Cibacron Blue Research Articles

Mass transfer process in the adsorption of Cibacron Blue (CB) on untreated pea pods (UPPs): kinetic and thermodynamic studies

ABSTRACT In recent decades, there has been extensive use of synthetic dyes in the dye-based industries, particularly the textile sector. Therefore, the aim of this study was to evaluate the adsorption of Cibacron Blue (CB) onto untreated pea pods (UPPs) in a batch system. The effects of the initial CB concentration (10–20 mg/L), solution pH (2–12), adsorbent dose (0–4 g/L), particle size (50–500 μm), and temperature (295–318 K) on the CB adsorption were investigated in batch configuration to determine the optimum conditions. Analyses of UPPs were characterized by Fourier Transform Infrared (FTIR) spectroscopy, Brunauer–Emmett–Teller (BET), and point-of-zero charge (pHpzc = 5.6). Under optimized conditions (pH: 2.5, particle size: 50 μm, time: 40 min, adsorbent dose: 2.5 g/L, and agitation speed: 250 rpm), up to 30.30 mg/g at 25 °C is removed from the solution. The adsorptions kinetics obey rather a pseudo-second-order kinetic model with a determination coefficient of R2 = 0.999. The adsorption isotherms have been used for the determination of thermodynamic parameters, i.e. the negative free energy ΔG° (−4.33 to 0.783 kJ/mol), negative enthalpy change ΔH° (−54.63 kJ/mol), and entropy (ΔS° = −0.1705 kJ/mol.K) indicate that the CB adsorption onto UPPs is spontaneous and exothermic in nature.

A pH-Mediated Highly Selective System Enabling Simultaneous Analysis of Circulating RNAs Carried by Extracellular Vesicles and Lipoproteins.

Extracellular vesicles (EVs) and lipoproteins (LPPs) serve as important carriers of circulating miRNAs in peripheral blood, offering immense potential for disease diagnosis and therapeutic interventions. Due to their shared physicochemical attributes, EVs and LPPs are frequently coisolated, potentially leading to misunderstandings regarding their distinct functional roles in physiological and pathological processes. Here, we report a highly selective magnetic system based on the pH-mediated affinity displayed by cibacron blue (CB) toward EVs and LPPs, enabling successful separation and collection of these two nanoparticles without cross-contamination for subsequent circulating RNA analysis. First, we found that CB-modified magnetic beads (CBMBs) exhibit a strong affinity toward LPP particles while displaying little interaction with EVs in standard samples under physiological pH conditions. We further demonstrate that the affinity between CB molecules and bionanoparticles in plasma samples is highly pH-dependent. Specifically, CBMBs show affinities for both LPP and EV particles under neutral and acidic conditions. However, at basic pH levels, CB molecules selectively bind only to LPP particles. Consequently, the remaining EV particles present in plasma are subsequently isolated by using titanium dioxide-modified beads (TiMBs) through phospholipid affinity. The simultaneous analysis of the transcriptomic contents of EV and LPP reveals clear differences in their small RNA profiles, with the differentially expressed RNAs reflecting distinct biological processes. Significantly, in a proof-of-concept study, we successfully demonstrated a strong correlation between miRNAs carried by both EV and LPP particles with the occurrence of ocular neovascularization during the progression of diabetic retinopathy. The involved miRNAs may serve as potential biomarkers for DR diagnostics and severity classification. To sum up, this pH-mediated separation system is not only user-friendly but also highly compatible, rendering it a potent tool for probing the molecular compositions, biomarkers, and underlying biological mechanisms of EVs and LPPs.

