Dean E. Wilcox

Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin

Abstract. The thermodynamics of Cu(II) and Ni(II) binding to bovine serum albumin (BSA) have been studied by isothermal titration calorimetry (ITC). The Cu(II) binding affinity of the N-terminal protein site is quantitatively higher when the single free thiol, Cys-34, is reduced (mercaptalbumin), compared to when it is oxidized or derivatized with N-ethylmaleimide. This increased affinity is due predominantly to entropic factors. At higher pH (~9), when the protein is in the basic (B) form, a second Cu(II) binds with high affinity to albumin with reduced Cys-34. The Cu(II) coordination has been characterized by UV-vis absorption, CD, and EPR spectroscopy, and the spectral data are consistent with thiolate coordination to a tetragonal Cu(II), indicating this is a type 2 copper site with thiolate ligation. Nickel(II) binding to the N-terminal site of BSA is also modulated by the redox/ligation state of Cys-34, with higher Ni(II) affinity for mercaptalbumin, the predominant circulating form of the protein.

Nitric oxide hemoglobin in mice and rats in endotoxic shock

Mice given ip bacterial endotoxin (LPS) at 10 mg/kg showed a statistically significant decrease in plasma glucose and an increase in hematocrit at 2 h after injection. Glucose was still decreased at 4 h, but the hematocrit had returned to control values. Nitrosylated hemoglobin (HbNO) was detected at 3, but not at 2 h. By 4 h it had increased 5-fold. When N-monomethylarginine (NMMA) at 100 mg/kg, ip was given 2 h after LPS in mice, the HbNO concentration at 4 h was significantly reduced, but the hypoglycemia was worsened because NMMA itself produced a significant hypoglycemia. Rats given iv LPS, 20 mg/kg, showed a fleeting, transient rise in mean arterial pressure (MAP) lasting only a few min. Thereafter, the MAP tended to drift slowly downward over 4 h, but when the MAP at 30 min intervals was compared to the pre-LPS MAP, there were no significant differences. Plasma glucose in unanesthetized rats was significantly elevated at 1 h, back to control at 2 h, and significantly decreased at 3 h. HbNO was detected as early as 1 h after injection. By 2 h the HbNO concentrations exceeded the highest levels found in mice, and they were still increasing as late as 5 h after injection. Unanesthetized rats showed toxic signs and 3/12 rats died within 4 hours of LPS administration. These results are consistent with a model for endotoxic shock in which LPS stimulates an inducible pathway for NO synthesis.

Red blood cells generate nitric oxide from directly acting, nitrogenous vasodilators

Human red blood cells (RBC) incubated under nitrogen with methylene blue and glucose at physiological temperature and pH can be used to test for the biotransformation of nitrogenous vasodilators to nitric oxide (NO). The NO generated was trapped as nitrosylated heme by reduced subunits (hemeII) on various hemoglobin valency species and quantified by electron paramagnetic resonance spectroscopy. It was possible to separate the various valency species of hemoglobin present in the mixture as (alpha 2 + beta 2)2, (alpha 2 + beta 3+)2, (alpha 3 + beta 2+)2, or (alpha 3 + beta 3+)2 by isoelectric focusing (IEF) unless cyanide (from nitroprusside) or azide was present in the mixture. These anions bind tenaciously to oxidized subunits (hemeIII) and prevent the separation of the various species by IEF. The fully oxidized tetramer, (alpha 3 + beta 3+)2, does not bind NO, but the other three species have hemeII units which can be nitrosylated. In the absence of cyanide or azide the valency species could be separated by IEF, and it was possible to quantify the degree of nitrosylation on each individual species. The various agents tested (nitrite, glyceryl trinitrate, hydroxylamine, hydralazine, nitroprusside, and azide) produced different patterns of valency species and degrees of nitrosylation of hemeII. When hemeIII ligands were present or in cases of very low yields, it was still possible to quantify the total concentration of NO-hemeII in the mixture. Thus, the method could still be used to test for NO formation. All of the so-called NO vasodilators tested yielded detectable amounts of NO in the system.

Application of isothermal titration calorimetry in bioinorganic chemistry

The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔCp°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (KITC, ΔHITC). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal–buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.

Oxidation of Zinc-Binding Cysteine Residues in Transcription Factor Proteins

Recent results on the oxidation of cysteine residues that bind zinc in transcription factors and their analogous peptides and in related proteins and model systems are reviewed. Two classes of oxidants, the transition metals and dioxygen, hydrogen peroxide, and related species, are considered, and the role of metal ions in suppressing or enhancing Cys oxidation is a major focus. Cysteines in the zinc-bound structures of transcription factors are less susceptible to oxidation than in the metal-free form, and this appears to correlate with reduced accessibility of the thiolates to oxidants. Substitution of other metal ions for Zn(II) increases the rate of Cys oxidation, apparently through increased oxidant accessibility. Reactions that result in reversible or irreversible oxidation of these zinc-binding cysteines under biological conditions are identified in the context of deleterious implications for gene expression.

