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

摘要

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's 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.