Electrochemical evaluation of the reaction rate between methyl viologen mediator and diaphorase enzyme for the electrocatalytic reduction of NAD+ and digital simulation for its voltammetric responses

The electrocatalytic reduction of NAD⁺ using diaphorase enzyme was studied. Methyl viologen was used as an electron transfer mediator between an electrode and the enzyme. A catalytic wave for the reduction of NAD⁺ when all the species were in the solution was measured with cyclic voltammetry at a gold-amalgam electrode which showed low background currents at negative potentials. Steady-state currents could be obtained under the conditions of slow scan rate, low methyl viologen concentration, and high NAD⁺ concentration as the electrode reaction was converted to an electrochemical-catalytic (EC′) reaction. The bimolecular rate constant for the reaction of the reduced methyl viologen with the oxidized diaphorase was estimated as 7.5×10³ M^?1 s^?1 from the slope of the current versus [MV²⁺] plot. Another slope of the current against the square root of the enzyme concentration also gave a close value of 6.7×10³ M^?1 s^?1. In the calculation of the rate constant, the stoichiometric factor when it is not one-to-one was considered. With the evaluated rate constant, digital simulation using the suggested reaction mechanism was compared with the experimentally obtained voltammograms. Satisfactory agreement indicates that the evaluation methods of the rate constant and the suggested mechanism are appropriate for the mediated enzyme-catalyzed electrochemical reactions.     

ATP-ase activity in the odontoblastic layer of rat incisor Determination with a radiochemical and a colorimetric method

The ATP-splitting enzyme activity in odontoblasts isolated from rat incisors has been studied by means of a radiochemical and a colorimetric micromethod. The results with the two methods were virtually identical. The reaction was linear with time for at least 45 min. The pH optimum was found to be 9.8 independently of the ATP concentration. Maximal substrate saturation occurred at a total ATP concentration of 3 mM. Ca²⁺ and Mg²⁺ ions activated ATP degradation. F- ions did not affect the activity at low concentrations, whereas higher concentrations were inhibitory. Na⁺ and K⁺ ions had no influence on ATP splitting enzyme activity, while PO4^3- ions were slightly inhibitory. Urea inhibited the enzyme activity at concentrations above 1.5 M, while EDTA and EGTA were strong inhibitors at very low concentrations. When incubating in the presence of low concentrations of specific inhibitors for nonspecific alkaline phosphatase, levamisole and R 8231, about 20% ATP degrading enzyme activity remained. In conclusion it is suggested that there are at least two ATP degrading phosphatases active at alkaline pH.     

A methanol/dioxygen biofuel cell that uses NAD+-dependent dehydrogenases as catalysts: application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at low overpotentials

Diaphorase catalyzes efficiently the oxidation of NADH to NAD⁺ by benzylviologen. This reaction was used to regenerate the NAD⁺ required for the biocatalyzed oxidation of methanol to CO₂ using NAD⁺-dependent dehydrogenases. A methanol/dioxygen biofuel cell was assembled using these bioreactions in the anode compartment. The open-circuit voltage of the cell was 0.8 V, and the maximum power output of the cell was 0.67 mW/cm² of graphite at 0.49 V. These results reflect the low overpotential at which NADH is oxidized and demonstrate a new approach to lowering voltage losses in biofuel cells due to activation overpotentials.     

Electron transfer between hydrogenase and 316L stainless steel: identification of a hydrogenase-catalyzed cathodic reaction in anaerobic mic

Electron transfer between the NAD-dependent hydrogenase from Ralstonia eutropha and AISI 316L stainless steel was studied with a view to identifying a possible role of hydrogenase in anaerobic microbially influenced corrosion (mic), as already suspected. A thin spectroelectrochemical cell was designed and qualified, taking great care to obtain a reproducible surface state of the stainless steel grid electrode. Successive electrolyses were performed at potential values in the range from −0.60 to −0.90 V(SCE), with different NAD⁺ concentrations, and different hydrogenase activities. Hydrogenase significantly enhanced the charge consumed when NAD⁺ was present in solution. In the absence of NAD⁺ it exerted, on the contrary, a masking effect, like an inert protein. The charge consumed during each electrolysis was compared with the charge derived from the spectrophotometric monitoring of NADH formation. Depending on the potential value, hydrogenase predominantly catalyzed either the reduction of water to hydrogen, or the reduction of NAD⁺ to NADH. The current due to the presence of hydrogenase and NAD⁺ remained low according to corrosion moities, but it was demonstrated here that the presence of hydrogenase on the surface of stainless steel induces an effective hydrogenase-catalyzed cathodic reaction that must be taken into account in mic.     

