A sensitive new fluorescence assay for measuring proton transport across liposomal membranes.
6-Methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) is a fluorophore that is collisionally quenched by halide anions and is widely used to measure chloride ion transport across cellular and liposomal membranes. We report a new finding that SPQ fluorescence is also quenched by the zwitterionic hydrogen ion buffers introduced by Good et al. [(1966) Biochemistry 5, 467-477]. Although buffer quenching interferes with chloride ion measurements using SPQ, it can be turned to good advantage for measurements of proton flux. The basis for this application is that, for most buffers, the anion quenches and the zwitterion does not. Accordingly, buffer quenching of SPQ can be used to assay proton transport across liposomal membranes. We describe application of the technique to liposomes in which proton transport was mediated by ionophores and by the purified, reconstituted uncoupling protein of brown adipose tissue mitochondria. Because SPQ detects changes in buffer anion concentration, it can be used to measure changes in total acidity, which is the parameter desired when measuring net proton transport. Furthermore, this technique can be used to measure proton transport under conditions in which pH changes are minimized with buffers, and, consequently, effects of pH on proton transport can be dissociated from the transport itself.
Orosz
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Department of Pharmacology, Medical College of Ohio, Toledo 43699-0008.
Garlid
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D000838:Anions; D002021:Buffers; D002352:Carrier Proteins / Q000378:metabolism; D002412:Cations; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007473:Ion Channels; D017136:Ion Transport; D007476:Ionophores; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins; D009994:Osmolar Concentration; D011522:Protons; D011806:Quinolinium Compounds; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D013696:Temperature; D000071256:Uncoupling Protein 1
D000838:Anions; D002021:Buffers; D002352:Carrier Proteins; D002412:Cations; D005456:Fluorescent Dyes; D007473:Ion Channels; D007476:Ionophores; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011522:Protons; D011806:Quinolinium Compounds; D000071256:Uncoupling Protein 1; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium