Triazine dye binding of human alpha-fetoprotein and albumin

Column chromatography was used to investigate the interaction of human alpha-fetoprotein and albumin with different immobilized dyes. Binding of alpha-fetoprotein to the dye conjugates was studied at pH 7.0. Between 56 and 93% of the total alpha-fetoprotein applied to the column was recovered in the break-through fractions of the respective runs. Of all the dyes, Cibacron Blue F3G-A adsorbs alpha-fetoprotein most strongly. This interaction clearly depends on the degree of dye substitution of the gel. A relatively weak interaction exists between alpha-fetoprotein and immobilized Procion Red HE-3B. This is used in the purification of alpha-fetoprotein by negative chromatography resulting in a 16.6-fold enrichment of this protein. Human albumin binds tightly to immobilized Cibacron Blue F3G-A as well as to Cibacron Brilliant Blue FBR-P and shows a lower affinity to Procion Blue MX-R. Procion Red dyes, which are structurally different from Cibacron Blue F3G-A are also capable of interacting with serum albumin. The results obtained are discussed in terms of the present theoretical conceptions about dye-protein interactions.     

Purification of neutral protease by dye affinity chromatography

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme^™, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25°C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8°C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25°C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.     

High resolution protein separations using affinity ultrafiltration with small charged ligands

Although the feasibility of affinity ultrafiltration was demonstrated more than 20 years ago, commercial applications have not developed due to the high cost and practical limitations of the large macroligands needed for highly selective separations. The objective of this study was to examine the use of small charged affinity ligands for protein purification by exploiting electrostatic interactions between the charged complex and an electrically-charged membrane. Experiments were performed using bovine serum albumin and ovalbumin with Cibacron Blue as the affinity ligand. Negatively charged versions of a composite regenerated cellulose membrane were generated by covalent attachment of a sulfonic acid functionality. Binding experiments were used to identify appropriate conditions for protein separations. The selectivity for the separation of BSA and ovalbumin was a function of the solution conditions, Cibacron Blue concentration, and membrane charge, with the addition of Cibacron Blue causing a 30-fold increase in selectivity. A diafiltration process was performed at the optimal conditions, giving a BSA product with a purification factor of more than 90-fold and a yield greater than 90%. These results clearly demonstrate the potential of using a small charged affinity ligand for high resolution protein separations.     

辛巴蓝F-3GA修饰的啤酒废酵母菌亲和吸附溶菌酶

利用j嗪染料辛巴蓝F-3GA修饰经戊二醛交联的啤酒废酵母菌,得到一种新型染料亲和吸附剂.辛巴蓝F-3GA的固载量为161.1 mg/g.以溶菌酶为研究对象,考察吸附时间、酶初始浓度、pH值、离子强度等因素对吸附率的影响.结果表明:当pH=7.0时,其对溶菌酶有较高的吸附量(229.1 mg/g),吸附性能明显优于未接枝...     

染料壳聚糖微球的制备及其对牛血清白蛋白(BSA)吸附性能的研究

采用反相悬浮交联法制备壳聚糖微球,对微球进行羟丙基氯化及氨基化,并偶联色素配体Cibacron Blue F3GA,得到一种新型染料亲和吸附剂.以牛血清白蛋白(BSA)为目标蛋白,考察了该染料亲和吸附剂的吸附性能,发现其对BSA有较高的吸附量(95.2mg/g),吸附行为满足Langmuir吸附等温式.负载牛血清白蛋白的微球容易洗脱,洗脱率高达99％.     

Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.     

Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems

Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.     

活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用

以3.0 μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出 一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和 溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观 解离常数为5.26×10-5 mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶 电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。     

Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification.

Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions.     

Dye attached poly(hydroxyethyl methacrylate) cryogel for albumin depletion from human serum

Cibacron Blue F3GA was immobilized on poly(hydroxyethyl methacrylate) cryogel and it was used for selective and efficient depletion of albumin from human serum. The poly(hydroxyethyl methacrylate) was selected as the basic component because of its inertness, mechanical strength, chemical and biological stability, and biocompatibility. Cibacron Blue F3GA was covalently attached to the poly(hydroxyethyl methacrylate) cryogel to produce poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel affinity column. The poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was characterized with respect to gelation yield, swelling degree, total volume of macropores, Fourier Transform Infrared spectroscopy, and scanning electron microscopy. It was found that the maximum amount of adsorption (343 mg/g of dry cryogel) obtained from experimental results is very close to the calculated Langmuir adsorption capacity (345 mg/g of dry cryogel). The maximum adsorption capacity for poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel column was obtained as 950 mg/g of dry cryogel for nondiluted serum. The adsorption capacity decreased with increasing dilution ratios while the depletion ratio of albumin remained as 77% in serum sample. Finally, the poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was optimized for using in the fast protein liquid chromatography system for rapid removal of the high abundant proteins from the human serum.     

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.     

IMMOBILIZED CIBACRON BLUE F3G-A ON CROSSLINKED POLY (VINYL ALCOHOL) FOR AFFINITY CHROMATOGRAPHY

Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, cibacron Blue F3G-A was immobilized,through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N'-Cibacron Blue F3G-A,which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinded poly(vinyl acetate),which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The cibacron Blue F3G-A-immobilized poly(vinyl alcohol)was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.     

