Membrane/Water Partition Coefficients of Bile Salts Determined Using Laurdan as a Fluorescent Probe

Abstract

The interaction of liposomal membranes composed of soybean phosphatidylcholine with the bile salts (BSs) cholate (Ch), glycocholate (GC), chenodeoxycholate (CDC), and glycochenodeoxycholate (GCDC) was studied. The BSs differed with regard to their lipophilicity, pKa values, and the size of their hydrophilic moiety. Their membrane interactions were investigated using Laurdan as a membrane-anchored fluorescent dye. The apparent membrane/water partition coefficient, D, at pH 7.4 was calculated from binding plots and compared with direct binding measurements using ultracentrifugation as a reference. The Laurdan-derived LogD values at pH 7.4 were found to be 2.10 and 2.25 for the trihydroxy BSs, i.e., Ch and GC, and 2.85 and 2.75 for the dihydroxy BSs, i.e., CDC and GCDC, respectively. For the membrane-associated glycine-conjugated GC and GCDC (pK_a values of ∼3.9), no differences in the Laurdan spectra of the respective BS were found at pH 6.8, 7.4, and 8.2. Unconjugated Ch and CDC (pK_a values of ∼5.0) showed pronounced differences at the three pH values. Furthermore, the kinetics of membrane adsorption and transbilayer movement differed between conjugated and unconjugated BSs as determined with Laurdan-labeled liposomes.

Figure 1

Chemical structure and pK_a values of the BSs used in this work. The trihydroxy compounds Ch and GC are more hydrophilic than the dihydroxy compounds CDC and GCDC. The pK_a values depend only on conjugation.

Figure 2

Binding diagram of GCDC at pH 7.4, 25°C, measured by separating free and liposome-associated BS by ultracentrifugation and equilibrium dialysis. (a) Initial section from which the binding parameters were determined. (b) Linear section from which membrane partitioning was determined (R = 0.9904). (c) Coexistence of membranes and mixed micelles.

Figure 3

Emission spectra of Laurdan-labeled SPC liposomes (0.24 mM lipid) upon increasing CDC concentrations below and above the point of onset of membrane solubilization between 1.4 and 2.0 mM CDC (pH 7.4, 25°C).

Figure 4

Calculated MSP_std values versus total CDC concentration (pH 7.4, 25°C). The vertical dotted lines show an MSP_Std of 0.8 with the corresponding total CDC concentration for lipid concentrations of 0.24, 0.69, and 0.92 mM.

Figure 5

Data evaluation for defined MSP_std values, reflecting identical BS/lipid ratios in the membranes. According to Eq. 7, the free CDC is given by the intercept at the y axis (total CDC) and the effective ratio R_e is given by the slope (pH 7.4, 25°C).

Figure 6

Binding diagram of CDC at pH 7.4, determined with Laurdan-containing liposomes. The slope at each BS concentration is the lipid-standardized partition coefficient D_L of the detergent between the membrane and buffer. (a) Initial section with very low BS bound to membrane. (b) Linear section from which membrane partitioning was determined (R = 0.9718). (c) Section of solubilization of membrane lipids to mixed micelles, as determined by the ultracentrifugation method.

Figure 7

MSP_std versus BS concentration at different pH values. (A) CDC. (B) GCDC. Lipid concentration of 0.25 mM, at 25°C.

Figure 8

Binding diagrams of CDC at different pH values over a wide concentration range (at 25°C). Arrows: onset of membrane solubilization determined via scattered light measurements. Inset: enlarged version of the linear parts of the binding plots at different pH values, from which the apparent partition coefficients were calculated.

Figure 9

Time-dependent changes of the MSP_std of Laurdan-containing liposomes (1 mM lipid) at different BS concentrations (mM). (A) GCDC. (B) CDC. pH 7.4, 25°C.

Figure 10

Time-dependent changes of the MSP_std of Laurdan-containing liposomes (1 mM lipid) at different pH values and 25°C in the presence of 0.80 mM CDC. The data represent 20 single measurements per minute of MSP_std.