Spectrophotometric Determination of Trace Elements

Marczenko, Zygmunt; Freiser, Henry

doi:10.1080/10408348108542732

Cited by 17 publications

(12 citation statements)

References 512 publications

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“…Thiol groups were determined using Ellman's reagent (23). Iron was determined as the 1,10-phenanthroline complex (24). Nuclease concentration was determined by absorbance measurement at 280 nm (25) ITo whom reprint requests should be addressed.…”

Section: Methodsmentioning

confidence: 99%

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Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.

Ermácora

Delfino

Cuenoud

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

144

123

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We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.Here, we report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD; Fig. 1, compound 1) an easily prepared cysteine-specific hydrophilic reagent useful for reversibly conjugating ethylenediaminetriacetic acid to any free thiol group in a macromolecule. This reagent is a versatile tool that promotes intramolecular and localized protein cleavage. Staphylococcal nuclease was genetically engineered to introduce a single cysteine at position 28, and this variant, K28C, was used to characterize EPD-Fe as a protein cleavage reagent. Experimentation with a protein of known three-dimensional structure (20, 21) allowed the accessibility and proximity of cleavage sites to be assessed. Sequence analysis of K28C-EDTA-Fe fragmentation products identified several cleavage sites located close in tertiary structure to the reagent attachment site. In the presence of sodium dodecyl sulfate (SDS) or guanidinium chloride, cleavage at sites remote in linear sequence was not observed and the reaction was limited to residues proximal in sequence to the Cys-28 attachment site.There is an increasing need for new probes to study the topology of protein nonnative states, such as the molten globule and other folding intermediates, that can be populated at equilibrium (1, 2). Modem NMR techniques are revolutionizing the analysis of small proteins in solution, but they can be used to characterize partially folded molecules only under favorable conditions (3). Antibodies, proteases, and chemical probes have been used in the study of protein folding (4-8), but they are not well suited for mapping partially folded structure.A new class of chemical probes was devised largely for footprinting studies of DNA (9, 10). These reagents generate reactive oxygen species that label surrounding structural elements by oxidative degradation. Several metal chelates, bound covalently (11-13) or by affinity (14-16) to a protein, are...

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Section: Methodsmentioning

confidence: 99%

“…Thiol groups were determined using Ellman's reagent (23). Iron was determined as the 1,10-phenanthroline complex (24 …”

mentioning

confidence: 99%

Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.

Ermácora

Delfino

Cuenoud

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

144

123

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“…After equilibration a sample (1 mL) was removed, filtered, and the liquid was retained for spectrophotometric analysis at 455 nm. [54] An additional volume of the [NiA C H T U N G T R E N N U N G (NO 3 ) 2 ]·6H 2 O aqueous solution was then added to obtain a higher Ni II concentration and the procedure was repeated. The whole experiment involved about 10 successive additions of the [NiA C H T U N G T R E N N U N G (NO 3 ) 2 ]·6H 2 O aqueous solution to obtain about 10 initial Ni II concentrations in the range 3 10 À4 -2 10 À2 m. Following the aforementioned procedure, adsorption isotherms were obtained at four pH values (6.0, 6.5, 7.0, and 7.5).…”

Section: Set-up Used In the Deposition And Potentiometric Titrations mentioning

confidence: 99%

“…The latter was determined spectrophotometrically at 455 nm. [54] Adsorption isotherms: In each experiment for obtaining an adsorption isotherm, the support (2 g) was suspended in an electrolyte solution (100 mL of 0.1 m NaNO 3 ) and the pH was adjusted to a given value by adding a small volume of a concentrated NaOH or HNO 3 aqueous solution of known concentration. A small volume of a concentrated [Ni-A C H T U N G T R E N N U N G (NO 3 ) 2 ]·6H 2 O aqueous solution at a pH equal to that of the suspension was then added to obtain a given initial Ni II concentration.…”

Section: Set-up Used In the Deposition And Potentiometric Titrations mentioning

confidence: 99%

Interfacial Impregnation Chemistry in the Synthesis of Nickel Catalysts Supported on Titania

Bourikas

Stavropoulos

Garoufalis

et al. 2010

Chemistry A European J

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The interfacial chemistry of the impregnation step involved in the preparation of nickel catalysts supported on titania is presented. Several methodologies based on deposition data, pH measurements, potentiometric mass titrations, and microelectrophoresis have been used in conjunction with diffuse reflectance UV/Vis/NIR spectroscopy, simulations, and semiempirical quantum chemical calculations. Three mononuclear inner-sphere complexes were formed at the compact layer of the "titania/electrolyte solution" interface: A monosubstituted, dihydrolyzed complex above a terminal oxo group, a disubstituted, dihydrolyzed complex above two terminal adjacent oxo groups, and a disubstituted, nonhydrolyzed complex above one terminal and one bridging adjacent oxo groups. The monosubstituted, dihydrolyzed complex predominates. The contribution of the disubstituted configurations is also important at very low Ni(II) surface concentration, but it decreases as the Ni(II) surface concentration increases. In addition, bi- and trinuclear inner-sphere complexes were formed. The receptor site involves one bridging and two terminal oxo groups in the first case and two bridging and three terminal oxo groups in the second case. The relative surface concentrations of these configurations increase initially with Ni(II) surface concentration and then remain practically constant. The understanding of these interfacial processes at a molecular level is very important to shift the catalytic synthesis from an art to a science as well as to obtain strict control of the impregnation step and, to some extent, of the whole preparative sequence. This study is very relevant to the synthesis of submonolayer/monolayer nickel catalysts supported on TiO(2) following equilibrium deposition filtration (otherwise called equilibrium adsorption).

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“…It has been known that Cu 2+ stoichiometrically forms a complex with APDC at a ratio of 1:2. 25,26 However, to form a complex quantitatively, an excess amount of chelating agent should be used. Therefore, the concentrations of APDC were chosen to be varied from 0.05 to 1% w/v to cover the highest concentration of 3 µg/ml Cu 2+ .…”

Section: Effect Of the Apdc Concentrationmentioning

confidence: 99%