6. It is your desire to purify the glycolytic enzyme, glyceraldehyde-3-phosphate

Question

6. It is your desire to purify the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the common sparrow. The enzyme has a MW of ~35kDa and a theoretical pI of ~9.00. In addition the enzyme carries out the following reaction: Glyceraldehyde-3-phosphate + NAD+ + Pi 1,3 bisphosphoglycerate + NADH

a. Based on this information would a Q-sepharose column be a good ion-exchanger for this protein (1 point)? Why or why not (1 point)?

b. Would you expect this protein to bind to a HiTrapTM Blue HP column? Why or why not (1 points)?

c. If you wanted to do an enzymatic activity assay on this protein using the absorbance properties of NAD+/NADH at 340 nm, what substrate would you put in the reaction mixture: Glyceraldehyde-3-phosphate or 1,3-bisphosphoglycerate (2 points)? Why (2 points)? (NOTE: You want absorbance at 340 nm to increase throughout the assay)

d. The last step of your purification protocol involves running your protein sample over a size exclusion column. At first you choose a column with a molecular weight cutoff of 80 kDa. Due to the size of your protein you expect it to come off somewhere in the middle of the run and foolishly decide to start collecting fractions half-way through the run. When you come back you see your data and realize the protein came off in the “void” volume (very early in the run) suggesting that your protein is much larger than 80 kDa. What is a possible explanation for these data? (1 point)

Explanation / Answer

Answer:

6. a. Q-sepharose column is an anion exchanger and has a broad working pH ranging from 4 to 10. The protein to be purified has a pI of 9 and possess a net negative charge when pH > pI (required for binding to the column).

For the Q-sepharose column, the ionic strength of the buffer should be kept low so as not to interfere with sample binding. Recommended operating pH is within 0.5 pH units of the buffer's pKa and at least one pH unit below the isoelectric point (pI) of the molecule of interest.

So, Q-sepharose column would not be a good ion-exchanger.

b. HiTrapTM Blue HP column is a tool for the separation of many proteins e.g. enzymes requiring adenyl-containing cofactors (including NAD+ and NADP+). Glyceraldehyde 3-phosphate dehydrogenase possess a NAD binding domain and thus the protein can bind to the HiTrapTM Blue HP column.

c. 340 nm is within the visible light region and this wavelength gives the highest absorbance maxima for NADH. At 340 nm NAD+ does not have a good absorbance.

So, the forward reaction with Glyceraldehyde-3-phosphate as the substrate is preferable.

d. The protein exists as a multimer (larger than a dimer) with a molecular weight greater tha 80 kDa. Thus, it comes out in the void.

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