Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

Sakasegawa, Shin-ichi; Hayashi, Junji; Ikura, Yoshiaki; Ueda, Shigeru; Imamura, Shigeyuki; Kumazawa, Takao; Nishimura, Atsuhisa; Ohshima, Toshihisa; Sakuraba, Haruhiko

doi:10.1016/j.ab.2011.04.043

Cited by 8 publications

(8 citation statements)

References 28 publications

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“…Additionally, it was shown that (d)ADP and (d)GDP were substrates of TmPPDK with comparable efficiency (Figure 3). In good agreement with our results, TmPPDK was described before to be highly specific for AMP when tested with ribonucleotide monophosphates [23]. However, little information is available on the substrate scope of PPDKs since mainly reactions with AMP as substrate were reported.…”

Section: Evaluation Of the Substrate Scopesupporting

confidence: 92%

“…Additionally, in the genome a pyruvate-phosphate-dikinase (TmPPDK) was found. This enzyme was described before to catalyze the phosphorylation of nucleoside monophosphates directly to nucleoside triphosphates using phosphoenol pyruvate and pyrophosphate as substrates [23].…”

Section: Resultsmentioning

confidence: 99%

“…For all enzymes showing phosphorylating activity towards nucleosides or nucleotides working pH ranges were determined, except for TmTK, TmPK, TmAcK and TmPPDK. For the latter enzymes, the optimal reaction pH has already been described ( [18,23,25,26], Table 1). Working pH ranges differed depending on the enzyme classes.…”

Section: Activity Testing Of the T Maritima Enzymes At Varying Phmentioning

confidence: 99%

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Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Hellendahl

Panse

Maiwald³

et al. 2023

Preprint

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Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, a database search on the carbohydrate and nucleotide metabolism of T. maritima was performed. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. The synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T. maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates and it was shown that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T. maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

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Section: Evaluation Of the Substrate Scopesupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Activity Testing Of the T Maritima Enzymes At Varying Phmentioning

confidence: 99%

See 1 more Smart Citation

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Hellendahl

Panse

Maiwald³

et al. 2023

Preprint

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“…More demanding is the application for monitoring the course of polymerase chain reaction, which is accompanied by PPi formation: the method must detect as low as 0.2 mM PPi in the presence of 0.1 mM nucleotide triphosphates. 8 Required sensitivity and selectivity are achieved by enzymatic methods of PPi determination, 9 but development of a relatively simple chemical sensor of lower cost and higher stability is very much desirable.…”

Section: Introductionmentioning

confidence: 99%

Selective fluorometric detection of pyrophosphate by 3-hydroxyflavone-diphenyltin(iv) complex in aqueous micellar medium

Villamil‐Ramos

Barba

Yatsimirsky

2012

Analyst

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3-Hydroxyflavonate-diphenyltin(IV) chloride (Ph(2)Sn(Ofl)Cl) was prepared and characterized structurally by single crystal X-ray diffraction. The compound possesses high fluorescence (λ(max) 464 nm with excitation at 400 nm) in neutral aqueous solutions containing 5 mM cetyltrimethylammonium chloride and does not lose the flavonol ligand even at high dilution. The fluorescence is selectively quenched by pyrophosphate (PPi) (linear range 0-5 μM, detection limit 0.1 μM). Determination of 1 μM PPi tolerates the presence of 100-fold excess of AMP, ADP, inorganic phosphate and acetate, and 10-fold excess of ATP. From concentration profiles of the quenching effect it is concluded that quenching occurs due to formation of the complex (Ph(2)Sn(Ofl))(3)PPi, which is transformed at higher PPi concentrations to a simple 1 : 1 complex Ph(2)Sn(Ofl)(PPi). No flavonol ligand exchange with PPi is observed. A series of related organotin(IV) complexes were prepared in situ by reacting 3-hydroxyflavone with Me(2)SnCl(2), PhSnCl(3) and n-BuSnCl(3). The derivatives of Me(2)SnCl(2) and PhSnCl(3) behave similarly to Ph(2)Sn(Ofl)Cl but with lower sensitivity to PPi. However, the n-butyltin(IV) derivative tentatively of the composition n-BuSn(Ofl)(2) undergoes fluorescence enhancement rather than quenching in the presence of PPi in the μM concentration range. The results of this study demonstrate that the use of metal flavonol complexes as optical sensors for anions is a promising approach.

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“…Until now, many interesting techniques for PPi detection have been reported, including chemiluminescence, surface-enhanced Raman scattering (SERS) assay, colorimetric sensing, as well as fluorescence spectroscopic measurement. − Although the above-mentioned methods are well developed for PPi quantification, some limitations still exist such as unsatisfied detection limit and sophisticated operation procedures. For example, the detection accuracy of fluorescent assays is impeded by the limited photophysical properties of the fluorescent dyes such as photobleaching, background autofluorescence, self-quenching, as well as poor water-solubility.…”

mentioning

confidence: 99%

Color-Coded Single-Particle Pyrophosphate Assay with Dark-Field Optical Microscopy

Han

et al. 2018

Anal. Chem.

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In this work, we demonstrate a convenient yet sensitive color-coded single-particle detection method for the quantification of pyrophosphate (PPi) by using single gold nanoparticle (GNP) as the probe. The design is based on GNP-dependent catalytic deposition of Cu onto the surface of GNPs with reduced nicotinamide adenine dinucleotide (NADH). Without PPi, Cu can be directly reduced to Cu through the gold-catalyzed oxidization of NADH. In the presence of PPi, the coating process is impeded due to the strong coordination capability of PPi with Cu. The selective coating of Cu shell onto the GNPs surface results in the extraordinary red-shift of localized surface plasmon resonance from individual GNPs. By quantitatively counting the fraction of yellow particles with color-coded dark-field optical microscopy, the trace amounts of PPi in solution can be accurately quantified. The limit-of-detection is as low as 1.49 nM with a linear dynamic range of 0-4.29 μM, which is much lower than the spectroscopic measurements in bulk solution. In artificial urine sample, good recovery efficiency was achieved. As a consequence, the method demonstrated herein will find promising applications for the ultrasensitive detection of target biomolecules under biological milieu in the future.

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Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

Cited by 8 publications

References 28 publications

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Selective fluorometric detection of pyrophosphate by 3-hydroxyflavone-diphenyltin(iv) complex in aqueous micellar medium

Color-Coded Single-Particle Pyrophosphate Assay with Dark-Field Optical Microscopy

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