| heal.abstract |
Seven chimeric biomimetic dye-ligands (BM) are purpose-designed and synthesized by specific structural modification of the parent anthraquinone dichlorotriazine dye Vilmafix blue A-R (VBAR). Each BM dye is composed of two enzyme-recognition moieties. The terminal biomimetic moiety bears a variable carboxylated structure linked to the triazine ring, thus mimicking the substrate of formate dehydrogenase (FDH). The anthraquinone moiety remains the same as that of the parent dye and recognizes the nucleotide-binding area of the target enzyme. Dyes are purified by liquid column chromatography (typically 99%), analyzed by liquid-paper chromatography, thin-layer chromatography, and high-performance liquid chromatography, and their λ(max) and ε values are determined. The ability of dyes to act as affinity ligands versus Candida boidinii FDH is evaluated by kinetic studies and determining K(D) values from both difference spectra and enzyme inactivation studies. The parent dichlorotriazine dye VBAR binds specifically and irreversibly to FDH (k3 0.19 min-1; K(D) 19.3 μM). The inactivation of the NAD+-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH, and ADP. Quantitatively inhibited FDH contained approx 1 mol of dye per mole of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM each of NAD+, NADH, and ADP or by extensive dialysis or gel filtration chromatography. The monochlorotriazine BM dyes do not inactivate FDH but inhibit competitively the inactivation by VBAR. When compared to VBAR and Cibacron blue 3GA (CB3GA), all BM dye-ligands exhibited lower K(D) values. FDH generally preferred binding to BM ligands which bore an aromatic terminal biomimetic moiety substituted with a monocarboxyl group rather than an α-ketoacid. Dye binding to FDH is accompanied by a characteristic spectral change in the range 550-800 nm. This phenomenon is perturbed after titration by increasing amounts of NAD+. Electrostatic interactions appeared to play a dominant role in the dye · FDH complex. The BM dye-ligand bearing a m-aminobenzoate at its terminal biomimetic moiety (BM1) exhibited the highest affinity (K(D) 1.6 μM, 8.0-fold decrease over CB3GA). BM1 differentiated between the binding sites of FDH, displaying uncompetitive inhibition with respect to NAD+ (K(i) 15.6 μM) and competitive with respect to formate (K(i) 18.1 μM). |
en |