QUIZ 2

1.
2.
3.

List 2 advantages of using microbial cells

as an enzyme source.
Distinguish extracellular and intracellular
enzyme.
List three (3)ways of how to improve the
production yield of enzyme.

General strategies for enzyme

purification

Questions should be asked before embarking on the

protein purification are:
What is the protein/enzyme required for?
What source should be used?
What is known about the protein/ enzyme?
How should the protein be assayed/detected?

Background knowledge required to plan a suitable strategy

What is the protein/enzyme required for?

For economic reasons cost & time are very

important in the manufacture of enzyme
The amount required & purity levels depend on
the end use of the manufactured protein.

Examples:
If the protein/enz is sold for research used (i.e. supplied by Sigma,
BCL etc) the quantities required are small, whilst in terms of purity
the removal of interfering activity will be essential.
For industrial applications, such as in food industry (i.e. enz
produced by Novo & Sturge for degrading starch into glucose &
maltose or for use in domestic detergents ), large quantities are
required & purity is usually of important to cost.
For therapeutic applications (i.e. tissue plasminogen activator for
treating mycocardial infection or monoclonal antibodies for cancer
imaging) purity is of the utmost importance & quantities required
are relatively small

The amount of purified protein will not only depend on the amount
of starting material, but also on yield.
Protein is lost at each step of a purification procedure.
In order to maximize yield the minimum no. of steps should be
used.
However, the final purity will be lower if minimizing the no. of steps

What source should be used?

Defined by the final application
Choose the most stable & abundant
Should also consider the availability &
quantity of the source.

Examples:
Mouse

liver for purifying small quantities of an

enzyme
Bovine liver would be more suitable for larger
amount
Animals that be able to keep in the laboratory are
preferable to animals from the wild
Cells which can be grown in culture, i.e. E.coli,
S.cerevisiae or mammalian cells are preferable to
animals

Reasons to use cell culture: Large

quantities of cell can be obtained

Culture conditions can be carefully
controlled giving more batch reproducibility
Need not to kill animals

What is known about the

protein/ enzyme?

Knowledge of the chemical & physical properties of the

protein & its cellular localization will aid the design of a
purification process.

If the protein has been previously isolated from a different

source, the knowledge can be applied to the protein from
the new source. Thus, its localization is likely to remain
the same, as is whether it is a glycoprotein or lipoprotein

Other characters: Size often remain similar

pI (isoelectric point): the pH at which a particular molecule or
surface carries no net electrical charge.

Hydrophobicity

Intracellular localization can be known by:

Assaying

subcellular fractionations
Microscopic examination using a lable specific for
the protein eg. Suitable labelled ligand or antibody

Alternatively, the activity of the protein may

identify its localization. Eg.
Enzymes

involved in transcription will be located

in the nucleus
Receptors for extracellular growth factors will be
found in the plasma membrane

Properties of glycoprotein or lipoprotein

can be exploited during purification,
e.g.,
glycoprotein

can be purified by lectin

affinity chromatography
Lipoprotein heparin affinity
chromatography

The choice of extraction method and buffer

composition used will be determined by:
Intracellular
Extracellular
Soluble
Insoluble

or membrane bound
Located in subcellular organelle

Extracellular protein, a high degree of purification is

achieved by removal of the cells
Membrane-bound proteins will require detergents or
organic solvents to solubilize them
Isolation of the subcellular organelle prior to extraction
will give a high degree of purification.
Enz or receptors activity can be exploited for affinity
purification on a substrate or ligand or an analogue.
Knowledge of the size & pI of the protein will be useful
for the selection of suitable matrices & conditions for
gel filtration & ion exchange chromatography.
A prior knowledge of the stability of the protein & its
sensitivity to temperature, extreme pH, proteases, air
& metal ions will also aid the design of a purification
procedure

How should the protein be

assayed/detected?
1.
2.
3.
4.

Equipments
Buffers
Assays
Determination of total proteins

1. Equipments

Cold cabinet/ cold room

Ice maker
Fridges
Spectrophotometer (205 805nm)
Other equipments for assaying: scintillation counter for
radiolabelled sample, ELISA (immunolabelled sample) and
HPLC
Electrophoresis
Equipments for cell disruption
Centrifuge (refrigerated)
Column chromatography

2. Buffers

Essential for enz stability

Control the pH of the solution to avoid denaturation or
inactivation of enz/protein
[buffer] will depend on the application, i.e proton, [ ] .
Usually 20-50 mM are adequate
Refer to Data fo Biochemical Research, Dawson,
R.M.C., Elliot, D.C., Elliot, W.H. & Jones, K.M. for buffer
preparation

Factors influence the choice of

buffer

Desired pH
Anionic/cationic buffer species

Variation of pH with ionic strength or temperature

Chemical reactivity

E.g., for ionic exchange chromatography, a cationic

buffer should be used (i.e. Tris , phosphate x)

E.g. 1amine, Tris can interfere with protein analysis.

