Cibacron Blue F3GA Research Articles

Glucose oxidase and catalase adsorption onto Cibacron Blue F3GA-attached microporous polyamide hollow-fibres

The aim of this study was to explore in detail the performance of polyamide hollow fibers to which Cibacron Blue F3GA was attached for adsorption of proteins. Model proteins were glucose oxidase, as a flavo-enzyme and contains two tightly bound flavine adenine dinucleotide cofactor, and catalase as a heme-containing metallo-enzyme. The hollow fiber structure was characterized by scanning electron microscopy. These dye-carrying hollow-fibers (35.8 μmol/g) were used in the glucose oxidase and catalase adsorption–elution studies. The non-specific adsorption values were 1.25 mg/g for glucose oxidase and 1.97 mg/g for catalase. Cibacron Blue F3GA attachment increased the adsorption capacity up to 248 mg/g. Langmuir adsorption model was found to be applicable in interpreting glucose oxidase and catalase adsorption by Cibacron Blue F3GA-attached hollow fibers. Significant amount of the adsorbed proteins (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0. In order to determine the effects of adsorption and elution conditions on possible conformational changes of studied protein structures, fluorescence spectrophotometry was employed. It was concluded that polyamide dye-affinity hollow-fibers can be applied for glucose oxidase and catalase adsorption without causing any significant conformational changes. Repeated adsorption/elution processes showed that these dye-attached hollow-fibers are suitable for glucose oxidase and catalase separation.

One step purification of alpha 1-acid glycoprotein from human plasma : Fractionation of its polymorphic allele products

Alpha 1-acid glycoprotein is a plasma protein that exhibits both microheterogeneity and polymorphism. Its purification from human plasma is usually performed using a sequence of different fractionation steps. Here we report a one-step isolation technique of this protein based upon pseudo-ligand affinity chromatography on immobilized Cibacron Blue F3GA at acidic pH. In addition, the use of two narrow pH elution buffers allows us to separate the two genetic products of this protein, which differ from each other by 21 amino acid substitutions. This technique will facilitate the study of the structural, biological and pharmacokinetic properties of each individual allele product.

Metal‐chelated polyamide hollow fibers for human serum albumin separation

AbstractWe modified microporous polyamide hollow fibers by acid hydrolysis to amplify the reactive groups and subsequent binding of Cibacron Blue F3GA. Then, we loaded the Cibacron Blue F3GA‐attached hollow fibers with different metal ions (Cu2+, Ni2+, and Co2+) to form the metal chelates. We characterized the hollow fibers by scanning electron microscopy. The effect of pH and initial concentration of human serum albumin (HSA) on the adsorption of HSA to the metal‐chelated hollow fibers were examined in a batch system. Dye‐ and metal‐chelated hollow fibers had a higher HSA adsorption capacity and showed less nonspecific protein adsorption. The nonspecific adsorption of HSA onto the polyamide hollow fibers was 6.0 mg/g. Cibacron Blue F3GA immobilization onto the hollow fibers increased HSA adsorption up to 147 mg/g. Metal‐chelated hollow fibers showed further increases in the adsorption capacity. The maximum adsorption capacities of Co2+‐, Cu2+‐, and Ni2+‐chelated hollow fibers were 195, 226, and 289 mg/g, respectively. The recognition range of metal ions for HSA from human serum followed the order: Ni(II) > Cu(II) > Co(II). A higher HSA adsorption was observed from human serum (324 mg/g). A significant amount of the adsorbed HSA (up to 99%) was eluted for 1 h in the elution medium containing 1.0M sodium thiocyanide (NaSCN) at pH 8.0 and 25 mM ethylenediaminetetraacetic acid at pH 4.9. Repeated adsorption–desorption processes showed that these metal‐chelated polyamide hollow fibers were suitable for HSA adsorption. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3346–3354, 2002

