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Chapter 2c Jan2010
Chapter 2c Jan2010
1.
2.
3.
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
Examples:
Mouse
Hydrophobicity
subcellular fractionations
Microscopic examination using a lable specific for
the protein eg. Suitable labelled ligand or antibody
or membrane bound
Located in subcellular organelle
Equipments
Buffers
Assays
Determination of total proteins
1. Equipments
2. Buffers
Desired pH
Anionic/cationic buffer species
Biological activity
Toxicity
-e.g. Tris
E.g. maleate
Good buffers
Theory of buffering
3. Assays
Enzyme activity
Example:Alcohol dehydrogenase:
NAD+ + EtOH ----> NADH + acetaldehyde
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
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
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.
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
Immunological assays
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
Preserving activity
Minimizing denaturation
Minimizing inactivation
Minimizing proteolysis
Other precautions
1. Minimizing denaturation
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
4. Other precautions
PURIFICATION OF ENZYMES
Extraction
Clarification
Precipitation
Chromatography methods
Metal chelate
Ion exchange
Chromatofocusing
Affinity
Gel filtration
Hydrophobic
interaction
covalent