Question

Cation-exchange chromatography is a popular technique for separating and purifying proteins. This is very similar to the separation purification of the mixture of amino acids you observed in Prac 3. You are asked to purify an enzyme with a pI of 6.5 from a mixture of different proteins You are provided with the following:

• A column packed with cation-exchange resin

• Phosphate buffer at pH 2.6

• Citrate buffer at pH 3.4

• Tris-HCl buffer at pH 8.2

• Sodium hydroxide at pH 12.0

• Pipettes and consumables such as test tubes and beakers

• 10 mL of a protein mixture (1 mg/mL) in citrate buffer at pH 3.4

Questions:

(1) Which buffer would you use to equilibrate your column?

(2) Design a purification procedure describing the loading of the sample onto the column, the sequential addition of buffers and the collection of elution fractions. (You must clearly indicate which buffer you would use for each elution step and the number of fractions you are going to collect.

(3) How would you determine which fraction contained your enzyme of interest?

(4) How would you determine the concentration of the enzyme in the fractions assuming that the enzyme was the only protein present in the fractions.

Answer 1

Ans 1. I will use Citrate buffer at pH 3.4 for equilibrium as it has the same pH as of the mixture and also pH lower than our protein of interest with pI 6.5 will get positively charged and get attached to the negatively charged cation-exchange resin.

Ans 2. After the column has come to equilibrium after the addition of the citrate buffer, the sample will be loaded into the column. After the sample is properly loaded into the column, sodium hydroxide at pH 12.0 will be first used to wash the column to remove all the proteins with pI less than 3.4 and pI close to 3.4. The second wash will remove some of our protein of interest and a third wash will completely remove all our protein of interest as it has pI around 6.5 so an increase in ionic strength will remove our protein of interest. Any further wash will lead to the removal of proteins with higher pI value. So, our protein of interest will illude out in 2nd and 3rd fraction wash with sodium hydroxide.

Ans 3. As the pI value, of our protein of interest, is 6.5 so it will be eluted out during 2nd and 3rd wash with sodium hydroxide as by the time 3rd wash is given the ion strength would have increased enough to remove our protein of interest from the column.

Ans 4. As the enzyme is the only protein in the fraction so its concentration can be determined biochemically using protein estimation protocols like Warburg-Christian, Lowry Assay, and Bradford Assay which can be used to determine the amount of protein present in the solution and this can be used to determine the concentration of the protein which will give the concentration of enzyme in the solution.