Question

1. If DE 52 column was equilibrated and eluted with 0.1 M NaOH, Predict and explain the results of a gel fileration chromatography and ion exchange chromatography

2. BSA (pI = 4.6), Lysozyme (pI = 11), hemoglobin pI (6.5)

Explain whether the above proteins will bind to the column equilibrated in 0.1 M Tris pH 8.0

3. For the ion-exchange resin, what could be an alternative approach for the elution of the sample components. What could be the result and explain why that result would be for the alternative aproach.

Answer 1

1) Ans: DE 52 column is a Diethylaminoethyl cellulose containing column. It contains positively charged Diethylaminoethyl groups which has the capacity to bind to negatively charged ions, If 0.1M NaOH is used to for equillibrating and eluting this column then most of the positively charged binding sites on the surface of the column would be neutralised and the positively charged groups will dissappear on the DE 52 column.

This leads to loss of efficiency of the column and there will be no separation of proteins on this column.

The pKa of the DE 52 column is 11.5 and 0.1 M NaOH solution has a pKa of 13.8. This makes the positively charged DE52 column loose its slightly acidic nature and loose its binding sites and make it in efficient for performing chromatographic separation.

2) Ans: pl - it is the isoelectric point - it is the pH at which there is no net charge on the protein.

If 0.1 M Tris pH 8.0 mobile phase is used to equillibrate the column then the protein with pl value below the pH of the medium will bind to the anion exchanger while the protein with pl value greater than the pH of the medium will be eluted out of the column as it shows weak interaction with the medium.

So the BSA with pl value of 4.6 and Heamoglobin with a pl value of 6.5 will bind to the medium while the lysozyme with pl value of 11 will be eluted from the column.

This is because both BSA and Heamoglobin have a pl value below the pH value of the medium, This makes them negatively charged and binds to positively charged stationary phase.

The BSA and Heamoglobin are present in ionised form at this pH while the lysozyme will still exist in unionised form at pH of 8, this makes BSA and hemoglobin to bind to positively charged surface while lysozyme will be eluted out of the column