Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence.
Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.
D001436:Bacteriorhodopsins; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002338:Carotenoids; D003619:Dansyl Compounds; D006834:Hydrazines; D013050:Spectrometry, Fluorescence; D014501:Uranium; D017974:Uranium Compounds
D003619:Dansyl Compounds; D006834:Hydrazines; D017974:Uranium Compounds; C027913:dansyl hydrazine; D002338:Carotenoids; D014501:Uranium; D001436:Bacteriorhodopsins; C012597:uranium dioxide