Dye affinity chromatography

The development of dye affinity chromatography may be attributed to the observation that some proteins exhibited anomalous elution characteristics when fractionated on gel-filtration columns in the presence of blue dextran. Blue dextran consists of a triazine dye (cibacron blue F3G-A) covalently linked to the high molecular weight sugar dextran. The discovery that some proteins bind the triazine dye soon led to its use as an affinity adsorbent by immobilization on an agarose matrix. A variety of other triazine dyes (Figure 6.16) also bind certain proteins and, hence, have also been used as affinity adsorbents. Dye affinity chromatography displays some positive general characteristics:

• The dyes are readily available in bulk and are relatively inexpensive.

• Chemical coupling of the dyes to the matrix is usually straightforward, often requiring no more than incubation under alkaline conditions at elevated temperature. The use of noxious coupling chemicals, such as cyanogen bromide, is avoided.

• The dye-matrix bead linkage is relatively resistant to chemical, physical and enzymatic degradation. In this way, ligand leakage from the column is minimized and is easily recognizable if it does occur - due to the dye colour.

• The protein binding capacity of immobilized dye adsorbents is also high and exceeds the binding capacity normally exhibited by natural biospecific adsorption ligands.

• Elution of bound protein is also relatively easily achieved.

A major potential disadvantage, however, is that it is not possible to predict accurately whether a specific protein will be retained on a dye affinity column, or what conditions will allow optimum bind-ing/elution. Such information must be derived by empirical study. An understanding of the specific interactions that allow many apparently unrelated proteins to bind to dye affinity ligands, whereas other proteins are not retained, is usually lacking. The presence of negatively charged sulfonate groups lends triazine dyes an ion exchange character. These dyes also contain aromatic groups that can lend them some degree of hydrophobicity. Hydrophobic interactions, along with charged-based interactions, therefore, may play some role in protein adsorption. The dyes can also hydrogen bond with proteins.

Procion Blue MX 3G

X O nh2

Procion Blue MX 3G

X O nh2

Procion Red H-3B

Procion Red H-3B

-O3S

-O3S

SO3-

Procion Yellow H-A

SO3-

Procion Yellow H-A

SO3-

NHCOCH3 NH2

Figure 6.16 Some mono- and di-chloro triazine dyes commonly used as affinity Ligands in dye affinity chromatography

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