Name: Vashista Shripat Date: 06|02|12 Course Code and Title: BIOL 3364 Clinical Biochemistry Practical #: 2 Title:

: Electrophoresis of Lactate Dehydrogenase (LDH) Isoenzymes. Aim:

I.D.#: 809003142

To examine the different isozymes of the enzyme Lactate Dehyrogenase in different tissues using Polyacrylamide Gel Electrophoresis. To examine the different proteins in blood serum using Polyacrylamide Gel Electrophoresis.

Theory: The enzyme lactate dehydrogenase or L-Lactate:NAD+ oxidoreductase (E.C. number: 1.1.1.27) is constitutively produced in all tissues in the body and is found in many organisms. It catalyzes the reaction

Figure 1. The Anaerobic Breakdown of Pyruvate to Lactate in order to regenerate NAD+

[http://en.wikipedia.org/wiki/Lactate_dehydrogenase, downloaded 4 March, 2012.]

which is the final step in anaerobic glycolysis and is responsible for the regeneration of NAD + so that glycolysis can continue to produce substrates and intermediates for the citric acid cycle and other biosynthetic pathways as well as ATP (Hames and Hooper, 2006).

lactate dehydrogenase

Figure 2. The connection between glycolysis and lactate.

[http://www.accessexcellence.org/RC/VL/GG/ecb/pyruvate_fermentation.php, downloaded 4 March, 2012]

Lactate dehydrogenase is a tetrameric enzyme composed of dissimilar protomers, H and M, and can thus exist in several different forms known as isoenzymes. Isoenzymes or isozymes are enzymes that differ structurally with respect to amino acid sequence or with respect to having different combinations of subunits but which catalyse the same chemical reaction. These enzymes generally have different kinetic or regulatory properties depending on the tissues in
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which they are produced and their subunits are coded for by the same gene loci in all tissues just in different proportions. The existence of isozymes allows metabolism to be modified or finetuned to suit the needs of a particular tissue. This is an advantage of having isozymes since the same gene loci are used to fulfil different metabolic needs in different tissues instead of using a different gene for the same reaction in different tissues just to meet different metabolic conditions. It conserves genetic space and allows for the maximization of just two genes. The synthesis of the H and M protomers are under the control of two different genetic loci that are expressed at different rates in different tissues. Often, in one kind of tissue, one type of protomer is expressed predominantly over the other type and these combine accordingly to produce an active enzyme. This leads to different tissues having different isoenzymes (Murray et al, 2000). The H and M protomers can be combined in the five following ways: HHHH, HHHM, HHMM, HMMM and MMMM.

Figure 3. The structure of the tetrameric 4M LDH 5 isozyme.

[http://biochemistryquestions.wordpress.com/2008/06/21/isoenzymes-or-isozymes/, downloaded March 4 2012.]

Table 1. The Subunits and Location of the Different LDH isozymes. Lactate dehydrogenase Isozyme LDH 1 LDH 2 LDH 3 LDH 4 Subunits 4H Location Mainly found in red blood cells and heart muscle. Mainly found in white blood cells. Mainly found in the lungs. The highest concentrations are found in the kidney, placenta and pancreas. The highest concentrations are found in the liver and skeletal muscles. Serum LDH (%) 33

3H1M 2H2M 1H3M

45 18 3

LDH - 5

4M

Isozymes, particularly those of lactate of dehydrogenase became of medical importance when it was discovered that human blood serum contained several LDH isozymes and that their proportions in relation to one another changed noticeably during certain medical conditions (Murray et al, 2000). When tissues containing LDH are damaged or broken down, LDH is released into the bloodstream causing LDH levels in the blood to rise. Elevated LDH levels can thus be used an indicator of tissue damage. However since LDH is produced in many tissues in the body, the total LDH does not give much information on the exact location of the damage. The different isozymes must first be distinguished from one another in order to determine which isozyme is present in higher levels than the others and then the location of the tissue damage can be determined. The level of LDH 2 is generally greater than LDH 1 in the bloodstream. However, after a heart attack, LDH 1 levels increase due to the heart muscle in which the isozyme is found becoming damaged and spilling its contents into the blood stream, and become higher than the LDH 2 levels. This is known as the flipped LDH pattern (MedlinePlus, 2012). This happens 24 48 hours after a heart attack with the total LDH level rising. The levels peak within 2 3 days and returns to normal levels within 5 10 days. Other conditions that can be diagnosed by the relative proportions of LDH levels in the blood are Hemolytic anemia, Hypotension, Infectious mononucleosis, Intestinal ischemia (blood deficiency) and infarction (tissue death), Liver disease such as hepatitis, Muscle injury, Muscular dystrophy, Pancreatitis, Lung tissue death Stroke and Ischemic cardiomyopathy.

