Journal articles

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.

Oligonucleotides employed in molecular biology have previously been purified by gel electrophoresis, gravity flow chromatography and more recently, high-performance liquid chromatography. However, these techniques have a number of problems and for this reason we investigated high-performance charge-transfer chromatography using the dye acriflavin coupled to silica as the stationary phase. Numerous oligonucleotides were purified using this technique and in this report we present data on four such oligonucleotides two homo-oligonucleotides and two hetero-oligonucleotides.

A simple methoxylated derivative of the triazine dye, Procion blue H-B, selectively precipitates rabbit muscle lactate dehydrogenase from solution. Optimum protein precipitation occurred at an enzyme subunit:dye ratio of approximately 2:1 and was fully reversible upon addition of competitive ligands such as NADH. With a crude extract of rabbit muscle, affinity precipitation with the dye followed by dissolution with NADH yielded homogeneous lactate dehydrogenase in 97% overall yield. © 1986.

The redox mediator hexacyanof errate(II/III) has been electrodeposited on nickel by anodization of porous nickel electrodes In aqueous electrolytes containing hexacyanoferrate(II) Ions. Cyclic voltammetry of the resultant nickel hexacyanoferrate films reveals the presence of two peaks corresponding to the hexacyanoferrate(II/III) redox couple. The redox films catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) with a decrease In overvoltage of ~150 mV compared to unmodified electrodes. The steady-state current response at +0.2 V vs.

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 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.

A number of terminal ring analogues of the anthraquinone dye C.I. Reactive Blue 2 were synthesised and characterised. The interaction of these dye analogues with horse liver alcohol dehydrogenase was investigated by difference spectroscopy and analytical affinity chromatography. Studies by difference spectroscopy showed that anionic terminal ring substituents or an unsubstituted phenyl ring promoted tight binding of the dye to the enzyme, whereas neutral or cationic terminal ring substituents reduced the affinity of the dye. Terminal ring analogues of C.I.

Polyampholytic acrylic co-polymers have been shown to form two-phase systems with polyvinyl alcohol under appropriate conditions of pH, ionic strength and temperature. These novel systems have been used for the purification of a number of proteins from crude extracts. The partition behavior of proteins appeared to be influenced predominantly by electrostatic interactions and could be manipulated by including electrolytes in the system.

A novel approach to an amperometrlc enzyme electrode for the analysis of glucose is described. The technique entails the electrochemical codeposltfon of the redox enzyme, glucose oxidase, In the conducting organic polymer, polypyrrole. Reagentless glucose electrodes have been generated by the synthesis of N-subStltuted pyrrole monomers containing redox-active side chains designed to accept electrons from the reduced form of the enzyme. Ferrocene-pyrrole conjugates were found to be efficient oxidants of reduced glucose oxidase, which on anodic polymerization form redox-active films.

Terminal ring isomers of the reactive anthraquinone dye C.I. Reactive Blue 2 were synthesised and the reaction product and potential hydrolytic by-products analysed by thin-layer chromatography and reversed-phase high-performance liquid chromatography using N-cetyltrimethylammonium bromide as an ion-pair reagent. These techniques provided excellent resolution of potential chromophoric contaminants and showed the predominant formation of a single dye product in each case.