Question

The following techniques will be used for purification of an enzyme from cultured cells; salt fractionation, affinity chromatography, ion-exchange chromatography, homogenization and gel filtration chromatography. The protocol states that the techniques should be used in the order listed above.

a) What is wrong with the order of techniques as they are listed?

b) How does each technique contribute to the process of obtaining a pure enzyme?

Answer 1

a) The order of the techniques should be :

• Homogenization
• Salt fractionation
• ion-exchange chromatography
• Gel filtration chromatography
• Affinity chromatography

b)

Homogenization - This method is done before fractionation. Homogenization helps in mixing of the cellular structures causing their lysis and easy access to the produced enzyme

Salt fractionation - It is a salting out method that allows to accumulate the secreted enzyme and form a complex or crystal that is determined the salt concentration in the electrolytic solutions. The salting out method is a major step of purification as it removes potential enzyme bond disruptors.

Ion exchange chromatography - Enzymes have net charge in solution due to the presence of certain proteins. The ion exchange chromatoraphy allows separating the enzyme from any other charged molecules that might be present in the mixture to produce a partially pure enzyme.

Gel filtration chromatography - The charged enzyme now is suspended to size exclusion chromatographic separation. This allows to separate the oligomeric forms of the enzyme structure and producing the exact sized molecules.

Affinity chromatography - This technique is used to separate proteins by binding the protein to the specific substrate and then eluting it out in purified protein. The proper size excluded enzyme protein now is subjected to bind to its substrate in the affinity chromatography column. The enzyme binds and remains in the column, eluting the rest of the non-binding components. Now the substrate bound enzyme can be separated and the purified enzyme can be obtained.