An EPR study of the peroxyl radicals induced by hydrogen peroxide in the haem proteins.

The reaction of hydrogen peroxide H(2)O(2) with horse heart metmyoglobin (HH metMb), sperm whale metmyoglobin (SW metMb) and human metHb (metHbA) was studied at pH 6-8 by low temperature (10 K) EPR spectroscopy with the emphasis on the peroxyl radicals formed during the reaction. The same type of peroxyl radical was found in both myoglobin systems, as was concluded from close similarities in the spectroscopic properties of the radicals and in their kinetic dependences. This is consistent with previous reports of the peroxyl radical being localised on the Trp14 of SW and HH myoglobins. There are two types of peroxyl radical found in the metHbA/H(2)O(2) system, one (ROO-I) having spectral parameters, kinetic and pH dependences similar to those of the peroxyl radical found in both myoglobin systems. The other peroxyl radical (ROO-II) found in metHbA treated with H(2)O(2) has slightly different, though distinguishable, spectral parameters and a significantly different kinetic dependence as compared to those of the peroxyl radical common for all three proteins studied (ROO-I). The concentration of ROO-I radical formed in the three proteins on addition of H(2)O(2) correlates with the effectiveness of incorporating molecular oxygen into styrene oxide reported before for these three proteins. It is shown that a different distance from Trp14 to haem iron in the three proteins might be the structural basis for the different yield of the peroxyl radical and the different efficiency of incorporation of molecular oxygen into styrene. The site of the peroxyl radical found only in metHbA (ROO-II) is speculated to be the Trp37 residue of the beta-subunit of HbA.

The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study.

The paramagnetic species in human metHb and horse metmyoglobin (metMb) have been studied at low temperature using EPR spectroscopy. The high-spin (HS) haem signal in aquometMb has a greater rhombic distortion than the HS metHb signal. Nevertheless, the individual line width (g=6) is smaller in metMb than in metHb, consistent with non-identical signals from the alpha and beta Hb subunits. Three low-spin (LS) haem forms are present in metHb, while metMb has only two. The major LS form in both proteins is the alkaline species (with OH(-) at the sixth co-ordination position). The minor LS forms are assigned to different histidine hemichromes in equilibrium with the normal HS species at low temperature. LS forms disappear when the haem is bound by a ligand, such as fluoride, which ensures 100% occupancy of the HS state both at room temperature and at 25 K. The small differences in effective g-factors of the histidine hemichromes are interpreted in terms of different distances between the distal histidine and haem iron. The pH dependence of the inter-conversion of the different paramagnetic species is consistent with a model whereby protonation of a residue with a pK of 5.69 (metHb) or 6.12 (metMb), affects ligand binding and transformation from the HS to the LS form. Chemical and spectroscopic considerations suggest that the residue is unlikely to be the proximal or distal histidine. We therefore propose a model where protonation of this distant amino acid causes a conformational change at the iron site. Identical effects are seen in frozen human blood, suggesting that this effect may have physiological significance.

A new method for quantitation of spin concentration by EPR spectroscopy: application to methemoglobin and metmyoglobin.

A new method of EPR spectral analysis is developed to quantitate overlapping signals. The method requires double integration of a number of spectra containing the signals in different proportions and the subsequent solution of a system of linear equations. The result gives the double integral values of the individual lines, which can then be further used to find the concentrations of all the paramagnetic species present. There is no requirement to deconvolute the whole spectrum into its individual components. The method is employed to quantify different heme species in methemoglobin and metmyoglobin preparations. A significantly greater intensity of the high-spin signal in metmyoglobin, compared to methemoglobin at the same heme concentration, is shown to be due to larger amounts of low-spin forms in methemoglobin. Three low-spin types in methemoglobin and two in metmyoglobin are present in these samples. When their calculated concentrations are added to those of the high-spin forms, the results correspond to the total heme concentrations obtained by optical spectroscopy.

Crystal structure of bis(4-methylimidazole)tetraphenylporphyrinatoiron(III) chloride and related compounds. Correlation of ground state with Fe-N bond lengths.

The crystal structure of the title compound is presented and shown to be one of a class of low-spin iron porphyrin complexes having a ground-state electronic configuration of (dxy)2(dxz)2(dyz)1. If their Fe-N bond lengths (average N-porphyrin plotted against average N-axial) are considered, this class of low-spin iron(III) porphyrins of general formula [Fe(III)Por(L)2]+X- and of 2B ground state is shown to be distinctly different crystallographically from a similar class of compounds with the same general formula but with a 2E or a (dxy)2(dxz,dyz)3 ground state. A third group of compounds with the same general formula have a (dxz,dyz)4(d)1 ground state and again are in a different region of the plot. Compounds showing intermediate properties can be forecast from the simple relationship presented in this work. The electron paramagenetic resonance data are shown to be dependent on the ground state, and those of configuration (dxy)2(dxz,dyz)3 and the 2B ground state obey a correlation previously suggested in the literature.

