To solve this question, you need to have the good understanding of isolectric po

Question

To solve this question, you need to have the good understanding of isolectric point of proteins.

Isolectric point is the pH at which the net charge on the protein is zero. For proteins with many basic amino acids, the isoelectric point will be high, while for proteins with many acidic amino acids the isoelectric point will be lower.

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Proteins in a buffer solution of lower pH than their isolectric point will bear net positive charge, while, proteins in a buffer solution of higher pH than their isolectric point will bear net negative charge.

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A (+) charged anion exchange column will have (+) charge on its stationary phase, therefore will bind with the proteins bearing (-) charge at the given pH of the buffer solution.

Similarly, (-) charged cation exchange column will have (-) charge on its stationary phase, therefore will bind with the proteins bearing (+) charge at the given pH of the buffer solution.

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Larger is the difference in the pH of buffer solution and the isolectric point, larger will be the charge on the protein and stronger will be the attraction between the charged protein and oppositely charged stationary phase of the column.

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(a) In this problem, pH of the buffer solution is lower than all the proteins, therefore, all the proteins will be (+) charged. Positively charged proteins will not bind to (+) charged anion exchange column. Therefore, all the proteins should be eluted together.

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(b) In SDS PAGE, the smaller sized molecules will move faster. More importantly, it should be remembered here that proteins are denatured in this technique i.e. they are converted in to separate subunits. Therefore, proteins with lowest molecular weight subunit will move fastest and protein with largest subunit size will move slowest. Largest sized subunit protein is E with a subunit mass of 30000 Da, therefore, it will move slowest in SDS PAGE chromatography.

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(c) Gel filtration chromatography deals with the separation of proteins from the mixture based on their size by using a column filled with a matrix of porous beads (stationary phase). Proeteins are not denatured in this techniques. The small sized molecules will enter all the available pores and will take a long route for elution, while, large sized molecules will not enter the small pores and will be eluted faster. Largest sized protein is B with a native mass of 120000 Da will be eluted fastest from gel filtration chromatography column. Lowest native mass is for protein C (20000 Da), therefore it will eluted last.

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(d) As discussed in part (a) of this question that at pH 4, all the proteins will be positively charged. Larger the positive charge on the protein, stronger will be the force of attraction between (-) charged stationary phase of the column. Protein (A) with isoelectric point of 8.1, has highest difference from pH of the buffer. This protein will have highest positive charge, therfore, it will be strongly bound and will be eluted last from the (-) charged cation exchange column.

Explanation / Answer

Consider five proteins with the properties shown in the table above and answer the following four questions. Record your answers and choose the set of correct answers for questions a --> d.

a) Which protein would elute first from a (+) charged anion exchange column in buffer at pH 4.0?

b) Which protein would migrate the slowest in an SDS-PAGE gel?

c) Which protein would elute last from a gel filtration chromatography column under non-denaturing conditions?

d) Which protein would elute last from a (-) charged cation exchange column in buffer at pH 4.0?

d) Which protein would elute first from a (+) charged anion exchange column in buffer at pH 4.0?

Subunit Native lsoelectric Proteinmass (Da) mass (Da)point (pl) 10,000 15.000 20,000 25,000 30,000 40,000 120,000 20.000 75,000 60,000 8.1 5.3 7.2 6.2

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