ENZYME PURIFICATION

Since enzymes are very unstable molecules, their isolation from the
tissues is to be done under controlled conditions of pH, ionic strength
and temperature. Enzyme assay is performed at each step of purification
so as to ensure that enzyme is not lost during purification. Following
steps are usually followed in isolation and purification of enzymes.

1. Extraction:
The plant or animal tissue from which the enzyme is to be extracted is
homogenized in a blender or with a mortar and pestle. Generally the
extraction is done under cold (below 4°C) conditions because most of
the enzymes are thermolabile and they are inactivated at high
temperatures. The extraction is done with a buffer of specific ionic
strength and EDTA (Ethylene Diamine Tetraacetic Acid) is generally
added to the extraction medium to solubilise the membranes and
chelate heavy metals which would otherwise inhibit enzyme activity.
Many enzymes and proteins contain disulphide (S-S) bonds due to the
presence of sulphur containing aminoacids which are easily oxidized
during enzyme extraction leading to the loss of enzyme structure and
activity. To overcome this problem thiols are also added. Sometimes
proteins such as casein or bovine serum albumin are also added in the
extraction medium. These proteins protect the enzyme from the
hydrolytic action of the endogenously present proteases which
become active during tissue homogenization.
2. Filtration and Centrifugation:
The tissue extract is filtered using cheese cloth or filter paper to
remove cell debris and fibres etc. The supernatant is centrifuged.
High speed centrifugation (around 20,000 g) removes cell debris and
also the bigger cell organelles such as chloroplast and mitochondria.
A very high speed centrifugation in ultracentrifuge at about 100,000g
removes all cell organelles and only proteins remain in the
supernatant. This supernatant can be used for further purification of
enzymes.

3. Precipitation:
Like any other proteins enzymes are also highly charged molecules
and can be precipitated out by proper charge breaking chemicals.
Once their charges are neutralized, they form aggregates and settle
down as precipitates. Acids, bases, salts and organic solvents are
often used to precipitate out proteins. Protein or enzyme precipitation
upon addition of concentrated solution of salt is called salting out. In
other words, salt dehydrates protein molecules which then forms
aggregates and precipitate out. For enzymes, the most commonly
used salt precipitant is ammonium sulphate. The enzymes settle down
as precipitate and can be removed by centrifugation. The precipitate is
dissolved in small amount of buffer and excess ammonium sulphate
and other contaminants are removed (dialysis). About four folds
enzyme purification is typically obtained with ammonium sulphate
precipitation.

4. Purification of enzymes:
The dialysed enzyme preparation is purified further by a variety of
techniques. The most common technique is electrophoresis.

Electrophoresis principle – Biological molecules possess ionizable

groups and can therefore be made to exist in solution as electrically
charged particles like cations and anions. Moreover molecules which
have similar charge will have different charge/mass ratio. In
combination these differences form a sufficient basis for a differential
migration when the ions in solution are subjected to an electrical
field.

The simplest type of electrophoresis is paper electrophoresis in which

a paper is used as a support for separating proteins.

The protein sample is spotted on a paper strip. The ends of the paper
are dipped in two electrode tanks filled with phosphate buffer of
specific pH and ionic strength. The electrodes are connected to power
supply. When current is applied to the electrophoresis apparatus
through these electrodes different proteins separate according to their
charge (electrophoretic mobility) on paper. They can be then located
and identified using suitable colour reaction of the protein.
Factors affecting electrophoresis:

1. Electric field –
● Voltage – potential gradient is directly proportional to
migration.
● Current – distance migrated by ions will be proportional to
both current and time.
● Resistance – offered by medium and buffer.
2. Sample –
Separation depends on charge, size and shape (frictional forces) of
sample molecules.

3. Buffer –
● Composition – it should not bind with compounds being
separated.
 ● Concentration – as ionic strength of the buffer increases, the
proportion of the current carried by the buffer will increase and
the share of the current carried by the sample will decrease, thus
slowing the rate of migration.
● pH – influences the ionization of the substances.
4. Supporting medium – filter paper, cellulose acetate,
polyacrylamide gel. The adsorption causes the tailing of the
sample.

ELECTROPHORESIS
Electrophoresis on the basis of differences in their net charges, size and
shape regardless of the conducting medium.

The method is employed for the separation of different proteins, as the

side chains of many of the constituent amino acids exist in a dissociated
form and contribute some number of positive and negative charges to the
macromolecules. Molecules such as polynucleotides are separated by
electrophoresis.

