Cyclodextrin biospecific-like displacement in dye-affinity chromatography

Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine d ye, and 2-hydroxypropyl-beta -cyclodextrin (HP-beta -CD), as a model of cyc lodextrin, were investigated by monitoring the spectral shift that accompan ies the binding phenomena. Matrix analysis of the difference spectral titra tion of CB F3GA with HP-beta -CD revealed only two absorbing species, indic ating a host-guest ratio of 1:1. The dissociation constant for this HP-beta -CD-CB F3GA complex, K-d, was found to be 0.43 mM. The data for HP-beta -C D forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatograp hy. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution st rategy. It provided a 15-fold purification factor with 89% recovery and sha rp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1 .8 column volumes). In addition, the new elution strategy showed a better p urification factor and sharper elution profile than traditional non-specifi c elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive e lution by inclusion complexes may be a promising strategy for eluting prote ins with high recoveries and purification factors in dye-affinity chromatog raphy. (C) 2001 Elsevier Science BN. All rights reserved.