Computer-aided identification of potential inhibitors against Necator americanus glutathione S-transferase 3

Hookworm infection is caused by the blood-feeding hookworm gastrointestinal nematodes. Its harmful effects include anemia and retarded growth and are common in the tropics. A current control method involves the mass drug administration of synthetic drugs, mainly albendazole and mebendazole. There are however concerns of low efficacy and drug resistance due to their repeated and excessive use. Although, Necator americanus glutathione S-transferase 3 (Na-GST-3) is a notable target, using natural product libraries for computational elucidation of promising leads is underexploited. This study sought to use pharmacoinformatics techniques to identify compounds of natural origins with the potential to be further optimized as promising inhibitors. A compendium of 3182 African natural products together with five known helminth GST inhibitors including Cibacron blue was screened against the active sites of the Na-GST-3 structure (PDB ID: 3W8S). The hit compounds were profiled to ascertain the mechanisms of binding, anthelmintic bioactivity, physicochemical and pharmacokinetic properties. The AutoDock Vina docking protocol was validated by obtaining 0.731 as the area under the curve calculated via the receiver operating characteristics curve. Four compounds comprising ZINC85999636, ZINC35418176, ZINC14825190, and Dammarane Triterpene13 were identified as potential lead compounds with binding energies less than −9.0 kcal/mol. Furthermore, the selected compounds formed key intermolecular interactions with critical residues Tyr95, Gly13 and Ala14. Notably, ZINC85999636, ZINC14825190, and dammarane triterpene13 were predicted as anthelmintics, whilst all the four molecules shared structural similarities with known inhibitors. Molecular modelling showed that the compounds had reasonably good binding free energies. More so, they had high binding affinities when screened against other variants of the Na-GST, namely Na-GST-1 and Na-GST-2. Ligand quality assessment using ligand efficiency dependent lipophilicity, ligand efficiency, ligand efficiency scale and fit quality scale showed the molecules are worthy candidates for further optimization. The inhibitory potentials of the molecules warrant in vitro studies to evaluate their effect on the heme regulation mechanisms.

Studies of the potential of a native natural biosorbent for the elimination of an anionic textile dye Cibacron Blue in aqueous solution

This work is devoted to the adsorption of Cibacron Blue (CB) an anionic textile dye, on bean peel (BP) an agricultural waste with neither activation nor carbonization. The adsorption was realized in batch configuration at ambient temperature in acidic medium. The adsorbent was characterized by FTIR, SEM and BET analyses; the equilibrium isotherms and kinetics were also studied. It has been found that this waste could be used as a low-cost biosorbent for CB elimination under optimal working conditions. The rate of CB elimination reaches 95% on bean bark (3.6 g/L) at pH 2.2 and a reject concentration of 25 mg/L. The pseudo-second-order describes suitably the experimental data and the external diffusion is the rate-determining step. The Freundlich isotherm fits better the CB adsorption with a correlation coefficient (R2) of 0.94 and an RMSE = 1.5115. The negative enthalpy (ΔH) and free enthalpy (ΔG°) indicate a physical and spontaneous nature of the CB biosorption onto the biomaterial.

Efficient native biosorbent derived from agricultural waste precursor for anionic dye adsorption in synthetic wastewater

In this work, a potential biosorbent is prepared from agricultural waste material (ash seed) without any treatment. This biomaterial could be successfully used as a low-cost adsorbent for the removal of an anionic dye named Cibacron Blue (CB), under optimal conditions. The prepared adsorbent was characterized by scanning electron microscope (SEM) coupled with EDX, Fourier transforms infrared spectrometer (FTIR), thermogravimetric analysis (TGA), and point of zero charge (pHpzc). The adsorption experiments were carried out in batch mode at room temperature. Results show that the rate of CB dye removal exceeds 95 % using a dose of 2 g/L ash seed at C0 = 25 mg/L and pH = 2.2. The kinetic data were analyzed using the pseudo-first-order, pseudo-second-order, Elovich’s, Bangham, intraparticle diffusion, and Boyd kinetic. Results show that the pseudo-second-order model fits better the experimental data. The isotherm data of the CB dye adsorption were analyzed with several theoretical models (Langmuir, Freundlich, and monolayer model coupled to real gaz (MMRG)). It was found that the adsorption process of the CB dye is well described by monolayer model coupled to real gaz (MMRG) compared to other models. The negative enthalpy (ΔH°) and free enthalpy (ΔG°) indicate the physical and spontaneous nature of CB adsorption. Therefore, the prepared material can serve as a potential adsorbent for removal of CB dye from industrial effluents.

