Protein separation methods

Protein Separation Methods
By: Zeravan Ali Sulaiman

Contents:
 Introduction.
 Methods of protein separation.
 Separation of protein depending on:
• Solubility.
• Ionic charge.
• Polarity.
• Molecular size.
• Binding specificity.
 References.

Introduction:
 Proteins are large, complex molecules that play many critical roles in the body.
They do most of the work in cells and are required for the structure, function,
and regulation of the body’s tissues and organs.
 Proteins are made up of hundreds or thousands of smaller units called amino
acids, which are attached to one another in long chains. There are 20 different
types of amino acids that can be combined to make a protein. The sequence of
amino acids determines each protein’s unique 3-dimensional structure and its
specific function.
 There are four distinct levels of protein structure.

 Proteins are at the center of the action in biological processes. They
function as enzymes, which catalyze the complex set of chemical
reactions that are collectively referred to as life.
 Proteins serve as regulators of these reactions, both directly as
components of enzymes and indirectly in the form of chemical
messengers, known as hormones, as well as the receptors for those
hormones.
 They act to transport and store biologically important substances
such as metal ions, O2 , glucose, lipids, and many other molecules.
 In the form of muscle fibers and other contractile assemblies,
proteins generate the coordinated mechanical motion of numerous
biological processes, such as the separation of chromosomes during
cell division and the movement of your eyes as you read this page.
Introduction:

 A protein must be purified before its structure and the mechanism
of its action can be studied. However, because proteins vary in size,
charge, and water solubility, no single method can be used to
isolate all proteins. To isolate one particular protein from the
estimated 10,000 different proteins in a cell is a daunting task that
requires methods both for separating proteins and for detecting the
presence of specific proteins.
 Any molecule, whether protein, carbohydrate, or nucleic acid, can
be separated from other molecules based on large differences in
some physical characteristic.
 The most useful physical characteristic for separation of proteins is
size, defined as either length or mass.
Introduction:

 The characteristics of proteins and other biomolecules that are utilized in the
various separation procedures are solubility, ionic charge, polarity, molecular size,
and binding specificity for other biological molecules.
 Some of the procedures we shall discuss and the protein characteristics they
depend on are as follows:
Methods of Protein Separation:
Characteristic Procedure
Solubility 1. Salting in
2. Salting out
Ionic Charge
1. Ion exchange chromatography.
2. Electrophoresis
3. Isoelectric focusing
Polarity
1. Adsorption chromatography
2. Paper chromatography
3. Reverse-phase chromatography
4. Hydrophobic interaction chromatography
Molecular Size
1. Dialysis and ultrafiltration
2. Gel electrophoresis
3. Gel filtration chromatography
4. Ultracentrifugation
Binding Specificity 1. Affinity chromatography

Separation of protein depending on solubility:
SOLUBILITIES OF PROTEINS:
 A protein’s multiple acid–base groups make its solubility properties
dependent on the concentrations of dissolved salts, the polarity of the
solvent, the pH, and the temperature.
 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.

1. Salting in:
The salting in phenomenon is that as the salt concentration of the
protein solution increases, the additional counterions more effectively
shield the protein molecules’ multiple ionic charges and thereby
increase the protein’s solubility.
2. Salting out:
At high ionic strengths, the solubilities of proteins, as well as those of
most other substances, decrease. This effect, known as salting out, is
primarily a result of the competition between the added salt ions and
the other dissolved solutes for molecules of solvation. At high salt
concentrations, so many of the added ions are solvated that the
amount of bulk solvent available becomes insufficient to dissolve
other solutes.
Separation of protein depending on solubility:

Separation of protein depending on Ionic
Charge:
1. Ion-Exchange Chromatography:
 proteins are separated based on differences in their charge. This
technique makes use of specially modified beads whose surfaces are covered
by amino groups or carboxyl groups and thus carry either a positive charge
(NH3+) or a negative charge (COO−) at neutral pH.
 The proteins in a mixture carry various net charges at any given pH.
When a solution of a protein mixture flows through a column of positively
charged beads, only proteins with a net negative charge (acidic proteins)
adhere to the beads; neutral and basic proteins flow unimpeded through the
column (Figure 1).
 The acidic proteins are then eluted selectively by passing a gradient of
increasing concentrations of salt through the column. At low salt
concentrations, protein molecules and beads are attracted by their opposite
charges. At higher salt concentrations, negative salt ions bind to the
positively charged beads, displacing the negatively charged proteins.

