The Interaction of Mammalian Interferons with Immobilized Cibacron Blue F3Ga: Modulation of Binding Strength

Mouse, hamster, rabbit, horse, and human interferons bind to immobilized Cibacron Blue F3GA under appropriate solvent conditions. Three forms of the immobilized ligand have been investigated: Cibacron Blue F3GA-Sepharose 4B, Blue Dextran-Sepharose 4B and Blue Sepharose CL-6B. The strength of binding of an interferon depends critically on the sorbent: Cibacron Blue F3GA-Sepharose 4B is the weakest in the series and Blue Sepharose CL-6B the strongest. The use of Blue Dextran-Sepharose 4B - a sorbent of intermediate binding properties - allows the complete separation of hamster, mouse and human fibroblast interferons in a single chromatographic step. Indeed, both the resolution, as well as the recovery, of those interferons is complete - regardless of the relative complexity of the chromatographed preparation (containing either crude or purified interferons). Thus, these ligands should prove of considerable use     

魔芋葡苷聚糖（KGM）凝胶为亲和层析载体与Sepharose4B的比较 …

将ＫＧＭ和Ｓｅｐｈａｒｏｓｅ ４Ｂ凝胶在相同条件下活化、偶联接上染料Ｃｉｂａｃｒｏｎ Ｂｌｕｅ Ｆ３ＧＡ制成了ＫＧＭ和Ｓｅｐｈａｒｏｓｅ ４Ｂ染料亲和吸附剂,并用来与牛血甭白蛋白（ＢＳＡ）作用,每毫升ＫＧＭ亲和吸附剂可吸附ＢＳＡ ２８ｍｇ,用ＮａＳＣＮ洗脱时间为８４．５％,而Ｓｅｐｈａｒｏｓｅ ４Ｂ染料样和吸附剂每毫升可吸附ＢＳＡ １５．３ｍｇ,用ＮａＳＣＮ洗脱时间收迷８１．７％,并对两种凝胶的染     

Comparative studies of the binding of some ligands to human serum albumin non-covalently attached to immobilized Cibacron Blue, or covalently immobilized on Sepharose, by column affinity chromatography.

A comparative study of the ligand-binding properties of human serum albumin was performed by the technique of affinity chromatography with the protein attached to immobilized Cibacron Blue F3GA (Blue Sepharose), or covalently immobilized on Sepharose. The binding strength of octanoate, decanoate and dodecanoate is much weaker when human serum albumin is attached to immobilized Cibacron Blue, indicating that the binding sites for fatty acids are involved in the attachment of human serum albumin to immobilized Cibacron Blue. The results revealed additional alterations of the ligand binding when human serum albumin was attached to immobilized Cibacron Blue, involving sites outside of the binding domains of fatty acids. Thus the stereoselective binding of L-tryptophan was abolished, and the resolution of the warfarin enantiomers was impaired. However, the binding strength of warfarin and salicylic acid was rather close to the values observed with human serum albumin covalently immobilized on Sepharose. It is suggested that the availability of the binding sites for L-tryptophan, warfarin and salicylic acid is partially blocked by the complex between albumin and the dye without direct participation in the complex-formation. An alternative interpretation involves an allosteric mechanism brought about by complex-formation between serum albumin and the immobilized Cibacron Blue.     

Binding of Cibacron Blue F3GA to flavocytochrome b2 from baker's yeast.

1. Flavin-free cytochrome b2 has been prepared by rapid Sephadex filtration at acid pH. The method, which yields an apo-enzyme with high reconstitution potential and has several advantages over previously used procedures, is described in detail. 2. Flavin-free cytochrome b2 thus prepared is retained by blue-dextran-bound Sepharose. It can be eluted by an increase in ionic strength, by dilute ethylene glycol and specifically by low concentrations of FMN. The holoenzyme is not retarded at all. 3. Both flavin-free and holocytochrome b2 bind Cibacron blue F3GA with appearance of distinct difference spectra. Cibacron blue is an inhibitor for the holoenzyme, it shows mixed type inhibition with respect to lactate. 4. It is concluded that there are two types of binding sites for Cibacron blue F3GA on flavocytochrome b2. Both possess ionic and hydrophobic character; one of them, which is the flavin binding site, is only available in the absence of the cofactor. Taken together these results may mean that the enzyme possesses a local flavin-binding structure similar to the 'dinucleotide fold'.     

