Isolation and Characterization of Avian Lactate Dehydrogenase

Summary
Isolation and Characterization of Avian Lactate Dehydrogenase experiment consist of extraction of LDH from chicken breast muscle, then purifying the LDH obtain from chicken breast through affinity column chromatograph, determine protein concentration by Bradford assay and then determining the amount of catalytic activity through activity assays. Through Bradford assays, copious amount of protein concentration was detected, thus showing purification methods with affinity was successful, and with LDH activity assay, results concluded that there was catalytic activity among the protein concentration. The isozyme was then determined and separated by Agarose gel electrophoresis. The molecular weight of the purified protein was then obtain through SDS-Page method. Westren blotting method was induced, but failed due to the wrong reagents were ordered.

Introduction
Lactate Dehydrogenase catalyzes the conversion of pyruvate to lactate. There are four different distinct classes of LDH enzymes. Two of the classes are cytochrome c- dependent enzyme, each acting on either L-Lactate or D-Lactate . The other two classes are NAD(P)-dependent enzyme that also either acts on L or D- Lactate. In muscles tissue, LDH is crucial to maintaining levels of NADH and NAD+, especially under anaerobic condition. In Glycolysis, which is a metabolic pathway that converts glucose molecule into pyruvate and in the absence of oxygen, the only source of ATP comes from Glycolysis, although this metabolic pathway consume copious amount of NAD+ to fuel this process. Lactate Dehydrogenase serves to replenish the depleted NAD+ so that the glycolysis could keep producing ATP. LDH is applied in various medical usages as a marker. Hemolysis, which is the breakdown of blood cells could be marked by LDH by anazlyzing the blood sample to see if there is an increase in LDH concentration.

LDH could also be a marker for myocardial infarction. In pathology, high levels of lacatate dehydrogenase in cerebrospinal fluid are an indication that the patient has bacterial meningitis. In many cases of viral meningitis, high LDH, in general, indicates the presence of encephalitis and poor prognosis. In HIV patient, LDH is often measured as a non-specific marker for pneumonia due to pneumocystis jiroveci (PCP). The objective of this experiment is to extract LDH from chicken breast tissue and purifying the LDH. Purification can be achieved by homogenization, ammonium sulfate fractionalization and affinity column chromatography. After purification have been achieved, determination if protein composition exist will be determined through Bradford assay. To see if catalytic activity is present, LDH activity assays will be analyzed. Then Agarose electrophoresis method was used to separate the isozymes of LDH. To acquire the molecular weight of the purified protein, SDS-Page method was used.

Methods

Extraction of LDH from chicken breast muscle
CFE Preperation
Cell Free Extract (CFE) of chicken breast was prepared through homogenization of the tissues within the chicken breast. This process is completed by removing the skin and fat froma chilled boneless chicken breast. Then the total of 51.32 grams of chicken was weighed out, cut into small pieces and placed in a blender along with 75 mL of cold extraction buffer. Four short bursts (around 5 seconds per burst) were applied to complete the homogenization process with a ten second pause between each pulse. The homogenized tissue solution was then poured into three 50 mL centrifuge tubes, and was centrifuged at 15000 RPM for 20 minutes. The supernatant poured through two layers of cheese cloth into a 50 mL falcon tube, and the volume was recorded. Three 0.5 mL aliquots were saved.

Ammonium Sulfate Precipitation
After centrifugation, the volume of each CFE was measured, and then per every mL, .39 grams of ammonium sulfate was weighed out. The CFE was then placed in a beaker that was suspended on top of ice in a larger beaker, which was placed on top of a stir plate. Over a course of 20 minutes, ammonium sulfate was added to the CFE while stirring slowly. After the completing adding all of the ammonium sulfate, solution was stirred for an addition 15 minutes. Followed by centrifugation at 15000 RPM for 20 minutes.

Purification of LDH
Dialysis of the Protien
Dialysis of the salted protein was completed by a lab T.A.

