Cibacron Blue 3GA Research Articles

Cloning, expression, and partial characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii

The gene encoding Sulfophobococcus zilligii DNA polymerase ( Szi DNA polymerase) was cloned and sequenced. The Szi DNA polymerase gene encompasses 2394 bp, and codes for a protein consisting of 797 amino acid residues. The deduced amino acid sequence of Szi DNA polymerase exhibited a high degree of similarity with archaeal family B-type DNA polymerase homologues found in both crenarchaeotes and euryarchaeotes (Group I), and contained all of the motifs conserved in the homologues for 3′ → 5′ exonuclease and polymerase activities. The Szi DNA polymerase gene was expressed under the control of the T7 lac promoter on the expression vector pET-28a(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified by heat treatment and Cibacron blue 3GA column chromatography. The optimum pH of the purified enzyme was determined to be in the range of 6.5–7.0. The enzyme was activated by the magnesium ion, and its activity was inhibited by EDTA and monovalent cations, such as potassium ion and ammonium ion. The half-life of the enzyme at 95 °C was calculated to be 4.5 h. Szi DNA polymerase possessed associated 3′ → 5′ and 5′ → 3′ exonuclease activities.

Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: A comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; ρ = 2.65 g ml −1) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 l h −1) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2 IU mg −1, after a single step elution with 0.15 M NaCl. The generic application of this approach has been evaluated.

Regulation of the P2X7 Receptor Permeability to Large Molecules by Extracellular Cl– and Na+

Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG+). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl- (Cl-(o)) regulates the outward current, whereas extracellular Na+ (Na+(o)) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG+ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl-(o) reduced the inward Na+ current by approximately 35% and the outward Na+ current by only 10%. By contrast, in the absence of Na+(i) and the presence of Na+(o) or NMDG+(o), the removal of Cl-(o) reduced the inward Na+ or NMDG+ currents by 35% but the outward NMDG+ current by >95%. The effect of Cl-(o) was half-maximal at approximately 60 mm. Reducing Cl-(i) from 150 to 10 mm did not reproduce the effects of Cl-(o). All currents were eliminated in P2X7R-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl-(o) primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG+ were stimulated in the presence of Na+(o), subsequent removal of Na+(o) resulted in a strongly outward rectifying NMDG+ current, indicating maintained high selectivity for Na+ over NMDG+. During continuous incubation in Na+-free medium, the permeability of the P2X7Rs to NMDG+ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG+ and only then stimulated with ATP, the NMDG+ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na+(o) memory" and that Na+(o) regulates the inward permeability of P2X7Rs to large molecules. The novel regulation of P2X7R outward and inward permeability to large molecules by Cl-(o) and Na+(o), respectively, may have an important protective function, particularly in secretory epithelial cells.

Piezoelectric immunochip for the detection of dengue fever in viremia phase

The global prevalence of dengue fever has grown so dramatically in recent years that it is endemic in more than 100 countries and has become a major international public health concern. Moreover, since the flu-like symptoms that accompany dengue fever are atypical and varied, the detection procedures currently used to identify it are cumbersome and time-consuming, making early stage epidemiological control and effective medical treatment of this epidemic almost impossible. In this study, a QCM-based detection system was developed in which two monoclonal antibodies against dengue E and NS-1 protein, respectively, were control orientated immobilized on QCM via protein A to produce an immunochip. Various sample pretreatment procedures were evaluated to ascertain the most suitable combination, and both the simulating samples and the clinical specimen were examined by the immunochip. The results revealed that the cibacron blue 3GA gel-heat denature (CB-HD) method was the most effective sample pretreatment technique. Due to the complex composition of the serum, the immunochip could only effectively quantify dengue viral antigens in a 1/1000 untreated simulated sample. With the help of the CB-HD method, the dilution folds were found to capable of being reduced from 1000 to 100, and the detection limit lowered to 1.727 microg/ml (E protein) and 0.740 microg/ml (NS-1 protein) in the original sample. While the cocktail immunochip could not quantify both antigens separately, the higher signal level rendered it a more effective qualification tool for suspect screening. Moreover, the results of the analysis of clinical specimens also proved the ability and future potential of cocktail immunochip in discriminating dengue-positive cases from negative serum specimens in the viremia phase.

Process intensification of fluidized bed dye-ligand adsorption of G3PDH from unclarified disrupted yeast: A case study of the performance of a high-density steel–agarose pellicular adsorbent

The development of a process intensified primary capture step for the direct selective recovery of intracellular proteins from very dense particulate-containing yeast extract has been explored. The purification of glyceraldehyde 3-phosphate dehydrogenase from bakers’ yeast was chosen as a potential demonstration of this approach. A high throughput (50%, w/v, yeast extracts at a superficial linear velocity of 450 cm h −1) was achieved by adoption of a high-density adsorbent (UpFront steel–agarose; ρ = 2.65 g ml −1) derivatized with selective ligand chemistries (Cibacron Blue 3GA). This should ultimately minimize adsorption time and maximize process efficiency of fluidized bed adsorption.