Phytoremediation of synthetic textile dyes: biosorption and enzymatic degradation involved in efficient dye decolorization by Eichhornia crassipes (Mart.) Solms and Pistia stratiotes L.

The effectiveness of four aquatic floating plants: Eichhornia crassipes, Pistia stratiotes, Lemna minor, Salvinia sp., and a submerged plant Hydrilla sp. on decolorization and detoxification of five structurally different textile dyes: CI Direct Blue 201 (DB 201), Cibacron Blue FR, Cibanone Gold Yellow RK, Vat Green FFB, and Moxilon Blue GRL were studied. The E. crassipes and P. stratiotes showed complete decolorization of all the dyes tested, while Salvinia sp. (79-86%), L. minor (16-24%), and Hydrilla sp. (6-13%) were recorded as the least tolerance for all the dyes even after 14 days of incubation. Therefore, E. crassipes and P. stratiotes were selected for further studies using DB 201 as the model dye. E. crassipes and P. stratiotes showed complete decolorization of DB 201 at 48 and 84 h of incubation, respectively, and decolorization was well effective in the pH range 6-9. The crude extract of intracellular enzymes obtained from the roots of E. crassipes (46%) and P. stratiotes (20%) showed significant involvement on decolorization of DB 201, compared with the activity of crude extracellular extract and isolated endophytic bacteria and fungi (p ≤ 0.05). Further, 18 and 22% of biosorption of DB 201 dye were recorded by E. crassipes and P. stratiotes, respectively, suggesting that decolorization mechanisms of DB 201 dye by E. crassipes and P. stratiotes were based on biosorption and intracellular enzyme activities. The FTIR spectra and seed germination assay confirmed biodegradation and detoxification of DB 201 dye by E. crassipes and P. stratiotes plants along with complete color removal. Thus, present study confers the potential applicability of E. crassipes and P. stratiotes plants for textile dye removal and release to the environment without further treatment.

Development of Cibacron Blue-Enhanced Affinity Nanofiber Fabric for Protein Adsorption

The separation of protein plays a critical role in the protein production process. A functionalized nanofiber fabric was prepared by immobilizing Cibacron Blue (CB) on a polyvinyl alcohol (PVA) nanofiber fabric produced by an electrospinning method. To evaluate its separation performance, the adsorption test of bovine serum albumin (BSA), a kind of protein, was conducted using this fabric. The CB molecules were immobilized by the covalent bonding of hydroxyl group of PVA and chlorinated triazine ring of CB under alkaline condition. The adsorption test of CB-enhanced affinity PVA nanofiber fabric was conducted by soaking it into the BSA solution as an adsorbate. As a result, after enhancing affinity by CB, the BSA adsorption amount of the PVA nanofiber fabric indicates a significant increase due to functionalization. Moreover, the adsorption characteristics were heavily dependent on the solution environment.

Mechanism of Antibacterial Cationic Peptide caP4 from Curcuma pseudomontana L. (Zingiberaceae) Against E. coli