Affinity Column Chromatography
To separate LDH from the rest of the protein, affinity column chromatography method was used. A column with a plastic stopcock was loaded with Cibacron blue affinity matrix by adding about 8 mL of resin solution to the column. Desalted protein solution was loaded in the column and a single fraction was collected and labeled flow through. After Flow through fraction has been collected, four different washes will be done. For fraction 1 through 4, columns were washed with 4 x 6 mL of Tris-PMSF buffer. 6 mL of solution was collected in each of the fraction For fraction 5 through 6, columns were washed with 2 x 6 mL of NAD buffer. 6 mL of solution was collected in each of the fraction.For fraction 7 through 9, columns were washed with 3 x 6mL of Tris-PMSF buffer. 6 mL of solution was collected in each of the fraction.For fraction 10 through 11, columns were washed with 2 x 6 mL of NADH buffer. 6 mL of solution was collected in each of the fraction.For fraction 12 through 14, columns were washed with 2 x 6 mL of Tris-PMSG buffer. 6 mL of solution was collected in each of the fraction.Then aliquot 3 x 1.5 mL samples for each of the fractions from 12 through 12 intro microfuge tubes.

Determination of Protein Concentration
Bradford Assay
The determination of protein concentration was determined through Bradford assays. Preparation of .1 mg/mL, .3 mg/mL, .5 mg/mL and 1 mg/mL solution of BSA. 200 uL of each solution was created and and 50 uL of each dilution was dispensed into cuvettes. Replication of CFE, desalted and faction 10 through 12 should be replicated twice per concentration. Samples were measured in a spectrophotometer at the absorbance of 595 nm.

Determination of LDH Activity
Activity Assays
Activity assays were ran to check for LDH activity in the fractions. All activity assay final volume were 2.5 mL after the addition of all the solutions. A single stock solution of 20 Mm Tris at pH 8.6, 2mM pyruvate and .16 mM of NADH was prepared. This solution was kept at 25 degree Celsius. Dispensed 1.25 mL of this solution into about 50 cuvettes and filled the cuvettes to 2.5 mL with deionized water.Two assays blanks were created to blank the spectrophotometer. One was pure deionized water and the other blank contained NADH. Fraction 10 through 12, CFE, and desalted should be replicated. The rest of the fraction was were ran once. Each cuvette contained 10 uL of sample.

LDH Reaction Kinetics and Inhibition
Kinetic Assays
To determine LDH parameters in absence and presence of oxamate inhibitor, activity assay was used.
A total of 45 cuvettes were prepared. The first 15 cuvettes were prepared with 50, 100, 200, 350 and 500 uM of pyruvate mixed with 10 mM of Tris-HCl at pH 8.6 and .08 mM NADH. These 15 assays buffer were prepared again, but this time 100 uM of oxamate was added to the cuvettes, then again with 200 uM of oxamate instead of 100 uM of oxamate.

UV-visible spectrophotometer was used to measure the absorbance of the cuvettes. To ensure proper data was obtained, spectrophotometer was set up for kinetics measurements at 340 nm and blanked with a solution of 10 mM of Tris at pH 8.6, 500 uM of pyruvate and .08 mM of NADH. The initial absorbance of the assay was around .498. Then 50 uM pyruvate with 200 uMoxamte was tested to ensure inhibition (reduced reaction in the absorbance with time). Fraction 11 was thawed and micro centrifuged. The same protein dilution that was used in the Bradford assays was applied to the kinetic assays. Thirty assays were ran, five for each varying pyruvate concentration without oxamate, then with 100 uMoxamate and then finally with 200 uMoxamate. Each of these assay had 10 uL of the diluted fraction added to 2.49 mL with a total volume of 2.5 mL. The kinetics for each sample were ran for two minutes each and the absorbencies were recorded at 340 nM per every 15 seconds till 2 minutes. Protein fraction was kept on ice the entire time.

