Inhibition of the sarcoplasmic reticulum Ca2+-ATPase by Reactive Red 120

The triazine dye, Reactive Red 120, was found to bind tightly (Kd = 30) nM) and with low stoichiometry to sarcoplasmic reticulum membranes. Our finding that this high-affinity binding caused noncompetitive inhibition of the Ca2+-ATPase indicates that the dye-binding site is distinct from both the active site and putative regulatory site. Detergent solubilization (monomerization) of the Ca2+-ATPase caused a 25-fold decrease in affinity for Reactive Red 120, while causing no decrease in affinity toward another dye, Reactive Blue 2. For the Reactive-Red-120-inhibited enzyme, the level of steady-state enzyme phosphorylation by ATP was not significantly different from that exhibited by the control Ca2+-ATPase. The rate of dephosphorylation in the presence and absence of ADP, however, was markedly decreased by the presence of the inhibitor. Distance measurements by fluorescence energy transfer from the active (FITC-reactive) site to the Reactive Red 120 site gave a value of 59 A. Similar experiments yielded an average distance of 35 A between the latter site and the tryptophan residues, most of which are postulated by the 'sequence model' (MacLennan et al. (1985) Nature 316, 696-700) to be located in a transmembrane domain.     

An affinity label for the regulatory dithiol of ribulose-5-phosphate kinase from maize (Zea mays).

Ribulose-5-phosphate kinase from maize (Zea mays) can exist in either a reduced, active form or an oxidized, inactive form. Reduced ribulose-5-phosphate kinase is rapidly and irreversibly inactivated by the dichlorotriazine dye Reactive Red 1 (Procion Red MX-2B), but the irreversible inactivation of the oxidized form of ribulose-5-phosphate kinase occurs at only 0.05% of this rate. The rate of inactivation of the reduced enzyme by Reactive Red 1 (apparent bimolecular rate constant 10(4)M-1 X s-1 at pH 7.4 and 25 degrees C) is several orders of magnitude greater than previous estimates of the rates of dye-mediated inactivation of other enzymes. The dye-dependent inactivation of the reduced enzyme is inhibited by Hg2+ or p-mercuribenzoate (thiol reagents that reversibly inhibit ribulose-5-phosphate kinase activity), or by ATP and ADP, the nucleotide substrates of the enzyme. Hydrolysed Reactive Red 1, which does not inactivate the enzyme, is a reversible inhibitor of ribulose-5-phosphate kinase. This inhibition is competitive with respect to ATP (Ki approximately 0.5 mM). The dye appears to act as an affinity label for the ATP/ADP-binding site by preferentially arylating a thiol residue generated during the reductive activation of the enzyme that is achieved by dithiothreitol or thioredoxin in vitro or during illumination of leaves.     

Decolorization and degradation of textile dyes with biosulfidogenic hydrogenases

Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480-620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds.     

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.     

Optimisation for enhanced decolourization and degradation of Reactive Red BS C.I. 111 by Pseudomonas aeruginosa NGKCTS

Soil samples collected from dye contaminated sites of Vatva, Gujarat, India were studied for the screening and isolation of organisms capable of decolourizing textile dyes. The most efficient isolate, which showed decolourization zone of 48 mm on 300 ppm Reactive Red BS (C.I.111) containing plate, was identified as Pseudomonas aeruginosa. Reactive Red BS (C.I.111) was used as a model dye for the study. The isolated culture exhibited 91% decolourization of 300 ppm dye within 5.5 h over a wide pH range from 5.0 to 10.5 and temperature ranging from 30 to 40°C. The culture was able to decolourize more than 91% of Reactive Red BS under static conditions in presence of either glucose, peptone or yeast extract. Addition of 300 ppm of Reactive Red BS, in each step, in ongoing dye decolourization flask, gave more than 90% decolourization within 2 h corresponding to 136 mg l⁻¹ h⁻¹ dye removal rate. The isolate had the ability to decolourize six different reactive dyes tested as well as the actual dye manufacturing industry’s effluent. The degradation of the dye was confirmed by HPTLC.     

Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized Reactive Red 120

Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).     

