Red HE-3B Research Articles

Treatability of textile dye-bath effluents by advanced oxidation: preparation for reuse

Treatability of textile dye-bath effluents by advanced oxidation with Fenton and Fenton-like reagents (FeII/H2O2 and FeIII/H2O2), in the presence and absence of UV light was investigated, using a reactive azo-dye (Procion Red HE7B), and typical dye bath constituents. Under the experimental conditions employed, it was found that with 20 min UV irradiation, complete color removal and 79% total organic carbon degradation is possible, when the system is operated at pH=3, and with a H2O2/Fe(II) molar ratio of 20:1. The increased dissolved solids content of the treated solution implies the necessity of an appropriate membrane system to make the effluent reusable in the dye/wash processes.

Direct lysozyme separation from egg white by dye membrane affinity chromatography

An affinity membrane from hydrophylised polyethylene hollow fibre as the support matrix was prepared. Red HE-3B was immobilised on the membrane and the adsorption behaviour of pure lysozyme solutions and homogenised egg white was investigated. Dye density (1.7 μmol ml−1) and maximum binding capacity (26 mg lysozyme ml−1) are comparable to those of commercial gel matrices. Dynamic binding capacity did not change when residence time was reduced from 3 to 1 min. A method for direct lysozyme separation from egg white was developed, with a productivity of 12 kg lysozyme m−3 h−1. The purity of the eluted lysozyme, as determined by HPLC, was 88%, with a recovery of 92%. Dynamic capacity was kept constant at 70% of the maximum binding capacity for at least 10 cycles through membrane regeneration with 0.1 M NaOH and 1 M NaCl. Functional properties of egg white, as judged by viscosity and foaming capacity measurements, did not change after the chromatographic lysozyme depletion. © 1999 Society of Chemical Industry

Treatability of textile dye-bath effluents by advanced oxidation: preparation for reuse

Treatability of textile dye-bath effluents by advanced oxidation with Fenton and Fenton-like reagents (FeII/H 2 O 2 and FeIII/H 2 O 2 ), in the presence and absence of UV light was investigated, using a reactive azo-dye (Procion Red HE7B), and typical dye bath constituents. Under the experimental conditions employed, it was found that with 20 min UV irradiation, complete color removal and 79% total organic carbon degradation is possible, when the system is operated at pH=3, and with a H 2 O 2 /Fe(II) molar ratio of 20:1. The increased dissolved solids content of the treated solution implies the necessity of an appropriate membrane system to make the effluent reusable in the dye/wash processes.

Pyrolysis of Tire Rubber: Porosity and Adsorption Characteristics of the Pyrolytic Chars

Tire rubber has been pyrolyzed at various temperatures under a nitrogen atmosphere. The resulting chars have been analyzed for their porosity using nitrogen gas adsorption and for their aqueous adsorption characteristics using phenol, methylene blue, and the reactive dyes Procion Turquoise H-A and Procion Red H-E3B. Nitrogen adsorption isotherms were modeled to the BET and Dubinin−Astakhov (DA) equations to determine effective surface areas, mesopore volumes, and micropore volumes. Results showed that pyrolysis of tire rubber was essentially complete at 500 °C and resulted in a char yield of approximately 42 wt %. Pyrolytic chars exhibited BET surface areas up to 85 m2/g and micropore volumes up to 0.04 mL/g. Owing to their poorly developed micropore structure, the pyrolytic chars exhibited limited aqueous adsorption capacity for compounds of small molecular weight, such as phenol. However, the chars possessed significantly greater adsorption capacity for species of large molecular weight which was attrib...

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.

