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A peptide corresponding to the large intercellular loop that contains a histidine-rich sequence from the first iron-regulated transporter of Arabidopsis thaliana, which transports [Fe.sup.2} ions as well as [Mn.sup.2}, [Co.sup.2}, [Zn.sup.2} and [Cd.sup.2} was prepared and studied to provide a chemical basis for its role in the widespread ZIP family of transmembrane metal-transporting proteins. The histidine-rich sequence was found to have a very high entropy-driven affinity for [Fe.sup.3}, which may have biological significance.

A study uses colorimetric (near-UV absorption spectroscopy) and calorimetric (isothermal titration calorimetry) methods to quantify the equilibrium and thermodynamics of arsenite and monomethylarsenite (MMA) coordinating to glutathione (GSH) and the dithiols dimercaptosuccinic acid (DMSA), dihydrolipoic acid (DHLA), and dithiothreitol (DTT). The findings shows that arsenite and MMA form moderately stable complexes (beta = 10(super 6)-10(super 7) with GSH.

The binding of Ni(II) and Cu(II) to histidine, to the tripeptides His, GlyGlyHis, and HisGlyHis, and bovine serum albumin is studied by isothermal titration calorimetry (ITC) to determine the experimental conditions and data analyses necessary to reproduce liteature values for the binding constants and thermodynamic parameters. Guidelines are developed for accurate ITC measurements of metal binding to biological macromolecules.

The ability of the [Zn.sup.2+], as well as the monomethlyarsenite (MMA) ions to act as the binding sites in the glucocorticoid receptor (GR) is discussed. MMA is shown to form more stable complexes with the biologically relevant thiol glutathione (GSH), as compared to arsenite.

The effect of oxygen in the EPR spectral properties of wood charcoals is measured and a model based on the properties of charcoals and EPR relaxation mechanisms is developed. EPR line broadening in these materials has correlated with char surface area and the concentration of adsorbed oxygen.

Isothermal titration calorimetry (ITC) is used to quantify the stoichiometry, affinity and thermodynamics of [Ni.sup.2+], [Cu.sup.2+] and [Zn.sup.2+] binding. The stoichiometries and affinities obtained by ITC are in good agreement with the equilibrium dialysis results.

A C[u.sup.2+] titration of Hpn was monitored by EPR spectroscopy to understand the metal coordination of the protein. The histidine-rich cytoplasmic protein Hpn represents a novel class of metal binding proteins with relative binding affinities: C[u.sup.2+] > N[i.sup.2+] > B[i.sup.3+] ~ Z[n.sup.2+].

Conditions have been developed for the comproportionation reaction of Cu2+ and copper metal to prepare aqueous solutions of Cu+ that are stabilized from disproportionation by MeCN and other Cu+-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu+ complexes (CuI(MeCN)3+, CuIMe6Trien+, CuI(BCA)23−, CuI(BCS)23−), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu+ interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu+ with the biologically important tri-peptide glutathione, GSH. These results identify Me6Trien as an attractive Cu+-stabilizing ligand for calorimetric experiments, and suggest that caution should be used with MeCN to stabilize Cu+ due to its potential for participating in unquantifiable ternary interactions. [ABSTRACT FROM AUTHOR]

Metal coordination and isomerization of a hydrazone-based molecular switch ( 1) was investigated with isothermal titration calorimetry (ITC) and UV/Vis spectroscopy. Three equilibria-initial binding, ligand redistribution, and isomerization ( 1→ 1′)-are required to characterize the interaction of metal ions with the molecular switch. The stoichiometry of the initial complex and the relative magnitudes of the three equilibria constants depend on the size and Lewis acidity of the metal ion and competition from the counterion. Strongly coordinating anions (e.g., Cl−) suppress the formation of the initial metal-switch complex and isomerization. The thermodynamics for the equilibrium M2++2 1⇌M( 1′)22+ have been determined for Zn2+, Cd2+, and Hg2+ from the ITC data. [ABSTRACT FROM AUTHOR]

