**5. Interplay between erythrocyte peroxidases and the erythrocyte membrane**

The individual contribution of CAT, GPx and Prx2 to erythrocyte protection against H2O2 damage has been a controversial issue for many years. It is clear that all three enzymes are involved in the prevention of H2O2 accumulation in the RBC through H2O2 conversion into water and O2 (**Figure 6**); however, the relative importance of the three enzymes is still a topic of discussion.

CAT was considered the main erythrocyte defense against OS, for many years [68, 88], but several studies [59, 69, 89] showed that GPx has also an important role in H2O2 decomposition. In fact, a study by Johnson et al. [59] showed that CAT and GPx-deficient RBCs were more sensitive to H2O2-induced OS than cells with only CAT deficiency, suggesting that GPx has an important role in erythrocyte defense. The same authors also showed [59, 70] that the action of both CAT and GPx was insufficient to explain the erythrocyte oxidative catabolism, and proposed [70] a model including Prx2 that, in accordance with their experimental data, could better explain the erythrocyte antioxidant defense system. Furthermore, the development of hemolytic anemia in Prx2 knock-out mice [72–74] and the high turnover rate of Prx2 with H2O2 [5, 75] strengthened the importance of the role of Prx2 in RBC antioxidant protection.

#### **Figure 6.**

*Interplay between erythrocyte's peroxidases. (1) Methemoglobin formation and release of O2 <sup>−</sup>. (2) Formation of band 3 protein aggregates triggered by methemoglobin linkage to the integral membrane protein band 3. (3) O2 <sup>−</sup> removal by SOD with H2O2 formation. (4) H2O2 removal by CAT. (5) H2O2 removal by GPx. (6) H2O2 removal by Prx2. (7) Linkage of CAT, GPx and Prx2 to the RBC membrane imposed by oxidative stress. CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; H2O, water; H2O2, hydrogen peroxide; Hb, hemoglobin; metHb, methemoglobin; Hb-O2, oxyhemoglobin; NADPH/NADP<sup>+</sup> , nicotinamide adenine dinucleotide phosphate; O2, oxygen; O2 <sup>−</sup>, superoxide anion; Prx2, peroxiredoxin 2; SOD, superoxide dismutase; Trx, thioredoxin; TrxR, thioredoxin reductase.*

**75**

*Interplay between Erythrocyte Peroxidases and Membrane*

The relative importance of the three enzymes appears to be related to the H2O2 levels in the RBC [59]. CAT is able to scavenge exogenous and high endogenous peroxide levels [59, 60, 70], while GPx and Prx2 appear to scavenge endogenous and low peroxide levels [59, 70, 73]. Thus, the elimination of the basal flux and low H2O2 levels is performed by GPx and Prx2, since CAT does not work efficiently at low H2O2 levels [60]. Whenever RBCs are exposed to higher H2O2 levels, CAT becomes essential for its rapid removal since this enzyme has a high turnover rate, unlike GPx and Prx2 that become less efficient (or even inactive), due to their slow

The enzymes GPx and Prx2 seem to have other functions in RBC antioxidant defense, beyond H2O2 scavenging. In fact, GPx and Prx2 are also able to detoxify organic peroxides [5, 59, 75], while CAT does not show this function [59]. As shown by Johnson et al. [59, 90], GPx-deficient RBCs are more susceptible to oxidation by organic peroxides than wild type cells [90]; and when CAT deficiency was added to GPx deficiency, no increased sensitivity to oxidation by organic peroxides occurred in these cells [59]. Thus, when erythrocytes are exposed to high H2O2 levels, organic peroxides will accumulate, since GPx and Prx2 become less efficient

Recently, it was reported that Prx2 can have multiple functions, as a peroxidase or as a chaperone, through the formation of high-molecular-weight complexes [78, 91]. It was shown that Prx2 acts as a chaperone in RBCs, by interacting directly with Hb to maintain its stability [79]. A study by our group [80] showed that when erythrocytes were saturated with CO, the enzyme Prx2 was present in the cytosol only in the monomeric form, suggesting that Prx2 was not acting as a peroxidase but, instead, exclusively as a chaperone for Hb's protection. Several authors have suggested [59, 92] that Prx2 can also have an important role in erythropoiesis. Johnson et al. [59] believe that the role of Prx2 as Hb chaperone is especially important in the different stages of erythropoiesis. According to Matte et al., Prx2 appears

