Ghost protein damage by peroxynitrite and its protection by melatonin.
We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 microM ONOO-) disappearance at pH 7. 4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ss-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 microM ONOO-). However, a lack of effect of ss-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that (ONOO-)-induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 microM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.
Di Mascio
P
P
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil. pdmascio@iq.usp.br
Dewez
B
B
Garcia
C R
CR
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000494:pharmacology; D008550:Melatonin / Q000494:pharmacology*; D051379:Mice; D009566:Nitrates / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects*; D013049:Spectrin / Q000187:drug effects
D000975:Antioxidants; D003598:Cytoskeletal Proteins; D016166:Free Radical Scavengers; D009566:Nitrates; D016877:Oxidants; D013049:Spectrin; C039642:peroxynitric acid; D008550:Melatonin