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Y D Clonis

Publications and source records attributed to Y D Clonis.

17 recordsLinked to original sources
PubMed1996

Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site.

The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.

Affinity Labels↗
PubMed1996

Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase.

Molecular modelling and kinetic inhibition studies, as well as KD determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose-designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower KD values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an alpha-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (KD 1.3 microM, which corresponded to a 219-fold decrease over the KD of a control dye). BM5 displayed competitive inhibition with respect to both NADH (Ki 2.7 microM) and oxaloacetate (Ki 9.6 microM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide-pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the ketocarboxyl biomimetic moiety. A match between the alternating polar and hydrophobic regions of the enzyme binding site with those of the biomimetic moiety is desirable. The length of the biomimetic moiety should be conserved in order for the keto acid to approach the enzyme active site and form charge-charge interactions.

Affinity Labels↗
PubMed1996

Biomimetic-dye affinity chromatography for the purification of mitochondrial L-malate dehydrogenase from bovine heart.

Seven biomimetic anthraquinone triazinyl dye-ligands, bearing as triazine-linked terminal moiety (keto)carboxylated structures mimicking substrates and inhibitors of malate dehydrogenase (MDH), were immobilised on cross-linked agarose Ultrogel A6R. These biomimetic ligands are terminal-ring analogues of commercial nonbiomimetic Cibacron blue 3GA (CB3GA) and parent Vilmafix blue A-R (VBAR). The biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing immobilised CB3GA and VBAR, were evaluated for their ability to purify mitochondrial malate dehydrogenase (mMDH) from bovine heart. All but two biomimetic-dye adsorbents displayed higher purifying ability for MDH, compared to nonbiomimetic-dye adsorbents. Furthermore, immobilised anthraquinone-dyes were able to discriminate between the mitochondrial and the cytoplasmic MDH isoenzymes, binding only to the former. One immobilised biomimetic-dye (BM5), bearing as biomimetic terminal moiety 4-aminophenyloxanylic acid, showed the highest purifying ability. This affinity adsorbent was exploited in the purification of mMDH from unpretreated bovine heart extract in one-step. The procedure afforded mMDH at 54% overall yield and of specific activity approx. 1300 U mg-1 (25 degrees C), using step-elution with a mixture containing 0.1 mM beta-nicotinamide adenine dinucleotide (NAD+) and 1.5 mM sulphite. Commercial analytical-grade bovine heart mitochondrial MDH, when assayed under identical conditions, gave a specific activity not exceeding 950 U mg-1. The well-known adsorbent Cibacron blue 3GA-agarose exhibited 8% lower recovery and 25% lower purification for mMDH. The product obtained from the procedure based on the BM5-adsorbent was free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase, and since it has also shown high specific activity, it should be suitable for analytical applications.

Acetone↗
PubMed1995

Biomimetic dye affinity chromatography for the purification of bovine heart lactate dehydrogenase.

Three biomimetic dye ligands bearing as a triazine-linked terminal moiety a carboxylated structure, which mimics substrates and inhibitors of L-lactate dehydrogenase (LDH), were immobilized on cross-linked agarose Ultrogel A6R. These biomimetic dyes are purpose-designed analogues of commercial monochlorotriazine Cibacron Blue 3GA (CB3GA) and parent dichlorotriazine Vilmafix Blue A-R (VBAR). The corresponding biomimetic adsorbents, along with non-biomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify LDH from bovine heart crude extract. When compared with non-biomimetic adsorbents, all biomimetic adsorbents exhibited a higher purifying ability. Further, one immobilized biomimetic dye, bearing mercaptopyruvic acid as biomimetic moiety, displayed the highest purifying ability. The concentration of immobilized dye affected both the capacity and the purifying ability of the affinity column, exhibiting an optimum value 2.2 mumol dye/g moist gel. This affinity adsorbent was exploited for the purification of LDH from bovine heart in a two-step procedure. The procedure consisted in a biomimetic dye affinity chromatography step (NAD+/sulphite elution, 25-fold purification, 64% step yield), followed by DEAE-agarose ion-exchange chromatography (1.4-fold purification, 78% step yield). The purified enzyme exhibited a specific activity of ca. 480 u/mg at 25 degrees C (content of impurities: pyruvate kinase and glutamic-oxaloacetic transaminase were not detected; malate dehydrogenase, 0.01%), compared with ca. 250 u/mg of commercial bovine heart LDH (malate dehydrogenase, 0.05%) suitable for analytical purposes.

