Electron spin resonance: spin labels.

Abstract

In general biological membranes possess no intrinsic paramagnetism and hence in the unlabeled state do not give rise to an electron spin resonance (ESR) spectrum. The introduction of a stable free radical (“spin label”) thus enables one to use ESR spectroscopy to study specific environments within the membrane. The spin label which is invariably used is the nitroxide radical, which has a three-line nitrogen hyperfine structure whose splitting varies with the orientation of the magnetic field relative to the nitroxide axes. It is this spectral anisotropy which has made spin label ESR such a powerful tool in the study of the molecular motions which are the characteristic feature of the highly dynamic structure of biological membranes. The nitroxide hyperfine splittings are partially averaged by the anisotropic motion, which gives a measure of the motional amplitude, and the linewidths are differentially broadened by an extent which depends on the rate of molecular motion. Other important features of the spin label spectra are the broadening by intermolecular label-label interactions and the ability to detect compartmentation of the label by quantitating spectral lineheights after selectively removing accessible spin-label signals by chemical reducing agents. Label-label interactions arise from two sources: the Heisenberg exchange interaction which is essentially a contact interaction and is therefore capable of measuring translational diffusion; and the dipole-dipole interaction which depends on the distance apart of the labels and is therefore capable of measuring intermolecular separations. Quantitation after treatment with reducing agents is chiefly concerned with the measurement of transport properties: of spin-label substrates or reducing agents, or the translocation of labelled lipid molecules.