methods of enzyme assay

Types of Enzyme assay
Two types (Based on sampling methods)
UV/VIS SPECTROSCOPY,FLURESCENCE METHOD/FLUORIMETRIC,RADIOISOTOPIC METHOD,CHEMILUMINESCENT,ION SELECTIVE ELECTRODE,OXYGEN ELECTRODE,IMMUNOCHEMICAL METHODS

1. Methods to assay enzymes
Introduction:
• Assay is an act of analyzing test or appraisal to determine the components of a substance or
object.
• Enzyme assays are laboratory methods for measuring enzymatic activity.
• They are vital for the study of enzyme kinetics and enzyme inhibition.
• The assay is the act of measuring how fast a given (unknown) amount of enzyme will
convert substrate to product (the act of measuring a velocity).
• Enzyme assays measure either the disappearance of substrate over time or the appearance
of product over time.
Types of Enzyme assay
Two types (Based on sampling methods)
1. Continuous assays
Continuous assays are most convenient, with one assay giving the rate of reaction with
no further work necessary. It gives a continuous reading of activity, multiple
measurements, usually of absorbance change are made during the reaction either at
specific time intervals (usually every 30 or 60 seconds) or continuously by a continuous-
recording spectrophotometer.
A few methods are spectrophotoometric, fluorometric, calorimetric and chemi-
luminescent.

2. Discontinuous assays:
In this assay where the samples are taken, the reaction stopped and then the
concentration of substrates/products determined.
The discontinuous assays are radiometric and chromatographic.

2. Features of a good Enzyme assay
• Simple and Specific
• Rapid (one doesn’t need to wait for hrs or weeks for the results to appear)
• Sensitive ( very little sample)
• Easy to use
• Economical

Measurement of enzyme activity by spectroscopy
• 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.
The following spectroscopic techniques are used: Fluorescence spectroscopy, UV/VIS
Spectroscopy, Spectrophotometric Assays, and Infrared spectroscopy.
UV/VIS SPECTROSCOPY:
UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400
nm.) is absorbed by the molecule. Absorption of the ultra-violet radiations results in the excitation
of the electrons from the ground state to higher energy state. The energy of the ultra-violet
radiation that are absorbed is equal to the energy difference between the ground state and higher
energy states (deltaE = hf).
Principle of UV spectroscopy
UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic
light is passed through a solution of an absorbing substance, the rate of decrease of intensity of
radiation with thickness of the absorbing solution is proportional to the incident radiation as well
as the concentration of the solution.
The expression of Beer-Lambert law is-
A = log (I0/I) = Ecl
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell

3. C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing
light of a given wavelength, the greater the extent of light absorption. This is the basic principle of
UV spectroscopy.


Instrumentation and working of UV spectroscopy
Instrumentation and working of the UV spectrometers can be studied simultaneously. Most of the
modern UV spectrometers consist of the following parts-
Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and
suitable light source as they cover the whole UV region.
Monochromator- Monochromators generally composed of prisms and slits. The various
wavelengths of the light source which are separated by the prism are then selected by the slits.
Sample and reference cells- One of the two divided beams is passed through the sample solution
and second beam is passé through the reference solution. Both sample and reference solution are
contained in the cells.
Detector- Generally two photocells serve the purpose of detector in UV spectroscopy. One of the
photocell receives the beam from sample cell and second detector receives the beam from the
reference.
Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The
main purpose of amplifier is to amplify the signals many times so we can get clear and recordable
signals.

4. Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to
the computer. Computer stores all the data generated and produces the spectrum of the desired
compound.
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.

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.


Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.
Here, the product of one reaction is used as the substrate of another, easily detectable reaction. For
example, figure 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.
FLURESCENCE METHOD/FLUORIMETRIC :
Compounds are said to be fluorescent when they absorb light of one wavelength and then emit
light of a longer wavelength.

5. At low concentrations, the intensity of fluorescence (fr) is related to the intensity of the incident
light (fo) of appropriate wavelength by the relationship:

Where is the molar absorption coefficient, c the molar concentration, l the length of the light-path
and q the quantum efficiency (i.e. the number of quanta fluoresced divided by the number of
quanta absorbed)
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 nonfluorescent.
Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction
reactions by an increase.
More sensitive than spectrophotometric assays, but can suffer from interference caused by
impurities and the instability of many fluorescent compounds when exposed to light.
Detection in small quantities
Non dangerous
Other example:-

RADIOISOTOPIC METHOD:
• The use of a radioactively-labelled substrate can be valuable in enzymatic analysis. The
isotopes most commonly used for labelling purposes are 3H (tritium), 14C(carbon),
32P(phosphorous), 35S(sulphur) and 131I(iodine). All of these isotopes emit beta-radiation
(electrons) as they decay.
• After the enzyme-catalysed reaction has progressed for a specified period, it is terminated.
The substrate is then separated from the product, usually by chromatography or
electrophoresis, and the product concentration is determined indirectly by measuring the
radioactivity of the product fraction.
• A typical example of enzymatic analysis by a radiochemical procedure is that involving the
cholinesterase-catalysed hydrolysis of [ 14C]-acetylcholine.

