Anthony E.G. Cass

Ferricinium ion as an electron acceptor for oxido-reductases

Abstract The ability of ferrocene monocarboxylic acid to act as a mediator to some oxido-reductases was investigated. Dc cyclic voltammetry was used to study the coupling of the electrode reaction, associated with the oxidation of ferrocene to ferricinium ion, to an enzymatic oxidation reaction. The reactions catalysed by pyruvate oxidase, xanthine oxidase, sarcosine oxidase, lipoamide dehydrogenase, glutathione reductase and alcohol dehydrogenase were investigated. The rates of reaction between the ferricinium ion and the reduced form of each of the enzymes, in the presence of their substrates, were measured.

The reaction of flavocytochrome b2 with cytochrome c and ferricinium carboxylate. Comparative kinetics by cyclic voltammetry and chronoamperometry

Abstract The kinetics of the l -lactate/flavocytochrome b 2 /cytochrome c system were investigated by two different electrochemical methods. Cyclic voltammetry and potential step chronoamperometry. Both of these methods gave the same value for the rate constant of 5 · 10 6 M −1 s −1 which agrees very well with the value previously determined by conventional spectrophotometric methods. The advantages and limitations of electrochemical methods are described. The replacement of cytochrome c by ferricinium carboxylate is described and it is shown that this compound is as effective an acceptor as cytochrome c : the second-order rate constant is 6.7 · 10 6 M −1 · s −1 .

Method for determining paracetamol in whole blood by chronoamperometry following enzymatic hydrolysis.

A method is proposed for the determination of paracetamol in whole undiluted blood, based on the enzymatic hydrolysis of the drug to p-aminophenol, which is then measured by chronoamperometry at a glassy carbon electrode. Hydrolysis of the paracetamol prior to electro-oxidation is shown to alleviate problems that arise from high background currents in the whole blood and so ensures a good linear correlation (r greater than 0.99) between the current and the paracetamol concentration. Recovery experiments and comparison with a reference method based on spectrophotometry suggest that the electrochemical assay only measures that proportion of paracetamol that is not bound to serum albumin.

Isolation of a membrane protein by chromatofocusing: Cytochrome b-561 of the adrenal chromaffin granule

Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.

1H Nuclear magnetic resonance spectroscopy of yeast copper-zinc superoxide dismutase. Structural homology with the bovine enzyme

High resolution nuclear magnetic resonance (n.m.r.) spectroscopy of the copper-zinc superoxide dismutase from Saccharomyces cerevisiae has revealed a substantial structural homology with the bovine enzyme. N.m.r. spectra of the apo enzyme and the holo-reduced and holo-oxidized enzymes are reported and assignments are made to the histidines in the active site and the single tyrosine residue. All the assignments are in agreement with the known amino-acid sequence and the geometry of the active site is virtually unchanged.Addition of halide ions to the reduced holo enzyme results in the perturbation of the chemical shift of three histidine C2 protons and the degree of perturbation is Cl−∼Br−>I−>F− for 1m solutions of the anions. The enzyme has also been shown to retain its structure at 75°C.

Ferrocene-modified horseradish peroxidase enzyme electrodes. A kinetic study on reactions with hydrogen peroxide and linoleic hydroperoxide

An enzyme electrode for the determination of hydrogen peroxide and linoleic hydroperoxide, a model compound for lipid hydroperoxides, is described. Horseradish peroxidase bearing covalently attached ferrocene groups as electron transfer mediators is used as the sensing molecule and is adsorbed to the surface of disposable printed carbon electrodes as the base sensor. The electrodes are suitable for the determination of hydrogen peroxide in the range 1–50 µmol l–1 and linoleic hydroperoxide in the range 5–100 µmol l–1. A comparison of the kinetic behaviour of the modified enzyme in solution by spectrophotometry, and on the electrode by amperometry, shows that the catalytic current is limited by mass transport when hydrogen peroxide is the substrate and enzyme kinetics when linoleic hydroperoxide is the substrate.

The binding of zinc(II) to bleomycin: An investigation using 1H NMR spectroscopy

The bleomycins (BLM) are a group of antibiotics isolated as the copper complexes from Streptomyces verticillus [1]. They are used in the treatment of squamous cell carcinomas. It has been suggested that their mode of action involves strand scission of DNA [2]. This strand scission is inhibited by metal ions such as Cu(II), Zn(II) and Co(II) [3] and a mechanism for the scission, involving the Fe(II) complex, has been suggested [4,5]. Knowledge of the structures of the metal ion complexes of BLM would appear to be a prerequisite to an understanding of its function. In this study the binding of zinc(II) to BLM was investigated using 1H nuclear magnetic resonance spectroscopy (NMR).

Assignment of the metal-histidine ligands from the tritium exchange rate of the histidine C-2 protons in the Cu (II), Zn(II)-superoxide dismutase from Saccharomyces cerevisiae

The metal-histidine ligands in the yeast Cu, Zn-superoxide dismutase have been assigned on the basis of the differential rate of tritium exchange of the histidine C-2 protons. The zinc ligands have been identified as His-71 and His-80 while His-46, His-48 and His-120 appear to be coordinated to the copper. The rate of tritium incorporation into His-63 was inconsistent with the coordination of this residue to copper and was suggestive further of a unique coordination of His-63 to zinc.

Heavy metal binding to biological molecules: Identification of ligands by observation of 199Hg-1H coupling

Many biological molecules bind metal ions either at specific sites as a prerequisite to the expression of function, or at suitable sites elsewhere in the molecules [ 1,2]. There have been many approaches towards elucidating the nature and the geometry of the immediate coordination sphere around the metal ion, and although X-ray diffraction methods yield the most unequivocal answer, many proteins have not yet succumed to this technique. Recently we have used high resolution nuclear magnetic resonance (NMR) spectroscopy to study the coordination in a variety of metalloproteins [3,4] . Although NMR spectroscopy has proved an extremely powerful method when the coordinating groups include histidyl residues, the distinction between coordinated and non-coordinated residues is generally based upon a comparison of the apoand the holo-protein, and may thus be open to the objection that the observed differences are due to indirect effects of metal binding, rather than to coordination per se. An ideal probe of metal coordination would involve the use of a metal ion which perturbs the ligands by a direct, through bond, effect and have a blank as near identical as possible. A metal ion with a nuclear spin, I, of l/2 is expected to be such a probe, for it should give rise to spin-spin splitting in the NMR spectrum of coupled, ligand, nuclei. The use of an isotope of the same metal with I = 0 would provide an ideal blank. The only example of