Human serum albumin (HSA) adsorption with chitosan microspheres

AbstractIn this study, chitosan microspheres were prepared and characterized for adsorption of human serum albumin (HSA) as affinity sorbent. The chitosan microspheres were obtained with a “suspension crosslinking technique” in the size range of 30–700 μm by using a crosslinker, i.e., glutaraldehyde. The chitosan microspheres used in HSA adsorption studies were having the average size of 170 ± 81 μm. Adsorption medium pH and the initial HSA concentration in the adsorption medium were changed as 4.0–7.0 and 0.5–2.0 mg HSA/mL, respectively, to investigate the HSA adsorption capacity of chitosan microspheres. Maximum HSA adsorption (i.e., 11.35 mg HSA/g chitosan microspheres) was obtained at pH 5.0 and 1.5 mg HSA/mL of the initial HSA concentration in the adsorption medium was obtained as the saturation value for HSA adsorption. A very common dye ligand, i.e., Cibacron Blue F3GA was attached to the chitosan microspheres to increase the HSA adsorption capacity. Actually, the HSA adsorption capacity was increased up to 15.35 mg HSA/g chitosan microspheres in the case of Cibacron Blue F3GA attached to chitosan microspheres used. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3035–3039, 2002

Preparation and chromatographic behavior of acetate-fiber filter rods with dye affinity ligands

The dyes cibacron blue F3GA and reactive red 120 have been immobilized on acetate fiber filter rods to produce potential affinity matrixes. The isothermal adsorption of bovine serum albumin or human serum albumin on these matrixes was investigated and proved to conform to the Freundlich equation. In the static adsorption of human plasma the adsorption capacity for human serum albumin was 12.5 mg g−1 fiber filter and one band was observed in SDS-PAGE electrophoresis of human serum albumin isolated by the immobilized cibacron blue F3GA filter rod. Ligand utilization efficiencies and breakthrough volumes were obtained for adsorption of HSA on the cibacron blue F3GA filter rod when the feed-rates were from 0.5 to 6 mL min−1. In the affinity chromatography of human plasma the yield of human serum albumin was 1.27 g per 100 mL plasma.

Purification of Wheat Flour High- Mr Glutenin Subunits by Dye-ligand Chromatography

Abstract Here is reported the first successful attempt of high- Mr glutenin subunits separation by dye-ligand chromatography of Alisei 1 (containing 1Bx7, 1Dx2+1Dy12 subunits), Alisei 2 (containing 1Bx7 and 1Dy12 subunits), and WRU 6979 (containing 1Dx5+1Dy10 subunits), flours, using Cibacron Blue F3GA-Sepharose CL6B as resin. High- Mr glutenin subunits interacted strongly with the Cibacron Blue F3GA ligand and their separation, together with high recovery, was successful by using sodium dodecyl sulphate as eluting agent. Experimental evidence and amino acid sequences of high- Mr glutenin subunits suggested a prevalent electrostatic interaction between high-Mr glutenin subunits and the Cibacron Blue F3GA dye.

Affinity partitioning of glucose-6-phosphate dehydrogenase and hexokinase in aqueous two-phase systems with free triazine dye ligands

The partitioning of glucose-6-phosphate dehydrogenase (G6PDH) (E.C. 1.1.1.49) and hexokinase (E.C. 2.7.1.1) in polyethylene glycol (PEG)–hydroxypropyl starch (PES) and PEG–phosphate aqueous two-phase systems was investigated with free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, as their affinity ligands. It was found that the free reactive triazine dyes, not bound to phase-forming polymers, preferentially partitioned in the top-PEG phase in the PEG–salt and PEG–PES systems. The effect of various parameters such as type and concentration of affinity ligands, pH of the system, molecular mass of PEG and phase composition on partitioning of the enzymes was estimated. Phosphate is a key factor affecting the enzyme partitioning in the PEG–PES system. Cibacron F3GA changed the partition coefficient of G6PDH from 0.73 to 1.59.

Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer.

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme.

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.