Direct purification of lysozyme using continuous counter-current expanded bed adsorption

We describe the application of a novel technique for the continuous counter-current chromatography of proteins. The unit operation has shown potential in extracting targeted species from unclarified feedstocks, delivering clarified streams of purified product. The adsorbent used in this equipment consists of a perfluorocarbon matrix, coated with poly(vinyl alcohol), and derivatized with the triazine dye Procion Red HE-7B. Purification of lysozyme from egg-whites and enriched bovine milk could be carried out continuously. The former was extracted in 90.5% yield at a rate of 7400 U/min, achieving a purification factor of 19.4. Lysozyme from the enriched milk sample was extracted continuously at a rate of 41 000 U/min, in 66.0% yield. The continuous product streams in both cases were fully clarified, thus enabling their direct application to a final polishing step, if desired.

Utilization of the Donnan effect for improving electrolyte separation with nanofiltration membranes

The possibility of utilizing the Donnan effect to enhance NaCl removal from a solution of a water-soluble organic dye (Procion Red H-E7B, ICI) using a nanofiltration membrane was examined. The increased salt removal is obtained by the addition of an ionized polyelectrolyte (Na salt of polyacrylic acid, PNa) having a molecular weight of 60 000 dalton. Salt rejection data and permeate flux were measured in three solution system: NaClH 2O, NaClH 2O-dye and NaClH 2O-dye-PNa. The solutions were fed through a parallel plate osmotic cell having an area of 200 cm 2, fitted with an MPF-44 nanofiltration membrane. Polyelectrolyte addition was found to provide a substantial enhancement of salt removal. For example, chloride rejection in a binary system with a salt weight concentration of 1% was around 50% (i.e. permeate salt concentration is about half that of the feed). Addition of 4.4% PNa decreased the salt rejection to a negative value of −68%, signifying that permeate salt concentration was almost 1.7 times higher than the feed concentration. However, polyelectrolyte addition reduced permeate flux and induced a flux limiting phenomenon, similar to that commonly encountered in ultrafiltration. A simple two parameter model is presented which successfully correlates the salt rejection data measured in this work, as well as results reported in the literature. The model enables prediction of salt rejection in multi-ionic systems from data measured easily in a single salt system and can be usefully applied for quantifying NF processes utilizing the Donnan effect.

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye–ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing.

Dye-ligand affinity chromatography on continuous beds.

Continuous beds derivatized with three triazine dyes (Cibacron Blue 3G-A, Pricon Red HE-3B and Pricon Red H-3B) were used for chromatographic purification of dehydrogenases from yeast enzyme concentrate. All three columns, which were prepared by a very simple and cost-effective method, provided strong binding of glucose-6-phosphate dehydrogenase and lactate dehydrogenase. The Cibacron Blue 3G-A column showed high affinity for alcohol dehydrogenase. Under the same conditions, the Pricon Red HE-3B column showed a lower affinity and the Pricon Red H-3B column showed none. The adsorbed dehydrogenases were eluted specifically from the columns in high yields (71-113% by desorption with the coenzymes NADP, NADH and NAD respectively). Non-specific binding of human serum albumin and transferrin to these columns was also investigated. Enzyme assays and analyses by capillary electrophoresis showed that the continuous beds derivatized with triazine dyes gave a high degree of purification.

Toxicity studies on native Procion Red HE-3B and released dye from affinity material exposed to degradative chemical conditions

Leached ligands from chromatographic packing material submitted to drastic regeneration conditions can contaminate pure biological preparations. These contaminants could have adverse effects from a toxicology point of view that are very poorly documented in liquid chromatography for protein separation. Investigations on toxicity level have been made on released material from immobilized Procion Red HE-3B, after formal identification of the nature of the leached chemical material. Toxicity investigations in vitro involved a number of tests on living cells (eucaryotic and procaryotic) covering different aspects. Behaviour of cells in regular cultures, polyploïdia induction, genotoxicity as well as mechanisms of endocytosis have been studied. Results showed no toxic effects within the range of concentration of dye and dye derivatives studied. Genotoxicity studies in particular did not show any toxic effect over a range of concentration much higher than the regular level of dye leakage from the sorbent.

