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 pigeon-liver nicotinamide-adenine dinucleotide kinase with cibacron blue F3GA.

The interaction of pigeon liver NAD kinase with Cibacron Blue F3GA was investigated. By using steady-state rate measurements, spectrophotometric titration and chromatography of the enzyme on immobilized dye, it was shown that binding occurs at two nucleotide sites with different affinities, and also at a site distinct from the substrate-binding region.     

Interaction of myosin subfragment 1 with Cibacron Blue F3GA

Cibacron Blue F3GA and its immobilized derivatives have been shown before to bind and inhibit nucleotide-dependent enzymes and, among them, myosin subfragment 1. Experiments have been carried out to examine the mechanism of the subfragment 1--dye interaction. Binding of subfragment 1 to immobilized dye (Affi-Gel Blue) does not involve the ATP binding site on myosin. Subfragment 1 hydrolyzes MgATP and CaATP while bound to the Affi-Gel Blue column. Inactivated subfragment 1, which contains [3H]ADP noncovalently trapped at the active site, binds and elutes from the Affi-Gel Blue column in the same manner as unmodified, active protein. Free Cibacron Blue inhibits the ATPase activity of subfragment 1. The inhibition is pH, salt, and time dependent. Complete inhibition correlates with the noncovalent binding of four to five dye molecules per mole of subfragment 1. Three to four of these dye molecules can be preferentially removed from subfragment 1 in the presence of 1 M KCl without relieving the inhibition. This inhibition, which can be traced to one dye molecule per subfragment 1, is reversible and is facilitated in the presence of MgADP and MgATP, suggesting that the dye does not bind at the active site of subfragment 1. Our observations are explained in terms of hydrophobic and electrostatic protein--dye interactions.     

Interactions of contractile proteins with free and immobilized cibacron Blue F3GA.

Differential binding of contractile proteins from skeletal muscle to Cibacron Blue F3GA-Sepharose affinity columns provides the basis for a new purification technique. Myosin subfragments bind at low ionic strength and are eluted by high salt (e.g., 1.5 m NaCl). Myosin light chain 2 also binds at low ionic strength, whereas light chain 1 is only partially retarded and light chain 3 does not bind. Myosin's marginal solubility in the low-salt buffers required for binding renders it unsuitable for Blue Sepharose chromatography. Neither G-actin nor F-actin bind. Crude preparations of myosin subfragment-1 or light chains undergo significant purification upon Blue Sepharose chromatography. Nee free chromophore inhibits the ATPase activities of myosin and actomyosin at micromolar dye concentrations, whereas the binding of subfragment-1 to actin (in myofibrils) and the tension of glycerinated fibers are inhibited at millimolar dye concentrations. The dye binds at multiple sites on myosin, and inhibits its actomyosin ATPase both competitively and uncompetitively.     

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.     

Studies on the kinetic sequence of in vitro ribosome assembly using cibacron blue F3GA as a general assembly inhibitor.

We have found that all E. coli ribosomal proteins strongly bind to an agarose affinity column derivatized with the dye Cibacron Blue F3GA. We have also shown that the capacity to bind the dye is lost when the proteins are organized within the structure of the ribosome or are members of pre-formed protein-RNA complexes. We conclude that the binding of ribosomal proteins to this dye involves specific protein-RNA recognition sites. These observations led us to discover that Cibacron Blue can be used to inhibit in vitro ribosome assembly at any stage of the assembly process. This has allowed us to determine a kinetic order of ribosome assembly.     

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.     

Dye-Ligand Affinity Chromatography: The Interaction of Cibacron Blue F3GA® with Proteins and Enzyme

Abstract

The dye Cibacron Blue F3GA has a high affinity for many proteins and enzymes. It has therefore been attached to various solid supports such as Sephadex, Sepharose, polyacrylamide, and the like. In the immobilized form the dye has rapidly been exploited as an affinity chromatographic medium to separate and purify a variety of proteins including dehydrogenases, kinases, serum albumin, interferons, several plasma proteins, and a host of other proteins. Such a diversity shown by the blue dye in binding several unrelated classes of proteins has generated considerable work in terms of studies of the chromophore itself and also the immobilized ligand. As a prelude to realizing the full potential of the immobilized Cibacron Blue F3GA, an understanding of the basic interactions of the dye with its surroundings must be gained. It has been recognized that the dye is capable of hydrophobic and/or electrostatic interactions at the instance of the ambient conditions. The study of interactions of the dye with salts, solvents, and other small molecules indicates the nature of the interactions of the dye with different kinds of groups at the interacting sites of proteins. The review will cover such interactions of the dye with the proteins, the interactions of the proteins with the immobilized ligand, and the media used to elute the bound protein in several cases, and thus consolidate the available information on such studies into a cogent and comprehensive explanation.     