There is an imperative need to develop methods that can rapidly and accurately determine individual exposure to radiation for screening (triage) populations and guiding medical treatment in an emergency response to a large-scale radiological/nuclear event. To this end, a number of methods that rely on dose-dependent chemical and/or physical alterations in biomaterials or biological responses are in various stages of development. One such method, ex vivo electron paramagnetic resonance (EPR) nail dosimetry using human nail clippings, is a physical biodosimetry technique that takes advantage of a stable radiation-induced signal (RIS) in the keratin matrix of fingernails and toenails. This dosimetry method has the advantages of ubiquitous availability of the dosimetric material, easy and non-invasive sampling, and the potential for immediate and rapid dose assessment. The major challenge for ex vivo EPR nail dosimetry is the overlap of mechanically induced signals and the RIS. The difficulties of analysing the mixed EPR spectra of a clipped irradiated nail were addressed in the work described here. The following key factors lead to successful spectral analysis and dose assessment in ex vivo EPR nail dosimetry: (1) obtaining a thorough understanding of the chemical nature, the decay behaviour, and the microwave power dependence of the EPR signals, as well as the influence of variation in temperature, humidity, water content, and O2 level; (2) control of the variability among individual samples to achieve consistent shape and kinetics of the EPR spectra; (3) use of correlations between the multiple spectral components; and (4) use of optimised modelling and fitting of the EPR spectra to improve the accuracy and precision of the dose estimates derived from the nail spectra. In the work described here, two large clipped nail datasets were used to test the procedures and the spectral fitting model of the results obtained with it. A 15-donor nail set with 90 nail samples from 15 donors was used to validate the sample handling and spectral analysis methods that have been developed but without the interference of a native background signal. Good consistency has been obtained between the actual RIS and the estimated RIS computed from spectral analysis. In addition to the success in RIS estimation, a linear dose response has also been achieved for all individuals in this study, where the radiation dose ranges from 0 to 6 Gy. A second 16-donor nail set with 96 nail samples was used to test the spectral fitting model where the background signal was included during the fitting of the clipped nail spectra data. Although the dose response for the estimated and actual RIS calculated in both donor nail sets was similar, there was an increased variability in the RIS values that was likely due to the variability in the background signal between donors. Although the current methods of sample handling and spectral analysis show good potential for estimating the RIS in the EPR spectra of nail clippings, there is a remaining degree of variability in the RIS estimate that needs to be addressed; this should be achieved by identifying and accounting for demographic sources of variability in the background nail signal and the composition of the nail matrix. [ABSTRACT FROM AUTHOR]

The thermodynamics of formation of the insulin hexamer, which is stabilized by two Zn2+ ions, were quantified by isothermal titration calorimetry (ITC). Because the insulin monomer is unstable to aggregation (fibrillation) during ITC measurements, an original method involving EDTA chelation of Zn2+ from the hexamer was employed. The two metal ions are chelated sequentially, reflecting stepwise Zn2+ binding and stabilization of the quaternary structure. Analysis of the ITC data reveals that two to three H+ bind to the hexamer upon its formation at pH 7.4, which is both enthalpically and entropically favored. The former is due to Zn2+ coordination to His residues from three subunits, and the latter is associated with desolvation that accompanies the protonation and the packing of the subunits in the hexamer. [ABSTRACT FROM AUTHOR]

Kinesins are molecular motors that require a divalent metal ion (for example, Mg2+) to convert the energy of ATP hydrolysis into directed force production along microtubules. Here we present the crystal structure of a recombinant kinesin motor domain bound to Mn2+ and ADP and report on a serine-to-cysteine substitution in the switch 1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn2+ to Mg2+. This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg2+. In human kinesin-1 and kinesin-5 as well as Drosophila melanogaster kinesin-10 and kinesin-14, this defect is rescued by Mn2+, providing a way to control both the enzymatic activity and force-generating ability of these nanomachines. [ABSTRACT FROM AUTHOR]

The article presents a study on the electron paramagnetic resonance (EPR) spectral analysis dosimetry of fingernal clippings. The study used fingernail parings irradiated with a Caesium (Cs) source at an approved facility in the Department of Radiology at Dartmouth Medical School in Hanover, New Hampshire. It recommends automated and authenticated algorithms based on a significant number and varied set of control measurements for the analysis of EPR data. Further studies to develop fingernail EPR dosimetry is validated by the study.

Summarizes the properties of binuclear metallohydrolases. Classification of metallohydrolases; Steps in metabolic and signaling biochemical pathways; Structural and mechanistic models of binuclear metallohydrolases.

