Electroanalytical Determination of Tungsten and Molybdenum in Proteins

Hagedoorn, Peter‐Leon; Slot, P. van 't; Leeuwen, Herman P. van; Hagen, Wilfred R.

doi:10.1006/abio.2001.5300

Cited by 18 publications

(15 citation statements)

References 37 publications

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“…The presence of the oxoanions did not change the elution profile of the monomeric protein. The metal content of the protein fraction and the low-molecularweight fraction (unbound oxoanion) was determined by catalytic adsorptive stripping voltammetry (8). It was found that molybdate and tungstate coeluted with the protein (Fig.…”

Section: Methodsmentioning

confidence: 99%

Tungsten Transport Protein A (WtpA) inPyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

et al. 2006

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Molybdenum and tungsten have similar ionic radii and chemical properties. Tungsten is the heaviest atom and the only third-row transition element that exhibits biological activity in enzymes. Molybdenum is the only second-row transition metal that exhibits biological activity when it is present in a cofactor of a metalloenzyme. Both metals are present mainly in enzymes that catalyze oxygen atom transfer reactions. In these enzymes they are coordinated by the two dithiolene sulfur atoms of a pterin molecule, called a molybdopterin cofactor (25). In the case of tungsten, the metal is always coordinated by two pterin moieties, forming a so called bis-pterin cofactor (9, 13). Molybdenum is an essential trace metal for many forms of life whereas tungsten is found mostly in archaea and in some bacteria. The molybdenum cofactor-containing enzymes can be divided into three families depending on the coordination chemistry of the Mo ligand: the sulfite oxidases; the xanthine oxidoreductases, also including the aldehyde oxidases; and the dimethyl sulfoxide reductases, which are found only in prokaryotes (18, 35). The tungsten cofactor-containing enzymes consist only of the aldehyde oxidoreductases (AORs), formate dehydrogenases (FDHs), and an acetylene hydratase (13). Based on sequence comparison the FDHs and acetylene hydratase are part of the molybdenum-containing dimethyl sulfoxide reductase family.The transport of molybdate has been well characterized in particular for Escherichia coli, which expresses a high-affinity ABC transporter for molybdate encoded by the modABC genes (27). The periplasmic molybdate binding protein ModA binds specifically molybdate and tungstate and not sulfate or other anions (27). Crystal structures of the E. coli and the Azotobacter vinelandii ModA indicate that the specificity for molybdate and tungstate is mostly determined by the size of the binding pocket. The Cambridge Structural Database (2) gives 1.75 Ϯ 0.04 Å and 1.76 Ϯ 0.02 Å for molybdate and tungstate, respectively, and 1.47 Ϯ 0.02 Å for sulfate. The ModA proteins cannot discriminate between molybdate and tungstate. The first tungsten-specific ABC transporter was identified in Eubacterium acidaminophilum (17). The periplasmic tungsten uptake protein (TupA) was cloned and expressed in E. coli and was shown to bind only tungstate with a high affinity. A crystal structure of TupA is not yet available, and it is not clear what the structural basis is of the specificity for tungstate over molybdate. Recently, a high-affinity vanadate transporter, which was highly selective for vanadate compared to tungstate, was identified in the cyanobacterium Anabaena variabilis ATCC 29413 based on the sequence similarity with the TupA protein from E. acidaminophilum (58% sequence similarity) (24). A. variabilis ATCC 29413 expresses an alternative V-dependent nitrogenase for the fixation of nitrogen, and therefore it requires vanadate (24). The specificity of this transporter for vanadate indicates that high sequence similarities are not conclusive fo...

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Section: Methodsmentioning

confidence: 99%

Tungsten Transport Protein A (WtpA) inPyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

et al. 2006

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“…In order to assess copper accumulation or deficiency in biological and environmental samples, sensitive, reproducible and accurate analytical methods are required. 2 With spectrophotometry, [3][4][5] electroanalytical methods, 6 chromatography, 7,8 and electrochemical analysis, 9,10 many new and modified techniques that can be used to determine metal ions in real samples, have been studied. While these methods have some pros and cons, spectrofluorimetry has merit in a sense that it is more sensitive, convenient, and simpler than other techniques.…”

Section: Introductionmentioning

confidence: 99%

Spectrofluorimetric Determination of Cu2+ Using New Fluorogenic Reagent

Sorouraddin¹,

Rashidi

Shabani

et al. 2005

Chin. J. Chem.

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A new synthesized fluorogenic reagent, 8-[(2-pyridine)methylideneamino] quinoline (PMAQ), was utilized for spectrofluorimetric determination of Cu(II) at trace levels. PMAQ, a good fluorogenic reagent, though insoluble in water, but is soluble in ethanol and 20% ethanol-water. The excitation and the fluorescence wavelengths of PMAQ were 310 and 434 nm respectively. When the reagent was complexed with Cu(II), the fluorescence intensity decreased proportionally with the concentration of Cu(II) at pH 4.5 by a static quenching effect. The highest sensitivity to Cu 2＋ determination was shown to be at PMAQ concentration of 1.0×10 -5 mol•L -1 . In order to enhance the quenching effect, the Cu(II)-PMAQ complex solution was kept at 22 ℃ for 20 min. Though the interferences by Co(II) and Fe(III) were very serious, they were however, completely eliminated by being masked with oxalate and ascorbate ions respectively. The linear dynamic range for Cu(II) determination was between 25-441 µg•L -1 with the detection limit of 18 µg•L -1 (RSD＝3.7%, n＝6). The proposed method was successfully applied to the determination of Cu(II) in real samples including human blood serum, commercial tea and wheat flour.

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“…Cell debris and unbroken cells were removed by centrifugation at 14,000 rpm at 5°C for 1 h using an Eppendorf centrifuge, and the supernatant was concentrated using a Microcon filter (Millipore) with a 3-kDa cutoff. The protein-containing samples were digested as previously described with 10% (wt/vol) perchloric acid (14). Precipitated proteins were removed by centrifugation at 14,000 rpm for 10 min.…”

Section: Methodsmentioning

confidence: 99%