Download 11-Electrophoretic method for the separation of LDH

Electrophoretic method for the separation of LDH
Isoenzymes On agarose gel
The final reaction of anaerobic (without oxygen) glycolysis is the conversion of
pyruvate to lactic acid and this reaction is catalyzed by the enzyme lactate dehydrogenase
(LDH). In skeletal muscle, where oxygen deprivation is common during exercise, the
reaction is efficient and large amounts of lactate can be formed. In tissues that
preferentially oxidize glucose aerobically to CO2 and water, such as cardiac muscle, the
reaction is not efficient and pyruvate is preferentially converted to acetyl CoA which
enters the citric acid cycle. In order to understand the differences in efficiency of this
reaction in skeletal and heart muscle, it is necessary to explore the structure of the LDH
enzyme in different tissues of the body.
Isoenzymes are different molecular forms of the same enzyme, and five major LDH
isoenzymes are found in different vertebrate tissues. 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. Each different combination of
subunits represents a distinct LDH isoenzyme. Because the H polypeptide has more
acidic amino acid residues than the M polypeptide, the electrophoretic mobilities of the
LDH isoenzymes are: LDH 1 > LDH 2 > LDH 3 > LDH 4 > LDH 5.
LDH isoenzyme 1 is the predominant form of the enzyme in cardiac muscle. The reverse
is true in skeletal muscle where there is more M than H polypeptide produced, and hence,
more of the isoenzyme 5 forms of the enzyme. The efficiency of the conversion of
pyruvate to lactate increases with the number of M chains. Therefore, the high
concentration of LDH 5 (4 M subunits) in skeletal muscle rapidly
converts pyruvate to lactate while the high concentration of LDH 1 (4 H subunits) in
heart tissue favors conversion of pyruvate to acetyl CoA which enters the citric acid
cycle.
In a myocardial infarction, enhanced enzyme level is proportional to the damage of the
heart muscle tissue and in serious cases its elevation could even be three fold. The
elevation of serum LDH activity might represent other diseases (e.g. anemia, tumors,
liver diseases) as well, so it is important to know from what tissue the LDH was released
into the bloodstream. Tissue specific differences in LDH isoenzymes can be readily
detected by the localization of LDH activity in an agarose gel after electrophoresis by an
activity staining process where the product of the enzymic reaction is a water insoluble
stain precipitating in the gel where the LDH proteins are located. Each of the LDH
isozymes can catalyze the following reaction:
LDH
Lactate + NAD+
Pyruvate + NADH + H+
In order to detect the LDH isozyme in an agarose gel after electrophoresis, the above
enzymatic reaction is coupled to a color producing reaction:
1) Lactate + NAD+
Pyruvate + NADH + H+
2) NADH + PMS
NAD+ + PMS-H
3) PMS-H + TNBT
PMS + TNBT-Formazan. (TNBT- Tetranitroblue tetrazolium)
(PMS - Phenazine methosulfate)
The highly colored TNBT-Formazan product localizes in the electrophoretic zones of
LDH activity and the amount of brown color formed is quantitatively related to the level
of LDH isoenzyme present.
PROTOCOL
Prepare a 1.2% agarose gel in 50 mM Tris–HCl buffer pH 8.2. Load 20 µl from serum
specimens into different slots gel. Use bromophenol blue as a tracking dye Carefully
cover the samples with tank buffer. Pour tank buffer into the reservoires and connect the
electric cables. Electrophorese at 170 V until the bromophenol blue has migrated to
within 1 mm of the positive electrode end of the gel.
Detection of LDH Isoenzymes
Turn off electricity,
and place the gel into the developing chamber which already
contains the developer solution ( H2O 18.4 ml, 1 M Tris 4 ml, tetrazolium-blue 12 ml,
phenazine-methosulphate 4 ml, Na-lactate 4 ml and NAD 1.3 ml). Incubate at 37 oC to
develop color reaction for 30 minutes.
Data Analysis
Rinse the gels with water and examine them on a light box. Locate the bands containing
LDH isozymes. The amount of brown color is roughly proportional to the amount of an
LDH isoenzyme present in a band.
The gels can be stored in standard destain solution containing 10% methanol and 5%
acetic acid.