from exceeding the linear range of a method, rather than true decrease in enzyme activity. If a test method depends upon a second enzyme or limiting reagent (post-reaction detection or coupled enzyme assays), it may become limiting as the concentration of the first enzyme increases (Fig. 5, curve b). In this case, increasing the concentration of the second enzyme or reagent will extend the linear portion of the curve, and the plateau will be observed at a higher signal level (Fig. 5, curve c). In the case of optical detection methods, deviations from the Beer-Lambert law are pronounced when absorbance measurements are greater than 2.0, since the transmitted light that the photometer measures is now only 1% incident light. Instrumental noise is often a limiting factor at these low light levels. With fluorescence, inner filter effects can occur at high concentrations of a fluorescent substrate or product, and these can actually lead to decreasing signal with increasing concentration (see Fig. 6, curve b). This can be diagnosed by checking the proportionality of the fluorescence of a sample after a large dilution (e.g., a 20-fold dilution yielding only 5-fold less fluorescence). These effects can also be corrected if the absorption of the sample is known at the excitation and

Figure 6 Ideal versus nonideal V versus S behavior. Normal hyperbolic kinetics (a), solubility or method limitations (b), interfacial partitioning (c), and positive coopera-tivity (d).

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