Gel Filtration Chromatography and Agarose Electrophoresis
Gel Filtration Chromatography
To determine the molecular weight of the LDH purified from the chicken breast, Gel Filtration Chromatography was the method of choice. Fraction 11 was concentrated to 10 mg/ml of lactate dehydrogenase and HPLC was performed. The column was Sepharose CL-6B with a mobile phase of 100 mMNaPhosphate (pH 6.8), 0.025% NaN3, and a flow rate of 1mL/min. The protein molecular weight standards were RNase-MW 14000, Ovalbumin-MW 45000, BSA-MW 66000, Alcohol dehydrogenase- MW 150000, Catalase-MW 232000, Thyroglobulin-MW 669000, Blue Dextran-MW 2000000, and DNP-aspartate –MW 299. The LDH had a concentration of 10 mg/mL and 100uL was injected with a flow rate of 1mL/min and ran for 30 minute Agarosee lectrophoresis

To determine the different type of isozymes of the LDH, agarose electrophoresis was the method of choice. Preperation of the agarose gel required 50 ml of 1% (w/v) agarose in Tris/gly/EDTA buffer. The agarose was heated until it completely melted, and was swirled until completely dissolved. After this step, the mixture was allowed to cool for five minutes and then placed in the casting tray, and then the comb was inserted. The gel was allowed to cool for approximately 15 minutes. Following the preparation of the gel, four samples were prepared. The first was 10uL of isozyme M plus 10 uL of 5x loading buffer for 20 uL total volume mixed with only 10 uL of this inserted into the well of the agarose gel. The second was 2.5 uL of 5x loading buffer mixed with 10uL of isozyme H with only 10uL of this inserted into the second well of the agarose gel.

The third was 2.5uL of 5x loading buffer mixed with 10uL of Cell Free Extract with only 10uL of this inserted into the third well. The fourth was 2.5uL of 5x loading buffer mixed with 10uL of fraction 11(LDH) with only 10uL of this inserted into the fourth well. After preparing the sample,the casting tray was oriented with the sample wells closest to the black electrode (-) for electrophoresis. The chamber was filled with electrophoresis buffer until the gel was suspended in buffer, with 1 cm of buffer above the gel. The samples were loaded into the wells. The unit was assembled and set to 100 V and ran for 30 minutes. The gel was stopped when the bromophenol blue marker dye was close to the edge (closest to red electrode (+)). After electrophoresis has been completed, the gel was placed in the staining box with activity stain and incubated for 15 minutes at 37C until bands developed and afterwards a photo was taken.

SDS-Page Analysis
SDS-Page
The separation gel buffer consisted of 1.5 M Tris, pH 8.8. The stacking gel buffer consisted of 0.5 M Tris-HCl, pH 6.8, 29% (w/v) acrylamide and 1% (w/v) bisacrylamide, 10% (w/v) SDS, 10% (w/v) ammonium persulfate, and TEMED. Protein sample loading buffer was composed of 50 mM Tris-HCl, 100 mM 2-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol, pH 6.8. Electrophoresis buffer was composed of 25 mM Tris, 250 mM glycine, 0.1% SDS, pH 8.3. The gel-staining solution was composed of 0.1% (w/v) Coomassie blue in 65:25:10 H2O-MeOH:HOAc. The gel-destaining solution was 60:30:10 H2O:MeOH:HOAc. All components of the separation gel except TEMED were added, the solution was mixed, and TEMED was added. The space between the glass plates was filled to approximately 1.5 cm below the top of the lower glass plate.

The gel was overlaid with 0.1% SDS in deionized water and 15 minutes was allowed for polymerization to occur. The stacking gel was prepared with its components. The space above the separation gel was filled with the stacking gel to the top of the lower glass plate and the gels were allowed to polymerize for 45 minutes. One volume of sample and one volume of denaturation buffer were combined and boiled for two minutes. Samples were applied to the wells and gels were ran at 135 mV until the tracking dye reached the bottom of the gel. The appropriate section of the gel was stained for approximately 45 minutes, removed, rinsed with deionized water, and placed in destaining solution for approximately 45 minutes. Western-Blotting

Due to a mis-communication, the wrong materials and reagents were obtained, and thus the experiment failed. Thus no experimental data was obtained from this part of the experiment. Materials

Sigma Reagents
Adenine, EDTA, Lithium-L-Lactate, Pyruvic acid, Oxamic acid, Sodium Azide, Nitroblue, Tetrazolium, Phenazinemethosulfate, Blue dextran Sigma, St Louis, MO, USA

