10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria

Eur J Biochem. 1992 Oct 1;209(1):267-73. doi: 10.1111/j.1432-1033.1992.tb17285.x.


The acridine orange derivative, 10N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 10(6) M-1 for cardiolipin-10N-nonyl-acridine-orange association and only 7 x 10(4) M-1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.

Publication types

  • Comparative Study

MeSH terms

  • Acridine Orange / analogs & derivatives*
  • Acridine Orange / metabolism
  • Animals
  • Cardiolipins / analysis
  • Cardiolipins / metabolism*
  • Cell Membrane / chemistry
  • Liposomes / metabolism
  • Male
  • Microscopy, Fluorescence
  • Mitochondria / chemistry*
  • Phosphatidylinositols / metabolism
  • Phosphatidylserines / metabolism
  • Rats
  • Rats, Wistar
  • Spectrophotometry
  • Thermodynamics


  • Cardiolipins
  • Liposomes
  • Phosphatidylinositols
  • Phosphatidylserines
  • N(10)-nonylacridine orange
  • Acridine Orange