Cobalt-mediated oxidative DNA damage and its prevention by polyphenol antioxidants.

Although cobalt is a required nutrient, it is toxic due to its ability to generate reactive oxygen species (ROS) and damage DNA. ROS generation by Co2+ often has been compared to that of Fe2+ or Cu+, disregarding the reduction potential differences among these metal ions. In plasmid DNA damage studies, a maximum of 15% DNA damage is observed with Co2+/H2O2 treatment (up to 50 µM and 400 µM, respectively) significantly lower than the 90% damage observed for Fe2+/H2O2 or Cu+/H2O2 treatment. However, when ascorbate is added to the Co2+/H2O2 system, a synergistic effect results in 90% DNA damage. DNA damage by Fe2+/H2O2 can be prevented by polyphenol antioxidants, but polyphenols both prevent and promote DNA damage by Cu+/H2O2. When tested for cobalt-mediated DNA damage affects, eight of ten polyphenols (epicatechin gallate, epigallocatechin gallate, propyl gallate, gallic acid, methyl-3,4,5-trihydroxybenzoate, methyl-4,5-dihydroxybenzoate, protocatechuic acid, and epicatechin) prevent cobalt-mediated DNA damage with IC50 values of 1.3 to 27 µM and two (epigallocatechin and vanillic acid) prevent little to no DNA damage. EPR studies demonstrate cobalt-mediated formation of •OH, O2•-, and •OOH, but not 1O2 in the presence of H2O2 and ascorbate. Epigallocatechin gallate and methyl-4,5-dihydroxybenzoate significantly reduce ROS generated by Co2+/H2O2/ascorbate, consistent with their prevention of cobalt-mediated DNA damage. Thus, while cobalt, iron, and copper are all d-block metal ions, cobalt ROS generation and its prevention is significantly different from that of iron and copper.

Polyphenol effects on CuO-nanoparticle-mediated DNA damage, reactive oxygen species generation, and fibroblast cell death.

The ability of ten polyphenolic antioxidants to prevent CuO nanoparticle (NPCuO) and H2O2-mediated DNA damage and cytotoxicity was investigated. Five of the polyphenols (MEPCA, PREGA, MEGA, ECG, and EGCG) prevent NPCuO/H2O2-mediated DNA damage (IC50 values of 7.5-800 µM), three have no effect (PCA, VA, and EC), and two (GA and EGC) result in increased DNA damage. Most polyphenols had similar antioxidant/prooxidant activity in the presence of NPCuO or free copper ions. Electron paramagnetic resonance (EPR) spectroscopy of reactive oxygen species (ROS) generated by NPCuO/H2O2 in the presence of representative polyphenols correlate with results of DNA damage studies: in the presence of NPCuO/H2O2, MEPCA prevents ROS formation, VA has no effect on ROS levels, and EGC increases ROS levels. EPR results with CuO nanoparticles washed to remove dissolved copper in solution (wCuO) in the presence of H2O2/ascorbate suggest that MEPCA prevents ROS formation on the nanoparticle surface in addition to preventing ROS formation from dissolved copper. In mouse fibroblast (L929) cells, combining NPCuO with H2O2 results in significantly greater cytotoxicity than observed for either component alone. After 3 h incubation with MEPCA or MEGA, the viability loss in L929 cells induced by NPCuO/H2O2 challenge was significantly rescued at physiologically relevant polyphenol levels (1 µM). These studies show that polyphenols can protect DNA and inhibit cytotoxicity generated by NPCuO under oxidative stress conditions.

Correlating Quantitative Measurements of Radical Production by Photocatalytic TiO2 with Daphnia magna Toxicity.

