TECHNIQUES
dextran or acrylic exchangers.
ION-EXCHANGE CHROMATOGRAPHY
-It’s a form of adsorption chromatography in
which ionic analytes display reversible
electrostatic interactions with a charged
stationary phase.
-The column is packed with a stationary phase
consisting of a synthetic resin that is tagged
with ionic functional groups.
-Analytes that have a charge opposite to that of
the resin bind tightly but reversibly to the
stationary phase.
-The strength of binding depends on the size of
the charge and the charge density (amount of
charge per unit volume of molecule) of the
analyte. The greater the charge, the stronger
the interaction.
-Neutral analytes or those with a charge
identical to that of the resin show little or no
affinity for the stationary phase and move with
the eluting buffer.
-The bound analytes can be released by
displacement, eluting the column with a buffer
of increased ionic strength or pH.
-The resins are prepared from a variety of
materials, including polystyrene, acrylic resins,
polysaccharides (dextrans), agarose, and
celluloses. A resin that has negatively charged
functional groups exchanges positive ions and is
a cation exchanger and if exchanges negative
ions it’s an anion exchanger.
-Before a proper choice of ion exchanger can be
made, the nature of the molecules to be
separated must be considered.
-For relatively small, stable molecules the
synthetic resins based on polystyrene are most
effective. For separations of larger molecules,
one must consider the use of fibrous cellulosic
ion exchanger and low-percent cross-linked
that contain small pores of a controlled size.
-Amphoteric molecules have both negatively
and positively charged groups and the net
charge on such molecules depends on pH.
-At the isoelectric point, the analyte has no net
charge and would not bind to any type of ion
exchanger.
-When one is dealing with large biomolecules,
the pH range of stability must be evaluated to
avoid denaturing the biomolecule.
-Below the pI, the molecule has a net positive
charge and would be bound to a cation
exchanger. Above the pI, the molecule has a net
negative charge and would be bound to an
anion exchanger.
-Buffer ions with a charge opposite to that on
the ion exchanger compete with the analyte for
binding sites and greatly reduce the capacity of
the column. Cationic buffers should be used
with anionic exchangers; anionic buffers should
be used with cationic exchangers.
-The pH chosen for the buffer depends first of
all on the range of stability of the
macromolecule to be separated. Second, the
buffer pH should be chosen so that desired
macromolecule will bind to the ion exchanger.
-Chromatofocusing involves the formation of a
pH gradient on an ion-exchange column.
Proteins bound to the ion exchanger are eluted
in the order of their isoelectric points.
GEL-EXCLUSION CHROMATOGRAPHY
-This method exploits the physical property of
molecular size to achieve separation. The
biomolecules range in molecular weight from
less than 100 to as large as several millions.
-In addition, the technique may be applied to
molecular weight determination and
quantitative analysis of molecular interactions.
-The stationary phase consists of inert particles
Microscopic examination of a particle reveals an
interior resembling a sponge. A solution
containing analytes of various molecular sizes is
allowed to pass through the column under the
influence of continuous solvent flow.
-Analytes larger than the pores cannot enter the
interior of the gel beads, so they are limited to
the space between the beads. As a result, they
are not slowed in their progress through the
column and elute rapidly in a single zone. Small
molecules capable of diffusing in and out of the
beads have a much larger volume available to
them and therefore, they’re delayed in their
journey through the column bed. Molecules of
intermediate size migrate through the column
at a rate somewhere between those for large
and small molecules.
-The order of elution of the various analytes is
directly related to their molecular dimensions.
The elution volume for a particular analyte is
proportional to its molecular size.
-Four basic types of gels are available, dextran,
polyacrylamide, agarose, and combined
polyacrylamide-dextran.
- The buffer pH should be chosen on the basis of
the range of stability of the macromolecules to
be separated.