Continuous superporous agarose beds in radial flow columns

Continuous superporous agarose beds constitute a new support material for chromatography, biocatalysis and electrophoresis. The bed consists of a single piece of agarose gel, homogeneously transected by flow-carrying pores, which easily can be varied in the range of 10-100 microm. In this work, large diameter beds (60 mm) were prepared and used in specially designed radial flow columns. The basic chromatographic properties of the beds were investigated by size-exclusion chromatography experiments. In an affinity chromatography application one bed was derivatized with Cibacron Blue 3GA and used for the purification of lactate dehydrogenase from a crude bovine heart extract. In a biotransformation application one bed was provided with immobilized beta-galactosidase and used in the production of lactose-free milk.     

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.     

Lactoferrin from human breast milk and from neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties

The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin-sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non-specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron-rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5-6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2-6.0 while the other binding site holds on to iron down to pH 3.6-3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12-25% and that for granulocyte lactoferrin less than 10%.     

Cyclodextrin biospecific-like displacement in dye-affinity chromatography

Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography.     

Plasma proteomics for biomarker discovery: a study in blue

The performance of Cibacron Blue dye (HiTrapBlue or Affigel Blue) in depleting albumin from plasma, as a pre-treatment for biomarker searching in the low-abundance proteome, is here assessed. It is shown that (i) co-depletion of non-albumin species is an ever-present hazard; (ii) the only proper eluant able to release quantitatively the proteins bound to the dye is boiling 4% SDS-25 mM DTT, an ion shock (2 M NaCl) being quite ineffective in releasing the low-abundance species tightly bound to the dye moiety; (iii) the mechanism of dye-protein interaction, after an initial ion-ion docking, is a robust hydrophobic interaction, which progressively augments at lower and lower pH values; (iv) at pH 2.2 in the presence of 0.1% TFA, the blue resin behaves, for all practical purposes, just as a reverse-phase chromatography column, since all residual proteins present in plasma are completely harvested. However Cibacron Blue technology should not necessarily be discarded: As long as also the plasma fraction adsorbed is properly released and analyzed, together with the flow through, one should be able to perform a viable analysis of the low-abundance proteome.     

Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins

Characteristic data are presented for Divicell, a macroporous bead cellulose with excellent flow parameters. The preparation of Divicell derivatives and their properties are described with respect to their application as chromatographic supports. The ion exchangers Divicell DEAE and Divicell CM were manufactured in two types with different exclusion limits and an available capacity for proteins of up to 100 mg/ml gel. Divicell Blue is a bead cellulose with covalently bound Cibacron Blue F3G-A and was found to be a very suitable adsorbent for the selective separation and purification of human serum albumin. Activation of Divicell with sodium periodate, epichlorohydrin and 5-norbornene-2,3-dicarboximido carbonochloridate provided activated supports used for immobilization of ligands in organic solvents and in aqueous solutions. Coupling of amines, diamines, amino acids, carbohydrates and proteins is described. The immobilized ligands retained their biological activity as determined by their specific adsorption of proteins. Divicell alkyl derivatives were tested in hydrophobic interaction chromatography with bovine serum albumin as a model. Examples are presented of the application of Divicell derivatives to the purification of biomacromolecules such as immunoglobulins and lectins by affinity chromatography. The results were comparable to those obtained using the corresponding Sepharose-derived absorbents.     

Solid-phase extraction and simultaneous determination of trace amounts of sulphonated and azo sulphonated dyes using microemulsion-modified-zeolite and multivariate calibration

A simple and rapid analytical method for the determination of trace levels of five sulphonated and azo sulphonated reactive dyes: Cibacron Reactive Blue 2 (C-Blue, trisulphonated dye), Cibacron Reactive Red 4 (C-Red, tetrasulphonated azo dye), Cibacron Reactive Yellow 2 (C-Yellow, trisulphonated azo dye), Levafix Brilliant Red E-4BA (L-Red, trisulphonated dye), and Levafix Brilliant Blue E-4BA (L-Blue, disulphonated dye) in water is presented. Initially, the dyes were preconcentrated from 250 ml of water samples with solid-phase extraction using natural zeolite sample previously modified with a microemulsion. The modified zeolite exhibited an excellent extraction for the dyes from solution. The parameters that influence quantitative recovery of reactive dyes like amount of extractant, volume of dye solution, pH, ionic strength, and extraction-elution flow rate were varied and optimized. After elution of the adsorbed dyes, the concentration of dyes was determined spectrophotometrically with the aid of principle component regression (PCR) method without separation of dyes. The results obtained from PCR method were comparable to those obtained from HPLC method confirming the effectiveness of the proposed method. With the aid of SPE by M-zeolite, the concentration of dyes could be reproducibly detected over the range 25-200 ppb for C-Yellow and L-Blue and from 50 to 250 ppb for C-Blue, C-Red, and L-Red. The multivariate detection limits of dyes were found to be 15 ppb for C-Yellow and L-Blue and 25 ppb for C-Blue, C-Red, and L-Red dyes. The proposed chemometric method gave recoveries from 85.4 to 115.3% and R.S.D. from 1.0 to 14.5% for determination of the five dyes without any prior separation for solutes.     

染料膜亲和色谱法中膜堆的制备及应用

将染料亲和配基偶联于大孔纤维素膜上,所得亲和膜用胶粘法制成亲和膜堆,膜堆的通透性远优于通常的亲和色谱柱。装有蓝色和红色亲和膜的膜堆可分别用于人血清白蛋白和碱性磷酸酯酶的分离纯化,其中碱性磷酸酯酶可在一步操作后纯化４０倍。