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Unformatted text preview: 4-1Experiment 4PREPARATION OF AN ENZYMEIntroductionLife science research is an integrated enterprise connecting molecular, biochemical, cellular, developmental, genetic and evolutionary biology. These disciplines have generated enormous amounts of information on protein structures and activities, on genetic loci coding for proteins, and on complex patterns of regulation of expression and cellular functions, which are available from databases. The rise of bioinformatics is a science devoted to the storage and pertinent access of this information, which drives life science research and biotechnology industry by creating experimental hypotheses from a knowledge-based approach. Protein purification and characterization remains a vitally active arena for broadening the informational databases and for increasing the understanding of native protein structure, metabolism, enzyme mechanisms, protein-protein interaction, signal transduction, receptor function, immune responses, and energy conversion.ObjectivesA. Perform Experiment 8B prior to the first day of this lab.1.Purify lactate dehydrogenase by ammonium sulfate fractionation and AMP-affinity chromatography.2.Assess the effectiveness of the purification steps and the purity of the LDH preparation by analyses of LDH activity and protein content.TheoryProtein PurificationTo confidently establish the properties of any protein, the protein must be purified. This means it must be separated from all other types of proteins found within cells, and, also from a variety of other molecules, such as nucleic acids, lipids, carbohydrates, amino acids and salts, etc. The beginning of any protein purification scheme involves the lysis of cells to create a homogenate of the cell contents and then a crude extract from the homogenate. Homogenates and resulting crude extracts may contain significant amounts of participating substrates, products, or cofactors of the enzyme, and compounds that activate or inhibit the enzyme. Afterward crude extracts are prepared, several steps are usually required to purify a protein. The number and variety of possible purification steps is large and the actual procedure for purifying a protein is determined empirically. Since so many proteins have been successfully purified, there are generalized strategies to follow. The initial steps of these strategies in large part depend on the cellular location of the protein to be purified: membrane proteins often require the addition of detergents that are non-ionic and non-denaturing to release them from the membranes and to keep them soluble in an aqueous solution, whereas proteins located in organs such pancreas or liver often requires the addition of protease inhibitors as these tissues contain high levels of proteases. Also, any protein may be located in several types of tissues and cells; often a tissue is chosen if it is known to contain the greatest cellular concentrations of the target protein. The earliest proteins purified were mostly soluble proteins located in the cytoplasm of cells and organelles that...
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- Fall '10
- LDH, NAD+