Design and applications of biomimetic anthraquinone dyes : II. The interaction of C.I. reactive blue 2 analogues bearing terminal ring modifications with horse liver alcohol dehydrogenase

A number of terminal ring analogues of the anthraquinone dye C.I. Reactive Blue 2 were synthesised and characterised. The interaction of these dye analogues with horse liver alcohol dehydrogenase was investigated by difference spectroscopy and analytical affinity chromatography. Studies by difference spectroscopy showed that anionic terminal ring substituents or an unsubstituted phenyl ring promoted tight binding of the dye to the enzyme, whereas neutral or cationic terminal ring substituents reduced the affinity of the dye. Terminal ring analogues of C.I. Reactive Blue 2 with ortho- or meta-orientated groups were bound more tightly than those with para-orientated substituents. The observed differences in the affinity of the dye analogues towards horse liver alcohol dehydrogenase could be accounted for by proposing that the terminal ring of C.I. Reactive Blue 2 is bound in a relatively apolar pocket lateral to the principal coenzyme binding site and located in the catalytic domain of the enzyme. The 2900-fold difference in affinity between the C.I. Reactive Blue 2 analogues and horse liver alcohol dehydrogenase in solution was not translated into significant differences in chromatographic behaviour when the dyes were immobilised to beaded agarose. However, an ortho-orientated sulphonate substituent promoted exceptionally tight binding of the enzyme to the immobilised dye. Immobilisation of the dye analogues via the reactive chlorotriazine was identified as the most likely explanation for the relatively minor influence of the terminal ring substituent of the immobilised dye on enzyme binding.