2011-05-19

Gelatin digesting units

Enzyme assay

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.

Enzyme units

Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.

Enzyme activity

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s, but this is an excessively large unit. A more practical and commonly used value is 1 enzyme unit (U) = 1 μmol min. 1 U corresponds to 16.67 nanokatals.

Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured in milk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU.

Specific activity

The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol minmg). Specific activity gives a measurement of the activity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of total protein. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal kg, but a more practical unit is μmol mg min. Specific activity is a measure of enzyme processivity, at a specific (usually saturating)substrate concentration, and is usually constant for a pure enzyme. For elimination of errors arising from differences in cultivation batches and/or misfolded enzyme etc. an active site titration needs to be done. This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should then be expressed as μmol min mg active enzyme.

Related terminology

The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L s ).

The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated.

Types of assay

All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:

Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

Continuous assays

Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.

Spectrophotometric

In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.

UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidized forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.

Direct versus coupled assays

Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the substrate of another, easily detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase.

Fluorometric

Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.

An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.

Calorimetric

Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way.

Chemiluminescent

Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected.

The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.

Light Scattering

Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.

Microscale Thermophoresis

Microscale Thermophoresis (MST) measures the size, charge and hydration entropy of molecules/substrates in real time. The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be measured with high time resolution in real time. The material consumption of the all optical MST method is very low, only 5 µl sample volume and 10nM enzyme concentration are needed to measure the enzymatic rate constants for activity and inhibition. MST allows to measure the modification of two different substrates at once (multiplexing) if both substrates are labeled with different fluorophores. Thus substrate competition experiments can be performed.

Discontinuous assays

Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.

Radiometric

Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are C, P, S and I. Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).

Radioactivity is usually measured in these procedures using a scintillation counter.

Chromatographic

Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than HPLC instruments (see High-performance liquid chromatography#Pump_pressure).

Factors to control in assays

List of enzyme assays

See also

References

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  2. How Many? A Dictionary of Units of Measurement By Russ Rowlett at the University of North Carolina at Chapel Hill
  3. Schnell, S., Chappell, M.J., Evans, N.D. Roussel, M.R. (2006). "The mechanism distinguishability problem in biochemical kinetics: The single-enzyme, single-substrate reaction as a case study". Comptes Rendus Biologies 329 (1): 51–61. doi:10.1016/j.crvi.2005.09.005. PMID 16399643.
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  10. Churchwella, M; Twaddlea, N; Meekerb, L; Doergea, D. (October 2005). "Improving Sensitivity in Liquid Chromatography-Mass Spectrometry". Journal of Chromatography B 825 (2): 134–143.
  11. Daniel RM, Peterson ME, Danson MJ, et al. (January 2010). "The molecular basis of the effect of temperature on enzyme activity". Biochem. J. 425 (2): 353–60. doi:10.1042/BJ20091254. PMID 19849667.
  12. Cowan DA (1997). "Thermophilic proteins: stability and function in aqueous and organic solvents". Comp. Biochem. Physiol. A Physiol. 118 (3): 429–38. doi:10.1016/S0300-9629(97)00004-2. PMID 9406427.
  13. Minton AP (2001). "The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media". J. Biol. Chem. 276 (14): 10577–80. doi:10.1074/jbc.R100005200. PMID 11279227. http://www.jbc.org/cgi/content/full/276/14/10577.

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