5. A biochemist disco vers and purifies a new protein, generating the data belo

Question

5. A biochemist disco vers and purifies a new protein, generating the data belo ProcedureTotal proteinActivity S Specific Fold increase activity in specific activity (mg) 1. Crude extract Chomogenate) 2. Precipitation (salt) 3. Precipitation (pH 4. Ion-exchange chromatography 5. Affinity chromatography 6. Size exclusion chromatography 4,500 3,500 600 100 80 nmol/min 6,000,000 1,670,000 1,450,000 810,000 325,000 270,000 a) From the information given in the table above, calculate the specific activity of the b) Calculate the fold increases by dividing specific activity at each step by specific c) Which of the procedures used for this enzyme is most effective (i.e. Gives the enzyme solution after each procedure. Give the units of specific activity. activity in the homogenate. 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 reasoning.

Explanation / Answer

The specific activity of enzyme is calculated as Specific activity = Activity/total protein. In your case it can be calculated simply by dividing the value in column 3 by the value in column 2. Fold increase can be calculated by dividing first specific activity value (in column 4) after each step by specific activity value after step 1.

a) and b). (Here you see the units of specific activity will be nmol/min/mg)

                   Specific activity and fold increase in each case is calculated and tabulated below,

                                   Specific Activity (in nmol/min/mg)          Fold Increase

                           1.             300                                                1 (here its is 300/300)

                           2.            3711                                               12.4 (here it is calculated as 3711/300)

                           3.            414.3                                                1.4 (here it is 414.3/300)

                           4.            1350                                                 4.5 (here it is 1350/300)

                           5.            3250                                                10.8 (here it is calculated as 3250/300)

                           6.            3375                                                 11.25 (here it is 3375/300)

c) By looking at the Table above, you can figure out that step 2 i.e. precipitation (salt) is the most effective step as it gives 12.4 fold increase in activity from the last step.

d) The same way by looking at the data in the Table you will find that step 3 i.e precipitation (pH) is the least effective step as it the fold increase decreses in this step.

e) The purity of the enzyme can only be acertained after testing it for its specific function.

f) SDS-PAGE is the commonly used method to check the purity of an enzyme (other than testing it against its specific function/reaction). You run your enzyme is thegel against the standard (100% pure) enzyme and compare the bands.

g) If you repeat the procedure, you want to elimate the purification step step that is least effective. In your case it is precipitation (by pH).

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