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A Microtiter-based Assay for the Determination of ID50s of b-lactamase Inhibitors Employing Reporter Substrates Detected at UV or Visible Wavelengths (MaxLine Application Note #20)

50s were measured using a concentration of reporter substrate which was approximately 5 times the Km of the reporter substrate for each of the enzymes (Table 2 ). This enables more accurate comparisons of ID50s for the different enzymes. This is particularly relevant if competitive inhibitors are being evaluated. Table 3 summarizes the contents of each well in the assay microtiter plate.


Step 11 Rapidly place the plate on the carriage of the SPECTRAmax 250 and click on the READ button.

Step 12 The axis of the well graphs can be changed while reading by clicking the reduction button in the plate window. Reading can be stopped at any point before the completion of the test by clicking on the STOP button in the control panel of the software. (This will save the data already collected.)

This protocol can also be used to measure ID50s following different preincubation times of enzyme and inhibitor. In addition, where no preincubation is required, the enzyme solution should be added last.


RESULTS
Figure 3
shows an assay microtiter plate set up to record the ID50s of 8 compounds. The rate of hydrolysis of reporter substrate is shown for each well (column 1 = blank, column 2 = control, columns 3 - 12 = range of inhibitor concentrations). This particular protocol has been set up to calculate the% inhibition of the enzyme at each concentration of inhibitor compared to the control. The blank value for each compound is automatically subtracted within the software program.

SOFTmax PRO will plot percent inhibition vs. concentration on a 4 Parameter logistic curve fit, and will then calculate the ID50 for each compound, in this case taken as the concentration of inhibitor (M) required to inhibit the enzyme by 50%.

Examples of the results produced are shown in Figure 3 and Figure 4.
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