Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD⁺ is converted to NADH by oxidation of ethanol and -lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD⁺, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or -lactic acid in the range 0.05–2 × 10^-9 mol.     

Facile synthesis of near infrared fluorescent trypsin-stabilized Ag nanoclusters with tunable emission for 1,4-dihydronicotinamide adenine dinucleotide and ethanol sensing

A facile chemical synthetic route was developed to prepare near-infrared fluorescent trypsin-stabilized Ag nanoclusters (Try-Ag NCs). The fluorescence emission wavelength of the produced Try-Ag NCs is tunable by simple adjusting pH value of the synthesis system, and the Try-Ag NCs offer a symmetric fluorescent excitation and emission peak. The fluorescence of Try-Ag NCs remains constant in the presence of various ions and molecules, and it can be effectively quenched by 1,4-dihydronicotinamide adenine dinucleotide (NADH) instead of its oxidized forms nicotinamide adenine dinucleotide (NAD⁺). This property enables the Try-Ag NCs to be a novel analytical platform to monitor biological reaction involved with NADH. In this work, the Try-Ag NCs was also applied to analyze ethanol based on the generation of NADH which was the product of NAD⁺ and ethanol in the catalysis of alcohol dehydrogenase. And the proposed platform allowed ethanol to be determined in the range from 10 to 300 μmol/L with 5 μmol/L detection limit.     

Cyclic regeneration of nicotinamide adenine dinucleotide with immobilized enzymes: Flow-injection spectrofluorimetric determination of ethanol

Ethanol (0.05–0.5%) in water is determined by injection of a 20-μl sample into a solution of 1.5 × 10^?3 M NAD⁺ in pH 8.0 phosphate buffer flowing from a reservoir. The solution passes through a minicolumn of yeast alcohol dehydrogenase immobilized on controlled-pore glass (CPG). The NADH formed is monitored spectrofluorimetrically in the flow system, before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on CPG in the presence of glutarate and ammonium ions, also in the flowing solution. The solution then returns to the reservoir. The regeneration of NAD⁺ allows the same coenzyme solution to be used for 50 ethanol determinations daily for 4 days.     

Adsorption phenomena in the NAD+/NADH system at glassy carbon electrodes

A variety of electrochemical approaches has been used to investigate the adsorption of NAD⁺, NADH and the NAD-NAD dimer from aqueous solution at glassy carbon electrodes (GCE) with supplementary studies of adsorption at pyrolytic graphite and platinum electrodes from aqueous media and at GCE from DMSO solution. The following hypotheses are advanced concerning the adsorption orientation: at carbon electrodes, on which NADH is not adsorbed, NAD⁺ produced by anodic oxidation of the NADH is first rapidly adsorbed in a planar configuration relative to the electrode surface, which is probably bound to the surface through the adenine moiety; there is then a relatively slow reorientation of the adsorbed NADH molecules to a perpendicular orientation relative to the electrode surface, which adsorbate is more tightly bound to the surface than the planar oriented adsorbate and which likely involves interaction between parallel adenine and pyridinium rings. Reduction (one-electron process) of NAD⁺ at the GCE produces the NAD-NAD dimer, which, at a clean electrode surface, involves a diffusion-controlled process and an adsorption-controlled process; the latter is due to formation of adsorbed dimer, which is more strongly adsorbed than NAD⁺. The dimer is oxidized at the GCE only if it is adsorbed. The factors controlling and involved in the adsorption processes have been examined with particular reference to the use of anodic voltammetry for the analytical determination of NADH.     

Albumin coated CuInS2 quantum dots as a near-infrared fluorescent probe for NADH, and their application to an assay for pyruvate

Water-soluble CuInS₂ quantum dots (QDs) stabilized with 3-mercaptopropionic acid were synthesized in aqueous solution and then coated with bovine serum albumin. The resulting particles display fluorescence with a peak at 680 nm that is effectively quenched by 1, 4-dihydro-nicotinamide adenine dinucleotide (NADH), but not by 1, 4-nicotinamide adenine dinucleotide (NAD⁺). The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and dehydrogenation of lactic acid using NAD⁺ or NADH as a cosubstrate. The new QDs were applied to monitor the course of lactate dehydrogenase-catalyzed reaction of pyruvate by detecting NADH via its quenching effect. This resulted in a convenient and selective detection scheme for pyruvate. The detection limit is as low as 25 nM.

