Alaouie Ali M, Smirnov Alex I
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Box 8204, Raleigh, NC 27695-8204, USA.
J Magn Reson. 2006 Oct;182(2):229-38. doi: 10.1016/j.jmr.2006.07.002. Epub 2006 Jul 21.
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.
设计并测试了一种用于生物样品电子顺磁共振（EPR）实验的超稳定可变温度附件。该附件由一个数控循环水浴组成，水浴将流体泵入连接到商用X波段EPR光谱仪的EPR谐振器上的高效铝散热器。重新平衡15分钟后，这个新附件的温度稳定性至少为±0.007K。对于放置在充满硅油的EPR管内的标准1厘米长毛细管样品，在283至333K的样品温度范围内，温度变化不超过±0.033K。通过对由合成磷脂1,2 - 二肉豆蔻酰 - sn - 甘油 - 3 - 磷脂酰胆碱（DMPC）组成的多层囊泡（MLV）的凝胶 - 液相转变进行比较自旋标记EPR和差示扫描量热法（DSC）研究，对这个新附件进行了测试。我们证明，通过EPR和DSC测量的MLV DMPC的凝胶 - 液相转变温度在±0.02K的实验误差范围内一致，尽管用于EPR研究的样品用1mol%的5PC（1 - 棕榈酰 - 2 - 硬脂酰 -（5 - 氧基）- sn - 甘油 - 3 - 磷酸胆碱）进行了标记。通过EPR测量的协同单元数N = 676±36，比从DSC获得的值（N = 458±18）高出近50%。这些高N值表明：（i）脂质域应至少包括几个自旋标记的脂质分子；（ii）自旋探针5PC分子不会像之前推测的那样被排除在与主体脂质相不同的域中。总体而言，我们的数据提供了DSC和EPR证据，表明在凝胶 - 液相转变研究中，自旋标记脂质对双层的扰动作用可忽略不计，因此可以通过自旋标记EPR准确测量相变的热力学参数。这可能表明，当以低浓度引入结构与脂质相似的自旋标记分子时，它们很容易被流体磷脂双层容纳，而脂质协同性不会有显著损失。