Selective adsorption of serum albumin on biomedical polyurethanes modified by a poly(ethylene oxide) coupling-polymer with cibacron blue (F3G-A) endgroups.

A tri-block-coupling polymer, "PEO-MDI-PEO" ["poly(ethylene oxide)-4,4'-methylene diphenyl diisocyanate-poly(ethylene oxide)", abbreviated "MPEO"], was used to react with a triazine dye, Cibacron Blue F3G-A (ciba), in an alkaline environment. The product of this nucleophilic reaction was a penta-block-coupling polymer, "ciba-PEO-MDI-PEO-ciba" (abbreviated "cibaMPEO"). The cibaMPEO-modified poly(ether urethane) (PEU) surfaces were prepared by dip-coating and detected by XPS. The surface enrichment of both ciba endgroups and poly(ethylene oxide) spacer-arms was revealed. On the modified surfaces, bovine serum albumin (BSA)-adsorbing experiments were carried out, respectively, in the low and high BSA bulk-concentration solutions, and accordingly, the methods of radioactive (125)I-probe and ATR-FTIR were, respectively, employed for the characterization. The competitive adsorption of BSA and bovine serum fibrinogen (Fg) in the BSA-Fg binary solutions was also studied using a (125)I-probe, and through which the reversibly BSA-selective adsorption on cibaMPEO-modified PEU surfaces was confirmed. Finally, the improvement of blood-compatibility on the modified surfaces was verified by the plasma recalcification time (PRT) test.

Bilirubin removal from human plasma by dye affinity microporous hollow fibers

Bioaffinity adsorption has a unique and powerful role as a support tool in the removal of toxic substances from human plasma. Synthetic hollow-fiber membranes have advantages as support matrices in comparison to conventional hemoperfusion columns because they are not compressible and they eliminate internal diffusion limitations. In this study, Cibacron Blue F3GA was covalently attached onto commercially available microporous polyamide hollow-fiber membranes for bilirubin removal from hyperbilirubinemic human plasma. Different amounts of Cibacron Blue F3GA were attached on the polyamide hollow-fibers by changing the dye-attachment conditions, i.e., initial dye concentration, addition of sodium carbonate, and sodium chloride. The maximum amount of Cibacron Blue F3GA attachment was obtained at 42.5 μmol g−1 when the hollow fibers were treated with 3 M HCl for 30 min before performing the dye attachment. The nonspecific bilirubin adsorption on the unmodified polyamide hollow-fiber membranes was 0.65 mg g−1 from human plasma. Higher bilirubin adsorption capacities, of up to 39.7 mg g−1, were obtained with the Cibacron Blue F3GA-attached polyamide hollow-fiber membranes. Further increase in bilirubin adsorption was obtained as 48.9 mg g−1. Bilirubin molecules interacted with these adsorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was much pronounced. Bilirubin adsorption increased with increasing temperature and maximum adsorption was observed at 37°C.

Affinity extraction of dye- and metal ion-binding proteins in polyvinylpyrrolidone-based aqueous two-phase system.

Affinity extraction of dye- and metal ion-binding proteins, respectively, in a polyvinylpyrrolidone (PVP40)-Reppal PES 100 two-phase system was investigated. Due to the ability of PVP to complex azo dyes and inorganic ions, covalent coupling of the ligands was not essential. Cibacron Blue F3GA was used as the ligand for extraction of lactate dehydrogenase (LDH) from porcine muscle, while copper ions were used for extraction of B. stearothermophilus LDH with a fusion tag of six histidine residues (His6-LDH) from recombinant Escherichia coli homogenate. The binding strength of the enzymes to their respective ligands was only slightly reduced in the presence of PVP. The partition coefficient of Cibacron Blue and Cu2+ ions in the two-phase systems composed of different concentrations of PVP and Reppal was in the range of 20-30, with maximal partitioning being observed in the 17% (w/w) PVP40-10% Reppal PES100 system. Only a minor leakage of the ligands to the bottom phase was observed with time. The partitioning of porcine LDH to the PVP phase was increased 100-fold, and a maximal recovery of 89% was obtained in the two-phase system loaded with 0.2% (w/w) Cibacron Blue. The enzyme was quantitatively recovered with further purification from the PVP-dye phase using a secondary extraction step with 170 mM phosphate or alternatively with 100 mM phosphate containing NADH or NaCl. A more than 10-fold increase in the partition coefficient of His6-LDH was achieved in the two-phase system loaded with 0.4% (w/w) copper sulfate compared to the system lacking the metal ions. The enzyme was also back-extracted into phosphate phase in the presence of imidazole.

