Nguyễn Thị Ngọc Linh

BTBCIU17041
ASSIGNMENT 2
ENZYMOLOGY

Topic: Liquid chromatography in protein purification.

Protein purification is vital for the characterization of the functions, structures, and interactions
of proteins. Liquid chromatography refers to a group of separation techniques which involved
several types such as size exclusion, ion exchange, hydrophobic and affinity chromatography.

1. Ion-exchange.
Due to the presence of the amino and the carboxyl groups in the side-chains of the polypeptide
chain, proteins have characteristics of ampholytes which considered having both positive and
negative charges. Positive charges are usually provided by arginines, lysines and histidines,
depending of the pH of the surrounding buffer. Negative charges are principally provided by
aspartate and glutamate residues and the C-terminal carboxyl group. The charged groups nearly
always reside on the protein surface.
The ion exchanger is composed of a base matrix
usually in the form of porous beads to provide
enough surface area for adsorption. These beads
can be 2 types :
Ÿ Anion exchanger: it is possitively charged.
Above isoelectric point, nagatively charged protein
bind to anion exchanger.
Ÿ Cation exchanger: it is negatively charged.
Below isoelectric point, possitively charged protein
bind to cation exchanger.
These anions and cations are immobilized.

Summary of steps:
Ÿ The protein mixture is poured into the column.
Ÿ The gravitational forces cause them to move down the column.
Ÿ Proteins with different net charges will travel along the column with different rate.
Ÿ The proteins with the same charges as the bead will go down faster than the other
proteins which have opposite charge
Ÿ Lastly, adding salt solution to break the electric interaction between the beads and the
desired proteins.

2. Size exclusion.
There is a great diversity of protein in the body and they all vary in size. Gel filtration
chromatography is a common method in proteins purification which based on the molecular size
and shape. This method have both the strength and weakness. About its strength, the function of
fragile proteins is not damaged by binding to a
chromatographic support, while the absence of
such binding limits the resolution of the
chromatography is considered as weakness.

Inside the column, there is gel bead which consist

of a hydrated polymer. The spherical beads is
insoluble and it containing pores that allow small
molecule to pass through. These pores has specific
size. Separation occurs when molecules of
different sizes are included or excluded from the pores within the matrix. Molecules that are too
large to enter the bead pores will only come in contact with the void volume and will therefore
elute first from the column. Besides, the smallest molecules will diffuse into the pores and come
into contact with the total column volume and will elute last. Hence, proteins are eluted from
the gel filtration column in decreasing order of size.

3. Hydrophobic chromatography
Protein hydrophobicity is a complex function of several properties, which include the amino acid
sequence, as well as protein tertiary and quaternary structure in a given solution.

Separation in hydrophobic interaction chromatography depends

upon the reversible adsorption of protein according to their
hydrophobicity. When hydrophobic proteins dissolved in an
aqueous solution, it will self-associate. This method exploit
hydrophobic regions of proteins which bind to hydrophobic
ligand on chromatography absorbents.

The polarity of the solvent can be controlled through the

addition of salts or organic solvents, which can strengthen or
weaken hydrophobic interactions between the hydrophobic
interaction chromatography adsorbent and the protein. The
influence of ions on hydrophobic interaction follows the well-
known Hofmeister series . Anions which promote hydrophobic
interaction the greatest are listed (in decreasing strength of
interaction) from left to right :
PO43- > SO 42- > CH3COO- > Cl- > Br- > NO3- > CLO4- > I- > SCN-

Ions which promote hydrophobic interactions are called lyotropes, while those which weaken
hydrophobic interactions are called chaotropes. Two of the most common lyotropic salts used
to promote hydrophobic interaction in aqueous solution are ammonium sulfate and sodium
chloride.

4. Affinity chromatography.
Affinity chromatography is known as the most powerful method for selective purification of a
molecule or group of molecules from complex mixtures based on highly specific biological
interaction between two molecules. It provides high selectivity, high resolution, high recovery of
activity and high capacity for target natural or recombinant proteins.These interactions, which
are typically reversible, are used for purification by placing one of the interacting molecules,
referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target
molecule is in the mobile phase.

An interacting protein has binding sites with complementary surfaces to its ligand The capture
step is generally followed by washing and elution, resulting in recovery of highly purified protein.