Experiment9 Separation and identification of lactate dehydrogenase （ LDH ） isoenzyme.

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Experiment9 Separation and identification of lactate dehydrogenase （ LDH ） isoenzyme

Master the method of agarose gel electrophoresis to separate and identify lactate dehydrogenase isoenzyme. 1. Aim and request

Lactate dehydrogenase （ LDH ） catalyses the reaction between lactate and pyruvic acid. In alkaline buffer solution, enzyme proteins are negatively charged. They will migrate toward anode in electric field. Because of the difference of net 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 medium and separate LDH isoenzyme by electrophoresis in electric field. After electrophoresis, cover the agarose gel plate with the dye solution. LDH catalyze the removal of hydrogen atoms from 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.  ⑵ Make an opening at the site 1.5cm from the edge of the plate. Put the plate on the electrophoresis device, add 3~5μl serum sample on the opening, turn on 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: LDH 2 ＞ LDH 1 ＞ LDH 3 ＞ LDH 4 ＞ LDH 5 5. Reference

⑴ Describe the principle of the LDH assay. ⑵ How many bands can be visualized in this experiment? Questions