Calcein leakage as a robust assay for cytochrome c/H₂O₂-mediated liposome permeabilization

Membrane-permeabilizing activity of cytochrome c (cyt c) in the presence of hydrogen peroxide associated with its functioning as peroxidase is considered relevant to initiation of the mitochondrial pathway of apoptosis. Here, we present evidence that the choice of a fluorescent dye for measuring cyt c/H₂O₂-induced dye leakage from liposomes by fluorescence de-quenching is of major importance. The popular fluorescent marker 5(6)-carboxyfluorescein appeared highly susceptible to cyt c-mediated peroxidative destruction and therefore unsuitable for the leakage assay with cyt c/H₂O₂. On the contrary, calcein, another conventional marker, proved resistant to oxidative stress and thus perfectly suitable for the assay. Based on the concentration dependences of the cyt c/H₂O₂-induced calcein leakage, the optimal conditions for the assay were found.