5. chemist discovers and purifies a new protein, generating the data below: Proc
ID: 210122 • Letter: 5
Question
5. chemist discovers and purifies a new protein, generating the data below: Procedure Total protein Activity Specific Fold increase nmol/min activity in specific (mg) 1. Crude extract20,000 6,000,000 (homogenate) 4,500 3,500 600 100 80 1,670,000 3. Precipitation| salt) 11,450,000 4. Ion-exchange 810,000 325,000 270,000 chromatography 5. Affinity chromatography 6. Size exclusion chromatography a) b) c) From the information given in the table above, calculate the specific activity of the enzyme solution after each procedure. Give the units of specific activity Calculate the fold increases by dividing specific activity at each step by specific activity in the homogenate. Which of the procedures used for this enzyme is most effective (i.e. Gives the greatest fold increase from one step to the next)? d) Which of the procedures is the least effective? e) Is the enzyme pure after step 6? Explain your reasoning f How can the purity of the enzyme be assessed? g) If you repeat this experiment what step would you eliminate? Please explain your reasoning.Explanation / Answer
1 Specific activity Fold increase
1. 6000000/20000 = 300 1
2. 1670000/4500 = 371 371/300 = 1.23
3. 1450000/3500 = 414 414/300 = 1.38
4. 810000/600 = 1350 1350/300 = 4.5
5. 325000/100 = 3250 3250/300 = 10.83
6. 270000/80 = 3375 3375/300 = 11.25
C. Affinity chromotography - this gives the greatest increase in specific activity (an index of purity and degree of increase in purification).
D. Precipitation pH -
E. Yes, further we do the purification step because specific activity still increasing.
F. SDS polyacrylamide gel electrophoresis is an excellent, standard way of checking homogeneity and purity.
G. Step 3 is eliminate. Not that much of purification is happened here.
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