6. • 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., which
measures the ionizing radiation.
• Very sensitive but hazardous
CHEMILUMINESCENT:
It 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.
ION SELECTIVE ELECTRODE:
An ion-selective electrode (ISE), also known as a specific ion electrode (SIE), is a transducer (or
sensor) that converts the activity of a specific ion dissolved in a solution into an electrical potential.
The voltage is theoretically dependent on the logarithm of the ionic activity, according to the
Nernst equation. Ion-selective electrodes are used in analytical chemistry and
biochemical/biophysical research, where measurements of ionic concentration in an aqueous
solution are required.
Working Mechanism
• The ion-selective electrode works based on the principle of a galvanic cell. It consists of a
reference electrode, ion-selective membrane and voltmeter.
• The transport of ions from an area of high concentration to low concentration through the
selective binding of ions with the specific sites of the membrane creates a potential
difference.
• This potential is measured with respect to a stable reference electrode having a constant
potential, and a net charge is determined. The difference in potential between the
electrode and the membrane depends on the activity of the specific ion in solution.
• The strength of the net charge thus measured is directly proportional to the concentration
of the selected ion.

7. • The electric potential can be calibrated by direct means, standard additions and titrations.
However, direct calibration is the most common means of measuring concentrations.

Enzyme electrodes definitely are not true ion-selective electrodes but usually are considered within
the ion-specific electrode topic. Such an electrode has a "double reaction" mechanism - an enzyme
reacts with a specific substance, and the product of this reaction (usually H+ or OH−) is detected
by a true ion-selective electrode, such as a pH-selective electrodes. All these reactions occur inside a
special membrane which covers the true ion-selective electrode, which is why enzyme electrodes
sometimes are considered as ion-selective. An example is glucose selective electrodes
Advanteages
- Exhibit wide response
- Exhibit wide linear range
- Low cost
- Color or turbidity of analyte does not affect results
- Come in different shapes and sizes
OXYGEN ELECTRODE
The oxygen electrode is an electrode that measures ambient oxygen concentration in a liquid using
a catalytic platinum surface according to the net reaction:
O2 + 4 e− + 4 H+ → 2 H2O
It improves on a bare platinum electrode by use of a membrane to reduce fouling and metal
plating onto the platinum.

8. Mechanism of Oxygen (pO2) Electrode:
The oxygen electrode laid the basis for the first glucose biosensor (in fact the first biosensor of any
type), invented by Clark and Lyons in 1962.

The basic concept of the glucose biosensor is based on the fact that the immobilized glucose oxidase (GOx)
catalyzes the oxidation of β-D-glucose by molecular oxygen producing gluconic acid and hydrogen peroxide.
In order to work as a catalyst, GOx requires a redox cofactor—flavin adenine dinucleotide (FAD).
FAD works as the initial electron acceptor and is reduced to FADH2.
Glucose + GOx − FAD
+
→ Glucolactone + GOx − FADH2
The cofactor is regenerated by reacting with oxygen, leading to the formation of hydrogen peroxides.
GOx − FADH2 + O2 → GOx − FAD + H2 O2
Hydrogen peroxide is oxidized at a catalytic, classically platinum (Pt) anode. The electrode easily recognizes
the number of electron transfers, and this electron flow is proportional to the number of glucose molecules
present in blood.
H2O2 → 2H
+
+ O2 + 2e
IMMUNOCHEMICAL METHODS
Enzyme immunoassays utilize enzymes, usually peroxidase or alkaline phosphatase, to detect and
quantify immunochemical reactions. Both antibodies or antigens can be labelled with an enzyme
in order to aid detection.
Types:
• Heterogeneous enzyme immunoassay
• Homogeneous enzyme immunoassay

Heterogeneous enzyme immunoassay:

