Ultra-stable temperature control in EPR experiments: thermodynamics of gel-to-liquid phase transition in spin-labeled phospholipid bilayers and bilayer perturbations by spin labels.

An ultra-stable variable temperature accessory for EPR experiments with biological samples has been designed and tested. The accessory is comprised from a digitally controlled circulator bath that pumps fluid through high-efficiency aluminum radiators attached to an EPR resonator of a commercial X-band EPR spectrometer. Temperature stability of this new accessory after a 15 min re-equilibration is at least +/-0.007 K. For a standard 1-cm-long capillary sample arranged inside an EPR tube filled with silicon oil, the temperature variations do not exceed +/-0.033 K over the sample temperature range from 283 to 333 K. This new accessory has been tested by carrying out a comparative spin-labeling EPR and differential scanning calorimetry (DSC) study of the gel-to-liquid phase transition in multilamellar vesicles (MLV) composed of a synthetic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We demonstrate that the gel-to-liquid phase transition temperatures of MLV DMPC measured by EPR and DSC agree within +/-0.02 K experimental error even though the sample for EPR study was labeled with 1 mol% of 5PC (1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3 phosphocholine). Cooperative unit number measured by EPR, N=676+/-36, was almost 50% higher than that obtained from DSC (N=458+/-18). These high values of N indicate that (i) the lipid domains should include at least several spin-labeled lipid molecules and (ii) the spin-probe 5PC molecules are not excluded into domains that are different from the bulk lipid phase as was speculated earlier. Overall, our data provide DSC and EPR evidence that in studies of the gel-to-liquid phase transition, the effect of bilayer perturbation by spin-labeled lipids is negligible and therefore thermodynamic parameters of the phase transition can be accurately measured by spin-labeling EPR. This might serve as an indication when spin-labeled molecules with structures similar to those of lipids are introduced at low concentrations, they are easily accommodated by fluid phospholipid bilayers without significant losses of the lipid cooperativity.