Immobilization-dependent fluorescence of colchicine

Abstract

Colchicine fluoresces when bound to tubulin but not in water, dioxane, or benzene. The basis of the fluorescence has now been investigated. Colchicine fluoresces in higher alcohols and shows a blue shift as a function of chain length. Glycerol produces a higher fluorescence efficiency and a further blue shift. Plots of 1/fluorescence versus T/η yield straight lines for both alcohols and glycerol/water mixtures. Fluorescence in glycerol/dimethyl sulfoxide mixtures, in which the dielectric constant remains unchanged, varies as a function of solvent viscosity. Even highly nonpolar solvents such as dioxane require a threshold viscosity for fluorescence to occur. When solvent polarity was decreased at constant viscosity, there was also an enhancement of colchicine fluorescence, but this effect appeared to be smaller than that obtained with increasing viscosity. Immobilization by covalent attachment of desacetylcolchicine to thyroglobulin, serum albumin, or lysozyme also promotes fluorescence from the drug. By contrast, the highly rigid analogue of colchicine, imerubine, fluoresces in water and is unaffected by viscosity changes. We concluded that a major contribution to colchicine fluorescence stems from immobilization of colchicine in the site and that this response to immobilization depends, in part, on the partially flexible nature of the drug. Since certain other flexible molecules such as auramine O, reduced flavines, and diarylalkanes also require increased viscosity or binding to macromolecules to fluoresce at room temperature, we propose that immobilization-enhanced fluorescence may be more common than heretofore believed.