A method used to separate and quantify the different isozyme fractions of LDH is Polyacrylamide Gel Electrophoresis or PAGE. This method will be employed in both Parts A and B of this practical. Electrophoresis works based on the principle that molecules with a net charge, when subjected to an electric field, will move towards an electrode of the opposite charge. The greater the net charge, the faster the molecules will move. Polyacrylamide Gel Electrophoresis goes a step further by using a gel as the medium through which the molecules, which in this case are protein molecules, travel in order to reach the electrode of opposite charge. The gel serves as a molecular sieve through which small molecules travel with relative ease while the movement of larger molecules are obstructed and retarded. The size and charge of the protein molecules therefore influence how fast they travel. Since they are travelling at different speeds, different types of proteins separate out from each other to produce bands or stacks with each band in the gel corresponding to a particular type of protein.

Figure 4. Transmission-Electron Microscopic Image of a Polyacrylamide Gel

[http://en.wikipedia.org/wiki/File:TEM_image_of_a_polyacrylamide_gel.jpg, downloaded 5 March 2012.]

The gel is made of polyacrylamide which is made from the polymerization of acrylamide. It is an inert material and the size of the pores in the gel can be controlled by using appropriate concentrations of acrylamide and the cross linking reagent methylene bisacrylamide. Higher concentrations of acrylamide produce smaller pores in the gel. The gel is placed between two glass plates separated by a small distance of 0.5 1.0mm. A comb is placed between the plates before the gel has set in order to make wells at the top of the gel. After the gel has set the comb is removed and the purified protein samples are introduced into the wells using a micropipette. An electric field is then applied to the gel and the protein molecules begin migrating through the gel in the appropriate direction depending on their net charge.

Figure 5. Apparatus for Polyacrylamide Gel Electrophoresis and an Electrophoretic Profile.

[http://biotech.matcmadison.edu/resources/proteins/labManual/chapter_5/procedure5_3.htm, downloaded 5 March, 2012.]

For this practical, discontinuous electrophoresis will also be used which uses the same principle as PAGE except that two different types of gels will be used. The upper of the layer of the gel has a low concentration of acrylamide and thus has larger pores through which the proteins will be able to travel more quickly and easily. This gel is known as the stacking gel. The lower layer of the gel is is known as the resolving gel. It has a higher concentration of acrylamide and retards the movement of the proteins moving through it causing them to move much more slowly. When the proteins reach the resolving gel from the stacking gel, the movement of the protein molecules become slower than the proteins still moving through the stacking gel. This causes the proteins to separate out into thin bands with a high resolution in pure protein. An accordion or stacking effect is seen due to the proteins travelling at different speeds and the movement of the proteins being further retarded by the stacking gel. The staining that is to be used in Part A involves use of a solution containing lithium lactate, NAD, methyl phenazonium methosphosphate (PMS) and nitroblue tetrazolium. The lactate IS used in this staining method as a substrate for the LDH enzymes so that it can be oxidized by the enzymes, and give up an electron to the coenzyme and electron carrier NAD +. The NADH produced will then give up the electron to the PMS which is acting like an electron carrier, which will then give up the electron to the yellow tetrazolium salt, reducing the salt and producing a red colour. The activity of the LDH isozymes on the lactate substrate would therefore give rise to a colour change in the tetrazolium salt, thus allowing the bands in the polyacrylamide gels to be identified. The bands which have a darker colour would be interpreted to have a greater concentration of the LDH enzyme.

Figure 6.Mechanism of Staining Reaction for LDH Bands.

[Clinical Biochemistry Lab Manual]

Coomasie Brilliant Blue is to be used to stain the proteins to stain the serum proteins in Part B. This dye stains the proteins by binding to them. It also gives the proteins an overall negative charge without denaturing them allowing them to be separated by polyacrylamide gel electrophoresis while retaining their activity. The movement of the proteins through the gel is thus influenced by both the size and charge of the proteins.

A. Demonstration that LDH exists in a number of electrophoretically separable forms. Comparison of the heart, liver, serum and skeletal muscle forms. B. Electrophoresis of Serum Proteins. MATERIALS Sucrose solution (40%w/v) 0.01M phosphate buffer pH 7.2 Lithium lactate NAD Nitroblue tetrazolium Methyl phenazonium methosulphate You are provided with the following solutions: Stock Acrylamide/bis (30% T, 2.67% C) 87.6 g acrylamide (29.2g/100mL) 2.4g NN-bis-methylene-acrylamide (0.8g/100mL) Make to 300mL with deionised water. Filter and store 30 at 4C in the dark (30 days maximum). 1.5M Tris-HCL, pH 8.8 27.23g Tris base 80mL deionised water. Adjust pH to 8.8 with 6N HCl. Make to 150mL with deionised water and store at 4C. 0.5M Tris-HCL, pH 6.8 6g Tris base 60mL deionised water. Adjust pH to 6.8 with 6N HCl. Make to 100mL with deionised water and store at 4C. 10% SDS Dissolve 10g SDS in 90mL ddwater with gentle stirring and bring to 100mL with ddH 2O. Running Buffer Tris Glycine Deionised Water Stain for Proteins: Coomassie Blue R-250 0.6g 2.88g 1000mL

