Search

Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving 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.

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) has been used to detect structural correlates of the well-known but poorly understood decay of tubulin function, by which tubulin loses its ability to polymerize and bind drugs in a complex, time-dependent way. The present results indicate that the decay of tubulin is accompanied by the appearance of hydrophobic areas, which bind a total of six Bis-ANS molecules with a dissociation constant of 19 microM. This binding seems to be a result of localized structural changes that are taking place in the tubulin molecule and can be used as a probe for these changes. In particular, circular dichroism measurements revealed no significant changes in the average secondary structure of the protein during the time required for complete binding of the Bis-ANS molecules. Preincubation of tubulin with the antimitotic drugs colchicine, podophyllotoxin, and vinblastine slows the rate of appearance of the hydrophobic region. Vinblastine has the maximal effect followed by colchicine and podophyllotoxin. In contrast, preincubation with maytansine has no effect. In addition, lowering the temperature decreases the rate of appearance of this region. These results correlate with the effect of drugs on the alkylation of tubulin sulfhydryl groups by iodoacetamide [Luduena, R.F., & Roach, M.C. (1981) Biochemistry 20, 4444-4450] and with the ability of inhibitors of microtubule assembly to permit the polymerization of tubulin into nonmicrotubule structures.

We have previously found that the sulfhydryl groups of tubulin are sensitive reporters of the effects of ligands on the tubulin molecule. In this study, we examined the effects of three anilinonaphthalenesulfonates on the interaction of tubulin with iodo[14C]acetamide and N, N'-ethylenebis(iodoacetamide). We found that 1,8-anilinonapthalensulfonate (1,8-ANS) and 2,6-anilinonaphthalenesulfonate (2,6-ANS) had no effect on the reaction with iodo[14C]acetamide. In contrast, bis(1,8-anilinonaphthalenesulfonate) (BisANS), an inhibitor of microtubule assembly, had a complex effect. Low concentrations of BisANS, where presumably only the high-affinity binding site was saturated, had little or no effect on alkylation. Higher concentrations of BisANS caused a strong enhancement of alkylation. None of these compounds had any effect on the reaction with N,N-ethylenebis(iodoacetamide). Our results suggest that the binding of BisANS, 2,6-ANS and 1,8-ANS to tubulin is complex and very different from that of the other anti-tubulin drugs. The correlation between the effects of drugs on alkylation of tubulin and the binding of BisANS is consistent with a model whereby the alkylatable sulfhydryls are located in apolar regions of the tubulin molecule.