In our previous published article ( https://doi.org/10.1007/s10989-019-09883-7 ), caP4???a cationic peptide of 2.97 kDa from Curcuma pseudomontana L. (Zingiberaceae) with sequences ASSCKPS and ASSKWVAPSEW showed significant antibacterial activity against Escherichia coli and Staphylococcus aureus. In the present study, circular dichroism (CD) data of caP4 showed 0% ??-helix, 21.6% ??-sheet, 31.2% ??-turns, and 47.2% random coils. Further, the study was focused on understanding the mode of action of caP4 against E. coli. At 8 ??g/ml (MIC), caP4 permeabilized the cell membrane with maximum recorded at 50 min. Scanning electron microscopy analysis of E. coli cells with caP4 at 16 ??g/ml showed disorientation of the membrane surface. Cetyl pyridinium chloride (CPC, 10 ??g/ml-IC50), Mitomycin C (10 ??g/ml-IC50), Colchicine (25 ??g/ml-IC50), Cytochalasin B (30 ??g/ml-IC50) and Cibacron blue (25 ??g/ml-IC50) were used as cell growth inhibitors with or without caP4 (8 ??g/ml) to check the action of peptide intracellularly. E. coli cells with caP4 showed 100% cell death at 100 min. E. coli cells pre-treated with caP4 for 30 min showed 100% and 96% cell death at 60 min and 56 min with CPC and Mitomycin C respectively, suggesting that caP4 is acting by entering into the cell membrane and thus inhibit protein synthesis similar to CPC and Mitomycin C, whereas Colchicine, Cytochalasin B, Cibacron blue did not show significant cell death with pre and post-treatment of caP4. Thus, the study showed the synergistic effect of caP4 with cell growth inhibitors CPC and Mitomycin C in enhancing the antibacterial activity. Thus the data provide a new insight to understand the mechanism of antibiotic activity of caP4 and could lay the foundation for developing plant based antibacterial drugs in future to combat drug resistant microbes.

Cibacron Blue F3GA Bağlı mPHEMA ile Sulu Sistemden Fenol ve Nitrofenollerin Uzaklaştırılması

Fenol ve nitrofenoller, çok düşük konsantrasyonlarda bile sulu ortamdaki diğer organizmalar için toksiktir. Bu çalışmada, sulu ortamdan bir adsorban olarak manyetik poli (2-hidroksietilmetakrilat) bağlı Cibacron Blue F3GA kullanılarak fenollerin ve nitrofenollerin uzaklaştırılması araştırıldı. Farklı zaman aralıklarında (15, 20, 45, 60, 90 ve 120 dakika) adsorban üzerindeki fenol ve nitrofenollerin adsorpsiyon miktarı, artan fenol ve nitrofenol konsantrasyonu ile denge süreleri araştırıldı. pH (2-12 arasında), adsorban dozaj, adsorpsiyon süresi ve başlangıç konsantrasyonu gibi parametreler araştırıldı. Maksimum adsorpsiyon denge süresi yaklaşık 20 dakika olarak bulunmuştur. Adsorban üzerindeki adsorbe edilen madde miktarının, 4-nitrofenol> 3-nitrofenol> ve 2-nitrofenol> fenol sırasıyla azaldığı gözlendi. Adsorban, adsorpsiyon öncesi ve sonrasında Fourier Transform Infra-Red, Taramalı Elektron Mikroskopisi-Enerji Dağıtıcı X-ışını teknikleri ve Diferansiyel Termal Analiz-Termal Gravimetrik Analiz Teknikleri kullanılarak incelenmiştir. Adsorpsiyon işleminin Langmuir izotermine uygun olduğu gözlenmiştir. Adsorpsiyon kapasitelerinde önemli bir azalma olmadan tekrar tekrar Cibacron Blue F3GA'ya takılı mPHEMA mikrokürelerin kullanılması mümkün olmuştur.

Rhamnolipids biosurfactants as potential modulators of phase and partitioning behavior in micellar systems of aliphatic alcohol ethoxylate surfactants