Increased use of titanium dioxide (TiO2 ) nanoparticles (NPs) in domestic and industrial applications has increased the risk for adverse environmental outcomes based on an elevated likelihood of organism exposure. Anatase TiO2 NP exposure to ultraviolet A (UV-A) radiation in aquatic environments generates radical oxygen species (ROS), which may ultimately be responsible for increased organism toxicity. We have identified and measured the 2 most relevant ROS species, hydroxyl and superoxide radicals, and described that ROS can be modeled using the highly reactive hydroxyl radical to provide an upper bound for toxicity. The TiO2 NPs were co-exposed to increasing natural organic matter (NOM) amounts (measured as concentration of dissolved organic carbon [DOC]) and simulated-sunlight UV-A intensities. Radical production rate was determined using fluorescence spectroscopy and was positively correlated with increases in TiO2 concentration and UV-A intensity, and negatively correlated with increased DOC concentration. Daphnia magna toxicity was also found to decrease with NOM addition, which is attributed to the decreased radical production rate with increased DOC concentrations. We demonstrate that the rate of ROS production from simulated-sunlight-irradiated TiO2 NPs can be quantified using relatively simple fluorescent techniques. We show that toxicity to TiO2 NP varies greatly with conditions, and that concentration alone is a poor predictor of toxicity. Describing toxicity/hydroxyl radical measurement may be a more accurate way to describe overall risk. We provide a framework for a simple model to describe toxicity/hydroxyl radical. These conclusions demonstrate the importance of considering exposure conditions as a means of risk management during TiO2 NP toxicity testing, waste management, and regulatory decisions. Environ Toxicol Chem 2021;40:1322-1334. © 2021 SETAC.

Stability constant determination of sulfur and selenium amino acids with Cu(II) and Fe(II).

Sulfur- and selenium-containing amino acids are of great biological importance, but their metal-binding properties with biologically-relevant metal ions are not well investigated. Stability constants of the methionine, selenomethionine, methylcysteine, and methylselenocysteine with Cu(II) and Fe(II) were determined by potentiometric titration. Stability constants of Cu(II) with these thio- and selenoether amino acids are in the range of 8.0-8.2 ([CuL]+) and 14.5-14.7 (CuL2) (L = amino acid). Fe(II) interactions with the same thio- and selenoether amino acids are much weaker, with stability constants between 3.5 and 3.8 ([FeL]+) and -4.9 and -5.7 (FeL(OH)). Stability of Fe(II) with penicillamine, a thiol-containing amino acid, is much higher (FeL = 7.48(7) and [FeL]2- = 13.74(2)). For both copper and iron complexes, thio- and selenoether amino acid coordination occurs through the carboxylate and the amine groups as confirmed by infrared spectroscopy, with no stability afforded by thio- or selenoether coordination. The first single-crystal structure of Cu(II) with a selenium-containing amino acid, Cu(SeMet)2, also confirms binding through only the amine and carboxylate groups. The measured Cu(II)-amino-acid stability constants confirm that nearly 100% of the available Cu(II) can be coordinated by these amino acids at pH 7, but very little Fe(II) is bound under these conditions. The relative instability of Fe(II) complexes with thio- and selenoether amino acids is consistent with their inability to prevent metal-mediated oxidative DNA damage. In contrast, the stability constants of these amino acids with Cu(II) weakly correlate to their ability to inhibit DNA damage inhibition.

Fluconazole induces ROS in Cryptococcus neoformans and contributes to DNA damage in vitro.

Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

Selective cation and anion guest binding in host selenazamacrocycles.

We report selenazamacrocycle hosts that are the first system to change guest binding affinity from cation to anion depending upon macrocycle oxidation/reduction. Selective cation (Fe2+) or anion (BF4-) binding occurs with both ions present and under identical reaction conditions. We also report the first macrocyclic complex with a Fe-Se bond.

DNA interactions of non-chelating tinidazole-based coordination compounds and their structural, redox and cytotoxic properties.

