Chromatographic techniques

Ion exchange chromatography:

Proteins differ from one another in the proportions of the charged amino acid
(aspartic and glutamic acids, lysine arginine and histidine) than can contain.
Hence proteins will differ in net charge at a particular pH. This difference is
exploited in ion exchange chromatography to separate enzymes. In this method,
enzyme of interest is bound on a solid support material (ion exchange resin)
bearing charged group of the opposite sign. Most of the enzyme purification is
done on anion exchange columns because most enzymes are negatively
charged at physiological pH. Elution of the bound enzyme is by exchanging the
charged enzyme by a corresponding cation/anion. Based on the charge
exchanged, it may be a cations exchange chromatography or anion exchange
chromatography. Ion exchanger consists of a water insoluble matrix namely
dextra or cellulose to which charged groups have been charged group have been
covalently bound. Cation exchangers have acidic group with a net negative
charge on the matrix and positively charged exchangeable counter ions.
Example is diethyl amino ethyl cellulose (DEAE.),
(C2H5)2 NH CH2 CH2 O Cellulose

Quaternary ammonium groups, such as triethylaminoethyl (TEAE) and QARE are

also used as anion exchangers. The most commonly used cation exchange
group is carboxymethl (CM). Phosphocellulose is another example of cation
exchanger.

Ion exchange adsorbents are usually eluted by means of a gradient or steps of

increasing KCl or NaCl concentrations, in presence of a constant concentration
of buffer. The charged form of the buffer should be of the same charge as the ion
exchange material, i.e. use Tris or another amine with DEAE, phosphate or
another acid with CM.

Chromatofocusing
A variation of ion exchange technique, it Chromatofocusing in which a linear pH
gradient is generated in the column with 2 or 4 pH units lower at the top as the
column is eluted using the acid form of an ampholyte at low ionic strength. When
a protein is added to this pH gradient with a buffer whose pH is similar to that
prevailing at the top of the column, it will migrate down the column as cation,
encountering an increasing pH, until it reaches a pH corresponding to this
isoelectric ponit. Just beyond this pint it will become an anion and will be able to
bind to the positive groups of the exchanger (in this example column is
anionexchanger). This process is repeated in the column by changing the pH of
the buffer and at the end protein is eluted at a pH slightly above is isoelectric
point. Proteins are claimed to to emerge in sharp, highly resolved peaks. This
technique has high capacity. Chromatofocusing gives a good resolution of quire
complex mixture of proteins, provided that there are discrete differences in their
isoelectric point. Proteins possessing very similar isoelectric points tend to be
poorly resolved.

Hydrophobic chromatography
Enzymes can stick to hydrophobic material by hydrophobic interaction with
nonpolar regions of their surface (by val, phe, etc). The hydrophobic groups used
in the column include alkyl (octyl Sepharose), phenyl (phenyl Sepharose) and
alkyl amino side chains. The capacity is high. Adsorption is strongest at high salt
concentration, so a sample may be applied immediately on redissolution after
(NH4)2SO4 precipitation. Proteins are eluted by decreasing the salt concentration.
Resolution is not as good as in ion exchange chromatography.

Affinity Chromatography
Affinity chromatography is a bio specific process which exploits the formation of
specific and reversible complexes between a pair of biomolecules. One of the
pair is called ligand and is usually immobilize on to a stationary phase while the
other called counter ligand, is adsorbed from the extract that is passing through
the chromatographic column containing the immobilized ligand. This technique
enables separate closely related proteins from a mixture. Method depend on a
specific interaction the enzyme of interest and specific ligands, which may be
substrate analogue binding to its respective enzyme (affinity chromatorgraphy), a
synthetic dyes which can bind to specific protein (dye lignad chromatography) a
lectin, chinch can bind to glycoprotein (lectin affinity chromatography) or an
antibodies binding to specific enzymes antigen (immunoardsorbent
chromatography).
Ligand and counter- ligand in affinity chromatography:
Ligand Counter Ligand Chromatography
Substrate, subnstrate,
1. Enzyme analogue, cofactor, Affinity chromatography
inhibitor
2. Glyco protein Lectin affinity
Lectin
enzyme chromatography
3. Enzyme (an Immunoabsorbent
Antibody
antigen) chromatography
4. Enzyme Dye Dye- ligand chromatography
Metal chelate
5. Metalloenzyme Metal ions
chromatography
The matrix or support is usually agarose or a cross linked derivative, because it
is very porous and admits large proteins to the pores, but has good strength and
stability. In general any matrix useful for ion exchange or gel filtration
chromatography is also good for affinity chromatography. The attachment of
ligand usually proceeds by treating the matrix with a reactive compound like
cyanogens bromide and gluteraldehyde, which either leaves reactive groups to
which ligand can be attached. The ligand is usually attached with a spacer arm
between it and the matrix to assure that the ligand will be fully accessible to the
desired protein. An example of non-specific space is 1.6 diaminohexzane.