 In a gradient of increasing salt concentration, weakly charged
proteins are eluted first and highly charged proteins are eluted last.
Similarly, a negatively charged column can be used to retain and
fractionate positively charged (basic) proteins.
Charge:
Figure 1: Ion-exchange
chromatography.

2. Electrophoresis:
 Electrophoresis is defined as the migration of charged molecules in a
solution through an electrical field. The most common type of
electrophoresis performed with proteins is zonal electrophoresis in which
proteins are separated from a complex mixture into bands by migration in
aqueous buffers through a solid polymer matrix called a gel.
 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.
Charge:

2. Electrophoresis:
 This is used as an analytical method. It permits to estimate number
of proteins in a mixture. This is also useful to determine isoelectric
point and approximate molecular weight.
 One is the stationary phase, which may be solid, liquid, gel or
solid/liquid mixture which is immobilized. 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.
Figure 2: Electrophoresis apparatus.

3. Isoelectric focusing:
 Also termed electrofocusing, is a modification of electrophoresis,
in which proteins are separated by charge in an electric field on a
gel matrix in which a pH gradient has been generated using
ampholytes. Proteins are focused or migrate to the location in the
gradient at which pH equals the isoelectric point, pI, of the protein.
At this point, the protein has no net charge. Resolution is among
the highest of any protein separation technique and can be used to
separate proteins with pIs that vary less than 0.02 of a pH unit.
 If a mixture of proteins is electrophoresed through a solution
having a stable pH gradient in which the pH smoothly increases
from anode to cathode, each protein will migrate to the position in
the pH gradient corresponding to its isoelectric point.
Charge:

3. Isoelectric focusing:
Figure 3: Isoelectric focusing.

1. Adsorption Chromatography:
 During adsorption chromatography of a mixture of solutes, separation of
its components occurs due to their different affinity to the solid
adsorbent.
 The only version of the method of adsorption chromatography used for
separation of proteins, the most complex organic substances, is
chromatography on hydroxyapatite. It has been used for purification of
some acidic and basic proteins.
 Protein purification by means of adsorption chromatography requires
protein movement along the adsorbent column due to adsorption–
desorption processes whereas unwanted proteins would remain at the
origin (due to ion exchange) or rapidly go down due to interaction with
the ion-exchange matrix of the same sign of charge. This can be achieved
in the case of equilibration of an adsorbent-ion exchanger with buffer at
pH that corresponds to pI a protein of interest.
Separation of protein depending on polarity:

2. Paper chromatography:
 Its played an indispensable role in biochemical analysis due to its
ability to efficiently separate small molecules such as amino acids,
oligopeptides, nucleotides, and oligonucleotides and its
requirement for only the simplest of equipment.
 The principle involved is partition chromatography wherein the
substances are distributed or partitioned between liquid phases.
One phase is the water, which is held in the pores of the filter paper
used; and other is the mobile phase which moves over the paper.
The compounds in the mixture get separated due to differences in
their affinity towards water (in stationary phase) and mobile phase
solvents during the movement of mobile phase under the capillary
action of pores in the paper.

2. Paper chromatography:
 Although paper chromatography has been supplanted by the more
modern techniques.
 Paper chromatography is specially used for the separation of a
mixture having polar and non-polar compounds. For separation of
amino acids.
Figure 4: Paper chromatography.

3. Reverse-phase chromatography:
 It has become a powerful tool widely used in the analysis and
purification of biomolecules because of the high resolution
provided by the technique. It is considered a very versatile
technique because it can be used for non-polar, polar, ionizable, and
ionic molecules.
 In RP-HPLC, the separation principle is based on the hydrophobic
interaction between the analytes and non-polar groups bound on the
stationary phase. Silica is the most common material used for
column packing, which consists mainly of silicon dioxide (SiO2)
and has octadecyl (hydrocarbons having 18 carbon atoms) and octyl
(hydrocarbons having 8 carbon atoms) groups chemically bound to
the surface.

