Quantitative characterization of reversible molecular associations via analytical centrifugation.
The ultracentrifuge provides several techniques for the quantitative characterization of reversible small molecule-macromolecule and macromolecule-macromolecule interactions in solution. The nature of the association to be studied determines the preferred technique. High speed centrifugation is the method of choice for characterizing reversible heteroassociations between species of greatly different mass (i.e., sedimentation coefficient). This technique provides a relatively rapid, artifact-free, and thermodynamically rigorous means of quantifying the amount of nonsedimenting or slowly sedimenting free ligand in equilibrium with rapidly sedimenting acceptor-bound ligand at one particular solution composition. Results obtained over a broad range of ligand and/or acceptor concentrations lead to model-independent binding isotherms that may subsequently be analyzed in the context of models for ligand-acceptor association. Lower speed centrifugation to sedimentation equilibrium is the method of choice for characterizing reversible selfassociations and for characterizing heteroassociations between components that cannot be well separated on the basis of sedimentation velocity. In the dilute limit, this technique can provide model-free information about the dependence of weight-average molecular weight of each component upon solution composition, which can subsequently be analyzed in the context of equilibrium models for self- or heteroassociation. At higher concentrations, the models must be generalized to allow for the effect of nonspecific (nonideal) interactions upon sedimentation and association. The use of tracers provides a means for greatly extending the range of solute concentrations and solution compositions over which both types of measurements may be applied, providing enhanced ability to discriminate between alternative proposed mechanisms for self- or heteroassociations.