AFFINITY CHROMATOGRAPHY
-This technique offers the ultimate in specificity-
separation on the basis of biological
interactions. The biological function displayed
by most macromolecules is a result of
recognition of and interaction with specific
molecules called ligands.
-Affinity chromatography requires the
preparation of an insoluble sorbent, to which
appropriate ligand molecules are covalently
affixed. Thus, ligand molecules are immobilized
on the stationary support, where a desired
macromolecule is allowed to percolate.
-Only macromolecules that recognize and bind
to immobilized L (ligand) are retarded in their
movement through the column.
-After the nonbinding molecules have washed
through the column, the desired
macromolecules are eluted by gentle disruption
of the L:M complex.
-This technique can be applied to the isolation
and purification of virtually all biological
macromolecules.
-The stationary supports used in gel-exclusion
chromatography are found to be quite suitable
for this technique because: 1) they’re physically
and chemically stable under most experimental
conditions, 2) they’re relatively free of non-
specific adsorption effects, 3) they have
satisfactory flow characteristics, 4) they’re
available with very large pore sizes and 5) they
have reactive functional groups to which an
appropriate ligand may be attached.
-Four types of media possess most of these
desirable characteristics: agarose, polyvinyl,
polyacrylamide and controlled-porosity glass
(CPG).
-The ligand can be selected only after the
nature of the macromolecule to be isolated is
known. If an enzyme is to be purified, a
substrate analog, inhibitor, cofactor, or effector
may be used as the immobilized ligand. The
actual substrate molecule may be used as a
ligand, but only if column conditions can be
modified to avoid catalytic transformation of
the bound substrate.
-The ligand must display a strong, specific, but
reversible interaction with the desired
macromolecule and it must have a reactive
functional group for attachment to the matrix.
Some ligands will work better than others, and
empirical binding studies can be performed to
select an effective ligand.
-To attach the ligand to the matrix is necessary
to: 1) activation of the functional groups on the
matrix, and 2) joining of the ligand to the
functional group on the matrix.
-In most cases, the immobilized ligand and
macromolecule interact through one or more of
the following forces: hydrogen bonding, ionic
interactions and hydrophobic effects.
-The unique high specificity of antibodies for
their antigens is exploited for the purification of
antigens. Immunoaffinity is one of the most
effective modifications of affinity
chromatography. There is often difficulty,
though, in eluting the bound protein without
denaturing it.
-The common methods of elution are: change of
buffer pH, increase of buffer ionic strength,
affinity elution, and chaotropic agents. The
choice of elution method depends on many
factors, including the types of forces responsible
for complex formation and the stability of the
ligand matrix and isolated macromolecule
HYDROPHOBIC INTERACTION
CHROMATOGRAPHY
- HIC separates proteins according to
differences in their surface hydrophobicity by
utilizing a reversible interaction between these
proteins and the hydrophobic surface of a HIC
medium.
- The interaction between hydrophobic proteins
and a HIC medium is influenced significantly by
the presence of certain salts in the running
buffer. A high salt concentration enhances the
interaction while lowering the salt
concentration weakens the interaction.
- The most hydrophobic protein elutes last,
requiring a greater reduction in salt
concentration to reverse the interaction.
-The hydrophobic ligands on HIC media can
interact with the hydrophobic surfaces of
proteins. In pure water any hydrophobic effect
is too weak to cause interaction between ligand
and proteins or between the proteins
themselves. However, certain salts enhance
hydrophobic interactions, and adding such salts
brings about binding (adsorption) to HIC media.
For selective elution (desorption), the salt
concentration is lowered gradually and the
sample components elute in order of
hydrophobicity.
- The final result of a HIC separation is based
therefore on interplay between the prevalence
and distribution of surface-exposed
hydrophobic amino acid residues, the
hydrophobicity of the medium, the nature and
composition of the sample, and the type and
concentration of salt used in the buffers.
