Crystal Structure of a Hydrated Molecular 1 : 2 Complex of Nicotinamide Adenine Dinucleotide (NAD+) and Gallic Acid: Polar Alignment of the Phenolic Partner Molecules

Nicotinamide adenine dinucleotide (oxidized form, free acid, NAD⁺) was utilized as an auxiliary for polar alignment of gallic acid (=3,4,5‐trihydroxybenzoic acid, GA) in the crystalline molecular complex NAD⁺?(GA)₂?(H₂O)₅. In this adduct, NAD⁺ takes up a novel U‐shaped conformation accepting a GA molecule to give rise to a pincer‐type π‐complex. With the assistance of further GA molecules, these pincer assemblies build up infinite donor? acceptor π‐stacks in the crystal. An extended network of H‐bonds among the multitude of pertinent active functional groups and the water molecules supports the crystal structure. These findings may imply noteworthy material properties, in particular nonlinear optical effects, and might also serve to trigger inspiring biochemical connections to be made. The crystal structure of anhydrous GA is also reported, which is centrosymmetric and non‐polar.     

Modification of glassy carbon electrode with a polymer/mediator composite and its application for the electrochemical detection of iodate

A modified glassy carbon electrode was prepared by depositing a composite of polymer and mediator on a glassy carbon electrode (GCE). The mediator, flavin adenine dinucleotide (FAD) and the polymer, poly(3,4-ethylenedioxythiophene) (PEDOT) were electrochemically deposited as a composite on the GCE by applying cyclic voltammetry (CV). This modified electrode is hereafter designated as GCE/PEDOT/FAD. FAD was found to significantly enhance the growth of PEDOT. Electrochemical quartz crystal microbalance (EQCM) analysis was performed to study the mass changes in the electrode during the electrodeposition of PEDOT, with and without the addition of FAD. The optimal cycle number for preparing the modified electrode was determined to be 9, and the corresponding surface coverage of FAD (Γ_FAD) was ca. 5.11 × 10⁻¹⁰ mol cm⁻². The amperometric detection of iodate was performed in a 100 mM buffer solution (pH 1.5). The GCE/PEDOT/FAD showed a sensitivity of 0.78 μA μM⁻¹ cm⁻², a linear range of 4–140 μM, and a limit of detection of 0.16 μM for iodate. The interference effects of 250-fold Na⁺, Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Cl⁻, NO₃⁻, I⁻, SO₄²⁻ and SO₃²⁻, with reference to the concentration of iodate were negligible. The long-term stability of GCE/PEDOT/FAD was also investigated. The GCE/PEDOT/FAD electrode retained 82% of its initial amperometric response to iodate after 7 days. The GCE/PEDOT/FAD was also applied to determine iodate in a commercial salt.     

Site-specific immobilization of flavin adenine dinucleotide on indium/tin oxide electrodes through flavin adenine amino group

A Mannich-type reaction was used to attach flavin adenine dinucleotide (FAD) covalently to aminosilane derivatized indium/tin oxide-coated glass plates. The aminosilane was activated with formaldehyde to give an intermediate that attached specifically to the adenine amino group of FAD. The presence of the intermediate also was demonstrated by coupling hydroquinone to the formaldehyde activated support. The immobilized FAD and hydroquinone were characterized by cyclic or differential pulse voltammetry. The immobilized FAD was shown to reduce the overpotential for NADH oxidation by 180 mV. In keeping with results for FAD on glassy carbon, FAD attached to indium/tin oxide at the adenine amino group did not lead to reconstitution of activity with apoglucose oxidase. On leave from University of Madras, India.     

A simple and highly sensitive DNAzyme-based assay for nicotinamide adenine dinucleotide by ligase-mediated inhibition of strand displacement amplification

Existing strategies for detecting nicotinamide adenine dinucleotide (NAD⁺) or other cofactors are commonly cumbersome and moderate sensitive. We report a novel DNAzyme-based visual assay strategy for NAD⁺ based on ligase-mediated inhibition of the strand displacement amplification (SDA). In the presence of NAD⁺, the SDA can be inhibited by the ligase reaction of two primers, which can initiate the SDA reaction in the case of no ligation, resulting in a dramatically decreasing yield of the SDA product, a G-quadruplex DNAzyme that can quantitatively catalyze the formation of a colored product. Therefore, the quantitative analysis for NAD⁺ can be achieved visually with high sensitivity. The developed strategy provides a simple colorimetric approach with high selectivity against most interferences and a detection limit as low as 50 pM. It also provides a universal platform for investigating cofactors or other related small molecules as well as quantifying the activity of DNA ligases.     

