NAD-硅胶亲和色谱固定相的合成和在核苷酸及其碱基分析中的应用

ＮＡＤ－硅胶亲和色谱固定相的合成和在核苷酸及其碱基分析中的应用于世林,苗凤琴,杨朝霞,冯茹（北京化工大学应用化学系,北京,１０００２９）关键词烟酰胺腺嘌呤二核苷酸（ＮＡＤ）,亲和色谱,核苷酸烟酰胺腺嘌呤二核苷酸（ＮＡＤ,辅酶Ⅰ）是多种脱氢酶的辅酶,通...     

烟酰胺腺嘌呤二核苷酸磷酸与生物光化学

生物光化学的核心内容是光合作用。在光合作用这个生物光化学的核心领域，烟酰胺腺嘌呤二核苷酸磷酸(NADPH)始终起着极其重要的作用，NADPH就是一个传递电子和能量的最关键活性生物分子。本文对海洋里的光合作用、细菌与光合作用以及光合作用的模拟等作了介绍。     

辅酶Ⅰ电化学配位反应机理的紫外光谱电化学研究

用薄层池原位紫外光谱电化学法研究了辅酶Ⅰ(NAD)在银电极上的电化学配位反应机理。实验结果表明：NAD能与溶出的银离子生成配合物，该配合物的配位反应是一种可逆过程，用光谱电化学的Nernst图解分析获得银离子与NAD配合物的主要存在形式是［Ag(NAD)₂］⁺，银离子是与NAD分子的腺嘌呤基团形成配合物的。配合物的银离子与银电极表面发生异相电子交换反应，该电化学反应是受吸附控制的准可逆电极过程。     

烟酰胺腺嘌呤二核苷酸(NAD~+)类似物的合成及性质

合成了3种新型结构稳定的烟酰胺腺嘌呤二核苷酸(NAD~+)类似物并通过~1H NMR、~(13)C NMR、~(31)P NMR和HRMS进行了表征。NAD~+类似物经80℃加热24 h后,通过~1H NMR确认其结构稳定;通过循环伏安法(CV)测定了其电化学性质,表明其仍然具有良好的氧化还原性能。     

烟酰胺腺嘌呤二核苷酸在纳米α-氧化铝上的吸附作用

用紫外-可见吸收光谱、X射线衍射、荧光光谱和衰减全反射傅里叶变换红外(ATR-FTIR）光谱等方法,研究了烟酰胺腺嘌呤二核苷酸(NAD+)在纳米α-Al2O3粒子上的吸附行为。 实验结果显示,NAD+的吸附量受pH值和离子强度影响较大,说明NAD+主要通过静电作用吸附在纳米α-Al2O3粒子上。 采用ATR-FTIR光谱分析了不同pH值溶液中及被吸附的NAD+,发现吸附后的NAD+与溶液中NAD+ 的ATR-FTIR光谱相似,但磷酸根的吸收峰向高波数位移,说明磷酸根参与了表面静电作用。 吸附过程符合Langmuir和Freundlich等温式。 荧光实验结果显示,随着吸附剂α-Al2O3用量的变化,NAD+构象也发生变化。     

酶催化动力学光度法测定汞

基于醇脱氢酶催化乙醇和氧化型烟酰胺腺呤二核苷酸的反应中，汞离子对于酶催化的抑制作用，通过测定还原型烟酰胺腺嘌呤二核苷酸吸光度的变化率得出其酶促反应速度，对应于汞而制得标准曲线。检出限为0.5μg/L。讨论了pH值和共存离子对测定的影响。本方法具有快速、灵敏、干扰少的优点。     

醇脱氢酶结构和作用机理研究进展

介绍了醇脱氢酶的种类, 酵母醇脱氢酶和肝醇脱氢酶等两类常用的醇脱氢酶的物理化学性质和活性位点结构. 归纳了对肝醇脱氢酶和酵母醇脱氢酶作用机理的研究, 重点评述了醇脱氢酶催化反应中的两个关键步骤质子转移和氢化物转移过程机理的研究进展.     

乳酸的液态酶荧光毛细分析法研究

基于乳酸脱氢酶(LDH)和烟酰胺腺嘌呤二核苷酸(NADH)氧化还原体系,提出了乳酸的液态酶荧光毛细分析方法(LE-FCA).在激发波长350nm、发射波长460nm奈件下,用LE-FCA法对乳酸进行了测定;其线性范围为0.2～1.0 mmol/L,r>0.9932,检出限为0.022 mmol/L,RSD<4.2%.LE-FCA能节省贵重的酶试剂,样品用量仅为18μL;可用于医药、卫生、工业、食品等含乳酸样品的测定.     

直接电化学法检测烟酰胺腺嘌呤二核苷酸

本实验所用丝网印刷电极(SPCE)对烟酰胺腺嘌呤二核苷酸(NADH)具有极好的直接电化学响应性能,在低电位(+0.200V)无需任何修饰的情况下,NADH在SPCE电极表面被氧化,据此可测定其含量。在最优实验条件下,NADH的氧化峰电流与其浓度在2.5×10-6~2.0×10-4 mol/L范围内呈线性关系,相关系数r=0.9992,检测限可达1.0×10-6 mol/L。该方法检测快速、灵敏度高、重现性好,具有良好的应用前景。     

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed‐phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose‐6‐phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.     

