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The final 27 page large lab report final that we spent the entire semester working on. ...

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Imberg 1 Purification and expression of Lactate Dehydrogenase from bovine heart and expressed LDH from Barracuda Scott Imberg & Deok Yong Kim College of Biological Sciences, Department of Biochemistry, Molecular Biology, & Biophysics University of Minnesota, Twin Cities, 55455 Keywords: Lactate Dehydrogenase (LDH), enzyme, chromatography, polymerase chain reaction (PCR), activity assay, specific activity, total units, Km, Vmax Abstract In this study, we purified Lactate Dehydrogenase (LDH) from bovine heart and the barracuda fish. We compared the different purification methods associated with both and determined which produces a more pure product. We used three different chromatography procedures to purify the LDH found in bovine heart. We used ion exchange, affinity exchange and size exclusion chromatography. In contrary, we used immobilize metal affinity chromatography to purify our expressed barracuda LDH. After each purification procedure, we measured the specific activity of our purified samples using a spectrophotometer by measuring the absorbance at 395 nm of light. In addition, we created Michaelis-Menten (Fig. 5) and Eadie-Hofstee (Fig. 5) plots to experimentally find the maximum velocity (Vmax), Km, and the turnover number of the substrate of LDH, NAD+. We then exposed our purified samples to various forms of gel electrophoresis. We preformed SDS-PAGE, native gel, and western blot procedures, and this helped us visualize the purified LDH. From our finding, we determined that the

purification methods implemented were effective, and the concentrated sample produced after the three different chromatography procedures produced the sample with the highest specific activity and fold purification. Moreover, we found the Km of NAD+ to be 200 µM, and the Kcat to be 23,000 min-1 which is higher than the literature K cat of NAD+. Introduction Our bodies contain about 75,000 different enzymes that help catalyze chemical reactions in us (Farley). One important enzyme found in most living organisms is Lactate Dehydrogenase (LDH). During anaerobic conditions from activities like high intensity exercise, LDH reversibly converts pyruvate into lactate and it oxidizes NADH to NAD+. The conversion of pyruvate to lactate depletes the pyruvate levels, and allows the glycolysis pathway to continue and provide us with energy in oxygen poor states (Schwert, et al). In starvation states, NAD+ is reduced back to NADH and lactate is converted back to pyruvate via the Cori Cycle in the liver. Due to the rise in

Imberg 2 pyruvate levels, the Cori Cycle favors the gluconeogenesis pathway so depleted glucose levels can be replenished (Katz & Tayek). LDH is a tetramer, commonly composed of the LDH-M and LDH-H subunits (Bishop, et al.). The LDH-M subunit is primarily used in the liver for Cori Cycle pathway, whereas LDH-H is used in other tissues for the conversion of pyruvate to lactate (Krieg, et al.). LDH-M and LDH-H composes a total of 5 possible tetramer isozymes, H4, M4, H3M1 , H2M2 and H1M3 (Bishop, et al.). The purpose of our study is to purify LDH and its isozymes, compare two purification techniques, to compare bovine and barracuda LDH properties, and to observe LDH’s enzymatic activity compared to reported values. We obtained LDH from two different sources—cow heart and the barracuda fish. We used three major chromatography techniques to purify our LDH from the cow heart. Before we could begin these techniques, we needed to prepare our LDH from our crude sample. We did this by the process known as salting out. We exposed our crude LDH sample to various concentrations of ammonium sulfate and we centrifuged our fractions. Salting out reduces the solubility of biomolecules in solution. (Baldwin). Ammonium sulfate causes biomolecules such as LDH to aggregate together and fall out of solution (Baldwin). After the salting out step, we loaded samples of our fractions into a spectrophotometer and recorded its absorbance over time. From there, we

were able to calculate the specific activity and total units of our fractions. The next part of the experiment was chromatography. We loaded our prepared LDH samples into three different columns: ion exchange affinity exchange, and size exclusion exchange. These three types of chromatography helped separate LDH based on its polarity, binding properties, and its size. In addition, the barracuda LDH was ran on an immobilized metal affinity chromatography (IMAC) column. We used this procedure to isolate the barracuda LDH because this sample was labeled with a His-tag. The IMAC procedure isolated the barracuda LDH based on its affinity to Nickel. After these chromatography procedures and exposer to restriction enzymes, our sample is ready for ligation and transformation. In DNA ligation, hydrogen bonds form between the sticky end overhangs on our insert and plasmid with the help of the enzyme DNA ligase (Brown). After ligation, our products were transfected into competent cells and plated on agar containing plates and allowed to incubate. After the incubation period, we collected our cells and amplified them using PCR. After the chromatography procedures, we found the Vmax and Km of our LDH fractions by measuring the activity of our samples and created a Michaelis-Menton, Lineweaver-Burk and Eadie-Hofstee plot. We ran our purified samples on an SDS-PAGE gel, native gel, and a Western blot to visualize their expression via electrophoresis. These procedures helped us determined if our ligation and