Bioinformatics aided microbial approach for bioremediation of wastewater containing textile dyes

Four textile azo dyes, Joyfix Red, Remazol Red, Reactive Red and Reactive Yellow, were studied for decolorization. Of nineteen soil bacterial isolates, two novel strains were found to highly decolorize Joyfix Red and were identified as Lysinibacillus sphaericus (KF032717) and Aeromonas hydrophila (KF032718) through 16S rDNA analysis. Laccase and Azoreductase enzyme modeling and enzyme–dye interaction performed using Schrödinger Suite imitated decolorization percentage. Results based on cumulative Glide score (Dry laboratory) and decolorization percentage of the other three dyes based on ultraviolet–visible (UV–vis) spectroscopy (Wet laboratory) were reliable. Biodegradation of Joyfix Red was confirmed by high-performance liquid chromatography (HPTLC) elution profile which showed four peaks at 1.522, 1.800, 3.068 and 3.804 min with that of parent dye which showed single peak at 1.472 min. Fourier transform infrared spectroscopy (FT-IR) analysis supported the biotransformation of Joyfix Red. Gas chromatography–mass spectroscopy (GC–MS) analysis showed sodium (3E,5Z)-4-amino-6-hydroxyhexa-13,5-triene-2-sulfonate was formed as end product during biodegradation. From these findings, it can be inferred that enzyme and dye interaction studies can assist in examining decolorization efficiency of bacteria and its enzyme, thereby enhancing the bioremediation process by reducing preliminary lengthy wet laboratory screening. This is the first report of a combinatorial in silico cum in vitro approach and its validation for the bioremediation of wastewater containing these textile azo dyes.     

Triazine dyes as inhibitors and affinity ligands of glycosyltransferases

The triazine dyes: Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Green 19, Reactive Blue 4, Reactive Brown 10 inhibited the activity of a purified preparation of 1,6fucosyltransferase (GDP-L-fucose:N-acetyl -glucosaminide 6--L-fucosyltransferase, EC 2.4.1.68) from human blood platelets. Cibacron Blue 3GA and Reactive Red 120 were examined for the nature of the inhibition and both were found to be competitive inhibitors of the enzyme, with K_i=11[emsp4 ]M and 2[emsp4 ]M, respectively. The two dyes inhibited also serum glycosyltransferases: 1,2fucosyltransferase (GDP-L-fucose: -D-galactosyl-R2--L-fucosyltransferase, EC 2.4.1.69), 1,4galactosyltransferase (UDP-galactose: N-acetyl-D-glucosamine 4--D-galactosyltransferase, EC 2.4.1.90) and 1,3N-acetylglucosaminyltransferase (UDP-GlcNAc: 4--D-galactosyl-D-glucose). Cibacron Blue 3GA was a more effective inhibitor of the glycosyltransferases that use UDP-linked sugar donors than Reactive Red 120 while the latter was a stronger inhibitor of the fucosyltransferases that use GDP-linked donor. All four glycosyltransferases could be affinity purified on Cibacron Blue 3GA-Agarose columns. The order of elution of glycosyltransferases from the columns with solutions of 0.25–1.0[emsp4 ]M potassium iodide also depended upon the structure of nucleotide sugar donor, i.e. whether it contained UDP or GDP. Thus, triazine dyes should interact with the sugar donor binding sites of glycosyltransferases. The main advantages of the use of triazine dyes as affinity ligands for isolation of glycosyltransferases are their universal applicability regardless of enzyme specificity, low cost, and insensitivity to high concentration of other proteins present in the solution.     

The purification and characterization of a Bacillus stearothermophilus methionine aminopeptidase (MetAP)

Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAESepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala- Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at 80 degrees C, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at 80 degrees C for 45 min. The Km and Vmax values of the enzyme were 3.0 mM and 1.7 mmol/min/mg, respectively. The B. stearothermophilus MetAP was completely inactivated by EDTA and required Co(2+) ion(s) for activation, suggesting the metal dependence of this enzyme     

kappa-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase

kappa-Carrageenan is a non-toxic polymer from seaweeds, which becomes reversibly insoluble upon the addition of K(+). Its conjugate with the dye, Cibacron Blue 3GA, was used to purify alcohol dehydrogenase from crude yeast extract by affinity precipitation. Response surface methodology was used to optimize conditions for affinity precipitation of the enzyme with the polymer-dye conjugate. Recovery of 58% of the enzyme activity with 13.6-fold purification was obtained.     

Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation

Non-porous poly(glycidyl methacrylate/ethyleneglycol dimetacrylate) (poly(GMA/EGDMA)) beads were prepared by suspension polymerization. The enzyme (i.e. laccase) was covalently immobilized onto plain and spacer-arm attached poly(GMA/EGDMA) beads. The amount of immobilized enzyme on the plain and spacer-arm attached beads was determined as 5.6 and 4.9 mg/g, respectively. The maximum activity (V(max)) and Michaelis constant (K(m)) of laccase immobilized on the spacer-arm attached beads, were found to be 77.6 U/min and 0.47 mM, respectively. Finally, the immobilized laccase was operated in a batch system, and textile dye Reactive Red 120 was successfully decolorized in the enzyme reactor.     

Affinity precipitation of lactate dehydrogenase with a triazine dye derivative: selective precipitation of rabbit muscle lactate dehydrogenase with a procion blue H-B analog

A simple methoxylated derivative of the triazine dye, Procion blue H-B, selectively precipitates rabbit muscle lactate dehydrogenase from solution. Optimum protein precipitation occurred at an enzyme subunit:dye ratio of approximately 2:1 and was fully reversible upon addition of competitive ligands such as NADH. With a crude extract of rabbit muscle, affinity precipitation with the dye followed by dissolution with NADH yielded homogeneous lactate dehydrogenase in 97% overall yield.     

Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity

Amaranth, Tropaeolin O, Reactive Blue 15, Congo Red, and Reactive Black 5 were completely decolorized with no dye sorption by Trametes versicolor. Cibacron Brilliant Red 3G-P, Cibacron Brilliant Yellow 3B-A, and Remazol Brilliant Blue R were partially decolorized with some dye sorbed to the biomass. The Microtox assay before decoloration showed that Amaranth and Tropaeolin O were not toxic [the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC₂₀) was greater than 100%]; Cibacron Brilliant Yellow 3B-A, Reactive Blue 15 and Cibacron Brilliant Red 3G-P were moderately non-toxic (100% > EC₂₀ > 75%); Remazol Brilliant Blue R was toxic (75% > EC₂₀ > 50%); and Congo Red and Reactive Black 5 were moderately toxic (50% > EC₂₀ > 25%). After decoloration the toxicity of the solutions containing Amaranth, Tropaeolin O and Reactive Black 5 was unchanged; Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P decreased to non-toxic levels; and Cibacron Brilliant Yellow 3B-A and Congo Red became very toxic (EC₂₀ < 25%).     

Accumulation of Acid Orange 7, Acid Red 18 and Reactive Black 5 by growing Schizophyllum commune

The effect of Acid Orange 7, Acid Red 18 and Reactive Black 5 on the growth and decolorization properties of Schizophyllum commune was studied with respect to the initial pH varying from 1 to 6 and initial dye concentration (10-100 mg/L). The optimum pH value was found to be 2 for both growth and color removal of these azo dyes. Increasing the concentration of azo dyes inhibited the growth of S. commune. It was observed that S. commune was capable of removing Acid Orange 7, Acid Red 18 and Reactive Black 5 with a maximum specific uptake capacity of 44.23, 127.53 and 180.17 (mg/g) respectively for an initial concentration of 100 mg/L of the dye. Higher decolorization was observed at lower concentrations for all the dyes. Finally it was found that the percentage decolorization was more in the case of Reactive Black 5 dye compared to the other two dyes used in the present investigation.     

Activation of the skeletal muscle Ca2+ release channel by the triazine dyes cibacron blue F3A-G and reactive red 120

Vesicle-45Ca2+ ion flux and planar lipid bilayer single-channel measurements have shown that the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (SR) is activated by micromolar concentrations of Cibacron Blue F3A-G (Reactive Blue 2) and Reactive Red 120. Cibacron Blue increased the 45Ca2+ efflux rate from heavy SR vesicles by apparently interacting with both the adenine nucleotide and caffeine activating sites of the channel. Dye-induced 45Ca2+ release was inhibited by Mg2+ and ruthenium red. In single channel recordings with the purified channel protein complex, Cibacron Blue increased the open time of the Ca2+ release channel without an apparent change in the conductance of the main and subconductance states of the channel.     