Preparation and Evaluation of Arylazide-Substituted Pyridine Adenine Dinucleotides for Photoaffinity Labeling Experiments

Two pyridine-modified NAD'analogs, 3–(3-azido benzo-yl) pyridine adenine dinucleotide 1 and N -(3-azido-5-car-boxyl) phenyl nicotinamide adenine dinucleotide 2 have been prepared and evaluated for photoaffinity labeling experiments. The syntheses were accomplished via a mammalian NADase-catalyzed base exchange reaction. The new NAD⁺ analogs retained the carbonyl or carhox-amido functional group at the 3 position of the pyridine ring. The analog 1 is the first pyridine-modified azido derivative of NAD⁺ that has shown coenzyme activity in a stereospecific hydride transfer reaction catalyzed by a dehydrogenase. Both NAD⁺ analogs have shown potential for the study of active sites of NAD⁺-utilizing enzymes.     

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.     

Synthesis of Nicotinamide Adenine Dinucleotide (NAD) Analogues with a Sugar Modified Nicotinamide Moiety

The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described.     

Immobilization enzyme fluorescence capillary analysis for determination of lactic acid

A new method for the determination of lactic acid based on the immobilization enzyme fluorescence capillary analysis (IE-FCA) was proposed. Lactic dehydrogenase (LDH) was immobilized on inner surface of a capillary with glutaraldehyde, and an immobilized enzyme lactate capillary bioreactor (IE-LCBR) was formed for the determination of lactic acid. After nicotinamide adenine dinucleotide (NAD⁺) is mixed with lactic acid solution, it was sucked into the IE-LCBR and was detected at λ_ex 353 nm/λ_em 466 nm. Optimized conditions are as follows: the temperature is 38 °C; the reaction time is 15 min; the concentrations of Tris buffer (pH 8.8) and NAD⁺ are 0.1 mol L⁻¹ and 4 mmol L⁻¹, respectively; the concentration of LDH used for immobilization is 15 kU L⁻¹. The concentration of lactic acid is directly proportional to the fluorescence intensity measured from 0.50 to 2.0 mmol L⁻¹; and the analytical recovery of added lactic acid was 99–105%. The minimum detection limit of the method is 0.40 mmol L⁻¹ and sensitivity of the IE-CBR is 4.6 F mmol⁻¹ L⁻¹ lactate. Its relative standard deviation (R.S.D.) is ≤2.0%. This IE-FCA method was employed for determination of lactate in milk drink.     

Studies on Synthesis and Chromatographic Properties of NAD Covalently Bound onto Phospholipid-Coated Aminated Silica

Abstract

Recently affinity chromatography with a biospecific stationary phase was widely used in separation and purification processes of all kinds of enzymes. In this paper, a series of synthetic reactions of solid-liquid phase on a silica surface are described. By using a wide-pore (30μm). microspheric silica (8μm) as the matrix and γ-aminopropyltriethoxy-silane as the activating agent, the nicotinamide adenine dinucleotide (NAD) was bonded through its amino groups with carboxylic groups of linked phospholipid by a covalent bond on the aminated supports. This bonded stationary phase provided high thermal stability which could be used for separating nucleotides with good resolution. At the same time, the effects of pH, organic modifier and ionic strength on the retention properties of nucleotides were also investigated.     

基于对烟酰胺腺嘌呤二核苷酸光致电化学响应的胆汁酸传感器

将硫堇电聚合在多壁碳纳米管修饰电极上,然后在其表面固定3α-羟类固醇脱氢酶制备了胆汁酸传感器。 由于该传感器具有光敏和电子受体功能的光电界面,同时偶合了光致电化学效应和酶促反应,能与3α-羟类固醇脱氢酶催化胆汁酸产生的电子供体NADH(烟酰胺腺嘌呤二核苷酸)产生光致电化学反应,通过测量反应产生的光电流实现了对胆汁酸的检测。 探讨了胆汁酸传感器对胆汁酸的光致电化学响应机理,研究了多壁碳纳米管、酶固定量和硫堇初始浓度对光电极性能的影响。 在优化了电解质溶液pH值、辅酶用量和偏压的测试条件下,该传感器对胆汁酸的测定范围为1.80~40.0 μmol/L,检出限为0.67 μmol/L。 应用该传感器对药品胆酸钠片和人尿中胆汁酸进行的检测结果显示,相对标准偏差小于5.64%,加标回收率为96%~104%。 该传感器的制备和对胆汁酸的检测不需要除氧,有经济、简便等优点。     

Dual probing of redox species,NAD(P)H and HOCl,with a benzo[a]phenoxazine based far red-emitting dye

We report a benzo[a]phenoxazine-based far red-emitting probe which selectively senses reduced nicotinamide adenine dinucleotide (NADH) and its phosphate ester NADPH, the most indispensable coenzymes found in all living cells, with “turn-off” fluorescence response at 650?nm. The original probe can be regenerated quantitatively through oxidation using hypochlorous acid, a reactive oxygen species, which was accompanied by restored fluorescence at 650?nm. This probe was also applied for fluorescence imaging of NADH in HeLa cells.