Inhibition of tubulin self-assembly in vitro by fluorodinitrobenzene

The self-assembly of bovine brain tubulin into microtubules is inhibited by low molar ratios of 1-fluoro-2,4-dinitrobezene (FDNB). Binding studies using [¹⁴C]FDNB indicate that the incorporation of between one and two dinitrophenyl groups is sufficient to inhibit assembly completely, although more dinitrophenyl groups can be incorporated using higher FDNB/tubulin ratios. Dinitrophenyltubulin, under assembly conditions, tends to assemble into amorphous aggregates. Thiolysis by 2-mercaptoethanol removes the dinitrophenyl moieties. Paper chromatography and high-voltage electrophoresis of acid-hydrolyzed modified tubulin containing one dinitrophenyl group identified the residue as $\text{S-}\text{dinitrophenylcysteine}$. The β-monomer of tubulin is preferentially modified at low FDNB/tubulin ratios. The reaction with FDNB is greatly reduced when tubulin is in polymerized form. FDNB reduces the colchicine binding activity but does not affect the Mg(II) content of the protein.