Chromatographic technique used for determining the biological activity of substances & to separate them from denatured or functionally different molecules. This type of chromatography is used to isolate enzymes & other proteins.

1. AFFINITY CHROMATOGRAPHY

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Introduction
A goal of biochemistry is to separate and identify chemical compounds.
chromatography is one of the most effective techniques for accomplishing
this.
In chromatography, substances are placed in a system consisting of two
physically distinguishable components -a mobile phase and a stationary
phase-and molecular species separate because they differ (many of them only
slightly) in their distribution between these two phase.
There are many kinds of chromatography:
Adsorption
Partition
Ion-Exchange
Molecular Sieve
Affinity
Column, paper, thin-layer and gas chromatography.

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Historical Perspective
of Affinity Chromatography
* The German pharmacologist Emil Starkenstein in paper published
in 1910 on the influence of chloride on the enzymatic activity of
liver-amylase was generally considered to be responsible for the
first experimental demonstration of the biospecific adsorption of
an enzyme onto a solid substrate, in this case, starch.
* Not long after, Willstatter et al. appreciably enriched lipase by
selective adsorption onto powdered stearic acid.
* Affinity chromatography as it is known today was introduced in
1968 by Cuatrecasas et al.

5. Affinity Chromatography
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The technique offers high selectivity, hence high
resolution, and usually high capacity for the proteins of
interest.
Purification that would otherwise be time-consuming,
difficult or even impossible using other techniques can
often be easily achieved with affinity chromatography.
The technique can be used to separate active biomolecules
from denatured or functionally different forms, to isolate
pure substances present at low concentration in large
volumes of crude sample and also to remove specific
contaminants.

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Affinity Chromatography
 Affinity chromatography separates
proteins on the basis of a reversible
interaction between a protein and a
specific ligand coupled to a
chromatography matrix.
 The kinds of Elution
 pH Elution
 Ionic Strength Elution
 Reduced Polarity of Eluent
 Competitive Elution
 Chemotropic Eluents

9. Elution
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11. Purification steps

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 Binding: buffer conditions are optimized to ensure that the target molecules
interact effectively with the ligand and are retained by the affinity medium as all
other molecules wash through the column.
 Elution: buffer conditions are changed to reverse (weaken) the interaction
between the target molecules and the ligand so that the target molecules can be
eluted from the column.
 Wash: buffer conditions that wash unbound substances from the column without
eluting the target molecules or that re-equilibrate the column back to the starting
conditions (in most cases the binding buffer is used as a wash buffer).
 Ligand coupling: covalent attachment of a ligand to a suitable pre-activated
matrix to create an affinity medium.
 Pre-activated matrices: matrices which have been chemically modified to
facilitate the coupling of specific types of ligand.

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Affinity Medium
Matrix
The Kinds of matrix
Ligand
Ligamd Immobilization
Spacer Arm

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Proper selection of a matrix or carrier for the ligands is of decisive
importance for the successful application of stereospecific adsorption.
1. Insolubility
2. Sufficient permeability
3. High rigidity and suitable particle form
4. Zero adsorption capacity
5. Chemical stability under the conditions required for adsorption, desorption
and regeneration
6. Chemical reactivity allowing ligands and spacers to be introduced
7. Resistance toward microbial and enzymatic attack
8. Good flow properties for rapid separation
9. An open pore structure ensures high capacity binding even for large
biomolecules.
Matrix

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No matrix support is ideal in all these respects.
Porous glass
Cellulose
Polyacrylamide
Agarose

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Ligand
 The selection of the ligand for affinity chromatography is influenced
by two factors:
o the ligand must exhibit specific and reversible binding affinity for the target substance
o and it must have chemically modifiable groups that allow it to be attached to the matrix
without destroying binding activity.
 The dissociation constant (kD) for the ligand-target complex should
ideally be in the range 10-4
to 10-8
M.

18. Ligang Immobilization
Science 1970, a large number of methods have been
developed for coupling ligands to matrix materials. The
most common procedure is to link a coupling agent to
the matrix material and then add the ligand.
It is important to mention that, after coupling of the
desired ligand, reactive Y groups may still be present
Deactivation may occur by spontaneous
 hydrolysis but, if this is not the case, coupling with a
low molecular weight
 substance. Glycine, neutral dipeptides, and
ethanolamine are deactivating
 substances that should be considered.
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Activation step
Coupling step

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 Methods for Immobilization
1. Cyanogen Bromide Coupling
2. Bisoxirane Coupling
3. Divinylsulfone Coupling

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1. Cyanogen Bromide Coupling
1,2-Diols are especially liable to react with cyanogen halides to
form cyclic imino carbonates. In the coupling step a substance
containing amino groups will form at least three products.
Activation Step:
Coupling Step:

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 This reaction is extremely useful in coupling enzymes, coenzymes,
inhibitors, antigen, antibodies, nucleic acids and most proteins to
agarose.
 Although most applications of cyanogen bromide coupling have
involved agarose and cross-linked agarose, other hydroxyl-
containing polymers may also be converted to biospecific
adsorbents by coupling of suitable ligands in the same manner.

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2. Bisoxirane Coupling
Bisoxiranes (bisepoxides) are particularly useful reagents for
introducing low molecular weight ligands through amino or
hydroxyl groups.

24. Continued…
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3. Divinylsulfone Coupling
 The vinyl groups introduced into the matrix are more reactive than are
the oxirane groups.
 They will thus couple to amines, alcohols, and phenols at lower
temperatures and at lower pH than the oxirane.

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Spacer Arm
 The binding site of a target protein is often located deep within the
molecule and an affinity medium prepared by coupling small ligands,
directly to matrix may exhibit low binding capacity due to steric
interference i.e. the ligand is unable to access the binding site of the
target molecule.
* The length of the spacer arm is critical.
* when using small ligands (Mr < 5 000) there is a risk of steric
hindrance between the ligand and the matrix that restricts the
binding of target molecules. In this case, select a pre-activated
matrix with a spacer arm. For ligands with Mr > 5 000 no
spacer arm is necessary.

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Ligand Design
1. Protein-Structure-Based Design
2. Protein-Function-Based Design

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Ligand Design
The rapid growth of bioinformatics and molecular docking
techniques and the introduction of combinatorial methods for
systematic generation and screening of large numbers of novel
compounds, has made feasible the rapid and efficient generation of
ligands for affinity chromatography.

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2. Protein-Function-Based Design
This approach is applied where the three dimensional structure of the
target protein is not available and is relies on the incorporation of
certain structural features on the ligand.
1. A certain required molecular shape.
2. Specific functional group.
3. A structural model derived from the combination of structural moieties which
are known substrates, inhibitors, effectors or cofactors.

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1. Protein-Structure-Based Design
The strategy for the rational design of affinity ligands involves
retrieving structural information about the target protein from
suitable databases and identifying a potential binding site on the
protein.
For example, for the design of ligands for
• L-Lactate Dehydrogenase
• Glutathione S-transferase
• Galactose Oxidase
• Galactose Dehydrogenase
• Elastase.

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