You wish to separate and purify enzyme A from contaminating

You wish to separate and purify enzyme A from contaminating enzymes B and C. Enzyme A is found in the matrix of the mitochondria. Enzyme B is embedded in the mitochondrial membrane and enzyme C is found in the peroxisome. Enzymes A and B have molecular weights of 60,000 Da. Enzyme C has a molecular weight of 100,000 Da. Enzyme A has a pi of 6.5. Enzymes B and C have pi values of 7.5. Design an experiment to separate enzyme A from the other two enzymes.

Solution

Gel filtration chromatography followed by Iso Electric Focusing gel are used to seperate Enzyme-A from B & C. Initially the Mixture (Contain Enzyme-A, B & C) is subjected to Gel filtration chromatography or Molecular seave chromatography, with sephadix beeds of pore size 70 kDa. During, the A & B enzymes enter into beads and C eluted out.

Later the A & B mixture collected from Gel filtration chromatography, then subjected to Iso Electric Focusing. Iso Electric Focusing, seperates proteins based on their pI values.

In this way we can seperate Enzyme A, contominated with B & C