Fluorescence Probing of Cell Membranes with Azacrown Substituted Ketocyanine Dyes

The three dyes with similar fluorescence properties but different lipophility—azacrown- and di-methylamino-substituted ketocyanines—are proposed as probes for the studies of biomembrane structure and dynamics. Their attractive feature is an extremely strong solvatofluorochromism, covering the range from 470 to 650 nm. Two photophysical mechanisms are responsible for these features, the general polarity effect associated with substantial increase of the probe dipole moment on electronic excitation and the excited-state stabilization due to hydrogen bonding to the central carbonyl groups. On the binding of these probes with erythrocyte membranes, three components in fluorescence spectra are resolved. They are attributed to probe molecules bound in two discrete types of binding sites inside the membrane: hydrophilic polar sites, in which carbonyl groups of the probes molecules are hydrogenbonded with hydrogen donor surrounding, and hydrophobic non-polar sites. The third component present in the emission spectra was attributed to the unbound probe in the near-membrane aqueous phase. Our results suggest that such a complex response of the probes is sensitive to the dynamics of hydration of the membrane interior, and this feature can be easily studied in simple ratiometric measurements. Among three studied compounds containing two crown-, two dimethylamino-, or both crown- and diethylamino- substituents, the latter proves to be most prospective in biomembrane research. This probe was tested in monitoring the phase transition of human erythrocyte membrane.