Mechanistic differences in the energy-linked fluorescence decreases of 9-aminoacridine dyes associated with bovine heart submitochondrial membranes.

(1) The pH dependence of the fluorescence intensities of 9-aminoacridines associated with energized submitochondrial membranes suggests that a mechanism(s) other than protonation of the dye molecules, as is the case with quinacrine, is responsible for the energy-linked fluorescence decreases of 9-aminoacridine and 9-amino-3-chloro-7-methoxyacridine (9-ACMA). (2) That the fluorescence polarization of quinacrine associated with submitochondrial membranes more than doubles upon energization of the membranes is attributed to: (i) the bulky side chain at the 9-position of the acridine moiety which hinders the molecular rotation of quinacrine and (ii) electrostatic forces resulting from the protonation of quinacrine . H+ which induce tight binding between the dye molecules and the membranes. (3) The protonation of quinacrine associated with energized membranes, from the monoprotonated to the diprotonated species, takes place in the membrane phase, as evidence by the observation of a 'break' in both the Arrhenius plot of the respiratory rate and the plot of fluorescence polarization as a function of temperature. (4) That the measured fluorescence polarization of both 9-aminoacridine and 9-ACMA associated with both energized and nonenergized membranes is nearly zero suggests that the emitting species of these dye molecules are those in the 'free' form and that the membrane-bound molecules have formed nonfluorescent complexes; consequently no polarization can be measured.