Enzyme Assays

• Enzyme assays are laboratory methods for measuring enzymatic activity.

• Enzyme activity is a measure of the ability of a given enzyme to convert its substrate(s)
into its product(s).
• Depending on the enzyme it is typically assayed by measuring either the amount of
substrate that is disappearing, or the amount of product that is appearing over a
specified period of time, such that the final result is expressed in terms of moles of
conversion per unit mass of protein per unit time, for example, mole of product per
minute or nmol/mg protein/sec.
• Under the usual in vitro assay conditions, the enzyme is present in limiting or “catalytic”
amounts in the neighborhood of 1012 to 107 M while [S] is generally 106 to 102 M.
• The initial velocity (vi) is always directly proportional to total enzyme [E]t, and this fact
can be used to quantitate the amount of enzyme present.
• It should be stressed that the relationship between vi and [E]t is linear only if true initial
velocities are measured. Since vi varies with [S], the assay period must be short enough
to ensure that only a small fraction of the substrate is utilized (5% or less).
To assay the enzyme quantitatively, it is necessary to know
1. the overall equation of the reaction
2. an analytical procedure for determining the disappearance of the
substrate or appearance of the products
3. whether the enzyme requires cofactors or coenzymes.
4. the dependence of the enzyme activity on substrate concentration
5. the optimum pH
6. a temperature zone in which the enzyme is stable and has high
activity.
Continuous vs. Discontinuous Assays
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.
1. 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 [MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di
phenyltetrazolium bromide to its insoluble purple formazan, (E,Z)-5-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) 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.
2. 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 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.
3. 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.

4. 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.
• 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.
• 
• 6. 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 10 nM 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.
 
1. 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 14C, 32P, 35S and 125I. 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.
• 2. 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.
 Factors to Control in Enzyme Assays
• Salt concentration: Most enzymes cannot tolerate extremely high salt concentrations. The ions
interfere with the weak ionic bonds of proteins.
• Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such
as the halophilic (salt loving) algae and bacteria.
• Effects of Temperature: All enzymes work within a range of temperature specific to the organism.
Increases in temperature generally lead to increases in reaction rates.
• There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction
rates.
• This is due to the denaturing (alteration) of protein structure resulting from the breakdown of the
weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme
active site.
• The "optimum" temperature for human enzymes is usually between 35 and 40°C. The average
temperature for humans is 37°C. Human enzymes start to denature quickly at temperatures
above 40°C. Enzymes from thermophilic archaea found in the hot springs are stable up to 100°C.
 Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH.
The pH can stop enzyme activity by denaturing (altering) the three dimensional shape of the enzyme by
breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in the
stomach works best at a pH of 2 and trypsin at a pH of 8.