Embryotoxic Effects of Sodium Azide Infusions in the Syrian Hamster. SANA, T. R., FERM, V. H., SMITH, R. P., KRUSZYNA, R., KRUSZYNA, H., AND WILCOX, D. E. (1990). 15, 754–759. Pairs of osmotic minipumps containing 400 mg/ml (6.15 M) sodium azide in distilled water were subcutaneously implanted in timed pregnancy Syrian golden hamsters. The total delivered dose was calculated as 6 X 10 mmol kg hr at the maximal pumping rate. Most dams exhibited obvious signs of toxicity during the period of pump implantation which was Days 7 through 9 of gestation. After removal of the pumps the dams were euthanized on Day 13 of gestation, and the uteri were removed for counting of the number of living, malformed, and resorbed fetuses. This dose rate resulted in a significantly increased incidence of resorptions of embryos over that in a control group implanted with pumps delivering only distilled water. The incidence of gross malformations exclusively in the form of encephaloceles was not different between control and azide-infused groups. The extent of nitrosylation of circulating hemoglobin was followed with time and found to involve only about 0.1% of the total blood pigment. Thus, this commercially important and widely distributed chemical with high acute toxicity is not considered to be teratogenic in hamsters, and it produces embryotoxicity only at dose rates that result in toxic signs in the dams. [ABSTRACT FROM PUBLISHER]

The article examines how monomethylarsenite competes with zinc ions for binding sites in the glucocorticoid receptor (GR). According to the authors, thermodynamic and structural results showed that monomethylarsenite, but not arsenite, is able to compete with zinc ions that are required to stabilize the functionally competent structure of the GR DNA-binding domain.

Focuses on a study in which metal ion binding on zinc finger motifs in transcription factors were examined. Study on zinc finger peptides; What affect the rate of Cysteines oxidation; Information on cysteines in proteins and peptides.

Abstract: The Xeroderma Pigmentosum A (XPA) protein is involved in the DNA damage recognition and repair complex formation steps of nucleotide excision repair (NER), and has been shown to preferentially bind to various forms of DNA damage including bulky lesions. DNA interstrand crosslinks are of particular interest as a form of DNA damage, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery, and mitomycin C (MMC) is one of several useful cancer chemotherapy drugs that induce these lesions. Purified XPA and the minimal DNA-binding domain of XPA are both fully capable of preferentially binding to MMC-DNA interstrand crosslinks in the absence of other proteins from the NER complex. Circular dichroism (CD) and gel shift assays were used to investigate XPA-DNA binding and to assess changes in secondary structure induced as a consequence of the interaction of XPA with model MMC-crosslinked and unmodified DNAs. These studies revealed that while XPA demonstrates only a modest increase in affinity for adducted DNA, it adopts a different conformation when bound to MMC-damaged DNA than when bound to undamaged DNA. This change in conformation may be more important in recruiting other proteins into a competent NER complex at damaged sites than preferential binding per se. Arsenic had little effect on XPA binding even at toxic concentrations, whereas cadmium reduced XPA binding to DNA to 10–15% that of Zn-XPA, and zinc addition could only partially restore activity. In addition, there was little or no change in conformation when Cd-XPA bound MMC-crosslinked DNA even though it demonstrated preferential binding, which may contribute to the mechanism by which cadmium can act as a co-mutagen and co-carcinogen. [Copyright &y& Elsevier]