Kinetic studies on interaction of (Na,K)-ATPase with Cibacron Blue F3GA as probe of the nucleotide fold

Cibacron Blue F3GA (Cb) effectively and reversibly inhibits the activity of (Na,K)-ATPase. Its inhibitory effect does not occur through occupation of the ouabain binding site, but presumably results from Cb-occupation of one catalytic site not competitively attracting ATP. Cb also inhibits ouabain binding to (Na,K)-ATPase. Its inhibitory effect is competitively antagonized by ATP proving accomodation of Cb in the ATP binding site. - If one admits Cb as a suitable analytical tool for the detection of a supersecondary structure folding pattern, the findings suggest that the ATP binding site is lined by β-pleated sheets flanked by α-helices thus providing an environment that funnels ATP to the catalytic site.     

Isoleucyl transfer ribonucleic acid synthetase. Competitive inhibition with respect to transfer ribonucleic acid by blue dextran.

The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.     

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.     

Hazards in the use of Cibacron Blue F3GA in studies of proteins.

Cibacron Blue F3GA from several commercial sources is shown to be heterogeneous. This crude dye inactivates both phosphoglycerate kinase and isoleucyl-tRNA synthetase. Purification of Cibacron Blue F3GA to homogeneity results in a dramatic decrease in inactivation of these enzymes. The inactivation is shown to be due to covalent modification of phosphoglycerate kinase and probably isoleucyl-tRNA synthetase by a minor component present in crude Cibacron Blue F3GA.     

Effect of albumin on binding and recovery of enzymes in affinity chromatography on Cibacron Blue

Bovine serum albumin appears to improve the specificity of Cibacron Blue F3GA in affinity chromatography of enzymes which interact with nucleotides. The action of bovine serum albumin may rest in its ability to selectively mask affinity sites in the dye, which are not specific for the nucleotide-binding region of the enzyme, while not seriously impairing binding nor its elution by nucleotides. Thus, the elution of Chlorella nitrate reductase from a Blue Sepharose chromatographic column by its coenzyme, NADH, fails, unless the column is first treated with bovine serum albumin. Such treatment also improves the recovery of some other nucleotide-binding enzymes tested.     

Analytical evaluation of the purity of commercial preparations of Cibacron Blue F3GA and related dyes

The composition and purity of three commercial preparations of the widely used affinity chromatography ligand Cibacron Blue F3GA have been evaluated by TLC and by paired-ion reversed-phase HPLC and were found to contain several chromophoric species. Stepwise synthesis of the reported dye structure showed that only one commercial preparation contained any actual Cibacron Blue F3GA, and that it was present only in minor amounts. In all three preparations the major component appears to be the dichlorotriazinyl precursor of Cibacron Blue F3GA. Commercial samples of the related dyes Procion Blue MX-3G and Procion Blue MX-R are also highly heterogeneous. In addition, our experiments suggest that TLC results must be evaluated carefully to ensure that catalytic surface activity of alumina and silica has not created ghost bands.     

Spectral changes induced in Cibacron Blue F3GA by salts,organic solvents,and polypeptides: Implications for Blue dye interaction with proteins

The interactions of Cibacron Blue F3GA with organic solvents, salts, oligopeptides, and polypeptides were studied by visible difference spectroscopy. The difference spectrum of the dye in an aqueous solution of NaCl (vs water) has a characteristic positive peak at 690 nm and negative double minima at 630 and 585 nm. Such a “salt-like” spectrum is also obtained for interaction of the dye with polycations such as oligolysines, polylysine, polyarginine, and protamine. In contrast, the difference spectrum of the dye in binary aqueous solvents containing dioxan or t-butyl alcohol at moderately high concentrations, measured against water, displays a positive peak and shoulder at 655 and 610 nm, respectively, with a small negative contribution below 550 nm. This spectrum is attributed to a nonpolar interaction of the dye with organic cosolvent molecules. The spectrum of the dye in 7 M urea is changed little from that in water, indicating similar interactions of the dye with water or urea molecules. The spectral characteristics described here for the interaction of the polyaromatic polysulfonate dye with positively charged groups, polar groups, and nonpolar moieties of neutral molecules provide a basis for describing the details of the interactions of Cibacron Blue F3GA with several proteins and for characterizing the dye binding environments in the proteins.     

On the existence of a nucleotide-binding regulatory site on brain hexokinase.

Binding of ADP to rat brain hexokinase provided protection against inactivation of the enzyme by glutaraldehyde or by chymotryptic digestion. Graphical analysis of the inactivation experiments was, in both cases, consistent with the existence of a single ADP binding site and a K_d ≈ 3mM for the hexokinase-ADP complex. Both Cibacron Blue F3GA and tetraiodofluorescein, previously found to have a general affinity for nucleotide binding sites, were competitive (vs. ATP) inhibitors of the enzyme, suggesting that they bound only to the site occupied by the nucleotide substrate, ATP. While alternate interpretations cannot be excluded, it is felt that these results are most consistent with the view that there is a single nucleotide binding site on the enzyme. They thereby may serve to stimulate a search for alternative explanations for the complex inhibitory pattern of ADP which had previously been attributed to the existence of two ADP binding sites on the enzyme (J. Ning, D.L. Purich, and H.J. Fromm, $\text{J. Biol. Chem. 244}$, 3840–3846 (1969).     

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     

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.     

Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of atleast 2.7 ? for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands.     

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.