Arne Torstensson

Electrocatalytic oxidation of reduced nicotinamide coenzymes by graphite electrodes modified with an adsorbed phenoxazinium salt, meldola blue

Abstract Meldola Blue (7-dimethylamino-1,2-benzophenoxazine) can be adsorbed on graphite to give chemically modified electrodes. The electrochemical redox reactions of the phenoxazine are fairly reversible at low coverages with an E′o of −175 mV vs. SCE at pH 7.0. The electrode was most stable in acid solutions, at pH 6.0 its electrochemical activity decreased by 15% during 2h. The adsorbed compound mediated electron transfer in the electrocatalytic oxidation of the nicotinamide coenzymes (NADH and NADPH). The formation of a charge transfer complex between Meldola Blue and the coenzyme is demonstrated by experiments with a rotating disk electrode. The complex decomposes in a rate limiting step (k+2=30 s−1) to the oxidized coenzyme and the reduced Meldola Blue. The latter can be reoxidized in a fast electrochemical step. The overall result is an electrocatalytic oxidation at a voltage which is about 500 mV lower than at an unmodified electrode.

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

Abstract A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β- d -glucose. The detection limit was 0.25 μM β- d -glucose for 50-μl samples. The electrode was kept at −50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.

Catalytic oxidation of NADH by surface-modified graphite electrodes

Abstract A voltage-facilitated immobilization procedure for phenazine methosulphate (PMS) and phenazine ethosulphate (PES) on graphite electrodes is described. The graphite is immersed in a solution of PMS or PES and a voltage of 1.1 V vs. SCE is applied for 15 min. The surface coverage was 5×10−9 mol cm−2. At pH 7.0 the E1/2 for immobilized PMS (−160 mV) and PES (−210 mV) were the same as for the soluble compounds. The modified electrodes catalysed electron transfer from dihydronicotinamide adenine dinucleotide (NADH). Linear sweep voltammetry produced anodic peaks of the NADH oxidation at −50 mV (PMS electrode) and −100 mV (PES electrode). The electrochemical step was faster than the preceding chemical step involving a reaction between NADH and the immobilized PMS or PES.

A kinetic study of the reaction between dihydronicotinamide adenine dinucleotide (NADH) and an electrode modified by adsorption of 1,2-benzophenoxazine-7-one

Abstract 1,2-Benzophenoxazine-7-one (BPO) was absorbed on graphite to give a chemically modified electrode. The electrochemical redox reactions of BPO itself are fairly reversible at coverages less than 2 × 10−9 mol cm−2 with an E°′ of −210 mV (vs. SCE) at pH 7.0. The E°′ decreased 60 mV per pH unit from pH 1 to 11. The adsorbed BPO mediated electron transfer in the electrocatalytic oxidation of the dihydronicotinamide adenine dinucleotide (NADH). The kinetic reaction was studied with a rotating disk electrode modified with BPO for different coenzyme concentrations and pHs. The formation of a charge-transfer complex between NADH and the mediator was indicated. The rate of reaction increased as the pH decreased; the slope of a log k+2 vs. pH plot was −0.55, indicating a complex proton dependence. A reaction scheme with two parallel paths is discussed.

Electrochemical oxidation of reduced nicotinamide adenine dinucleotide directly and after reduction in an enzyme reactor

Abstract Reduced nicotinamide adenine dinucleotide (NADH) was oxidized coulometrically at a rotating platinum gauze electrode at 0.7 V vs. SCE at pH 9. The NAD + formed was reduced enzymatically in a reactor containing immobilized alcohol dehydrogenase. Several recyclings were made with the same lot of NADH. The coulometric results were in reasonable agreement with spectrophotometric measurements. The overall conversion to enzymatically active NAD + showed a current efficiency of 99.3%. The electrode pretreatment described is essential for high current efficiency. A continuous method of oxidation of alcohol with electrolytic regeneration of cofactor and removal of aldehyde by dialysis was tested.

Biofuel anode based on d-glucose dehydrogenase, nicotinamide adenine dinucleotide and a modified electrode

A biofuel cell anode has been made from a modified graphite electrode and immobilized d-glucose dehydrogenase [β-d-glucose:NAD(P)+ 1-oxidoreductase, EC 1.1.1.4 7] so that energy could be drawn from the conversion of d-glucose to d-gluconic acid. An equivalent amount of dihydronicotinamide adenine dinucleotide (NADH) was formed from NAD+ and reduced the surface groups of the modified electrode. Reoxidationn of the latter produced the electrons necessary for a power output from the cell. Electrode modification was made by adsorption of N,N-dimethyl-7-amino 1,2-benzophenoxazinium onto the graphite. A current density of 0.2 mA cm−2 at a cell voltage of ∼0.8 V was obtained for more than 8 h with a simulated oxygen cathode. The internal resistance in the cell, in particular in the separator, appeared to be the main current-limiting factor.

Electrochemical Regeneration of NAD Covalently Bound to Liver Alcohol Dehydrogenase

Abstract An enzyme-coenzyme complex was immobilized on the surface of a glassy carbon electrode and investigated with cyclic voltammetry in ethanol-containing buffers. The complex consists of an Liver Alcohol Dehydrogenase molecule to which an NAD-analogue is covalently attached via its straight six-carbon, spacer. One cycle was observed but repeated recycling could not be carried out. presumably due to catalytic decomposition of the coenzyme at the electrode surface.

Spectroseopic, NMR, and theoretical studies on the intramolecular electron transfer in FAD

Abstract The charge transfer interaction between the two bases in FAD has been investigated by UV-VIS spectra. The charge transfer interaction in FAD is appreciably stronger than in NAD, and it appears feasible to modify FAD suitably for use in a bioelectrochemical anode. The molecule, from NMR data, seems to exist in intramolecularly folded conformations, that are extremely favorable for charge transfer interaction. Charge density calculations have been done on the flavin moiety, both in the oxidized and reduced form, by quantum-chemical methods. The charge density data suggests that it is possible to attach a semiconducting chain to one of the methyl groups in flavin without appreciably affecting the overall behaviour of the system. On the whole, this system appears to be a promising candidate for an electron transfer mediator in a bioelectrochemcial anode.

Interaction of FAD and NAD with aromatic amino acids

Abstract The interaction of FAD and NAD (both in the oxidized forms) has been studied by U.V.-vis spectroscopy as well as by p.m.r. studies. The interaction of these two coenzymes with the four aromatic amino acids phenylalanine, histidine, tyrosine and tryptophan has been studied in dilute aqueous solutions as a function of temperature. It appears that the interaction of NAD with these four aromatic amino acids is insignificant under the experimental conditions. On the other hand, it appears that the interaction of FAD with tryptophan is small but significant. The effects due to self-aggregation and intermolecular stacking seem small for FAD and negligible for NAD under the experimental conditions involved. The small but significant chemical shifts observed for the FAD-tryptophan system suggest a geometric arrangement in which the two aromatic bases overlap each other. The interaction is probably a charge transfer type, but the effect is too small to be conclusively decided from the U.V.-vis spectra. It is probable that similar interaction is present for other systems but is too weak to be detected under the experimental conditions employed. The strength of the interaction is probably directly related to the effectiveness of the overlap of the two bases involved.