PRINCIPLES OF ENZYME PURIFICATION
Enzyme purification is an important part of biological research. The technique of enzyme purification
belongs to that school of thought, which seeks to understand biological phenomena by breaking them
down into their component parts, thoroughly studying each component to gain insight into the complex
biological phenomenon.
The basic principle in protein purification is fractionation. A mixture of biological molecules is
fractionated according to some physical, chemical, or biological property. The fraction containing the
enzyme of interest is identified by enzyme assay and this fraction is subjected to further fractionation.
Ultimately, a pure fraction is obtained.
One very specific and effective purification technique is affinity chromatography. This technique employs
a stationary phase, to which a substrate, a substrate analog, or another molecule with a high affinity for
the enzyme of interest has been covalently bonded. Many such resins (chromatographic supports) are
available commercially (remember the resin utilized during Merrifield Synthesis). A sample containing
the biomolecule of interest is applied to the column. Association of the biomolecule and the column is
through non-covalent interactions only. The column is washed with mobile phase to remove extraneous
components in the sample, and finally, the biomolecule of interest is eluted from the column, usually by
some change in the mobile phase which will reduce the attraction of the enzyme to the column material
(the stationary phase).
One of the functions of the pancreas is the synthesis and excretion of proteolytic enzymes (or more
precisely, zymogens of proteolytic enzymes). Pancreatin is prepared from a pancreatic homogenate by
acetone precipitation. It therefore contains the zymogens of proteolytic enzymes (which spontaneously
activate after the death of the animal) and other proteins. In the present experiment trypsin will be purified
from pancreatin.
Pancreatin will be fractionated by affinity chromatography on a column of immobilized soybean trypsin
inhibitor. An extract of pancreatin, adjusted to pH 8, will be loaded on to a column, which contains a
protein covalently attached to agarose beads. The column will be equilibrated to pH 8. After loading, the
column will be washed with pH 8 buffer. During the loading and washing process trypsin will bind to the
trypsin-inhibitor agarose. Since the trypsin is bound to the soybean trypsin inhibitor, immobilized on
agarose beads, it (trypsin) will not wash through the column. In the elution process, a low pH buffer is run
through the column. At acid pH values the trypsin complex with soybean trypsin inhibitor will dissociate
and trypsin will be recovered from the column.
To determine the effectiveness of the purification process, enzyme assays must be carried out. The rate of
the reaction catalyzed by the enzyme is directly proportional to the amount of enzyme assayed. Thus,
enzyme concentrations are indirectly measured by the activity of the enzyme.
In an interesting ecological implication for the high specific affinity of trypsin for soybean trypsin
inhibitor, it is suggested that plant protease inhibitor proteins are plant defense mechanisms directed
against herbivore proteases. Hence, plant-based inhibitors prevent non-plant enzyme activity from
destroying plant proteins.