Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 μM. Kinetic analyses revealed that at a concentration below 0.5 μM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (K_a) and stoichiometry (r) for the enzyme-dye complex.     

Studies of the structure--function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran--sepharose and Cibacron blue 3G-A

Blue dextran--Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kiase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental additon of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvte kinase--Cibacron blue complex.     

Human serum albumin chromatography by Cibacron Blue F3GA-derived microporous polyamide hollow-fiber affinity membranes

An affinity dye ligand, Cibacron Blue F3GA was covalently attached onto commercially available microporous polyamide hollow-fibre membranes for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. Different amounts of Cibacron Blue F3GA were incorporated on the polyamide hollow-fibres by changing the dye attachment conditions, i.e. initial dye concentration, addition of sodium carbonate and sodium chloride. The maximum amount of Cibacron Blue F3GA attachment was obtained at 42.5 μmol g⁻¹ when the hollow-fibres were treated with 3 M HCl for 30 min before performing the dye attachment. HSA adsorption onto unmodified and Cibacron Blue F3GA-derived polyamide hollow-fibre membranes was investigated batchwise. The non-specific adsorption of HSA was very low (6.0 mg g⁻¹ hollow-fibre). Cibacron Blue F3GA attachment onto the hollow-fibres significantly increased the HSA adsorption (147 mg g⁻¹ hollow-fibre). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (230 mg HSA g⁻¹ hollow-fibre). Desorption of HSA from Cibacron Blue F3GA derived hollow-fibres was obtained using 0.1 M Tris–HCl buffer containing 0.5 M NaSCN or 1.0 M NaCl. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cibacron Blue F3GA derived polyamide hollow-fibre without significant decreases in the adsorption capacities.     

Dinucleotide fold proteins. Interaction of arabinose binding protein with Cibacron Blue 3G-A.

Cibacron Blue 3G-A, the dye moiety of Blue dextran-Sepharose, has been shown to not specifically bind a protein with a dinucleotide fold-like supersecondary structure, L-arabinose binding protein from Escherichia coli. This shows that Cibacron Blue 3G-A is not suitable as a definitive probe for the dinucleotide fold as suggested earlier (Thompson et al., 1975; Stellwagen, 1977). An explanation for the large predominance of proteins containing this protein supersecondary structure that bind to this dye is presented.     

Metal-ion-promoted binding of triazine dyes to proteins. The interaction of Cibacron Blue F3G-A with yeast hexokinase.

Bivalent metal ions, particularly Zn2+ and other members of the first-row transition series, promote irreversible inactivation of yeast hexokinase by Cibacron Blue F3G-A at a site competitive with both ATP and D-glucose. Difference spectroscopy indicates that the protein-dye dissociation constant is decreased from 250 micrometers in the absence of metal ions to less than 100 micrometers in the presence of appropriate concentrations of metal ions, with specificity displayed in the sequence of Zn2+ greater than Cu2+ greater than Ni2+ greater than Mn2+. Quantitative inactivation of yeast hexokinase leads to the incorporation of approx. 1 mol of Cibacron Blue F3G-A/mol of subunit of mol. wt. 51 000 in both the presence and the absence of metal ion. These results suggest the formation of a highly specific ternary complex involving enzyme, dye and metal ion at the active-site region of the enzyme, and correlate well with the known effects of metal ions in promoting the binding of hexokinase to immobilized Cibacron Blue F3G-A.     

Specific binding of Cibacron blue F3G-A to yeast phosphofructokinase.

The complex formation between the ATP-analogous reactive dye Cibacron blue F3G-A and yeast phosphofructokinase is accompanied by a red shift of the visible absorption spectrum. From the position of lambdamax of the dye-phosphofructokinase complex in the polarity scales obtained from model solvents it may be concluded that the chromophoric system is evidently located in a highly apolaric range of the enzyme protein. The spectrophotometric titration of yeast phosphofructokinase with Cibacron blue F3G-A yielded a sigmoidal binding curve, which can be described by the MONOD-WYMAN-CHANGEUX model.     

Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A.

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'.     

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.     

Active-site-directed inhibition of sucrose-phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin

Sucrose-phosphate synthase (SPS, E.C. 2.4.1.14) from spinach (Spinacia oleracea L.) was partially purified and the inhibition of the enzyme reaction by 1-deoxynojirimycin and Cibacron blue F3G-A analyzed. Cibacron blue was a high-affinity competitive inhibitor with respect to the substrate UDPglucose (K_i = 80 nM) and a mixed-type inhibitor with respect to fructose-6-phosphate. 1-Deoxynojirimycin was a mixed-type inhibitor of SPS with respect to UDPglucose [K_i(EI) = 5.8 mM] and a uncompetitive inhibitor with respect to fructose 6-phosphate. These results are discussed in relation to the mechanism of the reaction catalysed by SPS and the secondary structure of the enzyme.Abbreviations DN 1-deoxynojirimycin - Glc6P glucose-6-phosphate - Fru6P fructose-6-phosphate - SPS sucrose-phosphate synthase - UDPG1c UDPglucose We are grateful to M. Stitt (University of Heidelberg, Germany) for many helpful discussions and J. Harr and P. Bocion (both SANDOZ AGRO, Switzerland) for supporting the work.     

Kinetic investigation of the interaction of cibacron blue F3G-A with phosphofructokinase from yeast.

Yeast phosphofructokinase is strongly inhibited by Cibacron Blue F3G-A. The inhibition is competitive in respect to the phosphate donor. Fructose 6-phosphate and ATP are able to abolish the dye-inhibition. Replacement of the strong inhibitor ATP by ITP as phosphate donor gives qualitatively analogous effects. The influence of Cibacron Blue F3G-A on the kinetic pattern of yeast phosphofructokinase can be described in terms of the kinetic model of Freyer et al. [8] if one assumes that the dye binds to the ATP-binding sites in a competitive manner.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized <Emphasis Type="Italic">Coprinus cinereus</Emphasis> in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g⁻¹ h⁻¹ with a 50% conversion time (t _1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.     

7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching,suggesting binding at multiple protein sites

Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern–Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K_d 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K_d 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K_d 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K_d 0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.     

Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.

Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl (DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg²⁺. Kinetic data for chorismate in the forward reaction (K _m = 807 μM, V _max = 6.2 pkat · mg⁻¹) and for isochorismate in the reverse reaction (K _m = 675 μM, V _max = 5.9 pkat · mg⁻¹) were determined. Received: 18 November 1996 / Accepted: 28 December 1996     

Preparation of Homogeneous Alkaline Phosphatase 211011 Galf Intestine by Dye-Ligand Chromatography

The paper deals with a simple and effective procedure for the isolation of calf intestinal alkaline phosphatase (EC 3.1.3.1) with a yield of 35 per cent by employing immobilized Procion Red HD-33 and Cibacron Blue F3G-A, respectively, as dye-ligands. The resulting enzyme is homogeneous and has a specific activity of about 2500 units per mg of protein. Because dye liganded gels are of low costs and can be used several times without loss of binding properties, the presented method is in particular suited for large scale application.     

Affinity chromatography of serine proteases on the triazine dye ligand Cibacron Blue F3G-A

The interaction between complement component factor B and the triazine dye ligand Cibacron Blue F3G-A coupled to a cross-linked agarose matrix (Blue Sepharose) was found to involve the Bb part of the molecule, and to be inhibited by benzamidine. Human, chicken and rainbow trout factor B which had bound to Blue Sepharose could subsequently be eluted with benzamidine. Other serine proteases (C2, factor II, factor IX, trypsin, chymotrypsin, proteinase 3) also bound to Blue Sepharose but only those belonging to the trypsin family could be eluted with benzamidine. Trypsin treated with the active-site inhibitor phenylmethylsulfonyl fluoride did not bind to Blue Sepharose and pretreatment of Blue Sepharose with benzamidine did not influence binding of proteases. We conclude that trypsin-like serine proteases can be purified on Blue Sepharose and that the interaction of these serine proteases with Blue Sepharose involves the active site of the enzyme.     

