Cyclodextrin biospecific-like displacement in dye-affinity chromatography.
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. 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 strategy. It provided a 15-fold purification factor with 89% recovery and sharp 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 purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography.
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Spain.
J A
JA
López-Mas
S A
SA
Streitenberger
F
F
García-Carmona
A
A
Sánchez-Ferrer
D002846:Chromatography, Affinity / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry*; D003505:Cyclodextrins / Q000737:chemistry*; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification
D004396:Coloring Agents; D003505:Cyclodextrins; D007770:L-Lactate Dehydrogenase