Siedel I, Zimmermann H W
Institut für Physikalische Chemie, Universität Freiburg, Germany.
J Photochem Photobiol B. 1995 Mar;27(3):233-42. doi: 10.1016/1011-1344(94)07073-w.
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.
亲脂性阳离子荧光染料ESI4可特异性地对活细胞中的线粒体进行染色。通过定量显微荧光测定法，在浓度范围CD = 6×10⁻⁸至4×10⁻⁶ M内，利用该染料测定单个选定的HeLa细胞中线粒体的活体染色动力学。实验采用浓度跃变法进行。运用一个定量染色模型对实验结果进行评估，该模型考虑了染料在线粒体结合位点的结合以及释放情况。依据弛豫技术对模型的动力学方程进行了讨论。理论预测结果与实验结果吻合良好。利用该染色模型分别确定了染料结合速率常数k1 = 7.6×10² s⁻¹ M⁻¹和染料释放速率常数k2 = 1.6×10⁻³ s⁻¹，以及HeLa细胞中线粒体上染料分子的结合常数K = 4.8×10⁵ M⁻¹（根据弛豫实验得出的值）。对这些常数进行了详细讨论。此外，还进行了一些在抗霉素、尼日利亚菌素和寡霉素影响下的染料积累实验。