Imberg 3 transformation procedures were successful. The gel electrophoresis procedures separated our target proteins based on molecular weight and helped us determine the purity of our samples by comparing their weight to the expected values. Materials and Methods Plasmid Preparation: To prepare our plasmid for preparation, we resuspended our thawed pET15b/rKGA pellet in Buffer P1 by votexing. Next, we added Buffer P2 and N3. The tube was mixed well after each addition. We spun the tubes on a centrifuge and collected the supernatant. We placed the supernatant into a spin column and spun it again. We discarded the flow-through and added Buffer PE; the tube was spun once again. We added Buffer EB to the spin column, spun it and collected the flow through. Spectrophotometry: The spectrophotometer must be blanked with water before reading. The results from the spectrophotometer give us the ΔAbsorbance/ ΔMinuet. From these reading, the concentration of the protein of interest can be calculated using Beer-Lambert Law. Gel Electrophoresis: Gel Electrophoresis separates DNA based on its charge. Before we loaded our samples onto the 0.5X TBE gel, the proper PCR sample solutions needed to be made. We mixed 5 uL of our sample with 5 uL of water and 1 uL of 10X Loading Dye. After mixing, we loaded our samples into the wells in our gel, making sure to load a group of just

water and a 1 KB Plus Ladder for reference. The gels ran at 100 V until the dye front is half way to the bottom. After, we photographed our gel and recorded it in our lab book. Restriction Digest: We did two separate reactions for our vector and insert. Both the insert and vector were digested with 10X NEB CutSmart, BamHI, Ndel and sterile water. In our vector digest, we added CIP to remove phosphorylated ends of DNA and RNA so our vector can be used in cloning. After making the digest mixtures, we placed the tubes in a warm water bath so digestion can occur. After digestion, the tubes are stored in a freezer until needed. Preparing LDH from Crude homogenate (Ammonium Sulfate Precipitation): Our lab instructor prepared us a crude homogenate of beef heart containing LDH. We spun this homogenate using a centrifuge and collected the supernatant. After saving an aliquot of the solution, we measured the volume of supernatant and slowly mixed in ammonium sulfate to begin the salting out procedure by making a 40% saturated solution (40% SN). We added 0.242 g of ammonium sulfate per milliliter of supernatant. Once the ammonium sulfate is completely mixed in, we poured the solution into a centrifuge tube and spun it at 16,000 RPM for 20 minutes at 4ºC. We measured the volume of the supernatant after the spin and saved an aliquot sample. Next, we prepared a 65% saturated solution (65% SN) from our

Imberg 4 supernatant. We added 0.166 g of ammonium sulfate per milliliter of supernatant. We added the ammonium sulfate slowly while mixing vigorously. After mixing, we spun the tubes at the same settings as the previous spin settings, recorded the volume of supernatant after completion, and saved an aliquot sample. We resuspened the pellet left in the centrifuge tube from the 65% saturated solution in 0.03 M bicine and an 8.5 pH buffer. After resuspension, we saved an aliquot sample and placed the re-dissolved pellet in a dialysis bag at 4ºC, overnight, in a jar of buffer containing 0.03 M bicine at pH 8.5. LDH Activity Assay: We assayed each aliquot we collected in the salting out procedure using a spectrophotometer. Before the assay, a cocktail containing 1.9 mL of 0.14 M CAPS pH 10 buffer, 0.5 mL of 0.15 M lactate and 0.5 mL of 6 mM NAD+ needed to be prepared. We placed 900 μl of our cocktail into a 1 mL cuvette. We added enough water and aliquot sample so the total volume in the cuvette totaled 1 mL. We added the sample last, and as soon as it was added, we began to record the absorbance at 10 seconds intervals over a period of 1 minuet. We created a graph in excel and added a linear tread line through the points that were most linear (Fig. 2). The slope recorded is the Absorbance/minuet. From there, we were able to calculate the total units, the units/mL, the protein amount in mg/mL, and specific activity. Pouring Columns:

