Segregation of anionic lipophiles in bilayers monitored by binding of cationic dye NK-529.

Fluorescence emission properties of a cationic indodicarbocyanine dye, NK-529, bound to anionic and zwitterionic vesicles, are examined under a variety of conditions to monitor lateral distribution of anionic amphiphiles in bilayers as a function of their phase properties. The change in the fluorescence properties of NK-529 arises from the binding of the dye to the bilayer that is dominated by ionic interactions when possible, as well as from the self-quenching of the dye bound to bilayers when the surface density of the dye is high. The binding affinity of the dye to anionic interfaces is more than 100-fold higher compared to that in zwitterionic bilayers. The limiting phospholipid/dye ratio in anionic bilayers at low vesicle concentrations is about 3. Thus the density of the bound dye in anionic bilayers can be more than 40-fold higher than that in zwitterionic bilayers, and therefore under such conditions the bound dye is completely self-quenched in vesicles or micelles of anionic phospholipids. The change in the fluorescence emission intensity on incorporation of anionic amphiphiles in zwitterionic bilayers is used to monitor segregation of the anionic amphiphiles. The organizational features of bilayers that cause a change in the fluorescence properties of bound NK-529 show that the lateral distribution of anionic amphiphiles is appreciably influenced not only by the mole fraction of the amphiphile but also in the presence of other additives, and by the gel-fluid thermotropic transition. As shown in the following paper, the fluorescence changes related to self-quenching in anionic bilayers containing NK-529 can be used to understand the organizational changes that occur during the course of interfacial catalysis by phospholipase A2 on zwitterionic bilayers.