Purification of Enzymes
Why isolate enzymes?
understanding of the behavior of an enzyme in a
complex system be it a subcellular organelles :
- mitochondrion
- a cell
- or a whole organism
must understand its properties in as
simple a system as possible
simple system : consist of
a solution of enzyme in a medium containing only
small ions, buffer molecules etc.
special for enzyme bound to cell membrane 
need phospholipids or detergent
studies of isolated enzymes we can learn about:
- its specificity for substrates
- kinetic parameters
- its regulation
Understanding the role
of the enzyme in the
more complex systems
Possible if we can purify enzyme
and remove the contaminating
enzymes and other large molecules
Objectives in enzyme purification
 to isolate a given enzyme with the maximum
possible yield
 based on the percentage recovered activity
compared with the total activity in the original
extract.
 the recovered enzyme should posses :
- maximum catalytic activity
(no degraded or other inactivated enzyme)
- maximum possible purity
(contain no other enzymes
molecules)
or
large
The catalytic activity of enzyme is determined
under defined conditions 
- substrate concentration
- temperature
- pH , etc
The units of activity are usually expressed as:
- mol substrate consumed or product
formed per minute (unit /Internationa unit)
- mol subtrate consume or product formed
per second (Katal)
Strategy
Will involve:
- source of enzyme
- methods of homogenization
- methods of separation
Choice of source
The most important factors involve in starting
material choice are:
- abundance of enzyme
 the required enzyme occurs in large
amounts
example: enzyme catalyzing the fatty acid
synthesis  lactating mammary
gland
hydrolytic enzyme  kidney, etc
- availability
 some sources are not readily available for
various reason (economic; geographical,
etc)
- comparative
studies
 sometimes important to study a certain
enzyme from different species or another
tissues
source
Total homogenization
Partial
Homogenization
Subcellular
fractionation
Purified
organelles
extract
Large-scale
separation method
Extraction or
disperation
Affinity
separation
methods
Crude preparation
Small scale
separation
methods
pure enzyme
- subcellular location
 if enzyme occurs in only one location
within cell  the whole tissues can be
homogenized or extracted
 if enzymes exist in several location in the
cell  need subcellular fractionation.
Subcellular fractionation can be achieved
through
succession of centrifugation
steps
Methods of homogenization
The choice of method usually depends on the type
of tissue or organism which is used as source of
enzyme
- mammalian tissue
 lack of a rigid cell wall  relatively easy
 tissue is cut up into small pieces or minced
prior to homogenization
 using potter homogenizer or high speed
blender
 performed with isotonic solution  to avoid
the rupture of subcellular organelles
 maybe need to be added : proteinase
inhibitor ( ex. Dithiothreitol)  prevent
enzyme damage during extraction process
- plant, fungal and bacterial material
 the rigid cell wall surrounding cell  need
harsher method of extraction
example:
o grinding with abrasives like alumina or
sand
o freezing and thawing
o long period of blending
 can also use appropriate hydrolytic
enzymes
example: preparation of protoplast from
gram positive bacteria  by incubation w
lysozyme
Methods of separation
 make used of some properties by which the
substances may be distinguished
 properties of enzymes that can be exploited:
- size or mass
- charge
- solubility
- possession of specific binding sites
methods depend on size or mass
- centrifugation
 large molecules can be sedimented by high
centrifugal fields generated by an
ultracentrifugation
 used in the step of removing precipitation
or insoluble materials in solution
ex.
 Remove
cell
debris
after
homogenization
 Collect enzyme precipitated by
addition of ammonium sulphate
- gel filtration
 separation between molecules of different
sizes  based on their ability to enter the
pores within the bead of a beaded gel
 gel: sephadex (cross linked dextran) or
bio-gel P (cross linked polymers of
acrylamide)
- dialysis
 dialysis membrane (cellophane) act as a
sieve with holes large enough to permit
the passage of globular proteins up to
about 20 000 molecular weight
 it is widely used during purification to
remove salts, organic solvent or low
molecular weight inhibitor from solution
of enzyme
methods depend on charge
- ion exchange chromatography
 depends on the electrostatic attraction
between species of opposite charge
- electrophoresis
 based on differential movement of charged
molecules under the influence of an
applied potential.
 also depend on size and shape of molecules
 the position of protein can be determined
using Coomassie Blue : bind protein
- isoelectric focusing
 based on their equilibrium position in a
pH gradient
+
anode
-
pH gradient
cathode
methods based on changes in solubility
Solubility of a compound in a given solvent
depends on the balance of the forces between
solute >< solute  insoluble
solute >< solvent  soluble
enzyme purification  possible to alter the
balance between these forces
precipitate enzyme
3 of the important ways of changing solubility :
a. change in pH
Mostly used in
the initial steps
b.change the ionic strength
of enzyme
c. decrease dielectric constant
purification
ad.a. change in pH
 enzyme  least soluble at its IP (no
electrostatic forces
 adjustment of pH to appropriate value 
can be used to precipitate enzyme
 it is important to chect that the enzyme of
interest is not inactivated by these pH
changes
ad.b. change in ionic strength
- salting out  to precipitate enzyme by
addition of salt,
basic theory:
at a very high concentration of salt
concentration of water is significantly
decreased
decrease in solute-solvent interaction
insolubility of enzyme
salt  ammonium sulphate  advantages:
a. cheap
b. high solubility in water
c. lack of harmful effects on most enzymes
amm. Sulphate  weak acid, so when it added to
a weakly buffered solution  ammonia should be
added to maintain the pH at 7 or above
ad.3. decrease in dielectric constant
addition of a water-miscible organic solvent
decrease dielectric constant of a solution
precipitation of large charged molecules
methods based on the possession of specific
binding sites
enzymes normally display highly specific
interactions w/ their substrates  can be used for
affinity chromatography
the substrate or competitive inhibitor  interact
specifically with the enzyme of interest.
The substrate  is linked covalently to an inert
matrix (such as agarose).
Choice of methods
No actual sequence of methods employed.
The choice of methods depend on:
a. the scale of the preparation and yield of
enzyme required
b. time available for the preparation
c. the equipment and expertise available in lab