The use of high-resolution solid-state NMR spectroscopy and differential scanning calorimetry to study interactions of anaesthetic steroids with membrane

Abstract

We have used a combination of high-resolution solid-state 13C-NMR and DSC (differential scanning calorimetry) to study the distinctively different thermotropic and dynamic properties of the anaesthetic steroid alphaxalone and its inactive congener delta16-alphaxalone in dipalmitoylphosphatidylcholine (DPPC) model membranes. In the solid-state 13C-NMR, the techniques included cross polarization (CP) and/or magic angle spinning (MAS). The observed data revealed the following important results. (a) DSC as a bulk method showed that the active steroid lowers the main phase transition temperature and broadens the pretransition more significantly than the inactive congener. The 13C-CP/MAS experiments allowed us to detect the pretransition temperature in the alphaxalone-containing preparation, which was not discernible in DSC. (b) The chemical shift values varied with temperature, indicating different degrees of trans-gauche isomerization in the lipid acyl chains when the bilayer is in the liquid crystalline phase. (c) Only specific additional peaks appeared in the 13C-CP/MAS spectra when each of the steroids was present in the preparation. delta16-alphaxalone gives rise to more additional peaks than alphaxalone, indicating a different mobility of the corresponding molecular moiety in the phospholipid bilayer environment. (d) The relative intensities of these peaks also confirmed that alphaxalone is fully incorporated in the bilayer, whereas delta16-alphaxalone is only partially so. These results suggest that the differential effects of these two analogues in the membrane may, at least in part, explain the reason for their different biological activities.