Substrate Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity).
However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the
molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter
how much additional substrate is added. The graph of the reaction rate will plateau.
• Macmolecular crowding: The phenomenon of macromolecular crowding alters the properties of molecules
in a solution when high concentrations of macromolecules such as proteins are present.
• Such conditions occur routinely in living cells; for instance, the cytosol of Escherichia coli contains about
300–400 mg/ml of macromolecules. Crowding occurs since these high concentrations of macromolecules
reduce the volume of solvent available for other molecules in the solution, which has the result of increasing
their effective concentrations.
• This crowding effect can make molecules in cells behave in radically different ways than in test-tube assays.
• Consequently, measurements of the properties of enzymes or processes in metabolism that are made in the
laboratory (in vitro) in dilute solutions may be different by many orders of magnitude from the true values
seen in living cells (in vivo).
• The study of biochemical processes under realistically crowded conditions is very important, since these
conditions are a ubiquitous property of all cells and crowding may be essential for the efficient operation of
metabolism.
•
Enzyme Units and Specific Activities
• In most preparations the actual molar concentrations of enzyme is unknown. Consequently, the
amount of enzyme present can be expressed only in terms of its activity.
• In order to standardize the reporting of enzyme activities, the Commission on Enzymes of the
International Union of Biochemistry has defined a standard unit:
• Officially, enzyme activity is defined in terms of "Units", i.e., the amount of enzyme that converts
1 µmol substrate to product in 1 minute, but this is based on an assumption of purified enzyme,
assayed under conditions where the substrate is not limiting.
• However, in most real-world cases, one is not working with a totally purified enzyme, but rather a
relatively crude mixture, e.g., an extract of tissue or cells or body fluids. Thus, in the literature,
enzyme activities are most often reported as extent of conversion per unit time and most kinetic
calculations are based on this latter method of expressing activity.
• If you measure the activity of a crude extract you will get a fairly high total activity, but it's specific
activity, i.e., activity per unit protein will be low, because your enzyme will be a very small part of
the extract. If you repeat measurements at each step during a purification, the total activity will
decrease, but specific activity will go up, because your enzyme is constituting an increasing
proportion of the total protein in the sample.
• Specific Activity (SA) The concentration of enzyme in an impure preparation is expressed in terms
of units/ml.
• The Specific Activity (SA) of the preparation is reported as units/mg protein. As the enzyme is
purified, the specific activity will increase to a limit (that of pure enzyme).
• Since v varies with [S], pH, ionic strength, temperature, and like, a given preparation can have an
infinite number of specific activities. Consequently, specific activities are usually reported for
optimal assay conditions at a fixed temperature (usually 25, 30, or 37oC), with all substrates
present at saturating concentrations.
• International System (SI) of Units
• A new unit of enzyme activity, the katal, has been proposed.
The katal (symbol: kat) is the SI unit of catalytic activity.
• One katal is that amount of enzyme that catalyzes the conversion of one
mole of substrate per second under defined conditions. Thus,
• one International Unit = 1/60 katal = 16.67 nkatal.
• One katal = 6 x 107 Units.
• Specific activity can be expressed as katals/kg protein or katal/mg
protein. The molar activity of an enzyme is the katals/mole protein with
units of per sec.
Turnover Number
• The “turnover number” of an enzyme is the number of substrate
molecules transformed per unit time by a single enzyme molecule (or
by a single catalytic site) when the enzyme concentration is the rate
limiting factor.
• The enzyme carbonic anhydrase, an important enzyme found in high
conc. in the RBC, is among the most active of all enzymes with a
turnover number of 36,000,000 per minute per enzyme molecule.
• General Purification Scheme
• Enzymes are purified by employing successive chemical or physical fractionation procedures. The
object of each step is to retain as much of the desired enzyme as possible while getting rid of as
much of the other proteins, nucleic acids, and the like, as possible.
• The efficiency of each step is given by the “yield” or “recovery” (the percent of the total enzyme
activity originally present that is retained) and the ‘purification” or “purification factor” (the
factor by which the specific activity of the preparation has increased). The object is to optimize
both factors. Sometimes a good yield is sacrificed for the sake of an excellent purification step;
sometimes a good purification step is not used because the yield is too low.
• A hypothetical purification scheme is shown in Table 7.1. The crude cell-free extract may be
prepared by a number of means depending on the nature of the starting tissue or cells and the
size of the preparation. Some common cell-breakage methods include (i) autolysis, (ii) freeze-
thaw, (iii) sonic, (iv) oscillation, (v) mechanical grinding, (with or without an abrasive), (vi) ballistic
homogenization, or (vii) disruption in any one of a number of pressure cells (X-press, French
press). The resulting homogenate is usually centrifuged to remove unbroken cells and large
debris.
• There are no general rules concerning the order of the purification steps although heat treatment (where
possible) and ammonium sulfate precipitation are usually done early in the purification sequence.
• Gel filtration can follow ammonium sulfate precipitation and, thereby, serve to desalt the preparation as
well as fractionate the proteins according to size.
• If ion-exchange chromatography is to follow the ammonium sulfate step, then it is a good idea to dialyze
the preparation first, or pass the preparation through a rapid gel filtration column (e.g., Sephadex G-25).
• The removal of the ammonium sulfate will facilitate the binding of the proteins to the ion-exchange
column.
• Other steps not shown in the purification table that may be highly effective for certain enzymes include
(i) differential centrifugation (for mitochondria, chloroplasts, nuclei, mitosomes, ribosomes), (ii) pH
precipitation, (iii) organic solvent precipitation (e.g., ethanol, acetone), (iv) protamine sulfate or
streptomycin sulfate precipitation (to precipitate nucleic acids and acidic proteins), (v) affinity
chromatography, and (vi) preparative gel electrophoresis.
• 
The purity of the final preparation should be checked by several methods before concluding that the
preparation is homogenous. Suitable methods include (i) analytical disc gel electrophoresis at several
pH values and gel concentrations, and (ii) ultracentrifugation. A homogenous enzyme preparation
should elute from an ion-exchange or gel filtration column as a single symmetrical activity and protein
peak with a constant specific activity throughout. A homogeneous preparation is by no means
necessary for kinetic analyses, but the purer the enzyme, the less the complications from the
competing reactions that may use up the substrate or the product.
• Problems and Solutions
• 7.3.1 Enzyme Assays
• Problem #1
• A crude cell-free extract contained 20 mg of protein per milliliter. Ten microliters of this extract in a
standard total reaction volume of 0.5 ml catalyzed the formation of 30 nmoles of products in 1 min
under optimum assay conditions (optimum pH and ionic strength, saturating concentrations of all
substrates, coenzymes, activators, and the like).
• (a) Express vi in terms of (i) nmoles/assay, (ii) nmoles/liter/min,
• (iii) nmoles/liter/min, (iv) moles/liter/min, and (v) M/min.
• (b) What would vi be if the same 10 l of extract were assayed in a total volume of 1.0 ml?
• (c) What is the concentration of the enzyme in the assay mixture and in the extract (in terms of
units/ml)?
• (d) What is the specific activity (SA) of the preparation?
• Problem #2
• An enzyme preparation has a specific activity of 42 units/mg protein
and contains 12 mg of protein per milliliter.
• (a) Calculate the initial velocity of the reaction in a standard 1 ml
reaction mixture containing: (i) 20 l and (ii) 5 l of the preparation.
• (b) Should the preparation be diluted before an assay?
• Problem #3
• One microgram of a pure enzyme (MW = 92,000) catalyzed a reaction
at a rate of 0.50 moles/min (Vmax) under optimum conditions.
• (a) Calculate the specific activity (SA) of the enzyme in terms of
units/mg protein and units/mole.
• (b) Calculate the turnover number.
• (c) How long is one catalytic cycle?
DNS binds only to reducing sugars (eg. glucose) and cannot bind to non reducing
sugars (eg.sucrose).During the incubation period of this experiment, enzyme acts
upon sustrates to give products which are reducing sugars. Then you add DNS. Then,
the next step is heating the reaction mixture which is responsible for 2 steps, one for
heat inactivation of enzyme and other for efficient binding of reducing sugars to DNS
to give ANS which shows maximum absorbance at 540 nm.

