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

¹ Unidad de Biofísica

^* To whom correspondence should be addressed. E-mail: felix.goni{at}ehu.es.

Submitted on February 8, 2007
Revised on March 13, 2007
Accepted on 16 July 2007

	Abstract

We have examined the partitioning of the non-ionic detergent Triton X-100 at sub-solubilizing 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° 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, differential scanning calorimetry, infrared, fluorescence and ³¹P-NMR spectroscopies were applied in the 5-55° C temperature range. The results show that, irrespective of the chemical nature of the lipid, G° 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 Gº occurred as a result of the compensation of enthalpic and entropic components, which did vary 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 present data shed light on the relatively unexplored early stages of membrane solubilization and open new ways to understand the phenomenon of membrane resistance towards detergent solubilization.

Key Words: ITC, detergent partitioning into bilayers, lipid bilayer phases, membrane detergent solubilization, membrane domains, sphingomyelin