Animals↗
PubMed1995

Oxaloacetate decarboxylase: on the mode of interaction with substrate-mimetic affinity ligands.

The mode of interaction of the ketocarboxyl-group-recognizing enzyme oxaloacetate decarboxylase (OXAD) from Pseudonomas sp., with purpose-designed (keto)-carboxyl-terminal biomimetic monochlorotriazinyl-dyes (BM) and parent dichlorotriazinyl-dye Vilmafix blue A-R (VBAR) was investigated. Kinetic inhibition studies and determinations of KD values of the respective dye-enzyme complex from both difference spectra and enzyme inactivation studies were employed. Substratemimetic (biomimetic) dye-ligands bear a terminal (keto)carboxyl-moiety linked to the reactive chlorotriazine ring, thus mimicking the organic acid substrate of OXAD. Dichlorotriazine-dye VBAR bound specifically and irreversibly to OXAD (k3 0.22 min-1). The inactivation of OXAD by VBAR was enhanced in the presence of 1 mM Mn+2 (KD 67.2 microM) but in the absence of metal cation was decreased (KD 117 microM). The metal cation behaves as a partial competitive activator. Either of binary complexes dye.OXAD and OXAD.Mn+2 could be formed first, prior to addition of the third constituent to form the ternary complex, although the former route may be favored. The pKa of the catalytically important nucleophile, involved in the specific modification of OXAD, was calculated to 7.4. Biomimetic monochlorotriazine dyes have failed to inactivate OXAD but inhibited competitively the inactivation by VBAR. When compared to commercial VBAR and Cibacron blue 3GA (CB3GA), all BM ligands show lower KD values, therefore, higher affinity for the enzyme. OXAD preferred binding to BM dyes which exhibited a large aliphatic ketocarboxyl-terminal biomimetic moiety. Dye binding to OXAD was accompanied by a characteristic spectral change in the range 550-800 nm. Electrostatic interactions appeared to play a dominant role in the dye.OXAD complex. The BM ligand bearing an aminoethyloxamate as its terminal biomimetic moiety (BM7) displayed the highest affinity (KD 0.5 or 7.0 microM; approx 10-fold decrease over CB3GA). The BM7 ligand behaved as competitive inhibitor (Ki 98 microM) of oxaloacetate decarboxylase against oxaloacetate as variable substrate.

Anthraquinones↗
PubMed1995

The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands.

Seven chimeric biomimetic dye-ligands (BM) are purpose-designed and synthesized by specific structural modification of the parent anthraquinone dichlorotriazine dye Vilmafix blue A-R (VBAR). Each BM dye is composed of two enzyme-recognition moieties. The terminal biomimetic moiety bears a variable carboxylated structure linked to the triazine ring, thus mimicking the substrate of formate dehydrogenase (FDH). The anthraquinone moiety remains the same as that of the parent dye and recognizes the nucleotide-binding area of the target enzyme. Dyes are purified by liquid column chromatography (typically 99%), analyzed by liquid-paper chromatography, thin-layer chromatography, and high-performance liquid chromatography, and their lambda max and epsilon values are determined. The ability of dyes to act as affinity ligands versus Candida boidinii FDH is evaluated by kinetic studies and determining KD values from both difference spectra and enzyme inactivation studies. The parent dichlorotriazine dye VBAR binds specifically and irreversibly to FDH (k3 0.19 min-1; KD 19.3 microM). The inactivation of the NAD(+)-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH, and ADP. Quantitatively inhibited FDH contained approx 1 mol of dye per mole of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM each of NAD+, NADH, and ADP or by extensive dialysis or gel filtration chromatography. The monochlorotriazine BM dyes do not inactivate FDH but inhibit competitively the inactivation by VBAR. When compared to VBAR and Cibacron blue 3GA (CB3GA), all BM dye-ligands exhibited lower KD values. FDH generally preferred binding to BM ligands which bore an aromatic terminal biomimetic moiety substituted with a monocarboxyl group rather than an alpha-ketoacid. Dye binding to FDH is accompanied by a characteristic spectral change in the range 550-800 nm. This phenomenon is perturbed after titration by increasing amounts of NAD+. Electrostatic interactions appeared to play a dominant role in the dye.FDH complex. The BM dye-ligand bearing a m-aminobenzoate at its terminal biomimetic moiety (BM1) exhibited the highest affinity (KD 1.6 microM, 8.0-fold decrease over CB3GA). BM1 differentiated between the binding sites of FDH, displaying uncompetitive inhibition with respect to NAD+ (Ki 15.6 microM) and competitive with respect to formate (Ki 18.1 microM).

