Tubulin dimer dissociation detected by fluorescence anisotropy

We have demonstrated a concentration-dependent dissociation of bovine brain tubulin dimer covalently labeled with 5-(4,6-dichlorotriazin-2-yl)amino]fluorescein (DTAF) or with fluorescein isothiocyanate (FITC) by fluorescence anisotropy and size-exclusion HPLC. The fluorescence anisotropy values decreased to a limiting value upon dilution of tubulin from 10(-5) to 8 x 10(-8) M. A dissociation constant in 0.1 M Pipes, pH 6.9, 1 mM EGTA, and 1 mM MgSO4 at 20 degrees C was estimated to be (8.4 x 10(-7) +/- (0.4 x 10(-7) M. Control experiments using monomeric and other dimeric proteins, urea-denatured tubulin, and DTAF-tubulin diluted into solutions of bovine serum albumin or unlabeled tubulin were consistent with the finding that the changes in anisotropy upon dilution are due to protein dissociation. These results were supported by size-exclusion HPLC data where an increase in the elution volume of DTAF-tubulin and FITC-tubulin was observed with decreasing protein concentrations. Reversibility of the dissociation process and the lack of denaturation at high dilution were shown by the ability of reconcentrated protein to assemble into microtubules to about the same extent as undiluted protein. Fluorescent lifetimes and limiting anisotropy values were found to be approximately identical at different tubulin concentrations, indicating that the anisotropy changes reflect changes in size or rotational correlation time of the protein. Studies on the effects of tubulin ligands and promoters or inhibitors of assembly demonstrated that their effects on tubulin dimer-monomer equilibria are small but reproducible.(ABSTRACT TRUNCATED AT 250 WORDS)