By

N.Indra
 Living systems are shaped by an enormous variety of biochemical
reactions, nearly all of which are mediated by a series of remarkable
biological catalysts known as enzymes.

 Enzymology is the study of enzymes .

 Enzymes are unstable molecules with a definite physico-chemical
organization.

 Even a slight change in this organization reduce the activity of the

enzyme and sometimes the enzyme is totally inactivated.

 Therefore the enzymes have to be isolated under controlled

conditions.

 Since they are proteinaceous in nature, standard extraction and

purification procedures for enzymes are the same as those used for

proteins.
 Fresh tissue is crushed into a paste with an extraction medium in a
mortar and pestle, or in a tissue homogenizer, or in a blender or
bye by ultrasonic vibrations. the molarity and PH of the buffer is
suitably adjusted to achieve maximum solubility and activity of the
enzyme.

 EDTA is often included in the extraction medium to remove heavy

metals and for disrupting the membranes of cells and cell
organelles.
Detergents such as Triton-x also used sometimes to solubilize the
membranes.
 many enzyme proteins contain disulfide bonds (S-S) due to the presence
of cystein residues, which are easily broken during enzyme extraction
leading to loss of enzyme activity.
 To overcome this problem are added, thiols such as mercaptoethanol
whose sulfhydryl (-SH) group is able to maintain the S-S linkage in
enzyme.
 If the extract is not homogeneous, the homogenate is filtered to remove
cell debris, fibres, etc., otherwise filtration may be avoided.
All operations of extraction and purification or generally carried out in
cold( 0-4°C) , since most of the enzymes get inactivated at Higher
temperatures.
 Several steps are involved in preparation of crude extract of
enzymes, which is then used for purification of the enzyme.

 Generally the first step after forming a crude extract is a simple

filtration or centrifugation to remove the large material.

 Centrifugation is a process that involves the use of the centrifugal

force for the sedimentation of mixtures with a centrifuge.

 This process is used to separate two immiscible liquids with

more-dense components of the mixture migrate away from the

axis of the centrifuge, while less-dense components of the
mixture migrate towards the axis.

 Centrifugation alters the effective gravitational force on to

tube/bottle so as to more rapidly and completely cause the
precipitate ("pellet") to gather on the bottom of the tube.

 The remaining solution is properly called the "supernatant".

 The supernatant liquid is quickly decanted from the tube/bottle

without disturbing the precipitate.
 Because a protein contains multiple charged groups, its solubility
depends on the concentrations of dissolved salts, the polarity of the
solvent, the pH, and the temperature.

 Some or all of these variables can be manipulated to selectively

precipitate certain proteins while others remain soluble.

 The solubility of a protein at low ion concentrations increases as

salt is added, a phenomenon called salting in.

 The additional ions shield the protein’s multiple ionic charges,

thereby weakening the attractive forces between individual protein
molecules (such forces can lead to aggregation and precipitation).
However, as more salt is added, particularly with sulfate salts, the
solubility of the protein again decreases.
This salting out effect is primarily a result of the competition between the
added salt ions and the other dissolved solutes for molecules of solvent.
 At very high salt concentrations, so many of the added ions are solvated
that there is significantly less bulk solvent available to dissolve other
substances, including proteins.
Since different proteins have different ionic and hydrophobic
compositions and therefore precipitate at different salt concentrations,
salting out is the basis of one of the most commonly used protein
purification procedures.
Adjusting the salt concentration in a solution containing a mixture of
proteins to just below the precipitation point of the protein to be purified
eliminates many unwanted proteins from the solution .
Then, after removing the precipitated proteins by filtration or
centrifugation, the salt concentration of the remaining solution is increased
to precipitate the desired protein.
This procedure results in a significant purification and concentration of
large quantities of protein.
 Ammonium sulfate, (NH4)2SO4, is the most commonly used reagent for
salting out proteins because its high solubility (3.9 M in water at 0°C) allows
the preparation of solutions with high ionic strength.
The pH may be adjusted to approximate the isoelectric point (pI) of the
desired protein because a protein is least soluble when its net charge is
zero. Fractionation by salting out.
(a) The salt of choice, usually
ammonium sulfate, is added to a
solution of macromolecules to a
concentration just below the
precipitation point of the protein
of interest.
(b) (b) After centrifugation, the
unwanted precipitated proteins
(red spheres) are discarded and
more salt is added to the
supernatant to a concentration
sufficient to salt out the desired
protein (green spheres).
(c) (c) After a second centrifugation,
the protein is recovered as a
precipitate, and the supernatant
is discarded.
The isoelectric point(pIs) of some proteins are listed

