Study of Interaction of Fluorescent Cytochrome C with Liposomes, Mitochondria, and Mitoplasts by Fluorescence Correlation Spectroscopy

Abstract

Here, we studied the interaction of Cys-substituted (G56C) cytochrome c labeled with sulfocyanin-3 fluorescent dye (fCyt) with artificial and natural lipid membranes by using fluorescence correlation spectroscopy (FCS). Compared to mitochondria, mitoplasts were shown to have more fCyt binding sites with a lower affinity for this protein. The fCyt affinity for cardiolipin-containing liposomes depended on the content of cardiolipin in liposomes and decreased upon raising the ionic strength of the solution. A high value of the constant of the fCyt binding with mitochondria could be explained by the presence of specific binding sites for this protein on the mitochondrial outer membrane surface. This explanation is confirmed by observation of more efficient displacement of fluorescent cytochrome c by the unlabeled mutant variant K8T than by the WT protein, whereas in mitoplasts and liposomes the WT protein displaced fCyt more efficiently than K8T.