Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence

• Published in: Biophysical journal Vol. 45; no. 5; pp. 1001 - 1006
• Main Authors: Renthal, R., Cha, C.H.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 01.05.1984; Biophysical Society
• Subjects: Bacteriorhodopsins; Binding Sites; Biological and medical sciences; Biophysical Phenomena; Biophysics; Carotenoids; Cell structures and functions; charge; Chloroplast, photosynthetic membrane and photosynthesis; Dansyl Compounds; dansyl hydrazine; fluorescence; Fundamental and applied biological sciences. Psychology; Halobacterium halobium; Hydrazines; Molecular and cellular biology; purple membranes; Spectrometry, Fluorescence; Uranium; Uranium Compounds; Bacteriorhodopsin; Deexcitation; Surface charge; Asymmetry; Archaebacteria; Purple membrane; Bacteria; Halobacteriaceae; Halobacterium halobium; Fluorescence spectrometry
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(84)84245-9

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.