Cibacron Blue F3GA-Sepharose Research Articles

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.

Simplified elimination of rabbit anti-rat acute phase protein IgG that shows cross reactivity with albumin

Antibody preparations against rat acute phase proteins were tested for cross reactivity with other serum proteins, including rat albumin. Rabbit anti-rat a alpha1-acid glycoprotein and ceruloplasmin IgG purified on protein A-Sepharose did not show any cross reactivity with rat albumin, hemoglobin or transferrin. Rabbit anti-rat haptoglobin and -macroglobulin IgG purified on protein A-Sepharose showed a 39% and 30% cross reactivity with rat albumin and a 20% and 19% cross reactivity with rat hemoglobin. Because these proteins in whole serum were not adsorbed on Cibacron Blue F3-GA Sepharose, the albumin would be adsorbed on Cibacron Blue F3-GA Sepharose by the use of whole rat serum. Rabbit anti-rat haptoglobin and alpha2-macroglobulin IgG showing cross reactivity with albumin was simply eliminated.

Development of a competitive enzyme linked immunosorbent assay to measure alpha 2-macroglobulin in irradiated rat serum.

A competitive enzyme linked immunosorbent assay with antigen immobilized on the solid phase was developed to measure alpha 2-macroglobulin in rat serum. The cross reactivity with albumin and hemoglobin was eliminated by use of IgG fractions that were isolated after chromatography on Cibacron Blue F3-GA Sepharose immobilized rat whole serum and hemoglobin Sepharose. Blocking materials and pH of the coating buffer had no effect on the amount of alpha 2-macroglobulin that binds to the plate. When the coating amount of alpha 2-macroglobulin was 100 ng/well, at pH 7.4, 10 mM Tris-HCl containing 150 mM sodium chloride and the IgG amount added was 60 ng/well, then albumin and hemoglobin did not affect the assay at concentrations of 150 micrograms/ml or 200 micrograms/ml. This assay is useful for measuring the concentration of alpha 2-macroglobulin in normal and irradiated rat serum.

Low intracellular GTP level induces PKC-β-dependent cell differentiation in human myeloid leukemia cells

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.

Adsorption of proteins on sepharose affinity adsorbents of varying particle size

The binding of bovine serum albumin (BSA) and lysozyme to a series of samples of Cibacron Blue F3G-A Sepharose CL-6B with different narrow-range mean particle diameters has been investigated. Significant variation with particle size has been found for the parameters measured. For both proteins the maximum capacities obtained increased with decreasing particle size. For lysozyme, this percentage increase in capacity is less than the percentage increase in external surface area as the particle size decreases. For BSA, however, the percentage increase in capacity is more than four times the increase in surface area. The dissociation constants and forward rate constants describing the affinity interaction also differ with particle size. Again the effects are more pronounced for the larger protein, BSA, than for lysozyme. The effect of these differing parameters on the performance of fixed beds has been assessed using a computer simulation of the adsorption process.

A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties.

A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.

Evidence for a specific phosphoryl binding site in swine kidney phosphofructokinase.

Phosphofructokinase (PFK) from swine kidney was purified by a procedure which included affinity chromatography on Cibacron blue F3GA-Sepharose 4B and ATP-Sepharose 4B columns in order to examine its binding properties. The homogeneous enzyme was purified more than 3000-fold with a yield of 30% and it had a specific activity of 39.8 mumol/min/mg of protein at 25 degrees C. The molecular weight of the native enzyme was 360 000 and it contained 4 identical subunits of molecular weight 88 000. The principal catalytically reacting form of the enzyme had a S20,w of 13.7 S which corresponds to a molecular weight of 360 000 +/- 6 000. The initial velocity patterns in the forward and reverse directions suggested a sequential mechanism for the reaction. The Km values for fructose 6-phosphate, ATP, fructose, 1,6-bisP and ADP were 33 microM, 8.3 microM, 460 microM and 110 microM, respectively. The homogeneous native enzyme binds specifically to phosphoryl groups immobilized in cellulose phosphate columns. ATP and fructose 6-phosphate interacted with the enzyme and decreased its affinity for phosphoryl binding sites. Other metabolites including fructose 1,6-bisP, glucose 6-phosphate and various nucleotides, alone or in various combinations, were ineffective in promoting the dissociation of the enzyme. Allosteric effectors of the enzyme, such as citrate and AMP were also inactive. However, the cooperatively altered the concentration of ATP required to dissociate the enzyme from phosphoryl groups. The bound enzyme was enzymatically inactive. The enzyme was also inactivated when it was treated with pyridoxal 5'-phosphate and reduced with sodium borohydride and the inactive enzyme no longer bound to cellulose phosphate. These effects were not observed when treatment with pyridoxal 5'-phosphate was carried out in the presence of fructose 6-phosphate. These observations and the results of similar studies with swine kidney fructose 1,6-bisphosphatase (FBPase) show that both enzymes share the unique property of binding specifically to phosphoryl groups. FBPase interacts through its allosteric AMP binding site and PFK binds through its fructose 6-P binding site. This specific binding of both enzymes through these sites result in the inactivation of PFK and the desensitization of FBPase to allosteric inhibition by AMP. In the unbound state PFK may be active and FBPase can be inhibited by AMP. Taken collectively, these binding effects could play a role in the reciprocal regulation of these enzymes during gluconeogenesis in kidney.

