Interaction of lactate dehydrogenase with anthraquinone dyes: characterization of ligands for dye-ligand chromatography.
Anthraquinone dyes (ADs), originally developed for the textile industry, are useful nucleotide-specific ligands for the purification of proteins by affinity techniques. Their specific feature is to mimic the adenine nucleotides ATP, ADP, NAD, NADH, which enables them to interact with the nucleotide-binding sites of enzymes such as dehydrogenases, kinases and ATPases. In the present study, the interactions and/or inhibitory effects of seven ADs, including Cibacron Blue F3G-A, Remazol Brilliant Blue R, on the activity of lactate dehydrogenase (LDH) were investigated. The ADs used in this paper could be divided into two groups: (i) AD1-AD3 which do not contain a triazine moiety; (ii) AD4-AD7 which contain the triazine moiety. Enzyme kinetics and zonal affinity chromatography were used for the characterization of the interaction affinity between the dye and LDH. Enzyme kinetic measurements were carried 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 properties, three dimensional structure of the respective dyes) and their interaction efficiency with LDH was studied. LDH activity was inhibited by all ADs, excluding AD1 (precursor of the blue dyes) and inhibition was always competitive. Similarity in the mutual position of the acidic and basic groups in NADH and the respective AD molecule was found to be a crucial factor for influencing the inhibitory action of the substance. The existence of ADs in the protonated form should be considered as another factor, important for the ADs inhibitory action on this enzyme.
Bohácová
V
V
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Docolomanský
P
P
Breier
A
A
Gemeiner
P
P
Ziegelhöffer
A
A
D000880:Anthraquinones / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity
D000880:Anthraquinones; D004396:Coloring Agents; D007770:L-Lactate Dehydrogenase