Measurement of ligand binding to tubulin by sulfhydryl reactivity

Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.