Mixtures of 2.5 uM tubulin and the indicated drugs in 0.1 M MES, pH 6.9, 0.5 mM MgCl₂, 1% DMSO were incubated for 20 minutes at room temperature then injected into a gel filtration HPLC system (equilibration and elution by 0.1 M MES, pH 6.9, 0.5 mM MgCl₂). Our laboratory has published reports on the aggregation of tubulin induced by various drugs, including D10. The data here indicate that HEM has similar effects. By a plot resulting from system calibration with protein standards, T, tubulin ab-dimer’s retention time, corresponds to ~113 kDa, and the other 2 included peaks to ~270 kDa (2.4 tubulins, retention time b) and 440-480 kDa (3.8-4.2 tubulins, retention time a). The ordinates are arbitrary A₂₈₀ units. E, peak retention time of the excluded volume (calibration by Blue Dextran).

*The partially excluded peak marked * in "No Drug" is enigmatic. It may depend on handling of the tubulin stock solution, for it may not appear at all (i.e., it probably represents denatured tubulin). However, the extent of pre-injection column washing may also influence the subsequent appearance of this peak (i.e., it may be due to an injection artifact). The peak retention time of * is consistently ~30 s later than E, whereas the retention time of major peaks for super-stoichiometric drug concentrations is E.

Method details
Three columns in series were used—a TosoHaas TSK-GEL SW 7.5x75 mm guard column, and two Shodex Protein KW-803 8x300 mm. In these experiments, typical operating pressures were ~40 bar at 0.7 mL/min.

Injection volumes were 250 uL.

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