ESEEM Measurements of Local Water Concentration in D2O-Containing Spin-Labeled Systems

To calibrate electron spin echo envelope modulation (ESEEM) amplitudes with respect to the deuterium water content in spin-labeled biological systems, ESEEM of nitroxide TEMPO has been studied in frozen glassy D2O-dimethylsulfoxide mixtures of different composition. The interaction between the unpaired electron of nitroxide and the deuterium nuclei manifests itself in a cosine Fourier transform spectrum as the sum of a narrow line with the doublet quadrupole splitting and of a broad one. The narrow line arises from interaction with distant deuterium nuclei, the broad one arises from interaction with nearby nuclei belonging to nitroxide-water molecule complexes. The dependence on water concentration was found to be nonlinear for the intensity of the narrow line and close to linear for the intensity of the quadrupole doublet. Therefore, the intensity of the quadrupole doublet is suggested as a measure of concentration of free water around a spin label in biological objects. Fourier transform line shape was theoretically simulated for different model distributions of water molecules around the spin label. Simulations confirm the linear dependence of the quadrupole doublet intensity on water concentration seen in the experiment. The suggested approach was applied to analyze data for spin-labeled dipalmitoylphosphatidylcholine (DPPC) and DPPC-cholesterol D2O-hydrated model membranes. The concentration of free water near the spin-labeled fourth carbon atom along the lipid chain was estimated as 5.2 and 7.2 M for DPPC and DPPC-cholesterol membranes, respectively.