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.

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 200 800,000

chromatograhpy

5. Affinity chromatography 50 750,000

6. Size-exclusion 45 675,000

   chromatograhpy

__________________________________________________________

(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?

Explanation / Answer

Specific activity in activity/mg

after

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.

Affinity chromatography purification procedures used for this enzyme is most effective

c.

Precipitation (pH) purification procedures is least effective

d. since there is no change in specific activity , the enzyme may become pure

Enzyme activity can be measured  estimate the purity of the enzyme preparation

Enzyme activity = moles of substrate converted per unit time = rate reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s?1, but this is an excessively large unit. A more practical and commonly used value is enzyme unit(U) = 1 ?mol min?1. 1 U corresponds to 16.67 nanokatals.[1]

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