Raman spectroscopy characterization of interbilayer water of hydrated phospholipid multibilayers

The properties of phospholipid bilayers, which are important in various biophysical and biomedical studies, critically depend on the hydration of the lipid bilayer. Interbilayer water in multilamellar vesicles or planar multilayers is a very convenient object for studying the interfacial lipid-water interaction. However, many parameters of the interbilayer water remain incompletely studied, and in some cases different experimental methods yield different parameters of interbilayer water. Here, we developed a Raman spectroscopy method for characterizing interbilayer water in multilayer phospholipid samples. This method was applied to one saturated (DPPC) and one unsaturated (DOPC) phospholipid hydrated at high relative humidity and studied over a wide temperature range. It was found that although above the freezing point of water the OH stretching spectra of interbilayer water were similar to those of bulk water, only about one-fifth of the interbilayer water crystallized at the lowest experimental temperature (110 K). In combination with Raman spectra of aqueous suspensions of phospholipids of known compositions, the number of interbilayer H₂O molecules per lipid molecule (hydration number) was determined. The hydration number was found for the ordered (gel) and disordered (fluid) phases of hydrated phospholipid bilayers at different temperatures and several relative humidities. The results were compared with values of the hydration number obtained by other methods, and an interpretation was proposed that takes into account the fractions of the free and non-free (perturbed) interbilayer water.