title: Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase.
abstract: The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 . 10(5) or 6.0 . 10(5) molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4-diisothiocyano-1,2-diphenyl-ethane-2,2'disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.
mesh_terms: D001693:Biological Transport, Active / Q000187:drug effects; D002800:Cholinesterase Inhibitors; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D010777:Photochemistry; D013431:Sulfates / Q000378:metabolism
chemical_list: D002800:Cholinesterase Inhibitors; D005452:Fluoresceins; D008565:Membrane Proteins; D013431:Sulfates; D004801:Eosine Yellowish-(YS); D007460:Iodoacetamide