2. The purpose of enzyme assay is to determine how much of a given
enzyme, of known characteristics, is present in a tissue homogenate,
fluid or partially purified preparation.
Enzyme activity is measured by measuring the catalytic activity or the
rate at which it catalyzes the conversion of substrates to products.
Activity is proportional to the concentration of enzymes.
Specimens for enzyme assay are usually stored at temperatures below
4°C, both before and after the preparation of homogenates
3. Assay By Kinetic Determination Of Catalytic
Activity
to find out how much substrate is capable of converting to product in a given
time under specified conditions.
the initial velocity of an enzyme-catalyzed reaction taking place under
conditions where the Briggs-Haldane steady-state assumptions are valid is given
by
v0 = k2[E0][S0]
([S0 ] + Km),
here k2 is the rate constant relating to product formation
(i.e. kca1).
If the value of [So] used in a particular assay is fixed, the only variables are v0
and[Eo],
so vo =constant x [E0].
4. in practice it is more reliably valid when the substrate concentration is high enough to be
approximately saturating.
There are two main reasons for this.
1, integration of the appropriate rate equation shows that the linear, steady-state phase of the
reaction is more prolonged as the degree of enzyme saturation is increased (all other factors being
equal).
In simple terms, this is because the rate of utilization of substrate becomes less significant in relation
to the total concentration of substrate present as [So] is increased. For this reason more accurate
estimates of v0 can be obtained at high rather than at low [So] values
2, at high [So],
([S0] + Km) = [S0],
so v0 = k2[E0] and v0 does not vary with small changes in [S0].
This means that reproducible results can be obtained from an assay system without it being
necessary to fix the substrate concentration at an extremely precise value, provided [So] is sufficient to
almost saturate the enzyme. Hence the setting-up of a reliable assay system is more convenient the
higher the chosen value of [S0].
5. According to the Michaelis-Menten equation, an enzyme is only completely
saturated by substrate at infinite substrate concentration, so it is necessary to talk in
terms of near-saturation rather than complete saturation.
the vo/Vmax ratio to be calculated for each initial substrate concentration, provided
the Km value is known.
It should be clearly understood that the vo/Vmax ratios and fractional saturation
values are independent of [E0], provided [S0] » [Eo], However, the actual values of
v0 and Vmax do vary with [Eo], which is the whole point of enzyme assay by kinetic
methods
6. If a reaction involves more than one substrate, then the concentrations of
each must be fixed, preferably at near-saturating levels.
The rate of an enzyme-catalysed reaction may also depend on the
concentration of a cofactor (or cofactors).
As with substrates, each cofactor must be present at a fixed
concentration, and preferably in excess, if a reliable and reproducible
system for enzyme assay is to be obtained.
A procedure being used for enzyme assay could always be
improved by increasing the substrate concentration, without
limit, other factors such as the solubility of the substrate, the
cost of the substrate, and the possibility of substrate inhibition
have to be taken into consideration .
7. • The reaction should be carried out under fixed and suitable conditions of pH, ionic
strength and temperature.
pH
• Enzymes are often assayed at their optimal pH , but this is not essential. An enzyme
might not operate at its exact optimal pH in vivo, so there is no fundamental reason for
investigating its activity at this pH in vitro;
• the pH chosen for an assay system must be sufficiently near the optimum for an
appreciable rate of reaction to take place.
• It must also be one where the enzyme is relatively stable, for enzyme stability can vary
with pH.
• With some reversible reactions, the pH may influence the forward and back reactions
differently. For example, assays involving lactate dehydrogenase are best performed
in the direction of lactate production at pH 7, but in the direction of pyruvate formation
at pH 10.
Suitable conditions
8. Ionic strength
The presence of salts may affect enzyme-catalysed reactions by shifting the equilibrium of
any of the steps involved. They may also effectively reduce the concentration of a
substrate by complexing with it. Hence enzyme assays must be performed under carefully
controlled conditions of ionic strength and composition.
Temperature
The most common temperatures at which enzyme assays are performed are 25°C (as
recommended by IUB in 1961), 30°C (the 1964 IUB recommendation) or 37°C . Many
enzymes are somewhat unstable at 37°C, but this temperature provides conditions which
are the nearest approximation to those found in human beings and other mammals, and
reaction rates are faster than at 25°C or 30°C.
9. Unit of enzyme activity
Enzyme activity is measured as the amount of substrate lost (or product
gained) per unit time, and it should also be related in some way to the
amount of specimen used for assay.
In 1961 the enzyme commission of the IUB defined an Enzyme Unit (U),
later to be known as an International Unit (IU) (although the original term
continued to be widely used), as the amount of enzyme causing loss of 1
μmol substrate per minute under specified conditions.
Later, in 1973, the Commission on Biochemical Nomenclature introduced
the
katal (kat) as the Systeme International (SI) unit of enzyme activity:
this is defined as the amount of enzyme causing loss of 1 mol substrate per
second under specified conditions.