3, 5-Dinitrosalicylic acid (DNSA) is used extensively in biochemistry for the estimation of

reducing sugars. It detects the presence of free carbonyl group (C=O) of reducing sugars.
This involves the oxidation of the aldehyde functional group (in glucose) and the ketone
functional group (in fructose).
• Why is the absorbance reading on my device (spectrometer/colorimeter) unstable or nonlinear at
values above 1.0?
• FEBRUARY 25, 2020
• For most spectrometers and colorimeters, the useful absorbance range is from 0.1 to 1. Absorbance
values greater than or equal to 1.0 are too high. If you are getting absorbance values of 1.0 or above,
your solution is too concentrated. Simply dilute your sample and recollect data .
• Keep in mind that absorbance is the logarithm of the transmission (T) of light through a sample.
Transmission is the ratio of the intensity of light transmitted through the sample (I) to the intensity of
light transmitted through a blank (Io). So absorbance = log (Io/I).
• At an absorbance of 2 you are at 1%T, which means that 99% of available light is being blocked
(absorbed) by the sample. At an ABS of 3 you are at 0.1% T, which means that 99.9% of the available
light is being blocked (absorbed) by the sample. These ranges are outside the meaningful range of
most spectrometers and colorimeters.
• Note that there are spectrometers that will report meaningful values at absorbance ranges above 1.0