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Immobilized Metal Ion Affinity Chromatography 27
8-hydroxyquinoline (7,14). In this context, at pH > 4, the carboxyl groups of
aspartate and glutamate are fully deprotonated and able to donate electrons.
By including imidazole and ≥0.5 M NaCl in the binding buffer, any histidine
or electrostatic interactions will be quenched, leaving aspartate and glutamate
as the only amino acids able to donate electrons and interact with the IMCC.
This type of interaction can be further enhanced by using hard Lewis metal
ions as part of the IMCC so as to exploit the preference of hard Lewis metal
ions for hard bases such as those found in oxygen-rich compounds like the
carboxyl groups of aspartate and glutamate. This type of interaction has been
observed to occur predominantly in the pH region of 5.5–6.5 and may involve
some electrostatic component. Above this pH range, electrostatic influence
becomes more pronounced, and the IMCCs exhibit pseudo-cation exchange
behaviour.
The traditional use of IMAC has involved the inclusion of 0.5–1 M NaCl
in the binding buffer to prevent the protein from interacting with the IMCC
on the basis of non-specific electrostatic interactions. The contribution of such
interactions comes from charges presented to the protein by unoccupied chelate
sites, a variety of hydrolytic species that exist on the IMCC, as well as the
metal ion itself (6,15). The overall contribution results in a net negative charge
on the IMCC, which becomes increasingly negative as the pH becomes more
alkaline. This phenomenon occurs with any IMCC and will vary depending
on the metal ion and immobilized chelator involved. By encouraging this
phenomenon instead of quenching it, IMAC can be used in cation exchange
mode. In this mode, the binding buffers are of low ionic strength (