Novel tinidazole (tnz) coordination compounds of different geometries were synthesised, whose respective solid-state packing appears to be driven by inter- and intramolecular lone pairπ interactions. The copper(ii) compounds exhibit interesting redox properties originating from both the tnz and the metal ions. These complexes interact with DNA through two distinct ways, namely via electrostatic interactions or/and groove binding, and they can mediate the generation of ROS that damage the biomolecule. Cytotoxic studies revealed an interesting activity of the dinuclear compound [Cu(tnz)2(µ-Cl)Cl]27, which is further more efficient towards cancer cells, compared with normal cells.

Reactive oxygen species generation by copper(II) oxide nanoparticles determined by DNA damage assays and EPR spectroscopy.

Copper(II) oxide nanoparticles (NPCuO) have many industrial applications, but are highly cytotoxic because they generate reactive oxygen species (ROS). It is unknown whether the damaging ROS are generated primarily from copper leached from the nanoparticles, or whether the nanoparticle surface plays a significant role. To address this question, we separated nanoparticles from the supernatant containing dissolved copper, and measured their ability to damage plasmid DNA with addition of hydrogen peroxide, ascorbate, or both. While DNA damage from the supernatant (measured using an electrophoresis assay) can be explained solely by dissolved copper ions, damage by the nanoparticles in the presence of ascorbate is an order of magnitude higher than can be explained by dissolved copper and must, therefore, depend primarily upon the nanoparticle surface. DNA damage is time-dependent, with shorter incubation times resulting in higher EC50 values. Hydroxyl radical (•OH) is the main ROS generated by NPCuO/hydrogen peroxide as determined by EPR measurements; NPCuO/hydrogen peroxide/ascorbate conditions generate ascorbyl, hydroxyl, and superoxide radicals. Thus, NPCuO generate ROS through several mechanisms, likely including Fenton-like and Haber-Weiss reactions from the surface or dissolved copper ions. The same radical species were observed when NPCuO suspensions were replaced with the supernatant containing leached copper, washed NPCuO, or dissolved copper solutions. Overall, NPCuO generate significantly more ROS and DNA damage in the presence of ascorbate than can be explained simply from dissolved copper, and the NPCuO surface must play a large role.

Synthesis, characterization, DFT calculations, and electrochemical comparison of novel iron(ii) complexes with thione and selone ligands.

Thione- and selone-containing compounds and their metal complexes show promise as antioxidants, as antithyroid drugs, and for applications in lasers and blue light-emitting diodes. Although Cu(i/ii), Co(ii), Ag(i), and Zn(ii) coordination to thione and selone ligands has been broadly studied and Fe(ii) plays an important role in oxidative damage, very few iron-thione complexes and no iron-selone complexes are reported. Novel Fe(ii)-containing thione and selone complexes of the formulae FeL2Cl2, [FeL2(CH3CN)2](2+), and [FeL4](2+), and {FeL'Cl2}n, (L = N,N'-dimethylimidazole selone (dmise), and thione (dmit); L' = bis(thioimidazolyl)ethane (ebit) and bis(selenoimidazolyl)ethane (ebis)) have been synthesized and characterized. Structures of Fe(dmise)2Cl2, Fe(dmit)2Cl2, [Fe(dmit)4][BF4]2, [Fe(dmise)4][BF4]2, and {Fe(ebit)Cl2}n were determined by X-ray crystallography. All Fe(ii) centers adopt a distorted tetrahedral coordination geometry with Fe-S distances ranging from 2.339(1) to 2.397(1) Å and F-Se distances ranging from 2.453(1) to 2.514(1) Å. Density functional theory optimized structures of FeL2Cl2, [FeL2(CH3CN)2](2+), and [FeL4](2+) are consistent with experimental results and suggest that thiones and selones are π-donor ligands that coordinate through their zwitterionic resonance structures. Thione and selone coordination to Fe(ii) lowers the Fe(ii/iii) reduction potential, with a greater decrease for Fe(ii)-bound dmise than Fe(ii)-bound dmit. Dmit and dmise ligand-based oxidation potentials also significantly increase upon Fe(ii) binding compared, indicating that bound thione and selone ligands will undergo oxidation prior to Fe(ii). The synthesis of these complexes suggests that iron coordination by thione and selone ligands may occur in vivo and may contribute to the protective antioxidant properties of sulfur and selenium.

