FLUORESCENCE-DETECTED SEDIMENTATION IN DILUTE AND HIGHLY CONCENTRATED SOLUTIONS

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.