Graham Davis

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.

Bioelectrochemical fuel cell and sensor based on a quinoprotein, alcohol dehydrogenase

Abstract A biofuel cell, yielding a stable and continuous low-power output, based on the enzymatic oxidation of methanol to formic acid has been designed and investigated. The homogeneous kinetics of the electrochemically-coupled enzymatic oxidation reaction were investigated and optimized. The biofuel cell also functioned as a sensitive method for the detection of primary alcohols. A method for medium-scale preparation of the enzyme alcohol dehydrogenase [alcohol:(acceptor) oxidoreductase, EC 1.1.99.8] is described.

Carbon monoxide :acceptor oxidoreductase from Pseudomonas thermocarboxydovorans strain C2 and its use in a carbon monoxide sensor

The carbon monoxide:acceptor oxidoreductase from autotrophically-grown Pseudomonas thermocarboxydovorans strain C2 was purified to 95% homogeneity and found to contain fiavin, iron, acid-labile sulphide and possibly molybdenum; its molecular weight was 2.7 × 105. The enzyme catalyzed the oxidation of CO to CO2 with various electron acceptors including phenazine ethosulphate, methylene blue, hexacyanoferrate(III) and ferrocene derivatives (ferrocene monocarboxylic acid, 1,1′dimethylferrocene and horse heart cytochrome C) but not viologen dyes, NAD(P) and oxygen. The optimum pH and temperature for enzyme activity in the in vitro assay were 7.5 and 80°C, respectively. Carbon monoxide is the only known electron donor (apparent Km 5 × 10−7 M) and acetylene, cyanide, 8-quinolinol and sulphydryl reagents inhibited the enzyme. Second-order homogeneous rate constants for the reaction between the reduced enzyme and either cytochrome C (3.0 × 104 l mol−1 s−1) or the ferricinium ion of ferrocene monocarboxylic acid (4.0 × 105 l mol−1 s−1) were calculated by using cyclic voltammetry. The latter reaction was exploited by incorporating 1,1′-dimethylferrocene in a carbon electrode and retaining carbon monoxide oxidoreductase behind a membrane at the surface. The probe gave a linear current response to aqueous concentrations of carbon monoxide up to 65 μM and achieved a steady current in < 15 s. A similar configuration was used to detect gaseous carbon monoxide. With a fuel cell mode, 20 nmol could be detected.

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 .

Detection of ATP and creatine kinase using an enzyme electrode

Abstract An electrochemical method for the detection of ATP and creatine kinase (ATP:creatine N -phosphotransferase, EC 2.7.3.2) coupled through hexokinase to an amperometric glucose enzyme electrode is described. The assay can be used to monitor creatine kinase activity over the range 0.01–10 U ml −1 and forms the basis for the development of a single-use biosensor.