Applications of fluorescence detected sedimentation I Studying the thermodynamic impact of valence in non-ideal solutions II High affinity interactions and biological media

The recent addition of the fluorescence detection optical system (FDS) to the currently available absorbance and interference optical systems on the analytical ultracentrifuge has greatly expanded the wide application of this technique. The new FDS is able to detect very low concentrations of fluorophore allowing the study of high affinity interactions (Kd ∼ pM) not previously accessible with the absorbance optics. The types of high affinity interactions that can be explored include drug-protein interactions, lipid-receptor interactions, DNA-protein interactions and protein-protein interactions. Selective labeling of one or both of the macromolecules can allow elegant dissection of complex assemblies. The only limiting factor is the researcher\u27s ability to label the macromolecule of interest. In addition to high affinity interactions, selective labeling of the macromolecule of interest with either a synthetic fluorophore or GFP allows study of sedimentation, diffusion and association in complex mixtures such as lysates, serum and other biological milieu. The use of FDS to study of complex mixtures has application in both the pharmaceutical and food science industries. Finally, the ability to study macromolecules under non-ideal solution conditions will bridge what we know about macromolecular diffusion and assembly under ideal conditions and what has been learned about diffusion and assembly in the context of intact cells. New findings in this area will help to build upon what is already known about macromolecular crowding and aid in the refinement of current crowding theory. Examples of the applications listed above will be presented highlighting a diversity of systems and the power of this exciting new advancement in analytical ultracentrifugation

NUTS and BOLTS: Applications of fluorescence-detected sedimentation

Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors, absorbance and interference, impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU–FDS) enables two different types of sedimentation experiments. First, the AU–FDS can detect picomolar concentrations of labeled solutes, allowing the characterization of very dilute solutions of macromolecules, applications we call normal use tracer sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high-affinity interactions. Second, the AU–FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call biological on-line tracer sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieus such as cell lysates and serum. Examples that embody features of both NUTS and BOLTS applications are presented along with our observations on these applications