10. Coupled Kinetic Assays
if no easily measurable change takes place in a reaction, it may still be possible to
develop a kinetic assay for the enzyme by coupling the reaction to one where suitable
changes do occur.
For example, in the reaction catalysed by alanine transaminase,
L-alanine + 2-oxoglutarate ~ L-glutamate + pyruvate,
no reactant or product is coloured, nor does any one of them significantly absorb
ultraviolet light.
the reaction may be monitored spectrophotometrically if it is coupled to a second
reaction, in which the pyruvate formed in the first reaction acts as a substrate for
lactate dehydrogenase (LDH) and is reduced to lactate by the action of the coenzyme
(and co-substrate) NADH, as follows:
pyruvate + NADH + H+ ~ lactate + NAD+.
NADH absorbs light at 340 nm, but NAD+ does not , so the course of the reaction may
be followed by monitoring at this wavelength. If conditions are chosen carefully, the
rate of the second reaction will be an indicator of the rate of the first reaction.
11. Let us consider the general situation:,
where E1 is the enzyme catalysing the primary reaction and E2 the enzyme catalysing the
indicator reaction.
At zero time, the concentrations of B and C will be zero, and the concentration of A
should be fixed and non-limiting .
If there are any second substrates or cofactors for E1 or E2 (e.g. NADH is a co
substrate in the example where E2 is LDH), then these should also be present initially
at fixed and non-limiting concentrations.
As the reaction proceeds, the concentration of B (pyruvate in the example where E2 is
LDH) will start to rise from its initial value of zero. In general, the rate of change of [B]
is given by
d[B]/dt = v1 - v2 ,
where v1 is the velocity of the primary reaction and v2 the velocity of the
indicator reaction. As with the velocity of any single-enzyme system, v1 should reach a
constant value almost instantaneously and remain at this value over the period of interest
(the next few minutes). In contrast, v2 will have an initial value of zero (since [B] is initially
zero) and will rise as [B] rises
12. according to the Michaelis Menten equation
related to the reaction catalysed by the enzyme E2•
Therefore,
Integration of the expression for d[B]/dt shows that the time taken for this
steady-state to be established (or at least very nearly established) is
directly proportional to Km B Vmax B. Now, Kmax is a constant
characteristic of the enzyme E2, but Vmax is directly proportional to the
concentration of E2
13. Hence, the larger the concentration of E2, the quicker is the
establishment of a near steady-state for the overall system, with v1 =
v2•
If there is so little E2 present that Vmax is less than vi. then a steady-
state can never be set up, [B] will continue to rise and v2 can never
reach the value of v1.
It is therefore important that sufficient of the indicator enzyme is
added to a coupled assay system to ensure that there is a minimum of
delay before a steady-state is established, and that the rate of the
indicator reaction at steady-state is a reasonable approximation to the
rate of the primary reaction.
As with a single-enzyme system, the validity of a coupled assay
procedure can be checked by assaying different amounts of the same
enzyme preparation: the specific activity should be found to be the
same in each case.
14. Radioimmuno assay [ R I A ]
The sample to be assayed is mixed with antibody and with a small amount of
the labelled antigen. The following reactions take place, and are allowed to
come to equilibrium
The antibody, with its bound antigen, is then separated from the free antigen and the
distribution of radioactive isotope between the free and bound antigen fractions is
investigated. It will be realized that the labelled antigen competes with the antigen in the
sample for the available binding sites on the antibody, so the higher the concentration of
antigen in the sample, the less radioactive antigen will be able to bind to the antibody and the
greater will be the radioactive content of the free antigen fraction. In this way, the
concentration of antigen in the sample can be estimated.
Antibodies for the RIA of enzymes may be prepared in the form of antisera by
immunizing rabbits with the required enzyme. For example, the blood of a rabbit
immunized in the footpad with human pancreatic a-amylase contains sufficient
antibodies within a few weeks to be usable as an antiserum.
15. Immunoradiometric assay
A modified form of RIA, utilizes an immobilized antibody to
which the sample antigen can bind, together with a separate,
radio-labelled antibody which binds to a different site on the
antigen.
This technique can be applied to proteins, since these are large
enough to have two separate binding-sites for antibody, and it
will be noted that there is no requirement for a radio-labelled
sample of antigen.
16. TYPES OF ENZYME
ASSAYS
CONTINUOUS ASSAYS
With continuous assays
one can measure the
linearity of the assay
which can be used to
conduct a fixed timed
assay
A few methods are
spectrophotometric,
fluorometric,
calorimetric and
chemiluminescent
DISCONTINUOUS ASSAYS
when samples are3
taken from an enzyme
reaction at intervals and
the amount of product
formation or substrate
consumption is measured
in these samples.
Radiometric and
chromatographic
methods