Dinuclear copper(I) complexes with N-heterocyclic thione and selone ligands: synthesis, characterization, and electrochemical studies.

The synthesis, characterization, and structures of a series of homoleptic and heteroleptic copper(I) complexes supported by N-heterocyclic chalcogenone ligands is reported herein. The quasi-reversible Cu(II/I) reduction potentials of these copper complexes with monodentate (dmit or dmise) and/or bidentate (Bmm(Me), Bsem(Me), Bme(Me), Bsee(Me)) chalcogenone ligands are highly dependent upon the nature and number of the donor groups and can be tuned over a 470 mV range (-369 to 102 mV). Copper-selone complexes have more negative Cu(II/I) reduction potentials relative to their thione analogs by an average of 137 mV, and increasing the number of methylene units linking the heterocyclic rings in the bidentate ligands results in more negative reduction potentials for their copper complexes. This ability to tune the copper reduction potentials over a wide range has potential applications in synthetic and industrial catalysis as well as the understanding of important biological processes such as electron transfer in blue copper proteins and respiration.

Arsenic and Its Methylated Metabolites Inhibit the Differentiation of Neural Plate Border Specifier Cells.

Exposure to arsenic in food and drinking water has been correlated with adverse developmental outcomes, such as reductions in birth weight and neurological deficits. Additionally, studies have shown that arsenic suppresses sensory neuron formation and skeletal muscle myogenesis, although the reason why arsenic targets both of these cell types in unclear. Thus, P19 mouse embryonic stem cells were used to investigate the mechanisms by which arsenic could inhibit cellular differentiation. P19 cells were exposed to 0, 0.1, or 0.5 µM sodium arsenite and induced to form embryoid bodies over a period of 5 days. The expression of transcription factors necessary to form neural plate border specifier (NPBS) cells, neural crest cells and their progenitors, and myocytes and their progenitors were examined. Early during differentiation, arsenic significantly reduced the transcript and protein expression of Msx1 and Pax3, both needed for NPBS cell formation. Arsenic also significantly reduced the protein expression of Sox 10, needed for neural crest progenitor cell production, by 31-50%, and downregulated the protein and mRNA levels of NeuroD1, needed for neural crest cell differentiation, in a time- and dose-dependent manner. While the overall protein expression of transcription factors in the skeletal muscle lineage was not changed, arsenic did alter their nuclear localization. MyoD nuclear translocation was significantly reduced on days 2-5 between 15 and 70%. At a 10-fold lower concentration, monomethylarsonous acid (MMA III) appeared to be just as potent as inorganic arsenic at reducing the mRNA levels Pax3 (79% vs84%), Sox10 (49% vs 65%), and Msx1 (56% vs 56%). Dimethylarsinous acid (DMA III) also reduced protein and transcript expression, but the changes were less dramatic than those with MMA or arsenite. All three arsenic species reduced the nuclear localization of MyoD and NeuroD1 in a similar manner. The early changes in the differentiation of neural plate border specifier cells may provide a mechanism for arsenic to suppress both neurogenesis and myogenesis.

Sulfur and selenium antioxidants: challenging radical scavenging mechanisms and developing structure-activity relationships based on metal binding.