The rapid purification of lactate dehydrogenase from alcaligenes eutrophus in a two-step procedure

A NAD-linked L(+)-lactate dehydrogenase (EC 1.1.1.27) was isolated from Alcaligenes eutrophus N9A. During purification advantage was taken of the high affinity of MatrexTM Gel Green A for this enzyme in crude extracts. One ml of this medium adsorbed 2660 U lactate dehydrogenase if 129 ml crude extract containing 2,480 mg protein were applied onto the column, as determined by frontal analysis. The enzyme was purified 275-fold by chromatography on this medium. Subsequent chromatography on Cibacron Blue F3G-A Sepharose 6B-CL resulted in a 3-fold purification and in a homogeneous preparation of lactate dehydrogenase. Starting with a crude extract containing 12 g total protein, the overall purification factor was 712.5, corresponding to a recovery of 36.1% activity and a specific activity of 776.6 U/mg protein. The affinity of MatrexTM Gel Green A medium to lactate dehydrogenase from other sources like A. hydrogenophilus, A. eutrophus A7, A. faecalis, Escherichia coli, Lactobacillus lechmannii, and rabbit muscle was investigated. Advantages of this method for large scale purification of lactate dehydrogenase from Alcaligenes eutrophus are discussed and compared to large scale purification methods applied for other enzymes.

Versatility of mixed-function adsorbents in biospecific protein desorption: Accidental affinity and an improved purification of aspartate transcarbamoylase from wheat germ

Under appropriate experimental conditions (usually but not invariably including low ionic strength) wheat germ aspartate transcarbamoylase can be specifically desorbed by the substrate, carbamoyl phosphate, from hydroxyapatite, from N-(3-carboxypropionyl)aminooctyl-Sepharose, from 10-carboxydecylamino-Sepharose, from Cibacron Blue F3GA-Sepharose, and from Coomassie Blue R250-Sepharose. Experimental evidence suggests that (a) the enzyme is adsorbed at heterogeneous sites on each column, only some of which are susceptible to substrate-specific desorption; (b) in none of these cases is the initial adsorption essentially biospecific, i.e., these are not cases of classical affinity chromatography; (c) in the case of 10-carboxydecylamino-Sepharose, and therefore presumably also in the other cases, the desorption is biospecific, i.e., involves the formation of the catalytically significant enzyme-carbamoyl phosphate complex. Substrate-specific desorption in these cases appears to derive from “accidental” affinity between, on the one hand, clusters of active (ionic, hydrophobic, aromatic, etc.) groups on the protein and, on the other, complementary clusters on the adsorbent, some of these interactions being perturbed when the ligands binds to the protein. Biospecific desorption from 10-carboxydecylamino-Sepharose has been incorporated as the sole chromatographic step in a new, 8000-fold purification of the enzyme. It is suggested that biospecific desorption from essentially nonbiospecific adsorbents could explain some published purifications currently described as “affinity chromatography.”

Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A.

