Studies of Macromolecular Interaction by Sedimentation Velocity
As our knowledge expands and new systems are investigated, it becomes clear that elucidating precise biochemical regulatory mechanisms requires detailed understanding of macromolecular assembly processes. For example, the regulation of gene expression involves an intricate network of protein-protein and protein-nucleic acid interactions and the mechanism of some allosteric enzymes is linked to subunit assembly. In order to define these mechanisms one needs information on the identities of proteins in the complex, the affinities of these proteins for each other and the effects of regulators on the formation of these complexes. A direct way of studying macromolecular assembly is to monitor the resulting changes in mass as a consequence of the formation of these macromolecular complexes. One of the methods that enables one to directly monitor the mass of macromolecules is the transport technique. Among the transport methods sedimentation analysis is the technique of choice because of the sound fundamental principles on which the method is based and because of its resolving power. Excellent reviews on the applications of sedimentation equilibrium in studying macromolecular self-associations and heteropolymers formation are included in Part I of this volume. In this chapter the focus is on applications of sedimentation velocity. One of the advantages of sedimentation velocity over that of equilibrium is its speed of analysis, e.g., a run can be completed within an hour whereas equilibrium experiments may take much longer. Hence, if a biological sample is unstable the study on that system may have to be conducted using sedimentation velocity. However, a larger amount of sample will be required for velocity analysis than for equilibrium measurements, e.g., the calf brain tubulin and rabbit muscle phosphofructokinase systems require a few mg of protein to define a curve of sedimentation coefficient vs concentration.
KeywordsSedimentation Velocity Infinite Dilution Sedimentation Pattern Sedimentation Coefficient High Protein Concentration
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