title: Difference spectroscopic and kinetic studies on the interaction of lactate dehydrogenase with structurally related triazine dyes.
abstract: Difference spectroscopy and enzyme kinetics were employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle with the azo-dye Procion Red HE-3B and two of its structural variants in order to follow the significance of the sulphonated terminal rings for the strength and specificity of binding. Procion Red HE-3B possesses a significantly higher affinity to LDH compared to the dye Cibacron Blue F3G-A, a well characterized pseudo-biospecific ligand of dehydrogenases. Moreover, Procion Red HE-3B showed competition towards the cofactor NAD+/NADH. The enzyme-dye complex is mainly stabilized by hydrophobic interactions, but other binding forces cannot be excluded. LDH possesses one dye-binding site per subunit. As a binding region the active center of LDH, preferentially the hydrophobic nicotinamide pocket is involved. Removal of the negatively charged sulphonic acid group from the terminal rings of Procion Red HE-3B decreases the affinity to LDH significantly but does not change the type of binding. Addition of an anilino group to the terminal rings of Procion Red HE-3B does not affect the affinity to the active site significantly but enables the binding on other sites with lower affinity in dependence on the dye concentration.
authors.affiliation: Laboratoire de Technologie des Séparation, Université de Technologie de Compiègne, France.
authors.forename: F
authors.initials: F
authors.lastname: Cadelis
authors.forename: J
authors.initials: J
authors.lastname: Kirchberger
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.forename: G
authors.initials: G
authors.lastname: Kopperschläger
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D004396:Coloring Agents / Q000494:pharmacology*; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013053:Spectrophotometry / Q000379:methods; D014227:Triazines / Q000494:pharmacology*
chemical_list: D004396:Coloring Agents; D007527:Isoenzymes; D014227:Triazines; D007770:L-Lactate Dehydrogenase