Mechanism of the catalysis by Alcaligenes eutrophus H16 hydrogenase of direct electrochemical reduction of NAD+

The mechanism of direct electrochemical reduction of NAD⁺ into NADH catalysed by Alcaligenes eutrophus H16 hydrogenase was analysed in thin layer electrochemical cells with platinum and carbon electrodes. Two phases can be distinguished in the catalytic reaction occurring on platinum electrodes. In the potential range from approximately ?0.620 to ?0.675 V (SCE) direct electron transfer occurred via the diaphorase-like dimer of the hydrogenase. Below ?0.69 V versus SCE the hydrogenase used hydrogen species adsorbed onto the platinum electrode, but no molecular hydrogen was required for this second catalytic phase. The mechanism was quite similar to those which had been previously determined for Rhodococcus opacus hydrogenase. This confirmed the very great similarity of the two enzymes, even if the maximum NAD⁺ reduction rate of 0.36 mM min^?1 obtained here remains lower than those reached with R. opacus hydrogenase in a previous study. Careful analysis of the experimental data obtained on a carbon electrode led to the conclusion that no direct electron transfer was observed on this material under the operating conditions used. On the other hand, the voltammetric experiments performed on a carbon electrode in a thin layer cell showed clearly the occurrence of a catalytic current due to the hydrogenase-catalysed reduction of NAD⁺ by molecular hydrogen. This may be a useful tool for further analysis of the hydrogenase kinetics.     

Glucose utilization by resting cells of Fusobacterium polymorphum.

In the resting state, Fusobacterium polymorphum rapidly lost the ability to metabolize glucose. Washing of bacterial suspensions with water or saline resulted in a progressive decrease of glucose fermentation. A mixture of nicotinamide-adenine dinucleotide (NAD) adenosine monophosphate (AMP) and Mg²⁺ partially restored the metabolic capacity, but only under anaerobic conditions. Activity was maintained when early log phase bacteria were washed in a solution containing NAD⁺, AMP and Mg²⁺ and incubated with or without NAD⁺, AMP and Mg²⁺. When the bacteria were washed in a solution containing glucose and incubated without NAD⁺ or AMP of Mg²⁺, the activity was partially maintained.     

The oxidation of β-nicotinamide adenine dinucleotide (NADH) at poly(aniline)-coated electrodes: Part II. Kinetics of reaction at poly(aniline)–poly(styrenesulfonate) composites

Poly(aniline)–poly(styrenesulfonate) composite-coated glassy carbon electrodes are shown to produce a stable, reproducible amperometric response to NADH in citrate–phosphate buffer at pH 7. These responses have been studied as a function of electrode potential, film thickness and both NADH and NAD⁺ concentration. The results show that the oxidation of NADH occurs throughout the whole of the film and that NAD⁺, the reaction product, reversibly inhibits the reaction. Rate constants for the different processes have been obtained by kinetic modelling and compared with those previously determined for poly(aniline)–poly(vinylsulfonate) films. Preliminary comparisons imply that diffusion within ‘pores’ in these poly(aniline) composite matrices is important in determining the magnitude of the amperometric responses.     

Bioelectrocatalysis of NAD+ reduction

A thermokinetic approach to the catalysis of the electrochemical reduction of NAD⁺ by the hydrogenase of Alcaligenes eutrophus H16 is presented. The influence of temperature is weak, with an activation energy of 18 kJ/mol, whereas classical NAD⁺ hydrogenation with gaseous hydrogen shows an activation energy of 34 kJ/mol. The influence of the electric field on the thermal degradation of the enzyme is emphasized: the hydrogenase is clearly activated by the electric field and is shielded against thermal degradation. In contrast, in the absence of an electric field the thermal degradation of the hydrogenase follows rapid first-order kinetics with a high activation energy of the order of 70 kJ/mole. The catalysis of the bioelectrochemical reduction of NAD⁺ follows a quite different path to that of the hydrogenation reaction. In practice, the temperature is no longer an essential parameter to be considered in the integration of the NAD⁺ bioelectrochemical reduction within an enzymatic synthesis process. Adsorption phenomena and specific area of the electrode should be preferentially taken into account. The synthesis—regeneration coupling is tested in a thin-layer cell and gives a high NAD turnover number value of 448 cycles/h.     

