Self-treatment for gout.

After describing some of the symptoms of gout and considering some causes, such as an excess of ethanol, the source of the pain in the infected joint is discussed. This is known to be from urate crystals formed in the synovial fluid inside the joint. It is suggested herein that the pain is due to grinding from the crystals through the surface film of the joint, and possibly into the bone itself, which is relatively soft. The pain then stems in part from the resulting inflammation. The key hypothesis is that these urate crystals dissolve on warming. Hence, by warming the joint concerned in hot water, and moving the joint around to encourage diffusion, the urate concentration is reduced and crystals no longer form, provided the treatment is continued.

Hydrogen peroxide: a potent cytotoxic agent effective in causing cellular damage and used in the possible treatment for certain tumours.

H2O2, a highly reactive agent, can react under certain conditions with a variety of cellular components. These reactions include the lipid peroxidation of membrane and hydroxylation of proteins and DNA. The reactions can take place in the presence of oxygen and are fairly rapid, the H2O2 being converted to water and oxygen. Experiments were carried out in vitro to assess the ability of this agent to destroy cancer cells without generating dangerous by-products. The direct administration of aqueous H2O2 into solid tumours has the potential to cause tumour cell death. The efficacy of the use of H2O2 for treating 'solid' cancers will necessitate its delivery to the tumour site, for example by direct special multiple injection of H2O2 into a detectable tumour mass. We anticipate that, if suggested mode of delivery can be obtained, H2O2 can act as an anti-cancer drug with two distinct advantages over conventional chemotherapeutic agents: to produce minimal short- and long-term side-effects and is relatively cheap and cost effective.

Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2(-)) were investigated. Reduction of compound I by NO2(-) was rapid (k2 = 2.3 x 10(7) M(-1) x s(-1); pH = 7.2) and compound II was not an intermediate, indicating that NO2* radicals are not produced when NO2(-) reacts with compound I. The second-order rate constant for the reaction of compound II with NO2(-) at pH = 7.2 was 3.5 x 10(5) M(-1) x s(-1). The reaction of compound III with NO2(-) exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2(-) concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2(-) complex. The Km of compound III for NO2(-) was 1.7 x 10(-4) M and the first-order decay constant of the compound III/ NO2(-) complex was 12.5 +/- 0.6 s(-1). The second-order rate constant for the reaction of the complex with NO2(-) was 3.3 x 10(3) M(-1) x s(-1). Rate enhancement by NO2(-) does not require NO2* as a redox intermediate. NO2(-) accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2(-) with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.

Commentary: possible mechanisms for strand-breaks in DNA induced by stirring.

When aqueous solutions of DNA are stirred at room temperature, strand breaks occur extensively. Using various spin-traps, coupled with epr spectroscopy, we have shown that this does not proceed via homolysis. It is suggested that breaks occur by hydrolysis at strongly bent regions, momentarily induced by the stirring.

Characterization of the radical product formed from the reaction of nitric oxide with the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate.

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO(*)). The reaction between DBNBS and NO(*) yields a radical product which gives rise to an EPR signal consisting of three lines with an A(N) = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO(*) reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment-mass spectrometry (FAB-MS). Collectively, the FAB-MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO(*). A reaction scheme consistent with these results is presented.

The EPR spectrum for CuB in cytochrome c oxidase.

Incubation of cytochrome c oxidase (CcO) in its resting state in saturated ammonium sulfate, at room temperature overnight, gave EPR signals characteristic of a single Cu(II) center. From the g// and A// values it is concluded that this is a square-planar type 2 copper center, and superhyperfine splitting shows the presence of three nearly equivalent 14N nuclei in the plane. It is suggested that this center, also formed by incubating the enzyme in 10% methanol followed by direct irradiation, must be the CuB center. This type 2 copper EPR spectrum is identical to the EPR spectrum of CuB reported for the isolated cytochrome bo3 complex from Escherichia coli; and to the EPR spectrum reported for the sulfobetaine 12 heat-treated cytochrome c oxidase complex. It is argued that a small perturbation in the system causes decoupling of the magnetic coupling of the heme a3-CuB binuclear center and the appearance of the type 2 EPR signal.

A method for reducing the pain and danger of certain arthritic joints.

