New approach for studying macromolecular-ligand binding. Determination of the dissociation constant for macromolecule-bound ligand by gel filtration.

A graph is presented which permits facile determination of the dissociation constant constant for a macromolecule . ligand complex from gel filtration results; it can be used without a detailed understanding of the remainder of the text. When a solution of macromolecules and radioactive ligand is subjected to gel filtrations, the amount of ligand associated with the eluted macromolecule is easily measured. This quantity is then used for obtaining the corresponding dissociation constant from the graph. Values given in the graph had been determined from an equation, which is presented in the text, by means of a program which is run on a programmable calculator (Texas Instruments SR-52). It is also demonstrated how the validity of dissociation constants so obtained can be checked independently by means of a second calculation. A modification of these procedures permits determination of the dissociation constant for those situations where there is moderate dilution of the eluted sample relative to that applied to the gel filtration column, and the graph incorporates results from such calculations. The successful application of the present approach to the determination of the dissociation constants for a tubulin . [3H]GDP complex is described. Also, the potential importance of the present approach relative to other techniques for dissociation constant determination is discussed.