The effects of ²H₂O on the phase transition of large unilamellar vesicle (LUV) suspensions as detected by ultrasound spectroscopy and electron spin resonance

The importance of water in the molecular dynamics of large unilamellar vesicle (LUV) suspensions, in which increasing portions of the water were replaced by ²H₂O, was investigated. Determinations of the ultrasonic absorption coefficient per wavelength, αλ, were performed as a function of temperature and frequency for LUVs (LUVs: 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine, DPPC, and dipalmitoylphosphatidylglycerol, DPPG) in the vicinity of their phospholipid phase transition, using a double crystal acoustic interferometer. Electron spin resonance (ESR) and differential scanning calorimetry (DSC) were also employed to probe this system. When increasing portions of the aqueous content of the LUV suspensions were replaced by ²H₂O the phase transition temperature increased from 42.0°C to 42.9°C (indicating an increase in the activation energy of the transition), and the amplitude of αλ at the phase transition increased. However, αλ_max as a function of frequency at the phase transition did not change with the addition of ²H₂O, indicating that the relaxation time of the event responsible for the absorption of ultrasound was unaffected. The increase in the activation energy of the transition with the addition of ²H₂O suggested that the mobility of phospholipids near the membrane/aqueous interface was changed. Electron spin resonance (ESR) experiments on LUVs with nitroxide spin probes positioned at the membrane/aqueous interface (5-doxyl stearate and CAT₁₆) showed that LUVs in ²H₂O have a broader splitting, A_max, at the membrane/aqueous interface than do LUVs in H₂O. These results suggest that ²H₂O changes the mobility and/or structure of the phospholipids in the region of the membrane/aqueous interface. This difference in A_max was not seen for the probe PC-12-doxyl stearate, which resides at the C-12 position of the bilayer.