Serap Şenel

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

Abstract 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.

Nanospines incorporation into the structure of the hydrophobic cryogels via novel cryogelation method: An alternative sorbent for plasmid DNA purification

In this study, it was aimed to prepare hydrophobic cryogels for plasmid DNA (pDNA) purification from Escherichia coli lysate. The hydrophobicity was achieved by incorporating a hydrophobic ligand, N-methacryloyl-(L)-phenylalanine (MAPA), into the cryogel backbone. In addition to the conventional cryogelation process, freeze-drying step was included to create nanospines. Three different cryogels {poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine)-freeze dried, [P(HEMA-MAPA)-FD]; poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine, [P(HEMA-MAPA)] and poly(2-hydoxyethyl methacrylate)-freeze dried, [P(HEMA)-FD]} were prepared, characterized, and used for DNA (salmon sperm DNA) adsorption studies from aqueous solution. The specific surface areas of cryogels were determined to be 21.4 m(2)/g for P(HEMA)-FD, 17.65 m(2)/g for P(HEMA-MAPA) and 36.0 m(2)/g for P(HEMA-MAPA)-FD. The parameters affecting adsorption such as temperature, initial DNA concentration, salt type and concentration were examined in continuous mode. The maximum adsorption capacities were observed as 45.31 mg DNA/g, 27.08 mg DNA/g and 1.81 mg DNA/g for P(HEMA-MAPA)-FD, P(HEMA-MAPA) and P(HEMA)-FD, respectively. Desorption process was performed using acetate buffer (pH 5.50) without salt. First, pDNA was isolated from E. coli lysate and the purity of pDNA was then determined by agarose gel electrophoresis. Finally, the chromatographic performance of P(HEMA-MAPA)-FD cryogel for pDNA purification was tested in FPLC. The resolution (R(s)) was 2.84, and the specific selectivity for pDNA was 237.5-folds greater than all impurities.

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.

Zinc ion-promoted adsorption of lysozyme to Cibacron Blue F3GA-attached microporous polyamide hollow-fiber membranes

Abstract Dye-affinity and metal chelate affinity adsorption are increasingly used for protein separation. Synthetic hollow fiber membranes 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 explore in detail the performance of hollow fibers composed of modified polyamide to which Cibacron Blue F3GA and Zn(II) were attached for adsorption of lysozyme. The polymer matrix was characterized by scanning electron microscopy. These dye-affinity and Zn(II) chelated hollow-fibers were used in the lysozyme adsorption–elution studies. The effects of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye-attached and metal-chelated hollow-fibers were studied in a batch reactor. The effect of Zn(II) loading on lysozyme adsorption was also studied. The non-specific adsorption of lysozyme on the polyamide hollow-fibers was 1.8 mg g −1 . Cibacron Blue F3GA attachment significantly increased the lysozyme adsorption up to 63.2 mg g −1 . Lysozyme adsorption capacity of the Zn(II) chelated hollow-fibers (144.2 mg g −1 ) was greater than that of the Cibacron Blue F3GA-attached hollow-fibers. A 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. In order to examine the effects of separation conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We conclude that dye- and metal-chelate affinity chromatography with polyamide hollow-fibers can be applied for lysozyme adsorption without causing any significant conformational changes and denaturation. Repeated adsorption/elution processes showed that these novel dye-attached and Zn(II) chelated hollow-fibers are suitable for lysozyme adsorption.

Chiral recognition of proteins having L-histidine residues on the surface with lanthanide ion complex incorporated-molecularly imprinted fluorescent nanoparticles

In this study, lanthanide ion complex incorporated molecularly imprinted fluorescent nanoparticles were synthesized. A combination of three novel approaches was applied for the purpose. First, lanthanide ions [Terbium(III)] were complexed with N-methacryloyl-L-histidine (MAH), polymerizable derivative of L-histidine amino acid, in order to incorporate the complex directly into the polymeric backbone. At the second stage, L-histidine molecules imprinted nanoparticles were utilized instead of whole protein imprinting in order to avoid whole drawbacks such as fragility, complexity, denaturation tendency, and conformation dependency. At the third stage following the first two steps mentioned above, imprinted L-histidine was coordinated with cupric ions [Cu(II)] to conduct the study under mild conditions. Then, molecularly imprinted fluorescent nanoparticles synthesized were used for L-histidine adsorption from aqueous solution to optimize conditions for adsorption and fluorimetric detection. Finally, usability of nanoparticles was investigated for chiral biorecognition using stereoisomer, D-histidine, racemic mixture, D,L-histidine, proteins with surface L-histidine residue, lysozyme, cytochrome C, or without ribonuclease A. The results revealed that the proposed polymerization strategy could make significant contribution to the solution of chronic problems of fluorescent component introduction into polymers. Additionally, the fluorescent nanoparticles reported here could be used for selective separation and fluorescent monitoring purposes.

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

Abstract 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 and characterization of thiophilic cryogels with 2-mercapto ethanol as the ligand for IgG purification.

In this study, thiophilic cryogels were prepared by two different approaches and they were used in purification of IgG from aqueous solutions and human plasma. In the first approach, poly(2-hydoxyethyl methacrylate) [PHEMA] cryogel disks were prepared. The PHEMA cryogel disks were activated by divinylsulfone (DVS) and 2-mercapto ethanol was attached. In the second approach, poly(2-hydroxyethyl methacrylate-etylene glycol dimethacrylate) [P(HEMA-EGDMA)] beads were synthesized and 2-mercapto ethanol was attached to the beads as a thiophilic ligand. In order to increase the surface area, P(HEMA-EGDMA)/PHEMA composite cryogel disks were prepared by embedding the P(HEMA-EGDMA) beads into PHEMA cryogels. Both the thiophilic PHEMA (T-PHEMA) cryogel disks and the thiophilic P(HEMA-EGDMA)/PHEMA [T-P(HEMA-EGDMA)/PHEMA] composite cryogel disks were characterized by Fourier transform infrared spectroscopy, surface area measurements, elemental analysis, swelling tests and scanning electron microscopy. The effects of salt concentration, pH, temperature, initial IgG concentration were analysed for IgG adsorption from aqueous solutions in batch mode. When lyotropic salt, Na2SO4, was used, the adsorption capacity was 27.5 mg/g and 68.7 mg/g for T-PHEMA and the T-P(HEMA-EGDMA)/PHEMA composite cryogel disks, respectively. 1.0 M NaCl was used as desorption agent. The change in adsorption capacity was not remarkable for a repetition of adsorption-desorption cycle ten times. Both the thiophilic cryogel disks and the thiophilic composite cryogel disks were also successfully used for IgG isolation from human plasma. The purity assayed by SDS-PAGE was 89%. The adsorption capacities were 74.8 mg/g and 137.4 mg/g in human plasma samples for the T-PHEMA cryogel disks and the T-P(HEMA-EGDMA)/PHEMA composite cryogel disks, respectively.

Poly(hydroxyethylmethacrylate‐N‐methacryloyl‐(L)‐histidine‐methyl‐ester) Based Metal‐Chelate Affinity Adsorbent for Separation of Lysozyme

Abstract Comonomer and/or metal‐chelating ligand N‐methacryloyl‐(L)‐histidine‐methylester (MAH) was synthesized by using methacryloyl chloride and L‐histidine methyl ester. Spherical beads with an average diameter of 75–125 µm were produced by suspension polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Poly(HEMA‐MAH) beads had a specific surface area of 18.3 m2/g. Elemental analysis of MAH for nitrogen was estimated as 895 µmol/g of polymer. Then the beads were loaded with different metal ions (i.e. Zn2+, Cu2+, Ni2+) to form the metal chelate. The effect of pH, concentration of lysozyme, and metal type on the adsorption of lysozyme to the metal‐chelated beads was examined in a batch reactor. Purification of lysozyme from egg‐white was also investigated. Maximum lysozyme adsorption capacity of poly(HEMA‐MAH) beads was found to be 8.7 mg/g at pH 7.0 in phosphate buffer. Lysozyme adsorption capacity of Zn2+, Cu2+, and Ni2+‐chelated beads was higher than that of non‐chelated beads. The maximum capacities of Ni2+, Zn2+, or Cu2+‐chelated beads were 11.5, 12.6, and 37.1 mg/g, respectively. A significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 25 mM EDTA at pH 4.9. Repeated adsorption‐desorption process showed that this novel metal chelated beads are suitable for lysozyme adsorption. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 80% with recovery about 75%.

New metal chelate sorbent for albumin adsorption : Cibacron Blue F3GA-Zn(II) attached microporous poly(HEMA) membranes

Poly(2-hydroxyethyl methacrylate) [poly(HEMA)] membranes were prepared by UV-initiated photopolymerization of HEMA in the presence of an initiator (α-α′-azobis-isobutyronitrile, AIBN). The triazine dye Cibacron Blue F3GA was attached as an affinity ligand to poly(HEMA) membranes, covalently. These affinity membranes with a swelling ratio of 58% and containing 10.7 mmol Cibacron Blue F3GA/m2 were used in the albumin adsorption studies. After dye-attachment, Zn(II) ions were chelated within the membranes via attached-dye molecules. Different amounts of Zn(II) ions [650–1440 mg Zn(II)/m2] were loaded on the membranes by changing the initial concentration of Zn(II) ions and pH. Bovine serum albumin (BSA) adsorption on these membranes from aqueous solutions containing different amounts of BSA at different pH was investigated in batch reactors. The nonspecific adsorption of BSA on the poly(HEMA) membranes was negligible. Cibacron Blue F3GA attachment significantly increased the BSA adsorption up to 92.1 mg BSA/m2. Adsorption capacity was further increased when Zn(II) ions were attached (up to 144.8 mg BSA m2). More than 90% of the adsorbed BSA was desorbed in 1 h in the desorption medium containing 0.5M NaSCN at pH 8.0 and 0.025M EDTA at pH 4.9.

Synthesis of L-lysine imprinted cryogels for immunoglobulin G adsorption.

L-Lysine imprinted poly(2-hydroxyethyl methacrylate-co-N-methacryloyl-L-aspartic acid) [P(HEMA-co-MAAsp)] cryogels were synthesized and characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, surface area measurements, swelling, and squeezing tests. Specific surface area for imprinted cryogel was 34.2m(2)/g while the value was 21.3m(2)/g for non-imprinted cryogel. IgG adsorption from aqueous solution was examined in continuous mode examining the factors effecting adsorption capacity such as pH, concentration, flow rate, temperature, ionic strength, and incubation time. 0.5M NaCl was used as desorption agent. The IgG adsorption capacity was determined as 55.1 mg/g for 1.0 mg/mL IgG original concentration at 25.0°C while pH and flow rate were 7.0 and 0.5 mL/min, respectively. When human serum was used as IgG source, the removal of 90.4% of crude IgG was attained for 1/20 diluted plasma sample. The imprinted cryogel was used in ten successive cycles without significant loss in adsorption capacity. The cryogel was determined to be 1.79 times more selective to IgG than albumin and 1.45 times more selective than hemoglobin. The adsorption behavior well suited to Langmuir isotherm and the kinetics followed pseudo-second-order model. Thermodynamic parameters ΔH°, ΔS° and ΔG° for this adsorption process were also calculated.