Saturation-Recovery Electron Paramagnetic Resonance Discrimination by Oxygen Transport (DOT) Method for Characterizing Membrane Domains

The discrimination by oxygen transport (DOT) method is a dual-probe saturation-recovery electron paramagnetic resonance approachin which the observable parameter is the spin-lattice relaxation time (
T1
) of lipid spin labels, and the measured value is the bimolecular collision rate between molecular oxygen and the nitroxidemoiety of spin labels. This method has proven to be extremely sensitive to changes in the local oxygen diffusion-concentrationproduct (around the nitroxide moiety) because of the long
T1
of lipid spin labels (1–10 �s) and also because molecular oxygen is a unique probe molecule. Molecular oxygen is paramagnetic,small, and has the appropriate level of hydrophobicity that allows it to partition into various supramolecular structuressuch as different membrane domains. When located in two different membrane domains, the spin label alone most often cannotdifferentiate between these domains, giving very similar (indistinguishable) conventional electron paramagnetic resonancespectra and similar
T1
values. However, even small differences in lipid packing in these domains will affect oxygen partitioning and oxygen diffusion,which can be easily detected by observing the different
T1
s from spin labels in these two locations in the presence of molecular oxygen. The DOT method allows one not only to distinguishbetween the different domains, but also to obtain the value of the oxygen diffusion-concentration product in these domains,which is a useful physical characteristic of the organization of lipids in domains. Profiles of the oxygen diffusion-concentrationproduct (the oxygen transport parameter) in coexisting domains can be obtained
in situ
without the need for the physical separation of the two domains. Furthermore, under optimal conditions, the exchange rateof spin-labeled molecules between the two domains could be measured (
10
).