Purification of an Enzyme A biochemist discovers and purifies a new enzyme, gene

Question

Purification of an Enzyme A biochemist discovers and purifies a new enzyme, generating the purification table below.

(a) From the information given in the table, calculate the specific activity of the enzyme after each purification procedure.

(b) Which of the purification procedures used for this enzyme is most effective (i.e., gives the greatest relative increase in purity)?

(c) Which of the purification procedures is least effective?

(d) Is there any indication based on the results shown in the table that the enzyme after step 6 is now pure? What else could be done to estimate the purity of the enzyme preparation?

Procedure Total protein (mg) Activity (units) 1. Crude extract 20,000 4,000,000 2. Precipitation (salt)   5,000 3,000,000 3. Precipitation (pH)   4,000 1,000,000 4. Ion-exchange chromatography      200    800,000 5. Affinity chromatography        50    750,000 6. Size-exclusion chromatography        45    675,000

Explanation / Answer

Specific activity in activity(in units)/Total Protein(in mg)

Crude extract = 4,000,000/20,000=200

2. Precipitation (salt) =3,000,000/5,000 =600

3. Precipitation (pH) =1,000,000/4,000= 250

4. Ion-exchange=800,000/ 200 =4000

chromatograhpy

5. Affinity chromatography=750,000/50 =15000

6. Size-exclusion =675,000/45 =15000

b.Based on specific activity measurements, affinity chromatography at step 5 was the most effective step, where at the end of step 4, we had an enzyme prep with a specific activity of 4000uMol and after the 5th step it has increased to 15000uMol, a nearly four fold increase in the specific activity and possibly purity of the protein. c.Step 3 of precipitation based on pH was the least effective step. This is because when we calculate specific enzyme activity, which is calculated by the formula Specific activity = Enzyme activity measured in Units/amount of protein used (mg).

c. Based on this calculation for each step, we see that at the second step the specific activity was 600uMol, and after the third step, if the precipitation was indeed helpful in the purification of the enzyme it should increase the specificity whereas the estimated specificity after this step is 250uMol. This is why the precipitation step is the least effective.  

d. Again, based on the specific activity it seems like the protein has attained reasonable purity, but it can be confirmed only based on a SDS-PAGE method described above. Enzyme activity can be measured  estimate the purity of the enzyme preparation

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