The electron paramagnetic resonance characterisation of a copper-containing extracellular peroxidase from Thermomonospora fusca BD25.

The actinomycete Thermomonospora fusca BD25 contains a peroxidase with a high activity over a broad range of temperature and pH and a high stability against denaturing agents. Unusually this peroxidase (PO) is a non-haem enzyme. As prepared PO is characterised by two electron paramagnetic resonance (EPR) signals, detected at liquid helium temperature, a free radical signal (g=2.0045) and a broad signal at g=2.056. The peroxidase activity of the purified enzyme was assayed using H(2)O(2) and 2,4-dichlorophenol (DCP). The intensity of the free radical EPR signal correlated with the peroxidase activity in a variety of enzyme preparations. Furthermore, when DCP and H(2)O(2) were added to PO a significant increase of both the free radical signal and the broad signal at g=2.056 was observed. We associate the increase of the broad signal with the oxidation of the preparation since a similar increase can be achieved by the addition of ferricyanide. The high intensity of the broad signal in the ferricyanide treated PO allowed us to deconvolute the signal into several components using the difference in their relaxation characteristics: two distinct copper signals were detected, one of which was similar to a type 2 centre. Furthermore a symmetrical singlet was detected at g=2.059, consistent with the presence of an iron complex with a high degree of symmetry and weakly coordinated ligands.

A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure.

Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.

The globin-based free radical of ferryl hemoglobin is detected in normal human blood.

Normal human venous blood was studied by electron paramagnetic resonance (EPR) spectroscopy at -196 degrees C. The EPR signal of free radicals in frozen blood is shown to have the same radiospectroscopic parameters and properties as the signal of the globin based free radical, .Hb(Fe(IV)=O), formed in the reaction of purified methemoglobin (metHb) with H2O2 and therefore has been assigned as such. The globin-based radicals and metHb exhibited significant variation (fluctuations) in different frozen samples taken from the same liquid blood sample. In any given sample a high concentration of free radicals was associated with a low concentration of metHb and vice versa, i.e. the fluctuations were always of opposite sense. No such fluctuations were observed in the concentration of two other paramagnetic components of blood, transferrin and ceruloplasmin. The time course of free radical formation and decay upon the addition of H2O2 to purified metHb was studied at three different molar ratios H2O2/metHb. This kinetic study together with the results of an annealing experiment allow us to propose a mechanism for the formation and decay of the globin-based radical in blood. Within this mechanism, the source of H2O2 in blood is considered to be dismutation of O-2 radicals produced via autoxidation of Hb. We postulate that the dismutation is intensified on the phase separation surfaces during cooling and freezing of a blood sample. The fluctuations are explained within this hypothesis.

Redox cycling of human methaemoglobin by H2O2 yields persistent ferryl iron and protein based radicals.

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H2O2, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H2O2 resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H2O2 showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H2O2. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalytic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10(4) fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: approximately 20 mol H2O2 and abolished the protein radical steady state. The biological implications for these results are discussed.

An EPR investigation of human methaemoglobin oxidation by hydrogen peroxide: methods to quantify all paramagnetic species observed in the reaction.

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglobin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2 was added at 3- and 10-fold molar excess to 100 microM metHb in 50 mM phosphate buffer, pH 7.4, 37 degrees C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2 the EPR signals (-196 degrees C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (-196 degrees C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g parallel = 2.035 and g perpendicular = 2.0053), and the other an isotropic singlet (g = 2.0042, delta H = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic behaviour of both peroxyl (ROO.) and non-peroxyl (P.) free radicals were similar, we concluded that ROO. radicals are not formed from P. radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2 to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 x 10(-3) s-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haem centres with g = 4.35 was found. Their yield was proportional to the H2O2 concentration used and the centres were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at -196 degrees C were used.

[The decomposition of the EPR spectra of multicomponent systems irradiated at 77 K into paramagnetic center signals of different natures]. / Razlozhenie spektrov EPR obluchennykh pri 77 K mnogokomponentnykh sistem na signaly paramagnitnykh tsentrov razlichnoi prirody.