9. A heterogeneous enzyme immunoassay method is also called enzyme-linked immunosorbent assay
(ELISA). In this type of assay, one of the immunochemical reaction components (antigen or
antibody) is first non-specifically adsorbed to the surface of a solid phase. Tubes, wells of microtiter
plates, and magnetic particles may be used as the solid phases. The solid phase facilitates
separation of bound- and free-labelled reactants.
Homogeneous enzyme immunoassay:
A homogeneous enzyme immunoassay is a sort of enzyme multiplied immunoassay technique
(EMIT) that does not require a separation of bound and free labelled antibodies or antigens. It is
simple to perform and has been used for estimation of drugs, hormones and metabolites. Sample
containing the estimated antigen is mixed with a known quantity of the same antigen labelled with
enzyme (conjugate); and limited amount of specific antibody is added. The unlabelled antigen
from the sample competes with the conjugate for the antibody. Binding of antibody on the
conjugate results in loss of enzyme activity due to blocking the enzyme active site or change of its
conformation. The more unlabelled antigen is present in the solution, the less conjugate will bind
to the antibody, and more enzyme activity will be preserved in the solution. Therefore, the enzyme
activity is proportional to the antigen concentration in the sample.
The reaction scheme in these immunochemical assays can follow either competitive, or non-
competitive approach.
Competitive enzyme immunoassay:
This assay is always performed under condition of antigen excess. The enzyme-labelled antigen
(conjugate) is mixed with serum sample containing the unknown amount of antigen. The serum
antigen and enzyme-labelled antigen compete for binding sites of a limited quantity of specific
antibodies bound to the solid phase. Labelled and non-labelled antigen bind to the antibody in the
same proportion as is their proportion in the reaction mixture. In other words, the more non-
labelled antigen is contained in the mixture the less labelled antigen is bound.

10. Principle of competitive immunoassays – various proportions of antigen and antibody
Under these conditions the probability of the antibody binding the labelled antigen is inversely
proportional to the concentration of unlabelled antigen. The higher the amount of unlabelled
antigen in the sample, the more labelled antigen remains free (unbound). After an incubation, all
the unbound both enzyme-labelled and unlabelled antigens are removed by washing along with all
other serum constituents. In the subsequent indicator reaction for detection of enzyme activity a
chromogenic substrate for the enzyme label is added. The intensity of colour is inversely
proportional to the concentration of the antigen in serum sample. The results are obtained from a
calibration curve constructed with the standards of known concentration of antigen

11. Competitive enzyme immunoassay
Non-competitive enzyme immunoassay (sandwich methods):
This kind of enzyme immunoassay can be adopted for measurement of either antigens or
antibodies. It is a heterogeneous immunoassay using a solid phase coated with antibody or antigen,
which must always be in excess over the analyte being measured.
Non-competitive enzyme immunoassay for determination of antigen:
This non-competitive enzyme immunoassay is suitable for the measurement of large antigens with
several antibody-binding sites. Two different molecules of antibodies directed against various
epitopes are necessary for performing the assay. The first antibody is in excess adsorbed to a solid
phase. The serum sample or calibrators containing the desired antigen are added to the well with
immobilised antibody. The first imunochemical reaction occurs. Since the antibody is in excess, all
antigen molecules should bind. After an incubation, all the non-reacting material in the sample is
washed away. Then a second enzyme-labelled antibody (different from the first antibody) is added
in excess. In the second immunochemical reaction, another antigen epitope binds to the second
labelled antibody. “Sandwich complex“ consisting of solid-phase antibody – antigen –
enzymelabelled antibody is formed. After washing of all the unreacted enzyme-labelled antibody,

12. the substrate is added. The intensity of the finally measured coloured product of the enzyme
reaction is directly proportional to the amount of antigen.

Non-competitive enzyme immunoassay for determination of antigen
Example of fast immunochemical diagnostic methods
Detection of human chorionic gonadotropin (pregnancy test) Porous membrane that serves as a
support for the test is divided into four areas. Three different antibodies are employed. The first
sample area contains specific anti-hCG antibody labelled with microparticles of colloid gold or
blue latex (Ab1). When urine sample is applied molecules of this antibody flow to the second,
detection area. Another specific antibody against hCG (Ab2) is anchored in this area. If chorionic
gonadotropin is contained in the sample a combined immunocomplex with both labelled and
anchored antibody is formed (Ab2-hCG-Ab1) and a coloured band displays in the detection area.
Excess of the labelled antibody is caught in the third (control) area with immobilised antibodies

13. against labelled anti-hCG. A band in the control area is formed, indicating that the test works
properly. Thus, two bands are interpreted as a positive result. In case the urine sample contains no
hCG the labelled antibody is bound in the third area only. One band in the control zone only is
therefore read as a negative result. If no band is displayed in the control area the test is invalid.
Some tests are so designed that the control zone has a shape of a minus sign and the detection
zone crosses it perpendicularly. A plus sign therefore appears in case of positive result.

Tentative test for hCG in urine