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Unformatted text preview: 5-1 Ch. 5 Affinity Chromatography for the Isolation of LDHObjectives Develop pipeting skills and practice dilution techniques Separate LDH from a mixture using your prepared column Elute LDH using various solutions of NaCl and isolate the active fractions Understand how Cibacron Blue works for affinity chromatography Introduction Enzymes are responsible for controlling the biochemical reactions that take place in the human body. Almost all biochemical reactions are thermodynamically favorable, however, without the presence of enzymes many of these reactions would occur on such a slow time frame as to not be useful to the organism. Enzymes are called catalysts since they speed up the outcome of the reaction. Enzymes allow the biochemical reactions to proceed more rapidly by lowering the energy barrier (energy of the transition state) of the reaction pathway. In fact, enzymes can speed up the rate of a reaction up to 1020times. Enzymes have no effect on the position of the equilibrium, only how fast a reaction reaches equilibrium. Figure 1 represents a potential energy diagram for this process. EaENERGY H PRODUCTS REACTANTS An un-catalyzed reaction A catalyzed reaction There is no difference in energy between catalyzed and the un-catalyzed reaction COURSE OF THE REACTION Figure 1: Potential energy profiles for catalyzed and un-catalyzed reactionsChapter Six 5-2 There are already over 2000 known enzymes isolated from the human body alone, and biochemists are interested in determining what each one does. In order to study an individual enzyme, it must be separated, isolated and purified away from contaminants and other enzymes before determining its function. The enzyme you will be isolating is LDH, lactate dehydrogenase, which is isolated from rabbit muscle. You will be isolating and purifying LDH using a process called chromatography. Chromatography is a separation technique widely used by chemists in most fields. The basis to chromatography is that molecules in a mixture can be separated by their size (called size exclusion chromatography), by their charge (ion exchange chromatography), by their masses, etc. Affinity chromatographywill be your first introduction to chromatography, and it is in particular a type of adsorption chromatography (i.e. things are separated by being adsorbed to the column material). Affinity chromatography works by using a specific column material that has a ligand attached to it that the molecule of interest recognizes and to which it binds. In this lab you are interested in separating LDH from a mixture of proteins. The column material is specifically chosen so that LDH binds strongly to it. This chosen column material might have a ligand group attached to it that LDH would normally bind to in nature, like lactate, pyruvate, NAD+, or NADH. As the mixture of proteins is passed through the column material, the LDH will have a high affinity to bind to the column material while all other species elute (pass) through the...
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- Fall '11