Paper 10 (Enzymology)

Unit 1

Sub topic:
Criteria for purity of Enzyme preparation.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 Importance of purification:
 Purification is an all mandatory step in studying
macromolecules, but it is not necessarily easy.

 Task:

Typically, a substance that makes up <0.1% of a

tissue’s dry weight must be brought to ~98%
purity.
2015 RRA, BC Dept, FLS, JSS University, Mysuru
 Most purification procedures for a particular
enzyme are developed in an “empirical manner”,
the overriding principle being purification of the
enzyme to a “homogeneous state” with
acceptable yield.

 The aim of a purification procedure is to obtain

enzyme of the maximum possible purity and
maximum catalytic activity.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 Purification Strategies - a simple approach

 Apply a systematic approach to development of a purification strategy.

 The first step is to describe the basic scenario for the purification.

General considerations answer questions such as:

 What is the intended use of the product?

 What kind of starting material is available and how should it be handled?

 What are the purity issues in relation to the source material and intended use of
the final product?

 What has to be removed? What must be removed completely?

 What will be the final scale of purification?

 If there is a need for scale-up, what consequences will this have on the chosen
purification techniques?

 What are the economical constraints and what resources and equipment are
available?
2015 RRA, BC Dept, FLS, JSS University, Mysuru
2015 RRA, BC Dept, FLS, JSS University, Mysuru
2015 RRA, BC Dept, FLS, JSS University, Mysuru
 Guidelines
 Define objectives- for purity, activity and quantity required of
final product to avoid over or under developing a method.
 Define properties of target protein and critical impurities-
to simplify technique selection and optimization.
 Develop analytical assays- for fast detection of protein
activity/recovery and critical contaminants.
 Minimize sample handling at every stage- to avoid lengthy
procedures which risk losing activity/reducing recovery.
 Minimize use of additives- additives may need to be removed in
an extra purification step or may interfere with activity assays.
 Remove damaging contaminants early- for example,
proteases.
 Use a different technique at each step -to take advantage of
sample characteristics which can be used for separation (size,
charge, hydrophobicity, ligand specificity).
 Minimize number of steps - extra steps reduce yield and
increase time, combine steps logically.
KEEP IT SIMPLE! 2015 RRA, BC Dept, FLS, JSS University, Mysuru
 Purity of 95% may be acceptable if the remaining 5%
consists of harmless impurities. However, even minor
impurities which may be biologically active could cause
significant problems in both research and therapeutic
applications. It is therefore important to differentiate
between contaminants which must be removed
completely and those which can be reduced to
acceptable levels. Since different types of starting
material will contain different contaminant profiles they
will present different contamination problems.
2015 RRA, BC Dept, FLS, JSS University, Mysuru
How to judge the success of a purification
procedure?

Tests for purity:

 Analytical tests can only really be used to
demonstrate the presence of impurities
rather than prove their absence.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 By far the most convenient way to estimate the
concentration of a particular enzyme in a
preparation or fluid is to determine its catalytic
activity

 At each stage of purification procedure, an assay

of enzyme being purified should be performed on
all the fractions and its ‘specific activity’ in each
fraction should be determined.

 The total activity of this enzyme will be

unchanged as a result of purification step, unless
some has been lost during purification ; under no
circumstances can it be increased.
2015 RRA, BC Dept, FLS, JSS University, Mysuru
 Increase in specific activity will be a measure of
purification achieved.

 Purification until specific activity reaches a limiting value.

 Same specific activity value before and after a

purification step does not necessarily mean that enzyme
preparation is completely pure: it could simply mean
that contaminating proteins have passed through the
procedure in the same fraction as the enzyme.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 Similarly, X-ray crystallographic studies
cannot be taken as proof that only one
protein is present, for many mixed protein
crystals have been found.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

Some experimental criteria
 Gel filtration :- single and symmetrical peak. The peak
for enzymatic activity should be totally coincident with
protein peak. Any skewing can be taken as evidence for

heterogenous preparation.

OD
3.5

2.5
Axis Title

1.5 OD and corresponding

sp.activity
1

0.5

0 10, 0.1
Fraction2 number
0 4 6 8 10 12
Axis Title
2015 RRA, BC Dept, FLS, JSS University, Mysuru
Method Comments
Ultracentrifugation Not very satisfactory for detecting impurities
at the ≤ 5% level. Problems can arise from
associating-dissociating systems
Electrophoresis A good method for examining enzymes
composed of non identical subunits.
Electrophoresis in the presence of SDS A good method for detecting impurities that
differ in terms of the subunit Mr ; excellent
for detecting proteolytic damage. Problems
are from enzyme composed of non-identical
subunits, which give rise to multiple bands.
Capillary Electrophoresis A powerful analytical technique which can
be used in a variety of modes, including IEF.
Equipment required is specialized and
relatively expensive.
Isoelectric focusing A very sensitive method for detecting
impurities. Artefacts can arise suggesting
apparent heterogeneity.
Mass spectrometry A very powerful but specialized technique.
Subunit Mr values can be obtained very
precisely, confirming the authenticity of
primary structure. Post translational
modifications
2015 RRA, BCcan beFLS,
Dept, identified
JSS University, Mysuru
 Gel electrophoresis:- Enzyme may be a trace
protein, but can be mistaken for a major protein
band. A single band following non-dissociating
gel electrophoresis and SDS-PAGE is good
evidence for apparent homogeneity.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 End group analysis:- One N-terminal and One C-
terminal group.

