Cyanine and safranine dyes as membrane potential probes in cytochrome c oxidase reconstituted proteoliposomes.
Safranine and the cyanine dye, 3',3'-dipropylthiadicarbocyanine (diSC3-5), were examined as membrane potential probes in cytochrome c oxidase vesicles. The spectra of the vesicle-associated dyes resemble those of the same dyes in organic solvents, indicating that safranine and diSC3-5 probably dissolve in a hydrophobic region of the proteoliposomal membrane. This binding of safranine to proteoliposomes occurs with a dye-membrane dissociation constant in the micromolar range. The binding of safranine and of diSC3-5 to liposomes or proteoliposomes is accompanied by fluorescence enhancement. This enhanced fluorescence is quenched by respiration or by the establishment of a K+ diffusion potential by valinomycin (negative interior). An optimal dye/lipid ratio was required to secure maximum fluorescence quenching of the dyes, whether that quenching was active or passive. Calibrations of both the safranine and the diSC3-5 responses with K+ diffusion potentials were also affected by the dye/lipid ratio. At lower dye/lipid ratios, the calibration curve was linear at higher potentials but deviated from linearity at lower potentials. The converse was true at higher dye/lipid ratios. The non-linearity of the calibration curve at higher potential was attributed to a 'saturation' effect; it may also involve increased permeability of proteoliposomal membrane to protons. Destacking of dye at the lower dye/lipid ratio was probably responsible for the non-linearity of the calibration curves at lower potentials. When all these effects are taken into account, the steady-state value of delta psi generated during maximal proteoliposomal respiration was calculated to be between 140 and 160 mV (interior negative) when measured with either safranine or diSC3-5. We conclude that quantitative estimates of delta psi values can be made using these probes in cytochrome c oxidase reconstituted proteoliposomes provided that appropriate precautions are taken.
Singh
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AP
Nicholls
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D000818:Animals; D052160:Benzothiazoles; D002232:Carbocyanines; D002417:Cattle; D004396:Coloring Agents; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D008564:Membrane Potentials; D008722:Methods; D010619:Phenazines; D011188:Potassium / Q000378:metabolism; D011510:Proteolipids; D011804:Quinolines; D013050:Spectrometry, Fluorescence; D014634:Valinomycin / Q000494:pharmacology
D052160:Benzothiazoles; D002232:Carbocyanines; D004396:Coloring Agents; D008081:Liposomes; D010619:Phenazines; D011510:Proteolipids; D011804:Quinolines; D014634:Valinomycin; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine; D003576:Electron Transport Complex IV; D011188:Potassium; C009195:safranine T