The interaction of Cibacron Blue F3G A-Sepharose 4B with several serum albumins was studied. Although all albumins used were fond to bind to this adsorbent, human serum albumin was bound to a far greater extent than were the others. From the results of competition experiments and n.m.r. studies of Cibacron Blue and/or bilirubin binding to human serum albumin it is proposed that the mechanism of the interaction between human serum albumin and cibacron Blue is consistent wit Cibacron Blue binding to bilirubin-binding sites. In contrast with these findings with human serum albumin, there is little or no interaction of Cibacron Blue and the bilirubin-binding sites of albumins from rabbit, horse, bovine or sheep sera, although some interaction occurs between Cibacron Blue and the fatty acid-binding sites of these proteins. Structural analogues of Cibacron Blue have been used to investigate the binding of albumins to these ligands.

Investigations into the controlling factors of electrophoretic desorption: A widely applicable, nonchaotropic elution technique in affinity chromatography

Some of the important controlling factors involved in the electrophoretic desorption of material from affinity matrices (the electrophoresis current, the matrix thickness, and the electrophoresis buffer molarity and temperature) have been investigated. Subsequently, the optimum conditions have been used to desorb human serum albumin from Cibacron Blue F3G-A—Sepharose 6B. The results demonstrate some of the advantages of the technique, including the mild conditions, high yield, and small desorbate volume.

Interactions of contractile proteins with free and immobilized Cibacron Blue F3GA

Differential binding of contractile proteins from skeletal muscle to Cibacron Blue F3GA-Sepharose affinity columns provides the basis for a new purification technique. Myosin subfragments bind at low ionic strength and are eluted by high salt (e.g., 1.5 m NaCl). Myosin light chain 2 also binds at low ionic strength, whereas light chain 1 is only partially retarded and light chain 3 does not bind. Myosin's marginal solubility in the low-salt buffers required for binding renders it unsuitable for Blue Sepharose chromatography. Neither G-actin nor F-actin bind. Crude preparations of myosin subfragment-1 or light chains undergo significant purification upon Blue Sepharose chromatography. Nee free chromophore inhibits the ATPase activities of myosin and actomyosin at micromolar dye concentrations, whereas the binding of subfragment-1 to actin (in myofibrils) and the tension of glycerinated fibers are inhibited at millimolar dye concentrations. The dye binds at multiple sites on myosin, and inhibits its actomyosin ATPase both competitively and uncompetitively.

Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 m m) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 m m) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 m m OMP and 2 m m uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 m m OMP and 1 m m 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.

Radioaffinity assay, a new approach to binding assays applied to the measurement of serum albumin

The affinity adsorbent Cibacron Blue F3GA-Sepharose 4-B has been used to develop a binding assay for human serum albumin. The method is simple, accurate and precise. It agrees well with the ‘rocket” immunoelectrophoretic method and is proposed as an alternative technique for the re-estimation of albumin levels in the low range (less than 30 g/1) where the bromocresol green dye-binding method overestimates. Bilirubin, several drugs, gammaglobulin, haemoglobin and heparin do not interfere with the estimations.

The interaction of mammalian interferons with immobilized cibacron blue F3GA: modulation of binding strength.

Mouse, hamster, rabbit, horse, and human interferons bind to immobilized Cibacron Blue F3GA under appropriate solvent conditions. Three forms of the immobilized ligand have been investigated: Cibacron Blue F3GA-Sepharose 4B, Blue Dextran-Sepharose 4B and Blue Sepharose CL-6B. The strength of binding of an interferon depends critically on the sorbent: Cibacron Blue F3GA-Sepharose 4B is the weakest in the series and Blue Sepharose CL-6B the strongest. The use of Blue Dextran-Sepharose 4B--a sorbent of intermediate binding properties--allows the complete separation of hamster, mouse and human fibroblast interferons in a single chromatographic step. Indeed, both the resolution, as well as the recovery, of those interferons is complete--regardless of the relative complexity of the chromatographed preparation (containing either crude or purified interferons). Thus, these ligands should prove of considerable use when a facile chromatographic evaluation, both qualitative and quantitative of mammalian interferons is required.

Affinity chromatographic separation of arylsulfatase A and B using cibacron blue-sepharose

A simple and rapid affinity chromatographic method is described using Cibacron Blue F3GA-Sepharose for the separation of arylsulfatases A and B (aryl-sulfate sulfohydrolase, E C 3.1.6.1)