Use of the Xuorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on diVerent experimental models

The Xuorescent 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 speciWcity for cardiolipin; however, the limits and the mechanism of this speciWcity 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 eVects of some experimental factors on the selective Xuorescence of NAO in the presence of cardiolipin in artiWcial and natural membranes (mitochondria). The results show that the Xuorescence of NAO, due to interaction with cardiolipin, is signiWcantly modiWed 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 speciWc eVect of cardiolipin is to facilitate the dimerization of this Xuorescent dye, thus conWrming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The Wnding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.  2004 Published by Elsevier Inc.