The interaction of potential-sensitive molecular probes with dimyristoylphosphatidylcholine vesicles investigated by 31P-NMR and electron microscopy.

The effect of a number of commonly employed potential-sensitive molecular probes on the 31P-NMR properties of dimyristoylphosphatidylcholine vesicles at two field strengths has been investigated in order to obtain information on the location and effect of these probes on the membrane bilayer. In comparison to the control dye-free vesicle spectrum, the probes diS-C3-(5) and diS-C4-(5), when added to a vesicle suspension, cause a substantial broadening of the 31P resonance with no detectable chemical shift within an uncertainty of +/- 0.05 ppm at 24 MHz. The spin-lattice and spin-spin relaxation times are also reduced when the cyanines are present by well over 20% relative to those of the control vesicle preparation. The addition of anionic probes, including several oxonol derivatives and merocyanine 540, causes no chemical shift, line broadening, or changes in the relaxation times. Possible explanations for the failure of the anionic probes to alter the vesicle 31P-NMR properties include charge repulsion between these dyes and the phosphate group that prevents the probes from penetrating the bilayer to a depth sufficient to alter the local motion of the phosphate moiety. The 31P resonance broadening and reduction in the relaxation times caused by the two cyanines is at least in part due to an increase in vesicle size as judged by electron microscopy measurements, although an inhibition of the local phosphate motion as well cannot be completely eliminated. The cyanine-mediated increase in vesicle size appears to be due to an irreversible vesicle-fusion process possibly initiated by the screening of surface charge by these probes. The implications of these observations in relation to functional energy-transducing preparations is discussed.