le Maire M, Viel A, Møller J V
Laboratoire propre du Centre National de la Recherche Scientifique, Associé à l'Université Pierre et Marie Curie, Paris VI, Gif-sur-Yvette, France.
Anal Biochem. 1989 Feb 15;177(1):50-6. doi: 10.1016/0003-2697(89)90012-2.
We have tested the proposal (M. Potschka (1987) Anal. Biochem. 162, 47-64) that the elution position of macromolecules by gel chromatography is better correlated with the viscosity-based Stokes radius (R eta) than with the Stokes radius (RS) calculated from the frictional coefficient. By the use of different gel matrices (agarose, Sephadex, TSK silica gel columns) we found that the elution of dextran fractions and reduced proteins, denatured with guanidinium hydrochloride, agreed with their R eta, using water-soluble, globular proteins for gel calibration. However, the elution of large sodium dodecyl sulfate-protein complexes and other elongated macromolecules (fibrinogen, tropomyosin) was systematically retarded while polyethylene glycol and polyethylene glycol detergents eluted earlier than water-soluble, globular protein as a function of R eta. The same was the case for bacteriorhodopsin, solubilized by C12E8 or Triton X-100. It is concluded that for steric reasons size exclusion chromatography is more sensitive than hydrodynamic measurements to the detailed conformation of macromolecules (rods and random coils) and that for this reason gels with inert pores cannot be universally calibrated for all kinds of macromolecules.
我们已经验证了以下提议（M. Potschka (1987) Anal. Biochem. 162, 47 - 64）：凝胶色谱法中大分子的洗脱位置与基于粘度的斯托克斯半径（R eta）的相关性，比与根据摩擦系数计算出的斯托克斯半径（RS）的相关性更好。通过使用不同的凝胶基质（琼脂糖、葡聚糖凝胶、TSK硅胶柱），我们发现，使用水溶性球状蛋白进行凝胶校准时，葡聚糖级分以及用盐酸胍变性的还原蛋白的洗脱情况与其R eta相符。然而，大的十二烷基硫酸钠 - 蛋白质复合物和其他细长大分子（纤维蛋白原、原肌球蛋白）的洗脱被系统性延迟，而聚乙二醇和聚乙二醇去污剂根据R eta比水溶性球状蛋白洗脱得更早。用C12E8或Triton X - 100增溶的细菌视紫红质也是如此。得出的结论是，由于空间位阻原因，尺寸排阻色谱法比流体动力学测量对大分子（棒状和无规卷曲）的详细构象更敏感，因此具有惰性孔的凝胶不能对所有种类的大分子进行通用校准。
