysates with H(2)O(2) promoted the formation of Prx6 multimers. Similarly, treating purified preparations of the recombinant cyclic nucleotide-binding domain of the human hyperpolarization-activated cyclic nucleotide-modulated channels with H(2)O(2) promoted the formation of multimers. These studies revealed that the cellular environment defines the susceptibility of protein cysteines to H(2)O(2) and determines whether H(2)O(2) acts as a facilitator or a disrupter of disulfide bonds.