Role of the membrane in the formation of heme degradation products in red blood cells

Aims: Red blood cells (RBCs) have an extensive antioxidant system designed to eliminate the formation of reactive oxygen species (ROS). Nevertheless, RBC oxidant stress has been demonstrated by the formation of a fluorescent heme degradation product (excitation (ex) 321nm, emission (em) 465nm) both in vitro and in vivo. We investigated the possibility that the observed heme degradation results from ROS generated on the membrane surface that are relatively inaccessible to the cellular antioxidants. Main methods: Membrane and cytosol were separated by centrifugation and the fluorescence intensity and emission maximum were measured. The effect on the maximum emission of adding oxidized and reduced hemoglobin to the fluorescent product formed when hemin is degraded by hydrogen peroxide (H₂O₂) was studied. Key findings: 90% of the fluorescent heme degradation products in hemolysates are found on the membrane. Furthermore, these products are not transferred from the cytosol to the membrane and must, therefore, be formed on the membrane. We also showed that the elevated level of heme degradation in HbCC cells that is attributed to increased oxidative stress was found on the membrane. Significance: These results suggest that, although ROS generated in the cytosol are neutralized by antioxidant enzymes, H₂O₂ generated by the membrane bound hemoglobin is not accessible to the cytosolic antioxidants and reacts to generate fluorescent heme degradation products. The formation of H₂O₂ on the membrane surface can explain the release of ROS from the RBC to other tissues and ROS damage to the membrane that can alter red cell function and lead to the removal of RBCs from circulation by macrophages.