Because sulfur and selenium antioxidants can prevent oxidative damage, numerous animal and clinical trials have investigated the ability of these compounds to prevent the oxidative stress that is an underlying cause of cardiovascular disease, Alzheimer's disease, and cancer, among others. One of the most common sources of oxidative damage is metal-generated hydroxyl radical; however, very little research has focused on determining the metal-binding abilities and structural attributes that affect oxidative damage prevention by sulfur and selenium compounds. In this review, we describe our ongoing investigations into sulfur and selenium antioxidant prevention of iron- and copper-mediated oxidative DNA damage. We determined that many sulfur and selenium compounds inhibit Cu(I)-mediated DNA damage and that DNA damage prevention varies dramatically when Fe(II) is used in place of Cu(I) to generate hydroxyl radical. Oxidation potentials of the sulfur or selenium compounds do not correlate with their ability to prevent DNA damage, highlighting the importance of metal coordination rather than reactive oxygen species scavenging as an antioxidant mechanism. Additional gel electrophoresis, mass spectrometry, and UV-visible studies confirmed sulfur and selenium antioxidant binding to Cu(I) and Fe(II). Ultimately, our studies established that both the hydroxyl-radical-generating metal ion and the chemical environment of the sulfur or selenium significantly affect DNA damage prevention and that metal coordination is an essential mechanism for these antioxidants.

Redox-active and DNA-binding coordination complexes of clotrimazole.

DNA interactions of anticancer mononuclear Cu(2+), Co(2+), Zn(2+), and Ni(2+) complexes with the biologically active ligand clotrimazole (clotri) are reported. To fully characterize DNA binding modes for these complexes of the formulae [M(clotri)2Cl2]·nH2O (1-4), [M(clotri)2Br2]·nH2O (5,6), [M(clotri)3NO3]NO3·nH2O (9), and [M(clotri)3(NO3)2] (10), circular dichroism (CD) and linear dichroism (LD) spectroscopy, UV melting experiments, atomic force microscopy (AFM) and ethidium bromide (EtBr) displacement methods were used. Results indicate mixed electrostatic interactions, possibly through groove binding, that result in accretion and coiling of DNA. Electrochemical studies indicate that the Cu(2+) complex 9 readily reduces to the reactive-oxygen-species-generating Cu(+), which oxidatively damages DNA. There is a subtle correlation between log P values, calculated electrostatic potentials, and cytotoxicity of the complexes. The extent of cell-nucleus DNA-metal adduct formation in the HeLa cervix-uterine carcinoma cell line does not necessarily correlate with cytotoxicity, indicating that the nature of DNA lesions may be crucial to activity.

Oxidation of biologically relevant chalcogenones and their Cu(I) complexes: insight into selenium and sulfur antioxidant activity.

Hydroxyl radical damage to DNA causes disease, and sulfur and selenium antioxidant coordination to hydroxyl-radical-generating Cu(+) is one mechanism for their observed DNA damage prevention. To determine how copper binding results in antioxidant activity, biologically relevant selone and thione ligands and Cu(+) complexes of the formula [Tpm*Cu(L)](+) [Tpm* = tris(3,5-dimethylpyrazolyl)methane; L = N,N'-dimethylimidazole selone or thione] were treated with H2O2 and the products analyzed by (1)H, (13)C{(1)H}, and (77)Se{(1)H} NMR spectroscopy, mass spectrometry, and X-ray crystallography. Upon H2O2 treatment, selone and thione binding to Cu(+) prevents oxidation to Cu(2+); instead, the chalcogenone ligand is oxidized. Thus, copper coordination by sulfur and selenium compounds can provide targeted sacrificial antioxidant activity.

Prevention of iron- and copper-mediated DNA damage by catecholamine and amino acid neurotransmitters, L-DOPA, and curcumin: metal binding as a general antioxidant mechanism.