CAT, GPx and Prx2 are essentially cytosolic enzymes; however, the association of these enzymes to the erythrocyte membrane has been reported in different in vivo and in vitro studies [26, 31, 32, 93–97]. Erythrocytes from patients with hereditary spherocytosis showed CAT [95] and Prx2 bound to their membranes [26]. The association of CAT to the membrane appears to be a consequence of the metabolic stress triggered by the destabilization of membrane structure, due to an altered RBC membrane composition; the linkage of Prx2 might be involved in the protection of the RBC membrane against LPO [26, 31]. This linkage of Prx2 to the membrane appears to be through the N-terminal cytoplasmic domain of band 3, which is also the site of linkage of other cytoplasmic proteins, including metHb [98]. In recent in vitro assays performed by our group [32], we showed that H2O2-induced oxidative stress triggered the binding of Prx2 and GPx to RBC membrane. A recent study by Bayer et al. [96] about the interaction of Prx2 with the RBC membrane reported that the linkage of Prx2 to the membrane is independent of its redox state and that Prx2 competes with Hb for the same binding site in the RBC membrane. Thus, they demonstrated that Prx2 prevents metHb aggregation, and, probably acts as a chaperone for the denatured Hb [96]. Contrary to what was previously observed [26, 31, 32], Bayer et al. [96] found a decrease in Prx2 membrane binding, with increasing concentrations of H2O2. A decrease in Prx2 linkage to the RBC membrane with OS conditions was also observed in beta-thalassemic mice RBCs, probably due to the increase in metHb that

GSH reductase and Trx recycling systems, respectively [59, 60, 73].

and CAT is not able to detoxify these organic peroxides [60].

to be a regulator of iron homeostasis during erythropoiesis [92].

binds to the membrane, reducing the access of Prx2 to the same site [99].

GPx to the RBC membrane in response to in vitro H2O2-induced OS [32].

The linkage of GPx to the RBC membrane was first described by van Gestel et al.*,* using proteome analysis [94]. Later on, Rocha et al. showed the linkage of

*DOI: http://dx.doi.org/10.5772/intechopen.83590*

#### *Interplay between Erythrocyte Peroxidases and Membrane DOI: http://dx.doi.org/10.5772/intechopen.83590*

*Erythrocyte*

**membrane**

antioxidant protection.

hereditary spherocytosis patients [26]. Thus, there has been an increasing interest in Prx2 as a biomarker for different conditions where OS plays a crucial role. For example, a novel HPLC method to monitor the levels of reduced Prx2 form was

developed [87], which could prove useful for future clinical practice.

*Interplay between erythrocyte's peroxidases. (1) Methemoglobin formation and release of O2*

*of band 3 protein aggregates triggered by methemoglobin linkage to the integral membrane protein band 3.* 

*anion; Prx2, peroxiredoxin 2; SOD, superoxide dismutase; Trx, thioredoxin; TrxR, thioredoxin reductase.*

*<sup>−</sup> removal by SOD with H2O2 formation. (4) H2O2 removal by CAT. (5) H2O2 removal by GPx. (6) H2O2 removal by Prx2. (7) Linkage of CAT, GPx and Prx2 to the RBC membrane imposed by oxidative stress. CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; H2O, water; H2O2, hydrogen peroxide; Hb, hemoglobin; metHb, methemoglobin; Hb-O2,* 

*, nicotinamide adenine dinucleotide phosphate; O2, oxygen; O2*

*<sup>−</sup>. (2) Formation* 

*<sup>−</sup>, superoxide* 

tance of the three enzymes is still a topic of discussion.

**5. Interplay between erythrocyte peroxidases and the erythrocyte** 

The individual contribution of CAT, GPx and Prx2 to erythrocyte protection against H2O2 damage has been a controversial issue for many years. It is clear that all three enzymes are involved in the prevention of H2O2 accumulation in the RBC through H2O2 conversion into water and O2 (**Figure 6**); however, the relative impor-

CAT was considered the main erythrocyte defense against OS, for many years [68, 88], but several studies [59, 69, 89] showed that GPx has also an important role in H2O2 decomposition. In fact, a study by Johnson et al. [59] showed that CAT and GPx-deficient RBCs were more sensitive to H2O2-induced OS than cells with only CAT deficiency, suggesting that GPx has an important role in erythrocyte defense. The same authors also showed [59, 70] that the action of both CAT and GPx was insufficient to explain the erythrocyte oxidative catabolism, and proposed [70] a model including Prx2 that, in accordance with their experimental data, could better explain the erythrocyte antioxidant defense system. Furthermore, the development of hemolytic anemia in Prx2 knock-out mice [72–74] and the high turnover rate of Prx2 with H2O2 [5, 75] strengthened the importance of the role of Prx2 in RBC