pH 8.3

0.2% in 50% methanol, 10% Acetic Acid Filter before use

Destaining Solution 30% methanol, 10% Acetic acid for proteins Preparation of Polyacrylamide Gels 7.5% Separating Gel
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Deionised water 4.85mL 1.5M Tris-HCL, pH 8.8 2.60mL Acrylamide-BIS 2.50mL 10%Ammonium persulphate (fresh) 50uL TEMED 5uL 4.0% Stacking Gel Deionised water 6.10mL 0.5M Tris-HCL, pH 8.8 2.60mL Acrylamide-BIS 1.33mL 10%Ammonium persulphate (fresh) 50uL TEMED Procedure: 1mL of fresh rat blood was placed in a conical centrifuge and allowed to clot at room temperature for 15 20 minutes. The tubes were then allowed to stand on ice to shrink the clot. The heart and liver were removed from the rat and placed in ice to allow the blood to drain away. Both organs were gently blotted with filter paper and weighed. The heart was then cut up with scissors and homogenized in 10mL of cold phosphate buffer. The liver and muscle tissue were then homogenized in 10x the volume of buffer. The homogenates were kept separate. The blood, liver and heart homogenates were then centrifuged at 1000g for 10 minutes and the supernatants were decanted into three clean tubes. These three tube were stood on ice. The 0.1mL of the serum was diluted with 0.9mL of buffer to give a fourth solution. The polyacrylamide gel was meanwhile prepared. Into four ependorf tubes were then placed the 0.5mL serum and 0.1mL surcrose solution, 0.25mL heart supernatant and 0.15mL of sucrose solution, 0.25mL liver supernatant and 0.15mL sucrose solution and 0.25mL diluted serum and 0.1mL sucrose solution respectively. The contents of these tubes were then thoroughly mixed. Spacers were placed between the two glass plates of the electrophoresis apparatus and plastic sealant was placed securely around the sides. The separating gel and stacking gel were then prepared. These were mixed well and pipetted between the glass plates leaving some 5mm of space above the solution. The separating gel was then carefully poured to fill about 5/6 of the length of the glass plates. Any air bubbles were dislodged and the gel was allowed to polymerize for one hour. A comb was then placed between the pates and the stacking gel was then carefully poured until the teeth had been covered by solution. This was allowed to polymerise for 45 minutes. The comb and rubber strip were then carefully removed and the wells were then rinsed with a few drops of running buffer. Two drops of each sample was then carefully applied to each well and then a layer of running buffer was also added to each sample. Care was taken not to disturb the enzyme layer. The lower vessel was then filled with running buffer and the gel was placed in the electrophoresis apparatus. The upper buffer vessel was then carefully filled.

The leads were connected so as to have the anode as the lower buffer vessel. 0.5 1mL of bromophenol blue solution was added to the upper buffer. The electrical leads were then attached to the power pack set to 20V, constant voltage setting. With the power pack switched to Constant Current, a 60mA per gel was applied to the assemble and run until the blue marker band reached with 5mm of the bottom of the gel. This too approximately 45 minutes. Gels could be run in the cold to prevent overheating. The power pack was then switched off and the plates were removed from the apparatus. The gels were then carefully removed from them. The gels were then stained for LDH activity using nitroblue tetrazolium and for total proteins using 0.2% coomassie blue and 10% acetic acid. The ecess stain was removed using 7% acetic acid and the results were recorded. A sketch of the gel patterns was made. The gels were stained for LDH activity using the following solution: 5mL 0.5M lithium lactate, 20mL 0.2M Tris buffer, pH 9.2, 20-25mg NAD, 1mg nitroblue tetrazolium*, 0.5mg methyl phenazonium methosulphate (PMS)* ( added immediately before use). The gels were left in this solution for one hour in the dark. In order to stain for the serum proteins, the gel was placed in a staining tray with Coomassie blue for 30 minutes. The gel was destained repeatedly by shaking in destaining solution. The destaining solution was changed several times to allow proper destaining.