Sodium phosphate, Fischer Scientific, New Jersey, USA
NAD and NADH, RPI , Alabama, US
Agarose, Gibco BRL, Grand Island, NY, USA
SEC COLUMN: BioSep- Sec – s2000 from Phenomenex, California, USA UV-VIS
Hitachi L-4000 UV detector
Hitachi L-6200 Intelligent pump
Spectrometers: Biowave II, WPA from Biochrom, MA, USA
Bio-rad protein assay, Bio-rad, CA, USA

Result and Discussion
Lactate dehydrogenase enzyme that was to be studied was isolated from chicken breast muscle through a homogenization technique, which is the intensive blending of mutually related substances or groups to form a constant of different insoluble phases to obtain a suspension or emulsion. After this process, to reduce amount of available water so that protein could stay in solution, ammonium sulfate was used. Thus not allowing hydrophilic side chains to interact with the solvent. This could lead to aggregation and denaturation of the protein.

After extraction of proteins from chicken breast, to separate LDH from the rest of the other proteins, affinity chromatography is the best method of choice for purification. Since the proteins were all salted, a dialysis was run to desalt the proteins. From affinity chromatography, 15 fractions were obtained.

To analyze if the purification method was effective and that protein concentration existed, Bradford assay was the method used to determine protein concentration of the samples. Analyzing the data acquired from the Bradford Assays concluded that purification method using affinity chromatography was effective and that there is protein concentration present. The calibration curve from the Bradford assay gave good results compared to the absorption results from the experimental samples (figure 2)

y=.7071x-.008

Inputting absorbance for y and solving for x will give the protein concentration for the current fraction. Refer to (Figure 1 for complete data.) Fraction 1, with a protein concentration of .704285 ug/uL indicates a dense amount of protein concentration, but since this is the first fraction it might not be all LDH. As each fractions (2-8) wereanalyzed, protein concentration keeps dropping, until fraction 10 through 12, where most of the LDH proteins exist. To ensure the purification did not denature or destroy the enzyme, activity assays were used to check for catalytic activity. Fractions 1 throught 8 showed very little activity (Figure 3). This signifies there are very low amounts of LDH. Fraction 10 through 12 showed enough catalytic activity to signify that there is LDH activity. CFE was also analyzed and showed the highest of all activates (figure 4). Fraction 11 was chosen to be used in the kinetic assays of pyruvate to lactate.

The activity and inhibition of Lactate Dehydrogenase resulted in a measurement of activity for the conversion of pyruvate to lactate. The graph in figure 8 showed velocities measured in mM per minute versus the pyruvate concentration in mM. According to the graph, the conversion of pyruvate to lactate follows the Michaelis-Menton steady state kinetics, which is based on increased velocity as the pyruvate concentration increased. With the Michaelis – Menten kinetics Vmax, Km and Kcat was determined (refer to sample calculations).Figure 8 shows that then enzyme is reaching maximum velocity, which is when the enzyme is being saturated with substrate and velocity cannot be increased unless more enzyme was added. Then later, when oxamate was added at two different concentrations, the Ki could be then determined. The addition of this pyruvate analog, oxamate resulted in the slowdown of LDH activity and when the larger concentration of oxamate was added, LDH activities diminished rapidly. This type of inhibition is termed competitive inhibition; in this case competition comes from pyruvate and oxamate competing for the binding stie of LDH.

To calculate the rate of LDH converting pyruvate to lactate, the Vmax was calculated from the total enzyme concentration equation.

Vmax=Kcatx [EnzymeTotal]

In this equation Vmax indicates the maximum conversion possible of substrate to product based on the rate constant Kcat and the total enzyme concentration. From the Bradford and activity assays, fraction 11 showed dense amount of protein concentration and the most activity. With a concentration of .746165 mg/mL that was diluted thirteen times to .057474 mg/ml. Then 10 uL of that was added to the run for a totally of .000575 mg. This indicates that the assay concentration of .000575 mg in a 2.5 mL totally volume of 23e^-6 mM. The data that fraction 11 gave;Vmax could be obtained through a Lineweaver Burk Plot (Figure 12), which gave an intercept of ~ 21=1/vmax, giving .05 mM per minute for the vmax This value and the total enzyme concentration, 2e^-6 mM were plugged into the total enzyme concentration, rearranged and Kcat was obtained.

