Question

Describe how you could separate a mixture of cytochrome C, ferritin and an enzyme X (MW 450 kDa and pI 8.4) using only one chromatography column. Be sure to include the type of column, the type of gel or resin, and the conditions to ensure elution ( and separation) of the proteins.

Answer 1

Proteins are often separated on the basis of their molecular size or charge. Since ferritin also has MW of 450 kDa as the enzyme, therefore the best choice to separate them is on the basis of their charge. The pl value of cytochrome c is 10.8, ferritin 5.5 and therefore the value for the enzyme given as 8.4. The pl value is that pH at which the molecule has no net charge. Proteins will carry a positive charge if the pH of the environment is below the pl and that they will carry a negative charge if the pH is above the pl.

Gel electrophoresis is used to separete molecules of proteins. For separation of large molecules like proteins, polyacrilamide gel is used.

The method is as follows -

A stable pH gradient ( higher to lower) is used by adding appropriate ampholytes during a vertical column.

The protein mixture is placed on the gel

Protein molecules will migrate within the gel on application of an electrical field.

Migration will continue till they reach the place where their pH is equivalent to pl.