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Results: Figure 7. LDH isozyme banding patterns of heart, liver, muscle tissues and serum obtained from polyacrylamide gel electrophoresis

Figure 8. Picture of polyacrylamide gel electrophoretic profile of serum proteins

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Discussion: A. Electrophoresis of LDH isozymes. The bands produced by the Heart well corresponded to that of LDH-1, LDH-2 and LDH-3. The 4 bands produced by the Liver tissue corresponded to that of LDH-1, LDH-2, LDH-3 and LDH4. The band produced by the Muscle corresponded to LDH-5. The bands produced by the Serum corresponded to that of LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5. The band that was closest to the anode was LDH-1. The band that was second closest was LDH-2. The band that was third closest was LDH-3. The band that was fourth closest was LDH-4 and the band that was fifth closest and which was in fact near the region of the well and cathode was LDH-5. The bands were seen in this order because the H protomer is more negative than the M protomer at pH 7, therefore isozymes containing a higher proportion of H would have a more negative net charge and would consequently move faster towards the anode. LDH-1 is made of 4H subunits while LDH-2 has 3H subunits and an M subunit. LDH-3 has equal amounts of both subunits and LDH4 has 3M and just 1M while LDH-5 has no H subunits at all so was furthest from the anode. Another factor which influenced their movement down the gel was the size of the different isozymes. However the net charge on the isozymes seemed to have a greater influence. The liver produced bands with the highest intensity in colour. This was perhaps due to the fact that the liver was the most active of all the tissues and therefore had the most enzymes, followed by the serum and heart tissue. For the Heart tissue, the bands were evenly coloured although the band corresponding to LDH-1 should have had the greatest intensity in colour since theoretically the Heart has the highest proportion of this enzyme than all the other tissues. The isozymes of LDH differ in terms of catalytic, physical and immunological properties. There is a higher proportion of the H subunit in heart muscle since it is more specialized for the aerobic oxidation of pyruvate. There is a higher proportion of the M subunit in liver and muscle tissue because it is specialized more in anaerobic metabolism and reduction of pyruvate (Worthington Biochemical Corporation, 2012). The skeletal muscle tissue produced a single band with only LDH-5 emphasizing how specialized it was in anaerobic metabolism. It should be noted that the supernatants of the different tissue homogenates was mixed with sucrose to lyse the cells in the supernatants and to thus release the contents of the cells including their enzymes. The staining that was used in this section involved use of a solution containing lithium lactate, NAD, methyl phenazonium methosphosphate (PMS) and nitroblue tetrazolium. The lactate was used in this staining method as a substrate for the LDH enzymes so that it could be oxidized by the enzymes, and give up an electron to the coenzyme and electron carrier NAD+. The NADH produced would then give up the electron to the PMS which was acting like an electron carrier, which would then give up the electron to the yellow tetrazolium salt, reducing the salt and producing a red colour. The activity of the LDH isozymes on the lactate substrate would
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therefore give rise to a colour change in the tetrazolium salt, thus allowing the bands in the polyacrylamide gels to be identified. The bands which had a darker colour would be interpreted to have a greater concentration of the LDH enzyme.

Figure 9. Mechanism of Staining Reaction for LDH Bands.

[Clinical Biochemistry Lab Manual]

B. Electrophoresis of Serum Proteins (PAGE) The electrophoresis used for the Serum Proteins produced several bands. The bands nearest the anode were Albumin. The second band was 1-globulin. The second band was 2-globulin. The third was 1-globulin. The fourth was 2-globulin and the fifth was -globulin. Coomasie Brilliant Blue was used to stain the proteins. This dye stained the proteins by binding to them. It also gave the proteins an overall negative charge without denaturing them allowing them to be separated by polyacrylamide gel electrophoresis. The movement of the proteins through the gel was thus influenced by both the size and charge of the proteins. A phenomenon which occurred in this part of the lab was overstaining. Overstaining is the retention of excess stain within the gel which results in a high background stain. The background stain prevents the protein bands from being seen. One way to prevent this from happening would be to ensure that the gel is not allowed to stain for too long or to simply closely monitor the gels as they are staining so that they are not left for too long leading to overstaining.

For both parts A and B, a precaution that was taken was to introduce the samples into the wells slowly with the micropipette so as not to disturb the molecular linking of the gels. Another precaution that was taken was to introduce the layer of buffer over the samples in the wells slowly so as not to disturb the samples in the wells and to disrupt the gradual movement of the proteins through the gels.

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References: Hames, David, and Nigel Hooper. (2006). Principles in Biochemistry. New York: Taylor and Francis Group. Murray, Robert K., Daryl K. Ganner, Peter A. Mayes, and Victor W. Rodwell. (2000). Harper's Biochemistry. United States of America: Appleton and Lange. Worthington Biochemical Corporation. (2012). Lactate Dehydrogenase. http://www.worthington-biochem.com/ldh/default.html, downloaded March 4 2012. MedlinePlus. (2012). LDH Isoenzymes. http://www.nlm.nih.gov/medlineplus/ency/article/003499.htm, downloaded March 4 2012.

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