final LDH

Topics: Enzyme, Enzyme assay, Metabolism Pages: 3 (919 words) Published: January 31, 2014

Individual Purification Report
The 60% saturation of ammonium sulfate was performed to precipitate out LDH and other proteins. Components such as nucleic acids and sugars, which are more soluble, remained in the supernatant. In this step 0.13g of ammonium sulfate salt was slowly added per each ml of the 40% supernatant as the solution was stirring. Enzyme assay and protein assay were performed. The results indicated 4600±100 unit enzyme activity concentration and 75±2 mg of protein in the 60% pellet (Raw Data tables 1-A,2). The enzyme assay was performed on the 60% sup as well, which showed the enzyme activity concentration of the 60% sup was less than 1/3 of the 40% cut. The purification factor was 1.322±0.002. Although this step was not designed to remove protein, some contaminant protein was lost. This decreases the total amount of protein and increases the purity. The yield was calculated 61.7±0.5 (Raw Data Table 2). There was some loss of enzyme activity concentration despite the fact that everything had precipitated during this step, which could be due to surface air denaturation of the protein. Buffer solution was used to renature the protein. However, renaturation did not happen 100%. Using an affinity column, LDH was purified away from contaminant proteins. The 60% cut was loaded on to a column filled with AMP-agarose beads. There were two classes of contaminant proteins present, AMP binding and non-AMP binding proteins. First, a potassium phosphate buffer solution was used to rinse the column in order to make sure all the non-AMP binding proteins had passed through the column. Then another potassium phosphate buffer solution containing the modified version of our substrate, NAD-Pyruvate adduct, was used to elute LDH and get it away from the contaminants. LDH recognizes AMP and binds to it. Therefore, it remains in the mobile phase and binds the column. The binding in non-covalent fashion is temporarily. There is equilibrium of binding and...
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