Decolourization and degradation of C.I.Reactive Red 195 by Georgenia sp.CC-NMPT-T3

Azo dye Reactive Red 195 was selected for decolourization and degradation studies by Georgenia sp.CC-NMPT-T3. Optimization of parameters for dye decolourization was studied under static anoxic condition. Under optimized condition decolourization of Reactive Red 195 by Georgenia sp.CC-NMPT-T3 was found to be 95.93 % at 50 mg/L within five hours in static anoxic condition. The optimum pH and temperature for the decolourization was 7.0 and 40 degrees C respectively. The biodegradation was monitored by UV-Vis, and TLC and HPLC. Toxicity study demonstrated no toxicity of the biodegraded product. The results suggest that the isolated organism Georgenia sp.CC-NMPT-T3 as a useful tool to treat wastewater containing reactive dyes.     

Decolorization of synthetic and real textile wastewater by the use of white-rot fungi

Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor.     

Estrogen receptor purification by affinity chromatography using an orange triazine dye

A rapid two-step procedure was devised for the purification of the estrogen receptor from the calf uterus. A 900- to 1700-fold purification of the estrogen receptor was obtained using ammonium sulfate precipitation followed by dye affinity chromatography with Reactive Orange 14 immobilized to Sepharose. The Reactive Orange 14-Sepharose was used to purify the estrogen receptor in the presence or absence of estradiol as well as to purify the progesterone receptor. The purified estrogen receptor retained its estradiol- and DNA-binding properties and sedimented into sucrose gradients as the 5 S receptor dimer. The Reactive Orange 14-Sepharose is easily prepared and offers a higher yield and purity of the estrogen receptor than that afforded by estrogen- or heparin-Sepharose chromatography.     

Degradation analysis of Reactive Red 198 by hairy roots of <Emphasis Type="Italic">Tagetes patula</Emphasis> L. (Marigold)

Tagetes patula L. (Marigold) hairy roots were selected among few hairy root cultures from other plants tested for the decolorization of Reactive Red 198. Hairy roots of Tagetes were able to remove dye concentrations up to 110 mg L^−l and could be successively used at least for five consecutive decolorization cycles. The hairy roots of Tagetes decolorized six different dyes, viz. Golden Yellow HER, Methyl Orange, Orange M2RL, Navy Blue HE2R, Reactive Red M5B and Reactive Red 198. Significant induction of the activity of biotransformation enzymes indicated their crucial role in the dye metabolism. UV–vis spectroscopy, HPLC and FTIR spectroscopy analyses confirmed the degradation of Reactive Red 198. A possible pathway for the biodegradation of Reactive Red 198 has been proposed with the help of GC–MS and metabolites identified as 2-aminonaphthol, p-aminovinylsulfone ethyl disulfate and 1-aminotriazine, 3-pyridine sulfonic acid. The phytotoxicity study demonstrated the non-toxic nature of the extracted metabolites. The use of such hairy root cultures with a high ability for bioremediation of dyes is discussed.     

Degradation of reactive dyes by ozonation and oxalic acid-assimilating bacteria isolated from soil

Ozonation and treatment of wastewaters with oxalic acid-assimilating bacterium was attempted for the complete degradation of reactive dyes. Oxalic acid-assimilating bacterium, Pandoraea sp. strain EBR-01, was newly isolated from soil under bamboo grove and was identified to be a member of the genus Pandoraea by physicochemical and biochemical tests including 16S rDNA sequence analysis. The bacterium was grown optimally at pH 7 and temperature of 30 degrees C under the laboratory conditions. Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Black 5 (RB5) and Remazol Brilliant Blue R (RBBR) were used in degradation experiments. At the initial reactive dye concentrations of 500 mg/l and the ozonation time of 80 min, it was confirmed that 75-90 mg/l oxalic acid was generated from reactive dyes by ozonation. Microbial treatment using EBR-01 greatly decreased the amount of oxalic acid in the mixture after 48 h, but it was not removed completely. TOC/TOC(0) of reactive dye solutions was also decreased to 80-90% and 20-40% by ozonation and microbial treatment using EBR-01, respectively. The study confirmed that consecutive treatments by ozone and microorganisms are efficient methods to mineralize reactive dyes.