The protein mixture is applied to the column and the relevant enzyme is trapped
by the immobilized ligands while all other proteins pass through and are
discarded. The enzyme is the liberated from the column either by eluting with a
deforming buffer at a pH which changes the characteristics of the enzyme and no
longer allows it to bind to immobilized ligand. Another method is using a
competitive counter ligand, which displaces the immobilized ligand on the
enzyme.

Advantages of affinity chromatography included:

1. High selectivity compared to other purification techniques.
2. Extremely good purification up to several thousand folds in a single step and
recoveries greater than 90% can be expected provided conditions are
carefully selected.
3. Affinity chromatography had a high concentration effect, especially when the
enzyme of interest is a minor component of a complex mixture.
4. Affinity methods can also be used to remove unwanted materials from a
mixture.

Immunoadsorbent chromatography
Here immobilized ligand is antibodies to the desired enzyme. In principle, this
technique is the last word in specificity and tight binding, but of course there are
drawbacks. First of all, in order to prepare the antibodies it is required to purify
the protein first and the antibody preparation procedure is cumbersome. Another
problem is elution of the bound enzyme antibody will be difficult.

Dye ligand chromatography

Some reactive triazine-dyes (about 40 dyes) Cibacron F3GA, Procion Red H-
E3B, etc., WW have very affinity for protein and can there for use in enzyme
purification. These dyes will easily attach to agarose or other matrices and thus
can be used in an easy way. Elution is as with true affinity columns, either with
specific ligands which compete with the dye for the protein binding site, or with
high salt concentration or high pH.
Metal-binding chromatography
This can be a general approach for proteins with exposed histidines, cysteines or
carboxyl groups near each other, but in practice it is mainly for cloned proteins.
To cloned protein, a short sequence is added which facilities purification by
binding to specific metals. The commonly used method is to add a sequence of
six or so histidinesd at the C-terminus. These bind well to divalent cations of
transition metals such as nickel. A column is prepared by attaching nitrilotriacetic
acid to a solid support. This binds nickel ions tightly; the resin is washed with
5mM imidazole to remove unbond nickel. The fusion protein, perhaps denatured
in 6M urea to ensure that the hexa-His sequence is exposed, is applied to the
column. The column is washed with dilute imidazole, and then more
concentrated imidazole to elute the desired protein.

Some variations are: mercuric ions bound tightly to immobilized sulfhydryl

groups, which can bind proteins by their exposed sulfhydryl groups elution is
with excess free SH compound such as mercaptoethanol; and Fe+++ bound to
iminodiacetic acid, which binds phosphoproteins by the phosphate groups.

Gel filtration (size-exclusion or molecular sieve chromatography)

The gel filtration material is porous, with pores the size of protein molecules.
Large molecules, too large to enter any of the pores, pass down the column
through the space between the gel particles. Very small molecules enter all the
pores, and therefore spend much of their time not moving and elute out only
solely. Intermediate size molecules enter some of the pores, and are eluted
somewhere in between. Eluting buffer should be of high ionic strength to
counteract the few changes which may be present on the gel. Gel filtration is also
used to separate proteins from salts such as ammonium sulfate, using a small-
pored gel such as Sephadex G-25 or BioGel P10 which excludes all proteins; it is
much faster than dialysis. Gel filtration is widely used to separate protein
detergent miscelles from excess of detergent, during purification of membrane
bound enzymes.

The gel filtration materials are the Sephadexes (cross-linked dextrans),

sephacry (cross-linked acrylamide) and biogel (agarose).

High performance chromatographic techniques

HPLC stands for high performance liquid chromatography (through HP could
also be said to stand for high pressure). The stainless steel columns and robust
packing material of 104m or less which can with stand high pressure are used
and it enables the resolution in minutes. These columns yield good separation
and high resolution in short period. One advantage of fast operation is that they
can be run at room temperature without denaturing the protein, because the
protein comes off in 5 to 60 min. However, only small volumes can be purified.
For protein chromatography it was necessary to develop materials both strong
enough like silica, to stand the pressure and porous enough to have a high
surface area for adsorption, or for gel filtration. HPLC is a high resolution, but low
capacity method. High performance Size Exclusion Chromatography (HPSEC)
utilizes rigid beads of porous silica with bonded hydrophilic polar groups. High
Performance Ion Exchange Chromatography (HIPEC) utilizes amines as anion
exchanges and sulphonic or carboxylic acids as cation exchanges, each bonded
to a rigid support as silica. High Performance Liquid Affinity Chromatography
utilizes ligands bounds to supports such as epoxy-silica. Proteins can also be
separated by reverse phase HPLC (RP-HPLC) on alkylsilica columns, the eluting
solvents being buffered aqueous and organic mixtures.