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K Mosbach

Publications and source records attributed to K Mosbach.

At least 37 records · Page 2Linked to original sources
PubMed1998

Magnetic molecularly imprinted polymer beads for drug radioligand binding assay.

Molecularly imprinted polymer-magnetic iron oxide composite materials which exhibit recognition properties and can be withdrawn from solution by application of a magnetic field were prepared for the first time. Magnetic iron oxide was incorporated using a suspension polymerisation methodology with a perfluorocarbon liquid as the dispersing phase for the preparation of methacrylic acid-1,1,1-trimethylolpropane trimethacrylate copolymer beads molecularly imprinted with the beta-blocker (S)-propranolol. The resulting superparamagnetic imprinted polymer beads were capable of binding [3H]-(S)-propranolol more strongly than a non-imprinted, otherwise identical, polymer. In a competitive radioligand binding assay using a magnet to separate polymer from solution, (R)-propranolol and (R,S)-metoprolol exhibited cross-reactivities of 19 and 0.7%, respectively, compared with (S)-propranolol.

Binding, Competitive↗
PubMed1996

Molecularly imprinted polymers for bioanalysis: chromatography, binding assays and biomimetic sensors.

The field of molecular imprinting has undergone rapid expansion in recent years, and applications have been demonstrated for imprinted materials in separations, analysis and catalysis. The range of compounds that has been imprinted has also expanded. Imprinted polymers have been used for the chromatographic separation of amino acids, sugars, drugs and nucleotides. Drugs have been measured using imprinted polymers as antibody substitutes in radioligand binding assays. Applications are being found for imprinted polymers in biomimetic sensing devices and sensors. Particularly interesting developments from the past year include ligand binding assays for morphine in aqueous buffers with similar specificity to antibody-based assays and the imprinting of cholesterol using a novel procedure in which the template is covalently bonded to the monomer during polymerization, but after cleavage, rebinds the polymer only by non-covalent interactions.

Journal Article↗
PubMed1996

Artificial antibodies to corticosteroids prepared by molecular imprinting.

BACKGROUND: Molecular imprinting can be used to prepare antibody and receptor mimics. We have previously shown that acrylic acid polymers can be imprinted to recognize a variety of small molecules. Here, we show that molecularly imprinted polymers (MIPs) can selectively recognize steroid structures. RESULTS: Artificial antibodies mimicking the binding performance of natural anti-corticosteroid antibodies have been prepared using a molecular imprinting protocol with either cortisol or corticosterone as a target molecule. The binding characteristics of a range of structurally related ligands were estimated using a form of radioimmunoassay. The antibody mimics were found to be highly selective for the ligands used in their preparation and the cross-reactivities with compounds of related structure resembled those obtained in studies with natural antibodies. CONCLUSIONS: The binding properties of MIPs, prepared against corticosteroids, exhibit strong similarities to those of naturally raised antibodies. Such artificial antibodies may serve as a useful complement to their natural counterparts in studies of corticosteroid binding events.

Adrenal Cortex Hormones↗
PubMed1996

Towards artificial antibodies prepared by molecular imprinting.

The new technique of molecular imprinting has increasingly been adopted by research laboratories worldwide during the last few years. We have studied the use of such imprints against drugs as artificial antibody-binding mimics in competitive radioimmuno-style binding assays. The recognition sites "molded" in the polymers mimic the binding sites of natural antibodies in their interactions with the target antigen. Binding constants are as low as 4.0 nmol/L for a small number of well-defined sites, and cross-reactivities are similar to or better than those observed with biological antibodies. In some cases, the polymers have been used to determine drug concentrations in human serum specimens.

Antibodies↗
PubMed1995

Selection of peptides with surface affinity for alpha-chymotrypsin using a phage display library.

Peptides with affinity for the surface of alpha-chymotrypsin (EC 3.4.21.1) were selected from a hexapeptide phage display library consisting of approximately 10(7) different clones. Seven selections were performed and five individual phage clones analysed. Compared to the primary library, the five peptide phage clones all interacted more strongly with alpha-chymotrypsin, and DNA sequencing of the phage clones revealed five different amino acid sequences: Gly-Ala-Val-Ile-Thr-His, Arg-Asp-Ile-Val-Val-Ala, Val-Tyr-Ser-His-Ala-Ser, Gly-Ser-Tyr-Ser-Ala-Gly and Leu-Asp-Ile-Val-Val-Ala. Two of the peptides exhibited 83% identity (i.e. a difference of just one amino acid). The chemically synthesized peptides competitively reduced the binding of the corresponding peptide phage clone to alpha-chymotrypsin. Binding of some of the selected peptide phage clones to alpha-chymotrypsin was also reduced by several of the other non-corresponding synthesized peptides, suggesting that these peptides have common recognition areas on the enzyme. Three of the synthesized peptides were poor substrates of alpha-chymotrypsin and they did not inhibit enzyme activity. Our results suggest that it is possible to select peptides from peptide phage display libraries with affinity for different surface structures on the enzyme, not involved in the biologically active site.

