Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 215

teins and enzymes for the chelated metal ions may not be completely non-
physiological it has been aptly termed pseudo bioaffinity [12].
First row transition metals (Ni 2+ ,Zn 2+ ,Co 3+ ,Cu 2+ ,Fe 3+ ) are generally used as
ligands after chelation by iminodiacetic acid (IDA). IDA is a trident chelator that
binds metal ions through its nitrogen and two carboxylate oxygens. As the metal
ions coordinate 4 - 6 ligands,the remaining of coordination sites are occupied by
water molecules and buffer ligands which are available for replacement by
appropriate surface groups [140].
Although a number of amino acid side chain groups are known to bind to
chelated metal ions,it is now recognized that the histidine side chain dominate in
protein binding to chelated Ni 2+ ,Zn 2+ ,Co 2+ and Cu 2+ . In addition the N -terminal
amino groups as well as the -SH may contribute to the binding although the
latter may not be available in the reduced state on the surface of majority of
enzymes. Indeed a number amino acid residues may contribute to metal affinity
binding especially at higher pH even though at the commonly used pH (around
neutrality),their contribution may be quite small [140].
Considerable evidence has now accumulated to show that protein are re-
tained by a metal affinity supports according to the number of accessible histi-
dine residues [140,143]. The histidine imidazole nitrogens co-ordinate metals in
the unprotonated state hence in addition to the number of accessible histidines,
their ionization state,that is in turn dependent upon the nature of surrounding
amino acids,influence the retention of protein on metal chelate supports [143].
The association of proteins and enzyme with metal chelate is also strongly
influenced by metal ions involved in the coordination. Affinity of the metal for
imidazole usually forms the basis of protein retention and stability constants.
The affinity of imidazole for Cu 2+ is about 15 times higher than for Zn 2+ and
Co 2+ [144,145]. Although histidine containing proteins bind more strongly to
metal chelated supports bearing Cu 2+ ,protein binding capacities of various
immobilized metal ion supports may not be very different. In a more recent stu-
dy,Hale [81] has shown that proteins could be attached to Co

2+ supports in an
exchange inert or irreversible fashion when the cobalt is oxidised from the Co 2+
to Co 3+ state. It was however indicated that the protein bound Co 3+ support

may be removed by reduction of the metal to Co 2+ by appropriate reducing

agents.
Elution of proteins bound to immobilized metal supports can be easily per-
formed either with Lewis acids (H + ,Zn 2+ ),that compete with metal for protein
or with Lewis bases like imidazole that compete with protein for metal. Elution
can also be performed with strong chelators of metal ions like EDTA [140].
Histidine is one of the less abundant amino acids of proteins and most
globular proteins contain not more that 2 % histidine [146]. Considering that
less than half of histidine residues of proteins are located at the surface,most
proteins on the average have only about one exposed residue for every hundred
amino acids. It is therefore natural that many proteins do not bind to metal
chelates. Most of the work on binding and immobilization to metal chelate
supports is therefore related to proteins with enriched histidines. However it
has been shown that a single surface histidine may be adequate to retain a
protein strongly on metal chelate supports [140].