Question

Consult Table 5-1 to complete the following: (a) On a plot of absorbance at 280 nm versus elution volume, sketch the results of gel filtration of a mixture containing human cytochrome c and bacteriophage T7 RNA polymerase and identify each peak. (b) Sketch the results of SDS-PAGE of the same protein mixture showing the direction of migration and identifying each band.

Short answer

(a)

(b)

Step-by-step solution

Gel-filtration chromatography

Molecules are separated by shape and size in gel filtration chromatography (also known as size exclusion or molecular sieve chromatography). The stationary phase comprises Gel beads with pores covering a relatively narrow size range.

When an aqueous solution containing proteins of various sizes is run through the column containing such "molecular sieves" or pores, the large molecules that cannot pass through the pores are eluted out.As a result, bigger molecules travel through the column faster than small molecules passing through the pores.

Explanation for (a)

There is a direct correlation between the logarithm of a substance's molecular mass and its relative elution volume. Cytochrome c has a molecular mass of 11,617, and RNA polymerase (bacteriophage T7) has a molecular mass of 98,885.

As RNA polymerase is a large molecule, it will rapidly pass through the column and require less elution volume. In contrast, cytochrome c will pass through the pores and require more elution volume. When these 2 are plotted against absorbance at 280nm, we will get the first peak of RNA polymerase and the second peak of cytochrome c.

SDS-PAGE

Proteins in SDS-PAGE are separated based on their mass. The detergent sodium dodecyl sulphate (SDS) is used to denature proteins in polyacrylamide gel electrophoresis. SDS disrupts the hydrophobic connections that normally keep the proteins stable. The significant negative charge imparted by SDS masks the inherent charge of the proteins, and thus the proteins move towards the positive electrode.

Proteins treated with SDS have similar charge-to-mass ratios and shapes. As a result, SDS-PAGE separates proteins solely based on their molecular mass.

Explanation of (b)

The molecular mass of RNA polymerase is more than cytochrome c, and so when we run these two on the gel, cytochrome c migrates faster than RNA polymerase.