Institut für Angewandte Physik, Universität Tübingen , Auf der Morgenstelle 10, Tübingen D-72076, Germany.
Institut Max von Laue - Paul Langevin (ILL) , CS 20156, 71 Avenue des Martyrs, Grenoble Cedex 9, F-38042, France.
J Phys Chem B. 2017 Jun 15;121(23):5759-5769. doi: 10.1021/acs.jpcb.7b03510. Epub 2017 Jun 2.
Interactions and phase behavior of γ-globulins are of fundamental interest in biophysical and pharmaceutical research, as these are among the most abundant proteins in blood plasma. In this work, we report the characterization of the oligomeric state of bovine γ-globulin, the effective protein-protein interactions, and the colloidal stability in aqueous solution as a function of protein concentration and ionic strength. Classical biochemical techniques, such as size exclusion chromatography (SEC) and gel electrophoresis, together with small-angle X-ray and neutron scattering (SAXS/SANS), were employed for this study. The results show that bovine γ-globulin solutions are dominated by monomer and idiotype anti-idiotype dimer. Despite the flexibility and highly nonspherical shape of the protein, a simple model with a disk-type form factor and a structure factor of a square-well potential provide a satisfying description of the scattering data. The overall interactions are attractive and the strength decreases with increasing protein concentration, or adding buffer or salts. For higher protein volume fraction (>7%), the model would imply a strong particle-particle correlation which does not appear in the experimental data. This mismatch is most likely due to the smearing effect of the conformation change of proteins in solution. The stability of γ-globulin solutions is highly sensitive to protein concentration, ionic strength, and the type of added salts, such as NaCl, NaSO, and NaSCN. For solutions below 50 mg/mL and at low ionic strengths (<0.1 M), protein aggregation is most likely due to subpopulations of IgG molecules with attractive patches of complementary surface charge. This effect is reduced for higher protein concentration due to self-buffering effects. For high ionic strength (>1 M), typical salting-in (with NaSCN) and salting-out effects (with NaCl and NaSO) are observed. Results are further discussed in comparison with current studies in the literature on monoclonal antibodies.
γ-球蛋白的相互作用和相行为在生物物理和药物研究中具有重要意义，因为它们是血浆中最丰富的蛋白质之一。在这项工作中，我们报告了牛γ-球蛋白的低聚状态、有效蛋白质-蛋白质相互作用以及在蛋白质浓度和离子强度的函数下在水溶液中的胶体稳定性的特征。经典的生化技术，如尺寸排阻色谱（SEC）和凝胶电泳，以及小角度 X 射线和中子散射（SAXS/SANS），用于这项研究。结果表明，牛γ-球蛋白溶液主要由单体和独特型抗独特型二聚体组成。尽管该蛋白质具有灵活性和高度非球形形状，但具有圆盘型形态因子和方阱势结构因子的简单模型提供了对散射数据的令人满意的描述。总体相互作用是吸引的，并且强度随蛋白质浓度的增加而降低，或添加缓冲液或盐。对于较高的蛋白质体积分数（>7%），该模型将意味着强烈的颗粒-颗粒相关性，这在实验数据中没有出现。这种不匹配很可能是由于溶液中蛋白质构象变化的扩散效应。γ-球蛋白溶液的稳定性对蛋白质浓度、离子强度以及添加盐（如 NaCl、NaSO 和 NaSCN）的类型非常敏感。对于低于 50mg/mL 的溶液和低离子强度（<0.1M），蛋白质聚集最有可能是由于 IgG 分子的亚群具有互补表面电荷的吸引力斑块。由于自缓冲效应，这种效应在较高的蛋白质浓度下降低。对于高离子强度（>1M），观察到典型的盐析（用 NaSCN）和盐析（用 NaCl 和 NaSO）效应。结果与文献中关于单克隆抗体的当前研究进一步进行了讨论。
