Food Biochemistry and Food Processing (2 edition)

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BLBS102-c07 BLBS102-Simpson March 21, 2012 11:12 Trim: 276mm X 219mm Printer Name: Yet to Come


144 Part 2: Biotechnology and Enzymology

Table 7.6.Functional Groups Used on Ion Exchangers

Functional Group

Anion exchangers
Diethylaminoethyl (DEAE) –O–CH 2 –CH 2 –N+H(CH 2 CH 3 ) 2
Quaternary aminoethyl (QAE) –O–CH 2 –CH 2 –N+(C 2 H 5 ) 2 –CH 2 –CHOH–CH 3
Quaternary ammonium (Q) –O–CH 2 –CHOH–CH 2 O–CH 2 –CHOH–CH 2 N+(CH 3 ) 2

Cation exchangers
Carboxymethyl (CM) –O–CH 2 –COO−
Sulphopropyl (SP) –O–CH 2 –CHOH–CH 2 –O–CH 2 –CH 2 –CH 2 SO 3 −

pH, ionic strength) to maximise the conditions required for the
formation of strong complex between the ligand and the pro-
tein to be purified, (2) choice of washing conditions to desorb
non-specifically bound proteins and (3) choice of elution con-
ditions to maximise purification (Labrou and Clonis 1995). The
elution conditions of the bound macromolecule should be both

Figure 7.14.Schematic diagram depicting the principle of affinity
chromatography.

tolerated by the affinity adsorbent and effective in desorbing
the biomolecule in good yield and in the native state. Elution
of bound proteins is performed in a non-specific or biospecific
manner. Non-specific elution usually involves (1) changing the
ionic strength (usually by increasing the buffer’s molarity or
including salt, e.g. KCl or NaCl) and the pH (adsorption gen-
erally weakens with increasing pH), (2) altering the polarity of
the irrigating buffer by employing, for example ethylene glycol
or other organic solvents, if the hydrophobic contribution in the
protein-ligand complex is large. Biospecific elution is achieved
by inclusion in the equilibration buffer of a suitable ligand, which
usually competes with the immobilised ligand for the same bind-
ing site on the enzyme/protein (Labrou 2000). Any competing
ligand may be used. For example, substrates, products, cofac-
tors, inhibitors or allosteric effectors are all potential candidates
as long as they have higher affinity for the macromolecule than
the immobilised ligand.
Dye-ligand affinity chromatographyrepresents a powerful
affinity-based technique for enzyme and protein purification
(Clonis et al. 2000, Labrou 2002, Labrou et al. 2004b). The tech-
nique has gained broad popularity due to its simplicity and wide
applicability to purify a variety of proteins. The employed dyes
as affinity ligands are commercial textile chlorotriazine polysul-
fonated aromatic molecules, which are usually termed as triazine
dyes (Fig. 7.15). Such dye-ligands have found wide applications
over the past 20 years as general affinity ligands in the research
market to purify enzymes, such as oxidoreductases, decarboxy-
lases, glycolytic enzymes, nucleases, hydrolases, lyases, syn-
thetases and transferases (Scopes 1987). Anthraquinone triazine
dyes are probably the most widely used dye-ligands in enzyme
and protein purification. Especially the triazine dye Cibacron
Blue F3GA (Fig. 7.18) has been widely exploited as an affinity
chromatographic tool to separate and purify a variety of pro-
teins (Scopes 1987). With the aim of increasing the specificity
of dye-ligands, the biomimetic dye-ligand concept was intro-
duced. According to this concept, new dyes that mimic natural
ligands of the targeted proteins are designed by substituting the
terminal 2-aminobenzene sulfonate moiety of the dye Cibacron
Blue 3GA (CB3GA) for ( ́ηwith) a substrate-mimetic moiety
(Clonis et al. 2000, Labrou 2002, 2003, Labrou et al. 2004b).
These biomimetic dyes exhibit increased purification ability and
specificity and provide useful tools for designing simple and
effective purification protocols.
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