Biomedical Engineering Reference
In-Depth Information
Metal
chelator
Matrix
bead
(a)
C
M
Protein
Spacer arm
Immobilized metal
OH
CH
2
COO
-
CH
2
N
CH
CH
2
(b)
CH
2
COO
-
Figure 6.17
Schematic representation of the basic principles of metal chelate affi nity chromatography. Cer-
tain proteins are retained on the column via the formation of coordinate bonds with the immobilized metal ion
(a). The actual structure of the most commonly used metal chelator, iminodiacetic acid, is presented in (b)
are attracted to the metal ions, forming the weak coordinate bonds. Elution of bound proteins is
undertaken by lowering the buffer pH (this causes protonation of the histidine residues, which
are then unable to coordinate with the metal ion). Alternatively, a strong competitor complexing
agent (e.g. the chelating agent ethylenediaminetetraacetic acid (EDTA)) can be added to the elu-
tion buffer.
Metal chelate affi nity chromatography fi nds most prominent application in the affi nity puri-
fi cation of recombinant proteins to which a histidine tag has been attached (described later). As
protein binding occurs via the histidine residues, this technique is no more inherently useful for
the purifi cation of metalloproteins than for the purifi cation of non-metalloproteins (a common
misconception, given its name).
6.6.10 Chromatography on hydroxyapatite
Hydroxyapatite occurs naturally as a mineral in phosphate rock and also constitutes the mineral
portion of bone. It may also be used to fractionate protein by chromatography.
Hydroxyapatite is prepared by mixing a solution of sodium phosphate (Na
2
HPO
4
) with calcium
chloride (CaCl
2
). A white precipitate known as brushite is formed. Brushite is then converted to
hydroxyapatite by heating to 100
C in the presence of ammonia:
⎯→
Ca HPO
·
2H O
100 C/NH
⎯⎯⎯
Ca
(PO )
(OH)
3
2
4
2
10
4
6
2
brushite
hydroxyapatite
The underlying mechanism by which this substance binds and fractionates proteins is poorly
understood. Protein adsorption is believed to involve interaction with both calcium and phosphate
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