E.g. Lowry method or amino acid analysis

Biological activity

E.g. phosphate is a participant in biological reaction

& may either inhibit or inactivate an enzyme

Factors influence the choice of

buffer

Interaction with other components

E.g. phosphate complexes with di- & polyvalent metal ions
Thus, inhibiting meal ion dependant enzymes

Borate complexes with many organics & hydroxyl groups, especially

those on CHO
Penetration of biological membranes

Toxicity

E.g. barbitone &cacodylate buffers

Absorption at 280nm or less

-e.g. Tris

E.g. maleate

Expenses, if used on a large scale

solubility

Good buffers

Developed by Good & his colleagues

Biologically &chemically non-reactive, non-toxic,
do not absorb in the UV region, their pKa show
minimum dependence on temperature or ionic
strength
More expensive
Example?

Preparation of Buffers for Protein

Extraction
Proteins are extremely heterogeneous
biological macromolecules.
Their properties can be severely affected
by small changes in hydrogen ion
concentration, and thus a stable pH of the
protein environment is necessary

Theory of buffering

Preventing buffer contamination

To prevent bacterial or fungal growth:

a.

buffer can be filtered through a sterile

ultrafiltration device
b. can be mixed with 0.02% sodium azide
c. can be stored at 4.

Microbial contamination is common to

phosphate buffered saline (PBS), but this may
be avoided at 1M phosphate stock solution.

3. Assays

After each stage of purification, assays for the

protein of interest & its purity are required to assess
the efficiency of the purification.
Specific assays for enz is needed to identify which
fractions contain the enz prior to pooling for the next
step
In addition the yield for each step can be
determined.
Results from total protein assays when combined
with specific assays provide information on the
degree of purification achieved by each step & the
specific activity of the enz of interest.

Enzyme activity
Example:Alcohol dehydrogenase:
NAD+ + EtOH ----> NADH + acetaldehyde

Calculation enzyme activity

Assay mix:
0.99 ml 0.1 M Tris/HCl, pH 9.0, 100 mM EtOH, 0.5 mM
NAD+,
Assay started by addition of 0.01 ml ADH (enzyme), 50x diluted from
stock solution
Increase of absorbance at 340 nm followed:
A340 = 0.6/min.

Purification table

Measure for each purification step:

The volume of the enzyme solution (ml)
The protein content of the solution (mg.ml-1)
The activity of the enzyme solution (U.ml-1)

Total amount of enzyme (U):

Activity (U.ml-1) x volume (ml)

Specific activity (U.mg-1):

Activity (U.ml-1) / protein content (mg.ml-1)

Yield (%):

Total amount of enzyme after a purification step / total amount of enzyme before
that step

Purification factor:

Specific activity of enzyme after a purification step / specific activity before that
step

Purification table (example)

Step

Volume
Total
activity
(ml)
(U)

Total
Specific Yield
Purification
protein activity
factor
(mg)
(U/mg)
(%)_____________

CE (1) 500
3,000 15,000
0.2
100 -AS (2) 100
2,400 4,000
0.6
80
3.0
IEC (3)
45
1,440
500
2.9
48 14.5
GF (4)
50
1,000
125
8.0
33 40.0
______ __________________________________________________
Steps: (1) Crude cell extract; (2) ammonium sulfate fractionation;
(3) ion exchange chromatography; (4) gel filtration.

Specific activity = protein/enzyme (mg/unit)

Total protein (mg)
Degree of purification = specific activity at step2
specific activity at step1

Each step can be assessed by yield & degree of purification for its efficiency

Ideally an assay should be simple, highly specific & rapid in order to allow
many fractions to be screened for activity prior to the next stage of the
purification

Rapid assays minimize storage times btw steps, thus, minimizing

proteolysis (protein degradation).

Many enz can be rapidly assayed (<10 min) spectrophotometrically)

Immunological assays

can be used for many proteins provided an

appropriate antibody is available.
Most frequently used :
Radioimmunoassays (RIA)
Enzyme-linked assays (ELISA)
Not rapid as enz activity assays
Can detect denature protein
Highly specific & multiple samples can be screened
Purity is achieved when further purification steps do
not remove any bands in electrophoresis gel,
subsequent purification step do not increase the
specific activity.

