Endre Z H, Kuchel P W
Biophys Chem. 1986 Aug;24(3):337-56. doi: 10.1016/0301-4622(86)85039-6.
Metabolically active human erythrocytes were incubated with [alpha-13C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using 13C-NMR in which the longitudinal relaxation times (T1) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. The T1 values of labelled glycine were also determined in various-concentration solutions of bovine serum albumin and glycerol and also of the natural abundance 13C of glycerol in glycerol solutions. From the T1 estimates the rotational correlation time (tau r) was calculated using a formula based on a model of an isotropic spherical rotor or that of a symmetrical ellipsoidal rotor; for glycine the differences in estimates of tau r obtained using the two models were not significant. From the correlation times and by use of the Stokes-Einstein equations viscosity and translational diffusion coefficients were calculated; thus comment can be made on the likelihood of diffusion control of certain enzyme-catalysed reactions in the erythrocyte. Bulk viscosities of the erythrocyte cytoplasm and the above-mentioned solutions were measured using Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T1 measurements. We derived an equation from the theory of the viscosity of concentrated solutions which contains two phenomenological interaction parameters, a 'shape' factor and a 'volume' factor; it was fitted to data relating to the concentration dependence of viscosity measured by both methods. We showed, by using the equation and interaction-parameter estimates for a particular probe molecule in a particular solution, that it was possible to correlate NMR viscosity and bulk viscosity; in other words, given an estimate of the bulk viscosity, it was possible to calculate the NMR 'micro' viscosity or vice versa. However, the values of the interaction parameters depend upon the relative sizes of the probe and solute molecules and must be separately determined for each probe-solute-solvent system. Under various conditions of extracellular osmotic pressure, erythrocytes change volume and thus the viscosity of the intracellular milieu is altered. The volume changes resulted in changes in the T1 of [alpha-13C]glycine. Conversely, we showed that alterations in T1, when appropriately calibrated, could be used for monitoring changes in volume of metabolically active cells.
将具有代谢活性的人红细胞与[α-13C]甘氨酸一起孵育，这导致细胞内谷胱甘肽的特定富集。然后使用13C-NMR对细胞进行研究，测量游离甘氨酸和谷胱甘肽的纵向弛豫时间（T1）和核Overhauser增强。还在牛血清白蛋白和甘油的各种浓度溶液中以及甘油溶液中天然丰度的13C的甘油中测定了标记甘氨酸的T1值。根据T1估计值，使用基于各向同性球形转子模型或对称椭球形转子模型的公式计算旋转相关时间（τr）；对于甘氨酸，使用这两种模型获得的τr估计值差异不显著。根据相关时间并使用斯托克斯-爱因斯坦方程计算粘度和平动扩散系数；因此，可以对红细胞中某些酶催化反应的扩散控制可能性进行评论。使用奥氏毛细管粘度计测量红细胞细胞质和上述溶液的体相粘度。后一种粘度估计值与基于NMR-T1测量的粘度估计值之间存在很大差异。我们从浓溶液粘度理论中推导了一个方程，该方程包含两个唯象相互作用参数，一个“形状”因子和一个“体积”因子；它被拟合到与两种方法测量的粘度浓度依赖性相关的数据上。我们表明，通过使用该方程和特定溶液中特定探针分子的相互作用参数估计值，可以将NMR粘度和体相粘度相关联；换句话说，给定体相粘度的估计值，可以计算NMR“微观”粘度，反之亦然。然而，相互作用参数的值取决于探针和溶质分子的相对大小，并且必须针对每个探针-溶质-溶剂系统分别确定。在细胞外渗透压的各种条件下，红细胞会改变体积，因此细胞内环境的粘度也会改变。体积变化导致[α-13C]甘氨酸的T1发生变化。相反，我们表明，当进行适当校准时，T1的变化可用于监测代谢活跃细胞的体积变化。
