Food Biochemistry and Food Processing (2 edition)

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.