4. Determination of total protein

Essential for the estimation of the degree of

purification
Few techniques, direct or indirect
Ultraviolet

spectrophotometry
Biuret method
Lowry procedure
Bicinchomimic acid protocol
Dye-binding procedure

ASSIGNMENT 5
1.
2.
3.
4.
5.

Ultraviolet spectrophotometry
Biuret method
Lowry procedure
Bicinchomimic acid protocol
Dye-binding procedure

For the above techniques of protein

determination,

Discuss the principle of each technique

Explain the procedure of each technique

Preserving activity

Intracellular proteins will be subjected to many inactivating

conditions, once released from its native environment.
Within the cell, the pH is maintained at ~6.5-7.5, the
[protein] is high (~100mg/ml) & the reducing potential is
high.
On disruption into buffer, the [protein] is reduced & the
protein occur in an oxidizing environment.
In addition, tissue disruption will lead to the breakdown of
compartmentalization.
Particularly is the disruption of lysosomes, which will
release protease into the environment & causes an
acidification of solution.

Judicious choice of buffers must therefore

be made to minimize denaturation,
inactivation &/or proteolysis of intracellular
proteins
Extracellular proteins are not usually as
sensitive have evolved to maintain
activity in the less controlled environment
outside the cell.

How to preserve activity of

enzyme?
1.
2.
3.
4.

Minimizing denaturation
Minimizing inactivation
Minimizing proteolysis
Other precautions

1. Minimizing denaturation

Principal causes of denaturation:

Extremes of pH
Extremes of temperature
Organic solvents

Most proteins stable over a broader pH range, ~pH 5-9 or greater

Carry out the initial steps of purification at 4C
High temp (>40C) should be avoid unless the protein is known to
be heat-stable
Organic solvents (i.e. acetone & alcohol) and chaotropic agents
(i.e. urea & guanidine hydrochloride) should be avoided
Some proteins resistant to these denaturants & they can be
exploited during the purification to denature contaminant proteins.
Organic solvents can be used at low temp to precipitate protein.

2. Minimizing inactivation
Enz containing a free sulphyryl group in
the active site, may be rapidly oxidized
after disruption of the cell.
EDTA should not be included in the
buffers used for metal ion dependent
enz purification.
Many enz can be stabilized by including
cofactors or substrates in buffers.

3. Minimizing proteolysis

Speed & low temp (~40C) during the initial stages.

A coctail of protease inhibitors should be included to the
extraction buffer & other buffers used in the initial steps.
Inclusion of sucrose/maltose in the extraction buffer can
stabilize the lysosomal membane & minimize disruption.
Reducing reagents, i.e. 2-mercapto-ethanol or dithiothreitol
(DTT) should be added to all buffers.
2-mercapto-ethanol: 5-20mM, added to the buffer immediately
prior to use. It oxidize very rapid & lost its protective action within
24h.
DTT: last longer, suitable for use in storage buffers, less odorons,
1-5mM usually adequate, should add to buffers immediately prior
to use

Metal ions can inactivate sulphydryl groups, chelating agent,

i.e. EDTA (0.1-1.0mM) should be added to all buffers

4. Other precautions

Dilute protein solutions are often unstable, due to adsorption to

surface & dissociation of subunits.
Some proteins are more susceptible to adsorption to surface i.e.
glass.
Need to use siliconized containers or polypropylene container.
Most protein will bind irreversibly to polystyrene
Addition of BSA (0.1%), non-ionnic detergents (i.e. Tween 20 or
Triton X-100, 0.1%) may also minimize adsorption losses.

Glycerol is often included in buffers at 10-20% to minimize activity

losses & 50% for storage

May interfere with subsequent purification steps.

Alternative to glycerol are sugars, i.e. glucose or sucrose.

Bacteriostatic agents, i.e. sodium azide, 0.02% - 0.05% also
usually added to buffer.

PURIFICATION OF ENZYMES

No fix methods or rules in protein/enzyme purification

Disruption
Primary
separation
techniques

Extraction
Clarification
Precipitation
Chromatography methods
Metal chelate
Ion exchange
Chromatofocusing

Affinity

Gel filtration

Hydrophobic
interaction
covalent

Main types of purification methods

The following main types of purification methods for proteins can be distinguished:
1. Precipitation methods
2. Separation based on molecular size
3. Separation based on charge
4. Separation based on specific interaction with other biomolecules
5. Separation based on other principles
Examples:
1. Ammonium sulfate precipitation
2. Gel filtration
3. Ion-exchange chromatography
4. Bio-affinity chromatography
5. Hydrophobic interaction chromatography; hydroxyapatite chromatography