Purification of recombinant ricin A chain with immobilised triazine dyes

Immunotoxins, such as those based on ricin A chain, must be rigorously purified before they can be administered in vivo. The work described in this paper investigates the interaction between recombinant ricin A chain and several triazine dyes and other ligands that may be of value in its purification. All ligands displayed a high affinity (dissociation constants 32–20 μ M) and are displaced from their binding sites on the protein by polynucleotides, heparin and synthetic polyphosphates, but not by mono- or dinucleotides. Affinity chromatography on the immobilised dyes, Procion Red H-3B, Procion Red HE-3B, Procion Red HE-7B and Procion Yellow HE-4R, resulted in a one-step purification of recombinant ricin A chain from an Escherichia coli fermentation extract to 94–98% purity and with a > 95% yield. These materials are far superior to purification on the conventional dye, Cibacron Blue F-3GA, and show promise for the isolation of immunotoxins from immunoconjugation mixtures.

Purification of Human Cholesteryl Ester Transfer Protein by Affinity Chromatography on Immobilized Triazine Dyes

Human cholesteryl ester transfer protein was purified from lipoprotein-depleted serum or plasma in a three-step procedure utilizing commercially available triazine dyes immobilized on agarose. The method used consisted of successive chromatography steps on Reactive Red 120 agarose (Procion Red H-E3B, Cibachron Brilliant Red 4G-E), CM Sepharose, and Reactive Yellow 86 agarose (Procion Yellow M-8G). Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resultant protein preparation displayed two bands of variable intensity. The two components had apparent molecular weights of approximately 64,000 and approximately 65,000, respectively. Both bands reacted strongly to a monoclonal antibody directed against an epitope consisting of the last eight amino acids at the carboxy terminus of human CETP. Yields of cholesteryl ester transfer activity are 10-40% of the activity present in lipoprotein-depleted serum. The activity is approximately 50,000- to 100,000-fold purified relative to the starting material.

Novel affinity separations based on perfluorocarbon emulsions: Use of a perfluorocarbon affinity emulsion for the direct extraction of glucose-6-phosphate dehydrogenase from homogenised bakers' yeast

A perfluorocarbon affinity emulsion has been generated by homogenisation of perfluorodecalin with poly(vinylalcohol) and with subsequent cross-linking and derivatisation with Procion Red H-E7B. This affinity emulsion was tested for its applicability in direct extraction. The affinity emulsion exhibited limited fouling when contacted with a crude homogenate of bakers' yeast ( Saccharomyces cerevisiae) and could be washed clear from cell debris using an aqueous buffer. Adsorption isotherm experiments showed that the capacity of the affinity emulsion for glucose-6-phosphate dehydrogenase (G6PDH) was not severely affected by the presence of whole yeast cell and cell debris. Two different contacting techniques were examined for G6PDH purification directly from a yeast homogenate. The first technique, expanded bed affinity chromatography (EBAC), which is an essentially batch operation, was carried out under conditions optimised through frontal analysis and was compared with a second technique, PERCAS (perfluorocarbon emulsion reactor for continuous affinity separations). G6PDH could be successfully purified using both techniques with an average purification factor of 18 directly from a crude homogenate. An analysis of system productivity showed that PERCAS had a productivity of some 2.25 times higher than expanded bed affinity chromatography under similar process conditions.

NADP+-dependent glutamate dehydrogenase from the obligate methylotrophmethylobacillus flagellatum

NADP-dependent glutamate dehydrogenase (GDH; E.C.1.4.1.4) was purified from an obligate methylotroph Methylobacillus flagellatum using ammonium sulphate precipitation, DEAE-Sepharose and dye-ligand Procion red HE3B column chromatography and Sephacryl S-200 gel-filtration. The Mr of the native enzyme was estimated to be 300 000 (±5000). The enzyme consists of six identical subunits with an Mr of 47 000 (±3000) (SDS-PAGE). The enzyme has a pH optimum of 8.0 when participating in amination and 9.5 in deamination. Michaelis-Menten kinetics were observed for both reactions. The apparent Km values were 1.33 mM, 0.032 mM, 11.5 mM, 7.0 mM and 0.014 mM for α-ketoglutarate, NADPH, NH4+, glutamate and NADP+, respectively. The enzyme was highly specific for all the substrates and was insensitive to inhibitors. It plays an exclusively anabolic role in the cells.

