THE USE OF GEL CHROMATOGRAPHY FOR THE DETERMINATION OF SIZES AND RELATIVE MOLECULAR MASSES OF PROTEINS - INTERPRETATION OF CALIBRATIONS IN TERMS OF GEL-PORE-SIZE DISTRIBUTION
THE USE OF GEL CHROMATOGRAPHY FOR THE DETERMINATION OF SIZES AND RELATIVE MOLECULAR MASSES OF PROTEINS - INTERPRETATION OF CALIBRATIONS IN TERMS OF GEL-PORE-SIZE DISTRIBUTION
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DOI：
10.1042/bj2430399
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发表时间：
1987-04-15
影响因子：
4.1
通讯作者：
AGGERBECK, LP
中科院分区：
文献类型：
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作者：
LEMAIRE, M;GHAZI, A;AGGERBECK, LP
用凝胶排阻色谱法分离蛋白质的原理，可以解释为凝胶中大分子的分布是由不同半径的孔决定的。孔径分布为高斯分布的假设导致预测蛋白质的分子斯托克斯半径（RS）与函数erf-1（1-KD）之间的线性关系，其中KD是分配系数[Ackers（1967）J.Biol.Chem.242，3237-3238]。由于经典（琼脂糖和葡聚糖）凝胶和h.p.l.c.凝胶已经表明，这种线性关系在很宽的天然蛋白质大小范围内没有得到实验验证，我们重新研究了Ackers模型（上述参考文献）。我们表明，Ackers的（上述参考）推导是无效的，除了一个特定的高斯分布的孔径集中在原点。放宽这一限制以允许其他类型的高斯分布不能解释我们获得的非线性校准曲线。相反，我们表明，孔径分布可以从实验确定的函数KD = f（RS）计算，这种分布是双峰（非高斯）。一个分布集中在2 nm以下，而第二个分布的平均值约为6-8 nm。对于某些凝胶，该双峰分布中的最小值对应于分辨率差的区域，这需要被理解为在分子大小的测定中正确使用凝胶色谱法。
The separation of proteins by gel-exclusion chromatography has been explained in terms of partitioning of the macromolecules within the gel by a distribution of pores of various radii. The assumption that the distribution of pore sizes is Gaussian has led to the prediction of a linear relationship between the molecular Stokes radius (RS) of the protein and the function erf-1 (1-KD), where KD is the partition coefficient [Ackers (1967) J. Biol. Chem. 242, 3237-3238]. Since careful calibrations of classical (agarose and dextran) gels and h.p.l.c. gels have shown that such a linear relationship is not verified experimentally over a wide range of native protein sizes, we have reinvestigated the model of Ackers (above reference). We show that Ackers' (above reference) derivation is not valid except for a particular Gaussian distribution of pore sizes centred at the origin. Relaxation of this restriction to allow for other types of Gaussian distributions cannot account for the non-linear calibration curves that we have obtained. Instead we show that the pore-size distribution can be calculated from the experimentally determined function KD = f(RS) and that this distribution is bimodal (non-Gaussian). One distribution is centred below 2 nm, whereas the mean value of the second one is around 6-8 nm. The minimum in this bimodal distribution corresponds, for some gels, to a region of poor resolution, which needs to be appreciated for the proper use of gel chromatography in the determination of molecular size.