Jachimska Barbara, Wasilewska Monika, Adamczyk Zbigniew
Institute of Catalysis and Surface Chemistry, Polish Academy of Science, ul. Niezapominajek 8, 30-239 Cracow, Poland.
Langmuir. 2008 Jun 1;24(13):6866-72. doi: 10.1021/la800548p. Epub 2008 May 30.
In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.
在这项工作中，对两种球状蛋白——分子量为67 kDa的牛血清白蛋白（BSA）和分子量为69 kDa的人血清白蛋白（HSA）的物理化学性质进行了表征。这些蛋白质的整体特性包括扩散系数（流体动力学半径）、电泳迁移率以及作为不同pH值下蛋白质溶液浓度函数的动态粘度。流体动力学半径数据表明蛋白质分子存在缔合现象，很可能形成紧密的二聚体。利用流体动力学直径和电泳迁移率数据可以确定蛋白质表面未补偿（动电）电荷的数量。将电泳迁移率数据转换为zeta电位值，从而可以确定这些蛋白质的等电点（iep）。发现这两种蛋白质的等电点均为pH 5.1，这与先前的实验数据以及根据氨基酸组成和pK值得出的理论估计一致。为了进一步确定蛋白质溶液的稳定性，针对不同pH值，测量了动态粘度随其总体积浓度的变化。从这些测量中得出的特性粘度根据适用于硬球形颗粒的布伦纳模型进行了解释。当假设蛋白质尺寸为9.5×5×5 nm³（长椭球体）时，发现这些蛋白质的特性粘度实验值与该模型吻合良好。这些测量排除了形成缔合度高于2的线性聚集体的可能性。得出的结论是，动态粘度和动态光散射的结合不仅可以作为检测悬浮液中蛋白质聚集起始的便捷工具，还可以用于检测这些聚集体的形式和组成。