Abstract The impact of rhamnolipids (RL) biosurfactants presence on the phase-separation equilibrium, micellar properties and partitioning behavior of systems containing the aliphatic non-ionic surfactant Tergitol 15-S-7 (Tg7) was studied in this work. The cloud points of Tg7 solutions (1‐ 10% w/w) in water and sodium citrate (NaCit) buffers pH 7.00 were increased after the RL addition (0.25% w/w) while they were decreased in NaCit pH 5.00. The most marked changes were verified for those systems with low Tg7 concentrations ( 7°). As expected, the zeta potential (ζ) of non-ionic Tg7 systems was close to zero; however, ζ became clearly negative (≅ −20 mV) in NaCit pH 7.00 after the addition of RL. This finding suggested that RL, which is negatively charged at pH 7.00, self-incorporated into Tg7 micelles and conferred them its charge. Hydrodynamic diameter (Dh) was either increased (in citrate media) or decreased (in water) by the RL, depending on the changes caused in the critical packing parameter of mixed micelles. Interestingly, an exponential decay equation, useful for predictive purpose, was found to correlate the cloud point with the hydrodynamic diameter, thus evidencing the crucial role of micelle size/shape on the phase separation. The partitioning behavior of cibacron blue (CB) and malaquite green (MG), negatively and positively charged dyes respectively, in Tg7 aqueous micellar two-phase systems (AMTPS) was significantly affected by the RL 0.25% w/w. In AMTPS with pH 7.00, the RL induced opposite effects, either duplicating the partition coefficient of MG or reducing it to a half for CB. Attractive and repulsive electrostatic interactions between the negatively charged mixed micelles and the partitioned molecules would be responsible of these trends. These results demonstrate the role of RL as a modulator of partitioning behavior of charged molecules and its potential use to improve recoveries and selectivity in a purification protocol.

Molecular characterization and functional analysis of glutathione S-transferase kappa 1 (GSTκ1) from the big belly seahorse (Hippocampus abdominalis): Elucidation of its involvement in innate immune responses

Glutathione S-transferases (GSTs) are essential enzymes for the bioactivation of xenobiotics through the conjugation of the thiol group of glutathione (GSH). In this study, a kappa class of GST was identified from the big belly seahorse (Hippocampus abdominalis) (HaGSTκ1) and its biochemical and functional properties were analyzed. HaGSTκ1 has 231 amino acids encoded by a 696 bp open reading frame (ORF). The protein has a predicted molecular mass of 26.04 kDa and theoretical isoelectric point (pI) of 8.28. It comprised a thioredoxin domain, disulfide bond formation protein A (DsbA) general fold, and Ser15 catalytic site as well as GSH-binding and polypeptide-binding sites. Phylogenetic analysis revealed that HaGSTκ1 is closely clustered with the kappa class of GSTs from teleost fishes. The recombinant (rHaGSTκ1) protein exhibited activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl (4-NBC), and 4-nitrophenethyl bromide (4-NPB) but not 1,2-dichloro-4-nitrobenzene (DCNB). The optimum pH and temperature were 8 and 30 °C, respectively, for the catalysis of CDNB and the universal substrate of GSTs. The rHaGSTκ1 activity was efficiently inhibited in the presence of Cibacron blue (CB) as compared with hematin. Most prominent expression of HaGSTκ1 was observed in the liver and kidney among the fourteen different tissues of normal seahorse. After challenge with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), gram-negative Edwardsiella tarda, and gram-positive Streptococcus iniae, HaGSTκ1 expression was significantly modulated in the liver and blood tissues. Altogether, our study proposes the plausible important role of HaGSTκ1 in innate immunity and detoxification of harmful xenobiotics.

An efficient protein immobilization strategy: protein encapsulated in nano molecular cages

AbstractBACKGROUNDTo immobilize protein firmly and maintain its native conformation, a ‘nano molecular cage’ was designed to encapsulate bovine serum albumin (BSA) on the surface of poly(methyl methacrylate) (PMMA) magnetic microspheres by cross‐linking poly(ethylene imine) (PEI) chains.RESULTSThe amount of BSA adsorbed on PMMA@PEI microspheres decreased significantly from 70.6 to 7.6 mg g−1 microsphere after desorption under high ionic strength. A similar phenomenon could also be observed for BSA adsorbed on PMMA@PEI‐CB (Cibacron Blue F3GA) microspheres. In contrast, the amount of BSA encapsulated in molecular cages only decreased a little after desorption. Furthermore, conformational analysis by Fourier transform infrared spectroscopy (FTIR) showed that the contents of α‐helices and β‐sheets decreased slightly by 3.3 and 0.3% respectively on encapsulating BSA in molecular cages. Furthermore, the effects of various factors, including glutaraldehyde amount, cross‐linking time and temperature, on BSA encapsulation were investigated.CONCLUSIONThe results demonstrated that molecular cages could not only immobilize protein firmly but also maintain its native conformation. Therefore molecular cage encapsulation is suggested to be a potentially valuable protocol in enzyme immobilization. © 2019 Society of Chemical Industry