Comparison of adsorption performances of metal–chelated polyamide hollow fibre membranes in lysozyme separation

Commercially available microporous polyamide hollow fibres are modified by acid hydrolysis to activate the reactive groups and subsequently binding of the ligand, i.e. Cibacron Blue F3GA. Then the Cibacron Blue F3GA-derived hollow fibres were loaded with different metal ions (i.e. Zn(II), Cu(II), Ni(II)) to form the metal chelate. The internal polymer matrix was characterised by scanning electron microscopy. The effects of pH, initial concentration of lysozyme, metal type and temperature on the adsorption of lysozyme to the metal–chelated hollow fibres were examined in a batch reactor. The non-specific adsorption of lysozyme onto the polyamide hollow fibres was 1.8 mg/g. Cibacron Blue F3GA immobilisation increased the lysozyme adsorption up to 62.3 mg/g. Metal–chelated hollow fibres showed a significant increase of the adsorption efficiency. Lysozyme adsorption capacities of Zn(II), Cu(II) and Ni(II)-chelated hollow fibres were different. The maximum capacities of Zn(II), Cu(II) or Ni(II)-chelated hollow fibres were 144.2, 75.2 and 68.6 mg/g, respectively. Significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. Repeated adsorption–desorption process showed that this novel metal–chelated polyamide hollow fibres are suitable for lysozyme adsorption.

Preparation of cibacron blue F3GA‐attached polyamide hollow fibers for heavy metal removal

AbstractDye‐affinity adsorption has been used increasingly for heavy metal removal. Synthetic hollow fibers have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate internal diffusion limitations. The goal of this study was to investigate in detail the performance of hollow fibers composed of modified polyamide to which Cibacron Blue F3GA was attached for the removal of heavy metal ions. The Cibacron Blue F3GA loading was 1.2 mmol/g. The internal matrix was characterized by scanning electron microscopy. No significant changes in the hollow fiber cross‐section or outer layer morphology were observed after dye modification. The effect of the initial concentration of heavy metal ions and medium pH on the adsorption efficiency were studied in a batch reactor. The adsorption capacity of the hollow fibers for the selected metal ions [i.e., Cu(II), Zn(II) and Ni(II)] were investigated in aqueous media with different amounts of these ions (10–400 ppm) and at different pH values (3.0–7.0). The maximum adsorptions of metal ions onto the Cibacron Blue F3GA‐attached hollow fibers were 246.2 mg/g for Cu(II), 133.6 mg/g for Zn(II), and 332.7 mg/g for Ni(II). Furthermore, a Langmuir expression was calculated to extend the adsorption equilibrium. Nitric acid (0.1M) was chosen as the desorption solution. High desorption ratios (up to 97%) were observed in all cases. Consecutive adsorption/desorption operations showed the feasibility of repeated use of this novel sorbent system. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 3089–3098, 2002; DOI 10.1002/app.2338