Vmax=Kcatx [EnzymeTotal]

Kcat=Vmaxenzymetotalconcentration

Kcat=.05 mM/min2e-6mM

Kcat=417 persecond

The value for Kcat 417 per second indicates 417substrate molecules are converted to product every second per enzyme molecule. Km was determined through the following equation:

K-1+ K2K1=Km

Km was calculated to be .144 mM, and Km indicates the substrate concentration ([S]), when intial velocity is equal to one half Vmax. To make sure data was accurate:

Vo=Vmax2

Vo=Vmax [S]km+[S]

Vo=(21 x .144 mM)/(.144 mm+[.144M]

Vo=10.5

This proves that this experiment provided real data.
Ki, which is the imbibitions constant for enzyme kinetics could be determined by the examination of how different concentration of inhibitor oxamate affected the Lineweaver Burk. The double reciprocal plots in figure five showed that as the concentration of the inhibitor increased so did the slope of the assay. This indicate that the slope for LDH under no inhibition was ~3. The calculation of the inhibitor constant, , was based on the increase in the slope only for inhibition, because during competitive inhibtion the oxamate inhibitor directly competes for the active site on LDH with pyruvate and only affects the Km value directly with the maximal velocity unaffected and any plot of the double-receprocal M-M equation and direct comparison with the linear fits obtained in figure five shows that the reaction kinetics are driven by pure competitive inhibition, respectively.

By definition during no inhibition, is one, and when LDH is inhibited by oxamate this value increases. The slope for pure kinetics (no inhibition) was 2.98 ~3, and when 100uM of oxamate was added the slope changed to 9 and when 200uM oxamate was added the slope changed to 17.55~18. The way to calculate was to divide the change in slope with the slope with 0 uM oxamate to find ’s value at 100uM oxamate. This meant that when 0uM oxamate =3/3=1, and 100uM oxamate =9/3 =3, and 200uM oxamate = 18/3 = 6. The methods for calculating these can be found in the sample calculations in the appendix. The value of Ki determined for 100uM oxamate was 50, and for 200um oxamate was 40.

Using Agarose electrophoresis to analyze the LDH enzyme, the different isozymes were determined. The results indicated that bands corresponding to the 20 uL dilution of muscle isozyme and to 10uL dilution of heart isozyme. Bands for 10 uL dilution of CFE and to 10 uL dilution of LD were not readily observed. Data collected from the gel filtertaion indicates products appearance of purified LDH s at 10.5 min.

Lacatate dehyrodgenase proteins are either heterotetramers or homotertramer, composed of M and H protein subunits, which are encoded by two different genes. The H subunit is located on chromosome 12p12.2-p12.1. The M subunit is encoded by the LDHA located on chromosome 11p15.4. There are five types of LDH with different protein subunits found in a variation of tissues. Type 1 LDH proteins are based in heart tissues, composed of 4 H subunits. Type 2 LDH proteins are based in reticuloendotheial system, which is composed of three H subunits and 1 M subunit. Type 3 LDH proteins are located in the lungs and is composed of two H and two M subunits. Type 4 LDH proteins are located in the kidney, placenta and pancreas and are composed of one H subunit and three M subunits. Type 5 LDH proteins are located in the liver and striated muscle and are composed of four M subunits.

The molecular weight calculated from the SDS-PAGE is around 4 million daltons, divide that number by 1000 to get kilodaltons, which is 4000, meaning 1000 kilodaltons per an enzyme subunit. Literature values indicates that LDH is the molecular weight for LDH is 136,700 daltons in beef and 140,000 daltons in rabbits. Thus, in chicken breast LDH, the molecular weight of LDH is significantly higher compared to Rabbit muscle and beef heart muscle. One reason why chicken breast muscles is higher in molecular weight is that the breast area is dense in muscle, denser than the heart, thus having a higher molecular weight.