Figure 3.

HB binds to 100 kDa αβ-heterodimer. (A) Nonequilibrium binding of [³H]HB to tubulin. Dashed line, radiolabel; dotted line, A₂₈₀. A 0.1 mL sample containing 5 μM each of tubulin and [³H]HB was injected into a single Shodex KW-803 column with a guard column. Running buffer: 0.1 M Mes –0.5 mM MgCl₂ at 0.5 mL/min. First absorbance peak (12 min) is at the void volume (denatured tubulin aggregates). Second peak (20 min) emerges at 100 kDa.¹⁰ Third peak (29 min) contains unbound small molecule(s). (B) HPLC Hummel–Dreyer evaluation of the binding of [³H]HB to tubulin. (i) Radiolabel. (ii) A₂₈₀. Chromatography as in panel A. Equilibration with 1.0 μM [³H]HB. Injected sample: 50 μg tubulin (0.1 mL). (C) Differences in nonequilibrium versus equilibrium HPLC gel filtration occur with vinblastine or D10 but not HB, spongistatin 1, or maytansine. Two Shodex KW-803 columns with a guard column were used in series. Flow rate: 1.0 mL/min. Injection sample: 0.1 mL, containing 10 μM tubulin. “Unequilibrated columns” means the column system had been equilibrated with 0.1 M Mes–0.5 mM MgCl₂; “equilibrated columns” means the columns had been equilibrated with the buffer containing the indicated drug. Drug concentration, except with D10 (panel ii), was 20 μM in the injected sample and when the column system was equilibrated with drug (darker curve in i; panels iv, vi, and viii). The D10 concentration was 3 μM in the injected samples and equilibrated columns (panel ii, darker curve). (i) Vinblastine. Unequilibrated columns, lighter curve. Equilibrated columns, darker curve. (ii) D10. Unequilibrated columns, lighter curve. Equilibrated columns, darker curve. (iii) HB, unequilibrated columns. Inset: protein elution profile following injection of tubulin only into unequilibrated columns. (iv) HB, equilibrated columns. (v) Spongistatin 1, unequilibrated columns. (vi) Spongistatin 1, equilibrated columns. (vii) Maytansine, unequilibrated columns. (viii) Maytansine, equilibrated columns.