 The mobile phase composition is usually water or a water-miscible
organic solvent (methanol, acetonitrile). The analytes adsorbed on
the hydrophobic surface remain bound until the higher
concentration of the organic solvent promotes the desorption of the
molecules from the hydrophobic surface.
Figure 5: Diagram of Reverse-phase chromatography separation.

4. Hydrophobic interaction chromatography:
 In this type of chromatography, proteins are separated based on the
strengths of their hydrophobic interactions with an uncharged resin
containing hydrophobic groups. These interactions are facilitated by
increasing ionic strength. Consequently, proteins are often adsorbed
to a hydrophobic resin in the presence of high concentrations of
neutral salt (e.g.,NaCI).
 Selective elution of adsorbed proteins is achieved by altering the
eluent in a way that causes desorption based on the strength of the
hydrophobic interaction of individual proteins with the hydrophobic
matrix. This can be accomplished by lowering the ionic strength of
the eluent, lowering the polarity of the eluent by including
substances such as ethylene glycol, including detergent in the
eluent, or raising the pH of the eluant.

1. Dialysis and ultrafiltration:
 Dialysis is a process that separates molecules according to size through
the use of semipermeable membranes containing pores of less than
macromolecular dimensions. These pores allow small molecules, such as
those of solvents, salts, and small metabolites, to diffuse across the
membrane but block the passage of larger molecules. Cellophane
(cellulose acetate) is the most commonly used dialysis material, although
several other substances such as cellulose and collodion are similarly
employed. These are available in a wide variety of molecular weight
cutoff values (the size of the smallest particle that cannot penetrate the
membrane) that range from 0.1 to 500 kD.
 Dialysis has been largely supplanted by a related technique known as
ultrafiltration in which a macromolecular solution is forced, under
pressure or by centrifugation, through a semipermeable membranous disk,
which can be made from a variety of materials including cellulose acetate,
nylon, and polyvinylidene fluoride (PVDF).
Separation of protein depending on Molecular
size:

1. Dialysis and ultrafiltration:
Figure 6: Use of dialysis to separate small and large molecules.

2. Gel electrophoresis:
 Electrophoresis is defined as the migration of charged molecules in a
solution through an electrical field. The most common type of
electrophoresis performed with proteins is zonal electrophoresis in
which proteins are separated from a complex mixture into bands by
migration in aqueous buffers through a solid polymer matrix called a gel.
 Polyacrylamide gels are the most common matrix for zonal
electrophoresis of proteins, although other matrices such as starch and
agarose may be used. Gel matrices can be formed in glass tubes or as
slabs between two glass plates.
 Separation depends on the friction of the protein within the matrix and
the charge of the protein molecule as described by the following equation:
size:

 These gels are cast between a pair of glass plates by polymerizing a
solution of acrylamide monomers into polyacrylamide chains and
simultaneously cross-linking the chains into a semisolid matrix. The pore
size of a gel can be varied by adjusting the concentrations of
polyacrylamide and the cross-linking reagent.
 When a mixture of proteins is applied to a gel and an electric current
applied, smaller proteins migrate faster than larger proteins through the
gel. The rate of movement is influenced by the gel’s pore size and the
strength of the electric field. The pores in a highly cross-linked
polyacrylamide gel are quite small. Such a gel could resolve small
proteins and peptides, but large proteins would not be able to move
through it.
 In what is probably the most powerful technique for resolving protein
mixtures, proteins are exposed to the ionic detergent SDS (sodium
dodecylsulfate) before and during gel electrophoresis (Figure 7).

Figure 7: SDS-polyacrylamide gel electrophoresis.