It is the migration of charged particles through a liquid or semisolid

medium under the influence of an electric potential. It is a technique by
which molecules are separated from one another in an electric potential
gradient

Principle:

If two electrodes are inserted into a solution containing an electrolyte

and a suspension of macromolecules of varying net charge, the
macromolecules will be accelerated toward the electrode of opposite
sign with a force proportional to the magnitude of the charge or the
macromolecule (which is pH dependent) and the strength of the applied
electric potential. Since each of the migrating particle will encounter a
frictional resistance to its movement, each will soon attain some
maximum velocity of migration. Therefore if a mixture of these
macromolecules are exposed to a constant field strength. The maximum
velocities attained will be different for particles of different net charge.
Several other factors also influence the rate of electrophoretic migration
of proteins.

SDS PAGE – PolyAcrylamide Gel Electrophoresis

Sodium dodecyl sulfate (SDS) is a detergent. It binds non-covalently to

proteins. It also confers negative charge. In the presence of SDS, the
intrinsic charge of a protein is masked. During SDS PAGE, all proteins
migrate toward the anode (the positively charged electrode). SDS-treated
proteins have very similar charge-to-mass ratios, and similar shapes.
During PAGE, the rate of migration of SDS-treated proteins is
effectively determined by molecular weight.

Below is an example of the procedure for performing discontinuous

SDS-PAGE with a 14% separating gel and a 5% stacking gel.

Polyacrylamide is prepared just before use from a number of highly

toxic synthetic chemicals. It is prepared by copolymerizing acrylamide
monomer with a crosslinking agent usually N,N'- methyl bis acrylamide
in the presence of a catalyst accelerator chain initiator mix. This mixture
consists of freshly prepared ammonium persulphate as catalyst together
with a carrier of a suitable base TEMED – (TEtraMEthylene Diamide)
as initiator. The porosity of the gel is determined by the relative
proportion of acrylamide to the cross linking agent.

Procedure:

● Combine all reagents except the TEMED for the 14% separating
gel.

14% Separating Gel Components (4.195 mL)

1.184 milliliters deionized water

1.96 milliliters 30% acrylamide/Bis (Warning: Acrylamide

is a neurotoxin. Use gloves, do not ingest.)

1.0 milliliters 1.5 M Tris, pH 8.8

21 microliters 20% SDS

12 microliters 10% ammonium persulfate

18 microliters TEMED, pH 8.9

● When ready to pour the gel, quickly add the TEMED, mix using a
Pasteur pipette, and transfer the separating gel solution between the
glass plates in the casting chamber to about 3/4 inch below the
short plate.

● A small layer of butanol can be added on top of the gel prior to

polymerization to straighten the level of the gel and remove
unwanted air bubbles that may be present. Butanol will not mix
with the aqueous separating gel solution. Once the gel has
 polymerized, the butanol can be removed by absorption with
Kimwipes or filter paper. Rinse the top layer of the gel with dI
water prior to pouring the stacking gel.

● Insert the well forming comb into the opening between the glass
plates.
● Combine all reagents except the TEMED for the 5% stacking gel.

5.1% Stacking Gel (2.484 mL)

0.4 milliliters deionized water

1.8 milliliters 30% acrylamide/Bis

0.25 milliliters 0.5 M Tris, pH 6.8

12 microliters 20% SDS

17 microliters 10% ammonium persulfate

5 microliters TEMED

● When ready to pour the gel, quickly add the TEMED, mix using a
Pasteur pipette, and transfer the stacking gel solution between the
glass plates in the casting chamber.

● Both the separating and stacking gels should polymerize within six
minutes.

● Once the stacking gel has polymerized, the comb can be gently
removed. The polymerized gel between the short plate and spacer
plate forms the "gel cassette".
Fill the upper and lower chambers with running buffer. Connect leads to
power supply.

Load the protein sample mixed with tracking dye in one of the wells and
add standard protein in another well. Allow the electrophoresis till the
tracking dye reaches 0.5 cms from the bottom of the gel. Turn off the
power supply, remove the slide spacer and open the plates with a
spatula. Flush water between the gel and the plate. Slide the gel
completely into the tray containing the dye solution. Stain the gel
overnight, drain out the staining solution and add destaining solution –
give 2-3 changes. After complete destaining the gel is stored in 10%
acetic acid and photographed.