Enzyme units, activity and specific activity explained

There is much confusion over enzyme units, enzyme activity and specific activity. This guide
seeks to explain these concepts in simple terms so that you will easily be able to form an
opinion about the quality and value of competing product offerings.

The standard definition of the enzyme unit is given below:

1 unit (U) is the amount of enzyme that catalyses the reaction of 1 mol of substrate per
minute (definition A)

However, suppliers of enzymes seldom use this definition as it frequently requires labels to
be written in fractions of units or converted into milli-units. The following definition is more
commonly employed:

1 unit (U) is the amount of enzyme that catalyses the reaction of 1 nmol of substrate per
minute (definition B)

While the definitions may vary the user can still make valid product comparisons as long as
suppliers are explicit about their unit definitions and the assay conditions employed.

How do I compare enzymes from different suppliers?

There are several things to consider here: cost per unit, activity, specific activity and the
assay conditions that the supplier has used.

Cost per unit:

Let us assume that a vial contains 1 unit of enzyme, using definition A. The same vial would
be labelled 1000 units if definition B were employed. While the label is different, the
contents of the vial clearly are not. If the cost per unit from one supplier appears to be 500-
2000 fold cheaper than from another it is extremely likely that the unit definitions are
different. To ensure that you are comparing like with like, check the unit definitions and
express the number of units in each case as either nmol per min or μmol per min. Remember
to divide the nmol per min values by the cost per vial if the pack sizes are different.

If the calculated cost per unit varies more than 2-3 fold one of the products may represent
better value (i.e. you will get more assays per £ or $ spent), but this is not necessarily the case
(see below).

1 Innova Biosciences (www.innovabiosciences.com)

Factors that influence activity

In the above section we discussed unit definitions, which can markedly affect the number of
units quoted on otherwise identical vials from different suppliers. In this section we discuss
other factors that may lead to more subtle differences in the quoted values.

The conditions under which different suppliers carry out their assays may lead to genuine
differences in the reported activity values but in relation to the number of assays that you will
be able to carry out in your own laboratory these differences may be real or illusory. For
example, assays may be carried out between 20-37oC. While it is not unreasonable for a
supplier to use a temperature of 37oC it should be noted that the same assay carried out at
20oC would give significantly fewer units. The definition of enzyme units is better expressed
thus:

1 unit (U) is the amount if enzyme that catalyses the reaction of 1 nmol of substrate per
minute under standard conditions.

Unfortunately, the meaning of ‘standard conditions’ is somewhat vague. The assay

temperature and the concentrations of substrates, buffers, cofactors, and other additives may
all impact the measured number of enzyme units. For example, if the KM for the substrate is
0.1 mM, assays carried out at 1 mM substrate will give almost twice the number of units as
assays carried out at the KM concentration, even though the amount of protein added to the
assay is the same.

It follows therefore that vials of enzyme that are identical (in terms of quoted units) may not
necessarily give the same number of assays. Conversely, a vial of 100 units of an enzyme
measured by the supplier at relatively low temperature and low substrate concentration may
actually give more assays under your preferred conditions than a vial quoted as 150 units, but
measured by the supplier at 37oC with excess substrate.

It is unlikely that the assay conditions used by the supplier will be identical to your own
preferred conditions. However, you should be aware that if the supplier has run assays at
37oC and you plan to run assays at 20-25oC, as is the case in many drug screening labs, it
would not be surprising if you obtained fewer assays than expected. You might of course
increase the assay time to compensate for the lower activity under your conditions.

In summary, some of the differences in quoted units may be illusory and others will
genuinely reflect the different assay conditions used. All reputable suppliers will state, at an
absolute minimum, the definition of the enzyme unit, the temperature at which the assay was
preformed, and the full composition of the assay buffer. Without this information you will not
be able to replicate the supplier’s assay or extrapolate to your own assay conditions.

2 Innova Biosciences (www.innovabiosciences.com)

What is activity and how do you determine the amount of enzyme needed for an assay?

Activity is quoted as units per ml, in other words nmol per min per ml (assuming that unit
definition B has been adopted). Two vials of enzyme can contain the same number of units in
total but have different activities.

In practice, it is extremely rare for users to calculate the number of units required for an assay
either because it is difficult to replicate the assay conditions used by the supplier or because
different assay conditions are required in the user’s lab. Moreover, some activity may have
been lost in storage or during transportation so it is sensible to confirm the actual number of
units per ml in your own lab prior to setting up large-scale experiments. This is a simple task:
a small amount of enzyme is serially diluted (e.g. log dilutions) and a fixed volume of each
dilution is assayed for enzyme activity. A second set of dilutions can then be prepared in
order to home in on a suitable dilution.

What is specific activity?

Specific activity is the number of enzyme units per ml divided by the concentration of protein
in mg/ml. Specific activity values are therefore quoted as units/mg.

Specific activity is of no relevance as far as setting up assays is concerned, though it is an

important measure of enzyme purity and quality (see below). Activity values (units/ml) are
far more important for assay set up since the amount of substrate converted is determined by
the number of enzyme units added. Note: It is impossible to calculate the volume of enzyme
required for an assay from the specific activity value alone, since the specific activity values
for an undiluted enzyme and a 1/1000 dilution are identical. Both the units per ml and mg per
ml have been reduced by the same factor.

Quality control

There are two key parameters when considering quality of enzymes. Specific activity values
are important because different batches of a pure enzyme should always exhibit, within
experimental error, the same specific activity value. (Note: as specific activity is dependent
on enzyme unit definitions some care is required when comparing specific activity values
from different suppliers). Batches that are below the expected specific activity value may
contain impurities or may be electrophoretically homogeneous but with a population of
molecules that have become denatured. A second key parameter is purity by SDS-PAGE.
Equipment for running gels is ubiquitous thus if the supplier of an enzyme does not show a
gel for each batch then you might like to ask why not!

3 Innova Biosciences (www.innovabiosciences.com)

Innova Biosciences sells a range of ATPase assay kits and GTPase assay kits for drug
discovery and basic research.

These non radioactive colorimetric assay kits use a 96 well format, and all the necessary
reagents are supplied for measuring enzyme activity and are ideal for high throughput drug
screening.

Colorimetric assays for ATPases and GTPases are invariably based on the formation of
colored complexes between an inorganic phosphate and a dye molecule under acidic
conditions. Such assays are beset with problems of reagent precipitation and high
backgrounds caused by impure substrates (i.e. contaminated with inorganic phosphate) and/or
by non-enzymatic (acid) hydrolysis of the substrate.

At the heart of our assay kits is PiColorLock Gold, a superior phosphate detection reagent
which ensures high stability of the colored dye-phosphate complexes. Together with specially
purified substrates and proprietary stabilizers our kits offer the lowest possible assay
backgrounds and outstanding assay performance.

 Very easy to use

 Ultra-pure substrate
 Low Background
 Colorimetric Assay
 High Linearity
 Stable signal
 Contains all reagents required for the assay

4 Innova Biosciences (www.innovabiosciences.com)