title: Estimation of spin probe clustering in biological membranes.
abstract: An iterative spectral subtraction technique has been developed which accurately estimates the proportion of 'dilute' and 'clustered' I(12, 3) (i.e., 5-nitroxide stearate) in human erythrocyte ghosts at 37 degrees C, even if subtractant spectra free from probe-probe interactions cannot be measured due to technical limitations. Gordon et al. ((1985) J. Membrane Biol. 84, 81-95) earlier showed that I(12, 3) occupies a class of high-affinity sites in ghosts at probe/total lipid ratios (P/L) less than 1/2250. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membrane-bound clusters of variable size. Although this model allows determination of the dilute/clustered probe ratio, it requires subtraction of experimental spectra with a 'magnetically dilute' spectrum obtained using P/L less than 1/4600. The new methodology accurately profiles the % probe clustering in human erythrocyte ghosts over the entire P/L range, even if the lowest P/L for the subtractant spectrum contains substantial probe-probe interactions (i.e., P/L of 1/604 or 1/303). Application of either the subtraction technique in Gordon et al. (1985) or the iterative subtraction protocol described here should allow determination of probe clustering in a wide range of I(12, 3)-labeled biological membranes.
mesh_terms: D003497:Cyclic N-Oxides / Q000097:blood*; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000502:physiology*; D006801:Humans; D008560:Membrane Fluidity; D008954:Models, Biological; D013113:Spin Labels
chemical_list: D003497:Cyclic N-Oxides; D013113:Spin Labels; C005072:5-doxylstearic acid