Purification of immunotoxins containing ricin A-chain and abrin A-chain using Blue Sepharose CL-6B

We describe a method for separating antibody from immunotoxins by affinity chromatography on Cibacron blue F3GA coupled to Sepharose (Blue Sepharose). The antibody did not bind to the gel. The immunotoxins were bound by their ricin A-chain or abrin A-chain moiety and could be recovered in high yield and purity using mild elution conditions. The method is suitable for the large-scale purification of immunotoxins.     

Cadmium removal from human plasma by Cibacron Blue F3GA and thionein incorporated into polymeric microspheres

Poly(2-hydroxyethylmethacrylate–ethyleneglycoldimethacrylate) [poly(HEMA–EGDMA)] microspheres carrying Cibacron Blue F3GA and/or thionein were prepared and used for the removal of cadmium ions Cd(II) from human plasma. The poly(HEMA–EGDMA) microspheres, in the size range of 150–200 μm in diameter, were produced by a modified suspension copolymerization of HEMA and EGDMA. The reactive triazinyl dye-ligand Cibacron Blue F3GA was then covalently incorporated into the microspheres. The maximum dye incorporation was 16.5 μmol/g. Then, thionein was bound onto the Cibacron Blue F3GA-incorporated microspheres under different conditions. The maximum amount of thionein bound was 14.3 mg/g. The maximum amounts of Cd(II) ions removed from human plasma by poly(HEMA–EGDMA)–Cibacron Blue F3GA and poly(HEMA–EGDMA)–Cibacron Blue F3GA–thionein were of 17.5 mg/g and 38.0 mg/g, respectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of microspheres without significant loss in their adsorption capacity.     

Use of Cibacron blue F 3GA-CL-sepharose 6B for the purification of T4 DNA- and RNA-ligases

The sorption capacity of the dye cibacron blue F3GA, immobilized on CL-Sepharose 6B and other support matrices, in respect to DNA- and RNA-ligases T4 was being studies. Cibacron blue F3GA immobilized on CL-Sepharose 6B binds a three-fold amount of DNA-ligase in comparison to RNA-ligase. The enzyme chromatography on cibbacron blue F3GA-CL-Sepharose 6B revealed a stronger linkage between DNA-ligase T4 and the sorbent than between RNA-ligase T4 and the sorbent. Elution was performed with potassium chloride. DNA-ligase T4 was eluted with 0.25-0.5 M KCl and RNA-ligase T4 with 0.08-0.18 M KCl. Since deoxyexonuclease contaminants possess stronger bonds with the sorbent than ligases, elution of deoxyexonucleases occurs at higher concentrations of KCl. Chromatography of enzymes on cibacron blue F3GA-CL-Sepharose 6B allows one to obtain DNA- and RNA-ligases essentially free of DNase and RNase contaminants.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.     

Magnetic dye-affinity beads for human serum albumin purification

Cibacron Blue F3GA was covalently attached onto magnetic poly(vinyl alcohol) (mPVAL) beads (100-150 μm in diameter) for human serum albumin (HSA) adsorption from human plasma. Despite low nonspecific adsorption of HSA on mPVAL beads, Cibacron Blue F3GA attachment significantly increased the HSA adsorption. The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma. Desorption of HSA from mPVAL beads was achieved by medium containing 1.0 M KSCN at pH 8.0. To test the efficiency of albumin adsorption from human serum, before and after albumin adsorption was demonstrated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses. HSA molecules could be reversibly adsorbed and desorbed 10 times with the magnetic beads without noticeable loss in their HSA adsorption capacity.     

Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A.

The interaction of Cibacron Blue F3G A-Sepharose 4B with several serum albumins was studied. Although all albumins used were fond to bind to this adsorbent, human serum albumin was bound to a far greater extent than were the others. From the results of competition experiments and n.m.r. studies of Cibacron Blue and/or bilirubin binding to human serum albumin it is proposed that the mechanism of the interaction between human serum albumin and cibacron Blue is consistent wit Cibacron Blue binding to bilirubin-binding sites. In contrast with these findings with human serum albumin, there is little or no interaction of Cibacron Blue and the bilirubin-binding sites of albumins from rabbit, horse, bovine or sheep sera, although some interaction occurs between Cibacron Blue and the fatty acid-binding sites of these proteins. Structural analogues of Cibacron Blue have been used to investigate the binding of albumins to these ligands.     

