Relaxation technique for kinetic measurements of dye accumulation at the mitochondria of HeLa cells in situ.

The lipophilic cationic fluorescent dye ESI4 specifically stains the mitochondria in living cells. It was used to determine the kinetics of vital staining of mitochondria in single selected HeLa cells by quantitative microfluorometry in the concentration range CD = 6 x 10(-8) to 4 x 10(-6) M. The experiments were performed using the concentration jump method. They were evaluated with a quantitative staining model which considers dye binding as well as dye release at mitochondrial binding sites. The kinetic equations of the model are discussed in terms of the relaxation technique. The predictions of the theory agree well with the experimental results. The staining model was used to determine the rate constant k1 = 7.6 x 10(2)s-1 M-1 and k2 = 1.6 x 10(-3) s-1 for dye binding and dye release respectively, and the binding constant K = 4.8 x 10(5) M-1 of dye molecules at the mitochondria in HeLa cells (values according to relaxation experiments). The constants are discussed in detail. In addition, some experiments were performed on dye accumulation under the influence of antimycin, nigericin and oligomycin.