chapter  7
12 Pages

Dye ligand affinity chromatography

One of the most successful, yet perhaps the least understood method of biochromatography is variously known as dye ligand chromatography, pseudo-affinity chromatography or biomimetic affinity chromatography. The ligand is a dye molecule such as those developed for the colouring of clothing. Most important discoveries in science occur accidentally, and dye ligand chromatography is one of these. In the late 1960s, scientists purifying kinase enzymes were initially puzzled when, on gel filtration, their enzyme seemed to elute with the void volume marker molecule, Blue Dextran. But in the absence of marker, the elution position was later, at the expected volume (Haeckel et al., 1968; Kopperschläger et al., 1968). This was found to be due to an interaction between the enzymes and the dye itself, which was identified as the reactive dye known as Cibacron Blue F3G-A, from Ciba-Geigy (Fig. 7.1). By immobilising the dye on a suitable matrix, various Blue adsorbents were produced and marketed. These have been particularly suitable for adenine nucleotide-binding proteins, with NAD-utilising dehydrogenases suggested as the most likely to adsorb. However, it was soon found that many other proteins, including non-enzyme proteins were selectively bound to these Blue adsorbents, in particular human serum albumin (Gianazza and Arnaud, 1982) and some interferons (Jankowski et al., 1976). Since then, many hundreds of protein purification procedures have included a Cibacron Blue adsorption step.