Interactions involving the cyanine dye, diS-C3-(5), cytochromec and liposomes and their implications for estimations of Δψ in cytochromec oxidase-reconstituted proteoliposomes

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Abstract
The interference of cytochromec with absorption and fluorescence changes of the cyanine dye, diS-C3-(5), was investigated in the presence of liposomes and cytochromec-oxidase reconstituted proteoliposomes. The apparent cytochromec-dependent quenching of diS-C3-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochromec-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochromec-induced breakdown of dye was observed in the presence of hydrogen peroxide. The cytochromec mediated absorbance and fluorescence losses of diS-C3-(5) in liposomal or proteoliposomal systems are prevented by Ca2+ and La3+ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochromec is inhibited either directly (e. g. by BHT) or indirectly, by preventing the binding of cytochromec to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential inaa 3-reconstituted proteoliposomes is discussed.

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