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

Abstract

The use of 90°-out-of-phase first-harmonic absorption ( V 1 ′ - ) EPR to resolve the spectra from nitroxide spin labels with differing T 1-relaxation times is described. Non-linear V 1 ′ - EPR spectra recorded under moderate saturation have sharper lines compared with the in-phase V 1-EPR spectra, and amplitudes that preferentially enhance components with longer T 1-relaxation. Discrimination between V 1 ′ - 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 V 1 ′ - EPR spectroscopy is obtained at relatively low Zeeman modulation frequencies (20–30 kHz) and moderate saturation ( H 1 ∼ 0.2–0.3 G).