Int. J. Environ. Res. Public Health 2012, 9 484 [3H]NEM to tubulin with 50% inhibition occurring at 23 µM. This straightforward experiment shows As3+ binding to tubulin through -SH groups, initiating the cascade of MT disruption events. Figure 6. As3+-inhibition of MT protein polymerization in vitro as confirmed by EM examination. The in vitro MT polymerization was carried out as described in Figure 5. At the plateau level of the MT assembly (30 min after reaction), a 10 µL-drop of the reaction mixture was removed and processed for EM examination. (A) the absence of polymerized MTs at 0 min; (B) the typical morphology of in vitro assembled MTs at 30 min; (C) 40 µM As3+; (D) 40 µM As3+ plus 1 mM DTT. Photographs with a final magnification of 22,000 × observed under a Philips 300 transmission electron microscope. Figure 7. As3+ binding to tubulins through -SH groups as revealed by [3H]NEM binding assay. “Pure” tubulin free of MAP (1.5 mg/mL) prepared from bovine brain was pretreated with As3+ at indicated concentrations for 2 h at 0 C, mixed with [3H]NEM (2 µCi/mL), and incubated for an additional 2 h at 37 C. Proteins were precipitated with 5% TCA and filtered. Radioactivities coupled with collected proteins on the membrane were measured by β-counting. The significance of data presented was assessed by ANOVA analysis. * p < 0.05, ** p < 0.01 (n = 3).