The concentrations of solutions of hemiasterlins were initially based on weights of the preparations of hemiasterlins. However, the absorbances were only 1/4 - 1/2 those expected for compounds that included tryptophan (Trp) moieties. Na-methyl-Trp was selected as a standard for hemiasterlin A (Hem-A), and 1-methyl-Trp for hemiasterlin (Hem) and hemiasterlin C (Hem-C). The concentrations of hemiasterlins reported here are based on the standardization (using respective peak absorbance values), rather than weights of the hemiasterlin preparations. The discrepancy was observed subsequent to the experiments reported here, so note the substoichiometric (vs. tubulin) concentrations of hemiasterlins where applicable.
Details: Stock solutions (in DMSO) were diluted into 500 µL of 0.1 M MES, pH 6.9, 0.5 mM MgCl2, and additional dimethyl sulfoxide to obtain 520 µL final sample volume (3.85% dimethyl sulfoxide). Standards were at 10 µM. Hem-A lacks the N-1 methyl group, as do Trp and Na-methyl-Trp, but, as in the hemiasterlins, the alpha carbon of Na-methyl-Trp is methylated. The peak absorbance for Trp, Na-methyl-Trp, and Hem-A is at 280 nm, but the Î for Na-methyl-Trp is 10% lower than that for Trp, based on reagent weights. Not surprisingly, among the potential standards examined (1-, 4-, 5-, 6-, and 7-methyl-Trp), the spectrum of 1-methyl-Trp most resembled Hem (both peak at 289 nm), and was the best choice for Hem-C (peak at 285).
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