3. Gel filtration chromatography:
 In gel filtration chromatography, which is also called size exclusion and
molecular sieve chromatography, molecules are separated according to
their size and shape. The stationary phase in this technique consists of
beads of a hydrated, spongelike material containing pores that span a
relatively narrow size range of molecular dimensions.
 If an aqueous solution containing molecules of various sizes is passed
through a column containing such “molecular sieves,” the molecules that
are too large to pass through the pores are excluded from the solvent
volume inside the gel beads. These larger molecules therefore traverse the
column more rapidly, that is, in a smaller eluant volume, than the
molecules that pass through the pores (Fig. 8-1).
size:

Figure 8-1: Gel filtration chromatography.

 Proteins flow around the spherical beads in gel filtration chromatography.
However, the surface of the beads is punctured by large holes, and proteins will
spend some time within these holes. Because smaller proteins can penetrate into
the beads more easily than larger proteins, they travel through a gel filtration
column more slowly than larger proteins (Figure 8-2). (In contrast, proteins
migrate through the pores in an electrophoretic gel; thus, smaller proteins move
faster than larger ones.) The total volume of liquid required to elute a protein
from the column depends on its mass: the smaller the mass, the greater the elution
volume. By use of proteins of known mass, the elution volume can be used to
estimate the mass of a protein in a mixture.
Figure 8-2: Gel filtration chromatography.

4. Ultracentrifugation:
 When you put a particle in a centrifugal field, it is acted upon by the centrifugal
force, which is proportional to the molecular weight (M), to the square of the
speed (angular velocity, rpm) of the rotor (w2) and to the distance of the solution
from the center of rotation (r): Cent. Force ~ Mw2r
 If a container of sand and water is shaken and then allowed to stand quietly, the
sand will rapidly sediment to the bottom of the container due to the influence of
Earth’s gravity.
 The ultracentrifuge was developed around 1923 by the Swedish biochemist The
Svedberg. Using this instrument, Svedberg first demonstrated that proteins are
macromolecules with homogeneous compositions and that many proteins are
composed of subunits.
 In zonal ultracentrifugation, a macromolecular solution is carefully layered on
top of a density gradient prepared by use of a device resembling that diagrammed
in Fig. 9.
 zonal ultracentrifugation separates similarly shaped macromolecules largely on
the basis of their molecular masses.
size:

4. Ultracentrifugation:
Figure 9: Zonal ultracentrifugation .

Affinity Chromatography:
 A striking characteristic of many proteins is their ability to bind specific
molecules tightly but noncovalently. This property can be used to purify such
proteins by affinity chromatography (Fig. 10). In this technique, a molecule,
known as a ligand (in analogy with the ligands of coordination compounds),
which specifically binds to the protein of interest, is covalently attached to an
inert and porous matrix.
 When an impure protein solution is passed through this chromatographic
material, the desired protein binds to the immobilized ligand, whereas other
substances are washed through the column with the buffer. The desired protein
can then be recovered in highly purified form by changing the elution conditions
such that the protein is released from the chromatographic matrix.
 The great advantage of affinity chromatography is its ability to exploit the desired
protein’s unique biochemical properties rather than the small differences in
physicochemical properties between proteins that other chromatographic methods
must utilize.
Separation of protein depending on Binding
Specificity:

Affinity Chromatography:
Figure 10: Affinity chromatography. A
ligand (yellow) is covalently anchored to a
porous matrix. The sample mixture (whose
ligand-binding sites are represented by the
cutout squares, semicircles, and triangles)
is passed through the column. Only certain
molecules (represented by orange circles)
specifically bind to the ligand; the others
are washed through the column.

References:
1. Dayton, W.R., Separation Techniques.
2. VOET, DONALD, and JUDITH G. VOET. BIOCHEMISTRY, 4th Edition. JOHN
WILEY & SONS, INC., 2013.
3. Lodish, H., Berk, A. & Kaiser, C.A., 2007. Molecular cell biology, New York: W.H.
Freeman and Company
4. Catsimpoolas, N., 1975.Methods of protein separation, New York: Plenum Press.
5. Surovtsev, V., V. Borzenkov, and K. Detushev, Adsorption chromatography of proteins.
Biochemistry (Moscow), 2009. 74(2): p. 162-164.
6. Smith, D.M., Protein separation and characterization procedures, in Food analysis2017,
Springer. p. 431-453.

Protein separation methods

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