Reaction of N-Acetylcysteine with Cu2+: Appearance of Intermediates with High Free Radical Scavenging Activity: Implications for Anti-/Pro-Oxidant Properties of Thiols.

We studied the kinetics of the reaction of N-acetyl-l-cysteine (NAC or RSH) with cupric ions at an equimolar ratio of the reactants in aqueous acid solution (pH 1.4−2) using UV/Vis absorption and circular dichroism (CD) spectroscopies. Cu2+ showed a strong catalytic effect on the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical (ABTSr) consumption and autoxidation of NAC. Difference spectra revealed the formation of intermediates with absorption maxima at 233 and 302 nm (ε302/Cu > 8 × 103 M−1 cm−1) and two positive Cotton effects centered at 284 and 302 nm. These intermediates accumulate during the first, O2-independent, phase of the NAC autoxidation. The autocatalytic production of another chiral intermediate, characterized by two positive Cotton effects at 280 and 333 nm and an intense negative one at 305 nm, was observed in the second reaction phase. The intermediates are rapidly oxidized by added ABTSr; otherwise, they are stable for hours in the reaction solution, undergoing a slow pH- and O2-dependent photosensitive decay. The kinetic and spectral data are consistent with proposed structures of the intermediates as disulfide-bridged dicopper(I) complexes of types cis-/trans-CuI2(RS)2(RSSR) and CuI2(RSSR)2. The electronic transitions observed in the UV/Vis and CD spectra are tentatively attributed to Cu(I) → disulfide charge transfer with an interaction of the transition dipole moments (exciton coupling). The catalytic activity of the intermediates as potential O2 activators via Cu(II) peroxo-complexes is discussed. A mechanism for autocatalytic oxidation of Cu(I)−thiolates promoted by a growing electronically coupled −[CuI2(RSSR)]n− polymer is suggested. The obtained results are in line with other reported observations regarding copper-catalyzed autoxidation of thiols and provide new insight into these complicated, not yet fully understood systems. The proposed hypotheses point to the importance of the Cu(I)−disulfide interaction, which may have a profound impact on biological systems.

Unusual Chemistry in an Uncatalyzed Bromate-Aniline Oscillator: Ring-Contraction Oxidation of Aniline with Pulsative CO2 Production.

The bromate-aniline oscillatory reaction was discovered 4 decades ago, but neither the detailed mechanism nor the key products or intermediates of the reaction were described. We report herein a detailed study of this reaction, which yielded new insights. We found that oscillatory oxidation of aniline by acidic bromate proceeds, to a significant extent, via a novel reaction pathway with the periodic release of carbon dioxide. Several products were isolated, and their structures, not described so far, were justified on the basis of MS and NMR. One of the main products of the reaction associated with the CO2 release route can be assigned to 2,2-dibromo-5-(phenylimino)cyclopent-3-en-1-one. A number of known compounds produced in the studied reaction, including unexpected brominated 1-phenylpyrroles and 1-phenylmaleimides, were identified by comparison with standards. A mechanism is suggested to explain the appearance of the detected compounds, based on coupling of the anilino radical with the produced 1,4-benzoquinone. We assume that the radical adduct reacts with bromine to form a cyclopropanone intermediate that undergoes a Favorskii-type rearrangement. Further oxidation and bromination steps including decarboxylation lead to the found brominated phenyliminocyclopentenones. The detected derivatives of 1-phenylpyrrole could be produced by a one-electron oxidation of a proposed intermediate 2-phenylamino-5-bromocyclopenta-1,3-dien-1-ol followed by ß-scission with the abstraction of carbon monoxide. Such a mechanism is known from the combustion chemistry of cyclopentadiene. The proposed mechanism of this reaction provides a framework for understanding the observed oscillatory kinetics.

Kinetics of ABTS derived radical cation scavenging by bucillamine, cysteine, and glutathione. Catalytic effect of Cu(2+) ions.

Kinetics of reduction of the stable radical cation derived from 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) in reaction with the anti-rheumatic drug bucillamine (BUC) and two reference thiols - cysteine (Cys) and glutathione (GSH) was followed spectrophotometrically in acidic medium with 10-fold molar excess of the reductant. Decay of the radical is governed by pseudo-first order kinetics with small deviation in the case of GSH. H(+) ions displayed second order inhibition of the reaction with all the studied compounds. The reaction of BUC exhibits zero order kinetics to the radical at lower acidities with a moderate acceleration of the reaction rate by H(+) ions. A significant catalytic effect of Cu(2+) ions on the reactions with all the reductants was observed. The most sensitive to Cu(2+)-catalysis was the reaction of BUC with the radical cation, while Cu(2+) ions showed much lower effect on the reaction with GSH. The presence of EDTA strongly inhibited the reactions and equalized the reaction rates for all the reductants. A Cu(I) selective chelator bathocuproine disulfonate reduced the reaction rate with Cys, but accelerated the reaction with BUC at the lower acidities. The experimental results were rationalized in the framework of the mechanism of reductive chelation. The conclusions may have important consequences for interpretation of antioxidant capacity assays, such as TEAC, utilizing the ABTS derived radical cation.

Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes.

The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05-0.1-µM) calcium concentrations but only slightly at high (0.2-0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases.

Electrodiffusion kinetics of ionic transport in a simple membrane channel.

We employ numerical techniques for solving time-dependent full Poisson-Nernst-Planck (PNP) equations in 2D to analyze transient behavior of a simple ion channel subject to a sudden electric potential jump across the membrane (voltage clamp). Calculated spatiotemporal profiles of the ionic concentrations and electric potential show that two principal exponential processes can be distinguished in the electrodiffusion kinetics, in agreement with original Planck's predictions. The initial fast process corresponds to the dielectric relaxation, while the steady state is approached in a second slower exponential process attributed to the nonlinear ionic redistribution. Effects of the model parameters such as the channel length, height of the potential step, boundary concentrations, permittivity of the channel interior, and ionic mobilities on electrodiffusion kinetics are studied. Numerical solutions are used to determine spatiotemporal profiles of the electric field, ionic fluxes, and both the conductive and displacement currents. We demonstrate that the displacement current is a significant transient component of the total electric current through the channel. The presented results provide additional information about the classical voltage-clamp problem and offer further physical insights into the mechanism of electrodiffusion. The used numerical approach can be readily extended to multi-ionic models with a more structured domain geometry in 2D or 3D, and it is directly applicable to other systems, such as synthetic nanopores, nanofluidic channels, and nanopipettes.

Locking of Turing patterns in the chlorine dioxide-iodine-malonic acid reaction with one-dimensional spatial periodic forcing.

We use the photosensitive chlorine dioxide-iodine-malonic acid reaction-diffusion system to study wavenumber locking of Turing patterns with spatial periodic forcing. Wavenumber-locked stripe patterns are the typical resonant structures that labyrinthine patterns exhibit in response to one-dimensional forcing by illumination when images of stripes are projected on a working medium. Our experimental results reveal that segmented oblique, hexagonal and rectangular patterns can also be obtained. However, these two-dimensional resonant structures only develop in a relatively narrow range of forcing parameters, where the unforced stripe pattern is in close proximity to the domain of hexagonal patterns. Numerical simulations based on a model that incorporates the forcing by illumination using an additive term reproduce well the experimental observations. These findings confirm that additive one-dimensional forcing can generate a two-dimensional resonant response. However, such a response is considerably less robust than the effect of multiplicative forcing.

Preparation of D-galactofuranosyl nitromethanes: a revision and a new approach.

Sodium methoxide-promoted methanolysis of 7-deoxy-7-nitro-L-glycero-L-galacto-heptitol peracetate rapidly and nearly quantitatively accumulates 7-deoxy-6-O-methyl-7-nitro-L-glycero-L-galacto-heptitol. The prolonged treatment then provides 76% of D-galactofuranosyl nitromethanes and finally results in the equilibrium of 77% of ß-D-galactopyranosyl nitromethane and 7-9% of three other tautomeric D-galactosyl nitromethanes. Thermal treatment of 7-deoxy-7-nitro-L-glycero-L-galacto-heptitol in boiling water peaks at a 58% content of D-galactofuranosyl nitromethanes and ends in a similar equilibrium mixture of four D-galactosyl tautomers. The relevant kinetic parameters of the latter transformation are determined by a curve fitting using the nonlinear least-squares Marquardt-Levenberg algorithm.

Frequency and release flux of calcium sparks in rat cardiac myocytes: a relation to RYR gating.

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca(2+) releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca(2+) and Mg(2+) ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg(2+). Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg(4)RYR and CaMg(3)RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca(2)Mg(2)RYR and Ca(3)MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg(2+) unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.

Spatial and temporal Ca2+, Mg2+, and ATP2- dynamics in cardiac dyads during calcium release.

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP(2-) with Ca(2+) and Mg(2+) ions on spatiotemporal concentration profiles of Ca(2+), Mg(2+), and ATP(2-) in the dyadic cleft during Ca(2+) release. The model revealed that Ca(2+) concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca(2+)] in the dyadic space reached values similar to estimates of luminal [Ca(2+)] in approximately 1 ms, suggesting that during calcium release the Ca(2+) gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca(2+) bound to ATP(2-), and thus substantially decreased ATP(2-) concentration in the dyadic space. The released Ca(2+) could also replace Mg(2+) in its complex with ATP(2-) during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca(2+), Mg(2+), and ATP(2-) might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.