This document discusses affinity chromatography and biomimetic dyes. It begins by introducing affinity chromatography as a technique for separating substances based on their reversible interactions with immobilized ligands. It then provides historical background on affinity chromatography and describes various aspects of the technique including common matrices, ligand design and immobilization methods, and elution strategies. The document also discusses the use of biomimetic dyes as ligands for affinity chromatography, noting their low cost but lack of specificity. It describes strategies for designing new dye ligands with improved affinity and specificity for target proteins through mimicking natural ligands.

1. In the Name of GOD
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.

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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.
Affinity Chromatography

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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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15.  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.

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Ligand Immobilization
Sience 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.
Coupling Step:
Activation Step:
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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 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.

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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 Disign
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.
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.
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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Biomimetic Dyes

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Biomimetic Dyes as Affinity Chromatography
 In 1970, immobilized triazine dyes, particularly Cibacron
blue, have been used as affinity chromatography tools for
protein and enzyme purification.
 The low cost of these dyes, their ease of immobilization and
resistance to biological and chemical degradation, and the
high protein-binding capacity of the corresponding
adsorbents.
 Although textile dyes, in some case, interact with proteins
with remarkable degrees of specificity, their interaction with
a large number of seemingly unrelated proteins inevitably
compromises their protein binding specificity and endow
these molecules with a serious drawback.

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Another strategy which can cope with the drawback of textile dyes,
is to design new dye-ligands of improved affinity and specificity for
the target protein.
In principle this can be achieved by designing synthetic dyes which
mimic the structure and binding of natural biological ligands of the
targeted protein.
One way to cope with lack of specificity of immobilized dyes is to
use specific eluents which allow to elute specifically the target
protein with minimal contamination.
This approach is better known as affinity elution. An
alternative method of affinity chromatography, utilizing the
formation of
specific complex of the macromolecule under isolation with an
affinity ligand, is biospecific elution from non-specific adsorbents
and in some instances as “specific elution by the substrate”.

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First Generation
Biomimetic Dyes
Early in the 1980s, the time the first biomimetic dye was
under design, development and assessment, were not
available advanced molecular modeling software packages
for application on a personal workstation, Therefore,
biomimetic dye design was relying on the known binding
preference of the target enzyme for natural ligands, X-ray
crystallography data, and other available useful biochemical
information.
o Alkaline phosphatase
o Alcohol dehydrogenase
o Trypsin

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Second Generation
Biomimetic Dyes
Design by computer aided molecular modeling and use of bioinformatics.
In fact this computational technology, an area of bioinformatics, has marked
a new era in ligand design.
L-malate dehydrogenase
L-lactate dehydrogenase