Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume.
Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume.
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DOI：
10.1016/s0021-9258(18)43240-1
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发表时间：
1981-12
期刊：
影响因子：
--
通讯作者：
D. Atha;K. Ingham
中科院分区：
文献类型：
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作者：
D. Atha;K. Ingham
在不同大小的聚乙二醇（PEG）存在下测量各种蛋白质（14，000至670，000道尔顿）的表观溶解度。通过测量离心混合物上清液中的蛋白质浓度确定的所有溶解度曲线均表现出log S（g/升）对PEG浓度（%，w/v）的特征性线性依赖性。对于pH 4.5的PEG-4000中的人血白蛋白，即使沉淀相的外观从粘性流体变为白色无定形固体，该线性仍扩展至1，000倍的溶解度范围。随着PEG的Mr从20，000降低到400，斜率β从0.27降低到0.09，但对溶液条件（pH、T、盐）的变化不敏感，表明蛋白质和聚合物之间不存在特定的化学相互作用。这一结论得到以下观察结果的支持，即PEG浓度高达30%（w/v）对核糖核酸酶A的解链温度没有显著影响。此外，平衡透析测量，以及各种光谱测量，没有提供这种相互作用的证据。使用空间排阻模型（Edmond，E.，和Ogston，A. G.（1968）Biochem.J.109，569-576），并且假设固相的化学势是恒定的，β可以与使用PEG（r2）和蛋白质（r3）的等效球半径从共体积计算的相互作用系数相关。尽管PEG-20，000中的白蛋白获得了良好的一致性，但预测的β对r3的依赖性大于观察到的，预测的β对r2的依赖性与观察到的方向相反。然而，从白蛋白和PEG-1000的平衡透析测量确定的相互作用系数与预测值一致。因此，白蛋白对低浓度PEG的排斥可以通过简单的排斥体积模型来解释，而PEG对溶液中蛋白质的排斥似乎更为复杂。
The apparent solubilities of various proteins (14,000 to 670,000 daltons) were measured in the presence of polyethylene glycols (PEGs) of different sizes. All of the solubility curves, determined by measuring the protein concentration in the supernate of centrifuged mixtures, exhibited the characteristic linear dependence of log S (g/liter) on PEG concentration (%, w/v). For human albumin in PEG-4000 at pH 4.5, this linearity extended over a 1,000-fold range of solubility, even though the appearance of the sedimented phase changed from a viscous fluid to a white amorphous solid. The slope, beta, decreased from 0.27 to 0.09 with decreasing Mr of PEG from 20,000 to 400, but was insensitive to changes in solution conditions (pH, T, salts), suggesting the absence of specific chemical interactions between protein and polymer. This conclusion was supported by the observation that concentrations of PEG up to 30% (w/v) had no significant effect on the melting temperature of ribonuclease A. Furthermore, equilibrium dialysis measurements, as well as various spectral measurements, provided no evidence for such interactions. Using a steric exclusion model (Edmond, E., and Ogston, A. G. (1968) Biochem. J. 109, 569-576) and assuming that the chemical potential of the solid phase is constant, beta can be related to interaction coefficients calculated from co-volumes using the equivalent sphere radii of PEG (r2) and protein (r3). Although good agreement was obtained for albumin in PEG-20,000, the predicted dependence of beta on r3 was greater than observed and the predicted dependence of beta on r2 was of opposite direction to that observed. However, the interaction coefficient determined from the equilibrium dialysis measurements of albumin and PEG-1000 agreed with the predicted value. Thus, the exclusion of low concentrations of PEG by albumin can be explained by a simple excluded volume model, whereas the exclusion of protein out of solution by PEG appears to be more complex.