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EXPERIMENT 3.

2

SEPARATION OF LACTATE
DEHYDROGENASE (LDH) ISOENZYME BY
AGAROSE GEL ELECTROPHORESIS

P37- P39
Objective
 Master the method of agarose gel
electrophoresis to separate and identify
lactate dehydrogenase isoenzyme (LDH1-
5).
Principle
 Gels used as a support medium in electrophoresis to
reduce thermal convection; also act as a sieve to improve
resolution, Pore sizes in the gels could be controlled and
varied depending on the sample being studied.
 Agarose is a linear polymer of galactose coming from
agar. Agarose gels have a greater range of separation for
biomolecules: proteins or DNA fragments. It’s often run
in a horizontal configuration in an electric field.
•Isoenzymes are different molecular forms of the same enzyme
• Five major LDH isoenzymes are found in different vertebrate tissues.
• Each LDH molecule is composed of four polypeptide chains , There are
two types of polypeptide chains in LDH called M (for skeletal muscle)
and H (for heart muscle) which can be combined into the LDH tetramer
in 5 different ways.
• Because the H polypeptide has more acidic amino acid residues than
the M polypeptide, the electrophoretic mobilities of LDH isoenzymes
are:
LDH 1 > LDH2 > LDH 3 > LDH 4 >LDH 5
H4 > H3M > H2M2 > HM3 > M4. (subunit buildup )
CH3CHOCOOH NAD+
LDH
CH3COCOOH
NADH+H+
After electrophoresis, the above enzymatic reaction is coupled to a
color producing reaction:

NAD+ PMSH NBT (yellow, solubility)

NADH+H+ PMS Formazan (purple,insolubility)

NAD - Nicotinamide adenine dinucleotide
NADH - Nicotinamide adenine dinucleotide, reduced
PMS - Phenazine methosulfate
NBT - Nitroblue tetrazolium
Procedure
1. Prepare an 0.5% agarose gel, heating it in a microwave
for 2-4 min until the agarose is dissolved. Pour the gel
onto a slide, make a well with a plastic lid at the site 2
cm. Allow 20 ~ 30 min for solidification.
2. Carefully remove the plastic lid and Load 20μl sample
into the wells. Place the gel in a horizontal
electrophoresis apparatus. Connect the electrode and
the gel with filter paper. Electrophoresis is carried out at
a constant voltage of 90V for 50~60 min.
3. After electrophoresis, take out the gel, place it in a big
glass plate. Then add dye solution on the gel, keep it in
darkness in 45℃ water incubator for 15 min
4. Observe the shade of color and width of the bands by
naked eye to estimate their quantity.
Reference
The 5 types and their normal distribution and levels in healthy
adult serum are listed below.
LDH 1 - Found in heart and red-blood cells and is 17% - 27%
of the normal serum total.
LDH 2 - Found in heart and red-blood cells and is 27% - 37%
of the normal serum total.
LDH 3 - Found in a variety of organs and is 18% - 25% of the
normal serum total.
LDH 4 - Found in a variety of organs and is 3% - 8% of the
normal serum total.
LDH 5 - Found in liver and skeletal muscle and is 0% - 5% of
the normal serum total.
Clinical significance
When LDH is released by damaged
tissue, the isoenzyme zymogram of that
tissue is reflected in the isoenzyme
zymogram of LDH in the serum
•Observe and judge the shade of color and width of the bands by eye to estimate their
quantity or estimate their percentages by spectrophotometer scan.
•LDH isoenzyme in serum has the order as follows
Clinical significance
Examples:
 Following a myocardial infarction, the serum levels of
LDH1 rise within 24-48 hours reaching a peak by 2-3
days and return to normal in 5-10 days.
 Especially diagnostic is a comparison of the LDH-1/LDH-
2 ratio. Normally, this ratio is less than 1. A reversal of
this ration is referred to as a "flipped LDH". Following an
acute myocardial infarction the flipped LDH ratio will
appear in 12-24 hours and is definitely present by 48
hours in over 80% of patients.
 Persons suffering chest pain due to angina only will not
likely have altered LDH levels.