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Enzyme assays are used are used to study the rates of enzyme catalyzed reactions.
Enzyme assays have many applications in enzyme kinetics. Understanding the rates of reactions can help determine the mechanism that the reaction follows (a single-substrate or multiple-substrate mechanism). Figure 1 below demonstrates how enzymes change a reaction's mechanism by lowering the activation energy.
Fig. 1 - A Potential Energy Profile demonstrating the affect adding an enzyme has in a reaction's mechanism.
Enzyme activity is a measure of how much enzyme is present in a reaction. There are two ways to measure enzyme activity: monitoring the disappearance of substrate or the appearance of product. Measuring the appearance of product is usually more accurate because detecting small changes in [P] (when [P]=0) is easier to measure than detecting small changes in [S].
Through Michaelis-Menten kinetics, enzyme assays are used to calculate the enzyme's Km for a specific substrate, Vmax, and Ki for inhibitors. Enzyme assays can also reveal information about the substrates and inhibitors that may affect the enzyme. Figure 2 below uses a fixed-time assay to demonstrate the effect adding enzyme inhibitors have in a reaction. Details of enzyme-substrate reaction rates are further described by the Michaelis-Menten Kinetic Model.
Fig. 2 - A fixed-time assay Lineweaver-Burk analysis demonstrating the results of increasing the amount of inhibitor.
Micromoles of substrate converted to product per minute (μmol P/min), or International Units (IU), are commonly used units to express reaction rates. However, different measurements are used to quantify enzyme activity, including:
An enzyme assay must be considered valid to ensure accurate data and calculations. In order for an enzyme assay to be valid, it must meet multiple requirements:
When assaying enzyme activity many factors are considered to ensure proper results. Changes in these factors may lead to improper data and calculations.
Enzyme assays measure either the disappearance of substrate over time or the appearance of product over time. Multiple methods have been developed to measure the concentration of substrates or products in a reaction, but all enzyme assays fall into two types: fixed-timed and continuous.
The spectrophotometric assay is the most common method of detection in enzyme assays. The assay uses a spectrophotometer, a machine used to measure the amount of light a substance's absorbs, to combine kinetic measurements and Beer's law by calculating the appearance of product or disappearance of substrate concentrations. The spectrophotometric assay is simple, non-destructive, selective, and sensitive. For example, the NADH/NAD+ molecule is often used in enzymatic oxidation/reduction reactions. During these reactions NADH is often oxidized to NAD+, or NAD+ is reduced to NADH. NADH absorbs light at 340 nm, however NAD+ does not hold that property. A spectrophotometer can be used to measure the change in absorbance of 340 nm light, thus indicating a change in amount of NADH.
In many reactions, changes in substrates or products are not observable by spectrophotometric methods because they do not absorb light. These reactions can be measured by coupling them to enzymes that can be detected via a spectrophotometer. Light absorbing non-physiological substrates or products are synthesized for enzymes with substrates and products that do not absorb light. Visit the spectrophotometry page to learn more about the spectrophotometer assay.
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