Photolytic oxidation of reduced nicotinamide adenine dinucleotide

A photo-reaction between thionine dye and NADH has been observed which leads to the oxidation of the co-factor. Thionine (fluorescent), which is converted into semi/leuko thionine (non-fluorescent) during light reaction, fully recovers during the dark reaction. Analytical applications of this discovery are discussed. This reaction opens the door to the measurement of NADH using the fluorescence quenching of thionine and to the construction of several optical biosensors.     

Synthesis of phosphodiester-type nicotinamide adenine dinucleotide analogs

Fourteen phosphodiester-type β-nicotinamide adenine dinucleotide (NAD⁺) analogs were prepared starting from nicotinamide. The phosphodiester linkage was effectively assembled in 69-93% yields via condensation reaction between 2′,3′-di-O-acetyl nicotinamide mononucleotide and alcohols in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride. The analog β-nicotinamide ribose-5-(2-phenylethyl) phosphate showed beneficial effects on cell growth of model microorganisms.     

Thermodynamics of redox reactions involving nicotinamide adenine dinucleotide

Literature data on the thermodynamics of redox nicotinamide adenine dinucleotide (NAD) dependent reactions have been analyzed. It has been established that for the redox reaction of NAD

where all substances except H₂ are in the aqueous buffer with the ionization enthalpy equal to zero, the most reliable thermodynamic parameters should be considered as: ΔH(298.15 K; pH 7)=?27.4±1.7 kJ mole^?1; ΔG (298.15K; pH 7)=±17.8 kJ mole^?1. From the above thermodynamic parameters of the reaction ΔH, ΔG and ΔS for reactions of NAD with natural substrates, synthetic mediators and some inorganic compounds have been calculated.     

Sensitive assay for nicotinamide adenine dinucleotide phosphate and its reduced form based on the bioluminescence method

An assay of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by bioluminescence was investigated and applied for NADP⁺. The NADP⁺ is first reduced by glucose-6-phosphate dehydrogenase and then assayed in a mixture containing a NADPH/flavin mononucleotide oxidoreductase which in turn activates luciferase. Many interferences were observed and the method was modified accordingly. NADP⁺ and NADPH can be assayed separately or simultaneously within the range 1–100 pmol, which is sufficiently sensitive to be applied to biological materials. Many details and precautions must be taken into consideration.     

Differential pulse polarography as a tool for the determination of trace levels of nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) in aqueous solution

The differential pulse polarographic behavior of NAD⁺ and NADP⁺ has been investigated in phosphate buffer. The peaks obtained at pH 8.0 are recommended for the trace determination of these compounds. Linear calibration curves are obtained over the concentration ranges from 2.6 × 10⁻⁷ to 2.6 × 10⁻⁵M for NAD⁺ and from 2 × 10⁻⁶ to 4 × 10⁻⁵ for NADP⁺.     

Fluorescence quenching of thionine by reduced nicotinamide adenine dinucleotide

Reduced nicotinamide adenine dinucleotide (NADH) is shown to quench the fluorescence of thionine. Quenching of thionine is extremely efficient with a half quenching concentration of only 16.1 × 10⁻⁶ M NADH. A Stern—Volmer plot is linear over the NADH concentration range from 1 to 20μM. The corresponding Stern—Volmer quenching constant is 6.2 × 10⁴ M⁻¹ and the limit of detection for NADH measurements is 1.6 × 10⁻⁶ M. Process of quenching is attributed to the formation of an exciplex between thionine and NADH. Potential analytical features of this system are discussed.     

Microsomal electrodes for reduced nicotinamide adenine dinucleotide and its phosphate,glucose-6-phosphate and ascorbate

Rat liver microsomes have been immobilized in a membrane by gelatin entrapment. The resulting membranes can be attached to an oxygen electrode to provide a sensor for several compounds. NADPH and NADH are determined by utilizing the liver microsomal NADPH oxidase activity, which is, at least in part, due to the cytochrome P-450 system. The calibration graph for NADPH is linear up to 1 mM. A multi-enzyme system localized in liver microsomes allows the determination of 20–800 μM glucose-6-phosphate. The non-enzymatic lipid peroxidation in liver microsomes, which is induced by ascorbate, allows 0.5–2.5 mM ascorbate to be determined.     

