Titration of Drug Using GPC Spin ColumnESIMS Coelution of Multiple Ligands Noncovalently Bound to Protein Target

A titration study was undertaken to determine the extent of non-covalent binding between the inhibitor DBQ (MW 489 Da) and the protein CMVP A144L. This experiment was conducted by incubating a fixed amount (50 pmol) of the protein CMVP A144L with from 0-40 mol excesses of DBQ. The incubated materials were then subjected to GPC spin column analysis, whereby the eluate containing the tightly bound inhibitor was analyzed by ESI-MS under denaturing conditions to quantitate the mole ratio of inhibitor to protease (D/P). Figure 2.25 illustrates the [M+H]1+ region of the ESI mass spectrum for these titration experiments. (Note the characteristic 1:2:1 dibromo isotopic intensity distribution.) Table 2.5 summarizes the quantitative results for these titration experiments by tabulating the [drug]/[protein] (D/P) molar ratios during the incubation reaction and after elution from the spin column. Figure 2.26 graphically illustrates a plot of the D/P mole ratios of the reaction vs the bound mixtures. Note that at low reaction D/P mole ratios, from 0 to 1, the slope of the curve rises rapidly and continuously levels off from reaction D/P mole ratios from 1 to 40. These results suggest that there may be specific non-covalent binding at low D/P mole ratios up to about 0.5 D/P mole ratios, but as the reaction D/P ratio increases less specific non-covalent

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