Synthesis of 1,4‐diaminoanthraquinones for the purification of lactate dehydrogenase

Cibacron blue 3G‐A is a dye commonly used for the purification of enzymes using affinity chromatography, often utilized in undergraduate biochemistry laboratory studies. Unfortunately, companies only have this product available intermittently, and when available it is sold at a fairly high price. Due to these issues, an alternative molecule that could be easily synthesized and would be similarly efficient at enzyme purification is desired. A distinct feature of Cibacron blue is its anthraquinone core. Anthraquinones are naturally‐occurring aromatic compounds commonly used in the manufacture of dyes. This synthesis utilizes a method of converting 1,4‐diaminoanthraquinones into diazonium salts to then allow the addition of various other functional groups, including anilines and phenols. After synthesis has been conducted, this small library of aminoanthraquinone derivatives will be tested for biological activity by examining spectrophotometric activity in the presence of lactate dehydrogenase (LDH). This will determine whether the products synthesized are efficient at binding to LDH and whether they can be successfully utilized for purification methods. This experiment details a simple, two‐step synthesis of various 1,4‐disubstituted aminoanthraquinone derivatives, which may be found to be useful alternatives to Cibacron blue. In addition, these products could potentially have other biological activity, due to the highly‐activated anthraquinone core.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comprehensive analysis of the catalytic and structural properties of a mu-class glutathione s-transferase from Fasciola gigantica

Glutathione S‒transferases (GSTs) play an important role in the detoxification of xenobiotics. They catalyze the nucleophilic addition of glutathione (GSH) to nonpolar compounds, rendering the products water-soluble. In the present study, we investigated the catalytic and structural properties of a mu-class GST from Fasciola gigantica (FgGST1). The purified recombinant FgGST1 formed a homodimer composed of 25 kDa subunit. Kinetic analysis revealed that FgGST1 displays broad substrate specificity and shows high GSH conjugation activity toward 1-chloro-2,4-dinitrobenzene, 4-nitroquinoline-1-oxide, and trans-4-phenyl-3-butene-2-one and peroxidase activity towards trans-2-nonenal and hexa-2,4-dienal. The FgGST1 was highly sensitive to inhibition by cibacron blue. The cofactor (GSH) and inhibitor (cibacron blue) were docked, and binding sites were identified. The molecular dynamics studies and principal component analysis indicated the stability of the systems and the collective motions, respectively. Unfolding studies suggest that FgGST1 is a highly cooperative molecule because, during GdnHCl-induced denaturation, a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed. The protein is stabilized with a conformational free energy of about 10 ± 0.3 kcal mol−1. Additionally, the presence of conserved Pro-53 and structural motifs such as N-capping box and hydrophobic staple, further aided in the stability and proper folding of FgGST1.

Two glutathione transferase isoforms isolated from juvenile cysts of Taenia crassiceps: identification, purification and characterization.

We identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts of Taenia crassiceps (EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg-1 and 19 U mg-1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. The Km(CDNB) and Kcat(CDNB) values for TcGST1 and TcGST2 (0.86 μm and 62 s-1; 1.03 μm and 1.97 s-1, respectively) and Km(GSH) and Kcat(GSH) values for TcGST1 and TcGST2 (0.55 μm and 11.61 s-1; 0.3 μm and 32.3 s-1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20-55°C and pH 7.5 at 35-40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the μm range. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use of T. crassiceps as a model to investigate multifunctional GSTs.

