Biomedical subjects

C R Lowe

Publications and source records attributed to C R Lowe.

152 records · Page 9Linked to original sources

PubMedDate not supplied

Design, synthesis and characterisation of affinity ligands for glycoproteins.

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein-carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar-binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine-based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself.

Adsorption↗

PubMedDate not supplied

A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand.

An IgG-binding ligand library comprising 88 adsorbents based on a known lead compound (Li et al., 1998) was generated on an agarose solid phase. Individual members of the library were synthesized in two chemical steps using cyanuric chloride as the scaffold immobilized on the beaded support. The library was screened for binding of pure human IgG, whence selected ligands from the library were further assessed for specificity by the purification of IgG from human plasma. The potential of this strategy for the rapid identification and evaluation of chemical leads was demonstrated by the discovery of ligands with IgG binding capabilities. It was found that ligands comprising 3-aminophenol and an aminonaphthol moiety substituted on a triazine nucleus generally performed better than other ligands in the library. An immobilized ligand 22/8 adsorbent was able to purify IgG with high yield and a purity >99% from diluted human plasma.

Adsorption↗

PubMedDate not supplied

Design, synthesis and evaluation of biomimetic affinity ligands for elastases.

A low-molecular-weight biomimetic affinity ligand selective for binding elastase has been designed and synthesized. The ligand was based on mimicking part of the interaction between a natural inhibitor, turkey ovomucoid inhibitor and elastase, and modelled from the X-ray crystallographic structure of the enzyme-inhibitor complex. Limited solid-phase combinatorial chemistry was used to synthesize 12 variants of the lead ligand using the triazine moiety as the scaffold for assembly. The ligand library was screened for its ability to bind elastase and trypsin, and two ligands were studied further. Ligand C4/6 [2-alanyl-alanyl-4-tryptamino-6-(alpha-lysyl)-s-triazine] was found to bind porcine pancreatic elastase, but not trypsin, with a dissociation constant of 6 x 10(-5) M and a binding capacity of 21 mg elastase per ml gel. The adsorbent was used to purify elastase from a crude extract of porcine pancreas. Immobilized ligand C4/5 6 [2-alanyl-alanyl-4-tyramino-6-(alpha-lysyl)-s-triazine] was similarly chosen for optimal binding of elastase from cod and used to purify the enzyme from a crude extract of cod pyloric caeca. Ligand C4/6 was subsequently synthesized in solution and its structure verified by 1H-NMR.

Animals↗

PubMedDate not supplied

Glucose-sensitive holographic sensors.

Holographic sensors for monitoring glucose were fabricated from hydrogel films containing chemical ligands based on phenylboronic acid. The films were transformed into reflection holograms using a diffusion method coupled with exposure to laser light. The diffraction wavelength of the holograms was used to monitor the swelling of the hydrogel film in the presence of glucose. Fully reversible changes in diffraction wavelength were demonstrated, highlighting the potential for using these holograms as glucose sensors.

Biosensing Techniques↗

PubMedDate not supplied

The application of the acoustic spectrophonometer to biomolecular spectrometry: a step towards acoustic "fingerprinting".

A tunable acoustic biosensor for investigating the properties of biomolecules at the solid-liquid interfaces is described. In its current, format the device can be tuned to frequencies between 6.5 MHz and 1.1 GHz in order to provide a unique detection feature: a variable evanescent wave thickness at the sensor surface. The key to its successful implementation required the careful selection of antennae designs that could induce shear acoustic waves at the solid-liquid interface. This non-contact format makes it possible to recover resonant shear acoustic waves over 100 different harmonic frequencies as a result of the electrical characteristics of the spiral coil. For testing this multifrequency sensing concept the surface of a quartz disc was exposed to solutions of immunoglobulin G (IgG) to form an adsorbed monolayer, whence protein A and IgG were added again in order to form multilayers. Spectra at frequencies between 6 and 600 MHz were generated for each successive layer and revealed two characteristic phases: an initial phase at the low megahertz frequencies consistent with the conventional Sauerbrey relation, and a possible additional phase towards the high megahertz to gigahertz frequencies, that we believe relates to the structure of the biomolecular film. This two-phase behaviour evident from differences between high and low frequencies, rather than from any distinct frequency transition, was anticipated from the reduction in evanescent wave thickness down to nanometre dimensions, and thin film resonance phenomena that are known to occur for film and fluid systems. These measurements suggested that the single element acoustic biosensor we present here may form the basis from which to generate acoustic molecular spectra, or "acoustic fingerprints", in a manner akin to optical spectroscopy.

Acoustics↗

PubMedDate not supplied

De novo design, synthesis and screening of a combinatorial library of complementary ligands directed towards the surface of cutinase from Fusarium solani pisi.

The protein surface is the interface through which a protein molecule senses the external world. The composition of this interface, in charged, polar and/or hydrophobic residues is crucial for both the activity and stability of the protein. Protein immobilization on surfaces has been extensively explored as one of the most effective approaches for stabilization. The mechanism of stabilization, however, is still poorly understood, and usually the success of any method is more a matter of trial and error rather than the result of rational concepts. The importance of local unfolding processes in a number of biologically significant processes has been recognized and attracted increasing attention. Unfolding regions have been localized in different proteins including the recombinant cutinase from Fusarium solani pisi. The study of three structural surface regions associated with early cutinase unfolding events was the basis for the approach followed in this work. A 64-member solid-phase combinatorial library of ligands was synthesized on a triazine-substituted agarose matrix using a modified 'mix and split' procedure. The combinatorial library was assessed for binding to cutinase from Fusarium solani pisi in a biologically active form. Four lead ligands (3/5, 3/7, 4/5, 4/7) have been selected in which immobilized cutinase presented a relative activity of 30-60% as compared to the free enzyme.

Adsorption↗

PubMedDate not supplied

Detection of immuno-complex formation via surface plasmon resonance on gold-coated diffraction gratings.

The sensitivity of surface plasmon resonance techniques to changes in local interfacial refractive index has been exploited to detect immuno-complex formation in two model biochemical systems. A gold-coated diffraction grating has been used to excite surface plasmons at the gold/solution interface to which either human immunoglobulin G or the immunoglobulin fraction of sheep antiserum to human serum albumin was physically adsorbed. The complementary proteins, either affinity purified goat anti-human-IgG IgG or human serum albumin was subsequently specifically bound by immuno-complex formation. The binding reactions could be followed with respect to time.

Animals↗

PubMedDate not supplied

A microelectronic conductimetric biosensor.

The fabrication and operation of a microelectronic conductimetric biosensor is described. The device monitors the change in solution conductance occasioned by the catalytic action of enzymes immobilised over a planar conductance cell comprising serpentined and interdigitated metal conductor tracks. The output of the instrument was linear over a 3 min period on addition of urea to a sample cell overlaid with immobilised urease. The responses to any given urea concentration were reproducible to within approximately +/- 1%. The device responds to urea present in serum samples.

Electrochemistry↗