Direct cardiolipin assay in yeast using the red fluorescence emission of 10-N-nonyl acridine orange

The dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) has been recently identified as a specific probe for cardiolipin (Ka = 2 x 10(6) M-1). It also interacts, at lower affinity (Ka = 7 x 10(4) M-1), with other acidic Phospholipids [Petit, J. M., Maftah, A., Ratinaud, M. H. & Julien, R. (1992) Eur. J. Biochem. 209, 267-273]. In order to reduce the interference corresponding to monoacidic phospholipid binding, we have quantified cardiolipin by using a fluorimetric method based on the red fluorescence of the dye dimers formed at the diacidic phospholipid contact. Hence we have demonstrated that: (a) in yeast, the mitochondrion is the target of the dye whatever the cell metabolism; (b) membrane or protein organization and fatty acid unsaturation do not significantly modify the binding of 10-N-nonyl acridine orange. Using thin-walled vesicles, a linear relationship was established between the amount of cardiolipin and the red fluorescence emitted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at the same acidic phospholipid concentration, the fluorescence was much higher using cardiolipin-containing vesicles (fivefold that observed with phosphatidylserine-containing vesicles). Thus, 10-N-nonyl acridine orange was applied to cardiolipin quantification in yeast. This new method revealed that cells growing with a high glucose concentration contained 2.2 +/- 0.3 nmol cardiolipin/10(6) cells, whereas with lactate they contained about twice this amount (3.9 +/- 0.3 nmol cardiolipin).