Triton X-100 partitioning into sphingomyelin bilayers at subsolubilizing detergent concentrations: effect of lipid phase and a comparison with dipalmitoylphosphatidylcholine

Abstract

We examined the partitioning of the nonionic detergent Triton X-100 at subsolubilizing concentrations into bilayers of either egg sphingomyelin (SM), palmitoyl SM, or dipalmitoylphosphatidylcholine. SM is known to require less detergent than phosphatidylcholine to achieve the same extent of solubilization, and for all three phospholipids solubilization is temperature dependent. In addition, the three lipids exhibit a gel-fluid phase transition in the 38-41 degrees C temperature range. Experiments have been performed at Triton X-100 concentrations well below the critical micellar concentration, so that only detergent monomers have to be considered. Lipid/detergent mol ratios were never <10:1, thus ensuring that the solubilization stage was never reached. Isothermal titration calorimetry, DSC, and infrared, fluorescence, and (31)P-NMR spectroscopies were applied in the 5-55 degrees C temperature range. The results show that, irrespective of the chemical nature of the lipid, DeltaG degrees of partitioning remained in the range of -27 kJ/mol lipid in the gel phase and of -30 kJ/mol lipid in the fluid phase. This small difference cannot account for the observed phase-dependent differences in solubilization. Such virtually constant DeltaG degrees occurred as a result of the compensation of enthalpic and entropic components, which varied with both temperature and lipid composition. Consequently, the observed different susceptibilities to solubilization cannot be attributed to differential binding but to further events in the solubilization process, e.g., bilayer saturability by detergent or propensity to form lipid-detergent mixed micelles. The data here shed light on the relatively unexplored early stages of membrane solubilization and open new ways to understand the phenomenon of membrane resistance toward detergent solubilization.

FIGURE 1

DSC thermograms corresponding to the gel-fluid transition of phospholipid vesicles incubated with increasing amounts of Triton. (A) SM bilayers. (B) DPPC bilayers. When appropriate, lipid/detergent mol ratios are indicated for each thermogram. Third heating scan.

FIGURE 2

Temperature variation of the GP of the fluorescent probe Laurdan embedded in phospholipid bilayers. (A) SM bilayers. (B) DPPC bilayers. (•) Pure phospholipid. (○) Phospholipid/Triton (20:1 mol ratio). Average values of three independent experiments ± SE. The standard errors are smaller than the size of the symbols.

FIGURE 3

Comparison of representative ITC isotherms for the interaction of Triton with SM at different lipid phase states: (○) gel phase (10°C), (•) fluid phase (45°C), and (□) within the lipid phase transition (38°C).

FIGURE 4

Thermodynamic parameters for the interaction of Triton with SM vesicles studied as a function of temperature. (A) Partition constant, K. (B) Partition enthalpy ΔH. (C) Free energy and entropy change. Gel (g) and fluid (f) phases are indicated in the appropriate temperature ranges. Average values if three independent experiments ± SE. The standard errors are often smaller than the size of the symbols.

FIGURE 5

Thermodynamic parameters of the partition of Triton into DPPC membranes as a function of temperature. (A) Partition constant, K. (B) Partition enthalpy, ΔH. (C) Entropy and free energy change. Gel (g) and fluid (f) phases are indicated in the appropriate temperature ranges. Average values of three independent experiments ± SE. The standard errors are sometimes smaller than the size of the symbols.

FIGURE 6

Comparative values of TΔS and ΔG for the interaction of Triton with SM and DPPC membranes. (▪) SM. (•) DPPC. (○) pSM. Average values of three independent experiments. SM and DPPC data are redrawn from Figs. 4 and 5, except that error bars have been omitted for the sake of clarity. For pSM data, the standard error is smaller than the size of the symbols.