Molecular Insight into Affinity Interaction between Cibacron Blue and Proteins

Molecular dynamics (MD) simulations with all-atom models combining adsorption experiments are used to gain a better understanding of affinity interaction between proteins and Cibacron Blue (CB). These interactions with three different proteins are studied. The interactions with CB of different proteins are varying, although HSA and BSA have very similar structures. The influence of pH and salt concentration on these protein–CB bindings is investigated. Different parts of the CB molecule play miscellaneous roles in these interactions. The triazine part plays an important role in the hydrogen bonding between CB and bHb. The binding sites on protein surface differ by changing pH, salt concentration, and protein types. However, two parts seem to be very important to the binding of serum albumins.

Chemokine-Releasing Microparticles Improve Bacterial Clearance and Survival of Anthrax Spore-Challenged Mice

In this study the hydrogel microparticles (MPs) were used to enhance migration of neutrophils in order to improve outcome of anthrax infection in a mouse model. Two MP formulations were tested. In the first one the polyacrylamide gel MPs were chemically coupled with Cibacron Blue (CB) affinity bait. In the second one the bait molecules within the MPs were additionally loaded with neutrophil-attracting chemokines (CKs), human CXCL8 and mouse CCL3. A non-covalent interaction of the bait with the CKs provided their gradual release after administration of the MPs to the host. Mice were challenged into footpads with Bacillus anthracis Sterne spores and given a dose of MPs a few hours before and/or after the spores. Pre-treatment with a single dose of CK-releasing MPs without any additional intervention was able to induce influx of neutrophils to the site of spore inoculation and regional lymph nodes correlating with reduced bacterial burden and decreased inflammatory response in footpads. On average, in two independent experiments, up to 53% of mice survived over 13 days. All control spore-challenged but MP-untreated mice died. The CB-coupled particles were also found to improve survival likely due to the capacity to stimulate release of endogenous CKs, but were less potent at decreasing the inflammatory host response than the CK-releasing MPs. The CK post-treatment did not improve survival compared to the untreated mice which died within 4 to 6 days with a strong inflammation of footpads, indicating quick dissemination of spores though the lymphatics after challenge. This is the first report on the enhanced innate host resistance to anthrax in response to CKs delivered and/or endogenously induced by the MPs.

Assembly of ligands interaction models for glutathione-S-transferases from Plasmodium falciparum, human and mouse using enzyme kinetics and molecular docking

BackgroundGlutathione-s-transferases (GSTs) are enzymes that principally catalyze the conjugation of electrophilic compounds to the endogenous nucleophilic glutathione substrate, besides, they have other non-catalytic functions. The Plasmodium falciparum genome encodes a single isoform of GST (PfGST) which is involved in buffering the toxic heme, thus considered a potential anti-malarial target. In mammals several classes of GSTs are available, each of various isoforms. The human (human GST Pi-1 or hGSTP1) and mouse (murine GST Mu-1 or mGSTM1) GST isoforms control cellular apoptosis by interaction with signaling proteins, thus considered as potential anti-cancer targets. In the course of GSTs inhibitors development, the models of ligands interactions with GSTs are used to guide rational molecular modification. In the absence of X-ray crystallographic data, enzyme kinetics and molecular docking experiments can aid in addressing ligands binding modes to the enzymes. MethodsKinetic studies were used to investigate the interactions between the three GSTs and each of glutathione, 1-chloro-2,4-dinitrobenzene, cibacron blue, ethacrynic acid, S-hexyl glutathione, hemin and protoporphyrin IX. Since hemin displacement is intended for PfGST inhibitors, the interactions between hemin and other ligands at PfGST binding sites were studied kinetically. Computationally determined binding modes and energies were interlinked with the kinetic results to resolve enzymes-ligands interaction models at atomic level. ResultsThe results showed that hemin and cibacron blue have different binding modes in the three GSTs. Hemin has two binding sites (A and B) with two binding modes at site-A depending on presence of GSH. None of the ligands were able to compete hemin binding to PfGST except ethacrynic acid. Besides bind differently in GSTs, the isolated anthraquinone moiety of cibacron blue is not maintaining sufficient interactions with GSTs to be used as a lead. Similarly, the ethacrynic acid uses water bridges to mediate interactions with GSTs and at least the conjugated form of EA is the true hemin inhibitor, thus EA may not be a suitable lead. ConclusionsGlutathione analogues with bulky substitution at thiol of cysteine moiety or at γ-amino group of γ-glutamine moiety may be the most suitable to provide GST inhibitors with hemin competition.

