Fluorescent probes of membrane surface properties

We have studied the properties of two new fluorescent probes, 7-dimethylaminocoumarin derivatives, 4-[N,N-dimethyl-N-(n-tetradecyl)ammoniummethyl]-7-(N,N- dimethylamino)coumarin chloride (TAMAC) and 4-(n-dodecylthiomethyl)-7-(N,N-dimethylamino)coumarin (DTMAC) in model membrane systems. Both probes are sensitive to solvent polarity. The TAMAC probe has a quaternary ammonium function to position it at a fixed location with respect to the membrane interface. In membranes of dipalmitoleoylphosphatidylethanolamine (DiPoPE), both probes detect marked increases in surface hydrophobicity as the bilayer to hexagonal phase transition temperature is approached. This does not occur when the probes are embedded in dipalmitoleoylphosphatidylcholine (DiPoPC) in which case the fluorescence emission is found to be largely independent of temperature. A nitroxide quencher covalently linked to the 5 position of the sn-2 acyl chain of phosphatidylcholine quenches the fluorescence of DTMAC in DiPoPC mon than in DiPoPE, indicating the deeper insertion of this probe in DiPoPC. As the temperature is increased the DTMAC fluorophore moves even further out of the membrane. These findings indicate that DTMAC, which does not contain a group to fix its location along the bilayer normal, adjusts its position to small changes in environment polarity, so as to maintain an environment of a fixed dielectric constant. However, with greater changes in membrane inter-facial polarity the environment of the probe will be altered. Thus, in addition to the sensitivity of these probes to solvent polarity, the ability of a fixed nitroxide to quench DTMAC becomes another parameter with which to characterize membrane properties with these probes.