Exercise 4.7 - Separation of LDH Isozymes from Serum

LEVEL II

Figure 4.10 Preparation rack for tube gels

Figure 4.11 Filling gel tubes

Figure 4.12 Assembling tubes and bath chambers

Figure 4.13 LDH isozyme separation

Figure 4.14 Densitometry tracings of LDH patterns

Materials

• 1.0 M Tris-HCl pH 8.8
• Stock 30%T:0.8%C Acrylamide
• 10% (w/v) Ammonium persulfate
• 40% (w/v) Sucrose
• Tris-Glycine Buffer
• LDH Stain: Must be prepared just prior to use. For use, combine 8 parts A to 1 part B. Solution A:

  60% Sodium Lactate	2.4 ml
  Nitro blue tetrazolium	0.03 gm
  Phenazine Methosulfate	0.003 gm
  Sodium phosphate buffer to make	100 ml

  
  Solution B:
  

  0.5% (w/v) NAD (use 5 mg/ml HO)

  
  
• Non-heparanized hematocrit tubes and centrifuge
• Blood lancets and alcohol for swabbing 19

Procedure

The following directions are given for the preparation of tube gels. Many laboratories continue to use tube gels. The directions can be readily modified to use slab gels as detailed in Exercise 4.1 and Exercise 4.2
1. Assemble the appropriate sized glass tubes and a preparation rack. These may be cut from standard glass tubing or available commercially.

2. Insert the glass tubes in the preparation rack by laying a strip of Parafilm approximately 14" long on top of the black grommets located on the base of the preparation rack (Figure 4.10). Place Parafilm so that it just covers the first grommet and allow an approximate 2" overhang at the opposite end. Beginning at the first grommet, insert the tube through the hole in the upper section of the rack and press the tube down into the grommet, pushing the Parafilm ahead to form a tight leak-proof seal. Place your finger under the grommet and seat the tube and Parafilm against your finger. Do not stretch or pull the Parafilm while inserting the tubes. Leave a small amount of slack as you proceed from one tube to the next.

3. Prepare the separating gel (5% acrylamide) by mixing the following:

   Stock Acrylamide	3.0 ml
   Tris-HCl buffer	3.0 ml
   Ammonium persulfate	6.0 ml
   TEMED	20 µl

   Mix slighty more than 1 ml of solution for each tube being prepared. Once the reagents are mixed, complete the next two steps quickly. The mixture will begin to polymerize in approximately 10 minutes. Rinse syringe, beakers, and other implements after use to prevent the gel solution from solidifying on them.

4. Using a 10 cc syringe with a Loading Syringe Stub Needle, fill the gel tubes to within approximately 2 cm of the top with the separating gel mixture (Figure 4.11). Expel any remaining solution from the syringe when done.

5. Insert the syringe with stub needle into each gel tube as far as it will go; draw off as much gel solution as possible. This procedure will result in gels of uniform height. After removing excess gel solution from the gel tubes, rinse the syringe and stub needle. Carefully add a small layer of Tris-HCl Buffer to the top of the gel, without disturbing the gel itself.

6. Allow the separating gel solution to stand undisturbed for 30 minutes during polymerization.

   The following step involves the use of human blood. Extreme caution must be taken to guard against the dangers of infection. Use disposable lancets, wear gloves at all times and dispose of sharps as indicated by the instructor. Alternatively use blood from a laboratory animal.

7. Using a non-heparanized hematocrit capillary tube, obtain a sample of blood. Centrifuge the blood sample in a hematocrit head of a clinical centrifuge to separate the formed elements from the serum/plasma. A standard hematocrit tube contains just the right serum for a single gel tube analysis. Alternatively, blood can be centrifuged in regular centrifuge tubes, if there is a sufficient quantity of blood.

8. For each blood sample, prepare a 1:10 dilution of the sample by combining 1 part of sample serum with 9 parts of sucrose. Prepare at least 100 µ l of diluted serum for each specimen, enough for two gels.

9. Remove the storage solution from three gel tubes by inverting the gel tubes over a layer of absorbent paper. Shake the tubes abruptly once or twice in a downward motion to remove all the buffer. While the gel tubes are still inverted, blot the ends of the tubes to remove any remaining liquid.

10. Apply 50 µ l of diluted serum to each of two gel tubes. Do not touch the surface of the gel, but allow the sample to flow onto its surface.

11. Layer each gel tube with tris-glycine buffer to completely fill the tube. Be extremely careful not to disturb the serum layer.

12. Remove gel tube #1 from the preparation rack. Moisten the upper end of the tube with a little water and insert sample end up into the bottom of the bath tube adapter (position 1 -- ). Push the tube in so that its upper edge is flush with the upper edge of the adapter. Place all gel tubes into the bath in this manner. Observe the numbers on the upper bath lid and use them to identify the corresponding samples from the preparation rack. Handle the gel tubes with care to prevent mixing of sample and buffer layer. Use the hollow plastic bath stoppers to plug any empty tube adapters.