Protein separation with surfactant-coated octadecylsilyl silica involving Cibacron blue 3GA-conjugated nonionic surfactant

A novel medium for protein separation, namely affinity admicelle, was prepared by mixing of octadecylsilyl (ODS) silica gels, a polyoxyethylene-type nonionic surfactant (Triton X-100), and a surfactant-conjugated substrate (affinity ligand) in an aqueous solution. The ligand was synthesized by mixing a triazine dye (Cibacron Blue 3GA, CB) and a polyethylene glycol monooleyl ether (C 18EO 7, C 18EO 10, or C 18EO 20) having different length of polyoxyethylene moiety in weakly alkaline solutions. The amount of Triton X-100 sorbed on 1 g of ODS silica was 0.2 mmol. Affinity ligands having highly hydrophobic oleyl group were predominantly sorbed on ODS silica. The losses of Triton X-100 and affinity ligand were within 0.3% and negligible by washing the admicelles were with a 25-fold volume of 1 mM Tris–HCl solution (pH 7.4). The coating ODS silica with Triton X-100 was effective to prevent the irreversible sorption of albumin (bovine, serum). An NADH-dependent enzyme, alcohol dehydrogenase (ADH, yeast), was successfully collected on the admicelles involving CB-conjugated ligands (CB-C 18EO 20). The maximum collection of ADH to 90 mg/ml of affinity admicelles was 68 ± 4%. However, CB-C 18EO 7 and CB-C 18EO 10 having shorter polyoxyethylene unit were not available, suggesting the requirement of the spacer moiety in the affinity ligand. The recovery and purification factor based on the ratio of activity (unit)/protein (mg) from Whatman DE52-treated yeast extract was 27% and 12, respectively.

Modeling of the linear-gradient dye-ligand affinity chromatography with a binary adsorption isotherm

A mathematical model for the linear gradient elution chromatography of a binary protein system was established based on the axial dispersion theory and pore diffusion model. Bovine serum albumin (BSA) and hemoglobin (Hb) were used as model proteins and Cibacron blue 3GA (CB) modified Sepharose CL-6B (CB-Sepharose) was used as adsorbent. The binary adsorption isotherms of the proteins were expressed by the steric mass-action (SMA) formulism, and its parameters were determined by batch adsorption equilibrium experiments of each protein. With all the model parameters determined by independent experiments or calculated from available correlations, model simulations were performed and compared with the chromatograms of the single-component and binary protein systems. It was found that the model predictions agreed reasonably well with the experimental data obtained under various conditions. The results indicate that the binary SMA formulism with its parameters determined by single-component equilibrium experiments was useful for simulation of nonlinear chromatography.

Characterization of the ligandin site of maize glutathione S-transferase I.

Cytosolic GSTs (glutathione S-transferases) are a major reserve of high-capacity binding proteins and exhibit ligand-binding properties for a large variety of compounds. In the present study, the binding of two non-substrate anthraquinone dyes VBAR (Vilmafix Blue A-R) and CB3GA (Cibacron Blue 3GA) to maize (Zea mays) GST I was investigated. The results showed that the enzyme was specifically and irreversible inactivated by VBAR with a K(d) of 35.5+/-2.2 microM and a k(3) of 0.47 min(-1). Proteolytic cleavage of the VBAR-modified enzyme and subsequent separation of peptides gave only one modified peptide. Sequencing of the modified peptide revealed the target site of VBAR reaction to be Lys(41). CB3GA binds reversibly to GST I and behaves as a competitive inhibitor towards CDNB (1-chloro-2,4-dinitrobenzene) and glutathione. CB3GA binding to GST I is accompanied by a characteristic spectral change in the absorption at positive maximum (670 nm) which exhibited a hyperbolic dependence on dye concentration with a K(d) of 12.1+/-0.5 microM. Site-directed mutagenesis of selected residues (Trp(12), Phe(35), Lys(41), Asn(49), Gln(53), Ser(67) and Ile(118)) was employed, and the mutated enzymes were assessed for CB3GA binding. These results, together with molecular-modelling studies, established that the ligandin-binding site of GST I is located mainly in the hydrophobic binding site. The ability of VBAR to specifically inactivate GST I was exploited further to demonstrate the specific binding of several plant hormones and flavonoids to GST I. The inactivation of other GST isoenzymes by VBAR was also investigated, and it was concluded that VBAR may have wide applicability as an affinity label for probing structure-function relationships of GST isoenzymes.

