Kingsbury Jonathan S, Laue Thomas M
Therapeutic Protein Research, Genzyme Corporation, Framingham, Massachusetts, USA.
Methods Enzymol. 2011;492:283-304. doi: 10.1016/B978-0-12-381268-1.00021-5.
Analytical ultracentrifugation (AUC) is a powerful, first-principles method for characterizing macromolecules in solution. The recent development of fluorescence-detected sedimentation for the AUC (AU-FDS) has extended the sensitivity and selectivity of the instrument which, in turn, has enabled the study of both higher affinity interactions and the sedimentation of one component in complex, concentrated solutions. While still in its infancy, AU-FDS is becoming more widespread as shown by the increasing number of literature reports citing its use. While AU-FDS enables the analysis of systems not amenable to absorbance or interferometric detection, its use is not without limitations. In most cases, preparing samples for AU-FDS analyses requires chemical conjugation with fluorescent dyes, a step that may influence the size or shape of a molecule sufficiently to alter its transport during sedimentation. Careful preparation and characterization of the amount of free dye and the degree and site specificity of labeling is required for robust interpretation of AU-FDS data. In some cases, studies of the effect of labeling on the structure, activity, or association properties of the macromolecule may be warranted. However, these complications are of minor consequence compared to the unique information that can be obtained by AU-FDS. In particular, its ability to provide direct, physical characterization of the thermodynamic behavior of molecules in complex and concentrated solutions makes AU-FDS a powerful technology for understanding the physical underpinnings of living systems.
分析超速离心法（AUC）是一种强大的、基于第一原理的方法，用于表征溶液中的大分子。用于AUC的荧光检测沉降技术（AU-FDS）的最新发展扩展了该仪器的灵敏度和选择性，进而能够研究更高亲和力的相互作用以及复杂浓缩溶液中一种组分的沉降。虽然AU-FDS仍处于起步阶段，但正如引用其使用的文献报道数量不断增加所表明的那样，它正变得越来越普遍。虽然AU-FDS能够分析不适用于吸光度或干涉检测的系统，但其使用并非没有局限性。在大多数情况下，为AU-FDS分析制备样品需要与荧光染料进行化学偶联，这一步骤可能会充分影响分子的大小或形状，从而改变其沉降过程中的迁移。为了对AU-FDS数据进行可靠的解释，需要仔细制备和表征游离染料的量以及标记的程度和位点特异性。在某些情况下，可能有必要研究标记对大分子结构、活性或缔合性质的影响。然而，与通过AU-FDS可以获得的独特信息相比，这些复杂性的影响较小。特别是，它能够对复杂浓缩溶液中分子的热力学行为进行直接的物理表征，这使得AU-FDS成为理解生命系统物理基础的强大技术。
