Anion exchange chromatography 1. In the purification, the pH of the PBS buffer was 6.5, and...

Anion exchange chromatography

1. In the purification, the pH of the PBS buffer was 6.5, and the two proteins have different isoelectric points (Haemoglobin pI: pI 6.8; Catalase: pI 5.4). What are the charge states of the two proteins at this pH? (e.g. positively charged, negatively charged or neutral)

2. Based on the charge states of the proteins, which protein has a higher affinity to the DEAE-Sepharose resin at pH 6.5?

3. Why was the salt concentration of PBS buffer increased (from 17.5 mM to 175 mM) in order to elute out the catalase protein?

Expert Solution

1.Haemoglobin is positively charge at ph 6.5.

Catalase is negatively charged at ph 6.5.

Reason: pi is that pH at which there is not net charge on the molecule. If the ph of the solution is less than the isoelectric point of the molecule then the overall charge on the molecule is positive as in case of haemoglobin ( Pi 6.8)nd if the H of the solution is greater than Pi then the overall charge on the molecule is negative as in case of catalase (Pi 5.4) .

2. Catalase will show higher affinity for DEAE sepharose.

3. Buffer contains ions and ions show greater affinity for resin as conpared to catalase protein. Hemce, as we are increasing buffer concert ,ions increases and these ions substitute the catalse protein hence eluting the protein from resin

DEAE sepharose is anion exhanger resin that means it is positively charge and binds to negatively charged species.

At ph 6.5,Catalase is negatively charge hence, it will binds to positively charged DEAE sepharose resin whereas haemoglobin being negatively charged at ph 6.5 will repel the resin.

gladiator answered 4 months ago

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