BC 367 Experiment 3
Purification and Characterization of the Enzyme Lysozyme
decomposition of up to 5x10
moles of H
per minute per mole of enzyme (the turnover
number). Even though the turnover numbers of most other enzymes are substantially lower than
this, most of the reactions they catalyze occur at rates at least a million times faster under
physiological conditions in the presence of the enzyme than in its absence.
In this experiment you will purify an enzyme, using its known activity to monitor the
process. The isolation and purification of a specific protein or enzyme is generally a difficult
task. First, the enzyme must be liberated from its source tissue in an active form.
wide variety of tissue disruption techniques have been developed.
However, the enzyme to be
purified is usually only a small percentage of the total protein in the crude extract of the tissue.
The object of protein purification is to remove nonprotein contaminants as well as to isolate the
protein in question from other proteins.
The first objective is relatively easy to obtain, whereas
the latter is more difficult.
For example, it is not unusual for an enzyme to be 0.1% of the total
protein in a crude tissue extract. To purify this enzyme to homogeneity, 99.9% of the protein
must be removed, preferably with as little loss as possible of the desired enzymatic activity. This
can be a difficult task for two reasons:
1. Enzymes are relatively labile molecules. Most enzymes are denatured by foaming, by
heating, by organic solvents (particularly at room temperature), by drying at room
temperature, and by concentrated acids or bases. Furthermore, proteins in aqueous solutions
are excellent nutrient systems for microorganisms, and therefore, cleanliness of equipment
and avoidance of unnecessary contamination are vital for successful purification attempts.
2. Differences between some of the various protein molecules in the tissue extract may be
subtle. A single purification step is seldom adequate to purify an enzyme completely.
Typically, several different procedures that exploit different properties of proteins must be
Generally, either salt precipitation, organic solvent precipitation, or isoelectric
precipitation is used at an early point in the procedure. Chromatographic procedures, such as
ion-exchange, gel-filtration, or adsorption chromatography, are employed after the enzyme
has been partially purified by one of the precipitation techniques.
establishment of an enzyme purification procedure must be done largely by trial and error.
Determination of the progress of a purification process is important. Specific activity and
total activity are the critical parameters in enzyme purification.
The activity of an enzyme is
defined in some unit, usually micromoles of product formed per minute. The specific activity,
then, is defined as activity units per milligram of total protein. As the enzyme is purified, the