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The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.
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