Cibacron blue F3G-A and related dyes as ligands in affinity chromatography

Kopperschläger, Gèrhard; Böhme, H; Hofmann, E

doi:10.1007/3540118292_4

Cited by 37 publications

(32 citation statements)

References 170 publications

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“…The high purification factor achieved is an acceptable compensation for an initial loss of enzyme activity of approximately 35% (11.5 kU due to inactivation during operation and elution of diluted HSDH before the KCl pulse). It should be emphasized that a yield of approximately 65% is very common for enzyme purification by affinity chromatography based on interaction with triazine dyes [21].…”

Section: Purification Oj'hsdhmentioning

confidence: 99%

“…For this purpose, a range of Sephacryl-S-400-immobilized triazine dyes with similar coupling yield ( 5 -15 mg/g, corresponding to 7 -10 pmoljg, a usual range for these affinity materials [21]) was tested on their specificity to bind HSDH from crude extract (see Materials and Methods). Of the triazine dye ligands studied, HSDH interacted very strongly with Procion green HE-4BD, blue H-B, yellow HE-3B, red H-3B, red HE-3B and red HE-7B, as 1 M KCI was needed to eluate the enzyme in a sharp peak.…”

Section: Purification Oj'hsdhmentioning

confidence: 99%

“…KC1. The following dyes were immobilized: yellow HA, green HE-4BD, blue H-B, red HE-3B (for molar absorption coefficients, see [21]), yellow MX-R, yellow H-7G, yellow MX-6G, red MX-G, blue MX-3G, orange MX-G, scarlet MX-G, orange P-2R, yellow HE-3G, red H-3B, red HE-7B (molar absorption coefficient not available).…”

Section: Coupling Of Tviazine Dyes To Sephucryl S-400mentioning

confidence: 99%

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12α‐Hydroxysteroid dehydrogenase from Clostridium group P, strain C 48–50

Braun¹,

Lünsdorf

Bückmann³

1991

European Journal of Biochemistry

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NADP(H)-dependent 12a-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1 .O U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400 -Procion-orange-P-2R(5 cm x 28 cm) and Sephacryl-S-400 -Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x lo4 U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 Ujmg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5 -9.5 and a temperature optimum of approximately 55°C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability ( t l j z 4-10 d, 25°C; 2-5 d, 37°C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi hi mechanism with NADP(H) binding first to the free enzyme. K,, V,,, [forward (Vf) and reverse reactions (V,)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, K, = 35 pm, Kd = 35 pm; cholic acid, K, = 72 pm; deoxycholic acid, K , = 45 pm, Vf = 160 U/mg; NADPH, K, = 8.5 pm, Kd = 16 pm; 12-oxochenodeoxycholic acid, K, = 12 pm, V, = 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0,25 "C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (K, = 4.5 mM) are poorly accepted as coenzyme by HSDH.In the human intestinal flora, bacteria are present that metabolize biliary bile acids not taken up by the enterohepatic circulation. Besides hydrolysis of conjugated bile acids and reductive dehydroxylation at carbon-7, as major reactions, oxidation and epimerization of the 3a-, 7a-and 12a hydroxyl groups occur [l].These epimerizations proceed via the corresponding 0x0 intermediates, either by a single microorganism that expresses both a-and P-hydroxysteroid dehydrogenases or by cooperation of two different microorganisms, each expressing a single stereospecific hydroxysteroid dehydrogenase [l, 21.

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Section: Purification Oj'hsdhmentioning

confidence: 99%

Section: Purification Oj'hsdhmentioning

confidence: 99%

Section: Coupling Of Tviazine Dyes To Sephucryl S-400mentioning

confidence: 99%

See 1 more Smart Citation

12α‐Hydroxysteroid dehydrogenase from Clostridium group P, strain C 48–50

Braun¹,

Lünsdorf

Bückmann³

1991

European Journal of Biochemistry

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show abstract

“…Therefore, the higher adsorption capacity results in higher desorption concentration as shown in (Figure 4 and Figure 5). This column should afforded excellent BSA purification from the mixture with other proteins and contaminant [19].…”

Section: Dynamic Desorption Of Bsamentioning

confidence: 99%

Flexible Electrospun Cellulose Fibers as an Affinity Packing Material for the Separation of Bovine Serum Albumin

Miyauchi¹,

Miao²,

Simmons³

2011

J Chromatograph Separat Techniq

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Flexible, well-dispersed and continuous 100-nm diameter cellulose fibers were prepared from an ionic liquid solvent by a novel dry-jet wet-electro spinning process. The ribbon fibers formed were chemically activated and an affinity dye, cibacron blue (CB) was immobilized at 0.22 g CB/g dry fibers loading to the surface of these fibers. The resulting affinity matrix was packed into a chromatography column and the adsorption, desorption and specificity of this matrix for bovine serum albumin (BSA) was studied. These electrospun fibers had a BSA binding capacity of 230 mg/g, nearly twice that of CB-immobilized 100-μm beads and over ten-fold higher capacity that CB-immobilized cellulose fibers prepared by a conventional electrospinning process. The results of this work suggest that chromatography supports of flexible, well-dispersed, continuous nanofibers may offer advantages over conventional supports in affinity separations.

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“…Cibacron Blue F3G-A is well known as a pseudobiospecific ligand interacting with the ATP-binding site(s) of Pfk from S. cerevisiae and other yeast enzymes Kopperschläger et al, 1982;Kopperschläger, 1994). Since the kinetic data displayed differences in the ATP and AMP effect between the enzymes from P. pastoris and S. cerevisiae, dye-ligand affinity partitioning was applied to analyse the nucleotide binding site(s) of both enzymes.…”

Section: Pseudoaffinity Ligand-enzyme Interactionmentioning

confidence: 99%

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

Kirchberger

Bär

Schellenberger

et al. 2002

Yeast

Self Cite

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Abstract6-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 twophase 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.

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Cibacron blue F3G-A and related dyes as ligands in affinity chromatography

Cited by 37 publications

References 170 publications

12α‐Hydroxysteroid dehydrogenase from Clostridium group P, strain C 48–50

12α‐Hydroxysteroid dehydrogenase from Clostridium group P, strain C 48–50

Flexible Electrospun Cellulose Fibers as an Affinity Packing Material for the Separation of Bovine Serum Albumin

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

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