This Table shows assay data from a purification procedure. Which statement is mo

Question

This Table shows assay data from a purification procedure. Which statement is most likely TRUE? The gel-filtration step increased the specific activity but not the yield. Of the three chromatography steps, the affinity column step had the biggest drop in yield. After the CM-cellulose step, the purification factor reached over 1000, indicating effective purification. Most of the protein removed in the gel-filtration step was likely enzyme that had unfolded during the CM-cellulose step. All of the above are false. Based on the purification table from Question M4-HW-07, which statement below reports a value that is CORRECTLY calculated? The purification factor after the affinity column step was 350. The specific activity after the centrifugation step was 20 units. The yield after the CM-cellulose step was 82.5%. The yield after the gel filtration step was 1%. All of the above statements report incorrect values.

Explanation / Answer

This data in this above explained table has shown that the yield from a purification step as well as yield. Affinity column fraction keeps the activity in high standards while the nonspecific proteins are separated out. Another step followed by that is CM-cellulose fraction, is used for preparative separation of phospholipid classes. Here the separation step from has brought the yields to a narrowed down level which is the specific protein separation but the total activity is very high. The final step with Gel Filtration does the specific protein separation work which keep the activity low as the desired protein function and activity.

The first one A1. It has a decreased specific activity as there is only the desired protein with others non intended parts separated.

A2. This step from the Affinity chromatography is done to separate the proteins or macromolecules from the mixture, they are there to bind with the ligand. So from the cell fraction from centrifugation this steps has done the retention work of the protein molecules. Here the drop in yield is the highest most, so this option id chosen to be right.

A3. Purification factor is to be calculated by this formula specific activity of (x) __=purification factor for (x) specific activity of starting material. For each fraction, divide the enzyme concentration by the protein concentration. If we do the calculation it does not come to 1000 as it is mentioned in the comment.

Specific activity of CM-cellulose affect is 330000/600 = 550, and Specific activity from starting material is 350000/1000 = 350,

so now dividing both, 550/350 = 1.57 units.

A4. In CM cellulose step is where ion exchange takes place, as some protein and enzymes molecules re bigger in size so they are separated out by the CM cellulose exchange. These are the afcts about gel filtration part is this solely based on the separation of proteins, peptides, and oligonucleotides on the basis of size. Molecules move through a bed of porous beads, diffusing into the beads and the smaller molecules diffuse further into the pores, so as the option suggests that the bigger molecules are likely to be separated as they are unfolded during the CM-cellulose exchange step may be right or may not be. Because the CM cellulose step does not suggest the macromolecules to be opened up.

M4-HW-08

A1. From the reference drawn in the above mentioned answer, the purification factor consists of the formula:

Specific activity of (X) / specific activity of starting material = purification factor for (X) specific activity of starting material. For each fraction, divide the enzyme concentration by the protein concentration.

Specific activity of affinity material is 350 / specific activity of starting material (20) = 17.5.

The step after the affinity column step has not reached 350, as per the calculation.

A2. Specific activity is calculated from the value enzyme per milligram of total protein (expressed in mol min1mg1). Here the specific activity is the concentration of enzyme / concentration of protein = 400000/20000 = 20.

This option is correct.

A3. The yield (%) is calculated from the total activity (units) at each step / the total activity (units) in the homogenate, multiplied by 100.

And total activity is multiplying the activity by the total volume gives the total activity (i.e. units/ml x ml = units).

Here the yield will be approx. 25%, so it does not cope upto 82.5%.

A4. This too is an incorrect assumption,

Conclusion A2 is the correct answer choice.

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