Biochemical Simulations. Copyright (c) David A Bender, 1982 - 1997

Enzyme Assay

Units of enzyme activity

Enzymes are quantified in terms of their catalytic activity - the amount of substrate that is converted to product /unit time, under specified conditions.

1 unit of enzyme activity = 1 ?mol of product formed / minute

A number of factors affect the activity of an enzyme, and you will investigate most of these during this exercise:

The pH of the incubation

The pH of the incubation medium affects the ionisation of both the substrate and the amino acid side chains in the enzyme. Obviously, this will affect both the binding of the substrate to the enzyme and also the catalytic activity of the enzyme.

Any enzyme has an optimum pH at which it shows maximum activity. For some enzymes there is a broad range of pH around this optimum where activity is nearly maximum; for others there is only a narrow range of pH over which the enzyme has significant activity.

The pH optimum of an enzyme may be very different from the normal plasma pH of 7.4; some enzymes have pH optima as low as 2 - 3, or as high as 9 - 10.

If your results are to be meaningful, you have to determine the activity of an enzyme at or near its pH optimum.

The temperature of incubation

Like all chemical reactions, the rate of enzyme-catalysed reactions increases as the temperature increases. However, because enzymes are proteins, they are also denatured by heat. This means that above a critical temperature (which differs for different enzymes) there is a rapid loss of activity as the enzyme is denatured.

Although the program permits you to experiment with incubation temperature, this is only really useful if you want to determine the activation energy of the reaction. You should simply work at 30ºC for all of your experiments. It is usual to determine enzyme activity at 30ºC, although 37ºC is sometimes used for mammalian enzymes.

The time of incubation

For as long as the enzyme remains active, and there is substrate present to undergo reaction, the amount of product formed will increase with increasing time of reaction, levelling off as equilibrium is reached.

If the enzyme loses activity or is denatured, or if the supply of substrate is exhausted, then the formation of product will slow down and level off earlier during the incubation.

Obviously, in any studies in which you will be expressing the results /unit time (i.e. the rate of reaction) you have to know that the enzyme was acting at a constant rate over the time of incubation - i.e. that there was a (nearly) linear increase in the amount of product formed with increasing time.

The amount of enzyme present

As long as there is enough substrate present, you would expect the amount of product formed to increase in a linear fashion as the amount of enzyme increases. This is not always so.

Some enzymes form dimers or other aggregates at high concentration, and this can affect their catalytic activity, either increasing or decreasing it compared with the monomer at low concentration.

Other enzymes are unstable at low concentrations, and so are readily denatured. This means that there is less activity than you would expect at low concentrations of enzyme.

Sometimes there is also non-enzymic conversion of substrate to product, or there may be some product already present in the sample you are working with, so that there is some product present even when there is no enzyme present, or it has been incubated for zero time.

It is therefore useful to ensure, before you carry out too many experiments, that the enzyme you are working with behaves in a predictable manner as the amount of enzyme present in the incubation is varied.
Join now!


The concentration of substrate

A simple chemical reaction generally shows a linear relationship between the rate of formation of product and the concentration of substrate available. This is not so for enzyme catalysed reaction.

At high concentrations of substrate (region [A] in the graph) there is enough substrate available to ensure that as soon as the catalytic site of the enzyme is empty (i.e. as soon as the product has left) more substrate is available to bind and undergo reaction. The rate of formation of product now depends on the innate activity of the enzyme itself. ...

This is a preview of the whole essay