A mixture of three proteins (A,B,C) can be thereetically separated by anion exch

Question

A mixture of three proteins (A,B,C) can be thereetically separated by anion exchange chromotgraphy using cellulose coated with diethylamino-ethyl groups (pka 10.5). In the first step the mixture was applied to the resin at pH 7.5 and washed with the same buffer to elute protein(s) that did not bind. The resin was then washed with a buffer at pH 4.5

Protein A IS A HOMODIMER WITH A MOLECULAR WEIGHT OF 50 kda AND A pi OF 3.0 PROTEIN b IS A HETEROTETRAMER WITH A MOLECULAR WEIGHT OF 100 Kda AND A pi OF 6.0 PROTEIN C IS A MONOMER WITH A MOLECULAR WEIGHT OF 75 Kda AND A pI OF 9.0

WHICH OF THE FOLLOWING STATEMENTS IS INCORRECT?

A. PROTEINS A AND B ARE INITIALLY BOUND TO THE RESIN AT PH 7.5

B. THE RESIN ITSELF WILL REMAIN POSTITIVELY CHARGED OVER THE PH RANGE 3 TO 7.5

C. ONLY PROTEIN A WILL ELUTE WHEN THE RESIN IS WASHED WITH A BUFFER AT PH 4.5

D. PROTEIN C DOES NOT BIND TO THE RESIN AT PH 7.5

Explanation / Answer

Isoelectric point (Pl) is defined as the pH at which the protein has no net electric charge and is electrically neutral. When Pl is more than pH, the protein has net positive charge and when Pl is less than pH, protein has net negative charge. Ion exchange chromatography uses this principle for the separation or elution of proteins.

Here, the protein mixture contains 3 proteins, A, B and C. Protein A has Pl of 3, B has Pl of 6 and C has Pl of 9. When mixture was applied to the resin at pH 7.5, protein C will elute since it's Pl value is 9. Similarly in the second step, when the mixture was applied to resin at pH 4.5, protein B will elute. Protein A will remain attached and the resin will remain positively charged over the pH range 3 to 7.5.

Therefore, the answer for this question is option (C) only protein A will elute when the resin is washed with a buffer at pH 4.5.

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