BIOL 3364 Practical #2

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Name: Vashista Shripat I.D.#: 809003142

Date: 06|02|12

Course Code and Title: BIOL 3364 – Clinical Biochemistry

Practical #: 2

Title: Electrophoresis of Lactate Dehydrogenase (LDH) Isoenzymes.

Aim:

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.

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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).

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

lactate

dehydrogenase

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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 fine-

tuned 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.]

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Table 1. The Subunits and Location of the Different LDH isozymes.

Lactate dehydrogenase

Isozyme

Subunits Location Serum LDH

(%)

LDH – 1 4H Mainly found in red

blood cells and heart

muscle.

33

LDH – 2 3H1M Mainly found in white

blood cells.

45

LDH – 3 2H2M Mainly found in the

lungs.

18

LDH – 4 1H3M The highest

concentrations are

found in the kidney,

placenta and pancreas.

3

LDH - 5 4M The highest

concentrations are

found in the liver and

skeletal muscles.

1

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.

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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.

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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.

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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.

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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 N’N’-bis-methylene-acrylamide (0.8g/100mL)

Make to 300mL with deionised water. Filter and store 30 at 4°C 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 4°C.

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 4°C.

10% SDS

Dissolve 10g SDS in 90mL ddwater with gentle stirring and bring to 100mL with ddH2O.

Running Buffer

Tris 0.6g

Glycine 2.88g

Deionised Water 1000mL pH 8.3

Stain for Proteins:

Coomassie Blue 0.2% in 50% methanol, 10% Acetic Acid

R-250 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.

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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 LDH-

4. 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 LDH-

4 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 α1-globulin. The second band was α2-globulin. The

third was β1-globulin. The fourth was β2-globulin and the fifth was γ-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.