Adenosine Diphosphate↗
PubMed1994

The affinity technology in downstream processing.

The quality criteria imposed on several biochemicals are stringent, thus, high-separation purification technology is important to downstream processing. Affinity-based purification technologies are regarded as the finest available, and each one differs in its purifying ability, economy, processing speed and capacity. The most widely used affinity technology is classical affinity chromatography, however, other chromatography-based approaches have also been developed, for example, perfusion affinity chromatography, hyperdiffusion affinity chromatography, high-performance affinity chromatography, centrifugal affinity chromatography, affinity repulsion chromatography, heterobifunctional ligand affinity chromatography and the various chromatographic applications of 'affinity tails'. On the other hand, non-chromatographic affinity technologies aim at high throughput and seek to circumvent problems associated with diffusion limitations experienced with most chromatographic packings. Continuous affinity recycle extraction, aqueous two-phase affinity partitioning, membrane affinity filtration, affinity cross-flow ultrafiltration, reversible soluble affinity polymer separation and affinity precipitation are all non-chromatographic technologies. Several types of affinity ligands are used to different extents; antibodies and their fragments, receptors and their binding substances, avidin/biotin systems, textile and biomimetic dyes, (oligo)peptides, antisense peptides, chelated metal cations, lectins and phenylboronates, protein A and G, calmodulin, DNA, sequence-specific DNA, (oligo)nucleotides and heparin. Likewise, there are several support types developed and used; natural, synthetic, inorganic and composite materials.

Affinity Labels↗
PubMed1991

Monosized adsorbents for high-performance affinity chromatography. Application to the purification of calf intestinal alkaline phosphatase and human urine urokinase.

Affinity adsorbents comprising monodisperse spherical synthetic macroporous beads offer the prospect of high-capacity, high-resolution separation of proteins at low operating pressures. Purpose-designed biomimetic dyes were covalently attached to Dynospheres XP-3507 beads and exploited for the purification of calf intestine alkaline phosphatase and human urine urokinase from crude extracts. This study demonstrates that the combination of specifically designed affinity ligands with monosized support materials is a powerful approach to the resolution of proteins by high-performance affinity chromatography.

Adsorption↗
PubMed1988

The applications of reactive dyes in enzyme and protein downstream processing.

Downstream processing of proteins is often a key factor in the overall process of satisfying product specifications and meeting current commercial demands. In this context, affinity chromatography and other techniques based on the affinity concept have revolutionized protein purification technology, although they have failed to demonstrate their broader applicability at the process scale. On the other hand, reactive dyes offer many advantages as pseudoaffinity media and in many occasions have successfully circumvented problems associated with conventional affinity ligands. The main features of reactive dyes include their broad spectrum of interaction with proteins, low cost, ready availability, high reactivity, ease of immobilization, and both biological and chemical stability. Consequently, dye-ligand media now find application in both analytical and process-scale purification of proteins by techniques such as low- and high-pressure performance affinity chromatography, affinity partitioning, and affinity precipitation.

Chemical Phenomena↗
PubMed1987

Dye-ligand chromatography for the resolution and purification of restriction endonucleases.

The resolution of restriction endonucleases from the same microorganism is conventionally achieved by lengthy fractionation protocols. We now report effective single-step procedures that exploit dye-ligand chromatography for the resolution and purification of restriction enzymes. After suitable initial screening, we demonstrated that resolution of two restriction activities can be achieved in one chromatographic step, and further purification can subsequently be effected using selected dye-adsorbents. Accordingly, we resolved in one step, Hpa I from Hpa II, Hind II from Hind III, and Sac I from Sac II. Furthermore, a three-step chromatographic procedure has been developed to purify EcoRV suitable for commercial exploitation, as judged by the "overdigestion" and "cut-ligate-recut" quality control tests.

