Physicochemical properties of chitin-melanin and melanoprotein complexes from bee corpses

A technology for processing bee corpses and obtaining chitin-melanin and melanoprotein complexes has been developed. The obtained complexes of biopolymers were studied by the methods of absorption spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, thermogravimetry, and differential scanning calorimetry. The elemental composition of preparations was characterized. It was shown that the properties of the melanin-containing products of the processing of bee corpses are typical of chitin and melanin of animal origin. The results of EPR spectroscopy and thermal analysis are indicative of the diversity and structural complexity of the obtained products.     

Physicochemical properties of chitin-melanin and melanoprotein complexes from bee corpses

A technology for processing bee corpses and obtaining chitin-melanin and melanoprotein complexes has been developed. The obtained complexes of biopolymers were studied by the methods of absorption spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, thermogravimetry, and differential scanning calorimetry. The elemental composition of preparations was characterized. It was shown that the properties of the melanin-containing products of the processing of bee corpses are typical of chitin and melanin of animal origin. The results of EPR spectroscopy and thermal analysis are indicative of the diversity and structural complexity of the obtained products.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Measurement of oxygen concentrations in the intact beating heart using electron paramagnetic resonance spectroscopy: A technique for measuring oxygen concentrationsin situ

Electron paramagnetic resonance (EPR) spectroscopy can be applied to measure oxygen concentrations in cells and tissues. Oxygen is paramagnetic, and thus it interacts with a free radical label resulting in a broadening of the observed linewidth. Recently we have developed instrumentation in order to enable the performance of EPR spectroscopy and EPR oximetry in the intact beating heart. This spectrometer consists of 1–2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed lumped circuit resonator. This technique is applied to measure the kinetics of the uptake and clearance of different free radical labels. It is demonstrated that this technique can be used to noninvasively measure tissue oxygen concentration. In addition, rapid scan EPR measurements can be performed enabling gated millisecond measurements of oxygen concentrations to be performed over the cardiac cycle. Thus, low-frequency EPR spectroscopy offers great promise in the study of tissue oxygen concentrations and the role of oxygen in metabolic control.     

Identification of a single genome by electron paramagnetic resonance (EPR) with nitroxide-labeled oligonucleotide probes

Nitroxide-labeled nucleic acids are used as a molecular size sensor to identify as few as one genome under polymerase chain reaction (PCR) conditions by electron paramagnetic resonance (EPR) spectroscopy. DNA identification is based on differences in the EPR spectra of mono-nitroxide-labeled nucleic acids. The experimental data imply that rapid DNA identification can be achieved in many systems by EPR at the molecular level.     

On the EPR potentiality for revealing hydrocarbon pollution in sphagnum

Sphagnum mosses sampled in the background and industry-affected regions within an oil deposit situated in the southern zone of the Bolshezemelskaya tundra were analyzed by EPR spectroscopy. The EPR spectra of plants vary in intensity. As shown by heating samples, the intensity of EPR signals of free radicals is an exponential function of temperature. Experiments with preliminary evacuation demonstrated the presence of paramagnetic centers possessing different sensitivity to atmospheric oxygen. This specific oxygen effect is the most profound in plants taken from polluted areas. Therefore, EPR spectroscopy may be used for estimating the state of plants in oil-producing regions.     

Electron paramagnetic resonance spectroscopy of thyroid peroxidase

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.     

On the reaction of ferric heme proteins with nitrite and sulfite

Optical and EPR spectroscopy of ferric heme proteins of the porphyrin, oxyporphyrin, and isobacteriochlorin classes has indicated that nitrite reacts with these proteins at the heme iron. Sulfite has been conclusively proven to react only with proteins containing the isobacteriochlorin macrocycle. Quantitative EPR spectroscopy of these nitrite and sulfite adducts showed that most contained a substantial quantity of undetectable heme. It is suggested that protein-induced autoreduction of nitrite (but not sulfite) and a strained and/or uniaxial g-tensor are the principal ways by which the silent state is produced.     

EPR spectroscopy of free radical intermediates of antiarrhythmic-antihypoxic drug stobadine, a pyridoindole derivative

Mechanisms of antioxidant action of stobadine, a pyridoindole derivative with cardioprotective and antihypoxic properties, has been probed using EPR spectroscopy. Oxidation of stobadine by PbO2/tBuOOH in benzene results in the formation of nitroxide radical observable directly by EPR spectroscopy at room temperature, indicating conversion of indolic amino group to the corresponding nitroxide.     

