Winzor Donald J, Wills Peter R
Department of Biochemistry, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Anal Biochem. 2007 Sep 15;368(2):168-77. doi: 10.1016/j.ab.2007.04.030. Epub 2007 Apr 27.
Closer scrutiny has been accorded a recently reported procedure for characterizing weak protein dimerization by sedimentation equilibrium (INVEQ) in which the equilibrium distribution is analyzed as a dependence of radial distance on solute concentration rather than of solute concentration on radial distance. By demonstrating theoretically that the fundamental parameter derived from the analysis is simply the difference between the dimerization constant and the osmotic second virial coefficient for monomer-monomer interaction, this investigation refutes the original claim that independent estimates of these two parameters can be obtained by nonlinear curve fitting of the sedimentation equilibrium distribution. This criticism also applies to conventional analyses of sedimentation distributions by the commonly employed Beckman Origin and NONLIN software. Numerically simulated distributions are then analyzed to demonstrate limitations of the procedure and also to indicate a means of improving the reliability of the returned estimate of the dimerization constant. These features are illustrated by applying the original and revised analytical procedures to a sedimentation equilibrium distribution for alpha-chymotrypsin (pH 4.0, I 0.05 M).
最近报道了一种通过沉降平衡（INVEQ）表征弱蛋白二聚化的方法，该方法受到了更仔细的审查。在这种方法中，平衡分布被分析为径向距离对溶质浓度的依赖关系，而不是溶质浓度对径向距离的依赖关系。通过理论证明，从分析中得出的基本参数仅仅是二聚化常数与单体 - 单体相互作用的渗透第二维里系数之间的差值，这项研究驳斥了最初的说法，即可以通过沉降平衡分布的非线性曲线拟合获得这两个参数的独立估计值。这种批评也适用于使用常用的贝克曼Origin和NONLIN软件对沉降分布进行的传统分析。然后对数值模拟分布进行分析，以证明该方法的局限性，并指出一种提高返回的二聚化常数估计值可靠性的方法。通过将原始和修订的分析程序应用于α - 胰凝乳蛋白酶（pH 4.0，I 0.05 M）的沉降平衡分布来说明这些特征。
