Detecting proton flux across chromatophores driven by F₀F₁-ATPase using N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt

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• 作者：Cui Yuanbo ; Zhang Fan and Yue Jiachang
• 关键词：Proton flux ; F₀F₁-ATPase ; Fluorescence probe ; ATP hydrolysis ; F-DHPE
• 刊名：Analytical Biochemistry
• 出版年：2005
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：2005
• 卷：344
• 期：1
• 页码：102-107
• 文件大小：323 K

摘要

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-β antibody) or two antibodies (anti-β 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.