The effects of disruption of genes for peroxiredoxin-2, glutathione peroxidase-1, and catalase on erythrocyte oxidative metabolism

Abstract

Peroxiredoxin-2 (Prdx2), a potent peroxide reductant, is the third most abundant protein in the erythrocyte and might be expected to play a major role in the cell's oxidative defenses. However, in this study, experiments with erythrocytes from mice with a disrupted Prdx2 gene found that the cells were not more sensitive to exogenous H(2)O(2) or organic peroxides than wild type. Intraerythrocytic H(2)O(2) was increased, however, indicating an important role for Prdx2 in detoxifying endogenously generated H(2)O(2). These results are consistent with proposals that red cell Prdx2 acts stoichiometrically, not catalytically, in reducing peroxides. Additional experiments with mice with disrupted catalase or glutathione peroxidase (Gpx1) genes showed that Gpx1 is the only erythrocyte enzyme that reduces organic peroxides. Catalase(-/-) cells were readily oxidized by exogenous H(2)O(2). Cells lacking both catalase and Gpx1 were more sensitive to exogenous H(2)O(2) than cells lacking only catalase. A kinetic model proposed earlier to rationalize results with Gpx1(-/-) erythrocytes also fits the data with Prdx2(-/-) cells and indicates that although Gpx1 and Prdx2 both participate in removing endogenous H(2)O(2), Prdx2 plays a larger role. Although the rate of H(2)O(2) production in the red cell is quite low, Prdx2-deficient mice are anemic, suggesting an important role in erythropoiesis.

Figure 1

Absence of catalase makes red cell hemoglobin susceptible to oxidation by H₂O₂. To red cells from catalase deficient (−/−), heterozygotes (−/+), and wild-type mice at a hematocrit of 5 in PBS containing 10 mM glucose were added appropriate amounts of 1 mM H₂O₂ and buffer to achieve the indicated concentrations. MetHb was determined after 15 minutes at room temperature.

Figure 2

Hb oxidation in enzyme-deficient red cells. In this experiment, washed cellulose-filtered red cells from wild-type mice (triangles) or from mice with disrupted genes for catalase (open squares), Gpx1 (filled circles), or both (open circles) were suspended in KR buffer (Hb = 30 mg/ml) at 37°. Glucose oxidase was added (1mU/ml) to generate 0.137 μM H₂O₂ /min. MetHb was assayed at 0, 2, 4, 6 hours. Two runs are shown here to indicate the usual level of agreement between duplicate runs. Subsequent figures will present averages of two or more duplicate runs. Double KO red cells were more sensitive to an oxidative challenge than cells deficient in catalase alone.

Figure 3

Hb oxidation in enzyme-deficient red cells in response to bolus additions of H₂O₂. Washed red cells in KR buffer (Hct = 5) at 37° were mixed with 30 mM H₂O₂ and buffer to achieve the indicated [H₂O₂]. MetHb was determined after 15 minutes at 37°. Symbols as in figure 2.

Figure 4

Catalase(−/−) red cells are not sensitive to organic peroxides. Duplicate runs of washed red cells at a hematocrit of 5 were incubated in KR buffer with 10 mM glucose and different amounts of tert -butylhydroperoxide (top) or cumene hydroperoxide (bottom) at 37°. MetHb was assayed at the end of 1 hour.

Figure 5

Prdx2(−/−) red cells are not more sensitive to oxidation by exogenous H₂O₂. Washed cellulose-filtered red cells in KR buffer (Hct = 5) at 37° were mixed with 30 mM H₂O₂ and buffer to achieve the indicated [H₂O₂]. MetHb was determined after 15 minutes at 37°. (Patterned bars are wt; filled bars are Prdx(−/−) cells.)

Figure 6

Prdx2(−/−) red cells do not have increased sensitivity to organic peroxides. Wikd-type red cells (open circles) and Prdx2 red cells (filled circles) at a hematocrit of 5 were incubated in KR buffer with 10 mM glucose and different amounts of (a) cumene hydroperoxide or (b) tert -butylhydroperoxide at 37°. MetHb was assayed at the end of 1 hour.

Figure 7

Time course of catalase inhibition by 50 mM 3-AT in Prdx2(−/−) red cells (filled circles) and wt cells (open circles). The rate of catalase inactivation in wt is 23 nM/h and 37 nM/h in Prdx2(−/−) cells. (- - -) catalase activities in the presence of 3-AT predicted by the model. (—) least squares fit of the data.