10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria.
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.
Petit
J M
JM
Institut de Biotechnologie, Faculté des Sciences, Limoges, France.
Maftah
A
A
Ratinaud
M H
MH
Julien
R
R
D000165:Acridine Orange / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002308:Cardiolipins / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D008081:Liposomes / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013053:Spectrophotometry; D013816:Thermodynamics
D002308:Cardiolipins; D008081:Liposomes; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; C056258:N(10)-nonylacridine orange; D000165:Acridine Orange