Alterations of red cell membrane proteins and hemoglobin under natural and experimental oxidant stress |
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Authors: | N. Alloisio D. Michelon E. Bannier A. Revol Y. Beuzard J. Delaunay |
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Affiliation: | 1. Laboratoire de Chimie Biologique, Faculté de Médecine Grange Blanche, 69373 Lyon Cedex 8, France;2. Laboratoire de Biochimie, Centre Hospitalier Lyon Sud, 69310 Pierre Benite France;3. INSERM U 91, Hôpital Henri Mondor, 94010 Créteil France |
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Abstract: | We compared on red cell membrane proteins and hemoglobin (Hb) the effects of (i) natural oxidant stress that has been suggested to occur in a variety of oxidative hemolytic anemias, and (ii) experimental stress induced by hydrogen peroxide. SDS-polyacrylamide gel electrophoresis was used for protein analysis. Under natural conditions (thalassemias, hemoglobinopathies with Hb unstability), a high molecular weight polymer (HMWP) and variable amounts of globin mono- and dimers became apparent. Furthermore, a major 12 kDa polypeptide, its dimer, and conspicuous spectrin degradation products in the band 2.2–2.6 region occurred in a patient carrying the highly unstable Hb Hammersmith. Under experimental conditions, incubation of erythrocyte ghosts with H2O2 in the presence of minimal concentration (25 μM) of Hb generated a HMWP at the expense of membrane proteins, mainly spectrin. Incubation of a diluted (200 μM) membrane-free hemolysate with H2O2 induced a HMWP, an array of globin oligomers and a 12 kDa polypeptide similar to that mentionned above. Therefore, the damage to the red cell membrane present in various oxidative hemolytic anemias, including polypeptide polymerisation and breakdown, can be produced by experimental oxidant stress. These observations support the view that the alterations described in the patients result directly from oxidative reactions. However, we did not observe in the patient the sharp breakdown of polyunsaturated fatty acids that was triggered in vitro by H2O2 in the presence of Hb acting as a catalyst. In most cases, oligo- and polymers were resistant to β-mercaptoethanol, and the chemical nature of the underlying cross-links is discussed. To our knowledge, the 12 kDa polypeptide, that we consider as arising from globin proteolysis, has never been reported under pathological conditions. |
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Keywords: | Erythrocyte membrane protein Hemoglobin Hemolytic anemia Oxidant stress DEGS diethylene glycol succinate EDTA ethylene-diaminetetraacetic acid Hb hemoglobin PC phosphatidylcholine PE phosphatidylethanolamine PS phosphatidylserine SDS sodium dodecyl sulfate TEMED TMCS trimethylchlorosilane |
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