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Tubulin comprises 20 Cys residues, the sulfhydryls of which are all reduced in native tubulin. Tubulin bioactivity, measured by polymerization or drug binding, is reduced by reaction with a small number of these -SH groups, some implicated in drug binding to either the colchicine or vinca sites.
Sulfhydryls react with DTNB (dithio-bis-nitrobenzoic acid) to produce TNB-, the absorbance of which is easily monitored. Experiments (not shown) indicate that all 20 of tubulin's -SHs are reactive; moreover, denaturants (urea, guanidine HCl) increase the reaction kinetics, unfolding tubulin to expose -SHs. Thus, small perturbations in tubulin's structure are manifested in altered -SH reactivity.
In contrast to the case for denaturants, the kinetic suppression of this reactivity was used to examine drug effects on tubulin. Following incubation of tubulin ± drug, DTNB is introduced, and kinetic reaction curves like Plot A result. When slopes from such curves are plotted versus [drug], non-linear regression fitting produces binding curves, as in Plot B. Computer models return binding constants for the fitted data.
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Most, but not all, anti-microtubule drugs lower -SH reactivity.
DETAILS: In base buffer of 0.1 M MES, pH 6.9, 0.5 mM MgCl2, purified brain tubulin (0.7-4.0 mM) was incubated ± drug, 15 minutes, room T°, then DTNB added to 100 mM and A412 recorded ~30 minutes. Minus tubulin controls show no DA412. Approximately the first three -SHs react more rapidly than the remainder (which have similar reactivities), so slopes between 5 and 10 minutes were used in non-linear hyperbolic regression (one site binding saturation curve; Prizm software) versus [drug]. Bmax and Kd are returned from which the reported Kd is derived by subtracting ½[tubulin].
