Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis(8-anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin.
The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 microM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 degrees C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014404:Tubulin / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D014404:Tubulin; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan