Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values

The use of 90°-out-of-phase first-harmonic absorption (V1′-) EPR to resolve the spectra from nitroxide spin labels with differing T1-relaxation times is described. Non-linear V1′-EPR spectra recorded under moderate saturation have sharper lines compared with the in-phase V1-EPR spectra, and amplitudes that preferentially enhance components with longer T1-relaxation. Discrimination between V1′-spectral components can be increased further by means of selective paramagnetic relaxation enhancement agents. Examples are given of biophysical applications to double labelling in single-component membranes and phase separation in two-component membranes, to lipid-peptide complexes, and to binding of spin-labelled reagents. It is concluded that optimal resolution in V1′-EPR spectroscopy is obtained at relatively low Zeeman modulation frequencies (20-30 kHz) and moderate saturation (H1 ∼ 0.2-0.3 G).