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Ion-Exchange Chromatography
The next important technique of protein separation, identification and purification is
the ion-exchange chromatography. It is one of the powerful and perhaps most frequently used chromatographic technique for purification and separation of proteins,
polypeptides, nucleic acids, poly nucleotides, and other charged bio-molecules.
This technique is based on ionic interaction of solid support material (matrix) with
oppositely charged protein in a solution. Proteins are separated from the column
either by changing pH, concentration of ion salts or ionic strength of the buffer solution. This technique can be subdivided into two types namely cation and anion
exchangers that possess negatively charged groups attracting positively charged cations and positively charged groups that will attract negatively charged anions
respectively. Cation exchangers are also referred to as acidic ion exchangers because
their negative charges result from the ionisation of acidic groups and anion exchangers are also called as basic ion exchangers, as positive charges generally result from
the association of protons with basic groups (Wilson 2010).
Principle and Method
Proteins are complex ampholytes having both positive and negative charges. Positive
charges are usually provided by free N-terminal amine below pH 8 and 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. Virtually all these residues are ionized above pH 6. At
higher pH values (>8) cysteines may become ionized too. At a certain pH point,
called isoelectric point (pI) of protein, the net charge is zero (Righetti and Caravaggio
1976) and it depends on the proportions of ionizable amino acid residues in its
structure. In an ion exchange chromatography separation a protein at a pH above its
pI will bind to a positively charged medium or anion exchanger and, at a pH below
its pI it will bind to a negatively charged medium or cation exchanger (Jadaun
et al. 2017).
Anion exchanger matrices such as quaternary amines or diethylaminoethyl
groups coupled via a linker to a cellulose matrix are used for binding of anionic
proteins. For the binding of cationic proteins, sulfopropyl or carboxymethyl groups
can be used. The bound proteins of interest are eluted by changing the pH of the
eluting buffer or increasing ionic strength by sodium chloride gradient to break the
interaction of protein with charged matrix.
Recent Advances in Analysis of Food Proteins
Ion-Exchange Chromatography
The next important technique of protein separation, identification and purification is
the ion-exchange chromatography. It is one of the powerful and perhaps most frequently used chromatographic technique for purification and separation of proteins,
polypeptides, nucleic acids, poly nucleotides, and other charged bio-molecules.
This technique is based on ionic interaction of solid support material (matrix) with
oppositely charged protein in a solution. Proteins are separated from the column
either by changing pH, concentration of ion salts or ionic strength of the buffer solution. This technique can be subdivided into two types namely cation and anion
exchangers that possess negatively charged groups attracting positively charged cations and positively charged groups that will attract negatively charged anions
respectively. Cation exchangers are also referred to as acidic ion exchangers because
their negative charges result from the ionisation of acidic groups and anion exchangers are also called as basic ion exchangers, as positive charges generally result from
the association of protons with basic groups (Wilson 2010).
Principle and Method
Proteins are complex ampholytes having both positive and negative charges. Positive
charges are usually provided by free N-terminal amine below pH 8 and 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. Virtually all these residues are ionized above pH 6. At
higher pH values (>8) cysteines may become ionized too. At a certain pH point,
called isoelectric point (pI) of protein, the net charge is zero (Righetti and Caravaggio
1976) and it depends on the proportions of ionizable amino acid residues in its
structure. In an ion exchange chromatography separation a protein at a pH above its
pI will bind to a positively charged medium or anion exchanger and, at a pH below
its pI it will bind to a negatively charged medium or cation exchanger (Jadaun
et al. 2017).
Anion exchanger matrices such as quaternary amines or diethylaminoethyl
groups coupled via a linker to a cellulose matrix are used for binding of anionic
proteins. For the binding of cationic proteins, sulfopropyl or carboxymethyl groups
can be used. The bound proteins of interest are eluted by changing the pH of the
eluting buffer or increasing ionic strength by sodium chloride gradient to break the
interaction of protein with charged matrix.
Recent Advances in Analysis of Food Proteins