SELECTIVE REMOVAL OF LEAD IONS BY POLYETHYLENE GLYCOL METHACRYLATE GEL BEADS CARRYING CIBACRON BLUE F3GA

Polyethylene glycol methacrylate (PEG-MA) gel beads carrying Cibacron Blue F3GA (42.6 μmol/g polymer) were prepared for the removal of Pb(II), Cu(II), and Cd(II) from aqueous solutions that contained different amounts of these ions (10–100 mg/L) and at different pH values (2.0–7.0). Adsorption rates were high, and adsorption equilibria were reached within 20 minutes. Adsorption of these metal ions on the unmodified PEG-MA gel beads was zero. The maximum adsorptions of heavy metal ions onto the Cibacron Blue F3GA–attached microbeads from single solutions were 23.3 mg/g (112.4 μmol/g) for Pb(II), 12.4 mg/g (110.3 μmol/g) for Cd(II), and 7.0 mg/g (110.2 μmol/g) for Cu(II). When the heavy metal ions competed (in the case of the adsorption from their mixture) the amounts of adsorption were 14.96 mg/g (72.2 μmol/g) for Pb(II), 0.72 mg/g (11.3 μmol/g) for Cu(II), and 1.10 mg/g (9.8 μmol/g) for Cd(II). Under competitive conditions, the system showed high selectivity for Pb(II) ions. The formation constants of Cibacron Blue F3GA–metal ion complexes were investigated through the application of the Ružić method. The calculated values of formation constants were 8.5 × 105 L/mol1 for Pb(II) dye, 2.5 × 105 L/mol for Cu(II) dye, and 8.2 × 104 L/mol for the Cd(II) dye complex. PEG-MA gel beads carrying Cibacron Blue F3GA can be regenerated through washing with a solution of nitric acid (0.1 mol/L). The maximum regeneration value was as high as 98.5%. These PEG-MA gel beads are suitable for more than 3 adsorption-desorption cycles without experiencing considerable loss of adsorption capacity.

Urea–formaldehyde resin monolith as a new packing material for affinity chromatography

A urea–formaldehyde resin (UF) continuous bed has been prepared through in-situ condensation polymerization in a confined tube. The monolith is an agglomerate of 2-μm irregular particles. Nitrogen adsorption shows that the monolith has a bimodal pore size distribution. It has low resistance to flow. A dyed monolith is obtained through modification of the UF monolith with Cibacron blue F3GA. Although its dye concentration and dynamic capacity are low compared to Sepharose type affinity media, the dyed monolith can separate some proteins in the affinity mode of liquid chromatography.

Packed-bed columns with dye-affinity microbeads for removal of heavy metal ions from aquatic systems

A dye ligand Cibacron Blue F3GA, was covalently coupled with polyhydroxyethylmethacrylate (PHEMA) microbeads in the 150–200 μm particle size range. The sorbent carrying 22.3 μmol Cibacron Blue F3GA per gram of polymer was then used to remove Pb(II), Cd(II), Cu(II) and Zn(II) from aqueous solutions in a packed-bed column system. Heavy metal ion adsorption capacity of the column was investigated as a function of heavy metal ion-bearing solution flow rate and the inlet heavy metal ion concentration. The maximum metal ion uptake values found were: 80.60, 96.98, 78.36, 103.98 μmol/g polymer for Pb(II), Cd(II), Cu(II) and Zn(II), respectively. The heavy metal adsorption capacity of the microbeads decreased with an increase in the circulation rate of aqueous solution. The order of affinity based on molar uptake was Zn(II)>Cd(II)>Pb(II)>Cu(II). Removal percentages of heavy metals related to flow time were determined for different flow rates and initial metal ion concentrations. It was observed that PHEMA microbeads carrying Cibacron Blue F3GA can be regenerated by washing with a solution of nitric acid (0.05 M). The desorption ratio was as high as 98.5%.

Therapeutic affinity adsorption of iron(III) with dye‐ and ferritin‐immobilized pHEMA adsorbent

AbstractMicroporous poly(2‐hydroxyethylmethacrylate) (pHEMA) films carrying cibacron blue F3GA, Congo red, and ferritin were prepared and used for iron(III) removal from human plasma. pHEMA films were produced by a photopolymerization of 2‐hydroxyethylmethacrylate in the presence of azobisisobutyronitrile. The reactive dye ligands cibacron blue F3GA, Congo red, and bioligand ferritin were then covalently attached to the pHEMA films. The maximum dye loadings were 1.07 and 0.80 μmol/cm2 for cibacron blue and Congo red, respectively. The maximum amount of ferritin attached was 1.04 × 10−3 μmol/cm2. Characterizations of the films were achieved by contact‐angle, water‐uptake, and scanning electron microscopy studies as well as atomic force microscopy images. The aqueous water‐uptake properties and contact angles (air underwater) of the pHEMA films did not change after derivatization with cibacron blue F3GA, Congo red, and ferritin. These hydrophilic films (contact angle = 45.3°), having a swelling ratio of 58% (w/w) and carrying cibacron blue F3GA, Congo red, and ferritin, were used in Fe(III) removal studies. The maximum amounts of Fe(III) removed from human plasma by cibacron blue F3GA‐, Congo red‐, and ferritin‐attached pHEMA films were 3.80 μg/cm2 for cibacron blue F3GA, 4.41 μg/cm2 for Congo red, and 8.1 μg/cm2 for ferritin‐attached films. Fe(III) ions could be repeatedly adsorbed and desorbed with these affinity pHEMA films without a noticeable loss in their Fe(III) adsorption capacity. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 186–194, 2001

Protein structure information from mass spectrometry? Selective titration of arginine residues by sulfonates

Noncovalently bound complexes between basic sites of peptides/proteins and sulfonates are studied using Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Reactive sulfonate dyes such as Cibacron Blue F3G-A are known to bind to protonated amino groups on the exterior of a protein. In this work, we examine a wide range of other sulfonates with distinctly simpler structure and more predictable reactivity. Naphthalene-sulfonic acid derivatives were found to bind to arginine only, as opposed to expected binding to all basic sites (Arg, Lys and His). Detailed control experiments were designed to unambigously confirm this selectivity and to rule out nonspecific adduct formation in the gas phase. The data show that the number of complex adducts found equals the number of accessible arginine sites on the surface of folded peptides and proteins, plus the N-terminus. Lys and His are not complexed nor are buried residues with hindered access. MALDI-MS can therefore provide fast information related to the exposed surface of these biomolecules. Additional titration experiments with 1-anilino-naphthalene-8-sulfonic acid (ANS) revealed that this fluorescent dye, which was often hypothesized to bind to so-called molten globule states of proteins, behaved exactly like all other naphthalene-sulfonic acids. ANS binding thus occurs largely through the sulfonate group.

Removal of chlorophenols from aquatic systems with dye-affinity microbeads

Cibacron Blue F3GA carrying pHEMA microbeads were investigated as dye-affinity sorbents for removal of chlorophenols (i.e. phenol, m-chlorophenol, p-chlorophenol and 2,4,6-trichlorophenol). PHEMA microbeads were prepared by modified suspension polymerization of HEMA in the presence of an initiator (azobisisobutyronitrile). These microbeads, with a swelling ratio of 55% and carrying 16.5 μmol Cibacron Blue F3GA/g polymer, were then used in the removal of chlorophenols from aqueous media. Adsorption rates of chlorophenols were very high, and equilibrium was achieved in about 20 min. The maximum adsorptions of chlorophenols onto the Cibacron Blue-carrying microbeads were 88.8 μmol/g for phenol, 94.6 μmol/g for 2,4,6-trichlorophenol, 97.6 μmol/g for p-chlorophenol and 109.1 μmol/g for m-chlorophenol. The affinity order was as follows: m-chlorophenol>p-chlorophenol>2,4,6-trichlorophenol>phenol. The adsorption of chlorophenols decreased with increasing pH. Desorption of chlorophenols was achieved using methanol solution (30%, v/v). The Cibacron Blue F3GA-carrying microbeads are suitable for repeated use for more than five cycles without any noticeable loss of adsorption capacity.