You have purified LDH using affinity chromatography. Using the Bradford assay, y

Question

You have purified LDH using affinity chromatography. Using the Bradford assay, you determined the total protein concentration of the column fraction to be 7.5 mg/ml, in a final volume of 2.6 ml.

You diluted some of your column fraction to 1 mg/ml using SDS PAGE sample loading buffer, and loaded 15 µl on to an SDS PAGE gel. You also loaded 10 µl of a 1mg/ml LDH standard in an adjacent lane.

The stained SDS-PAGE revealed that your LDH column fraction contained a number of bands besides LDH. Upon scanning, you found that the LDH band in your column fraction had an optical density of 42,000 pixels. The optical density of the LDH standard band was 58,000 pixels.

a. What is the percentage purity of your LDH column fraction?

b. How many total mg of LDH are there in your column fraction?

Explanation / Answer

a. Lactate dehydrogenase (LDH) was purified from bovine heart tissue. This was done primarily through selective centrifugation with salting out and through ion-exchange chromatography. The final product from ion-exchange chromatography had a specific activity of 0.492 U/mg, with a percent recovery of 1.6% and a fold purification of 0.0228. Based on the gel electrophoresis results of that sample, it seems to be relatively pure because it has large bands at the same distance as the LDH control. The molecular weight of the two main bands on the purified sample are approximately 30,000 and 60,000 Daltons.

The two primary methods used for purifying LDH in this experiment were salting out and ion-exchange chromatography. Salting out, or ammonium sulfate precipitation, involves adding an extremely polar compound (ammonium sulfate in this case) to a protein solution. The ammonium sulfate causes the solvent (water) molecules to react with them instead of the protein molecules. The protein molecules then become attracted to each other and clump together. Different proteins will do this at different concentrations of salt, some higher, some lower. Centrifugation after salting out will cause a pellet to form made of protein. Dialysis is necessary after salting out to make sure that the salt leaves the solution and does not contaminate it during further purification steps.

Ion-exchange chromatography with salt elution is the other important step in the purification process. It separates proteins based on their charges, using a column containing the positively charged anion exchanger Q-sepharose agarose (the solid state), and a buffer (the fluid state), which can include a salt. Negatively charged lactate dehydrogenase adheres to the positively charged Q-sepharose, and will not elute (come out of the column) until it is displaced by NaCl.

b. LDH is a tetramer of 35 kd subunits. There are 2 types of subunits: and H form which predominates in the heart and an M form that predominates in the muscle and liver. These subunits can associate to form five types of tetramers (H4, H3L1, H2L2, H1L3, L4) all with LDH activity but with different substrate affinities and different responses to allosteric effectors. In general, enzymes that catalyze the same reaction but which differ in structure are referred to as isozymes or isoenzymes. You will appreciate the tertiary structure of LDH

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