Kunji Edmund R S, Harding Marilyn, Butler P Jonathan G, Akamine Pearl
The Medical Research Council, Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 0XY, UK.
Methods. 2008 Oct;46(2):62-72. doi: 10.1016/j.ymeth.2008.10.020. Epub 2008 Oct 24.
Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.
尺寸排阻色谱法是一种用于测定蛋白质或蛋白质复合物流体力学体积的成熟技术。当应用于膜蛋白时，为了获得蛋白质的准确测量值，需要确定保持蛋白质在水相中可溶所需的去污剂胶束的贡献。在一系列去污剂中，这些去污剂仅在烷基链长度上有所不同，去污剂胶束对流体力学体积的贡献是可变的，而蛋白质的贡献是恒定的。通过使用这种方法，可以通过外推法估计膜蛋白的几个参数，例如膜中点处的半径、平均半径、斯托克斯半径和排阻体积。蛋白质的分子量可以通过尺寸排阻色谱过程中游离去污剂胶束和蛋白质-去污剂胶束的行为以及去污剂-蛋白质比率的测定所产生的两种独立测量来确定。确定蛋白质-去污剂胶束的尺寸可以通过定义蛋白质表面的可及性来促进膜蛋白的纯化和结晶。
