Borate-nucleotide complex formation depends on charge and phosphorylation state

Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 microM nucleotide and 500 microM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3) showed that borate complexation with nicotinamide nucleotides was significantly influenced by the charge on the nicotinamide group and the number of phosphate groups on the adenine ribose. Borate binding decreased in the order of NAD(+), NADH, NADP(+) and NADPH. To investigate the relationship between complex formation and phosphorylation, association constants (K(A)) of borate-adenine (AMP, ADP, ATP), -guanine (GMP, GDP, GTP), -cytidine (CMP, CDP, CTP) and -uridine (UMP, UDP, UTP) complexes were compared. The results showed that the number of nucleotide phosphate groups was inversely proportional to the relative abundance of the borate complexes, with the K(A) of borate-nucleotide complex decreasing in the order mono-, di- and tri-phosphates (AMP approximately GMP approximately CMP approximately UMP > ADP approximately GDP approximately CDP approximately UDP > GTP > ATP approximately CTP approximately UTP). At pH 7.4, using ammonium bicarbonate buffer, only borate-NAD(+) complex was observed. This indicates that the borate-NAD(+) complex may be the most physiologically relevant of those studied.     

Capillary electrophoresis apparatus equipped with a bioluminescence detector using a batch- or flow-type detection cell

We developed a capillary electrophoresis (CE) apparatus equipped with a batch- or flow-type bioluminescence (BL) detection cell. Firefly luciferin-luciferase BL reaction was used to analyze samples of nucleotides, such as ATP, dATP, ADP, GTP, UTP, CTP, ITP, and TTP. In the CE apparatus with the batch-type cell, ATP was detected at concentrations of 5-100 microM, while the other nucleotides were not detected at concentrations less than 500 microM. The electropherogram of ATP included two BL peaks; the latter peak showed peculiar broadening, which continued up to ca. 2.5 h. In the CE apparatus with the flow-type cell, ATP, dATP, and ADP were detected with single peaks with detection limits of 1, 75, and 100 microM, respectively. The other nucleotides, GTP, UTP, CTP, ITP, and TTP, were not detected at concentrations less than 0.5 mM. A mixture of 10 microM ATP and 100 microM dATP was examined using the CE apparatus with the flow-type BL detection cell. ATP and dATP were separated using running buffer at pH 10 containing 1 mM phenylboronic acid. The interaction between ATP and phenylboronic acid delayed the migration time of ATP.     

Bis- and tris-naphthoimidazolium derivatives for the fluorescent recognition of ATP and GTP in 100% aqueous solution

Naphthoimidazolium groups can form unique ionic hydrogen bonds with anions as imidazolium moieties, and in addition, they are fluorescent, so no further elaborative synthesis is needed to introduce a fluorescent group. In this paper, three naphthoimidazolium derivatives were synthesized and studied for the recognition of nucleotides. Compound 1 composed of a single naphthoimidazolium group and quaternary ammonium group did not show any significant fluorescent changes with various anions and nucleotides, such as ATP, GTP, CTP, TTP, UTP, ADP and AMP. A tripodal compound 3 bearing three naphthoimidazolium groups and three quaternary ammonium groups, respectively, showed large fluorescence enhancements with UTP, CTP and TTP and moderate fluorescence enhancements with ATP and pyrophosphate and a fluorescence quenching effect with GTP. On the other hand, compound 2 bearing two naphthoimidazolium groups and two quaternary ammonium groups displayed a selective fluorescence enhancement with ATP and a selective fluorescence quenching effect with GTP in 100% aqueous solution.     

Ion-pair reversed phase HPLC determination of nucleotides,nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes

Chromatographia - Three groups of metabolites were analyzed in extracts of rat hepatocytes by an HPLC method: (i) nucleotides (ATP, ADP, AMP, GTP, GDP, UTP, UDP, IMP, UMP), (ii) nucleosides and...     

Study of cavity size and nature of bridging units on recognition of nucleotides by cyclophanes

We synthesized a few novel cyclophanes CP-1 to CP-4 containing anthracene units linked together through different bridging and spacer groups and have investigated their interactions with various nucleosides and nucleotides. Of these systems, CP-1 and CP-3 showed selectivity for 5'-GTP and 5'-ATP as compared to other nucleotides and nucleosides, whereas negligible selectivity was observed with CP-2 and CP-4. Interestingly, CP-1, CP-2 and CP-3 exhibited significant binding interactions with the fluorescent indicator, 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS), resulting in the formation of non-fluorescent complexes. Titration of these complexes with nucleosides and nucleotides resulted in the displacement of HPTS, leading to the revival of its fluorescence intensity. It was observed that 5'-GTP induced the maximum displacement of HPTS from the complex [CP-1·HPTS] with an overall fluorescence enhancement of ca. 150-fold, while 5'-ATP induced ca. 45-fold. Although the displacement of HPTS from the complexes [CP-2·HPTS] and [CP-3·HPTS] was found to be similar to that of [CP-1·HPTS], these complexes showed lesser selectivity and sensitivity. In contrast, negligible displacement of HPTS was observed from the complex [CP-4·HPTS] under similar conditions. These results indicate that CP-1, having a well-defined cavity and good electron acceptor (viologen), is capable of forming selective and stable complexes. Though CP-2 and CP-3 retain the good electron acceptor (viologen), their reduced aromatic surface and larger cavity, respectively, resulted in lesser sensitivity. In contrast, CP-4 having a large cavity and a poor acceptor (1,2-bis(pyridin-4-yl)ethene) showed negligible selectivity, thereby indicating the importance of cavity size, bridging unit and aromatic surface on biomolecular recognition properties of cyclophanes.     

Fluorescent sensing of triphosphate nucleotides via anthracene derivatives

A nucleotide is composed of a nucleobase, a five-carbon sugar, and phosphate groups. Recognition of these three sites can provide useful information for the development of selective fluorescent receptors for a specific nucleotide. In this paper, anthracene derivatives with two imidazolium groups at the 1,8- and 9,10-positions, quaternary ammonium groups, or the boronic acid group were examined for the recognition of nucleotides, such as ATP, GTP, CTP, TTP, UTP, ADP, and AMP, via fluorescence changes. The anthracene group provides the interaction between the bases of the nucleotides. The imidazolium and quaternary ammonium groups induce hydrogen bonding interactions with the phosphate groups of the nucleotides. The boronic acid group can interact with the ribose of the nucleotides.     

Sensitive determination of nucleotides and polynucleotides based on the fluorescence quenching of the Tb3+-Tiron complex

 A sensitive method using fluorescence quenching for the determination of nucleotides (ATP, ADP, AMP, CTP, UTP) and polynucleotides[poly(A), poly(I), poly(U)] is proposed. It is based on the ability of nucleotides and polynucleotides to inhibit the formation of a strongly fluorescent complex of Tb³⁺ ion with Tiron. The possibilities of spectrofluorimetric measurements of these systems were studied under optimal conditions (pH 6.9 in hexamethylene tetramine-HCl buffer, 1.2×10^-6 mol/L of Tb³⁺, 4.0×10^-6 mol/L of Tiron, λ_ex=317 nm, λ_em=546 nm). The results showed that the Tb³⁺-Tiron complex could be used as a fluorescence test for the phosphate moieties of nucleotides and polynucleotides. The detection limits are 0.3, 1.2, 3.7, 0.2, 0.3, 1.1, 0.6 and 0.9 ng/mL for ATP, ADP, AMP, CTP, UTP, poly(A), poly(I), and poly(U), respectively. The relative standard deviations (6 replicates) are within 4.0% in the middle of the linear range. The fluorescence quenching mechanism of these systems is also discussed. Received: 16 July 1996 / Revised: 13 November 1996 / Accepted: 13 November 1996     

Hydrophilic interaction chromatography of nucleotides and their pathway intermediates on titania

Nucleotides and their pathway intermediates play important roles in all living species. They are essential cellular components in energy transfer, metabolic regulatory processes and biosynthesis. Titania (TiO(2)) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of nucleotides. Herein a bare titania column (150 mm x 4.6 mm I.D., 3 microm) with UV detection at 254 nm was used for the separation of a set of nucleotides (AMP, ADP, ATP, UMP, UDP, UTP, GMP, GDP, GTP, CMP and CTP) and their intermediates (NAD, NADH, UDP-Glu and UDP-GluNAc). Addition of phosphate to the eluent suppresses the ligand-exchange interactions with the titania surface such that hydrophilic interaction chromatography (HILIC) separations may be performed. Increasing the %ACN resulted in increasing retention and efficiency (up to 13,000, 9500 and 4500 plates/m for AMP, ADP and ATP, respectively). The effects of pH, buffer concentration and other eluent anions (fluoride and acetate) were also studied. Fifteen nucleotides and their intermediates were separated in 26 min (R(minimum)>1.3) using an one-step gradient.     

A cholic acid-based fluorescent chemosenor for the detection of ATP

A novel ditopic cholic acid-based fluorescent chemosensor for ATP, 1a, was designed and synthesized. Its interactions with phosphates, AMP, ADP, ATP, CTP, GTP, and TTP have been investigated. When ATP was added to a 1:1 aqueous CH3CN solution of the sensor at pH 7.4, a significant decrease in fluorescence of 1a was observed, whereas other guest molecules showed a much smaller effect. The complex between 1a and ATP was confirmed through combined UV, 1H, 13C and 31P NMR spectroscopic methods. The uniqueness of the new sensor is that it binds with ATP 33-124 times more selectively than other nucleotides, as evidenced from the respective binding constants. 1a is a highly sensitive sensing probe; as little as 30 nM ATP can cause 15% fluorescence quenching of the sensor.     

Behaviour of nucleotides and oligonucleotides in potentiometric HPLC detection

Potentiometric HPLC detection was studied of mononucleotides (UMP, AMP, UDP, ADP, CTP, UTP, GTP, ATP) and oligonucleotides (a synthetic mixture d(T)_12–18 5′OH, a mixed 21-mer, 33-mer, and 60-mer). Coated-wire electrodes were used. The coatings were of the liquid membrane type containing PVC, DOS and synthetic macrocyclic amine- and podand ureum receptors. Electrodes based on these receptors gave very sensitive responses to triphosphate nucleotides and to oligonucleotides. The molar response of the oligonucleotides was related to their molar mass. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. The sensitive potentiometric response of these highly charged ions is discussed.     

Delayed luminescence analysis (DLA) of purine and pyrimidine ribose and deoxyribose nucleotide triphosphates in picomole quantities

A discovery is reported of a new system that enables one to quantitate the amounts of separated nucleotide triphosphates in picomole quantities. This system of delayed luminescence analysis (DLA) is sensitive to both purine and pyrimidine ribose and deoxyribose nucleotide triphosphates. A crude luciferin-luciferase (substrate-enzyme) preparation from firefly lanterns, in the presence of nucleotide triphosphate, is utilized to generate light that is detected by a liquid scintillation counter with the coincidence of the photomultiplier tubes turned off. Light is produced in a delayed fashion, the maximum emission being dependent on the type of nucleotide. Purine nucleotides (GTP, ITP, dATP, dGTP) give maximal light emission at approximately 2 mins; with the pyrimidine nucleotides the time required for maximal light emission was 5 min for UTP, dUTP, and TTP, 10 min for CTP, and 12 min for dCTP. A linear relationship on a log-log plot of light emission vs. concentration of nucleotide is demonstrated with ITP, dATP, UTP, and CTP.     

On-off-on fluorescence detection for biomolecules by a fluorescent cage through host-guest complexation in water

Detection of nucleoside derivatives has paramount importance because they are the essential biomolecular units for all life. Herein, we report a host-guest approach by using a fluorescent tetraphenylethenebased octacationic cage as host and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt(HPTS) as guest and fluorescent indicator to form non-fluorescent 1:1:1 host-(endo-exo)guest complex in water. This new host-(endo-exo)guest complex can be successfully used for detecting nucleosides(e.g., ...     

Fluorescence enhancement of terbium(III) by nucleotides and polyhomonucleotides in the presence of phenanthroline

Summary The fluorescence enhancement of terbium(III) by nucleotides (AMP, ADP, ATP, GMP, GDP, GTP) and polyhomonucleotides [poly(A), poly(G), poly(C), poly(U)] in the presence of phenanthroline (phen) was studied. Investigation of the composition of the terbium(III)/ANP(AMP, ADP, ATP)/phen complexes and conditions of optimization suggest a 1:2 molar ratio of terbium(III) and phen for the ternary complexes. The results showed that the presence of phen enhanced the net fluorescence of terbium(III)/ANP, poly(A), poly(C) or poly(U) from several fold to more than one-hundred fold, while it has little effect on the fluorescence of terbium(III)/GNP(GMP, GDP and GTP) or the poly(G) system. The possibility of spectrofluorimetric measurements of these compounds were studied under optimal conditions (pH 7.0 in tris-HCl buffer; E_x=298 nm, E_m=543.5 nm). The detection limits were 2.0×10^–7, 6.0×10^–7 and 1.0×10^–6 mol/l for AMP, ADP and ATP, respectively. The relative standard deviations (6 replicates) were within 2.0% in the middle of the linear range.     

Purine and pyrimidine compounds in murine peritoneal macrophages cultured in vitro.

Extracts of murine peritoneal macrophages were analysed by ion-pair reversed-phase high-performance liquid chromatography during incubation at 37 degrees C in vitro. Four-step gradient elution was applied to an ODS column (250 x 4.6 mm I.D.) at a flow-rate of 1.3 ml/min, allowing the separation of hypoxanthine, inosine, guanosine, adenosine, IMP, CDP, AMP, GDP, UDP, ADP, CTP, GTP, UTP and ATP within 50 min. Samples of 0.4 . 10(6)-0.5 . 10(6) cells were washed twice with RPMI 1640 medium and extracted with perchloric acid. Nucleotide concentrations of murine peritoneal macrophages did not change during incubation for 4 days in vitro.     

Rapid determination of creatine, phosphocreatine, purine bases and nucleotides (ATP, ADP, AMP, GTP, GDP) in heart biopsies by gradient ion-pair reversed-phase liquid chromatography.

A simple binary solvent method has been developed for the simultaneous determination of creatine (Cr), phosphocreatine (PCr), ATP, ADP, AMP, GTP, GDP, IMP, NAD, inosine, adenosine, hypoxanthine and xanthine. This allows separation of the most important nucleotides present in myocardial biopsies as, for example, in studies using 31P NMR spectroscopy. In NMR spectra ATP and PCr are the only visible high-energy phosphates, therefore the status of other nucleotides and bases cannot be determined. The nucleotides, AMP degradation products, PCr and Cr in pig and rat heart muscle were resolved with 35 mM K2HPO4, 6 mM tetrabutylammonium hydrogensulfate buffer, pH 6.0, and a binary acetonitrile gradient on medium-bore, 250 mm or 125 mm x 3.9-4.6 mm I.D. steel octadecyl-bonded (C18) columns at a flow-rate of 1.5 or 1.0 ml/min. This method, optimized for use with older high-performance liquid chromatography pumps (100 microliters displacement heads), resolves the major porcine and rat myocardial nucleotides and degradation products within 22 min. The amounts found in normoxic porcine muscle are: Cr 9.21 +/- 0.75; hypoxanthine 1.40 +/- 0.14; PCr 7.20 +/- 1.2; IMP 1.34 +/- 0.13; beta NAD 1.82 +/- 0.23; AMP 0.10 +/- 0.04; GDP 0.05 +/- 0.02; ADP 1.23 +/- 0.09; GTP 0.19 +/- 0.01; ATP 4.45 +/- 0.32 mumol/g wet weight. The method, incorporating adenosine tetraphosphate as an internal standard, allows the documentation of changes in both the high-energy phosphates and their degradation products in a single analysis of myocardial samples as small as 200 micrograms (wet weight).     

Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition.

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.     

Separation of nucleoside mono-, di- and triphosphates by capillary electrophoresis via cadmium complexation

Summary The CE separation of twelve nucleotides (5′-mono-, di-, triphosphates of adenosine, guanosine, cytidine and uridine) was improved by adding cadmium ion to the ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM). Cadmium ion acts as a complexing agent for some nucleotides (ATP, CTP, GTP, UTP, GDP). In order to accelerate the separation, the electroosmotic flow was reversed by flushing the fused-silica capillary with 0.2 % aqueous solution of the polycationic surfactant hexadimethrine bromide. A good separation of the twelve nucleotides studied was then achieved on a dynamically coated capillary in less than 5 min by using an ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM) to which 2 mM cadmium ion has been added. High peak efficiencies were obtained (210 000 theoretical plates) and the resolution between two adjacent peaks was always greater than 1.5.     

über Struktur und Aktivität der den H2O2-Zerfall katalysierenden Cu2+-Komplexe VII

It has been stated in a preceding paper [3] that only parts of a ligand coordinated to a metal ion can be oxidized by H₂O₂ (= peroxidative activity). Considering the reversal of this statement to be true, it is shown by means of the peroxidative activity of the Cu²⁺-complexes of ATP, ITP, CTP, UTP, and TTP that in these complexes the heteroaromatic groups contribute to the coordination of Cu²⁺ ion. By analogy with the Cu²⁺-ATP-complex, where a macrocyclic phosphate-metal-adenine chelate is formed [4], and based on his experimental results, the author considers the existence of such a macrocyclic chelate in the copper complexes of ITP, GTP, CTP, UTP, and TTP as established. The coordination sites of the heteroaromatic groups in these complexes are discussed.     

ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene

A simple and sensitive method for ATP detection using a label-free DNA aptamer as the recognition element and ethidium bromide (EB) as the signal reporter is reported. The ATP-binding aptamer undergoes a conformational switch from the aptamer duplex to the aptamer/target complex upon target binding, which induces the fluorescence change of intercalated EB emission. Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between the ATP aptamer and ATP. Using EB alone as a signal reporter, the ATP detection limit was estimated to be approximately 0.2 mM. When a light harvesting cationic tetrahedralfluorene was used as an energy donor to sensitize the intercalated EB emission, a 10-fold increase in detection limit and a 2-fold increase in detection selectivity was demonstrated. The sensitivity and selectivity of the tetrahedralfluorene sensitized assay is comparable to or better than most fluorescent ATP assays with multiple labels.     

Labile zinc-assisted biological phosphate chemosensing and related molecular logic gating interpretations

Herein, molecular fluorescence 'OFF-ON' behavior with aqueous addition of biological phosphate and Zn(2+) is studied with Zn(2)(slys)(2)Cl(2) [H(2)slys = 6-amino-2-{(2-hydroxybenzylidene)amino}hexanoic acid], a fluorescent water-soluble complex, using various spectroscopic tools (e.g., (31)P NMR, UV-vis, emission, and CD spectroscopy) at the micromolar level. Adduct-dependent fluorescence intensity changes can be interpreted as a two-input (cation/anion) implication molecular logic gating system. A displacement study of PPi from the dizinc complex is also reported. Diphosphate and triphosphate addition/displacements were also studied. (31)P NMR spectroscopy shows gradual NMR peak shifts from bound ADP/GDP to free ADP/GDP with increasing [PPi]. In the emission spectrum, fluorescence quenching is shown: CD signal maxima decrease with addition of PPi. These displacement events are also tested with triphosphates (ATP, GTP), and their binding strength/displacement ability over ADP/GDP is quantified: PPi > ATP ≈ GTP (3.35 ± 0.77 × 10(4) M(-1) for PPi, 7.73 ± 1.79 × 10(3) M(-1) for ATP, 9.21 ± 2.88 × 10(3) M(-1) for GTP over 1·ADP). Many anions and cations were also screened for selectivity. Tubulin polymerization was assayed in the presence of 1 and its copper analogue which reflected a slight inhibition in polymerization.