Toxicology of Selected Nitric Oxide-Donating Xenobiotics, with Particular Reference to Azide

Nitric oxide (NO) has been discovered recently to be a ubiquitous, endogenous mediator, which is responsible for a variety of normal physiological functions. However, NO also has been implicated in several pathophysiological processes. For example, the pulmonary toxicity of various nitrogen oxides, including NO, found in photochemical smog has been studied for decades; endogenous NO also is associated with bleomycin-induced lung damage, as well as other adverse effects. Recently, a variety of xenobiotics have been shown to owe their biological activity in vivo to their biotransformation to NO. Thus, the therapeutic vasodilatation produced by drugs such as nitroglycerin and sodium nitroprusside is now believed to result from their release of NO, which then mimics the effects of endogenously synthesized NO. The toxic effects of NO prodrugs are, therefore, a matter of concern, especially the extent to which, if any, NO contributes to their toxicity. As reviewed here, NO does not appear to contribute importantly to the toxicity of the NO donors nitrite, hydroxylamine, or nitroprusside. However, it is by no means clear whether or not the NO generated in vivo from sodium azide contributes in a major way to its toxicity. Azide is almost as acutely toxic as cyanide, with which it shares a number of biological effects; yet, azide also has certain cardiovascular actions in common with nitrite. Unlike either cyanide or nitrite, some evidence suggests a tendency for azide to produce low-grade cumulative toxicity. In laboratory animals, azide frequently produces nonasphyxial convulsions, whereas most human deaths appear to be the result of cardiovascular collapse. Neither of these azide-induced syndromes appears to be due to the inhibition of cytochrome c oxidase. Azide is widely used as a preservative in aqueous laboratory reagents and as the propellant in automobile air bags and aircraft escape chutes. Both of these inflable systems are generally safe, and will prevent untold numbers of injuries and deaths. However, to protect workers who handle these devices and others who may come into contact with the sodium azide propellant in these systems, our rudimentary knowledge of azide toxicity needs to be expanded.

Thermodynamics of Zn2+ binding to Cys2His2 and Cys2HisCys zinc fingers and a Cys4 transcription factor site.

The thermodynamics of Zn(2+) binding to three peptides corresponding to naturally occurring Zn-binding sequences in transcription factors have been quantified with isothermal titration calorimetry (ITC). These peptides, the third zinc finger of Sp1 (Sp1-3), the second zinc finger of myelin transcription factor 1 (MyT1-2), and the second Zn-binding sequence of the DNA-binding domain of glucocorticoid receptor (GR-2), bind Zn(2+) with Cys(2)His(2), Cys(2)HisCys, and Cys(4) coordination, respectively. Circular dichroism confirms that Sp1-3 and MyT1-2 have considerable and negligible Zn-stabilized secondary structure, respectively, and indicate only a small amount for GR-2. The pK(a)s of the Sp1-3 cysteines and histidines were determined by NMR and used to estimate the number of protons displaced by Zn(2+) at pH 7.4. ITC was also used to determine this number, and the two methods agree. Subtraction of buffer contributions to the calorimetric data reveals that all three peptides have a similar affinity for Zn(2+), which has equal enthalpy and entropy components for Sp1-3 but is more enthalpically disfavored and entropically favored with increasing Cys ligands. The resulting enthalpy-entropy compensation originates from the Zn-Cys coordination, as subtraction of the cysteine deprotonation enthalpy results in a similar Zn(2+)-binding enthalpy for all three peptides, and the binding entropy tracks with the number of displaced protons. Metal and protein components of the binding enthalpy and entropy have been estimated. While dominated by Zn(2+) coordination to the cysteines and histidines, other residues in the sequence affect the protein contributions that modulate the stability of these motifs.

Dosimetry based on EPR spectral analysis of fingernail clippings.