Glutathione and its related enzymes in the gonad of Nile Tilapia (Oreochromis niloticus).

Glutathione (GSH) concentration, the activity of its metabolizing enzymes, glutathione transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and the antioxidant enzyme catalase (CAT) in O. niloticus ovary and testis were examined. GSH concentration of O. niloticus testis exhibited high concentration (129 ± 21 nmol/g tissue) compared with GSH concentration (49.2 ± 8.3 nmol/g tissue) in the ovary. GST, GPx, GR, and CAT activities of O. niloticus testis exhibited high values compared with their corresponding values in ovary homogenates. However, protein concentration in ovary homogenates exhibited higher values (175 ± 40.6 mg) compared with testis homogenates (27.1 ± 3.7 mg). O. niloticus ovary was less effective in excretion of xenobiotices compared with the testis, where its function is mainly in increasing the protein content of the eggs; however, in O. niloticus testis, the glutathione cycle operated in accelerated way in the direction of reduced GSH production in order to protect the maturation stages in a save way. A simple reproducible procedure for the purification of GST from O. niloticus ovary was established. The enzymes proved to be homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its molecular weight was calculated to be 25.1 kDa. GST of O. niloticus ovary exhibited maximum activity at pH 7.5. The Michaelis-Menten constant (K(m)) of the purified ovary GST for GSH and CDNB was 0.076 mM and 1.0 mM, respectively. Cibacron blue was the most potent inhibitor of ovary GST activity (IC50 value, concentration of inhibitor that will give 50% inhibition, equal 0.002 μM). The specific activity of GST toward different electrophilic substrates was determined. GST activity toward benzyl isothiocyanate was the highest compared with phenethyl isothiocyanate and allyl isothiocyanate.

Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum

The key objective of this study was to investigate the effect of germination on biochemical and enzymatic antioxidant activities of Trifolium alexandrinum seeds. The T. alexandrinum was chosen for GST purification and characterization due to its highest GST activity and antioxidant capacity, beside its economical importance. Seeds of T. alexandrinum were germinated for 6 days. Changes in glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) in germinating and dormant seeds of T. alexandrinum were evaluated. Also the changes in the total phenolic and flavonoid contents as well as the antioxidant capacities were monitored for 6 days of germination using spectrophotometeric methods. Simple reproducible procedures for the purification of T. alexandrinum GST from both dormant and 6 days germinated seeds were established using DEAE-Sepharose and Sephadex G-100 columns chromatography. The results showed the presence of three isoenzymes for both dormant and germinated seeds designated as GST1, GST2 and GST3. The major GST of the dormant seed was GST2 while for the germinated one was GST3. Dormant GST2 was expressed as a heterodimer with molecular weight of 27.5 and 28.5 KDa while germinated GST3 was expressed as a homodimer with molecular weight of 27 KDa. Characterization of both dormant GST1 & GST2 and germinated GST2 and GST3 including optimum pH, kinetic parameters [kmGSH of 1.17±0.39, 1.22±0.13, 0.84±0.19, 0.96±0.24mM, respectively and kmCDNB of 0.65±0.095, 0.57±0.11, 0.59±0.14, 1.02±0.18 mM, respectively]. The substrate selectivity using different electrophilic compounds and inhibitor effects were carried out. In opposite to other isoenzymes, germinated GST3 exhibited enzymatic activity towards ethacrynic acid with specific activity of 0.073±0.005 µmol/min/mg protein while cibacron blue was the most potent inhibitor for these isoenzymes.