Zorrilla Silvia, Hink Mark A, Visser Antonie J W G, Lillo M Pilar
Instituto de Química Física Rocasolano, Consejo Superior Investigaciones Científicas (CSIC), Serrano 119, 28006 Madrid, Spain.
Biophys Chem. 2007 Feb;125(2-3):298-305. doi: 10.1016/j.bpc.2006.09.003. Epub 2006 Sep 27.
Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.
荧光相关光谱法（FCS）被用于测量标记的脱辅基肌红蛋白（示踪剂）在核糖核酸酶A和人血清白蛋白（拥挤剂）的浓缩溶液中的平移扩散，作为在拥挤的生理环境中蛋白质扩散运动的定量模型系统。示踪蛋白在蛋白质拥挤溶液中的扩散系数与其在水溶液中的扩散系数之比，已根据局部表观粘度进行了解释，局部表观粘度是每个示踪剂-拥挤剂系统的一个分子参数特征。在这项工作中研究的所有蛋白质溶液中，局部平移粘度值均大于溶液的本体粘度，且大于在相同拥挤溶液中为脱辅基肌红蛋白估计的旋转粘度。在此，我们提出了一种直接从浓缩蛋白质溶液的本体粘度估算示踪剂平移和旋转扩散的局部表观粘度的方法。作为这项研究的结果，借助FCS和时间分辨荧光去极化技术对模型拥挤蛋白质溶液中的蛋白质种类进行鉴定以及对流体动力学变化和相互作用进行研究，有望得到极大简化。