Exposure of fingernails and toenails to ionizing radiation creates radicals that are stable over a relatively long period (days to weeks) and characterized by an isotropic EPR signal at g = 2.003 (so-called radiation-induced signal, RIS). This signal in readily obtained fingernail parings has the potential to be used in screening a population for exposure to radiation and determining individual dose to guide medical treatment. However, the mechanical harvesting of fingernail parings also creates radicals, and their EPR signals (so-called mechanically-induced signals, MIS) overlap the g ∼2.0 region, interfering with efforts to quantify the RIS and, therefore, the radiation dose. Careful analysis of the time evolution and power-dependence of the EPR spectra of freshly cut fingernail parings has now resolved the MIS into three major components, including one that is described for the first time. It dominates the MIS soon after cutting, but decays within the first hour and consists of a unique doublet that can be resolved from the RIS. The MIS obtained within the first few minutes after cutting is consistent among fingernail samples and provides an opportunity to achieve the two important dosimetry objectives. First, perturbation of the initial MIS by the presence of RIS in fingernails that have received a threshold dose of radiation leads to spectral signatures that can be used for rapid screening. Second, decomposition of the EPR spectra from irradiated fingernails into MIS and RIS components can be used to isolate and thus quantify the RIS for determining individual exposure dose.

Generation of valency hybrids and nitrosylated species of hemoglobin in mice by nitric oxide vasodilators.

The hemoglobin fraction of blood samples from C57BL/6 male mice was purified by column chromatography and subjected to isolectric focusing (IEF) across a pH gradient. Densitometric scanning of the IEF gel showed the presence of a single peak corresponding to the fully reduced tetramer, H, or (alpha 2+ beta 2+)2. Twenty minutes after an ip injection of 1.1 mmol/kg NaNO2 blood samples treated the same way showed four peaks corresponding to the species: H = 43%, X or (alpha 2+ beta 3+)2 = 10%, Y or (alpha 3+ beta 2+)2 = 33%, and M or (alpha 3+ beta 3+)2 = 14%. In contrast blood samples from control CD-1 male mice showed the presence of three IEF distinct peaks which were all believed to be H valency forms, and six distinct peaks were seen after treatment in vivo with NaNO2. Thus, the C57BL/6 mice yield patterns similar to those observed after in vitro treatment of human red cells with NaNO2 (H. Kruszyna, R. Kruszyna, R. P. Smith, and D. E. Wilcox, 1987b, Toxicol. Appl. Pharmacol. 91, 429-438), and the CD-1 mice are a much less satisfactory model. The appearance and disappearance of the species X, Y, and M over time after ip injection of 1.1 mmol/kg NaNO2 or hydroxylamine HCl were followed in C57BL/6 mice by the technique of IEF. In each case the patterns were consistent with previously established patterns for the respective methemoglobinemias as determined by absorption spectrophotometry, and they were consistent with the suggestion that two pathways exist for the oxidation of H and for the reduction of M which proceed through X and Y, respectively. By using electron paramagnetic resonance (EPR) spectroscopy, we were also able to follow with time the concentration of nitrosylated heme (NO-heme) on reduced subunits in both mouse strains. The peak for the NO-heme coincided in time with the peak methemoglobinemia as determined by either IEF or absorption spectrophotometry. EPR was also used to determine NO-heme in CD-1 mice after injection of a series of NO-vasodilators with and without methylene blue (MB). Low, but clearly detectable amounts of NO-heme were found in the blood of animals given all xenobiotics tested including NaNO2, hydroxylamine HCl, glyceryl trinitrate, hydralazine, sodium nitroprusside, and sodium azide. MB has little effect on the response, and no NO-heme could be detected in control mice.(ABSTRACT TRUNCATED AT 400 WORDS)

ELECTRON PARAMAGNETIC RESONANCE DOSIMETRY FOR A LARGE-SCALE RADIATION INCIDENT

Abstract With possibilities for radiation terrorism and intensified concerns about nuclear accidents since the recent Fukushima Daiichi event, the potential exposure of large numbers of individuals to radiation that could lead to acute clinical effects has become a major concern. For the medical community to cope with such an event and avoid overwhelming the medical care system, it is essential to identify not only individuals who have received clinically significant exposures and need medical intervention but also those who do not need treatment. The ability of electron paramagnetic resonance to measure radiation-induced paramagnetic species, which persist in certain tissues (e.g., teeth, fingernails, toenails, bone, and hair), has led to this technique becoming a prominent method for screening significantly exposed individuals. Although the technical requirements needed to develop this method for effective application in a radiation event are daunting, remarkable progress has been made. In collaboration with General Electric and through funding committed by the Biomedical Advanced Research and Development Authority, electron paramagnetic resonance tooth dosimetry of the upper incisors is being developed to become a Food and Drug Administration-approved and manufacturable device designed to carry out triage for a threshold dose of 2 Gy. Significant progress has also been made in the development of electron paramagnetic resonance nail dosimetry based on measurements of nails in situ under point-of-care conditions, and in the near future this may become a second field-ready technique. Based on recent progress in measurements of nail clippings, it is anticipated that this technique may be implementable at remotely located laboratories to provide additional information when the measurements of dose on-site need to be supplemented. The authors conclude that electron paramagnetic resonance dosimetry is likely to be a useful part of triage for a large-scale radiation incident.