Protein pI
Pepsin <1.0
Ovalbumin (hen) 4.6
Serum albumin (human) 4.9
Tropomyosin 5.1
Insulin (bovine) 5.4
Fibrinogen (human) 5.8
γ-Globulin (human) 6.6
Collagen 6.6
Myoglobin (horse) 7.0
Hemoglobin (human) 7.1
Ribonuclease A (bovine) 9.4
Cytochrome c (horse) 10.6
Histone (bovine) 10.8
Lysozyme (hen) 11.0
Salmine (salmon) 12.1
Enzyme purification involves the following three techniques:

 Dialysis
 Chromatography
 Electrophoresis
 Dialysis is the process of
separating molecules in solution by the
difference in their rates of diffusion through a
semi permeable membrane, such as dialysis
tubing.
 The most common application of dialysis is
for the removal of unwanted small molecules
such as salts, reducing agents, or dyes from
larger macromolecules such
as proteins, DNA, or polysaccharides.
 Chromatography is a laboratory technique for the separation of a
mixture.
 The mixture is dissolved in a fluid called the mobile phase, which
carries it through a structure holding another material called
the stationary phase.
 The various constituents of the mixture travel at different speeds,
causing them to separate.
 The separation is based on differential partitioning between the mobile
and stationary phases.
 Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus affect the
separation.
Four types of Chromatography

 Adsorption or column Chromatography

 Ion exchange Chromatography
 Gel filtration Chromatography
 Affinity Chromatography
 Adsorption chromatography is the oldest types of chromatography
technique.
 It makes use of a mobile phase which is either in liquid or gaseous
form.
 The mobile phase is adsorbed onto the surface of a stationary solid
phase.
 Adsorption Chromatography involves the analytical separation of a
chemical mixture based on the interaction of the adsorbate with the
adsorbent.
 The mixture of gas or liquid gets separated when it passes over the
adsorbent bed that adsorbs different compounds at different rates.
 Adsorbent – A substance which is generally porous in nature with a
high surface area to adsorb substances on its surface
by intermolecular forces is called adsorbent.
 Some commonly used adsorbents are Silica gel H, silica gel G, silica
gel N, silica gel S, hydrated gel silica, cellulose microcrystalline,
alumina, modified silica gel, etc.
 Ion Exchange Chromatography Separates Anions and Cations.

 In ion exchange chromatography, charged molecules bind to

oppositely charged groups that are chemically linked to a matrix
such as cellulose or agarose.

 Anions bind to cationic groups on anion exchangers, and

cations bind to anionic groups on cation exchangers.

 Perhaps the most frequently used anion exchanger is a matrix

with attached diethylaminoethyl (DEAE) groups, and the most
frequently used cation exchanger is a matrix bearing
carboxymethyl (CM) groups.
 In gel filtration chromatography (also called size exclusion or molecular sieve
chromatography), molecules are separated according to their size and shape.
 The stationary phase consists of gel beads containing pores that span a relatively narrow size
range.
 The pore size is typically determined by the extent of cross linking between the polymers of
the gel material.
 If an aqueous solution of molecules of various sizes is passed through a column containing
such “molecular sieves,” the molecules that are too large to pass through the pores are
excluded from the solvent volume inside the gel beads.
 These large molecules therefore traverse the column more rapidly than small molecules that
pass through the pores.
 Because the pore size in any gel varies to some degree, gel filtration can be used to separate a
range of molecules; larger molecules with access to fewer pores elute sooner (i.e., in a smaller
volume of eluant) than smaller molecules that have access to more of the gel’s interior volume.
 This method relies on the knowledge of the properties of the
enzymes or proteins that you want to isolate.
 Either: (a) Antibodies must be available that have been raised
against the enzyme. or
 (b) The enzyme must be known to bind to some substrate, such
as a ligand, non-covalently in a reversible way (e.g. a
glycosylated enzyme will bind to lectin).
 It is the most specific chromatographic method because it relies
on the specific binding of the target protein with its own specific
ligand.
 Electrophoresis, the migration of ions in an electric field. Polyacrylamide
gel electrophoresis (PAGE) of proteins is typically carried out in
polyacrylamide gels with a characteristic pore size, so the molecular
separations are based on gel filtration (size and shape) as well as
electrophoretic mobility (electric charge).
 However, electrophoresis differs from gel filtration in that the
electrophoretic mobility of smaller molecules is greater than the mobility
of larger molecules with the same charge density.
 The pH of the gel is high enough (usually about pH 9) so that nearly all
proteins have net negative charges and move toward the positive
electrode when the current is switched on
 Molecules of similar size and charge move as a band through the gel.
 Most of the commercially available enzyme preparations, purified as
above, are concentrated and sterile-filtered after purification.

 This is done to reduce both, the volume and the microbial

contamination of the sample.

 Often before storage and transport, the sample is freeze dried with
additives such as sugar substrates and dextrans.