We loaded our columns for ion chromatography using Q-Sepharose. We needed to be sure that no bubbles were present in our matrix, because bubbles can affect the speed at which the proteins elute out of the column. We drew up as much Q-Sepharose as we could in a Pasture pipette and squirted it into our closed column. We did this about two more times. After that, we opened the stopcock and allowed the matrix to settle. We adjusted the volume of Q-Sepharose so that it is right underneath a specific marker. We added 0.03 M bicine, pH 8.5, so the column would not run dry and place parafilm over the top of the column for storage. Ion Exchange Chromatography: Q-Sepharose is an effective ion exchanger because it is positively charges and binds negatively charged proteins, like LDH. Before we loaded our sample into the column, we labeled 20 tubes to collect the fractions and removed the excess buffer from our column. With the stopcock off, we slowly added 1.5 mL of our 65% dialyzed sample of LDH, with 10 mL of initial buffer wash containing 0.03 M bicine, pH 8.5. After addition, we opened the stopcock and collected 3 mL fractions. Once when the column was about ready to run dry, 15 mL of elution buffer I containing 0.03 M bicine, pH 8.5 with 0.2 M NaCl was loaded to the column. After elution buffer I ran through, 15 mL of elution buffer II containing the same bicine buffer and 0.4 M NaCl. Once when elution buffer II ran through, 15 mL of elution buffer III congaing the same bicine buffer with 0.6

Imberg 5 M NaCl was added. Next, 15 mL of elution buffer IV (0.03 M bicine, pH 8.5, 0.6 M NaCl) was added after elution buffer III ran through the column. Finally 15 mL of elution buffer V (0.03 M bicine, pH 8.5, 0.8 M NaCl) was added after elution buffer IV ran through. After collecting our fractions, we recorded the OD280 of our fractions using a Nanodrop. Furthermore, we did an activity assay on the fractions with an OD280 of above 0.1. After the activity assay, we pooled the fractions together with the highest activity, recorded the pooled activity, saved an aliquot sample (pooled IEX), and placed them in a bag for dialysis (dial IEX). Affinity Chromatography - Cibacrom Blue: In this procedure, we used Cibacron blue to mimic the substrates of LDH. Before we began the procedure, we set up 50 test tubes to collect our fractions. We loaded 1.5 mL of the dialyzed sample from IEX into a Fast Protein liquid Chromatography (FPLC) machine with 0.02 M sodium phosphate and 1 M NaCl/0.02 M sodium phosphate buffer solutions. We looked at the DuoFlow report and found where our active fractions eluted. We did an activity assay to determine which fractions had the most activity and pooled together the fractions with the most activity (pooled AFC). We saved a sample and did an activity assay of our pooled fractions. Our pooled fractions were concentrated to be used in the next lab protocol (conc’t AFC). Size Exclusion Chromatography:

Larger proteins will elude first because they pass directly around the beads in the column. Smaller proteins get trapped in the beads and will elude out later in the procedure. After setting up 50 tubes to collect our fractions, we loaded 100 μl of Blue Dextran and 900 μl of our concentrated AFC sample into the FPLC. We assayed our samples and pooled together the active samples (pooled SEC). We saved a sample of the pooled fractions, and the rest of the pooled fractions will be concentrated down to be used in the next lab procedure (conc’t SEC). Recombinant LDH purification and NiNTA spin column: A pellet of crude barracuda LDH was thawed and suspeneded in 1 mL lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, pH8). To our sample, 150 µl lysozyme (10 mg/mL) was added and mixed. Thee sample was placed in 4°C on a rotator for one hour. After, it was spun in a centrifuge for 30 minuets at the same temperature at top speed. The supernatant was transferred to a new microfuge tube and a sample was saved (SN). In order to equilibrate the Ni-NTA spin column, 600 µl of the same lysis buffer was added to our spin column and it was spun for 2 minuets at 2000 RPM. The column was placed in a new microfuge tube where 600 µl of our SN sample was added and spun for 2 minuets at 2000 RPM. The flow through was saved (flow-through QIA) and the column was added to new microfuge tube. The spin column was washed with 600 µl wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM Imidazole, pH8)

Imberg 6 and spun for 2 minuets at 2000 RPM. The flow through was saved (1st wash QIA) and the column was placed in new microfuge tube. The same process with the wash buffer was repeated and we saved a sample (2nd wash QIA). The protein in the column was then eluted with 200 µl Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM Imidazole, pH8) and spun for 2 minuets at 2000 RPM. We saved the flow through (Elution QIA). Affinity Chromatography - Immobilized Metal Affinity Chromatography (IMAC): One mL of the SN sample was added to the DuoFlow system with Buffer A (50 mM NaH2PO4/300 mM NaCl) and Buffer B (50 mM NaH2PO4/300 mM NaCl/500 mM Imidazole, pH 8). The flow-through DuoFlow sample was collected from fractions 7-9 and the wash-through DuoFlow sample was collected from fractions 9-12. About 500 µl of each sample was saved and labeled. The elution DuoFlow sample eluted in fractions 14-18, all fractions were collected and placed in conical tube. The samples collected were diluted to a volume of 500 µl. Enzyme Kinetics: Enzyme kinetics are affected by concentrations of substrates and enzymes, temperatures and inhibitors. Each enzyme has a maximum rate of reaction when the enzyme is saturated by substrate. This rate is known as the V max. The Km is the amount of substrate required to give ½ the V max. To find the Vmax and K m, You must make a Velocity