Preparation,characterization and electrocatalytic properties of polynuclear mixed-valent ruthenium oxide/hexacyanoruthenate film modified electrodes

Polynuclear mixed-valent ruthenium oxide/ruthenocyanide (ruthenium oxide/hexacyanoruthenate or mvRuO/RuCN) films were prepared using consecutive cyclic voltammetry directly from the mixing of Ru³⁺ and Ru(CN)₆^4? ions from solutions of two divalent cations (Ba²⁺ and Ca²⁺), and seven monovalent cations (H⁺, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, and Ga⁺). The films exhibited three redox couples with Ba(NO₃)₂ or BaCl₂ aqueous solutions, and the formal potentials of the redox couples showed a cation and pH effect. An electrochemical quartz crystal microbalance (EQCM), cyclic voltammetry, UV–visible spectroscopy, and the stopped-flow method (SFM) were used to study the growth mechanism of the mvRuO/RuCN films. The results indicated that the redox process was confined to the immobilized ruthenium oxide/ruthenocyanide. The EQCM results showed a Ba²⁺ ion exchange reaction for the two most negative redox couples. The electrocatalytic reduction properties of SO₅^2?, and S₂O₈^2? by the ruthenium oxide/ruthenocyanide films were determined. The electrocatalytic oxidation of NADH and dopamine were also determined, and revealed two different types of properties. The electrocatalytic oxidations of SO₃^2?, S₂O₃^2?, and N₂H₄ were also investigated. The electrocatalytic reactions of the ruthenium oxide/ruthenocyanide films were investigated using the rotating ring-disk electrode method.     

Calcium-stimulated ATPase of dog submandibular gland

A Ca^2?-stimulated ATPase present in a microsomal fraction prepared from canine submandibular glands was investigated. The Ca²⁺ concentration for half maximal activation of the enzyme was about 0.3 mM. Addition of Mg²⁺ to incubation media containing Ca²⁺ decreased the ATPase activity. The presence of neither Na⁺ nor K⁺ is required for Ca²⁺-activation of the enzyme. Also, Ca²⁺ will not substitute for Mg²⁺ in the Mg²⁺-dependent (Na⁺ + K⁺)-ATPase reaction. The Ca²⁺-activation was not appreciably affected by ouabain (10^?4M), but was inhibited by about 50 per cent by 5 × 10^?3M ethacrynic acid. These studies provide a possible enzymatic basis for the calcium uptake by salivary gland microsomes that has been reported by other workers.     

Transport phenomena inside the pores involved in the kinetics and mechanism of growth of porous anodic Al2O3 films on aluminium

The mass and charge transport phenomena inside the pores during the growth of porous anodic Al₂O₃ films, which are involved in the kinetics and mechanism of the oxide growth, were investigated and a relevant system of mathematical equations was formulated describing these phenomena in each general case. The solution of the above system of equations in the simplified case of a stirred bath and films with cylindrical pores, where it could be solved, showed that, during the oxide growth, the concentrations of H₂SO₄ and Al₂(SO₄)₃ are highest and remain constant at pore bases and vary linearly between the pore base and film surface. Equations, correlating the electrolytes concentrations at pore bases during the oxide growth and the bath bulk concentrations with the charge transport numbers of the different ions, were also derived. These, referring to cylindrical pores, can be considered to be approximately applicable to the case of the real shape of elongated pores. Also, it was shown that the charge is transported inside the pores mainly by the migrating H⁺ and Al³⁺ ions while the contribution of the SO^2?₄ ions to the charge transport is insignificant.     

New EQCM,voltammetric and radiotracer evidences proving the role of Cu+ ions in the behavior of the Cu2+–Cu system

Combined EQCM and voltammetric studies in conjunction with radiotracer adsorption investigations were carried out in order to obtain direct information on the interfacial behavior of the Cu²⁺–Cu system under equilibrium and dynamic conditions with the aim of clarifying the role and participation of Cu⁺ ions formed by the interaction of cupric ions and copper metal. The appearance and accumulation of Cu⁺ ions in the solution phase is clearly demonstrated through radiotracer experiments using labeled Cl^? ions. The formation of bulk CuCl phase is preceded by the formation of a CuCl surface layer. Data obtained from combined voltammetric and EQCM studies allow us to separate the potential regimes where the electrosorption, the formation of Cu⁺ or Cu²⁺ ions, and dissolution of CuCl are the predominant processes.     

