Wesley R. Harris

Binding of Vanadate to Human Serum Transferrin

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin.

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.

Behavior of vanadate and vanadyl ion in canine blood.

Radiolabeled vanadium as either vanadyl ion or vanadate ion was injected intravenously into adult beagle dogs, and blood samples were collected at various times up to 48 hr post injection. For each sample, the distribution of vanadium between the cells and the plasma was determined, and the plasma was analyzed by electrophoresis to identify specific vanadium-binding proteins. Initially, vanadyl ion left the bloodstream more rapidly than vanadate, but the rates equalized after about 5 hr. A significant fraction of the vanadium in blood was associated with the cellular component following injection of both forms of vanadium. About 77% of the plasma vanadium was eventually bound by the serum iron transport protein transferrin, regardless of the vanadium species initially injected. For both vanadyl and vanadate, about 30 hr were required to reach the maximum degree of transferrin binding.

Time-dependent leaching of coal fly ash by chelating agents.

The rates of leaching of several transition-metal ions from coal fly ash by pH 7.4 solutions of the chelating agents citric acid, EDTA, Listidine and glycine have been measured as part of an investigation of the potential health effects of inhaled fly ash. The results are compared to leaching of the same fly ash by 0.5M HCl, 0.10 M pH 7.4 Tris buffer, 0.5 M NH/sub 4/OH, and canine serum. For the trace elements, Zn, Mn, G, Ni, and Cu, the initial leaching rates with 0.5 M HCl range from 350 to 850 ppm/d. The rates drop by 1-2 orders of magnitude within 24 h and then level off at 1-10 ppm/d. The initial rates with EDTA and citric acid are also high, 100-400 ppm/d, but they fall off even more rapidly than the HCl leaching rates. The leaching of vanadium is exceptionally rapid, with initial rates of 1000-3000 ppm/d.

Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].

Determination of Arsenic(III) and Arsenic(V) in Coal and Oil Fly Ashes

Abstract Total arsenic has been determined for fly ashes generated by conventional combustion of pulverized western coal, oil, a coal-oil mixture, a coal-water mixture, and from the fluidized-bed combustion of North Dakota lignite. For most of the ashes the total arsenic levels were between 100 and 200ppm, but the coal-water mixture ash contained 348 ppm. Leaching with 0.5 N H2SO4 or a pH 5 1 M citrate solution resulted in the removal of 78 to 97% of the total arsenic from the particles. This clearly indicates a high surface enrichment of arsenic on the particles, in agreement with numerous previous studies on coal fly ash. The pH5 citrate solution was more effective for the removal of arsenic from the particles without significant oxidative loss of arsenic(III). Speciation of arsenic in the citrate leachate by hydride atomic absorption spectrometry indicated that in most cases less than 2% of the soluble arsenic was arsenic(III). The only exception was the leachate of the coal-oil mixture ash, which cont...

Correlation of nitroaromatic compounds with the mutagenic activity of coal fly ash

Stack-collected fly-ash particles from a commercial pulverized-coal power plant were extracted with 60/40 w/w benzene-methanol to remove as much of the organic fraction as possible. The extract was sequentially fractionated on a series of high-performance liquid chromatography columns, and the Salmonella bacterial mutagenicity assay using both normal and nitroreductase-deficient strains was used to localize the most mutagenic fractions. Selected fractions were analyzed by a variety of techniques, including gas chromatography with dual-flame ionization and thermionic nitrogen-phosphorus detectors, gas chromatography-mass spectrometry, direct-probe low-resolution or low-voltage mass spectrometry, and high-resolution mass spectrometry. Mutagenicity data indicated that nitroorganic compounds were the primary mutagens in all samples submitted for chemical analysis. A series of homologous alkylated nitrophenanthrenes appear to be important mutagens in one major fraction, while alkylated nitrofluorenones appear to be the dominant mutagens in a second major fraction. No nitro compounds were identified in a third major fraction. In addition to the nitro compounds, substantial amounts of fluorenones were also found, although these are not believed to contribute to the direct-acting mutagenic activity of the samples.

Leaching of metal ions from fly ash by canine serum.

w Fly ashes from the combustion of a variety of fossil fuels have been leached with normal canine serum for 24 h, and the concentrations of selected elements in the serum leachate have been determined by atomic absorption spectroscopy. Results are compared with similar leaching experiments involving either 0.5 M HC1 or pH 7.4 tris(hydroxymethy1)aminomethane buffer. Chelation by serum ligands is the dominant process in serum leaching of all metal ions except manganese. Serum leaching efficiency, defined as the percent of acid-soluble material removed by serum, is consistently between 40 and 90% for Co, Ni, Cu, and Zn and is consistently below 30% for Al, Fe, V, and Pb. Serum leaching of vanadium is lower than had been expected, whereas a combination of high bulk concentration and effective serum leaching leads to extremely high nickel concentrations in some of the serum leachates.

STRUCTURE-REACTIVITY RELATION FOR THE COMPLEXATION OF Ni, Cd, Zn, AND Fe

Abstract A structure-reactivity relationship is presented which predicts formation constants based solely on the structure of the organic ligand. This relationship has been evaluated using literature values of formation constants for Ni, Cd, Zn, and Fe. In most cases the root-mean-square deviation between observed and calculated log KML values is less than 0.8 log units. The relationship includes an adjustable parameter for each type of donor group coordinated to the metal ion, and the contribution of each donor atom is assumed to be independent of the other ligating groups. A separate term is included to account for the added stability due to the formation of chelate rings.

Mutagenicity in salmonella of nitroorganic compounds in extracts of fly ash from a lignite‐fired atmospheric fluidized‐bed combustor

The mutagenicity of a benzene/methanol extract of fly ash from an atmospheric fluidized-bed combustor burning Beulah, N.D., lignite was tested in Salmonella. Six strains were used, including three that were mutants in a nitroreductase gene locus. The numbers of revertants from his- to his+ as a function of the amount of fly ash extracted were determined. The results showed that the major mutagens in the crude extract were nitro compounds, from the fact that reversion rates in the nitroreductase-deficient strains were significantly lower than in the parent strains from which they were derived. The responses of the three parental strains, TA1538, TA98, and TA100, were quite similar; thus no conclusions could be made about frameshift versus base-substitution mutagens. Mutagenicity of 15 fractions of the extract was also tested, and one major peak of activity was detected. This activity eluted from a high-performance liquid chromatograph outside the range of retention times associated with mononitroaromatics. No further identification of specific nitroorganic compounds has been made.