高效毛细管电泳—电化学法检测烟酰胺腺嘌呤二核苷酸

报道了一种测定烟酰胺腺嘌呤二核苷酸的毛细管电泳电化学检测方法。采用３０ｃｍ×２５μｍ的石英毛细管分离ＮＡＤＨ与脲酸，以微型碳糊电极测定经分离后的ＮＡＤＨ的含量。在ｐＨ７．５的磷酸盐缓冲溶液中，标准工作曲线的范围为１．０－１００μｍｏｌ／Ｌ；最代检测浓度为０．６０μｍｏｌ／Ｌ。     

辅酶NADH与色氨酸共振能量转移的荧光动力学研究

采用时间相关单光子计数技术, 结合紫外-可见吸收光谱和稳态荧光光谱, 对不同环境下的色氨酸和辅酶还原型烟酰胺腺嘌呤二核苷酸(NADH)之间的共振能量转移荧光动力学进行了研究. 单体色氨酸、 牛血清白蛋白以及乳酸脱氢酶蛋白与NADH之间相互作用的光谱数据表明, 只有存在NADH结合位点的乳酸脱氢酶和NADH之间发生了荧光共振能量转移. 进一步通过加入丙酮酸来阻断乳酸脱氢酶和NADH之间的荧光共振能量转移通道, 时间分辨荧光光谱和衰减相关光谱(DAS)证实, 蛋白结合位点的存在是NADH和色氨酸之间发生荧光共振能量转移的前提条件. DAS揭示了乳酸脱氢酶平均荧光寿命的减小主要是源于色氨酸中7.35 ns的荧光寿命成分与NADH之间的荧光共振能量转移, 同时给出了NADH和色氨酸之间的能量转移效率, 为研究NADH和蛋白之间的相互作用提供了新思路.     

黄素腺嘌呤二核苷酸在热石墨柱电极上电化学行为及吸附溶出分析

研究了黄素腺嘌呤二核苷酸(FAD)在热石墨柱电极上的电化学行为.发现对吸附有FAD的电极加热同时进行循环伏安扫描,随电极温度升高,氧化还原峰电位负移,峰电流减小,峰形变差.采用方波伏安进行吸附溶出分析,如果只在吸附步骤对电极加热,室温溶出,则随电极温度升高,溶出峰电流显著增加.这主要归因于直接对电极加热引起的强制热对流,这种对流能提高传质,促进吸附,增强溶出响应.5 min预富集对电极加热到72 ℃,检出限可以达到1×10-8 mol/L(S/N=3),比室温27 ℃时降低了一个多数量级,相应灵敏度也明显提高.     

Detection of cationic guest molecules by quenching of luminescence of a self-assembled host molecule consisting of terbium(III) and calix[4]arene-p-tetrasulfonates

By self-assembly in aqueous solution, calix- (CAS) and thiacalix[4]arene-p-tetrasulfonate (TCAS) formed luminescent complexes Tb^III·(CAS)₂ and Tb^III·TCAS, respectively, which were utilized as a host for cationic guests. Addition of 1-ethylpyridinium guest quenched luminescence of Tb^III·(CAS)₂ in accordance with the Stern-Volmer (SV) relation with a low detection limit (D.L.) of 5.94 × 10⁻⁸ M (S/N = 3, M ≡ mol dm⁻³). On the other hand, 1-ethylquinolinium quenched luminescence of Tb^III·TCAS most efficiently, affording a very low D.L. (6.71 × 10⁻¹⁰ M). The agreement of the SV coefficients obtained with luminescent intensity (K_SV,all = 6.74 × 10⁶ M⁻¹) and lifetime (K_SV,Tb = 6.50 × 10⁶ M⁻¹) implied that dynamic quenching of ⁵D₄ excited state of Tb^III was predominant in the quenching processes. The quenching rate was estimated to be k_q,Tb = 9.94 × 10⁹ M⁻¹ s⁻¹, which was as fast as diffusion-limited rate. Quenching of Tb^III·(CAS)₂ was also applied to detection of NAD⁺, with a D.L. of 2.78 × 10⁻⁷ M.     

Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly(vinyl sulphonate) films for use in a lactate biosensor

The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD⁺ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.     

Computational Approach to Explore the B/A Junction Free Energy in DNA

Protein–DNA interactions induce conformational changes in DNA such as B‐ to A‐form transitions at a local level. Such transitions are associated with a junction free energy cost at the boundary of two different conformations in a DNA molecule. In this study, we performed umbrella sampling simulations to find the free energy values of the B–A transition at the dinucleotide and trinucleotide level of DNA. Using a combination of dinucleotide and trinucleotide free energy costs obtained from simulations, we calculated the B/A junction free energy. Our study shows that the B/A junction free energy is 0.52 kcal mol^?1 for the A‐philic GG step and 1.59 kcal mol^?1 for the B‐philic AA step. This observation is in agreement with experimentally derived values. After excluding junction effects, we obtained an absolute free energy cost for the B‐ to A‐form conversion for all the dinucleotide steps. These absolute free energies may be used for predicting the propensity of structural transitions in DNA.