Interaction of l-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography

Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of l-glutamate to α-ketoglutarate. Its kinetic constants for l-glutamate were measured equal to 2 mM for K m and 85.8 s −1 for k cat. BLAST search and amino acid sequence alignments revealed low homology to other l-amino acid oxidases (18–38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic α-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting l-glutamate oxidase activity. All but Cibacron ® Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye–ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a K i of 10.5 μM, indicating that the dye–enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg 1/2 ml 1/2/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for l-glutamate oxidase.

Protein separation with surfactant-coated polystyrene involving Cibacron Blue 3GA-conjugated Triton X-100

Through mixing of porous polystyrene particles (Amberlite XAD-4), non-ionic surfactants, and surfactant-conjugated substrates (affinity ligand) in an aqueous solution led to the formation of a novel medium (affinity admicelle) for protein separation. The ligand (CB–Triton) was synthesized by mixing a triazine dye (Cibacron Blue 3GA (CB)) and a polyoxyethylene-type non-ionic surfactant (Triton X-100) in weakly alkaline solutions. Triton X-100 and CB–Triton were competitively sorbed onto XAD-4. Albumin (bovine serum), alcohol dehydrogenase (yeast), and lysozyme (chicken egg) having specific interaction to CB were collected onto the affinity admicelle. On the other hand, the collection of ovalubmin (chicken egg white), having no binding ability to CB, was negligibly small. Lysozyme in 100 μl of chicken egg white, diluted with 900 μl of 10 mM Tris–HCl (pH 7.4), was successfully collected on 18 mg of CB–Triton admicelles and, then, it was eluted with 1 ml of aqueous solution of 100 mM phosphate (pH 7.4). The recovery based on the activity for the lysis of micrococcus and the concentration factor were 60% and 40 ( n=3), respectively.

Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing.

Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability.

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

κ-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.

Dense pellicular agarose–glass beads for expanded bed application:: Fabrication and characterization for effective protein adsorption

Dense pellicular composite solid matrices had been prepared by coating 4% agarose gel onto glass beads by a water-in-oil emulsification method. Two pellicular matrices, namely agarose-coated small glass bead (AG-S, 70–190 μm, mean size 120 μm) and agarose-coated large glass beads (AG-L, 130–280 μm, mean size 200 μm), were fabricated. The densities of AG-S and AG-L were as high as 1.77 and 1.98 g/ml, and the average depth of the agarose pellicle was estimated to be 20–30 μm, respectively. The matrices were modified with Cibacron Blue 3GA (CB) and lysozyme adsorption equilibrium and kinetic properties of the dye–ligand adsorbents were evaluated with the Langmuir equation and pore diffusion model, respectively. Stability of the matrices in basic solution and in recycled use was confirmed. It is found that the apparent pore diffusion coefficient of lysozyme in the pellicular beads was over two times higher than that in a homogeneous matrix due to their pellicular assembly. At nearly same the liquid velocity, the expanded bed of AG-S had approximately twofold higher plate number than that of AG-L, so the expanded bed of the former was much more efficient than that of the latter. Moreover, due to the rapid mass transfer property of the pellicular adsorbent CB-AG-S, its expanded bed showed a dynamic capacity over twice that of a commercial homogeneous medium designed for expanded bed application.

Affinity separations using microfabricated microfluidic devices:In situ photopolymerization and use in protein separations

The use of microfabricated microfluidic devices offers significant advantages over current technologies including fast analysis time and small reagent requirements. In the context of proteomic research, the possibility of using affinity-based separations for prefractionation of samples using microfluidic devices has significant potential. We demonstrate the use of microscale devices to achieve affinity separations of proteins using a device fabricated from borosilicate glass wafers. Photolithography and wet etching are used to pattern individual glass wafers and the wafers are fusion bonded at 650°C to obtain enclosed channels. A polymer has been successfully polymerizedin situ and used either as a frit for packing beads or, when derivatized with Cibacron Blue 3GA, as a separation matrix. Both of these technologies are based onin situ UV photopolymerization of glycidyl methacrylate (GMA) and trimethylolpropane trimethacrylate (TRIM) in channels.

Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes

Immobilized metal affinity membranes (IMAMs) were prepared by immobilizing copper ions on microporous regenerated cellulose membranes through different types of chelating agents (dentate and triazine dye). The resulting chelator utilization percentages were 95% for iminodiacetic acid, 56% for N, N, N-tris(carboxymethyl)ethylenediamine, 52% for Cibacron blue 3GA, and 140% for Cibacron red 3BA. On the other hand, triazine dyes were slightly superior to dentate chelators on metal ion utilization for protein adsorption. In batch single-protein adsorptions, the protein adsorption capacity decreased with increasing molecular size and number of accessible surface histidine residues [lysozyme>bovine serum albumin (BSA)>γ-globulin], while the binding strength order was the opposite (γ-globulin>BSA>lysozyme). Moreover, the proportions of specific and nonspecific bindings were evaluated by varying pH and salt concentration conditions. A large fraction of the adsorption capacity was found to come from the nonspecific interactions for the prepared IMAMs. Lastly, batch three-protein adsorptions were performed and weak adsorption competition was observed.

Constitutional isomers of Reactive Blue 2 – selective P2Y-receptor antagonists?

The anthraquinone derivative Reactive Blue 2 (RB 2) is one of the most widely used P2-receptor antagonists, still claimed to be P2Y-selective. RB 2 is defined as a mixture of two constitutional isomers and commercially available in different identity and purity. A sample of RB 2, offered for sale by RBI, purchased from Biotrend, Köln, Germany, was chromatographically purified and identified by 1H- and 13C-NMR studies as a 35:65 mixture of the terminal ring F meta and para constitutional isomers. The two constitutional isomers of RB 2 were synthesised and tested alongside with the ortho isomer Cibacron Blue 3GA (CB 3GA) on contractions of the rat vas deferens (RVD) elicited by α,β-methylene ATP (α,β-MeATP), mediated by P2X 1-receptors, and relaxations of the carbachol-precontracted guinea pig taenia coli elicited by adenosine 5′- O-(2-thiophosphate) (ADPβS), mediated by P2Y 1-like-receptors. All compounds inhibited the α,β-MeATP induced contraction of the RVD and the ADPβS induced relaxation of the carbachol precontracted guinea-pig taenia coli. The IC 50-values at P2X 1-R were 9.1 μM for CB 3GA, 28.4 μM for RB 2, 19.7 μM for RB 2 meta, and 35.5 μM for RB 2 para. The IC 50-values at P2Y 1-like-R were 17.4 μM for CB 3GA, 7.7 μM for RB 2, 12.0 μM for RB 2 meta, and 2.6 μM for RB 2 para. The results clearly show that neither RB 2 as a mixture nor the pure ortho and meta isomer are P2Y 1-like- versus P2X 1-selective antagonists. In contrast the pure para-isomer of RB 2 is a moderately P2Y 1-like- versus P2X 1-selective antagonist.

A novel chloride conductance activated by extracellular ATP in mouse parotid acinar cells

Salivary gland fluid secretion is driven by transepithelial Cl− movement involving an apical Cl− channel whose molecular identity remains unknown. Extracellular ATP (ATPo) has been shown to activate a Cl− conductance (IATPCl) in secretory epithelia; to gain further insight into IATPCl in mouse parotid acinar cells, we investigated the effects of ATPo using the whole-cell patch-clamp technique. ATPo and 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (Bz-ATP) produced concentration-dependent, time-independent Cl− currents with an EC50 of 160 and 15 µm, respectively. IATPCl displayed a selectivity sequence of SCN− > I−= NO3− > Cl− > glutamate, similar to the Cl− channels activated by Ca2+, cAMP and cell swelling in acinar cells. In contrast, IATPCl was insensitive to pharmacological agents that are known to inhibit these latter Cl− channels, was independent of Ca2+ and was not regulated by cell volume. Moreover, the IATPCl magnitude from wild-type animals was comparable to that from mice with null mutations in the Cftr, Clcn3 and Clcn2Cl− channel genes. Taken together, our results demonstrate that IATPCl is distinct from the channels described previously in acinar cells. The activation of IATPCl by Bz-ATP suggests that P2 nucleotide receptors are involved. However, inhibition of G-protein activation with GDP-β-S failed to block IATPCl, and Cibacron Blue 3GA and 4,4′-diisothyocyanostilbene-2,2′-disulphonic disodium salt selectively inhibited the Na+ currents (presumably through P2X receptors) without altering IATPCl, suggesting that neither P2Y nor P2X receptors are likely to be involved in IATPCl activation. We conclude that IATPCl is not associated with Cl− channels previously characterized in mouse parotid acinar cells, nor is it dependent on P2 nucleotide receptor stimulation. IATPCl expressed in acinar cells reflects the activation of a novel ATP-gated Cl− channel that may play an important physiological role in salivary gland fluid secretion.

ATP regulation of epithelial Cl- channels - new challenges?

ATP regulation of epithelial Cl- channels - new challenges?

ATP regulation of epithelial Cl- channels--new challenges?

ATP regulation of epithelial Cl- channels--new challenges?