Membrane association and detachment of cytochrome c (cyt c) in millisecond to second time domain were investigated by stopped-flow fluorescence spectroscopy monitoring the efficiency of energy transfer from a pyrene-fatty acid containing phospholipid derivative, 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphoglyce rol (PPDPG, mole fraction X = 0.01) to the heme of the cyt c. Large unilamellar liposomes composed of egg phosphatidylcholine (eggPC) with varying content of the acidic phospholipid phosphatidylglycerol (eggPG) were employed. Unexpectedly, the rate of binding of cyt c to membranes was attenuated upon increasing the mole fraction of the acidic phospholipid (XPG). For example, at 50 microM phospholipid and 5 microM cyt c, when XPG was increased from 0.20 to 0.40 the half-time for the single-exponential decay in fluorescence increased from 4.7 to 8.6 ms. A similar observation was made for the membrane binding of another cationic protein, histone H1. We suggest that the formation of cooperative hydrogen-bonded networks by deprotonated and protonated PG in the vesicle surface retards the binding of cyt c to the liposome surface. However, once formed, the complex of cyt c with acidic phospholipids is stabilized by increasing XPG. Accordingly, significantly prolonged half-times of dissociation of cyt c from liposomes by NaCl, ATP, and different cationic proteins are measured upon increasing XPG. Differences between the latter cationic membrane binding ligands most likely reflect the varying relative contributions of hydrophobicity and Coulombic forces to their attachment to liposomes. Our data on the release and binding of cyt c to liposomes as a function of XPG and in the presence of ATP also provide the first direct experimental evidence for multiple lipid binding sites in cyt c.

An azobenzene containing a fatty acid derivative, 4-octyl-4′-(5-carboxypentamethyleneoxy)azobenzene (8A5), was incorporated into large unilamellar vesicles (LUVs) of L-R-dimyristoylphosphatidylcholine (DMPC), and its effects before and after photoisomerization (in trans and cis forms, respectively) on fast osmotic processes were observed by stopped-flow light scattering measurements. Differential scanning calorimetry (DSC) measurements revealed 8A5, in particular the cis form, to strongly perturb the membrane structure. More specifically, fast osmotic shrinkage of the LUVs was detected by the changes in the light scattering at 500 nm following the mixing of DMPC LUVs and an aqueous solution of betaine at high flow velocity. A rapid and novel type of the scattering signal was observed in the vicinity of the phase-transition temperature, suggesting the thermodynamic lateral reordering in the membrane due to the imposed osmotic pressure difference.