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...
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