Quantifying Solute Partitioning in Phosphatidylcholine Membranes

Abstract

Time-resolved fluorescence measurements were used to characterize and quantify solute partitioning into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid vesicles as a function of solute concentration and temperature. The solutes, coumarin 152 (C152) and coumarin 461 (C461), both belong to a family of 7-aminocoumarin dyes that have distinctive fluorescence lifetimes in different solvation environments. The two solutes differ in the 4-position where C152 has a trifluoromethyl group in place of C461's -CH group. In vesicle containing solutions, multiexponential fluorescence decays imply separate solute populations in the aqueous buffer, solvated in the vesicle headgroup region and solvated in the acyl chain bilayer interior, respectively. Fluorescence amplitudes, corrected for differences in radiative rates, are used to calculate absolute partition coefficients and average number of solutes per vesicle as a function of coumarin:lipid ratio and average number of solutes per vesicle. Results show that C152 has an ∼10-fold greater affinity than C461 for lipid bilayers, despite both solutes having similar hydrophobicities as inferred from their log(P) values. Temperature-dependent partitioning data are used to calculate enthalpies and entropies of C152 partitioning as a function of concentration. These values are used to extrapolate to the infinitely dilute limit. Above and below the lipid gel-liquid crystalline temperature, partitioning is exothermic with negative changes in entropy. In the vicinity of the transition temperature, these quantities change sign with ΔH becoming endothermic (+70 kJ/mol) and entropically favored (ΔS = +240 J/(mol·K)).

Coumarin Partitioning in Model Biological Membranes: Limitations of log P as a Predictor

“…Moreover, our results also identify where in the membrane solutes accumulate. This work extends the scope of previous studies that quantified the partitioning a 7-aminocoumarin, Coumarin 152 (or C152, 7-dimethylamino-4-(trifluoromethyl)­chromen-2-one) into lipid vesicle bilayers. − Specifically, the results presented below illustrate how small changes in solute structure can induce large changes in solute partitioning into biological membranes. Furthermore, the small differences in molecular architecture can create a large difference in the partitioning behavior which is not captured using a log P model.…”

“… a Lifetimes are ±0.2–0.3 ns. Numbers in parentheses next to lifetimes are amplitudes of that lifetime. b Quantum yields (ϕ r ) and radiative rates ( k r ) reported by Pal et al and Gobrogge et al − , c Quantum yields (ϕ r ) and radiative rates ( k r ) measured in this work. d Quantum yields (ϕ r ) and radiative rates ( k r ) reported by Pal et al e Fluorescence excitation and emission spectra for all three solutes in bulk solvents are shown in the supporting information (Figures SI-4–SI-6). How the quantum yields and radiative rates were calculated can be seen in the supporting information. …”

“…The solution was sonicated for 30 min at ∼50 °C. The solution was heated (50 °C) and passed through an Avanti Mini Extruder 10 times with a membrane pore size of 200 nm. − …”

“…The difference in excitation and emission wavelengths between the bulk solvent wavelengths used for C15X in vesicle solution did not change fluorescence lifetimes of that system. Additional details about this assembly can be found in refs , , , , .…”

“…It is worth noting the variability in log P values depends on how they are calculated and/or measured. , The molecular weight and hydrogen bonding opportunities also fall within acceptable Ro5 limits, implying that each solute should show a strong affinity for biological membranes in comparison to water. Previous studies have reported that C152 partitions primarily into the polar glycerol-backbone region of phosphatidylcholine (PC) bilayers, and that the partitioning behavior is strongly temperature dependent. − …”