Cardiolipin binds nonyl acridine orange by aggregating the dye at exposed hydrophobic domains on bilayer surfaces

10-N-Nonyl acridine orange (NAO) has been used at low concentrations as a f luorescent indicator for cardiolipin (CL) in membranes and bilayers. The me chanism of its selective fluorescence in the presence of CL, and not any ot her phospholipids, is not understood. The dye might recognize CL by its hig h pK (pK(2) > 8.5). To investigate that, we established that NAO does not e xhibit a pK in a pH range between 2.3 and 10.0. A second explanation is tha t the dye aggregates at hydrophobic domains on bilayers exposed by the CL. We found that a similar spectral shift occurs in the absence of CL in a con centrated solution of the dye in methanol and in the solid state. A model i s proposed in which the nonyl group inserts in the bilayer at the hydrophob ic surface generated by the presence of four chains on the phospholipid. (C ) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.