Thursday, December 17, 2015

Protein separation techniques: Protein fractionation

Fig. Protein purification steps
Proteins differ in their molecular size and charge. They can be separated on the basis of their following properties:
1.       molecular size
2.       solubility
3.       electrical charge
4.       adsorption properties
5.       specific bioaffinity.

Many techniques for protein purification exist, but the emphasis here is on some of the most popular procedures and the principles involved in their use.  Protein fractionation is required to separate and characterize a protein in detail.

Differential Centrifugation

A typical crude broken cell preparation contains disrupted cell membranes, cellular organelles and a large number of soluble proteins all dispersed in an aqueous buffered solution. The membranes and the organelles can be separated from the soluble proteins by differential centrifugation.

This type of centrifugation involves the use of different speeds and different durations. For eg. If the protein of interest is in the mitochondrial fraction, the crude cell lysate is first centrifuged at 1000g for removing nuclei, debris etc. The supernatant contains among other elements the mitochondria, which can be pelleted at 3300g for 10 minutes.

Salt Fractionation

Proteins show a variation in solubility that depends on the concentration of salts in the solution. This method is frequently used to separate serum proteins into albumins and globulins.  Albumin is soluble in water whereas globulins are not.  Globulins are soluble in weak salt solutions, going into solution at salt concentrations of 0.1 mol/L. This phenomenon called "salting in". This is thought to be due to electrostatic attraction between salt ions and the charged groups on the protein, which decreases the intermolecular electrostatic attraction of proteins & increases the interactions of protein molecules with water, a polar solvent, thus making them soluble.  Salts with divalent ions are more effective than those with monovalent ions.

As the salt concentration is increased, however, salt ions compete for the water molecules of hydration of the hydrated groups of proteins, resulting in the decreased solubility and precipitation of protein out of the solution.  The larger proteins are usually precipitated first. This phenomenon is referred to as “salting out” of protein.  Ammonium sulphate is commonly used for salting out proteins. Globulins are the proteins precipitated by half saturation of a solution with ammonium sulphate, while Albumin is precipitated on fully saturating the solution.


Fig. Electrophoresis apparatus

Electrophoresis is the movement of charged particles through an electrolyte in an electric field. The positively charged particles move towards the cathode and the negative ions to the anode. The rate of migration of particles of like charge will depend among other things on the number of charges it carries. Different rates of migration separate a complex mixture such as plasma proteins into a number of fractions according to mobility. Electrophoresis is not used to purify proteins because some alteration in protein structure and ultimately function. This is used as a analytical method. It permits to estimate number of proteins in a mixture. This is also useful to determine isoelectric point and approximate molecular weight.


This technique was originally used to separate chlorophyll from plant extracts on silica, hence the name chromatography, which means separation of colored compounds. It is the name given to any technique in which the members of a group of similar substances are separated by a continuous redistribution between two phases. One is the stationary phase, which may be solid, liquid, gel or solid/liquid mixture which is immobilised. The second mobile phase may be liquid or gaseous and flows over or through the stationary phase. The choice of stationary or mobile phases is made so that the compounds to be separated have different distribution coefficients.
The movement of any substance being chromatographed is the outcome of

a.   Forces resulting from its interaction with the mobile phase which tend to pull it along with the mobile phase
b.   Forces resulting from its attraction to the stationary phase, which tend to retard its movement.

Since the forces are different for different substances chromatography will be able to separate out the components of a mixture of substances.

Based on the properties of the stationary phase and the acting forces, several different types of chromatographic procedures have been described.
1. Adsorption chromatography: Adsorption equilibrium between a stationary solid and a mobile liquid phase. This is used to separate the non ionic, water insoluble compounds such as vitamins, triglycerides drugs etc.
2.   Partition chromatography: Partition equilibrium between a stationary liquid and a mobile liquid phase. This is used to analyze drugs, insecticides, pestisides,amino acids .
3.  Gas-Liquid chromatography: Partition equilibrium between a stationary liquid and a mobile gaseous phase. The mobile phase is generally an inert gas - steroid separation.
4.   Ion-exchange chromatography: Ion exchange equilibrium between an ion-exchange stationary resin phase and a mobile electrolyte phase. So amino acids proteins which have ionized groups can be separated on the basis of this.
5.    Gel permeation chromatography: Equilibrium between a liquid phase inside and outside a porous molecular sieve depending on molecular size - preparative procedure.
6.    Affinity chromatography: Equilibrium between a macromolecule and a small molecule (Ligand) for which it has high affinity - monoclonal antibody purification.

Types of solid support media
a.      Dextran (Sephadex),
b.      Agarose (Sepharose),
c.       Silica Gel,
d.      Cellulose (Sephacel)

Dextran is a polysaccharide built from glucose residues. When the dextran is cross-linked by epichlorhydrin to form a gel, the polysaccharide chains of the gel from a 3-dimensional network. The product is insoluble unless it is broken down chemically. Dextran gels are commercially known under the name of Sephadex. The different types of sephadex beads differ in their degree of cross-linking. The various types are characterized by the letter G, followed by a number. Based on their extent of cross-linkage, proteins of different MW are separated from each other.

Principle of the method:

In the column, proteins of different molecular size penetrate into the internal pores of the beads to different degrees and thus travel down the column at different rates. In other words, very large protein molecules cannot enter the pores of the beads and they are excluded, while small proteins can enter the pores of the beads freely and are retarded. This is called molecular sieving or gel permeation. The different peaks of separation can be monitored by UV detectors and recorded. The peak containing required material can be identified depending upon the properties of the substance for e.g. measurement of enzyme activity, Ag-Ab binding, etc.

Bed volume
Volume of the gel which is available to those molecules which are smaller than the fractionation range for the gel.

Void volume
Volume of the liquid in the interstitial spaces between beads of the gel. It is available to those molecules whose size is bigger than the fractionation range of the gel eg. Blue Dextran (2 million MW).                                                                                                 

Applications of protein purification: 
1.       Protein concentration
2.       Monoclonal antibody purification
3.       Removal of endotoxins from a protein solution
4.       Removal of proteins from a sugar solution.

Fig. Paper chromatography
Amino acid separation by paper chromatography:
In this, the amino acids partition themselves on the paper between an organic solvent of choice and water.

Procedure: There are two techniques, which may be employed for the development of paper chromatograms: ascending or descending methods.  In ascending technique, the paper is kept in a glass jar containing the solvent such that the lower edge of the paper is in contact with the solvent. The sample is applied to the position just above the surface of the solvent. As the solvent moves vertically up the paper by capillary action, separation of the amino acids is achieved. In the descending techniques, the solvent moves downwards under gravity.

Component Detection: Spraying of the paper with ninhydrin will stain amino acids.  The identification of a given amino acid may be on the basis of its relative fraction (Rf) value, which is defined as:

This value is constant for a particular compound under standard conditions.  Quantification of the amino acid may be carried out after eluting it with a suitable solvent.

Clinical applications:
1.   Screening and diagnosis of inherited disorders of amino acid metabolism e.g. Phenylketonuria and Tyrosinaemia.
2. Peptide fingerprinting for the diagnosis of diseases like sickle cell anemia.
3.   One dimensional thin layer chromatography (TLC) can detect aminoacidopathies and is the most adequate for screening.

Specimens reference solution (mixture of known amino acids at known concentration) and controls are applied to thin layer plates, run in an appropriate solvent-vapor system and then dried and stained with ninhydrin

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