The Sensitivity Shift Procedure

In most enzymatic reactions it is not uncommon for a sample to contain a concentration of substrate 100- to 1000-fold greater than that of the reaction product. This situation often arises with enzymes that require high concentrations of substrate (low Km) for reactivity and have low rates of product formation. It also occurs soon after the start of the reaction, when only small amounts of product have been formed. 

The analysis of a sample from an incubation mixture that contains significant differences in substrate and product concentrations presents some problems, since the detector must be set so that the substrate peak is on scale, but also it must be set to detect small amounts of product. Usually different sensitivity settings are required to put both components on scale. For these cases we have adopted a procedure that might be called the sensitivity shift. In this procedure, the injection is made with the sensitivity set at a value that allows for the detection of one of the compounds. If this is the product, the detector is set at its maximum sensitivity. As soon as the product has emerged, the sensitivity of the detector is changed (either manually or electronically by computer) to a value that allows the substrate peak to appear completely on scale. 

Assuming that the settings on the detector are proportional, there should be no difficulty in relating the areas of both peaks to the calibration curve. These problems can be avoided with electronic integrators, which measure total absorbance and are not affected by sensitivity settings. 

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It is of interest to note the use of the sensitivity shift procedures, illustrated in Figure 9.55. Arrows on the figure indicate where detector sensitivity was increased to allow for the detection of low levels of the product. 

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