NADP +-dependent glutamate dehydrogenase from the obligate methylotroph methylobacillus flagellatum

NADP-dependent glutamate dehydrogenase (GDH; E.C.1.4.1.4) was purified from an obligate methylotroph Methylobacillus flagellatum using ammonium sulphate precipitation, DEAE-Sepharose and dye-ligand Procion red HE3B column chromatography and Sephacryl S-200 gel-filtration. The M r of the native enzyme was estimated to be 300 000 (±5000). The enzyme consists of six identical subunits with an M r of 47 000 (±3000) (SDS-PAGE). The enzyme has a pH optimum of 8.0 when participating in amination and 9.5 in deamination. Michaelis-Menten kinetics were observed for both reactions. The apparent K m values were 1.33 mM, 0.032 mM, 11.5 mM, 7.0 mM and 0.014 mM for α-ketoglutarate, NADPH, NH 4 +, glutamate and NADP +, respectively. The enzyme was highly specific for all the substrates and was insensitive to inhibitors. It plays an exclusively anabolic role in the cells.

Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification

Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions.

Inactivation of wheat-germ aspartate transcarbamoylase by the triazinyl dye, Procion Red HE3B

Aspartate transcarbamoylase from wheat germ is irreversibly inactivated by the triazinyl dye Procion Red HE3B. Since triazinyl dyes may mimic nucleotides, and UMP is a known allosteric modifier of this enzyme, the reaction was studied to elucidate whether the dye is an ‘affinity label’ for the enzyme. The reaction is apparently first order in the first 5–10 min, but is more complex in the longer term and does not go to completion. Kinetic analysis of the initial phase suggests that there are two parallel reactions, one saturable (dye binds reversibly before reaction) and one non-saturable (bimolecular). The apparent rate constant Kapp (i.e. the sum of the rate constants for the parallel reactions) varies only slightly over the pH range 7–10. In the presence of a number of active centre ligands, as well as the allosteric ligand UMP, there is a clear increase in Kapp. This finding is contrary to the reduction in rate of inactivation (protection) normally provided by ligands against active-site directed reagents, suggesting that in the saturable reaction, there is a conformational change upon dye-binding that increases the exposure of the essential residue(s) with which the dye reacts. These results show that, although it probably inactivates by reaction with specific amino-acid residues, the dye is not bound at the substrate-binding or allosteric sites, i.e. it is not an affinity-labelling reagent in the usual sense.

Immunochemical quantification of Procion Red HE-3B used as ligand in affinity chromatography

The quantification of Procion Red HE-3B used as a ligand in affinity chromatography for proteins is reported. It's based on an enzyme-linked immunosorbent assay using antibodies against the dye. Polyclonal antibodies were classically prepared for conjugation of the dye on KLH and injection into rabbits. The development of the assay was based on the competitive inhibition between hemoglobin-dye complex and free dye. The sensitivity of this method was about 1000-times higher than a classical spectrophotometric assay, and was modulated by some chemical substituents attached on the native dye. It was demonstrated that the assay was applicable to the determination of dye traces that may be released from dye affinity sorbents. Moreover, the quantification of the dye was successfully applied to proteins that are being purified from a dye affinity column.

Investigation of affinity partition chromatography using formate dehydrogenase as a model

The enzyme formate dehydrogenase (FDH) was purified from the crude extract of Candida boidinii by affinity partition chromatography. The partition coefficient, K, of the enzyme was selectively increased by adding polyethylene glycol—Procion Red HE3b as an affinity ligand to the mobile phase in the chromatographic column. The increased K value led to early elution of the enzyme—ligand complex and separated the target protein from the main peak of the contaminants.

P-319 - Ca2+-dependent interaction of annexins from bovine lung with Procion Red HE-3B

P-319 - Ca2+-dependent interaction of annexins from bovine lung with Procion Red HE-3B