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Lab Report 1 _ Step-Wise Purification and Characterization of a Lactate Dehydrogenase Isozyme from unknown tissue sample of Sus domestica Tyler Mitchell October 28, 2008 A02 : Darwin Group : 2 Mike , Won Abstract Given an unknown sample of crude homogenate our group sought to identify what isozyme of Lactate Dehydrogenase was present. We concluded our sample was from the heart of Sus Domestica.
The LDH tetramer found in the heart tissue consists of 4 H isozyme subunits of protein coded by the LDHA gene. 4 different steps of purification were attempted and samples taken at the conclusion of each major step were tested for activity. It was found that the 65% cut pellet, the second intermediate, had the highest LDH activity of all purifications. Our SEC purified product was also highly active but second to the 65% CP. 2) Abstract. This should be a brief summary of the major conclusions from your experiments, and should not be more than a paragraph in length.
Do not include experimental details on procedures here, only your important results and how they support your overall conclusions. A good abstract usually has a single introductory sentence to let the reader know what field you are working on. Introduction Lactate Dehydrogenase (LDH) is an enzyme present in a large number of plants and animals. It is a tetramer that consists of differing numbers of subunits from the LDHA and LDHB genes. The LDHA subunit is known as M and the LDHB subunit is known as H. There are 5 total isozymes of LDH, most are confined to a particular area of the body.
LDH catalyses the reversible formation of lactate from pyruvate with the parallel conversion of NADH to NAD+ this provides energy to tissues (by feeding NAD+ to the glycolysis process) when muscle conditions have become anaerobic such as sprinting. The lactate is then removed from the muscles and converted to glucose in the liver (using a different LDH tetramer) to recirculate and feed muscles. This movement of glucose to pyruvate to lactate in the muscle and then from lactate to pyruvate to glucose in the liver is called the Cori cycle.
The sample we examined was taken from an unknown tissue of Sus Domestica and the LDH was purified using progressively selective methods with testing done to samples that were taken after each major step in the purification scheme. The main purpose was to identify the LDH isozyme contained in the tissue, identify the tissue it was recovered from and characterize its activity throughout the various purification steps. The strategy employed used consecutively selective steps to end in an isolate of nearly pure LDH product.
Our approach was closely related to that used in the isolation of glutathione reductase from rainbow trout liver. Initially those authors prepared a homogenate and centrifuged it, followed by an ammonium sulfate fractionation, affinity chromatography and a gel filtration also known as size exclusion chromatography . Our scheme was identical; however a step of gel electrophoresis was added at the end with standard preparations of LDH H4 and LDH M4 to identify the exact isozyme present. Introduction: You will need to give some background information on LDH, enough to make the reader familiar with the enzyme you are working with.
Also, give a brief introduction to enzyme purification in general, not going so much into specific purification techniques but rather the multi-step purification strategies that are used. To do this, you should cite a scientific journal article where another enzyme (not LDH) was purified. You will need to do some searching on PubMed for this. Ask your TA or the instructor for help if you are not familiar with using PubMed. What purification steps were used in the article to purify that enzyme? Don’t forget to state what your ‘purpose’ is. You may want to give a brief outline of your purification strategy, but don’t go in any background on how he individual purification techniques work. Materials and Methods Crude Homogenate Enzyme Extraction A crude homogenate of unknown tissue type was prepared in 50mM phosphate buffer @ pH 7. 5. Approximately 100ml was obtained and put on ice. This lysate was centrifuged at 20,000g for 10 minutes @ 4? C. The supernatant was extracted and labeled as a Clarified homogenate (CH) for further processing. A sample of 0. 5ml was taken for later testing. Ammonium Sulfate Precipitation of Clarified Homogenate (CH) A 40% ammonium sulfate cut was preformed upon the recovered CH fraction. . 242g of ammonium sulfate per ml of CH was added to beaker kept in an ice bath. The ammonium sulfate was added over the course of 3 minutes while the preparation was on a stir plate and then allowed to stand for 15 minutes. The preparation was then centrifuged @ 15,000g for 15 minutes @ 4? C. The supernatant was recovered and pellet discarded. Another 0. 166g of ammonium sulfate per ml of recovered supernatant was added in the ice bath over the course of 3 minutes and allowed to stand for 15 minutes. This mixture was then centrifuged at 15,000g for 15 minutes @ 4? C.
