Principles of Biochemistry

8.2.4.1.4 Purification

Purification of the extracted enxyme can be done using dialysis or chromatography.

a) Dialysis for Enzyme Purification

Dialysis is the process that is used to remove small molecules from enzyme. For this, enzyme precipitate obtained in previous step is dissolved in a small quantity of buffer solution in which the enzyme was originally extracted. The solution is taken in a dialysis bag (may be a cellophane tube) and after sealing securely, the bag is suspended in either distilled water or a buffer of known molarity and ionic composition.

b) Chromatography for Enzyme Purification –

Chromatographic separation of proteins is the most common method of enzyme purification. Following four types of chromatography are available for this purpose:

(i) adsorption or column chromatography;

(ii) ion exchange chromatography;

(iii) gel filtration chromatography and

(iv) affinity chromatography.

b) Adsorption Chromatography for Enzyme Purification –

In adsorption chromatography, the enzyme solution suspected to contain other proteinaceous impurities is passed through a column of inert material packed in a glass or steel tube. Most commonly used column materials include finely divided solids such as charcoal, silica, alumina, calcium phosphate, hydroxyapatite, etc.

The effluent solution is continuously collected in small fractions of 1.0 to 2.0 mi. The protein in each fraction is estimated by measuring the absorption at 280 nm using a UV spectrophotometer. The enzyme is also assayed in each fraction. Various spleen enzymes such as basic RNAase, acidic RNAase, acidic DNAse, phosphodiesterase, phos phomonoesterase, etc. are often separated from each other using adsorption chromatography.

For large scale chromatographic separation of enzymes, the process is accelerated by using motors and other mechanical devices for packing the column, for loading the enzyme on the column and for eluting the enzyme.

c) Electrophoresis for Enzyme Purification –

Electrophoresis is a technique in which molecules (enzymes, proteins, amino acids, nucleotides and nucleic acids) are separated by differences in their net charge in the presence of an externally applied electric field.

This technique is routinely used in enzyme purification and isozymes separation in the laboratories, although it has found only limited application at large scale, since the technique is time consuming and is a bit expensive.

Final Step in Processing Enzymes - Most of the commercially available enzyme preparations, purified as above, are concentrated and sterile filtered, after purification. This is done to reduce both, the volume and the microbial contamination of the sample. Often, before storage and transport, the sample is freeze dried with additives such as sugar substrates and dextrans.