Abstract

Red blood cells (RBCs) circulate through the lung and capillaries during their 120-day life span, transporting oxygen and carbon dioxide. They are exposed continuously to reactive oxygen species (ROS) derived from hemoglobin autoxidation. Protection of the RBC membrane from ROS is critical since on oxidation the cell loses its ability to deform in narrow capillaries. We investigated by Western blotting and confocal microscopy membrane recruitment of the key antioxidant enzymes, CuZn-superoxide dismutase (CuZnSOD), catalase, peroxiredoxin-2 (Prx2) and glutathione peroxidase-1 (Gpx1).
Prolonged air exposure of human RBCs, which likely increases their O2●¯ levels, resulted in less membrane-associated CuZnSOD but more membrane-associated catalase and Gpx1. Prx2 membrane levels remained unchanged.
Challenge with H2O2 or cumene hydroperoxide recruited Gpx1 to the membrane to metabolize lipid peroxides. Localization of catalase, which is always present at the membrane, remained unchanged but membrane-associated Prx2 decreased after peroxide treatment.
We also investigated membrane recruitment of the antioxidant enzymes in diabetic vs control rat RBCs. Notably, CuZnSOD increased at the RBC membrane in control animals at 2 versus 4 weeks. We attributed this to the observed increase in PTP1B activity, suggesting that CuZnSOD membrane localization is regulated by phosphorylation of the cytoplasmic domain of Band-3 (CDB3). Catalase decreased at RBC membranes of 4-week diabetic rats whereas Prx2 levels increased. Gpx1 membrane levels did not change in diabetic RBCs suggesting that its membrane binding sites are always saturated to protect against lipid oxidation. Our results provide evidence that CuZnSOD, catalase, Prx2 and Gpx1 respond to damaging ROS in RBCs.