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.     

Fluorescein derivative-based, selective and sensitive chemosensor for NADH

Fluorescein mercury acetate (FMA) derivative can be used as a chemosensor for NADH that plays key roles in cellular energy metabolism and dehydrogenase enzymatic reactions. The sensor recognizes NADH by two mercury metal ions in the compound using the metal-anion interaction and its subsequent binding-induced fluorescence changes of FMA. The FMA can detect an aqueous NADH concentration of approximately 1.0 μM and has a high selectivity over various anions including NAD⁺, the oxidized form of NADH.     

Equilibrium (NAD+/NADH) potential on poly(Neutral Red) modified electrode

The electrochemical regeneration of nicotinamide adenine dinucleotide (NAD⁺/NADH) has been one of the central subjects of bioelectrochemistry during past three decades. We report on the unique chemical electrocatalyst for NAD⁺/NADH regeneration based on electropolymerized Neutral Red. Using poly(Neutral Red) modified electrodes, the reversible polarographic waves of nicotinamide adenine dinucleotide reduction–oxidation and the equilibrium (NAD⁺/NADH) potential were observed. This was impossible using all known catalytic and mediator systems. The unique poly(Neutral Red) based electrocatalyst allowed us to determine the standard (NAD⁺/NADH) potential more precisely (E^′≅0.59 V SCE, pH 6.0).     

Molecularly selective field-effect transistors for determining nicotinamide adenine dinucleotide and its phosphates

Molecularly selective field-effect transistors are developed for determining oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD⁺ and NADH) and their phosphates (NAD(P)⁺ and NAD(P)H). Copolymers of acrylamide and acrylamidophenylboric acid imprinted with corresponding nucleotides are used as sensing elements of sensors. After washing out a nucleotide, a cavity corresponding to the surface of the imprinted nucleotide is retained and ensures the selectivity of the sensor. At the same time, sensors containing any of the nucleotides of interest in solutions demonstrate almost equal sensitivities of 10^?7 M. The properties of the sensors are explained by the structure of the nucleotides and the composition of the membranes.     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

NAD<Superscript>+</Superscript> metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity

Background

NAD⁺ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD⁺-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD⁺ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD⁺ to nicotinamide ratio and the NAD⁺ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD⁺ metabolism requires analytical definition and quantification of the key metabolites.

Results

Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD⁺ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD⁺ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD⁺ or nicotinamide levels in ways that would increase Sir2 activity.

Conclusions

The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.

     

Amperometric bienzyme electrode for l-carnitine

An electrochemical sensor with two soluble enzymes allows assay of 0.1–10 mM, l- carnitine with an accuracy of 2%. The assay takes about 2 min. The first enzyme, carnitine dehydrogenase, catalyzes the oxidation of l-carnitine by NAD⁺. The NADH formed is oxidized by hexacyanoferrate(III) in a reaction catalyzed by diaphorase, and the hexacyanoferrate(II) produced is oxidized by electrolysis at 0.3 V. The sensor can be used for several days.     

Biomimetic Oxidation of Aldehyde with NAD+ Models: Glycolysis-Type Hydrogen Transfer in an NAD+/NADH Model System

Just like in biological systems , the GAPDH-catalyzed oxidation of aldehyde to carboxylate proceeds in conjunction with 1,4-selective reduction of NAD⁺ to NADH model compounds [Eq. (1)]. The combination of GAPDH- and LDH-type transfer reactions is also described here as a system mimic for the NAD⁺/NADH redox cycle in anaerobic glycolysis. GAPDH=D -glyceraldehyde-3-phosphate dehydrogenase, LDH=L -lactate dehydrogenase.     

A New Amperometric Carbon Paste Enzyme Electrode for Ethanol Determination

Abstract

In this study, a new amperometric carbon paste enzyme electrode for determination of ethanol was developed. The carbon paste was prepared by mixing alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized form, NAD⁺), poly(vinylferrocene) (PVF) that was used as a mediator, graphite powder and paraffin oil, then the paste was placed into cavity of a glass electrode body. Determination of ethanol was performed by oxidation of nicotinamide adenine dinucleotide (reduced form, NADH) generated enzymatically at +0.7 V. The effects of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature were investigated. The linear working range of the enzyme electrode was 4.0×10^?4–4.5×10^?3 M, determination limit was 3.9×10^?4 M and response time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of enzyme, NAD⁺ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 37°C, 2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode at +4°C was 7 days. Enzyme electrode was used for determination of ethanol in two different wine samples and results were in good agreement with those obtained by gas chromatography.     

