Question

Question: Purification tables are often used to keep track of the yield and purification of a protein. The specific activity is a ratio of the amount of the protein of interest, in this case Mb, obtained at a given step (μmol or enzyme units) divided by the amount (mg) of total protein. The yield is the ratio of the amount of the protein of interest obtained at a given step (μmol or enzyme units) divided by the original amount present in the crude extract, often converted to percent yield by multiplying by 100. The fold purification is the ratio of the specific activity of the purified protein to that of the crude preparation.

(a) For the purification table below, calculate the specific activity, % yield, and fold purification for the empty cells.

(b) Which step—DEAE or affinity chromatography—causes the greatest loss of Mb?

(c) Which step causes the greater purification of Mb?

(d) If you wanted to use only one purification step, which technique would you choose?

Short answer

a)

b) DEAE-cellulose chromatography causes great loss of Myoglobin.

(c) Third step I.e. affinity chromatography provides more purification of Myoglobin.
(d) It would be best to choose affinity chromatography.

Step-by-step solution

Protein Purification tables

Purification table of protein contains specific activity, yield percent and purification level of a given purification protocol; and the purpose of determining them is to see if the individual steps of protein purification are effective in isolating the protein while still maintaining it's activity.

Explanation for (a)

The specific activity is obtained by dividing the amount of target protein (myoglobin) by the total amount of protein.

Percent yield gives the proteins activity that is retained after each purification step. It is calculated by dividing the amount of target protein which is obtained at a given step by the actual amount that is present in the crude extract. The value obtained is then multiplied by 100.

The fold purification measures the effectiveness of each step in purifying a target protein. It can be calculated by dividing the specific activity of the purified protein by the activity of the crude preparation.

Explanation for (b)

The yield from the DEAE chromatography step is only 47%, but the affinity chromatography step yields is 80% from the step preceding it. As a result, the most of the Myoglobin is lost during the DEAE chromatography step.

Explanation for (c)

The affinity chromatography step gives a 92-fold purification from its previous step, while The DEAE chromatography step gives 1.3-fold purification. As a result, the affinity chromatography stage offers a maximum level of Myoglobin purification.

Explanation for (d)

Affinity chromatography would be the best choice if you wanted to use only one step of purification as it gives a greater yield and purification level.