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Enzyme Activity and Assays

Introductory article
Article Contents

Robert K Scopes, La Trobe University, Bundoora, Victoria, Australia


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...
Measurement of Enzyme Activity

...
The objective of
measuring enzyme activity is normally to determine the
amount of enzyme present under defined conditions, so
that activity can be compared between one sample and
another, and between one laboratory and another
...
In many cases the activity is
measured in the opposite direction to that of the enzyme’s
natural function
...

The factors that affect the activity of an enzyme include
substrate concentrations(s), pH, ionic strength and nature
of salts present, and temperature
...
There are
many compounds that may act as inhibitors which repress
the activity, so they should not be present
...
However, it is worth noting that the converse,
namely the involvement of nonsubstrate activators, must
be attended to with many enzymes, since they can be totally
inactive without an activator present
...
Š
K+ ‡ ‰
...
Summary

exactly what the concentration is (some preparations of
unusual substrates may be impure, or the exact amount
present may not be known)
...
In most cases an enzyme assay has already been
established, and the substrate concentration, buffers and
other parameters used previously should be used again
...
The most extreme deviations
are with those enzymes known as ‘allosteric’, in which a
sigmoid shape of response is found (Figure 1)
...
Each
allosteric enzyme has its own specific characteristics, so we
cannot generalize about their behaviour
...
But
the limits may be wide, e
...
pH 5 to 10, or narrow, e
...
over
1 pH unit
...
The activity
can also be affected by the nature of the buffer used
...
Alternative buffers
for a given pH should be tested
...
However, if tested at low concentration, it is

‰/Š

where [S] is the substrate concentration, v is the rate
measured, Vmax is the maximum theoretical rate at infinite
substrate concentration, and Km is the Michaelis constant
...
The substrate concentration that is used in enzyme
assays is chosen according to parameters such as the Km,
the solubility of the substrate, whether high concentrations
may inhibit, and the cost of the substrate
...
Methods for Purifying Enzymes

Substrate concentration

Figure 1 Comparison of a conventional Michaelis–Menten enzyme with
an allosteric enzyme: the rate variation with substrate concentration
...
els
...
For standard enzyme
assays, a value of pH is chosen that is close to the optimum,
unless some other component of the assay mixture cannot
operate at that pH
...
In more cases than not, the optima in the
two directions will be different, especially if there is uptake/
evolution of a proton in the reaction
...


Effect of temperature
Temperature affects enzyme activity in much the same way
as it affects other chemical reactions
...
Thus it is important when
carrying out an enzyme assay to ensure that the temperature remains constant, and also that you know exactly
what it is
...


Effect of ionic strength, salts
This is a complex subject as each enzyme responds in a
unique way to ionic strength I (salt content)
...
Most are
inhibited at high ( 4 0
...
For most enzymes
there is a variation of activity with I, so the value should be
fixed and recorded (it is implicit in the total composition of
the assay buffer)
...
15 to 0
...


Initial Rates and Steady State Turnover
Definition of the initial rate of an enzymatic
reaction
An enzyme assay is set up with appropriate buffers and
with the substrates present, but no products
...
The initial rate of
reaction occurs at this moment (after a short lag, which
may be only milliseconds)
...
This
decline is often not observable because the thermodynamics of the reaction being catalysed greatly favours
2

product formation
...
It is the aim of a successful
enzyme assay to measure the product formation before it
has accumulated sufficiently to affect the initial rate
...


Steady state turnover
This term refers to the situation in which there is a steady
and unchanging flow of substrate through to product
...
More useful, though, is the use of
the term steady state in a metabolic pathway, in which the
net flow through an enzyme is determined by both the
concentration of its substrate, which is constantly being
replenished by the previous enzyme, and of its product,
which is steadily being removed by the next enzyme, all
enzymes having the same flux, or steady state net forward
rate
...
Thus ideally a method for measuring
either product or substrate in the presence of the other is
required
...
Stopped
assays involve stopping the reaction after a fixed time, then
measuring how much product has been formed
...

In many cases a selective method can distinguish between
substrate and product, so that no separation step is
required
...
Otherwise separation of unused
substrate from product may be needed
...

Separation methods include chromatographic (thin-layer
chromatography, TLC; high-performance liquid chromatography, HPLC), solubility and partition procedures
...
In some cases the enzyme can be
stopped by addition of a complexing agent such as

ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www
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Enzyme Activity and Assays

ethylenediaminetetraacetic acid (EDTA), which removes
metal ions essential for activity; even chilling on ice may be
sufficient
...


Continuous assays
The alternative to a stopped assay is a continuous one in
which the progress of the reaction is followed as it occurs
...
On the other hand
not all enzymes have an assay method that can be observed
continuously
...
g
...
In many
examples of hydrolase assays, an artificial substrate which
releases a coloured or fluorescent product is used
...
This can be overcome
using a coupled continuous method
...
A great
advantage of coupled assays is that the product is removed,
so helping to keep the measured rate constant over a long
period by avoiding product inhibition and reversal of
reaction
...


Measurement of Protein Concentration
The determination of how much protein is present is very
important when purifying an enzyme, since purity depends
on the removal of unwanted proteins, and can be assessed
by relating the activity to total protein present (the specific
activity)
...

With the exception of direct spectrophotometry, protein
methods are based on a chemical reaction, and comparison
of the colour produced with a standard protein
...

A second important feature is sensitivity; one does not
want to sacrifice a large proportion of a precious protein
just to measure how much is (was) there
...

Three chemical methods have been used most widely
...
This method has a very
low variability between proteins, but suffers from a very
low sensitivity: several mg are needed for good colour
development
...
Stopped assay
...
Continous, direct assay
...
Continous coupled assay
...
Continuous coupled assay
...


ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www
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Enzyme Activity and Assays

protein to spare
...
This also uses alkaline copper, but produces a
stronger colour by oxidation of residues in the protein with
a phosphotungstomolybdate reagent (Folin reagent)
...
Some
20 years later the simplest and now probably the most
widely used method came into force, the ‘Bradford’
(Coomassie blue) method
...

Even more sensitive than the Lowry method, a microBradford procedure can accurately measure as little as 1–2
mg of protein
...
Variability between proteins is a little
better than with the Lowry method, but variability can still
introduce errors of over 10% when comparing with BSA
...
But the final method which will be
mentioned is direct ultraviolet spectrophotometry
...
The traditional method is to measure at
280 nm
...
A 1 mg mL 2 1
solution of protein may have an absorbance at 280 nm
anywhere between zero and 3 1 , though the typical
protein will be in the range 0
...
5
...
66, but there is no point in ‘comparing’ the result with
BSA in this method
...
0 is commonly used, and this does give some idea
of the relative amounts of protein in different fractions
...

Alternatively, if the exact Tyr/Trp content and molecular
weight is known, which is the case for many proteins whose
gene has been sequenced, an estimate of the extinction
coefficient can be made, within about 10% accuracy
...
Since the peptide bond content is roughly proportional to mass of the protein, this method has relatively
little variability between proteins
...
By also making a measurement at
280 nm, the contribution in the far range by the aromatic
amino acids (Tyr, Trp) can be corrected for
...
From these readings and calculations one can
then determine the extinction coefficient at 280 nm, which
can be used for future measurements on the protein
...


Methods for Purifying Enzymes
This section can only be a brief outline of the many
methods that are available for purifying enzymes
...


Traditional methods
These have developed over a period of more than a century,
and have reached degrees of sophistication that can enable
rapid purification on scales varying from micrograms to
kilograms
...

Precipitation methods

O-PQRSPQTR STUVVQSQURP VTW / +X +Y /
/`__NabW ‡ cc__NaWd
ZT[\PQTR ,P ]^_ R+ ˆ
ef

‰]Š

where MW is the molecular weight of the protein, NTyr is
the number of tyrosine residues, and NTrp is the number of
tryptophan residues per molecule
...
The main one that
is used is salt, especially salts with divalent anions,
commonly ammonium sulfate
...
The proteins
precipitating through one short range of salt concentration

ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www
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Enzyme Activity and Assays

are expected to include the majority of the desired enzyme
...
This is one of the oldest procedures, but
still finds use in many situations
...


through
...
In this method a column is packed with bead
particles that are porous, with the pores ranging in size
from too small to allow the desired protein through, to
much larger ones which the protein can access
...
On running a gel filtration column, the largest
proteins emerge first, and the smallest last
...


Adsorption methods
There are many adsorbents for proteins
...
Adsorption is generally carried
out in columns as chromatography
...
By using subtle gradients of buffer, a high degree of
resolution between different proteins can be obtained, and
so a high degree of purification of the desired enzyme
...
The
interacting ‘ligand’ is chemically attached to neutral beads
which constitute the adsorbent in the column
...
Antibody columns are highly specific, but there
are many problems in using them on a routine basis
...
A further class is the ‘pseudoaffinity’ or
‘biomimetic’ adsorbents, which consist of ligands that do
not necessarily resemble any known feature of the enzyme,
but which have been discovered by empirical experimentation to have a specific interaction with it
...

‘In solution’ methods
The third category of protein separation methods consists
of a group in which the proteins remain in solution while
the separation takes place
...
Electrophoresis separates principally according to
the charges on the protein molecules, and in some systems
by their relative size also
...
The main problem is in the
design of suitable equipment that is easy and safe to use
...
Expression in a host organism
has many advantages
...
It
is the latter that has made protein purification a very simple
process, even for the inexperienced
...

The host organism is most often Escherichia coli
...
But often this protein does not fold correctly, and is
precipitated in insoluble ‘inclusion bodies’
...
A further cleaning up by one of the traditional
techniques such as ion exchange chromatography or gel
filtration completes the process
...

One trend for commercial enzyme production is to source
the enzyme from a thermophilic bacterium, so that the
enzyme is very heat-stable
...
coli extract can then be
heat-treated, which denatures most of the host proteins
...
A short stretch of
amino acids added either to the N- or C-terminal can be
used; typically a six-histidine stretch may be added,
allowing selective adsorption on an immobilized metal

ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www
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Enzyme Activity and Assays

column, which coordinates with the histidines
...
If necessary, the tag
can be removed by specific proteolysis, but usually the
presence of a short tag does not affect the enzyme’s
properties
...
The main advantage of these
whole-protein tag systems is that the expression level of an
otherwise poorly expressed protein is much enhanced
...


Summary
For consistent and reproducible results, an enzyme assay
should be carried out in well-defined conditions that can be
duplicated in other laboratories
...
This will define how
much enzyme is present in a sample compared with others,

6

but generally will not indicate the flux of metabolites
through that enzyme in vivo
...
Classic methods of
precipitation, adsorption and in solution are used, but if
the enzyme is produced recombinantly, modification of the
polypeptide at the gene level can greatly simplify the
purification process
...
Analytical Biochemistry 176: 1–14
...
Oxford: IRL Press
...
) (1996) Enzymology Labfax
...

Price NC and Stevens L (1989) Fundamentals of Enzymes
...

Purich DL (ed
...

New York: Academic Press
...
Methods in
Enzymology 182: 38–49
...
New
York: Springer-Verlag
...
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