13. Pour enough tris-glycine buffer into the lower bath so that the bath is approximately half full.

14. Assemble the upper and lower chambers, ensuring that the lower portions of the gel tubes are immersed in the lower bath and that all trapped air bubbles are removed from the ends of the tubes.

15. Carefully pour additional tris-glycine buffer into the upper bath, so that the level is sufficient enough to make contact with the cathode when it is inserted (Figure 4.12). Be particularly careful not to disturb the buffer inside the gel tubes. Again, check the gel tubes to insure that there are no air bubbles trapped in the upper part of the gel tubes.

16. Place the cover onto the upper bath and connect the electrodes to the baths and the power supply.

17. Turn on the power source and, if necessary, allow it to warm up. Check the bath to insure that the polarity is normal. Connect the safety interlock jack to the pins on the bath. Adjust the power source to deliver 5 milliamps of current per gel tube in the bath. For example, if 6 gel tubes are being run, the total current should be 30 milliamps.

18. Continue electrophoresis for 20 minutes or until a clearly defined albumin band is seen near the bottom of the tube. If prestained protein markers are used in the standard, the timing can be precisely controlled by visually checking its progress.

19. When electrophoresis is complete, disconnect the safety inter lock and turn the power source off. Pour the upper bath buffer into a storage container. Place the upper bath on the U-stand and remove the first gel tube.

20. Remove the separating gel for staining. Fit the 10 cc syringe with the blunt-tipped gel removing needle and half fill the syringe with water. While holding the gel tube with one hand, carefully insert the needle at the sample application end of the tube, between the inside wall of the tube and the gel. While slowly pushing the needle in and keeping it flat against the tube wall to avoid scratching the gel, force a stream of water through the needle and rotate it completely around the circumference of the gel. Remove the needle and insert it from the other end of the tube (the separating gel end) using the same technique described above. Once the needle is inserted and rotated completely, the gel will come loose and slide from the tube.

21. Rinse the gel with distilled or deionized water to remove any enzymes on the surface and place the gel in a stoppered test tube.

22. Repeat steps 21-22 for the rest of the tubes. Label each gel carefully.

23. Place 2 ml of freshly prepared LDH stain solution into 10 x 75 mm amber tubes and add a gel to each tube. Keep stoppered and away from light.

24. Incubate the gels in stain solution at 37 ° C for 60 minutes.

25. When the color of the bands has developed, drain off the stain solution from the tubes and fill them with 7% acetic acid. Continue to protect the gels from light for one hour. At the end of this time, the acetic acid will have inhibited further color development and preserved the protein gels.

26. Transfer each gel to a clear glass test tube containing fresh 7% acetic acid and stopper the tube. The gels are now ready for photography or densitomer quantitation. Figure 4.13 and 4.14 demonstrate LDH separation patterns for a normal individual and a comparison with a pattern consistent with myocardial necrosis.

27. If the gels are scanned, integrate the area of each band and calculate the amount of each isozyme as a % of the total LDH present.

Notes

The term isozyme was introduced by C.L. Markert and F. Moller in 1959 20 to describe multiple enzyme forms with similar or identical substrate specificity, and occurring within the same organism. Markert later proposed to modify the term by such adjectives as allelic, nonallelic, multimeric, conformational, and conjugated. These adjectives imply a broader definition of the term isozyme and thus include many genetic variations, as well as physiological modifications of the protein structures.

Serum lactic dehydrogenase (LDH) is an ideal enzyme for the analysis of isozymes, and is also a model system for electrophoretic analysis. The enzyme actually consists of five electrophoretically separable isoenzymes, identified as LDH--1, LDH--2, LDH--3, LDH--4, and LDH--5 in the order of their relative rates of electrophoretic migration; LDH--1 migrates most rapidly. Since these isoenzymes are usually associated with characteristic tissue or organ sources, their relative concentrations in serum may provide useful information in the differentiation of diseases of various body systems, such as myocardial infarction, liver necrosis, pulmonary infarct, primary muscle dystrophy, pernicious anemia, and malignancy. Consequently several easily available commercial kits can be used for their analysis.

LDH isozymes are important indicators of cellular differentation as well. Analysis of the peptide synthesis for LDH isozymes presents what is now a classical analysis of differential gene activity.

LDH1 is composed of a single peptide species A, while LDH5 is composed of the single peptide species B. LDH2-4 are the permutations of combining species A and B into a functional tetramer. Thus, whether LDH1 or LDH5 are synthesized depends entirely on the gene transcription and translation for species A and B respectively. If both are turned on and are equimolar within the cell, then LDH3 will be the predominant form (AABB). In short, the LDH tetramer is a self-assembling molecule that depends upon the concentration of reactants for its tetrameric form. The distribution of isozymes can thus be analyzed statistically to determine the extent to which gene A and/or gene B are functioning.

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