Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values.
The use of 90 degrees-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 approximately 0.2-0.3 G).
Livshits
V A
VA
Max-Planck-Institut für biophysikalische Chemie, Abt. Spektroskopie, 37077 Göttingen, Germany.
Marsh
D
D
D000042:Absorption; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006096:Gramicidin / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry*; D013113:Spin Labels
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D013113:Spin Labels; D006096:Gramicidin; C010942:1,2-distearoyllecithin; D004134:Dimyristoylphosphatidylcholine