Preparation and characterisation of Cibacron Blue F3G-A poly(ethylene) hollow-fibre affinity membranes

Poly(ethylene) hollow-fibre membranes with immobilised Cibacron Blue F3G-A were obtained in four different ways from epoxy-activated fibres. Membranes with a maximum capacity of 26 mg lysozyme ml^–1and a dye density of 52 mol ml^–1were obtained when ammonia was used to open the epoxy group before dye immobilisation. Pure water flux of the modified membranes at 1 bar pressure was 1.0 cm min^–1, thus meaning only a reduction of 1.5-fold with regard to the unmodified membranes. The support-dye bond was stable as judged by the unmodified capacity of the membranes and the negligible amount of dye leaked after 520 h of exposure to 6 M urea in 0.5 M NaOH.     

Interaction of cibacron blue 3G-A and related dyes with nucleotide-requiring enzymes

Cibacron Blue 3G-A (I), the chromophore in Blue Dextran, its structural isomer Cibacron Brilliant Blue BR-P (II), and two other structural analogs (III, IV) were used to probe the nucleotide binding sites of selected kinases and dehydrogenases. Inhibition studies indicate that the portion of the dye molecule necessary for effective inhibition of nucleotide binding is a structure similar to 1-amino-4(4′-aminophenylamino)-anthraquinone-2,3′-disulfonic acid (ASSO; III). The strong inhibition exhibited by these dyes is likely to be due to interaction with specific nucleotide binding sites, irrespective of the presence of a “dinucleotide fold” in the proteins' supersecondary structure.     

Effect of anthraquinone dyes and evaluation of the kinetic mechanism of yeast phosphoglycerate kinase.

Cibacron Blue 3G-A (CB3G-A, I) was investigated as a ternary complex analogue of lactate dehydrogenase and phosphoglycerate kinase as had been suggested earlier (Stellwagen, E. (1977) Accts. Chem. Res. 10, 92-98). CB3G-A and Procion Brilliant Blue (PBB, II), a structural isomer of the Cibacron dye without the sulfonated benzoyl moiety, were attached covalently to Sepharose CL-6B. The two enzymes were adsorbed to the columns and then eluted by substrates in various combinations. CB3G-A and PBB interact similarly with the two enzymes in spite of the structural differences between the dyes indicating that the specific structure of CB3G-A is not a ternary complex analogue. Inhibition studies of phosphoglycerate kinase by CB3G-A suggest that 2 molecules of dye bind per monomer and are consistent with multiple substrate binding sites. It is suggested that the kinetic mechanism of phosphoglycerate kinase is best described as steady state random.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

Dye-incorporated poly(EGDMA-HEMA) microspheres as specific sorbents for aluminum removal

Aluminum [Al(III)] adsorption onto dye-incorporated poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microspheres was investigated. Poly(EGDMA-HEMA) microspheres, in the size range of 150–200 μm, were produced by a modified suspension polymerization of EGDMA and HEMA. The reactive dyes (i.e., Congo Red, Cibacron Blue F3GA and Alkali Blue 6B) were covalently incorporated to the microspheres. The maximum dye load was 14.5 μmol Congo Red/g, 16.5 μmol Cibacron Blue F3GA/g and 23.7 μmol Alkali Blue 6B/g polymer. The maximum Al(III) adsorption on the dye microspheres from aqueous solutions containing different amounts of Al(III) ions were 27.9 mg/g, 17.3 mg/g and 12.2 mg/g polymer for the Congo Red, Cibacron Blue F3GA and Alkali Blue 6B, respectively. The maximum Al(III) adsorption was observed at pH 7.0 in all cases. Non-specific Al(III) adsorption was about 0.84 mg/g polymer under the same conditions. High desorption ratios (95%) were achieved in all cases by using 0.1 M HNO₃. It was possible to reuse these dye-incorporated poly(EGDMA-HEMA) microspheres without significant losses in the Al(III) adsorption capacities.