Shaptsr 5 Sedimentation equilibrium in the analytical ultracentrifuge

For many years analytical ultracentrifugation was the major source of information on the heterogeneity and molecular size of macromolecules. In the field of protein chemistry the question of solute heterogeneity is now usually addressed by gel electrophoretic and gel chromatographic techniques, and the molecular wetght is either calculated from the amino acid sequence or obtained by mass sPectromety. Because such molecular weight values refer only to the covalentlylinked polypeptide chain(s), they provide no information about the macro molecular state of the functional protein or en4rrne. In its simplest application moleorlar werght measurement by analytical ultracentrifugation is therefore used to characterize quaternary structure, which affords an example ofa selfassociation equilibrium that has gone to completion. For many proteins, however, the monomeric and polyrneric forrrs coexist in association equilibrium, the relative proportions of the two macromolectrlar states varying with total solute concentration in accordance with k Chokliels principle: the polymeric state is favoured by an increase in concentration, whereas dilution favours the monomeric state. Analytical ulhacentrifugtion has great potential for characterizing the self-association equilibrium by virtue of these concentrationdependent changes in the average macromolecular state of the solute. The requirement that a rapidly reequilibrating solute system be characterized without perturbing the equilibrium state is readily accommodated by either of the two commonly used techniques in analJrrical ultracentrifugation -sedimentation velocity and sedimentation equilibrium. In the former the ultracentrifuge is operated at a sufficiently high angular velocity for the