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.
Belozersky Institute of Physico-Chemical Biology, Moscow 119991, Russia; Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119991, Russia.
Alexander M
AM
Firsov
Belozersky Institute of Physico-Chemical Biology, Moscow 119991, Russia.
Elena A
EA
Kotova
Belozersky Institute of Physico-Chemical Biology, Moscow 119991, Russia. Electronic address: antonen@genebee.msu.ru.
Yuri N
YN
Antonenko
D045304:Cytochromes c / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008081:Liposomes; D018384:Oxidative Stress; D010539:Permeability
D005452:Fluoresceins; D008081:Liposomes; C024098:6-carboxyfluorescein; D045304:Cytochromes c; D006861:Hydrogen Peroxide; C007740:fluorexon
Calcein leakage; Carboxyfluorescein oxidative damage; Cytochrome c; Fluorescence de-quenching; Liposome permeabilization; Peroxidase