Behlke J, Ristau O
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Biophys Chem. 2000 Sep 15;87(1):1-13. doi: 10.1016/s0301-4622(00)00173-3.
Analysis of protein-protein interactions in highly concentrated solutions requires a consideration of the non-ideality in such solutions which is expressed by the virial coefficients. Different equations are presented to estimate effects of the thermodynamic non-ideality on the macromolecular interaction of self-associating proteins in sedimentation equilibrium experiments. Usually the influence of thermodynamic non-ideal behavior are described by concentration power series. The convergence of such power series is limited at high solute concentration. When expressing the thermodynamic non-ideality by an activity power series this disadvantage can be minimized. The developed centrifuge equations are the basis for a global analysis to estimate equilibrium constants and the corresponding thermodynamic activities of the reactants. Based on fit analysis of synthetic concentration profiles it was established that marked deviations from the expected association constants are observed for proteins with strong association forces between solute molecules. Considerable differences were also observed in weakly interacting systems. This was due to the excluded volume of the protein which is similar in magnitude to the binding constant. For interactions with moderate affinities values extremely close to the true binding values were obtained, as confirmed by experimental results with concanavalin A.
对高浓度溶液中蛋白质 - 蛋白质相互作用的分析需要考虑此类溶液中的非理想性，这种非理想性由维里系数表示。本文提出了不同的方程，用于估算在沉降平衡实验中热力学非理想性对自缔合蛋白质大分子相互作用的影响。通常，热力学非理想行为的影响通过浓度幂级数来描述。此类幂级数在高溶质浓度下的收敛性有限。当通过活度幂级数来表示热力学非理想性时，这一缺点可以最小化。所推导的离心机方程是进行全局分析以估算平衡常数和反应物相应热力学活度的基础所在。基于对合成浓度分布的拟合分析发现，对于溶质分子间具有强缔合作用力的蛋白质，会观察到与预期缔合常数存在显著偏差。在弱相互作用体系中也观察到了相当大的差异。这是由于蛋白质的排阻体积与结合常数大小相近所致。对于具有中等亲和力值的相互作用，获得了极其接近真实结合值的值，伴刀豆球蛋白A的实验结果证实了这一点。
