QUANTITATIVE-ANALYSIS OF PROTEIN INTERACTIONS WITH LIGANDS - QUANTITATIVE CHARACTERIZATION OF REVERSIBLE MOLECULAR ASSOCIATIONS VIA ANALYTICAL CENTRIFUGATION

The determination of the strength and stoichiometry of reversible associations between interacting species has long been, and will remain for the foreseeable future, one of the most commonly encountered objectives in biochemical research. Consequently, many techniques have been devised for such determinations, and the literature devoted to the subject of binding measurement is correspondingly large. Other minireviews in this series are devoted to techniques for measuring binding to immobilized substrates; centrifugal methods are necessarily limited to the study of interactions between soluble species.2 2 Binding assays employing low speed centrifugation to accelerate filtration or to effect pelleting as a means of separating free from membrane-bound ligands are not reviewed here. The purpose of the present minireview is threefold: to illustrate, by example, the several different types of binding measurements that may be carried out using the ultracentrifuge; to emphasize that binding measurements carried out using the ultracentrifuge are in many cases free from artifacts and ambiguities of interpretation that complicate other types of measurement; and to call attention to some new techniques that promise unprecedented ability to discriminate between possible alternative association mechanisms. Methods developed prior to the mid-1970's are reviewed in more detail in Fujita (1) and Van Holde (2). In general, measurements of reversible association may be divided into two categories, termed direct and indirect. In a direct measurement of the association of two species (for example, ligand and acceptor) one attempts to individually measure the amount of uncomplexed and complexed forms of either or both of the two species, preferably while they remain in equilibrium with each other. In an indirect measurement one quantifies an average property of the solution as a whole that varies with the extent of binding or association. Composition-dependent variations in spectroscopic, hydrodynamic, thermodynamic, or functional properties are then interpreted in the context of models relating fractional changes in the measured property to fractional changes in association. Below we described the use of the ultracentrifuge for both direct and indirect measurements of association. Each method of measurement described will be illustrated by reference to a single published application of the method. © 1990.