Bacteria↗
PubMed1987

High-performance liquid chromatography for the purification of restriction endonucleases, application to BanII, SacI, and SphI.

Conventional fractionation methods are time consuming, thus they prolong the time required to process low-stability restriction enzymes. We now report a rapid and effective two-step chromatographic method that affords high purity endonucleases in a short time. Accordingly, an inexpensive chromatographic adsorbent such as phosphocellulose or dyed agarose in the first step is coupled to a high-performance ion exchanger, namely, MonoQ, in the second step. The purification schemes reported here are now in routine use to prepare high-purity BanII, SacI, and SphI as judged by the "overdigestion" and "cut-ligate-recut" stringent quality tests.

Bacillus↗
PubMed1986

Design and application of bio-mimetic dyes in biotechnology.

The last decade or so has been the introduction of multi-coloured reactive dyes as substitutes for natural biological ligands in the purification of proteins by affinity chromatography. This paper reviews the evidence for the remarkable selectivity of the interaction of reactive dyes with proteins and describes our recent work with dye analogues. Terminal ring, bridging ring and anthraquinone ring analogues of Cibacron Blue F3G-A were synthesised de novo and shown to interact selectively with the NAD+-binding site of horse liver alcohol dehydrogenase but with affinities differing by several orders of magnitude. It is anticipated that these novel dye ligands will lead to affinity adsorbents with improved affinity, capacity and specificity.

Animals↗
PubMed1981

The interaction of yeast hexokinase with Procion Green H-4G.

1. A number of reactive triazine dyes specifically and irreversibly inactive yeast hexokinase at pH 8.5 and 33 degrees C. Under these conditions, the enzyme is readily inactivated by 100 microM-Procion Green H-4G, Blue H-B, Turquoise H-7G and Turquoise H-A, is less readily inactivated by Procion Brown H-2G. Green HE-4BD, Red HE-3B and Yellow H-5G and is not inactivated at all by Procion Yellow H-A. 2. The inactivation of hexokinase by Procion Green H-4G is competitively inhibited by the adenine nucleotides ATP and ADP and the sugar substrates D-glucose, D-mannose and D-fructose but not by nonsubstrates such as D-arabinose and D-galactose. 3. Quantitatively inhibited hexokinase contains approx. 1 mol of dye per mol of monomer of mol.wt. 51000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentration (20 mM) of ATP or D-glucose. 4. Mg2+ protects the enzyme against inactivation by Procion Green H-4G but enhances the rate of inactivation by all the other Procion dyes tested. In the presence of 10 mM-Mg2+ the apparent dissociation constant between enzyme and dye is reduced from 199.0 microM to 41.6 microM. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. 5. Mg2+ promotes binding of yeast hexokinase to agarose-immobilized Procion Green H-4G but not to the other dyes tested. Elution could be effected by omission of Mg2+ from the column irrigants or by inclusion of MgATP or D-glucose, but not by D-galactose. These effects can be exploited to purify hexokinase from crude yeast extracts. 6. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.

Affinity Labels↗
PubMed1981

Affinity chromatography on immobilised triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes.

A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted. Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes. This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography.

Adenylosuccinate Synthase↗
PubMed1980

Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes.

A number of reactive dichlorotriazine dyes specifically and irreversibly inactivate pig heart lactate dehydrogenase, yeast glucose 6-phosphate dehydrogenase and yeast hexokinase at sites competitive with NAD+, NADP+, and ATP respectively. Monochlorotriazine dyes, including Cibacron Blue F3G-A, do not inactivate lactate dehydrogenase but display high affinity and thus inhibit the inactivation by dichlorotriazine dyes. These data are interpreted in terms of the ability of nucleotide-binding enzymes to bind polysulphonated aromatic chromophores.

Affinity Labels↗
PubMed1980

Affinity chromatography on immobilised nucleotides. The synthesis, specificity and applications of immobilised inosine 5'-monophosphate.

The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx8IMP, and inosine 2',3'-O-[1-(6-aminohexyl)-levulinic acid amide]-acetyl 5'-monophosphate, (AhxLvn)2',3'IMP, is described. These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule. The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14) but not for the NAD+-dependent enzymes, L-alanine and L-acetate dehydrogenases. Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large-scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20-1 culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.

Animals↗