Spin-labeling studies of the conformational changes in the vicinity of D36, D38, T46, and E161 of bacteriorhodopsin during the photocycle.

Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin-labeled bacteriorhodopsin mutants is used to study structural changes during the photocycle. After exchange of the native amino acids D36 and D38 in the A-B loop, E161 in the E-F loop, and T46 in the putative proton channel by cysteines, these positions were modified by a methanethiosulfonate spin label. Time-resolved EPR spectroscopy reveals spectral changes during the photocycle for the mutants with spin labels attached to C36, C161, and C46. A comparison of the transient spectral amplitudes with simulated EPR difference spectra shows that the detected signals are due to changes in the spin label mobility and not to possible polarity changes in the vicinity of the attached spin label. The kinetic analysis of the EPR and the visible data with a global fitting procedure exhibits a structural rearrangement near position 161 in the E-F loop in the M state. The environmental changes at positions 36 and 46, however, occur during the M-to-N transition. All structural changes reverse with the recovery of the BR ground state. No structural changes are detected with a spin label attached to C38.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Interaction of human myeloperoxidase with nitrite.

EPR (electron paramagnetic resonance) and optical spectroscopy show that human neutrophil myeloperoxidase is converted from ferric high-spin to low-spin by the addition of nitrite. The Soret peak shifts from 429 to 447 nm and new peaks appear in the visible region at 573 and 627 nm; the EPR g-values change from 6.84, 5.02, 1.95 to 2.55, 2.31, 1.82. Small differences are seen in the EPR (but, not optical) spectra of myeloperoxidase isoenzyme I compared to isoenzymes II and III. The reaction with nitrite is detectable by EPR in intact neutrophils and is thus a possible in vivo monitor of NO/nitrite production by these cells.     

Antioxidant Properties of Fruits and Vegetables Shots and Juices: An Electron Paramagnetic Resonance Study

The antioxidant activity of some commercially available fruit and vegetable juices was evaluated with regard to their radical scavenging activity against the stable free radical 4-hydroxy-2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPOL) monitored by electron paramagnetic resonance (EPR) spectroscopy. TEMPOL is a stable nitroxide free radical characterized by a well-defined EPR spectrum consisting of three peaks. The integral intensity of the EPR spectra of TEMPOL was decreased upon juice addition, and the decrease was dose dependent. EPR spectroscopy using stable free radicals provides a simple, rapid, and sensitive method for the determination of antioxidant activity of fruit and vegetable juices. The method was standardized by using the standard antioxidant compound Trolox, and the antioxidant activity of the juices was expressed as Trolox equivalents. When concentrated juices of fruits and vegetables (shots) were considered, the evaluated antioxidant activity was almost twofold higher than that of the conventional, non-concentrated ones. Fruits and vegetables shots also showed very good stability during storage. This finding indicates that natural antioxidant compounds contained in commercially available concentrated juices are not eliminated or inactivated when the juices are kept refrigerated according to the instructions of the manufacturer.     

Synthesis,structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthesized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated. The compounds are potential candidates for targeted spin probes in EPR imaging.     

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn₄Ca cluster of photosystem II. Since the discovery of the multiline signal from the S₂ state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn₄Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn₄Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.     

Reversible carbon monoxide binding and inhibition at the active site of the Fe-only hydrogenase

Carbon monoxide binding and inhibition have been investigated by electron paramagnetic resonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. Simulation of the EPR spectrum of the as-isolated state indicates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clusters. Addition of carbon monoxide to CpI in the presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristic axial EPR signal [g(eff(1)), g(eff(2)), and g(eff(3)) = 2.0725, 2.0061, and 2.0061, respectively] was observed. Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resulted in samples that exhibited the native oxidized EPR signature that could be converted to the reduced form upon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopically defined states of CpI and those observed structurally by X-ray crystallography, we have examined the CpI crystals using EPR spectroscopy. EPR spectra of the crystals in the CO-bound state exhibit the previously described axial signal associated with CO binding. The results indicate that the addition of carbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized by loss of enzyme activity and the distinctive axial EPR signal.     

Spin-echo EPR of Na,K-ATPase unfolding by urea

Denaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II -SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D2O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function.     

Spectroscopic characterization and ligand-binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1.

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated 'narrow' (gx,y,z = 6.24, 5.42, 2.00) and 'broad' (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = -23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.