title: Role of the membrane in the formation of heme degradation products in red blood cells.
abstract: OBJECTIVE
  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) 321 nm, emission (em) 465 nm) 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.
  
  
  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 (H(2)O(2)) was studied.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These results suggest that, although ROS generated in the cytosol are neutralized by antioxidant enzymes, H(2)O(2) generated by the membrane bound hemoglobin is not accessible to the cytosolic antioxidants and reacts to generate fluorescent heme degradation products. The formation of H(2)O(2) 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.
authors: Molecular Dynamics Section, National Institute on Aging, National Institutes of Health, 251 Bay View Blvd, Baltimore, MD 21224, United States.
authors: Enika
authors: E
authors: Nagababu
authors: Joy G
authors: JG
authors: Mohanty
authors: Surya
authors: S
authors: Bhamidipaty
authors: Graciela R
authors: GR
authors: Ostera
authors: Joseph M
authors: JM
authors: Rifkind
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005453:Fluorescence; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress
chemical_list: D006454:Hemoglobins; D006418:Heme; D006861:Hydrogen Peroxide