 Ratio of cofactor to apoprotein: a fixed defined

proportion.

 Sequencing in sequanators.

 What if a particular enzyme is known to be the

contaminant?

 A logical step is to carry out an assay for that enzyme

and demonstrate its absence.

2015 RRA, BC Dept, FLS, JSS University, Mysuru
 An example to define the criteria of purity

FRACTION TOTAL ACTIVITY SPECIFIC FOLD RECOVERY

n PROTEIN (units) ACITIVITY PURITY (%)
(mg) (units/mg) x

1.0 100
1.Homogenate 10,000 4,000 0.4

8.75 87.5
2. Ammonium 1,000 3,500 3.5
sulphate
precipitation

125 62.5
3. G-50 50 2,500 50

500 50
4. Ion Exchange 10 2,000 200

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 Volume (ml) this refers to the measured total solution volume at
the particular stage in the isolation.

 Total protein (mg) - the primary measurement is of protein

concentration, i.e. mg ml-1, which is obtained using a protein assay.
Multiplying the protein concentration by the total volume gives the
total protein (i.e. mg/ml x ml = mg).

 Total activity (units) - the activity, in units ml-1, is obtained from

an activity assay. Multiplying the activity by the total volume gives
the total activity (i.e. units/ml x ml = units).

 Specific-activity(units/mg) - the specific activity is obtained by

dividing the total activity by the total protein. Alternatively, the
activity (units/ml) can be divided by the protein concentration
(mg/ml), in which case the ‘ml’s cancel out, leaving units/mg.
2015 RRA, BC Dept, FLS, JSS University, Mysuru
 Purification (fold) - “Fold” refers to the number of multiples of a starting
value. In this case it refers to the increase in the specific activity, i.e. the
purification is obtained by dividing the specific activity at any stage by the
specific activity of the original homogenate. The purification “per step” can
also be obtained by dividing the specific activity after that step by the
specific activity of the material before that step.

 Yield (%) - the yield is based on the recovery of the activity after each step. The
activity of the original homogenate is arbitrarily set at 100%. The yield (%)
is calculated from the total activity (units) at each step divided by the total
activity (units) in the homogenate, multiplied by 100. The yield can also be
calculated on a “per step” basis by dividing the total activity after that step
by the total activity before that step and multiplying by 100.

 The efficiency of a step - is calculated as:-

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 PURIFICATION TABLE FOR ADENYLATE KINASE
STARTING WITH 6kg PIG MUSCLE
TOTAL TOTAL TOTAL SPECIFIC YIELD PURIFICATION
VOLUME PROTEIN ACTIVITY ACTIVITY FACTOR
STEP (cm3) (mg) (katal) (katal/kg) (%) PER STEP
1.Extraction 16600 435000 0.0413 0.095 100 1.0
2.pH 15700 112000 0.0365 0.0325 88.3 3.42
3.Phoshocellulose 1380 1716 0.0223 13.02 54.0 40.06

4.Gel filtration 211 462 0.0200 43.17 48.4 3.32

5.Crystallization - 344 0.0160 46.50 38.7 1.077

2015 RRA, BC Dept, FLS, JSS University, Mysuru

2015 RRA, BC Dept, FLS, JSS University, Mysuru
 Note that the specific activity of the protein (the
enzyme xanthine dehydrogenase) in the immuno-
affinity purified fraction (fraction 5) has been
increased 152/0.108, or 1407 times the specific
activity in the crude extract (fraction 1). Thus,
xanthine dehydrogenase in fraction 5 versus fraction
1 is enriched more than 1400-fold by the
purification procedure.

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 No single technique, however sensitive, cannot establish
enzyme purity because the contaminating protein might
behave in an identical fashion to the desired enzyme under
the conditions used.

 However, if several characteristics are investigated, and

conditions varied, the chances of contaminant avoiding
detection are reduced.

 Therefore, evidence of purity must be obtained by use of

several of the procedures before concluding that enzyme
sample is pure!!!

2015 RRA, BC Dept, FLS, JSS University, Mysuru

 References:-
 ENZYMES

-Trevor Palmer.

 PROTEIN PURIFICATION-HANDBOOK

- Amersham Biosciences.

 FUNDAMENTALS OF ENZYMOLOGY

- Nicholas C. Price and Lewis Stevens.

 A GUIDE TO PROTEIN ISOLATION

- Clive Dennison

 BIOCHEMISTRY

- Voet and Voet

2015 RRA, BC Dept, FLS, JSS University, Mysuru