Two major components of insensitive munition formulations, nitroguanidine (NQ) and 3-nitro-1,2,4-triazol-5-one (NTO), are highly water soluble and therefore likely to photo-transform while in solution in the environment. The ecotoxicities of NQ and NTO solutions are known to increase with UV exposure, but a detailed accounting of aqueous degradation rates, products, and pathways under different exposure wavelengths is currently lacking. Here, we irradiated aqueous solutions of NQ and NTO over a 32-h period at three ultraviolet wavelengths (254 nm, 300 nm, and 350 nm) and analyzed their degradation rates and transformation products. NQ was completely degraded by 30 min at 254 nm and by 4 h at 300 nm, but it was only 10% degraded after 32 h at 350 nm. Mass recoveries of NQ and its transformation products were ≥80% for all three wavelengths, and consisted of large amounts of guanidine, nitrate, and nitrite, and smaller amounts of cyanamide, cyanoguanidine, urea, and ammonium. NTO degradation was greatest at 300 nm with 3% remaining after 32 h, followed by 254 nm (7% remaining) and 350 nm (20% remaining). Mass recoveries of NTO and its transformation products were high for the first 8 h but decreased to 22–48% by 32 h, with the major aqueous products identified as ammonium, nitrate, nitrite, and a urazole intermediate. Environmental half-lives of NQ and NTO in pure water were estimated as 4 and 6 days, respectively. We propose photo-degradation pathways for NQ and NTO supported by observed and quantified degradation products and changes in solution pH. Image 1 • Aqueous NQ yielded guanidine, cyanamide, urea, cyanoguanidine, nitrate, and nitrite. • Nitrosoguanidine suspected as an intermediate in NQ photo-transformation. • NTO produced mineralized gases, nitrate and nitrite, and transient urazole. • NQ and NTO estimated to have surface environmental half-lives on the order of days. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d 10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn 2+ and Cu + binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn 2+ binding was measured by chelation titrations of Zn 7 MT-3, while Cu + binding was measured by Zn 2+ displacement from Zn 7 MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant ( K ) and the change in enthalpy (Δ H ) and entropy (Δ S ) for these metal ions binding to MT-3. Zn 2+ was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn 7 MT-2 with Cu + revealed that both MT isoforms have similar Cu + affinities and binding thermodynamics, indicating that Δ H and Δ S are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu 4 + -thiolate clusters when Cu + displaces Zn 2+ under physiological conditions. Comparison of the Zn 2+ and Cu + binding thermodynamics reveal that enthalpically-favoured Cu + , which forms Cu 4 + -thiolate clusters, displaces the entropically-favoured Zn 2+ . These results provide a detailed thermodynamic analysis of d 10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The enthalpic and entropic components of Cu 2+ and Cu + binding to the blue copper protein azurin have been quantified with isothermal titration calorimetry (ITC) measurements and analysis, providing the first such experimental values for Cu + binding to a protein. The high affinity of azurin for Cu 2+ is entirely due to a very favorable binding entropy, while its even higher affinity for Cu + is due to a favorable binding enthalpy and entropy. The binding thermodynamics provide insight into bond enthalpies at the blue copper site and entropic contributions from desolvation and proton displacement. These values were used in thermodynamic cycles to determine the enthalpic and entropic contributions to the free energy of reduction and thus the reduction potential. The reduction thermodynamics obtained with this method are in good agreement with previous results from temperature-dependent electrochemical measurements. The calorimetry method, however, provides new insight into contributions from the initial (oxidized) and final (reduced) states of the reduction. Since ITC measurements quantify the protons that are displaced upon metal binding, the proton transfer that is coupled with electron transfer is also determined with this method. Preliminary results for Cu 2+ and Cu + binding to the Phe114Pro variant of azurin demonstrate the insight about protein tuning of the reduction potential that is provided by the binding thermodynamics of each metal oxidation state.

ITC measurements involving metal ions are susceptible to a number of competing reactions (oxidation, precipitation, and hydrolysis) and coupled reactions involving the buffer and protons. Stabilization and delivery of the metal ion as a well-defined and well-characterized complex with the buffer, or a specific ligand, can suppress undesired solution chemistry and, depending on the stability of the metal complex, allow accurate measurements of higher affinity protein-binding sites. This requires, however, knowledge of the thermodynamics of formation of the metal complex and accounting for its contribution to the experimentally measured values (KITC and ΔHITC) through a post hoc analysis that provides the condition-independent binding thermodynamics (K, ΔG(o), ΔH, ΔS, and ΔCP). This analysis also quantifies the number of protons that are displaced when the metal ion binds to the protein., (© 2016 Elsevier Inc. All rights reserved.)

Conditions have been developed for the comproportionation reaction of Cu(2+) and copper metal to prepare aqueous solutions of Cu(+) that are stabilized from disproportionation by MeCN and other Cu(+)-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu(+) complexes (Cu(I)(MeCN)3(+), Cu(I)Me6Trien(+), Cu(I)(BCA)2(3-), Cu(I)(BCS)2(3-)), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu(+) interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu(+) with the biologically important tri-peptide glutathione, GSH. These results identify Me6Trien as an attractive Cu(+)-stabilizing ligand for calorimetric experiments, and suggest that caution should be used with MeCN to stabilize Cu(+) due to its potential for participating in unquantifiable ternary interactions.

It was recently reported that a bacterium, Halomonas species GFAJ-1, isolated from arsenic-rich Mono Lake and further selected for growth under conditions of high arsenate and low phosphate, is able to grow using arsenic instead of phosphorus. This claim, and subsequent studies to evaluate GFAJ-1, has brought new attention to the question of whether arsenic can play an essential or sustaining role for living organisms. If true, this would be in stark contrast to the well known toxicity of this element and its ability to cause a number of diseases, including cancer of the skin, lung, bladder, liver, and kidney. However, while deadly at high doses, arsenic oxide is also an approved and effective chemotherapeutic drug for the treatment of acute promyelocytic leukemia (APL). This review examines the evidence that arsenic may be a beneficial nutrient at trace levels below the background to which living organisms are normally exposed. It also examines whether arsenic can be used to sustain organisms growing under high arsenic conditions, specifically the results from recent studies of arsenic biochemistry motivated by the report of GFAJ-1. Both of these topics are considered in the context of the toxicity of this element and its ability to cause cancer and other diseases, yet its Janus-faced ability to effectively treat APL.