Binding of cytochrome c to liposomes as revealed by the quenching of fluorescence from pyrene-labeled phospholipids.
Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring the decrease either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could be reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the absence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoylphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place.(ABSTRACT TRUNCATED AT 250 WORDS)
D003574:Cytochrome c Group / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008954:Models, Biological; D010712:Phosphatidic Acids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D003574:Cytochrome c Group; D008081:Liposomes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines