Immobilized Cibacron Blue F3GA Research Articles

Lipase purification by affinity precipitation with a thermo-responsive polymer immobilized Cibacron Blue F3GA ligand

A thermo-responsive polymer (PNNB) was synthesized with lower critical solution temperature 27.5°C and over 95% recovery. The adsorption of porcine pancreatic lipase on Cibacron Blue F3GA-conjugated PNNB (PNNB-CB) closely followed the bi-Langmuir adsorption isotherm. The maximum adsorption capacity was found at pH 5.0, with a ligand density of 18.4 μmol/g polymers. The optimized eluent was a 0.01 M phosphate buffer solution at pH 8.0 containing 20% ethylene glycol. Six adsorptiondesorption recycles indicated excellent reusability of the affinity adsorbent. PNNB-CB was applied to separate porcine pancreatic lipase from its crude material giving a lipase activity recovery of 81.6% with a 16-fold purification factor. Lipase could be purified to single-band purity, according to gel electrophoresis. The purification strategy is therefore feasible and efficient for purifying proteins of interest.

Adsorption of Waste Beer Yeast Modified with Cibacron Blue F-3GA for Lysozyme

Abstract A new dye affinity adsorbent was prepared by modifying glutaraldehyde-crosslinked waste beer yeast with Cibacron B1ue F-3GA. The amount of immobilized Cibacron Blue F-3GA on the crosslinked yeasts was 161.1 mg g−1. The effects of contact time, initial lysozyme concentration, medium pH, and ionic strength on the adsorption of lysozyme on modified yeasts were studied. The dye affinity adsorbents were used for purification of lysozyme from chicken egg white. The lysozyme adsorption capacity of the modified yeast was 229.1 mg g−1, and its adsorption performance was better than the unmodified yeast. The high desorption ratio (89.1%) was achieved by using 1.0 mM NaSCN as eluent, and the ratio of activity-preserving was 91.0%. In the extraction of lysozyme from chicken egg white, the purification fold was 26.7 and average recovery of enzyme activity was 73.5%. The adsorbents could be reused without significant decreases in the adsorption capacities.

Albumin adsorption on cibacron blue F3G-A immobilized onto oligo(ethylene glycol)-terminated self-assembled monolayers.

Self-assembled monolayers can be tailored with specific ligands to a certain protein and at the same time prevent the non-specific adsorption of other proteins. Cibacron Blue F3G-A (CB-thiol) was successfully immobilized onto tetra(ethylene glycol)-terminated alkanethiol (CB-thiol). The affinity of human serum albumin (HSA) to immobilized Cibacron Blue F3G-A was studied using mixed thiolate self-assembled monolayers on gold with different n-alkyl chain lengths and functional terminal groups (CH(3)-; OH- and tetra(ethylene glycol)). Surfaces were characterized using X-ray photoelectron spectroscopy and water contact angle measurements. Albumin adsorption and exchangeability of the adsorbed albumin molecules with other albumin molecules in solution were evaluated using (125)I-radiolabeled HSA. Competitive adsorption between albumin and fibrinogen to the different self-assembled monolayers (SAMs) was also investigated. Results showed that the incorporation of CB-thiol on the monolayers does not increase the HSA adsorption and reversibility on the SAMs. However, although specific adsorption of HSA to the immobilized Cibacron Blue F3G-A was not demonstrated, the presence of CB-thiol decreases the affinity of fibrinogen to the OH-terminated SAMs.

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.

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.

Performance of Different Metal–Dye Chelated Affinity Adsorbents of Poly(2-Hydroxyethyl Methacrylate) in Lysozyme Separation

The triazine dye Cibacron Blue F3GA was covalently immobilized as an affinity ligand onto microporous poly(2-hydroxyethyl methacrylate) (pHEMA) membranes. Three different metal ions [i. e., Fe(III), Zn(II), or Cu(II)] were then chelated with the immobilized Cibacron Blue F3GA molecules. Lysozyme adsorption onto these affinity adsorbents from aqueous solutions containing different amounts of lysozyme at different pH was investigated in a batch system. Lysozyme adsorption capacity of all of the metal-dye-immobilized membranes was greater than that of the dye-immobilized membranes. The nonspecific adsorption of the protein on the pHEMA membranes was negligible. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The maximum capacity (qm ) of the Fe(III)–Zn(II),–or Cu(II)–dye chelated membranes for lysozyme adsorption (384, 326, and 306 μg/cm2) was greater than that of the dye-immobilized membrane (224 μg lysozyme/cm2), respectively. The dissociation constant (kd ) values were found to be 2.51 × 10−7 M with dye-immobilized membrane, and 2.32 ×10−7×10−7and 2.40 ×10−7 M with the Fe(III)–Zn(II),–and Cu(II)–dye-chelated membranes, respectively. More than 95% of the adsorbed lysozyme was desorbed in 60 min in the desorption medium containing 0.5 M KSCN at pH 8.0.

Preparation and characterisation of Cibacron Blue F3G-A poly(ethylene) hollow-fibre affinity membranes

Poly(ethylene) hollow-fibre membranes with immobilised Cibacron Blue F3G-A were obtained in four different ways from epoxy-activated fibres. Membranes with a maximum capacity of 26 mg lysozyme ml−1and a dye density of 52 μmol ml−1were obtained when ammonia was used to open the epoxy group before dye immobilisation. Pure water flux of the modified membranes at 1 bar pressure was 1.0 cm min−1, thus meaning only a reduction of 1.5-fold with regard to the unmodified membranes. The support-dye bond was stable as judged by the unmodified capacity of the membranes and the negligible amount of dye leaked after 520 h of exposure to 6 M urea in 0.5 M NaOH.

Examination of protein adsorption in fluidized bed and packed bed columns at different temperatures using frontal chromatographic methods.

The influences of various experimental parameters on the dynamic adsorption capacity (DAC) and the dynamic adsorption rate (DAR) of a biomimetic affinity silica-based adsorbent in fluidized and packed bed columns operated under plug flow conditions and at different temperatures have been investigated with different inlet concentrations of hen egg white lysozyme (HEWL) and human serum albumin (HSA). The DACs as well as the DARs of both the fluidized and packed beds were examined at 10% saturation (i.e., at the QB value) and the experimental data compared with the corresponding data obtained from batch equilibrium adsorption procedures. Parameters examined included the fluid superficial velocity and protein concentration and their effect on the binding capacity and column efficiency. Consistent with various results reported from this and other laboratories on the behavior of biospecific affinity adsorbents derived from porous silica and zirconia particles, adsorbents prepared from Fractosil 1000 were found to exhibit appropriate rheological characteristics in fluidized bed systems under the experimental conditions. Moreover, changes in temperature resulted in a more significant effect on the breakthrough profiles of HSA compared to HEWL with the immobilized Cibacron Blue F3G-A with Fractosil 1000 adsorbent. This result suggests that temperature effects can possibly be employed profitably in some processes as part of a strategy to enhance column performance with fluidized bed systems for selective recovery of target proteins. At relatively low superficial velocities of the feed, the DARs with HEWL and HSA were similar for both the fluidized and packed bed column systems, whereas, at high superficial velocities, the DARs for these proteins were larger with the packed bed columns.

Bilirubin removal from human plasma in a packed-bed column system with dye-affinity microbeads

A dye-ligand, Cibacron Blue F3GA, was covalently coupled with the poly(EGDMA-HEMA) microbeads. The affinity sorbent carrying 16.5 μmol Cibacron Blue F3GA per gram polymer was then used to remove bilirubin from human plasma in a packed-bed column system. Bilirubin adsorption from human plasma on the unmodified poly(EGDMA-HEMA) microbeads was 0.32 mg/g, while much higher adsorption values, up to 24.2 mg/g, were obtained with the dye-attached microbeads. The bilirubin adsorption capacity of the microbeads decreased with an increase in the recirculation rate of plasma. Bilirubin adsorption increased with increasing temperature, and the maximum adsorption achieved at 37°C (32.5 mg bilirubin/g polymer). Bilirubin molecules interacted directly with the immobilized Cibacron Blue F3GA molecules. Contribution of albumin adsorption on bilirubin adsorption was also significant.

Dye–ligand and metal chelate poly(2-hydroxyethylmethacrylate) membranes for affinity separation of proteins

Cibacron Blue F3GA was covalently immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) membranes via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl group of pHEMA. Then, Fe 3+ ions were complexed by chelation with the immobilized Cibacron Blue F3GA molecules. Different amounts of Fe 3+ ions were loaded on the membranes by changing the concentration of Fe 3+ ions and pH of the reaction medium. Membranes with or without Fe 3+ were used in the adsorption of glucose oxidase, catalase and bovine serum albumin. The adsorption capacities of these membranes were determined by changing pH and the concentration of the proteins in the adsorption medium. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The maximum capacities ( q m) of the Fe 3+ complexed membranes for glucose oxidase, catalase and bovine serum albumin (8.70·10 − 3 μmol m − 2, 2.15·10 − 3 μmol m − 2 and 2.21·10 − 3 μmol m − 2) were greater than those of the untreated membranes (6.79·10 − 3 μmol m − 2, 1.34·10 − 3 μmol m − 2 and 1.94·10 − 3 μmol m − 2) respectively. The nonspecific adsorption of the enzymes and the protein on the pHEMA membranes was negligible.

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.

Two-step chromatographic purification of human plasma α1-acid glycoprotein Its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent

α 1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (F1 s*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1 s*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A ∗AD, S/A ∗X 0 and F1/A ∗C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X 0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.

Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner.

Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.

PH titration curves of the desialylated human α 1-acid glycoprotein variants by combined isoelectrofocusing—electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent

Using a two-dimensional isoelectrofocusing (IEF)—electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human α 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then desialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5–9.5) and a narrow gradient (pH 5–8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5–5.5 and pH 7.5–9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose—Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.

Interacting Properties of Bovine Lactoferrin with Immobilized Cibacron Blue F3GA in Column Chromatography

Bovine lactoferrin, isolated from colostral milk, interacted strongly with immobilized Cibacron blue F3GA column. Lactoferrin, adsorbed on the dye column, could not be eluted by 8 M urea, 1% Triton X-100, and 75% ethylene glycol, but was eluted by .1 M sodium hydroxide, 1 M potassium thiocyanate, 3 M potassium chloride and free Cibacron blue F3GA. Electrostatic forces between the sulfonic groups of Cibacron blue F3GA and the basic side-chain groups in lactoferrin molecule probably are responsible for the observed interaction. The elution profile for lactoferrin differed from those of lactoperoxidase and serum albumin, which might allow efficient isolation of lactoferrin from whey via affinity chromatography.

High-performance anion-exchange chromatographic study of desialylated human α 1-acid glycoprotein variants : Development of a fractionation method for the protein slow variants

The three main desialylated variants (F1, S and A) of human α 1-acid glycoprotein (AAG) a serum acute-phase reactant, were analysed by high-performance anion-exchange chromatography in order to determine their optimum separation conditions. The analysis consisted of three steps, as follows: (1) A desialylated commercial AAG was separated into one “fast”- and one “slow”-migrating fraction by preparative isoelectrofocusing. The “fast” and “slow” fractions were shown to contain the F1 variant and a mixture of the S and A variants, respectively. (2) The pH titration curves of these two fractions were then measured by strong anion-exchange chromatography with several buffer systems of increasing pH. From the data obtained, it was not possible to select the optimum conditions to separate the “fast” variant F1 from the “slow” variants A and S. However, the S and A variants were shown to ionize very differently. (3) The specific fractionation of the S and A variants was therefore carried out by anion-exchange chromatography under operating conditions based on the data obtained from the study of their pH titration curves. This was performed both with the “slow”-migrating fraction obtained by preparative isoelectrofocusing of commercial AAG and with an AAG (containing only variants S and A) purified from an individual serum on immobilized Cibacron Blue F3G-A. Identification of the fractionated proteins was achieved by analytical isoelectrofocusing.

Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with Blue Sepharose CL-6B affinity chromatography

Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. • Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaC1 were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. • A high performance liquidd immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.

Antimicrobial supplementation of growing pigs: the effect of porcine sera fractions on in vitro muscle cell proliferation.

Sera obtained from pigs before and after subtherapeutic levels of ASP250 supplementation (pre and post serum pools) have been subjected to comparative fractionation by using gel filtration and affinity chromatography on immobilized Cibacron Blue F3G-A. Comparable serum fractions obtained from pre- and post-ASP250 blood sera were assayed in muscle cell culture bioassays designed to measure their effect on proliferation. Pre- and post-ASP250 sera were subjected to gel filtration and divided into the following fractions: fraction 1, Kav less than .17; fraction 2, Kav = .17 to .41; fraction 3, Kav = .41 to .59. Post-ASP250 fractions 2 and 3 increased proliferation rate in cultured muscle cells to a greater extent than comparable pre-ASP250 fractions (P less than .001). Chromatography of fraction 3 on immobilized Cibacron Blue F3G-A showed that both pre- and post-ASP250 fraction 3 contained a putative inhibitor of myogenic cell proliferation as well as mitogenic factors. However, negative growth factor activity was greater in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). Additionally, positive growth factor activity was lower in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). These data suggest that levels and(or) activities of both positive and negative muscle growth factors in serum may be altered by the addition of antimicrobials to the diets of growing pigs.

Affinity chromatography of human plasma gelsolin with polyphosphate compounds on immobilized Cibacron Blue F3GA

Human plasma gelsolin was specifically eluted from a Cibacron Blue F3GA column with 1 m M adenosine, guanosine, cytidine and uridine di- and triphosphates, except for cytidine 5′-diphosphate. Inorganic polyphosphates also eluted gelsolin, but neither nicotinamide adenine dinucleotide nor nicotinamide adenine dinucleotide phosphate did so. The results suggest that a terminal pyrophosphate structure might be essential for the specific elution. After elution with 1 m M adenosine 5′-triphosphate, additional gelsolin was eluted by washing the column with a high salt concentration. This indicates that human plasma gelsolin may bind to Cibacron Blue F3GA in at least two different ways.

Large-Scale Purification of Bovine Brain Lactate Dehydrogenase by Affinity Chromatography on Immobilized Colchicine

Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction.