Interaction between isoamyl adenine and rice cytokinin oxidase

Cytokinin (CTK) dehydrogenase is responsible for regulating the endogenous CTK content by oxidative removal of the side chain. Herein, we have applied fluorescence method to study the interaction between CTK dehydrogenase and CTK in vitro and obtain some parameters of their interaction. We found that addition of isopentenyl adenine can quench the fluorescence of CTK dehydrogenase, and the quenching mechanism was to be a static quenching procedure. We have measured the number of binding sites n and the apparent binding constant K and have calculated the thermodynamics parameter ΔH, ΔG, and ΔS by fluorescence quenching method. Based on thermodynamics parameter’s results, we concluded that their binding reaction was both entropy driven and the enthalpy driven, and the Van der Waals force and hydrogen bond force played a major role in the interaction. Based on the synchronous fluorescence spectrometry results, we demonstrated that the binding site between isopentenyl adenine and CTK dehydrogenase is in the microenvironment of both tryptophan and tyrosine. The fluorescence signal of coenzyme, flavin adenine dinucleotide, decreases gradually with the addition of isopentenyl adenine. And this method can be used for isopentenyl adenine routine assay. Under optimized experimental parameters, the linear segment increases from 0.6 µM to 100 µM with a regression equation of ΔF = 0.04 + 0.15c_ip (r = 0.999, c_ip in µM) with the detection limit of 0.15 µM iP.     

pH Resistant Ratiometric Measurement of Nicotinamide Adenine Dinucleotide Levels by Time-resolved Fluorescence Spectroscopy

Circular permutation fluorescent protein is a novel method to construct biosensors. The ratio of two excitation channels is employed to quantitatively calibrate the level of analysts. SoNar is one of them, which can be used to monitor cellular NADH/NAD⁺ levels. However, the 490 nm excitation channel of these biosensors is sensitive to pH environments, which is negative in real applications. In this work, we demonstrated that the fractional intensity ratio extracted from time-resolved fluorescence spectroscopy could be used to quantify NADH levels with one excitation (420 nm) and one emission channels. The 420-nm excitation channel was pH resistant. Comparing to average lifetime, the fractional intensity ratio had a 3.2-fold dynamic range, which was much wider than average lifetimes.     

Nicotinamide complex of silver(III) with expanded coordination number

Abstract

In strongly alkaline media ([OH^?]?≥?0.12 M), nicotinamide (nica) forms a complex with square-planar Ag(OH)₄^? [nica]?≥?0.05 M. The complex decomposes in seconds to nicotinamide N-oxide. The correlation of maximum absorbance of the complex with concentrations of nicotinamide and hydroxide requires that the complex is either the five-coordinate Ag(OH)₄(H_-1nica)^2? or the six-coordinate Ag(OH)₅(nica)^2?. Comparison with the reactions of Ag(OH)₄^? with nicotinate ion (nic^?) and acetamide under similar conditions indicates that nicotinamide coordinates with Ag(OH)₄^? by the amido group rather than the nitrogen on the pyridine ring or the amido oxygen. Kinetics of the Ag(III)-nica redox reaction are consistent with direct reaction between nicotinamide and uncoordinated Ag(OH₄)^?. Oxidation takes place at the pyridine ring, yielding nicotinamide N-oxide. Silver(III) is reduced to monovalent silver.     

Study on the interaction of a palladium complex with DNA

The complex [Pd(bipy)Cl₂] (1) (bipy = 2,2′-bipyridyl) has been synthesized and characterized by NMR spectroscopy, elemental analysis and X-ray diffraction method. The first step hydrolysis reaction kinetics for the complex was studied by UV-absorption spectroscopy; the speed constant (k ₁) was found to be 3.0×10^?4 s^?1. The fluorescence spectra have been collected to investigate the interaction of complex (1) with fish sperm DNA (FS-DNA) and the results indicate that the complex (1) has an effective intercalation within DNA. The reaction of complex (1) with adenine in ethanol/water results in the compound [Pd₂(bipy)₂(ade)₂]Cl₂·3H₂O (2) (ade = adenine) whose crystal structure was determined by X-ray diffraction method. The structure is orthorhombic, Pmmn, a = 12.993(4) Å, b = 14.512(5) Å, c = 9.837(3) Å, V = 1854.8(11) ų, Z = 2 (C₃₀H₃₀Cl₂N₁₄O₃Pd₂), final R ₁ = 0.0675. The palladium complex is a binuclear cation, where two ade ligands bridge two Pd(II) centers, while each Pd(II) is also chelated by one bipy ligand.     

Mechanochemically Induced Conversion of Crystalline Benzamide Polymorphs by Seeding

Benzamide has been known for its polymorphism for almost 200 years. Three polymorphic forms are described. To date, it was only possible to crystallize a metastable form in a mixture together with the thermodynamically most stable form I. A complete transformation of form I into the metastable form III by mechanochemical treatment has been achieved. Catalytic amounts of nicotinamide seeds were used to activate the conversion by mechanochemical seeding. NMR experiments indicated that the nicotinamide molecules were incorporated statistically in the crystal lattice of benzamide form III during the conversion. The transformation pathway was evaluated using in situ powder X‐ray diffraction.     

Fabrication and characterization of Meldola's blue/zinc oxide hybrid electrodes for efficient detection of the reduced form of nicotinamide adenine dinucleotide at low potential

We report the synthesis and the electrochemical properties of hybrid films made of zinc oxide (ZnO) and Meldola's blue dye (MB) using cyclic voltammetry (CV). MB/ZnO hybrid films were electrochemically deposited onto glassy carbon, gold and indium tin oxide-coated glass (ITO) electrodes at room temperature (25 ± 2 °C) from the bath solution containing 0.1 M Zn(NO₃)₂, 0.1 M KNO₃ and 1 × 10⁻⁴ M MB. The surface morphology and deposition kinetics of MB/ZnO hybrid films were studied by means of scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical quartz crystal microbalance (EQCM) techniques, respectively. SEM and AFM images of MB/ZnO hybrid films have revealed that the surfaces are well crystallized, porous and micro structured. MB molecules were immobilized and strongly fixed in a transparent inorganic matrix. MB/ZnO hybrid films modified glassy carbon electrode (MB/ZnO/GC) showed one reversible redox couple centered at formal potential (E⁰′) −0.12 V (pH 6.9). The surface coverage (Γ) of the MB immobilized on ZnO/GC was about 9.86 × 10⁻¹² mol cm⁻² and the electron transfer rate constant (ks) was determined to be 38.9 s⁻¹. The MB/ZnO/GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the oxidation of nicotinamide adenine dinucleotide (NADH). The linear response range between 50 and 300 μM NADH concentration at pH 6.9 was observed with a detection limit of 10 μM (S/N = 3). The electrode was stable during the time it was used for the full study (about 1 month) without a notable decrease in current. Indeed, dopamine (DA), ascorbic acid (AA), acetaminophen (AP) and uric acid (UA) did not show any interference during the detection of NADH at this modified electrode.     

A low perfusion rate microreactor for continuous monitoring of enzyme characteristics: application to glucose oxidase

This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector. The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety of further applications of the microreactor are suggested. Figure Preparation of apoGOx and restoration of enzyme activity using a soluton of FAD     

气相和溶液中金属离子与腺嘌呤配位选择性的理论研究

在B3LYP/6-31G(d,p)水平,全优化Mg2+、Ca2+、Mn2+、Co2+、Zn2+、Cu2+、Ni2+、Cd2+ (用有效实势(ECP) 方法处理)与腺嘌呤(A) N(1)、N(7)位点配位的两类配合物气相结构,通过相互作用能和自由能分析分别得出两位点对所研究金属离子的选择性顺序;后采用Onsager模型,优化其在水溶液(=78.39)中的结构,讨论溶剂效应对上述顺序的影响;通过相对自由能分析分别得出气相和溶液中,同种金属对两位点的选择性规律。溶液中金属离子与N(7)位配位的相对优选顺序为:Co2+ > Mg2+ > Cd2+ > Ca2+ > Zn2+ > Mn2+ > Ni2+ > Cu2+。     

Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS

Water-soluble B vitamins participate in numerous crucial metabolic reactions and are critical for maintaining our health. Vitamin B deficiencies cause many different types of diseases, such as dementia, anaemia, cardiovascular disease, neural tube defects, Crohn’s disease, celiac disease, and HIV. Vitamin B3 deficiency is linked to pellagra and cancer, while niacin (or nicotinic acid) lowers low-density lipoprotein (LDL) and triglycerides in the blood and increases high-density lipoprotein (HDL). A highly sensitive and robust liquid chromatography–tandem mass spectroscopy (LC/MS-MS) method was developed to detect and quantify a vitamin B3 vitamer (nicotinamide) and vitamin B6 vitamers (pyridoxial 5′-phosphate (PLP), pyridoxal hydrochloride (PL), pyridoxamine dihydrochloride (PM), pridoxamine-5′-phosphate (PMP), and pyridoxine hydrochloride (PN)) in human hair samples of the UAE population. Forty students’ volunteers took part in the study and donated their hair samples. The analytes were extracted and then separated using a reversed-phase Poroshell EC-C18 column, eluted using two mobile phases, and quantified using LC/MS-MS system. The method was validated in human hair using parameters such as linearity, intra- and inter-day accuracy, and precision and recovery. The method was then used to detect vitamin B3 and B6 vitamers in the human hair samples. Of all the vitamin B3 and B6 vitamers tested, only nicotinamide was detected and quantified in human hair. Of the 40 samples analysed, 12 were in the range 100–200 pg/mg, 15 in the range 200–500 pg/mg, 9 in the range of 500–4000 pg/mg. The LC/MS-MS method is effective, sensitive, and robust for the detection of vitamin B3 and its vitamer nicotinamide in human hair samples. This developed hair test can be used in clinical examination to complement blood and urine tests for the long-term deficiency, detection, and quantification of nicotinamide.     

Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD_ox), semiquinone form (FADH), and anionic hydroquinone form (FAD_redH⁻). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD_ox to the anionic semiquinone form (FAD⁻) with a quantum efficiency of about 2% and a back recovery time of about 10 s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD_ox to FADH, FADH to FAD_redH⁻, and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.