QUANTITATIVE BINDING AND AGGREGATION OF R123 AND R6G RHODAMINES AT THE SURFACE OF DPPG AND DPPS PHOSPHOLIPID-VESICLES

The ability of host molecules, such as vesicle-forming phospholipids, to alter significantly the spectroscopic behaviour of guest molecules was used to study the microenvironment and binding properties of two important dyes. The interaction and aggregation processes which occur between cationic dyes of the rhodamine family (R123 and R6G) and negatively charged vesicles of DL-a-dipalmitoylphosphatidyl-L-serine (DPPS) and L-a- dipalmitoylphosphatidyl-DL-glycerol (DPPG) phospholipids were used as a convenient scheme for the investigation of the electrostatic stabilization of biosystems. Fluorescence was used as a monitor for the processes of binding and aggregation of the cationic ligands on the vesicles in aqueous solution, and the phenomena were treated quantitatively using a resolution procedure of complex fluorescence spectra. Organized macrosystems of DPPG and DPPS vesicles obtained by sonication presented distinctive numbers of sites (n) for interaction with R123 and R6G dyes. Values of n = 14 (R123) and n = 24 (R6G) were obtained by titration with DPPS vesicles, while two kinds of sites were found with DPPG vesicles. The first class of sites (n(a) = 53 (R123) and n(a) = 24 (R6G)) was determined in the presence of low concentrations of DPPG vesicles; the second class of sites (n(b) = 36 (R123) and n(b) = 6 (R6G)) was found for larger concentrations of DPPG vesicles, These interactions involve reversible binding of the dyes to the vesicles in a multiple equilibria process well described by the identical and independent site model, At high vesicle concentrations, the quantitative binding vs. aggregation of the rhodamines at the surface of the vesicles can thus be seen as a disaggregation process; the rationale for these results is that esterified rhodamines in these heterogeneous solutions seek out electrostatic attraction and binding on the anionic head groups of the lipids with a definite number of sites.