Effect of Cibacron Blue F3GA on phosphoglycerate kinase of Lactobacillus plantarum and phosphoglycerate mutase of Leuconostoc dextranicum

Blue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2.7.2.3] competitively with respect to ATP (Thompson et al. (1975) Proc. Natl. Acad. Sci. U.S. 72, 663--667; Beissner and Rudolph (1979) J. Biol. Chem. 254, 6273--6277). However, we have found that phosphoglycerate kinase of Lactobacillus plantarum was inhibited by Cibacron Blue F3GA, the blue chromophore of blue dextran, noncompetitively with respect to ATP, but competitively with respect to 3-phosphoglycerate. Further inhibition studies with Cibacron Blue F3GA suggest that one molecule of the dye was bound per molecule of phosphoglycerate kinase at a saturated level of either substrate, but two molecules of the dye were bound per molecule of the kinase with an unsaturated level of either substrate used as a fixed substrate. Furthermore, phosphoglycerate mutase [EC 2.7.5.3] of Leuconostoc dextranicum was also inhibited by Cibacron Blue F3GA competitively with respect to 3-phosphoglycerate and noncompetitively with respect to 2,3-bisphosphoglycerate. These results suggest that the 3-phosphoglycerate-binding site on both phosphoglycerate kinase and phosphoglycerate mutase can interact with Cibacron Blue F3GA.     

Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.     

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.     

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.     

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.     

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.     

Development of the magnetic beads for dye ligand affinity chromatography and application to magnetically stabilized fluidized bed system

Magnetic poly(2-hydroxyethylmethacrylate) [mPHEMA] beads were prepared by suspension polymerization of HEMA in the presence of Fe₃O₄ nano-powder. Cibacron Blue F3GA was covalently immobilized to the mPHEMA beads via nucleophilic substitution reaction between chloride of its triazine ring and hydroxyl groups of HEMA under alkaline conditions. The mPHEMA/Cibacron Blue F3GA beads (100–140 μm in diameter) carrying 68.3 μmol Cibacron Blue F3GA per gram polymer were used for β-casein adsorption studies. Adsorption studies were performed under different conditions in a batch system (i.e., pH, β-casein initial concentration, temperature, and ionic strength) and then in a magnetically stabilized fluidized bed (MSFB) system. The swelling ratio of the mPHEMA was 62.1%. The maximum adsorption capacity for batch system was 20.2% lower as compared to the value obtained in MSFB. The mPHEMA/Cibacron Blue F3GA beads could be repeatedly applied for β-casein adsorption without significant losses in the adsorption capacity.     

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.     

Separation of polypeptide chains of ricin and the interaction of the A chain with Cibacron Blue F3GA

From ricin bound to the galactomannan guar gum in a column, the nonbinding toxic A chain could be eluted by reduction with 2-mercaptoethanol and later the B chain by lactose. The presence of a nucleotide-binding domain on the toxic chain A could be demonstrated from its interaction with the blue dye Cibacron Blue F3GA.     

Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A

1. Phosphofructokinase from baker's yeast is partitioned between the phases of an aqueous two-phase system, containing dextran (Mr = 500000) and poly(ethyleneglycol) (Mr = 6000), in favour of the dextran-rich phase. By covalent binding of the dye Cibacron blue F3G-A to poly(ethyleneglycol) the enzyme can be extracted to the phase rich in this polymer, i.e. affinity partitioning. 2. The affinity partitioning effect, measured as the logarithmic increase of the partition coefficient by introducing polymer-bound Cibacron blue, depends on several factors. The influence of dye-polymer concentration, polymer concentration, polymer molecular weight, kind of salt and salt concentration, pH and temperature has been studied. 3. The effect of ATP, ADP, AMP, ITP, fructose 1,6-bis-phosphate and fructose 6-phosphate show large differences in the binding strength of these substances to the Cibacron blue binding sites. AMP cannot compete with Cibacron blue while ATP is strongly competing. 4. The use of affinity partitioning for enzyme isolation and determination of ligand binding is discussed, as well as possible mechanisms concerning this type of liquid/liquid extraction.