FIG. 1.

Effect of storage time on Prx-2 oxidation and recycling kinetics. (A) Basal Prx-2 oxidation in RBC freshly isolated or after storage for 7 or 35 days. Each symbol represents an RBC preparation from a distinct donor. p-Value shown determined by one-way ANOVA (nonparametric assuming non-Gaussian distribution). (B) H₂O₂ (10 μM) was added to human RBCs stored for different times (7–35 days) in isotonic saline buffer (pH 7.4) and Prx-2 oxidation measured at 5, 15, 30, or 60 min as described in Materials and Methods section. Representative Western blots and quantitation of Prx-2 oxidation expressed as the fraction of the Prx-2 dimer relative to total Prx-2 are shown. Data are mean±SEM (n=6). *p<0.001 compared with respective basal (time 0) by one-way RM-ANOVA with Tukey's post hoc test. (C) Time-dependent changes in Prx-2 relative to the maximal amount formed (at 5 min). Solid lines show linear regression fits (p<0.001 for all slopes being not zero) for data between 5 and 30 min and were y = −0.021x+1.09 (day 7, ■); y = −0.02x+1.09 (day 14, ◯); y = −0.014x+1.07 (day 28, △); y = −0.0064x+1.04 (day 35, ▼). Slopes were significantly different between day 14 and 28 (p<0.01), between day 7 and 35 (p<0.001), and between day 14 and 35 (p<0.0001). (D) Hemolysis induced by addition of H₂O₂ (10 μM) to day 7 or 35 RBCs (after 60 min in isotonic saline buffer (pH 7.4). Each data point represents a distinct RBC preparation. *p<0.02 by t-test. (E) H₂O₂ (10 μM) was added to phosphate-buffered saline (■), day 7 RBC (▼), or day 35 RBC (◯) and H₂O₂ concentrations were measured at indicated times. Data shown are mean±SEM (n=3–4). No differences between day 7 and 35 RBC-dependent consumption of H₂O₂ were observed. H₂O₂, hydrogen peroxide; Prx-2, peroxiredoxin-2; RBC, red blood cell; RM-ANOVA, repeated measures-analysis of variance.