vs Substrate graph. We did this by taking an activity assay of our concentrated SEC sample with varying levels of substrate and plotting out your results. The maximum value that the graph reaches is the V max and the Km is simply half of that value. We created a Lineweaver-Burk plot by graphing Substrate-1 verses Velocity-1. This gave us a near linear graph. In a LineweaverBurk plot, where the line crosses the yintercept is equivalent to Vmax-1 and where the line crosses the x-intercept is equivalent to -Km-1. Moreover, we created an Eadie-Hofstee plot where the reaction rate is plotted as a function of the ratio between rate and substrate concentration. SDS-PAGE: Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) looks at proteins rates of migration through a porous, gel matrix when exposed to an electric charge. Sodium dodecyl sulfate (SDS) is a strongly negatively charged detergent that can give proteins a net overall negative charge. Once all proteins have the same charge, the smaller proteins will migrate further down the gel and the larger proteins will stay higher up. In this procedure, we did two SDS-PAGE reactions. One looked at the Beef Heart LDH and the other looked at the recombinant LDH. We prepared 10 μl each bovine heart purification sample with 10 μl of 2X loading dye. These samples were boiled for 10 minutes and loaded on our gel apparatus that was filled with 1X SDS buffer. We used M3913 ladder and Benchmark as

Imberg 7 molecular markers and the 2X loading dye as a negative control. We placed the top on the gel electrophoresis unit and ran it at 150 volts for about 40 minutes. We rinsed our gel, heated it, rinsed it again, and stained it with SimplBlue Safe Stain. After staining, our gels could be photographed. Native Gel: We mixed 10 μl of our samples with 10 μl of 2X native sample buffer. The samples were vortexed and spun in a centrifuge. We inserted the gel into the electrophoresis unit, ensuring the tape was removed. We loaded all of our samples into the gel and ran the gel at 150 V for about 30 minuets. Once when the blue line reached the bottom of the gel, we continued to run for an additional 20 minuets. The gel was removed from plastic and placed in staining dish. We rinsed the gel with diH2O and drained it. We staind our gel with the LDH activity stain (0.1M Tris, pH 9.4, 1% Lactate, 0.05% NAD+, 0.1M Tris, pH 9.5, 0.005% NBT, 0.0005% PMS). The gel was covered with tinfoil and placed in a 37 degree C incubator for 40 minuets, checking the gel every 5 minuets. The reaction was stopped by rinsing with diH 2O before the bands stained too dark. The gel was placed on a white background and photographed. Western Blot: We mixed 10 μl of every beef heart LDH and expressed LDH samples that were collected with 10 μl of 2XSDS sample buffer. We boiled each sample for 10 minutes, except for the benchmark ladder. Our beef heart LDH samples were loaded on one gel, and our

expressed LDH samples were loaded on a different gel. Each sample was loaded on an Any-kD acrylamide gel from BioRad. The gels were ran at 150 V for about 40 minutes. The gels were removed from the plastic plates, and the proteins on the gel were electrophoretically transferred to a labeled nitrocellulose membrane. The beef heart membrane was placed in a blocking solution of Tris buffered Saline with Teen 20 (10% powdered milk in TBST [10mM Tris, 150 mM NaCl, 0.05% Tween 20]) and stored at 4°C overnight. The expressed protein membrane was placed in a Tris buffered Saline solution and stored at 4°C overnight. Antibody Detection (Beef heart): The beef heart membrane was rinsed 3X with 15 mL diH 2O and then rinsed another 3X with TBST. Two µl of primary antibody, monoclonal anti-LDH human antibody in mouse, was mixed with 10 mL of TBST. The solution was added to and mixed with the membrane for 30 minutes. Next, the membrane was washed 3X with 20 mL TBST. Four µl of secondary antibody, AP-goat antimouse IgG conjugate, and 10 mL TBST were mixed with the membrane for 30 minutes. The membrane was washed 3X with 20 mL TBST and then rinsed with diH2O. After draining, 10 mL of color development solution was added to the membrane and stained on a rocker until bands were visible (about 20 minutes). The reaction was stopped with diH 2O; the membrane was dried and subsequently photographed. Antibody Detection (Expressed LDH):

Imberg 8 The expressed LDH membrane was incubated for 1 hour at room temperature in a 1/1000 dilution of NiNTA HRP Conjugate stock solution. The membrane was washed 3X in 40 mL TBST buffer. After washing, the membrane was stained with 30 mL of HRP staining solution (18 mg 4-chloro1-naphthol, 6 ml methanol, 1X...