Characterization of copper hexabromoplatinate films and their electrocatalytic properties with dopamine,NADH, and S2O32−

Copper(II) hexabromoplatinate (CuPtBr₆) films have been prepared by mixing Cu²⁺ and PtBr₆^2? ions in an aqueous KBr solution. An electrochemical quartz crystal microbalance (EQCM), a rotating ring-disk electrode, UV–visible absorption spectroscopy and cyclic voltammetry were used to study the deposition and growth mechanism of the copper(II) hexabromoplatinate films. The electrochemical and EQCM properties of the films indicate that a single redox process was confined to the immobilized copper(II) hexabromoplatinate films. The deposition of a copper hexabromoplatinate film occurs when Cu²⁺ is reduced to Cu⁺. In the aqueous KBr solution, Pt^IVBr₆^2? is reduced electrochemically to Pt^IIBr₆^4?, and the Cu⁺ reacts with the Pt^IIBr₆^4? and Pt^IVBr₆^2?species. The electrocatalytic oxidation properties of dopamine, NADH, and S₂O₃^2? were determined using the copper(II) hexabromoplatinate films. The electrocatalytic reaction of dopamine with a copper(II) hexabromoplatinate film was investigated using the rotating ring-disk electrode method.     

Self-assembled monolayers with biospecific affinity for NAD(H)-dependent dehydrogenases: Characterization by surface plasmon resonance combined with electrochemistry ‘in situ’

Gold surfaces with high biospecific affinity for pyridine-nucleotide-dependent dehydrogenases have been prepared on the basis of hydrophilic alkanethiol self-assembled monolayers bearing an artificial pyridine-nucleotide analogon as pendant ligand group. This site-specific bioaffinity binding provides a mild immobilization method to attach enzymes to metallic electrodes with a particular orientation which allows complexation of the ligand into the enzyme's NAD⁺-binding pocket. The investigation of protein adsorption events by surface plasmon resonance spectroscopy reveals that a satisfactorily high amount of protein (between 30 and 40%) of a densely packed protein layer can be tightly adsorbed on ligand-anchored alkanethiol self-assembled monolayers, whereas an appreciably lower amount adsorbs on their ligand-free analogous layers. The combination of surface plasmon resonance spectroscopy with electrochemistry ‘in situ’ allows us to monitor simultaneously the amount and electrocatalytic activity of the immobilized biological material. The ratio (K) between the protein's electroenzymatic response and the average thickness of the immobilized protein layer can be taken as an indication of the state of the protein on the electrode surface. It was found from these measurements that electrodes modified with a monolayer of lactate dehydrogenase lose electroenzymatic activity upon successive shots of lactate due to progressive detachment of the protein from the ligand-anchored monolayer. The catalytic activity of the enzyme, however, remains unaltered during the electrochemical runs, as shown by the constant calculated K values (K ≈ 1.0 μA cm^?2nm). The ratio obtained between K values for enzymatic electrodes prepared from ligand-anchored and ligand-free alkanethiol monolayers suggests that lactate dehydrogenase is anchored to the ligand monolayer through two NAD⁺-binding pockets which are not involved in the electroenzymatic reaction.     

Direct electron transfer between Alcaligenes eutrophus Z-1 hydrogenase and glassy carbon electrodes

In this work we report on the ability of the Alcaligenes eutrophus Z-1 hydrogenase immobilized on glassy carbon electrodes to support the electro-enzymatic reduction of NAD⁺ at ?700 mV vs. standard calomel electrode without external promoters or electron mediators.The electron-transfer process seems to be coupled to a relaxation of the quaternary structure of the enzyme molecules in contact with the electrode surface.     

Effect of mono- and divalent ions on diffusion and binding in bovine tooth enamel

A radiochemical method was used to determine quantitatively the effect of RbCl, CaCl₂ and MgCl₂ on the transport of [³H]-sorbitol, [¹⁴C]-glycerol, ⁴⁵Ca²⁺, ³⁶Cl^? and ⁸⁶Rb⁺ through dental enamel. The findings indicate that the transport of ⁴⁵Ca²⁺ is influenced strongly by surface exchange reactions. The influence of Ca²⁺ and Mg²⁺ on the transport of ⁸⁶Rb⁺ and ³⁶Cl^? indicates that, at a concentration of ~10mmol/l, these ions alter the fixed charge of enamel from negative to less negative, or positive values. Previously adsorbed ⁴⁵Ca²⁺ ions are desorbed at higher Ca²⁺ and Mg²⁺ concentrations. Rb⁺ ions at 10 mmol/l have no such effect. The results indicate that Ca²⁺ and Mg²⁺ can be adsorbed reversibly on the crystal surfaces or in their Stern layers in the pores of dental enamel.     

