Nile red, an uncharged, highly polarity-sensitive, fluorescent dye emitting in the red, has been used to study the interaction of the alpha and beta subunits of pure tubulin. Time-resolved deconvolution of the fluorescence of liganded nile red reveals two life times that correspond to two different emission maxima and quenching constants that suggest a solvent exposed and a solvent- shielded type of binding. Low tubulin concentrations cause a red shift and increased fraction of short life time fluorescence; high concentrations of microtubule stabilizing agent, especially glutamate reverse the process and cause a blue shift, increased life time and intensity. The equilibrium for this reaction corresponds to the hydrodynamic equilibrium constant for dimerization. Subtilisin susceptibility of the beta subunit C terminus also reflects the monomer-dimer equilibrium in which this portion of tubulin is more exposed in the monomer. These are therefore relatively easy methods to assess the monomer/dimer equilibrium of tubulin and we expect to apply this analysis to overall microtubule assembly questions.