Amino Acid Sequence↗
PubMed1995

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

Amino Acid Sequence↗
PubMed1995

Separation of amino acids, peptides and proteins on molecularly imprinted stationary phases.

Stationary phases, to be used in high-performance liquid chromatography, were tailor-made for the separation of amino acids, peptides and proteins. The stationary phases were prepared by molecular imprinting, applying two different approaches. Low-molecular-mass compounds were imprinted in bulk polymers by copolymerization of functional monomers and cross-linkers in the presence of the compound of interest, the print molecule. These polymers were, after extraction of the print molecule, successfully applied as chiral stationary phases, showing high resolution and load capacity. The development of a surface-imprinting approach for the preparation of stationary phases selective for proteins is also discussed.

Amino Acid Sequence↗
PubMed1995

Horse liver alcohol dehydrogenase can accept NADP+ as coenzyme in high concentrations of acetonitrile.

The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCl pH 7.4, was investigated. The enzyme could accept NADP+ as coenzyme if it was first bio-imprinted with NADP+, i.e. precipitated from an aqueous buffer with 1-propanol in the presence of NADP+ dried, and then assayed in a system with less than 10% buffer. When assayed in a system with 25% buffer, no activity with NADP+ as coenzyme was observed. The activity was measured with a coenzyme regeneration assay with cinnamoyl alcohol and octylaldehyde as substrates. Other methods to prepare a binary complex of horse liver alcohol dehydrogenase and NADP+, i.e. if the enzyme was immobilized on silica and NADP+ added afterwards or if it was deposited on Celite together with NADP+, failed to show any bio-imprinting effect. The activity of horse liver alcohol dehydrogenase bioimprinted with NADP+ and assayed in acetonitrile/buffer mixtures with less than 10% buffer showed the same or higher activity than an enzyme preparation prepared by bio-imprinting with NAD+. The hypothesis is that the conformation of the active site of horse liver alcohol dehydrogenase is modified to one that is complementary to the ligand present during precipitation and drying. This is possible because of the restricted motility of the enzyme conformation in high concentrations of organic solvent. In the presence of more than 10% buffer the mobility increases in such a way that the imposed conformation with activity towards NADP+ disappears.

Acetonitriles↗
PubMed1994

Sugar binding polymers showing high anomeric and epimeric discrimination obtained by noncovalent molecular imprinting.

Noncovalent molecular imprinting of sugar compounds in ethylene glycol dimethacrylate-methacrylic acid copolymers yielded materials containing highly selective sugar binding sites. Investigation of a range of polymers demonstrated that the resulting polymer imprints have a high degree of both anomeric and epimeric selectivity favoring the original print molecule. In HPLC assays, a polymer imprinted using p-nitrophenyl-alpha-D-galactoside could separate the alpha and beta anomers of the same compound with near baseline resolution. A polymer imprinted using p-nitrophenyl-beta-D-galactoside showed similar results but with the beta anomers retained longer as expected. Anomeric discrimination of closely related sugars was also possible, with the degree of separation depending on the structural resemblance to the print molecule. Similarly, a polymer imprinted with p-nitrophenyl-alpha-L-fucoside could separate alpha/beta mixtures of p-nitrophenyl-L-fucoside with baseline resolution. Using radioligand displacement assays a polymer imprinted using octyl-alpha-D-glucoside was shown to bind methyl-alpha-D-glucoside with a 40-fold higher affinity than that for the beta-anomer. The epimeric selectivity was even more impressive: methyl-alpha-D-mannoside and methyl-alpha-D-galactoside had 130- and 240-fold lower affinity, respectively, than methyl-alpha-D-glucoside. The results are discussed in relation to possible uses of such polymers in separation and analysis.

Carbohydrates↗
PubMed1994

Selection of peptides with affinity for single stranded DNA using a phage display library.

