Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H 2 O 2 . Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 ؋ 10 7 M ؊1 s ؊1 for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant protein in the erythrocyte, and its absence in knock-out mice gives rise to hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely sensitive to oxidation by H 2 O 2 , as dimerization was observed after exposure of 5 ؋ 10 6 cells/mL to 0.5 M H 2 O 2 . In contrast to Prx2 in Jurkat T lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine thiol to a sulfinic/ sulfonic acid) in erythrocytes. Reduction of dimerized Prx2 in the erythrocyte occurred very slowly, with reversal occurring gradually over a 20-minute period. Very low thioredoxin reductase activity was detected in hemolysates. We postulate that this limits the rate of Prx2 regeneration, and this inefficiency in recycling prevents the overoxidation of Prx2. We also found that Prx2 was oxidized by endogenously generated H 2 O 2 , which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H 2 O 2 noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H 2 O 2 concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress. ( IntroductionThe peroxiredoxins (Prxs) constitute a family of homodimeric peroxidases that reduce H 2 O 2 and alkyl hydroperoxides to water and alcohol, respectively. They rely on a conserved cysteine residue to catalyze peroxide reduction. There are 6 known mammalian isoforms (Prx1-6), classified as typical 2-Cys, atypical 2-Cys, or 1-Cys Prxs based on the mechanism and number of cysteines involved during catalysis. 1 When peroxiredoxin 2 (Prx2; a typical 2-Cys Prx) reacts with peroxide, the peroxidatic cysteine at the active site on one subunit is oxidized to a sulfenic acid. A second conserved cysteine at the C-terminal end of the other subunit (the resolving cysteine) then reacts with the sulfenic acid to form a disulfide bridge. Reduction of the disulfide by thioredoxin (Trx) regenerates Prx2 and completes the cycle. Trx is in turn regenerated by thioredoxin reductase (TrxR), with reducing equivalents derived from NADPH. 2 An intriguing feature of mammalian 2-Cys Prxs is that in the presence of high levels of peroxide, the peroxidatic Cys becomes overoxidized to the sulfinic (SO 2 H) or sulfonic (SO 3 H) acid form. 3 This abolishes the enzyme's peroxidase activity, although overoxidized Prx can be slowly reverted to the reduced state by sulfiredoxin. 4 It has been suggested that overoxidation allows intracellular accumulation of H 2 O 2 , which can then function as a signal transducer for various pathways. 5,6 Compared with other somatic cells, erythrocytes are exposed to oxidative stress from a wide variety of sources. They contain high levels of O 2 and hemoglobin (Hb), which continually autoxidizes to produce O 2 Ϫ and H 2 O 2 . They also have membranes...

Peroxiredoxin 2 (Prx2) is an antioxidant enzyme that uses cysteine residues to decompose peroxides. Prx2 is the third most abundant protein in erythrocytes, and competes effectively with catalase and glutathione peroxidase to scavenge low levels of hydrogen peroxide, including that derived from hemoglobin autoxidation. Low thioredoxin reductase activity in the erythrocyte is able to keep up with this basal oxidation and maintain the Prx2 in its reduced form, but exposure to exogenous hydrogen peroxide causes accumulation of the disulfide-linked dimer. The high cellular concentration means that although turnover is slow, erythrocyte Prx2 can act as a noncatalytic scavenger of hydrogen peroxide and a sink for hydrogen peroxide before turnover becomes limiting. The consequences of Prx2 oxidation for the erythrocyte are not well characterized, but mice deficient in this protein develop severe hemolytic anemia associated with Heinz body formation. Prx2, also known as calpromotin, regulates ion transport by associating with the membrane and activating the Gárdos channel. How Prx2 redox transformations are linked to membrane association and channel activation is yet to be established. In this review, we discuss the functional properties of Prx2 and its role as a major component of the erythrocyte antioxidant system.

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with HO, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to HO or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.