There are various reasons why you might want to purify an enzyme, including:

• to characterise it - determine its kinetics (including its kcat, the activity per mol of enzyme) and inhibitor sensitivity without interference from other enzymes that may compete for the substrate, remove the product or act on the inhibitor. From such studies you can begin to determine its mechanism of action;
• to raise antibodies against it, in order to:
  • determine amounts of enzyme protein in tissues under various conditions by immunoassay;
  • localise the enzyme in tissues and sub-cellular compartments by immunohistological techniques;
• to crystallise it, in order to determine its structure by X-ray crystallography and other techniques;
• to produce an enzyme preparation that can be used industrially to catalyse just one reaction with no undesired by-products. Even when an industrially useful enzyme has been prepared by genetic modification of a micro-organism so that it is over-expressed, it is normally necessary to undertake at least a partial purification to obtain a useful product.

Enzyme purification is an art rather than a science, in that there is no way of knowing when you begin which techniques are most likely to be useful. Every protein is different, and successful purification depends on the application of different techniques, and assessing each step in the procedure for its effectiveness in removing other proteins and its efficiency of recovery of your enzyme.

In this simulation you will purify an enzyme using a variety of techniques.

At each stage of your purification you will see the volume of your sample, the enzyme activity /mL and the protein concentration; you should calculate the recovery of enzyme through each stage, and the degree of purification you have achieved to date.

To calculate the recovery of enzyme you need to note the initial activity of the enzyme (and the volume of the initial sample) - this is shown on the results screen when you perform the ammonium sulphate fractionation - and the activity of the enzyme (and sample volume) at the present stage of the purification. Simply multiply the activity /mL by the volume of the sample to obtain the total amount of enzyme, then express this as a percentage of the total activity in your original sample.

To calculate the purification of the enzyme at each stage you need to calculate its specific activity - i.e. the activity per mg protein, and express this as a multiple of the activity /mg protein in your original sample.

By the final stage you should have achieved purification to homogeneity (as determined by gel filtration chromatography). At this stage, having estimated its molecular mass by gel filtration, and knowing the amount of protein present in the sample, you can calculate the kcat (turnover number) of your enzyme. This is the activity (µmol of substrate converted / minute) divided by the amount of enzyme present in your sample (in mol, calculated from the amount of protein present divided by the molecular mass of the enzyme).

The techniques you will use are:

• ammonium sulphate fractionation
• ion exchange chromatography
• affinity chromatography
• gel filtration, to allow you to estimate the molecular mass of the enzyme
• SDS polyacrylamide gel electrophoresis - this shows the individual subunits of the enzyme (if there are multiple subunits in your enzyme)

Each technique may only be used when the appropriate previous steps have been performed, but you can always go back and repeat one of the earlier steps, then carry the purification through using this new set of results.

Each time you run this program you will see a different enzyme, with a randomly selected molecular mass and subunit composition.