INTERACTION OF LACTATE-DEHYDROGENASE WITH ANTHRAQUINONE DYES - CHARACTERIZATION OF LIGANDS FOR DYE-LIGAND CHROMATOGRAPHY

Anthraquinone dyes (ADs), originally developed for the textile industr y, are useful nucleotide-specific ligands for the purification of prot eins by affinity techniques. Their specific feature is to mimic the ad enine nucleotides ATP, ADP, NAD, NADH, which enables them to interact with the nucleotide-binding sites of enzymes such as dehydrogenases, k inases and ATPases. In the present study, the interactions and/or inhi bitory effects of seven ADs, including Cibacron Blue F3G-A, Remazol Br illiant Blue R, on the activity of lactate dehydrogenase (LDH) were in vestigated. The ADs used in this paper could be divided into two group s: (i) AD1-AD3 which do not contain a triazine moiety; (ii) AD4-AD7 wh ich contain the triazine moiety. Enzyme kinetics and zonal affinity ch romatography were used for the characterization of the interaction aff inity between the dye and LDH. Enzyme kinetic measurements were carrie d out at three different pH values: 6.5, 7.5 and 8.5. The relationship between physical and chemical properties of ADs (e.g., acid-basic pro perties, three dimensional structure of the respective dyes) and their interaction efficiency with LDH was studied. LDH activity was inhibit ed by all ADs, excluding AD1 (precursor of the blue dyes) and inhibiti on was always competitive. Similarity in the mutual position of the ac idic and basic groups in NADH and the respective AD molecule was found to be a crucial factor for influencing the inhibitory action of the s ubstance. The existence of ADs in the protonated form should be consid ered as another factor, important for the ADs inhibitory action on thi s enzyme. (C) 1998 Elsevier Science B.V. All rights reserved.