Enzyme assay
Laboratory methods for measuring enzymatic activity.
Enzyme assays are lab techniques used to measure how much enzymatic activity is present, making them essential for studying enzyme kinetics and inhibition.
The amount of an enzyme can be expressed in molar terms like any chemical, or in units of activity. Enzyme activity measures the quantity of active enzyme and depends on physical conditions, which must be stated. It is calculated as the moles of substrate converted per unit time, which equals the reaction rate multiplied by the reaction volume. The SI unit is the katal (1 katal = 1 mole per second), but this is impractically large. More common is the enzyme unit (U), defined as 1 micromole per minute, which equals 16.67 nanokatals. Activity in katals usually refers to the enzyme’s natural target substrate. It can also be given for standardized substrates, like gelatin (measured in gelatin digesting units, GDU) or milk proteins (measured in milk clotting units, MCU). These units reflect how fast one gram of enzyme digests the respective substrate; 1 GDU roughly equals 1.5 MCU. Adding more substrate increases reaction rate until active sites become saturated, after which the rate plateaus.
Specific activity is another common unit: the activity per milligram of total protein, typically expressed as micromoles per minute per milligram. It indicates enzyme purity in a mixture. Specific activity equals the reaction rate times the reaction volume divided by the total protein mass. The SI unit is katal per kilogram, but micromoles per minute per milligram is more practical. For a pure enzyme, specific activity is usually constant at a saturating substrate concentration and reflects processivity. To correct for errors from batch differences or misfolded enzymes, active site titration can be performed—for example, using an irreversible inhibitor to count active sites. Specific activity is then expressed per milligram of active enzyme. If the enzyme’s molecular weight is known, the turnover number (micromoles of product per second per micromole of active enzyme) can be calculated, representing how many times each enzyme molecule completes its catalytic cycle per second.
Reaction rate is the concentration of substrate lost or product formed per unit time (moles per liter per second). Purity is calculated as 100% times the specific activity of the sample divided by the specific activity of the pure enzyme.
- Field
- Biochemistry
- Known for
- Measuring enzymatic activity, enzyme kinetics, and enzyme inhibition
Lore & Background
Enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods exist, and many enzymes can be assayed in several ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments: initial rate experiments, progress curve experiments, transient kinetics experiments, and relaxation experiments. Initial rate experiments are the most commonly used, as they are simplest to perform and analyze. Enzyme activity is calculated using the formula a = n/t = r × V, where a is enzyme activity, n is moles of substrate converted per unit time, r is the rate of reaction, and V is reaction volume. The SI unit is the katal (1 katal = 1 mol s⁻¹), but a more practical unit is the enzyme unit (U) = 1 μmol min⁻¹. Specific activity is another common unit, expressed as μmol min⁻¹ mg⁻¹, giving a measurement of enzyme purity. Continuous assays, such as spectrophotometric and fluorometric assays, provide a continuous reading of activity, while discontinuous assays require stopping the reaction and measuring concentrations.
Reader's Guide
Enzyme assays are fundamental tools in biochemistry, enabling the quantification of enzyme activity and the study of enzyme kinetics and inhibition. They allow researchers to determine how fast an enzyme converts substrate to product, which is essential for understanding metabolic pathways, drug development, and disease mechanisms. The use of enzyme units (U) and specific activity provides standardized ways to compare enzyme preparations and assess purity. Assays can be continuous or discontinuous, with spectrophotometric methods being particularly common due to their convenience and ability to monitor reactions in real time. Coupled assays extend the applicability of spectrophotometric methods to enzymes whose reactions do not directly produce a change in light absorbance. The choice of assay type depends on the enzyme and the experimental goals, with initial rate experiments being the most straightforward. Overall, enzyme assays are indispensable for both basic research and applied fields such as biotechnology and clinical diagnostics.
Did You Know?
- Specific activity is expressed as μmol min⁻¹ mg⁻¹ and gives a measurement of enzyme purity.
- Initial rate experiments are the most commonly used type of enzyme kinetics experiment because they are simplest to perform and analyze.
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