Journal articles

The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine.

Optical sensors are widely utilized in drug discovery to analyze biomolecular interactions in vitro. Aside from additional time and cost demands, other issues associated with labeled screening methods include signal interference that can arise from the label per se and/or the screened compounds. This report describes an enzyme inhibition-based holographic sensor as a potential label-free detection system, using acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7; abbreviated herein AChE) as the model enzyme.

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz.

The molecular basis for differences in germinant recognition of Bacillus megaterium QM B1551 spores containing the GerVB and/or GerUB receptor proteins has been examined by site-directed mutagenesis and the construction of cross-homologue chimeras. Focusing on nonconserved residues predicted to reside in transmembrane domains 9 and 10, we demonstrate that GerVB residues Ser319 and Leu345 are of particular importance in defining the specificity and apparent affinity of the receptor for germinants.

We present a flexible new sensor system that combines the joint advantages of (i) discretely functionalized, code-bearing, microparticles and (ii) label-free detection using grating-coupled surface plasmon resonance. This system offers the possibility of simultaneously investigating the real-time binding kinetics of a variety of molecular interactions. One single multiplexed assay could employ a wide range of immobilization chemistries, surface preparation methods, and formats.

The development of smooth hydrophilic surfaces that act as substrates for supported lipid bilayers (SLBs) is important for membrane studies in biology and biotechnology. In this article, it is shown that thin films of poly(dimethylsiloxane) (PDMS) formed on a sensor surface can be used as a substrate for the deposition of reproducible and homogeneous zwitterionic SLBs by the direct fusion of vesicles. Poly(dimethylsiloxane) solution (1% w/v) was spin coated on Love acoustic wave and surface plasmon resonance devices to form a thin PDMS layer.

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used.

The effectiveness of a new immobilized cationic triazine dye was investigated alongside two new amphoteric triazine dyes and two well known anionic triazine dyes, Procion Red H-3B and Procion Blue H-B, as chromatographic media for binding four familiar proteases-trypsin, chymotrypsin, thrombin and carboxypeptidase-B-as well as a typical oxidoreductase, lactate dehydrogenase, and human serum albumin.

The glucose-dependent cross-linking of phenylboronate gels was investigated in order to understand the principle underlying glucose-induced hydrogel contraction. Glucose conformation upon binding to phenylboronates in aqueous solution was also examined and a 1:2 glucofuranose: boronate model was suggested by the results obtained.