Separation
and purification of proteins using ion exchange chromatography is based
primarily on differences in the ionic properties of surface of amino acid.
Proteins bind to ion exchangers by electrostatic forces between the protein’s
surface charges (mainly) and the dense clusters of charged groups on the
exchangers. The charges are of course balanced by counterions such as metal
ions, chloride ions, and sometimes buffer ions. A protein must displace the
counterions and
become attached; generally, the net charge on the protein will
be the same sign as that of the counterions displaced-hence “ion exchange”. The
protein molecules in solution are also neutralized by counterions; the overall effect
in a given region of the absorbent must be electrically neutral. If we use Tris buffer, it is assumed that a
protein with a net negative charge is preequilibrated in Tris-chloride buffer;
the counterions associated with the protein are thus HTris+. The
adsorbent (DEAE-cellulose) also equilibrated with the buffer has the cl- counterions.
The protein displaces the chloride ions, occupies a site in the adsorbent, and
Tris-Cl is discharged. A change in experimental conditions (such as an increase
in the counterion concentration or decrease in the protein net ion charge) will
cause another exchange of ions, this time releasing the protein from the ion
exchange matrix in favor of the counterion. This process of successive exchanges
of ions allows separation of proteins with different charge properties.
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