Concentrations of labile iron and copper are elevated in patients with neurological disorders, causing interest in metal-neurotransmitter interactions. Catecholamine (dopamine, epinephrine, and norepinephrine) and amino acid (glycine, glutamate, and 4-aminobutyrate) neurotransmitters are antioxidants also known to bind metal ions. To investigate the role of metal binding as an antioxidant mechanism for these neurotransmitters, L-dihydroxyphenylalanine (L-DOPA), and curcumin, their abilities to prevent iron- and copper-mediated DNA damage were quantified, cyclic voltammetry was used to determine the relationship between their redox potentials and DNA damage prevention, and UV-vis studies were conducted to determine iron and copper binding as well as iron oxidation rates. In contrast to amino acid neurotransmitters, catecholamine neurotransmitters, L-DOPA, and curcumin prevent significant iron-mediated DNA damage (IC(50) values of 3.2 to 18 µM) and are electrochemically active. However, glycine and glutamate are more effective at preventing copper-mediated DNA damage (IC(50) values of 35 and 12.9 µM, respectively) than L-DOPA, the only catecholamine to prevent this damage (IC(50) = 73 µM). This metal-mediated DNA damage prevention is directly related to the metal-binding behaviour of these compounds. When bound to iron or copper, the catecholamines, amino acids, and curcumin significantly shift iron oxidation potentials and stabilize Fe(3+) over Fe(2+) and Cu(2+) over Cu(+), a factor that may prevent metal redox cycling in vivo. These results highlight the disparate antioxidant activities of neurotransmitters, drugs, and supplements and highlight the importance of considering metal binding when identifying antioxidants to treat and prevent neurodegenerative disorders.

Investigating the copper coordination, electrochemistry, and Cu(II) reduction kinetics of biologically relevant selone and thione compounds.

Selenium- and sulfur-containing compounds can act as antioxidants by binding copper. To determine how this copper coordination results in the observed antioxidant activity, biologically relevant Cu(+) and Cu(2+) complexes with the formulae [Cu(dmit)(3)](+) (3), [Cu(dmise)(4)](+) (4a), and [Tpm(iPr)Cu(MISeox)](2+) (6) (dmise = N,N'-dimethylimidazole selone; dmit = N,N'-dimethylimidazole thione; MISeox = bis(1-methylimidazolyl)diselenide; Tpm(iPr) = tris(1,3-diisopropylpyrazolyl)methane) were synthesized, characterized, and their structures determined by single-crystal X-ray crystallography. In addition, kinetic studies using UV-vis spectroscopy indicate that dmise reduces Cu(2+) to Cu(+) three times faster than dmit. Coordination of dmise and MISeox to copper also results in more negative Cu(2+/+) reduction potentials (-373 mV and -503 mV) compared to dmit (-217 mV). These results highlight the different complexation behaviors and reactivities of analogous selone- and thione-containing compounds, traits which likely influence their antioxidant activity.

Interactions of Cu(I) with selenium-containing amino acids determined by NMR, XAS, and DFT studies.

Cu(I) coordination by organoselenium compounds was recently reported as a mechanism for their prevention of copper-mediated DNA damage. To establish whether direct Se-Cu coordination may be involved in selenium antioxidant activity, Cu(I) coordination of the selenoamino acids methyl-Se-cysteine (MeSeCys) and selenomethionine (SeMet) was investigated. NMR results in D(2)O indicate that Cu(I) binds to the Se atom of both MeSeCys and SeMet as well as the carboxylic acid oxygen atom(s) or amine nitrogen atoms. X-ray absorption spectroscopy (XAS) and density functional theory (DFT) results confirm Se-Cu coordination, with the identification of a 2.4 Å Se-Cu vector in both the Se- and Cu-EXAFS data. XAS studies also show Cu(I) in an unusual three-coordinate environment with the additional two ligands arising from O/N (2.0 Å). DFT models of 1:1 Cu-selenoamino acid complexes suggest that both selenoamino acids coordinate Cu(I) through the selenium and amino groups, with the third ligand assumed to be water. These compounds represent the first structurally characterized copper(I) complexes with sulfur- or selenium-containing amino acids.

Metal retention in human transferrin: consequences of solvent composition in analytical sample preparation methods.

