Determination of free and bound microtubular protein and guanine nucleotide under equilibrium conditions

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Abstract
The dissociation constant for GDP binding to the E site of [porcine] tubulin isolated by chromatography on Sepharose 6B is 6.1 .times. 10-8 M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value. The discrepancy with earlier results is analyzed. Use of a recently described column centrifugation procedure established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP and the dissociation constant is 2.2 .times. 10-8 M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. It was used to determine that the dissociation constant for GDP bound to tubulin is 5.5 .times. 10-8 M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment of ligand loss during exclusion chromatography is discussed.