title: Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide.
abstract: Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H(2)O(2)). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H(2)O(2) (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H(2)O(2) (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.
authors.affiliation: Instituto de Física, Universidade Federal de Goiás, Goiânia, Brasil.
authors.forename: S A
authors.initials: SA
authors.lastname: Mendanha
authors.forename: J L V
authors.initials: JL
authors.lastname: Anjos
authors.forename: A H M
authors.initials: AH
authors.lastname: Silva
authors.forename: A
authors.initials: A
authors.lastname: Alonso
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D002392:Catechin / Q000494:pharmacology; D003497:Cyclic N-Oxides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D006454:Hemoglobins / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D015227:Lipid Peroxidation / Q000187:drug effects*; D008560:Membrane Fluidity / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism*; D024502:alpha-Tocopherol / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D003497:Cyclic N-Oxides; D006454:Hemoglobins; D008565:Membrane Proteins; D017392:Thiobarbituric Acid Reactive Substances; C005072:5-doxylstearic acid; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; D002392:Catechin; D006861:Hydrogen Peroxide; D024502:alpha-Tocopherol; D001205:Ascorbic Acid