Main principles of the way to decompose an EPR spectrum of a multicomponent system, irradiated at 77 K, into separate radiation-induced paramagnetic centre signals are given. The decomposition is possible due to the computer assistant spectra processing, and is based on different properties of different paramagnetic centres, namely, on different thermostability of the centres, on different rate of relaxation, and on different photosensitivity. Concrete examples of the EPR spectrum decomposition into different free radical signals are given for cases of murine liver and spleen irradiated at 77 K. Radiochemical yields of different free radicals, induced by gamma radiation at 77 K in whole biological tissues, were defined. The data on nature and properties of the paramagnetic centres induced by radiation in biological tissues are shortly reviewed.

[Ascorbic acid radicals induced by the action of radiation in tissues from rat organs frozen at 77 K]. / Radikaly askorbinovoi kisloty, indutsirovannye deistviem radiatsii v zamorozhennykh pri 77 K tkaniakh organov krys.

Subsequent annealing technique and computer assistant analysis of EPR spectra were used to isolate an asymmetric EPR signal Rs(g = 2,0051; delta H = 0.8 mT) from the EPR spectrum of rat spleen gamma-irradiated at 77 K. Radicals with the same EPR spectrum were registered in: 1) water solution of ascorbic acid (2.10(-2) M, pH 3.4) frozen and irradiated at 77 K and 2) water-glycerol solution of ascorbic acid (10(-2) M, pH 10.3) frozen rapidly at the moment of intensive autooxidation. These model experiments allow to conclude that Rs signal is caused by the radicals of semidehydroascorbic acid. Radiochemical yield of these radicals as well as of all the radicals induced by gamma radiation in the whole rat tissues were measured. The EPR signal (Rs) is equivalent to the well known "artifact" signal of lyophilized tissues. The explanation of the mechanism of the radicals formation taking place under annealing of the frozen and irradiated tissues was suggested.

[Modification by cystamine of radiation-induced free radical damage to biomolecules in tissues of mouse organs]. / Modifikatsiia tsistaminom radiatsionnykh svobodnoradikal'nykh povrezhdenii biomolekul v tkaniiakh organov myshei.

The method of low-temperature ESR-spectroscopy was used to study a modifying effect of cystamine on the yield of radiation-induced free radicals in different biomolecules of liver and spleen tissues of mice. Intraperitoneal administration of cystamine (150 mg/kg) 15 min before isolation and freezing of the tissues was shown to reduce by 11 per cent the yield of radicals of H-adducts of thymine DNA bases, to decrease by 23 per cent the yield of radicals of triacyglycerol and phospholipid radiolysis, and to increase by 24 per cent the yield of radicals of lipid fatty acid residues in splenic tissue. According to the criterion used, cystamine has no modifying action on the yield of free-radical damages to liver biomolecules.

[Paramagnetic centers formed in mouse blood gamma-irradiated at 77K]. / Paramagnitnye tsentry, voznikaiushchie v krovi myshei, gamma-obluchennoi pri 77K.

The method of low-temperature ESR-spectroscopy was used to study the primary products of radiation injury of mouse blood. It was shown that when blood samples were exposed to gamma-radiation at 77 K radicals of water, lipids and proteins and also oxyhemoglobin adducts (Fe3+--O2(2-)) were formed. It was shown that oxyhemoglobin electron adducts were protonated during stepwise annealing to form complexes (Fe3+--HO2-). High spin methemoglobin is probably the final product of transformations of the oxyhemoglobin complexes.

[Radiation-induced free radicals in DNA, DNA level in tissues and tissue radiosensitivity]. / Radiatsionno-indutsirovannye svobodnye radikaly DNK, soderzhanie DNK v tkaniakh i tkanevaia radiochuvstvitel'nost'.

Radiochemical yields (G-values) of H-adducts of thymine bases of DNA (TH) in frozen (77 K) gamma-irradiated mouse and rat tissues were measured. The content of DNA and the number of TH radicals formed in DNA mass of 10(12)D at a dose of 1 Gy (beta parameter) were determined for each of the studied tissue. It was shown that beta parameter, which indicated DNA in situ radiosensitivity, was different for different tissues: it was higher for radiosensitive tissues. The possible causes of the effect observed are discussed.

[Free-radical disorders in the tissues of mice exposed to gamma irradiation and neutrons in vitro. The radiochemical yields]. / Svobodnoradikal'nye narusheniia v tkaniakh myshei pri deistvii gamma-oblucheniia i neitronov in vitro. Radiatsionno-khimicheskie vykhody.

The ESR method was used to study free-radical disturbances in tissues of mouse liver and spleen exposed in vitro to gamma-radiation and fission neutrons. It was shown that both types of radiation induced damages to basic chemical components of the cell, namely, DNA, lipids, proteins, and water. The radiochemical yields of each radical registered were obtained and RBE of neutrons were evaluated with a reference to the formation in tissues of radicals of each type. Membrane lipids were shown to be markedly injured by neutrons.