Detecting proton flux across chromatophores driven by F0F1-ATPase using N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt.
N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (F-DHPE) is a lipid fluorescence dye sensitive to pH changes and is used in this study for detecting proton flux through F0F1-ATPase within chromatophores driven by ATP hydrolysis. F-DHPE is easily labeled to the outer surface of chromatophores. In the range of pH 7.0 to 9.0, fluorescence intensity is sensitive to pH changes. The sensitivity is especially great in the range of pH 8.2 to 9.0, so pH 8.6 was chosen as the appropriate experimental condition. It is shown that added ATP not only acts as a fluorescence quencher but also can be hydrolyzed by F0F1-ATPase to pump protons into chromatophores, resulting in fluorescence restoration. A stimulator (NaSO3) and various types of inhibitors (NaN3, 5'-adenylyl imidodiphosphate [AMP-PNP], and N,N'-dicyclohexylcarbodiimide [DCCD]) of F0F1 confirmed that fluorescence restoration is caused by ATP-driven proton flux. When loaded with one antibody (anti-beta antibody) or two antibodies (anti-beta antibody and sheep to rabbit second antibody), F0F1-ATPase exhibits lower proton pumping activities, as indicated by fluorescence restoration. The possible mechanism of the inhibition of antibodies on proton pumping activity is discussed.
Yuanbo
Cui
C
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Fan
Zhang
Z
Jiachang
Yue
Y
D000906:Antibodies / Q000276:immunology; D020130:Bacterial Chromatophores / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D017494:Proton Pumps / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000648:ultrastructure
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; C505938:N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; D010714:Phosphatidylethanolamines; D017494:Proton Pumps; D006180:Proton-Translocating ATPases