Interaction of Fluorescent Probes with Submitochondrial Particles during Oxidative Phosphorylation

Addition of several N-arylaminonaphthalenesulfonate dyes to submitochondrial particles from bovine heart resulted in an enhancement of fluorescence of from 50- to 150-fold and a considerable shift in the wave length of maximum emission toward the ultraviolet. The results indicate 40 to 55 dye-binding sites per mg of submitochondrial particles. A preliminary search revealed that mitochondrial ATPase may be one of the components of the mitochondrial membrane responsible for the fluorescent responses of the dyes. Addition of substrates or ATP to a reaction containing submitochondrial particles and dye led to a further enhancement of fluorescence which was about equal in magnitude to that observed upon formation of the dye-particle complex. The second enhancement was prevented or diminished by the addition of inhibitors of respiration or uncouplers of oxidative phosphorylation. Substrate- and ATP-mediated fluorescence enhancement was stimulated by coupling factors in submitochondrial particles which required coupling factors for oxidative phosphorylation. Evidence is presented which shows that these dyes may be used as highly sensitive indicators of energy coupling in submitochondrial particles.