**74**

**Figure 6.**

*(3) O2*

*oxyhemoglobin; NADPH/NADP<sup>+</sup>*

The relative importance of the three enzymes appears to be related to the H2O2 levels in the RBC [59]. CAT is able to scavenge exogenous and high endogenous peroxide levels [59, 60, 70], while GPx and Prx2 appear to scavenge endogenous and low peroxide levels [59, 70, 73]. Thus, the elimination of the basal flux and low H2O2 levels is performed by GPx and Prx2, since CAT does not work efficiently at low H2O2 levels [60]. Whenever RBCs are exposed to higher H2O2 levels, CAT becomes essential for its rapid removal since this enzyme has a high turnover rate, unlike GPx and Prx2 that become less efficient (or even inactive), due to their slow GSH reductase and Trx recycling systems, respectively [59, 60, 73].

The enzymes GPx and Prx2 seem to have other functions in RBC antioxidant defense, beyond H2O2 scavenging. In fact, GPx and Prx2 are also able to detoxify organic peroxides [5, 59, 75], while CAT does not show this function [59]. As shown by Johnson et al. [59, 90], GPx-deficient RBCs are more susceptible to oxidation by organic peroxides than wild type cells [90]; and when CAT deficiency was added to GPx deficiency, no increased sensitivity to oxidation by organic peroxides occurred in these cells [59]. Thus, when erythrocytes are exposed to high H2O2 levels, organic peroxides will accumulate, since GPx and Prx2 become less efficient and CAT is not able to detoxify these organic peroxides [60].

Recently, it was reported that Prx2 can have multiple functions, as a peroxidase or as a chaperone, through the formation of high-molecular-weight complexes [78, 91]. It was shown that Prx2 acts as a chaperone in RBCs, by interacting directly with Hb to maintain its stability [79]. A study by our group [80] showed that when erythrocytes were saturated with CO, the enzyme Prx2 was present in the cytosol only in the monomeric form, suggesting that Prx2 was not acting as a peroxidase but, instead, exclusively as a chaperone for Hb's protection. Several authors have suggested [59, 92] that Prx2 can also have an important role in erythropoiesis. Johnson et al. [59] believe that the role of Prx2 as Hb chaperone is especially important in the different stages of erythropoiesis. According to Matte et al., Prx2 appears to be a regulator of iron homeostasis during erythropoiesis [92].

CAT, GPx and Prx2 are essentially cytosolic enzymes; however, the association of these enzymes to the erythrocyte membrane has been reported in different in vivo and in vitro studies [26, 31, 32, 93–97]. Erythrocytes from patients with hereditary spherocytosis showed CAT [95] and Prx2 bound to their membranes [26]. The association of CAT to the membrane appears to be a consequence of the metabolic stress triggered by the destabilization of membrane structure, due to an altered RBC membrane composition; the linkage of Prx2 might be involved in the protection of the RBC membrane against LPO [26, 31]. This linkage of Prx2 to the membrane appears to be through the N-terminal cytoplasmic domain of band 3, which is also the site of linkage of other cytoplasmic proteins, including metHb [98]. In recent in vitro assays performed by our group [32], we showed that H2O2-induced oxidative stress triggered the binding of Prx2 and GPx to RBC membrane. A recent study by Bayer et al. [96] about the interaction of Prx2 with the RBC membrane reported that the linkage of Prx2 to the membrane is independent of its redox state and that Prx2 competes with Hb for the same binding site in the RBC membrane. Thus, they demonstrated that Prx2 prevents metHb aggregation, and, probably acts as a chaperone for the denatured Hb [96]. Contrary to what was previously observed [26, 31, 32], Bayer et al. [96] found a decrease in Prx2 membrane binding, with increasing concentrations of H2O2. A decrease in Prx2 linkage to the RBC membrane with OS conditions was also observed in beta-thalassemic mice RBCs, probably due to the increase in metHb that binds to the membrane, reducing the access of Prx2 to the same site [99].

The linkage of GPx to the RBC membrane was first described by van Gestel et al.*,* using proteome analysis [94]. Later on, Rocha et al. showed the linkage of GPx to the RBC membrane in response to in vitro H2O2-induced OS [32].

Studies of stored RBCs in blood bank conditions also reported the recruitment of Prx2, CAT and GPx to the RBC membrane due to OS modifications resulting from the metabolic stress of long-term erythrocyte storage [93, 97].

Data in literature suggest that the linkage of these RBC cytosolic enzymes to the membrane is triggered by metabolic stress, possibly, to protect the erythrocyte membrane and counteract the effects of OS.
