200 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
to maximize the conditions required for the forma-
tion of a strong complex between the ligand and the
protein to be purified, (2) choice of washing condi-
tions to desorb nonspecifically bound proteins, and
(3) choice of elution conditions to maximize purifi-
cation (Labrou and Clonis 1995). The elution condi-
tions of the bound macromolecule should be both
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 nonspecific or biospecific manner. Nonspecific
elution usually involves (1) changing the ionic
strength (usually by increasing the buffer’s molarity
or including salt, e.g., KCl or NaCl) and pH (adsorp-
tion generally 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 elu-
tion is achieved by inclusion in the equilibration
buffer of a suitable ligand, which usually competes
with the immobilized ligand for the same binding
site on the enzyme/protein (Labrou 2000). Any com-
peting ligand may be used. For example, substrates,Figure 8.14.Schematic diagram depicting the principle of affinity chromatography.