7. Draw the elution /fractionation profile of all the homogenate proteins separa

Question

7. Draw the elution /fractionation profile of all the homogenate proteins separated using cation exchange chromatography. List the proteins in order of their elution. Why are these proteins eluted in this order. What is the basis for these proteins being released from the ion exchange column. protein isoelectric point (pl) protein identity protein mol wt (kdaltons) 290 9.2 7.7 6.4 protein A RNA polymerase rotein B alcohol dehydrogenase protein C RNase A protein D pyruvate kinase protein E tRNA synthetase 323 98 200 190 4.8 5.0

Explanation / Answer

Ans. Note the following points-

I. Cation exchange chromatography (matric is negatively charged) separates cations (positively charged proteins, or other molecules) based on their relative charges. At a pH below (more acidic) its pI, the protein is positively charged. Greater is the difference between pH of medium and pI of the protein, larger fraction of the protein is positively charged.

II. In cation exchange chromatography, the protein with greater positive charge is retained on the negatively charged matrix of stationary more strongly. So, a protein with maximum positive charged in eluted last.

III. Isoelectric point (pI) is the pH at which an amino acid exists in electrically neutral species.

At pH = pI : No net gain or loss of proton from the specified amino acid.

At pH < pI : (pH below pI): The carboxylate ( COO-) and amine (NH2) group (if any) of the specified amino acid accepts proton to counteract increase in pH. Protein is positively charged.

At pH > pI : (pH above pI): The carboxyl acid (COOH) and amine (NH3+) group donates proton to counteract decrease in pH. Protein is negatively charged.

IV. Basis of Elution: When pH of elution buffer is increased to the pI of a protein, the protein becomes neutral. So, a neutral molecule can’t be retained on the negatively charged stationary phase any longer, and it’s eluted from the column.

V. Order of Elution: Say, the initial pH of the column is 2.0. When pH needs to increase, it’s done gradually. That is, pH 2 to 3, 3 to 4, 4 to 5 and so, gradually till the elution is achieved.

Therefore, the first pI to reach is pI of 4.8, that of protein D (pyruvate kinase), it’s eluted first. When pH is increased to 5.0, protein E (tRNA synthetase) becomes neutral and is eluted second; and so on. So, the proteins are eluted gradually in the order of increase in their pI.

So, Order of elution (first to last eluted) is – D > E > C > B > A

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