Changes in the circular dichroic spectrum of colchicine associated with its binding to tubulin.
Circular dichroism has been used to study the interaction of colchicine with the tubulin alpha beta dimer at 26 degrees C. Tubulin purified from bovine brain microtubule protein exhibits negligible circular dichroism at wavelengths above 310 nm. Free colchicine exhibits a negative circular dichroic band at 340 nm characterized by an extremum in molar ellipticity [theta] of -3.35 (+/- 0.27) x 10(4) deg.cm2/dmol. This negative band either vanished or was greatly reduced, i.e., [theta] = 0 (+/- 0.57) x 10(4) deg.cm2/dmol, when colchicine was bound to tubulin. This was demonstrated by circular dichroic studies on stable [3H]colchicine-tubulin complexes which were separated from all unbound colchicine by means of gel filtration. Also, tubulin was titrated with colchicine, and at low colchicine concentrations, the observed ellipticity at 340 nm could be extrapolated to zero at 0 M colchicine; saturation occurred at a molar ratio of colchicine to tubulin of about 1:1. The association constant characterizing the drug-protein interaction was estimated to be about 0.9 micro M-1. As controls, three other acidic proteins were studied at a molar ratio of colchicine to protein of 2:1, and their presence had no effect on the circular dichroic properties of colchicine. These results are consistent with the idea that a conformational change in colchicine accompanies its binding to tubulin. The spectrum of the complex between 250 and 300 nm was quite similar to that expected from simple additivity of the spectra of drug and protein except between 255 and 265 nm. The technique described herein should be applicable to other protein-drug systems.
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002942:Circular Dichroism; D003078:Colchicine; D046911:Macromolecular Substances; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D014404:Tubulin
D046911:Macromolecular Substances; D014404:Tubulin; D003078:Colchicine