An enzymatic-fluorimetric method for monitoring of ethanol in ambient air

A method is described for the continuous monitoring of ethanol in ambient air. The system consists of a scrubber coil for enrichment of the analyte from air in an aqueous solution and a directly connected fluorescence detector. Because of using a reagent solution containing alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) for absorption, ethanol can react directly with ADH and NAD⁺ during air sampling, producing NADH, which can be measured by fluorescence detection. The influence of reagent concentrations, gas flow rate and scrubber solution flow rate on the performance of the instrument was tested. Possible ozone interferences can be avoided by placing a KI coated filter in front of the scrubber inlet. The response time of the system was found to be 2.3 min and the detection limit about 1 ppb_V. The applicability of the developed method was demonstrated during a field campaign in Brazil.     

Enzymatic methods for the determination of ethanol based on linear and cyclif flow-injection systems

Linear and cyclic systems are described for the determination of ethanol (ca. 0.17–30×10^?3 M). In the linear system, the solution passes either through a minicolumn of yeast alcohol dehydrogenase (YADH) immobilized on controlled-pore glass or through minicolumns of the immobilized YADH and of yeast aldehyde dehydrogenase immobilized on cyanogen bromide-activated Sepharose-4B. The NADH formed is monitored either spectrophotometrically or spectrofluorimetrically. In the cyclic system, the solution passes through the same enzyme columns, and the NADH produced is monitored similarly before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on cyanogen bromie-activated Sepharose-4B in the presence of α-ketoglutarate and ammonium ions also present in the flow system. the sample throughout for both systems is ca. 40 h^?1 and 50 h^?1 for spectrophotometric and spectrofluorimetric detection, respectively. An on-line double-injection technique is described as an alternative to the cyclic system for limiting the consumption of NAD⁺.     

Enzyme electrodes with substrate and co-enzyme amplification

The enzyme couples horseradish peroxidase/glucose dehydrogenase, glucose oxidase/glucose dehydrogenase, and cytochrome b₂/lactate dehydrogenase are applied in enzyme electrodes. Based on amplification by the recyclization reactions catalyzed by these two-enzyme systems, NADH, NAD⁺, glucose, lactate and pyruvate, are determined with 8–40-fold increased sensitivity compared to the unamplified reactions. Detection limits are 1.0 × 10^?6 M NADH, 1.2 × 10^?6 M NAD⁺, 8 × 10^?7 M glucose, and 3 × 10^?7 M lactate or pyruvate.     

Graphene Paste Electrode: Analytical Applications for the Quantification of Dopamine,Phenolic Compounds and Ethanol

This work reports the analytical applications of a graphene paste electrode (GrPE) for the quantification of dopamine, ethanol and phenolic compounds. Dopamine was detected by differential pulse voltammetry‐adsorptive stripping with medium exchange at submicromolar levels even in the presence of high excess of ascorbic acid and serotonin. The electrocatalytic activity of graphene towards the oxidation of NADH and the reduction of quinones allowed the sensitive amperometric determination of ethanol and phenols using GrPE modified with alcohol dehydrogenase/NAD⁺ or polyphenol oxidase, respectively, with successful applications in real samples like alcoholic beverages and tea.     

An Enzymatic Microdetermination Method for Hydroxylase Cofactor

Abstract

An enzymatic microdetermination method is described to determine the concentration of biologically active hydroxylase cofactor in crude samples. A small amount of hydroxylase cofactor was oxidized and reduced in a cyclic fashion by phenylalanine 4-monooxygenase (EC 1.14.16.1, PheOHase) and dihydropteridine reductase (EC 1.6.99.7, DPRase) in the presence of excess phenylalanine (Phe) and NADH. The amount of NAD⁺, accumulated as a result of this cyclic reaction (first cycling), was in proportion to that of the hydroxylase cofactor. The NAD⁺ was further amplified by an enzymatic amplification reaction, NAD cycling (second cycling) and determined fluorometrically. This double cycling method with more than a 20,000 fold amplification provided extremely high sensitivity, down to 0.02 pmol per assay, and was successfully applied to crude microsamples of pg wet weight.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.