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Biochemistry

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JoVE Journal Biochemistry
Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy
 

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Article DOI: 10.3791/58896-v • 12:15 min • February 21st, 2019
February 21st, 2019 •
1, 2, 1
1School of Biomedical Sciences & the Astbury Centre for Structural Molecular Biology, University of Leeds, 2Department of Chemistry and Nano-Science Center, University of Copenhagen

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Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

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Liposome Measurements Study Proton-pumping Membrane Enzymes Electrochemistry Fluorescent Microscopy Respiratory Membrane Enzymes Cytochrome BO3 Lipid Bilayer Environment Enzymatic Reactions Glass Syringe Chloroform Stock Lipid Polar Extract Escherichia Coli Ubiquinone 10 Lipids Fluorescent Dye Labeled Lipid FDLL Homogenize Vortexing Evaporation Nitrogen Flow, Argon Flow Vacuum MOPS Buffer PH 7.4 Lipid Film Resuspension Ultrasonic Bath HPTS Dye OGP Surfactant
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