The electrochemical reduction and determination of reduced nicotinamide adenine dinucleotide in acidic media

The apparent reduction of reduced nicotinamide adenine dinucleotide (NADH) in acidic media at a static mercury drop electrode was investigated. A simple, quick pretreatment procedure was developed to convert the NADH to its acid-hydrated form. This adsorbs on the mercury surface during a film deposition time and the film is then reduced. The adsorption is diffusion-controlled and hence the peak currents for square-wave and linear-scan voltammetry are proportional to Ct^1/2_pAf and Ct^1/2_pAv, respectively, where t_p is the effective film deposition time, C the concentration of NADH, A the electrode area, f the square-wave frequency, and ν the linear scan rate. Several electrochemical techniques were compared for the determination of NADH; the method of choice is square-wave voltammetry, although staircase or linear scan voltammetry can also be used. The detection limit is less than 7 nM, and the range of linear response covers 2–3 orders of magnitude of NADH concentration.     

Ultrasensitive detection of the reduced form of nicotinamide adenine dinucleotide based on carbon nanotube field effect transistor

We developed a simple, ultrasensitive, and quantitative detection method for the reduced form of nicotinamide adenine dinucleotide (NADH), based on carbon nanotube field effect transistors (CNTFETs). Following the injection of NADH at different concentrations, we obtained different electrical signals from a semiconductor characterization system mimicking biological catalysis of NADH dehydrogenase (CoI). Here, FET was fabricated via photolithography, attaching silicon wells, as the detection chamber, on the channel area of the single wall carbon nanotube (SWCNT). SWCNTs were functionalized with phenazine derivant, a counterpart of the key functional prosthetic group of CoI enzyme. In the presence of NADH, electrons transferred to phenazine derivant through SWCNT, by analogous means of the electron transport chain formed by a series of iron-sulfur (FeS) clusters in CoI. Using this method, the limit of detection was as low as 1 pM, and the range of linear response was 10 pM to 500 nM. Significantly, this approach possesses great potential for applications in real-time detection of NADH at extremely low concentrations, and rigorous analysis for NADH in electrochemical fields.     

Novel nicotinamide adenine dinucleotide analogues as selective inhibitors of NAD-dependent enzymes

Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD⁺) have been synthesised from d-ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.     

On the nuclear magnetic resonance spectrum of reduced nicotinamide adenine dinucleotide

The two C-4 protons of reduced nicotinamide adenine dinucleotide (NADH) produce an AB NMR spectrum at 100 MHz as well as at 220 MHz. This observation allows an upper limit of 50 sec^?1 to be placed on the mean rate of interconversion of the two folded forms of NADH invoked to account for the magnetic non-equivalence of the C-4 protons. The interpretation of non-equivalence of the C-4 protons in terms of the various equilibria among folded and unfolded forms of NADH and its possible significance in the mechanism of action of dehydrogenase enzymes is discussed. It is suggested that one folded form of NADH is strongly favored thermodynamically over the other and that the resulting magnetic non-equivalence of the C-4 protons is of doubtful significance in explaining the stereospecificity of dehydrogenase enzymes toward the nicotinamide ring.     

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide

Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD⁺), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5′ end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3′ end. Only in the presence of NAD⁺, the probe was linked at 5′ and 3′ ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM–500 nM with a detection limit of as low as 1 pM NAD⁺, and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD⁺ level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD⁺, taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.

聚硫堇/石墨烯复合材料修饰电极对NADH的电催化氧化研究

采用循环伏安法将硫堇在石墨烯修饰的玻碳电极表面聚合,得到了一种新的聚硫堇/石墨烯修饰电极,此电极兼备了石墨烯和聚硫堇的特性.实验表明:该修饰电极能有效降低NADH的过电位;对NADH的检测范围为2.4×10~(-6)~4.89×10~(-3) mol·L~(-1);检出限为6.826×10~(-7) mol·L~(-1);对尿酸和抗坏血酸的干扰有很好的消除作用;此电极稳定性、重现性较好,有很高的实际应用价值.     

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.     

Enhanced acid stability of a reduced nicotinamide adenine dinucleotide (NADH) analogue

A methyl substituent at C-5 of the nicotinamide ring is found to confer increased acid stability in a reduced nicotinamide model (5-20 fold) and in a reduced dinucleotide coenzyme (2-3 fold), while retaining reactivity towards hydride transfer.