A hexapeptide phage library was used to affinity select peptides which interact with single stranded heptaoligonucleotides consisting of cytosine (oligo-C). Selections were performed in two different buffer systems, 50 mM 2[N-Morpholino]ethanesulphonic acid(MES)-buffer, pH 5.5, and 50 mM Tris(hydroxymethyl)-aminomethane, 150 mM NaCl, pH 7.5, respectively. Selection was more successful in 50 mM MES-buffer and three clones with affinity for oligo-C were further investigated. The peptides, Pro-Pro-Pro-Leu-Tyr-Phe, Arg-Phe-Cys-Asp-Thr-Ser and Arg-Ser-Arg-Leu-Ile-Trp, all interacted more strongly with oligo-C compared to the original peptide phage library and wildtype phage. The selected clones also showed different specificity in the interaction with oligo-C, -G, -A and -T.

Amino Acid Sequence↗
PubMed1994

Metabolic studies on Saccharomyces cerevisiae containing fused citrate synthase/malate dehydrogenase.

We have constructed two different fusion proteins consisting of the C-terminal end of CS1 fused in-frame to the N-terminal end of MDH1 and HSA, respectively. The fusion proteins were expressed in mutants of Saccharomyces cerevisiae in which CS1 and MDH1 had been deleted and the phenotypes of the transformants characterized. The results show that the fusion proteins are transported into the mitochondria and that they restore the ability for the yeast mutants CS1-, MDH1-, and CS1-/MDH1- to grow on acetate. Determination of CS1 activity in isolated mitochondria showed a 10-fold increase for the strain that expressed native CS1, relative to the parental. In the transformant with CS1/MDH1 fusion protein, parental levels of CS1 were observed, while one-fifth this amount was observed for the strain expressing the CS1/HSA conjugate. Oxygen consumption studies on isolated mitochondria did not show any significant differences between parental-type yeast and the strains expressing the different fusion proteins or native CS1. [3(-13)C]Propionate was used to study the Krebs TCA cycle metabolism of yeast cells containing CS1/MDH1 fusion constructs. The 13C NMR study was performed in respiratory-competent parental yeast cells and using the genetically engineered yeast cells consisting of CS1- mutants expressing native CS1 and the fusion proteins CS1/MDH1 and CS1/HSA, respectively. [3(-13)C]Propionate is believed to be metabolized to [2(-13)C]succinyl-CoA before it enters the TCA cycle in the mitochondria. This metabolite is then oxidized through two symmetrical intermediates, succinate and fumarate, followed by conversion to malate, oxalacetate, and other metabolites such as alanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1994

Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase.

We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined. It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed. In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.

Aspartate Aminotransferases↗
PubMed1994

Molecular imprinting.

Molecular imprinting is an emerging methodology for the creation of selective recognition sites in synthetic polymers. This technique entails the polymerization of functional monomers in the presence of an important molecule (template). Recent studies have shown that the polymers obtained exhibit a surprisingly high degree of stereo- and regiospecific selectivity, making the commercial use of such tailor-made separation materials in several areas, such as the chiral separation of bioactive molecules and as antibody mimics, a realistic possibility.

Binding Sites, Antibody↗
PubMed1994

Chiral recognition of N alpha-protected amino acids and derivatives in non-covalently molecularly imprinted polymers.

Highly crosslinked synthetic polymers, selective for various N alpha-protected amino acids and derivatives, were prepared by non-covalent molecular imprinting. Methacrylic acid and ethylene glycol dimethacrylate were copolymerized in the presence of the print molecules, which were subsequently extracted from the polymers. The recognition of the polymers for the print molecules and molecules of similar structures was investigated by using the polymers as stationary phases in HPLC. The functional groups of the print molecules interact via hydrogen bonds with the positioned carboxyls of the polymer. It was shown that the N alpha-protecting group, the C alpha-protecting group and the amino acid side chain are also recognized by the binding sites in the polymer.

Amino Acids↗
PubMed1993

Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

Alkaline Phosphatase↗
PubMed1993

Drug assay using antibody mimics made by molecular imprinting.

Ligand-binding assays are used for determination of minute amounts of substances in the bloodstream. Such assays require a receptor that specifically binds the substance of interest. The receptor used is often an antibody, but antibodies require special handling and a costly production procedure. We have used molecular imprinting, a method for creating selective recognition sites in synthetic polymers, to prepare polymers that mimic antibody combining sites. Molecular imprints made against theophylline and diazepam showed strong binding and cross-reactivity profiles similar to those of antibodies. Here we describe a new radiolabelled ligand-binding assay, the molecularly imprinted sorbent assay, which uses antibody mimics. This assay accurately measures drug levels in human serum, with results comparable to those obtained using a well established immunoassay technique. Antibody mimics, which are stable and readily prepared by molecular imprinting, may provide a useful general alternative to antibodies.

Antibodies↗