The supernatant was discarded and the pellet containing the LDH was resuspended in 4ml of phosphate buffer @ pH 7. 5. A 100? l sample was taken for later testing and this intermediate was labeled as the 65% cut pellet (65%CP) and put on ice. Affinity Chromatography Purification of 65% Cut Pellet (65%CP) A 10cm long by 1cm diameter affinity chromatography column was prepared by pouring 12ml of a 1:1 slurry of Cibacron Blue-agarose and phosphate storage buffer with 0. 02% sodium azide in it. The storage buffer was washed away with approximately 20ml of phosphate buffer to equilibrate the column.
Warm the 65%CP to room temperature and load the column. Allow the column to elute at a rate of 4 to 5 drops per minute or 1 drop every 10-15 seconds. Establish the baseline A280. Wash the column with 18ml of phosphate buffer, taking an A280 reading every 3rd ml. Wash until baseline is nearly achieved. Elute the proteins from the column using 1M NaCl and collect all fractions. Spot test the fractions, combine the most active fractions, measure the volume and place the affinity purified intermediate on ice. If size exclusion chromatography is planned, add 0. 1g of ammonium sulfate per ml of consolidated fractions and refrigerate this intermediate. Size Exclusion Chromatography of the Affinity Purified (AP) intermediate Load a 30cm length by 1cm diameter column with Sephadex G100 that was previously hydrated in phosphate buffer with 0. 02% sodium azide @ pH 7. 5. Allow Sephadex to settle for at least 24 hours. Run phosphate buffer through the column to remove the sodium azide. Centrifuge the AP fractions @ 15,000g for 15 minutes @ 4? C. Resuspend the pellet in 0. 4ml of pH 7. 5 phosphate buffer, measure the final volume and take a 40 ? l sample to freeze for later testing. Add 200 ? Blue Dextran and 100 ? l Orange G to the resuspended pellet. Load the sample onto the column. Collect fractions when dextran hits the bottom of the column. Continue adding phosphate buffer and collecting fractions until Orange G is completely eluted. Prepare spot test to determine LDH activity, combine active fractions and measure volume of final product, SEC purified LDH. Store at 4? C. Spot Test for LDH enzyme activity The spot test for LDH activity will determine the activity of LDH in collected fractions. The test is based upon a cyclic reaction between LDH, PMS and NBT, a positive spot test is indicated by a blue reaction.
The excess of lactate causes the LDH to run in “reverse” causing the reduction of NAD to NADH, this leads to chain reaction where phenazine methosulfate (PMS) is reduced and then the reduced PMS then reduces nitro-blue tetrazolium (NBT) which changes to a blue color upon this reduction. This assay is only qualitative. A master mix of spot test reagents is prepared using these reagents per 1 reaction: 25mM lactate in 100mM CAPS buffer90 ? l 10mM NAD+ 10 ? l 2mM NBT2. 5 ? l 2mM PMS0. 65 ? l Total100 ? l In a 96 well plate, add 2 ? l of 65%CP to one well as a (+) control and nothing to a second well for a (-) control.
Add 2 ? l of each fraction collected and wait 5 minutes for results. Strong blue color indicates activity. Bradford Total Protein Assay Prepare a 1X solution of Bio-Rad 500-006 dye. Create standards of bovine serum albumin (BSA) from 0-10 ? l of BSA and 100-90 ? l of dH2O. Mix each of the different purification samples in 2 ? l : 98 ? l dH2O and 10 ? l : 90 ? l dH2O, add 900 ? l to each preparation, vortex and allow to stand for 5 minutes. Measure A595 for BSA. Measure A595 for LDH unknowns. Concentration can be calculated through Isozyme identification using agarose gel electrophoresis rotein determination (Bradford Assay), activity assays, and isozyme identification (agarose gel electrophoresis). : Tekman B, Ozdemir H, Senturk M, Ciftci M. Purification and characterization of glutathione reductase from rainbow trout. Comparative biochemistry and physiology. Toxicology & pharmacology. 2008 Aug;148(2):117-21. Cited in PubMed; PMID-18508412 Marchat L, Loiseau PM, Petek F. Purification and characterization of lactate dehydrogenase isoenzymes 1 and 2 from Molinema dessetae (Nematoda:Filarioidea). Parasitology Research. 1996 September 82(8):672-80. Cited in