Procedures for Analytical Ultracentrifugation

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Procedures for Analytical Ultracentrifugation The ultracentrifuge provides direct and reliable information about the molecular weights and the interactions of macromolecules as they exist in solution. Sedimentation equilibrium approaches are based on equilibrium thermodynamics and thus can provide reliable estimates of the parameters of molecules in solution. Interferometric observations of concentration distributions provide a high degree of precision for such estimations. The recent commercial introduction of automated interferometry in a modern ultracentrifuge provides a powerful tool with a high "signal to noise ratio" for studying macromolecular distributions. The proposed research will examine ways to improve the precision of such measurements. This should lead to the ability to carry out experiments under a wider range of conditions and to better distinguish amongst alternative explanations (models) of the systems investigated both for equilibrium and velocity experiments. Software and new approaches to equilibrium sedimentation experiments, if successful, will make experiments easier to carry out, and either faster or more precise and thus able to provide more meaningful information. These improvements would, for example, facilitate determinations of levels of impurities in protein preparations or estimations of the strength of interactions between molecules. The programs to be developed should make it easier (and sometimes possible) to obtain information from difficult interacting systems, such as the complexes formed between cytokines and their receptors. These systems often exhibit a sequence of complex formations where it is difficult to determine stoichiometries. These various approaches will be applied to problems of topical interest, largely, but not completely, from biotechnology enterprises to try to answer interesting questions.