Introduction: Lactate dehydrogenase (LDH) is an enzyme that interconverts pyruvate to lactate and back by transferring a hydride ion from NADH to form NAD+ and back. This reaction is important in humans because pyruvate, the end product of glycolysis, can be converted to lactate in the absence of oxygen in muscles. As lactate builds up in the muscle, it is then transferred to the liver to proceed through the Cori Cycle. The Cori cycle uses LDH to proceed through the reverse reaction and produce pyruvate which can then be used to produce glucose for transfer through the blood stream to oxygen deprived muscle cells. This cycle helps to remove lactate from muscles, regenerate ATP, and to also replenish NADH for further use but would not …show more content…
(put in a couple of sentences about the questions studied in this experiment and how these questions of products figure into the overall project and why they are important) Ammonium sulfate precipitation takes advantage of differential solubility of proteins. As the concentration of ammonium sulfate increases in the solution, different protein will precipitate out at different concentrations. This simple purification of a protein keeps most of the other proteins in solution at specific concentrations. On the molecular level, salts like ammonium sulfate work in low concentrations to solubilize proteins in solution by stabilizing the charged groups found on proteins. When the ammonium sulfate concentration reaches a point of maximum protein solubility, there becomes less water availability for protein-water interaction so protein begins to precipitate …show more content…
In this experiment, affinity chromatography worked by using an affinity column to bind LDH to a ligand, Cybacron blue, that is covalently bonded to an insoluble column resin. As LDH passed through the column, it binded to Cybacron blue due to Cybacron blue characteristics that mimic that of pyridine nucleotides. LDH has a substrate binding domain that has a high affinity for pyridine nucleotides. To elute out the LDH from the Cybacron blue, a buffer solution must be added as a way to interfere and release binding of the protein. Different buffer solutions were coordinated at different times to release the LDH. TRIS-PMSF washes were completed to remove anything that was not LDH from the column. NAD and NADH washes were used as a way to bind LDH to a mobile substrate. NAD and NADH also have pyridine characteristics so LDH is entropically favored to bind to these mobile