METHODS OF ELECTROPHORESIS
-Electrophoresis is an analytical tool that allows
biochemists to examine the differential
movement of charged molecules in an electric
field. The migration of molecules is influenced
by: 1) the size, shape, charge, and chemical
composition of the molecules to be separated;
2) the rigid, mazelike matrix of the gel support;
and 3) the applied electric field.
-The charged particle moves at a velocity that
depends directly on the electric field and
charge, but inversely on a counteracting force
generated by the viscous drag.
-Polyacrylamide and agarose gels are widely
used as support media for larger molecules.
Polyacrylamide Gel Electrophoresis (PAGE)
-Electrophoresis through polyacrylamide gels
leads to enhanced resolution of sample
components because the separation is based on
both molecular sieving and electrophoretic
mobility. This technique is called nondenaturing
and is used when an investigator requires that
the protein analyzed still retains its biological
activity.
-The order of movement is small molecules
followed by large molecules.
-The resolving power and molecular size range
of a gel depend on the concentrations of
acrylamide and bis-acrylamide. Lower
concentrations give gels with larger pores,
allowing analysis of higher molecular weight
biomolecules. In contrast, higher concentrations
of acrylamide give gels with smaller pores.
-Gel electrophoresis is usually carried out a t
basic pH, where most biological polymers are
anionic; hence, they move down toward the
anode.
-The discontinuous gel electrophoresis cause
formation of highly concentrated bands of
sample in the stacking gel and greater
resolution of the sample components in the
lower gel.
Sodium Dodecyl Sulfate- Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
-The molecular weights of proteins may be
estimated if they are subjected to
electrophoresis in the presence of a detergent,
sodium dodecyl sulfate (SDS), and a disulfide
bond reducing agent, mercaptoethanol. This
method is often called denaturing
electrophoresis.
- SDS disrupts the 2º, 3º and 4 º structures to
produce linear polypeptide chains coated with
negatively charged SDS molecules. The bound
detergent molecules carrying negative charges entropy and is thus energetically unfavorable.
mask the native charge of the protein. When salt is added to the solution, the surface
-Empirical measurements have shown a liner tension of the water increases, resulting in
relationship between the log molecular weight increased hydrophobic interaction between
and the electrophoretic mobility. protein and water. The protein responds to this
This modification of gel electrophoresis finds its situation by decreasing its surface area in an
greatest use in characterizing the sizes and attempt to minimize contact with the solvent—
different types of subunits in oligomeric as manifested by folding (the folded
proteins. conformation is more compact than the
- SDS-PAGE is limited to a molecular weight unfolded one) and then self-association leading
range of 10000 to 200000. to precipitation. Both folding and precipitation
free up bound water, increasing the entropy of
AMMONIUM SULFATE PROTEIN PRECIPITATION the system and making these processes
The solubility of globular proteins increases energetically favorable. Timasheff and his
upon the addition of salt an effect termed colleagues provide a detailed discussion of
salting-in. At higher salt concentrations, protein these complex effects.
solubility usually decreases, leading to
precipitation; this effect is termed salting-out.
Salts that reduce the solubility of proteins also
tend to enhance the stability of the native
conformation. In contrast, salting-in ions are
usually denaturants.
The mechanism of salting-out is based on
preferential solvation due to exclusion of the
cosolvent (salt) from the layer of water closely
associated with the surface of the protein
(hydration layer). The hydration layer, typically
0.3 to 0.4 g water per gram protein (Rupley et
al., 1983), plays a critical role in maintaining
solubility and the correctly folded native
conformation. There are three main protein-
water interactions: ion hydration between
charged side chains (e.g., Asp, Glu, Lys),
hydrogen bonding between polar groups and
water (e.g., Ser, Thr, Tyr, and the main chain of
all residues), and hydrophobic hydration (Val,
Ile, Leu, Phe). In hydrophobic hydration, the
configurational freedom of water molecules is
reduced in the proximity of apolar residues. This
ordering of water molecules results in a loss of