12 Affinity

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AffinityChromatography

The method dates back to 1910.

• Modern method was first published in

1967, by Axen, et al. -- ‘Cyanogen

Bromide Method for the

Immobilization of Ligands on Ararose.’

• Ohlson (1978) was the first to

demonstrate the use of a rigid,

microparticulate support - beginnings

of instrumental method.

Affinitychromatography

In general, the methodinvolves the interaction of a ligand with the solute of interest.

It can be viewed as beingcomparable to ion-exchange.

Components of an affinity phase

ligandspacer matrix

Ligand Site of interaction.

Spacer What binds the ligand to the support.

Matrix Supporting phase. AffinityChromatography

Two general types of ligands

Specific! ! Binds only to one species.! ! !Antibody/antigen

General! ! Group specific! ! ! Binds to specific groups! ! ! on target species.

Ligand types

• Enymes ! ! Substrate, inhibitor, cofactor

• Antibody ! ! Antigen, virus, cell

• Lectin ! ! ! Polysaccharide, glycoprotein, cell receptor

• Nucleic Acid Complementary base sequence, histone, nucleic acid,polymerase, binding protein

• Hormones ! ! Receptor, carrier protein

Spacer

o Carbon chain interposed between ligand and matrix.

o Used when active site is located deep within a samplemolecule.

o If too long, it can interact with sample species on itsown (hydrophobic interactions).

o If too short, ligand can’t reach active site on samplemolecule.

o Commercial phases have spacers that are optimizedof specific separations.



Support or matrix

• The material that ligand or spacer is bound to.

• It should be rigid, stable and have a high surface area.

• Agarose is the most popular, cellulose, dextran andpolyacrylamide have been used.

• Sepharose is a bead-form of agarose gel.


Agarose gel• A polymer of D-galactose and 3,6-anhydro-L-galactose.• It can be used at pressures up to 1 psi and over a pH

range of 4-9.• Cross-linking can be used to extend the pressure range.

Examples of specific phases

Protein A-Sepharose Cl-4B! Fc region of IgG and related molecules

Con A-Sepharose ! ! Terminal -D glucopyranosyl, -D mannopyranosyl or similar residues

Poly (U)-Sepharose 4B Nucleic acids, especially mRNA, which contains poly (A) sequences; poly(U)- binding proteins

Lysine-Sepharose 4B ! ! Plasminogen; ribosomal RNA

Blue Sepharose Cl-6B! ! Broad range of enzymes which have nucleotide cofactors; serum albumin; etc.

5 AMP-Sepharose 4B! ! Enzymes which have NAD+ as cofactor and ATP-dependent kinases

2'5'ADP-Sepharose 4B! ! Enzymes which have NADP+ as cofactor

Affinity chromatography

Creation of the phase.

The ligand should be covalently bound to the support tocreate a stable phase - immobilization.

Two steps

Activation of the support with a reactive compound

Attachment of the ligand.


Attachment can result in the ligand no longerbeing active.

! Possible reasons! ! Alteration of the 3-D structure.

! ! Site is no longer accessible.


Ideal - binding siteis readily available.

Alteration of ligandstructure.

Poor orientation orspacing.




Once a column in prepared, the separation is conducted in fourbasic steps.

! Sample introduction

! Adsorption of components of interest

! Removal of impurities

! Elution of components.


Sample introduction

You must make sure that yourcolumn has adequate capacity.

ligandspacer matrix


Absorption

• Using a slow flow, yoursample is then allowed topass through the column.

• The flow helps drive your

sample components towards‘fresh’ sites.


Washing

Next, you can remove impurities bypassing several volumes of freshsolvent through the column


Elution

• The component of interest mustthen be removed and collected.

• This also acts to regenerate thecolumn.


Elution methods

! Biospecific

• An inhibitor is added to themobile phase (free ligand).

• The free ligand will compete forthe solute.

• This approach is most often usedwhen a low molecular weightinhibitor is available.




Elution methods

! Nonspecific

• A reagent is added that denatures the solute (pH,KSCN, urea, ionic strength...)

• Once denatured, the solute is released from theligand.

• If the solute is to be further used, it must not beirreversibly altered.

Example chromatogram

L o w

e r p H

I n t r o d u c

e s a m p l e

Elution phase Washing phase

Time


Column: 50 mm x 30 mmcontaining 60 ml ofProtein A Sepharose

Sample: 5 liter cell culturesupernate with mouse IgGa2

and 0.5% fetal calf serum.

Starting buffer:0.1 M Na2HPO4, pH 7

Elution buffer:

0.1 M citric acid, pH 4Flow rate: 66.6 ml/min

(c) 1997, Pharmacia Biotech


Purification of IgG frombovine serum

Column: Protein G Sepharose

Sample: 100 ul Bovine serum

Flow rate: 0.6 ml/min

Starting buffer:20 mM sodium phosphate, pH 7

Elution buffer0.1M glycine-HCl, pH 2.7



Purification of IgG (anti-CEA)on Proteine G Sepharose 4

Starting buffer20 mM sodium phosphate,pH 7

Elution buffer0.1 M glycine-HCl,pH 2.7

Flow rate 0.8 ml/min


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