Quantification of Cytochrome c Interaction with Cardiolipin and Phosphatidic Acid Using Phosphatidylcholine Labeled with the NBD Fluorescent Probe

Abstract

Cytochrome c interaction with anionic phospholipids is a key stage in the development of apoptotic processes occurring in the cell and beginning on the inner membrane of mitochondria. Such phospholipids include the well-known cardiolipin, phosphatidylserine, phosphatidic acid, and several other phospholipids. The stability of the formed cytochrome c/phospholipid complex is determined by the presence of a negatively charged characteristic group and unsaturated fatty acid residues in phospholipid. In this study, NBD-PC fluorescent probe whose fluorescence intensity decreased in the presence of cytochrome c was used to determine the stability of the formed complexes. To quantify the interaction of cytochrome c with liposomal phospholipids, the titration curves of the fluorescent probe with cytochrome c were analyzed by the Langmuir method, which enabled the obtaining of the equilibrium dissociation constant (K_d) and the maximum normalized decrease in fluorescence intensity (B_max) proportional to the number of binding sites for cytochrome c with lipids. As a result of the conducted experiments, it was found that the force of cytochrome c interaction with liposomal membranes containing 10% cardiolipin was approximately twice as strong as that of liposomes containing 10% phosphatidic acid. When the content of anionic phospholipids was increased to 20%, the interaction with cardiolipin enhanced by a factor of 1.5, and that with phosphatidic acid by a factor of approximately 3 which, under those conditions, puts phosphatidic acid on the same level as cardiolipin. The obtained results indicate that phosphatidic acid, along with cardiolipin, may be involved in apoptotic processes.