Lab Activity 10
Purification of LDH from Chicken
Part I
IUG, Fall 2012
Dr. Tarek Zaida
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Background
• LDH catalyzes the nicotinamide cofactordependent interconversion of lactate and
pyruvate:
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• LDH is found in almost all organisms because
it plays an important role in carbohydrate
metabolism during conditions in which
pyruvate production from glycolysis exceeds the
ability of the cell to metabolize the pyruvate,
• LDH converts the pyruvate to lactate, and
thereby regenerates the oxidized NAD required
for further glycolysis.
• LDH also allows the conversion of lactate to
pyruvate;
• both the pyruvate and NADH produced can then
be used for other processes.
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LDH activity is readily measurable:
• The extinction coefficient at 340 nm of NADH
is much higher than that of NAD.
• If the only substrates added to the reaction
are NAD and lactate (or NADH and pyruvate),
the change in absorbance at 340 nm should
be proportional to the change in NADH
concentration due to the LDH activity present
in the cuvette.
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• In any protein purification protocol it is
necessary to take advantage of the way in
which the protein of interest (in this case,
LDH) differs from the other proteins in the
mixture.
• Most tissues contain thousands of proteins;
you need to use the properties of LDH to
separate it from all of the other proteins
present.
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• Most tissues contain proteases (enzymes
that degrade other proteins).
• Avoiding proteolytic damage to your protein
can be difficult.
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• Three techniques are commonly used to keep
proteolysis to a minimum:
1) perform the purification in the presence of
protease inhibitors,
2) perform the purification at low temperatures
(4°C or on ice), and
3) perform the purification in the minimal
amount of time possible.

Because it is not easy to do the last of these (the
purification procedure will take more than one lab period),
you should keep your sample on ice or in the refrigerator as
much as possible.
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Definitions
• Tris-(hydroxymethyl) aminomethane
hydrochloride (Tris-HCl) is a commonly used
buffer, and is intended to help control the pH of
the solution.
• Tris is inexpensive and generally inert in
biochemical experiments.
• One drawback is the fact that the pKa of Tris
changes by –0.031 pH units per °C, and therefore
the pH of a Tris-buffered solution is very
temperature-dependent.
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• 2-Mercaptoethanol (b-ME) is a reducing agent;
it prevents formation of disulfide bonds
between free cysteine residues.
• It also inhibits some proteases.
• Phenylmethylsulfonyl fluoride (PMSF) is an
irreversible inhibitor of serine proteases.
• PMSF is toxic; avoid getting PMSF on your skin.
• Ethylenediamine tetraacetic acid (EDTA) is a
chelating agent; it is used to remove metal ions
from solution.
• Some proteases are dependent on metal ions
(especially calcium ions), so EDTA acts as an
inhibitor of some proteases.
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Experiment
Reagents
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Procedure
1. Tissue preparation– Cut ~50 g of muscle
tissue from the tissue source (record the
exact weight of tissue used). Cut the tissue
into small pieces with scalpel or razor blades.
Discard the connective tissue and fat.
2. Soluble protein extraction – Place the
minced tissue and 75 ml of cold Extraction
Buffer in a blender, and put the top on the
blender.
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Disrupt the tissue by homogenizing. Use 4 x 30 second
bursts, with at least 10 seconds in between each burst
to allow the temperature of the homogenate to
decrease.
3. Centrifugation– Put the homogenized tissue/buffer
mixture into four pre-chilled 50 ml centrifuge tubes
(note: the mixture will be the consistency of a
thick milk shake, so a spatula will help).
Balance the tubes (i.e . make sure that each pair of
tubes have the same mass). Make sure that the tubes
are not too full (you do not want to spill your
mixture inside the rotor).
Centrifuge your homogenate for 20 minutes at 15,000
rpm.
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4. Filtration– Pour the supernatant (i.e . the
fluid on top) through two layers of
cheesecloth into a pre-chilled beaker. The
cheesecloth removes lipids from the
solution; the filtration step is much easier if
you put the cheesecloth into a funnel).
• Discard the cell debris pellets. Measure and
record the volume of the supernatant.
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