Preparation,characterization, and electrocatalytic properties of copper hexacyanoferrate film and bilayer film modified electrodes

Copper(II)hexacyanoferrate films have been prepared from various electrolyte aqueous solutions using consecutive cyclic voltammetry. The cyclic voltammograms recorded the direct deposition of copper(II)hexacyanoferrate films from the mixing of Cu²⁺ and Fe(CN)₆^3? ions from solutions of ten cations: Li⁺, Na⁺, K⁺, Rb⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, H⁺ and Al³⁺. An electrochemical quartz crystal microbalance (EQCM) and cyclic voltammetry were used to study the in situ growth of the copper(II)hexacyanoferrate films. The copper(II)hexacyanoferrate film showed a single redox couple that exhibited a cation effect (Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺) and an anion effect (F^?, Cl^? and Br^?) in the cyclic voltammograms and formal potential of the redox couple. The electrochemical and EQCM properties of the film indicate that the redox process was confined to the immobilized copper(II)hexacyanoferrate, and the interaction between the copper(II)hexacyanoferrate film with K⁺ (monovalent cation) and Ca²⁺ (divalent cation). The electrocatalytic oxidation properties of NADH, NH₂OH, N₂H₄, SO₃^2? and S₂O₃^2? were also determined. The electrocatalytic reduction properties of SO₅^2? and S₂O₈^2? by monolayered iron, nickel, and cobalt hexacyanoferrate films, and by bilayered metal–copper hexacyanoferrate films were determined. Two-layered modified electrodes and hybrid films composed of a copper(II)hexacyanoferrate film with iron(II)hexacyanoferrate, cobalt(II)hexacyanoferrate, or nickel(II)hexacyanoferrate film were prepared, and these films caused the electrocatalytic reduction of SO₅^2? and S₂O₈^2?.     

The ion selectivity of monensin incorporated phospholipid/alkanethiol bilayers

Monensin was incorporated into phospholipid/alkanethiol bilayers on the gold electrode surface by a new, paint-freeze method to deposit a lipid monolayer on the self-assembled monolayers (SAMs) of alkanethiol. The advantages of this assembly system with a suitable function for investigating the ion selective transfer across the mimetic biomembrane are based on the characteristics of SAMs of alkanethiols and monensin. On the one hand, the SAMs of alkanethiols bring out their efficiency of packing and coverage of the metal substrate and relatively long-term stability; on the other hand, monensin improves the ion selectivity noticeably. The selectivity coefficients K_Na+,K+, K_Na+,Rd+, and K_Na+,Ag+ are 6×10^?2, 7.2 × 10^?3 and 30 respectively. However, the selectivity coefficient K_Na+,Li+ could not be obtained by a potentiometric method due to the specific interaction between Li⁺ and phospholipid and the lower degree of complexion between Li⁺ and monensin. The potential response of this bilayer system to monovalent ions is fairly good. For example, the slope of the response to Na⁺ is close to 60 mV per decade and its linearity range is from 10^?1 to 10^?5m with a detection limit of 2 × 10^?6m. The bilayer is stable for at least two months without changing its properties. This monensin incorporated lipid/alkanethiol bilayer is a good mimetic biomembrane system, which provides great promise for investigating the ion transfer mechanism across the biomembrane and developing a practical biosensor.     

Self-assembled monolayers with biospecific affinity for lactate dehydrogenase for the electroenzymatic oxidation of lactate

Surface modified gold electrodes with high biospecific affinity for NAD(H)-dependent lactate dehydrogenase have been prepared by covalent attachment of several traizine dyes to stepwise functionalized mixed alkanethiol self-assembled monolayers. The biospecific affinity of such ligand-anchored monolayers to bind submonolayer amounts of enzyme was demonstrated from the course of the protein adsorption events monitored by surface plasmon resonance. Electroenzymatic activity measurements of lactate dehydrogenase modified surfaces for the reaction of lactate oxidation, carried out ‘ex situ’ at different stages of protein layer growth, allowed the optimization of the preparative procedure to yield reproducible enzymatic electrodes with a low amount of unspecifically bound protein. A short adsorption time, as well as a high concentration of enzyme in the solution used for protein layer growth, led to lactate dehydrogenase-modified gold electrode surfaces with a high electroenzymatic activity arising mainly from biospecifically bound species. The lowest amount of unspecifically adsorbed protein was found for ligand-anchored monolayers prepared from mixed alkanethiol underlayers with an excess of positively charged groups. The lack of electroenzymatic activity shown by lactate dehydrogenase modified electrodes in the absence of soluble coenzyme (NAD⁺) indicates that none of the investigated ligand-anchored monolayers could provide an efficient electronic pathway from the metal to the active site of the enzyme. Therefore, the monolayers acted just as an anchoring system for lactate dehydrogenase.