(Section 6.7.1). The cobalt commercial product called Dynabeads Myone TALON™has
the practical advantage that it only binds adjacent histidines or histidines in certain
arrangements and is commonly used for the isolation of recombinant proteins.11.8.5 Dye–ligand chromatography
A number of triazine dyes that contain both conjugated rings and ionic groups
fortuitously have the ability to bind to some proteins. The termpseudo-ligandhas
therefore been used to describe the dyes. It is not possible to predict whether a
particular protein will bind to a given dye as the interaction is not specific but is
thought to involve interaction with ligand-binding domains via both ionic and
hydrophobic forces. Dye binding to proteins enhances their binding to materials such
as Sepharose 4B and this is exploited in the purification process. The attraction of the
technique is that the dyes are cheap, readily coupled to conventional matrices and
are very stable. The most widely used dye is Cibacron Blue F3G-A. Dye selection
for a particular protein purification is empirical and is made on a trial-and-error
basis. Attachment of the protein to the immobilised dye is generally achieved at pH
7 to 8.5. Elution is most commonly brought about either by a salt gradient or by
affinity (displacement) elution.11.8.6 Covalent chromatography
This form of chromatography has been developed specifically to separate thiol(–SH)-
containing proteins by exploiting their interaction with an immobilised ligand
containing a disulphide group. The principle is illustrated in Fig. 11.11. The most
commonly used ligand is a disulphide 2^0 -pyridyl group attached to an agarose matrix
such as Sepharose 4B. On reaction with the thiol-containing protein, pyridine-2-
thione is released. This process can be monitored spectrophotometrically at 343 nm,
thereby allowing the adsorption of the protein to be followed. Once the protein has
been attached covalently to the matrix, non-thiol-containing contaminants are eluted
and unreacted thiopyridyl groups removed by use of 4 mM dithiothreitol or mercap-
toethanol. The protein is then released by displacement with a thiol-containing
compound such as 2050 mM dithiothreitol, reduced glutathione or cysteine. The
matrix is regenerated by reaction with 2,2^0 -dipyridyldisulphide. The method has
been used successfully for many proteins but its use is limited by its cost and the
rather difficult regeneration stage. It can, however, be applied to very impure protein
preparations.11.9 Gas chromatography
11.9.1 Principle
The principles ofgas chromatography(GC) are similar to those of HPLC but the
apparatus is significantly different. It exploits differences in the partition coefficients470 Chromatographic techniques