The analysis of metal-binding proteins requires careful sample manipulation to ensure that the metal-protein complex remains in its native state and the metal retention is preserved during sample preparation or analysis. Chemical analysis for the metal content in proteins typically involves some type of liquid chromatography/electrophoresis separation step coupled with an atomic (i.e., inductively coupled plasma-optical emission spectroscopy or -mass spectrometry) or molecular (i.e., electrospray ionization-mass spectrometry) analysis step that requires altered-solvent introduction techniques. UV-VIS absorbance is employed here to monitor the iron content in human holo-transferrin (Tf) under various solvent conditions, changing polarity, pH, ionic strength, and the ionic and hydrophobic environment of the protein. Iron loading percentages (i.e. 100% loading equates to 2 Fe(3+):1 Tf) were quantitatively determined to evaluate the effect of solvent composition on the retention of Fe(3+) in Tf. Maximum retention of Fe(3+) was found in buffered (20 mM Tris) solutions (96 ± 1%). Exposure to organic solvents and deionized H(2)O caused release of ~23-36% of the Fe(3+) from the binding pocket(s) at physiological pH (7.4). Salt concentrations similar to separation conditions used for ion exchange had little to no effect on Fe(3+) retention in holo-Tf. Unsurprisingly, changes in ionic strength caused by additions of guanidine HCl (0-10 M) to holo-Tf resulted in unfolding of the protein and loss of Fe(3+) from Tf; however, denaturing and metal loss was found not to be an instantaneous process for additions of 1-5 M guanidinium to Tf. In contrast, complete denaturing and loss of Fe(3+) was instantaneous with ≥6 M additions of guanidinium, and denaturing and loss of iron from Tf occurred in parallel proportions. Changes to the hydrophobicity of Tf (via addition of 0-14 M urea) had less effect on denaturing and release of Fe(3+) from the Tf binding pocket compared to changes in ionic strength.

Iron binding of 3-hydroxychromone, 5-hydroxychromone, and sulfonated morin: Implications for the antioxidant activity of flavonols with competing metal binding sites.

The iron binding properties and antioxidant activities of compounds with hydroxy-keto binding sites, 3-hydroxychromone, 5-hydroxychromone, and sulfonated morin were investigated. For these compounds, prevention of iron-mediated DNA damage and kinetics of Fe(II) oxidation were studied in aqueous solutions close to physiological pH (pH 6). 3-Hydroxychromone and sulfonated morin inhibit iron-mediated DNA damage at lower concentrations than 5-hydroxychromone. All three compounds bind iron, but 3-hydroxychromone and sulfonated morin promote Fe(II) oxidation much faster than 5-hydroxychromone. These results indicate that DNA damage inhibition by flavonols with competing hydroxy-keto binding sites is primarily due to iron binding at the 3-hydroxy-keto site. Iron oxidation rate also plays a significant role in antioxidant activity. In addition to iron binding and oxidation, reactive oxygen species scavenging occurs at high concentrations for the hydroxychromones. This study emphasizes the importance of iron binding in polyphenol antioxidant behavior and provides insights into the iron binding antioxidant activity of similar flavonols such as quercetin and myricetin.

Competitive binding of Fe3+, Cr3+, and Ni2+ to transferrin.

Competitive binding of Fe(3+), Cr(3+), and Ni(2+) to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M(n+) to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe(3+) for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 µM) or nickel (10.3 µM) to load completely into Tf. Low Fe(3+) uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni(2+) loading into Tf. Competitive binding kinetic studies were performed with Fe(3+), Cr(3+), and Ni(2+) to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe(3+) loading increased in the presence of nickel or chromium, with maximal Fe(3+) loading into Tf in all cases reaching approximately 24%. Addition of Cr(3+) to 50% preloaded Fe(3+)-Tf showed that excess chromium (15.0 µM) displaced roughly 13% of Fe(3+) from Tf, resulting in 7.6 ± 1.3% Cr(3+) loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.