Experiment9 Separation and identification of lactate dehydrogenase

（ LDH） isoenzyme

Master the method of agarose gel electro

phoresis to separate and identify lactate deh

ydrogenase isoenzyme.

1. Aim and request

Lactate dehydrogenase （ LDH ） catalyses

the reaction between lactate and pyruvic acid. In

alkaline buffer solution, enzyme proteins are ne

gatively charged. They will migrate toward anod

e in electric field. Because of the difference of n

et charge number, their migration velocities are

distinct.

2. Principle

The LDH with more ‘H-subunit’ will have

greater migration velocity. The LDH with

more ‘M-subunit’ will have smaller migration

velocity.

Use agarose gel plate as the supporting mediu

m and separate LDH isoenzyme by electrophor

esis in electric field. After electrophoresis, cov

er the agarose gel plate with the dye solution.

LDH catalyze the removal of hydrogen atoms f

rom lactate.

The product NADH ＋ H+ reacts with the artificial hydrogen transmitter phenazine methosulfate(PMS) and finally with the last hydrogen acceptor nitroblue tetrazolium(NBT). NBT is deoxidized to purple compound. Then every zones of LDH isoenzyme can be seen. Estimate their relative content by darkness of color.

lactic acid

pyruvate

NAD+

NADH+H+

PMS-2H

PMS

NBT

NBT-2H(hyacinthine）

⑴ Agarose gel plate preparation. Spread 5 ml hot

agarose solution evenly onto a clean glass plate (air

bubbles must be removed), and cool it naturally. ⑵Make an opening at the site 1.5cm from the edg

e of the plate. Put the plate on the electrophoresis de

vice, add 3~5μl serum sample on the opening, turn o

n the power and let the electrophoresis continue for

30 minutes.

3. Procedure

⑶ After electrophoresis, cover the plate

with the dye solution. ( this step is finished

by the teacher).

⑷ Incubate the plate in warm bath for

several minutes until the color appears

Record what you have observed on your

lab reports.

4. Results

LDH isoenzyme in healthy adult serum has the order as follows:

LDH2 ＞ LDH1 ＞ LDH3 ＞ LDH4 ＞ LDH5

5. Reference

⑴ Describe the principle of the LDH assay.

⑵ How many bands can be visualized in

this experiment?

Questions