It has been found that by hanging upside-down from the feet, the pain arising from an arthritic hip joint is largely eliminated. If this method is not used, the pain returns rapidly. It is suggested that the direct cause of the pain and damage is the presence of small, hard crystals of apatite or related compounds present in the synovial fluid of the joint. Under the normal pressures involved in standing, walking or running, these damage the joint involved by a process of grinding. If this joint is opened by hanging and swivelling, the crystals are induced to move out of the joint region, thereby alleviating the problem.

Spectroscopy of aqueous solutions: protein and DNA interactions with water.

This review emphasises the need to use spectroscopy in order to understand the behaviour of water, and summarises the background of the subject. The various forms of spectroscopy that are especially informative are described, with particular reference to near-infrared (NIR) spectrophotometry. The key results are outlined, first those obtained with small molecules and ions, and second those involving proteins, DNA and cell membranes. Finally, some interpretations are offered which include the novel but possibly controversial concept of free OH and free lone-pair groups.

Cis- and trans-conformations for peroxynitrite anions.

The biochemically important anion, peroxynitrite, is commonly thought to have a cis-conformation which is stable. This is thought to have a cyclic structure, which blocks the formation of the trans-conformation. Furthermore, it is often suggested that the latter structure isomerises to the far more stable nitrate ion. Here I suggest that the cis-structure has comparable stability to the trans-structure, and they are in rapid equilibrium at room temperature. This is supported by the very large linewidths for certain vibrational bands at room temperature, which become narrow, well-separated bands at 4 K. Lifetimes are about 10(-12) s for each isomer.

Dibromonitroso benzene sulphonate spin-adducts--why no hyperfine coupling to bromine?

Both 79Br and 81Br nuclei have very large magnetic moments and hence can give rise to large hyperfine splittings. However, for the title compound, dibromonitroso-p-benzene sulphonate (DBNBS), which is a widely used spin-trap, liquid-phase spectra of radical adducts are characterised by narrow EPR features with no bromine splitting, despite the expected delocalisation of spin-density onto bromine. I find that for such radicals in the solid-state the 14N parallel features are very broad, although the seven expected hyperfine features from the 79Br and 81Br nuclei were not clearly defined. I use these results to offer an explanation for the complete absence of any hyperfine splitting from bromine in the isotropic spectra.

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.

Effects of AQ4N and its reduction product on radiation-mediated DNA strand breakage.

Supercoiled plasmid pBR322 DNA was irradiated in phosphate buffer by 60Co gamma-rays at a dose rate 19.26 Gy/min and total dose of 10 Gy in the presence of a bioreductive antitumour prodrug namely 1,4-bis [¿2-(dimethylamino-N-oxide)ethyl¿ amino] 5, 8-dihydroxyanthracene-9,10-dione (AQ4N) and its DNA affinic reduction product 1,4-bis[¿2(dimethylamino)ethyl¿ amino] 5,8-dihydroxyanthracene-9,10-dione (AQ4) under air and nitrogen. AQ4N and AQ4 were found to protect against radiation-induced plasmid single and double strand breakage as assessed by agarose gel electrophoresis. The differences between the two agents, and between atmospheres of air or nitrogen were negligible. It was also found that the protection efficiencies of the compounds were pH dependent and showed maximum protection at pH 6. These results indicate that protection of DNA by AQ4 and AQ4N against radiation damage is an indirect effect since both agents are equally effective despite major differences in their DNA affinity. It is likely that radiation-induced phosphate buffer radicals are intercepted by AQ4 and AQ4N in a pH-dependent process.

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.

Free radical activity following contraction-induced injury to the extensor digitorum longus muscles of rats.

The purpose of the study was to investigate the role of free radicals in the injury induced by a protocol of repeated pliometric (lengthening) contractions to the extensor digitorum longus (EDL) muscle in situ in rats. Previous data have indicated that prior treatment with the antioxidant polyethylene glycol-superoxide dismutase reduced the damage that was apparent at 3 days following this type of exercise. Three hours and 3 days following the protocol, the magnitude of the semiquinone-derived free radical signal observed by electron spin resonance spectroscopy (ESR) was not different for exercised and non-exercised skeletal muscles. A reduction in the protein thiol content of muscle was evident at 3 h, and was still apparent at 3 days. Three hours after the protocol, the total muscle glutathione content and the percentage in the oxidized form were unchanged, but by 3 days the percentage of muscle glutathione present in the oxidized form was elevated. The susceptibility of muscle to lipid peroxidation in vitro was reduced 3 days after the pliometric contractions. These data indicate that oxidation of protein thiols and glutathione may be involved in the secondary damage following pliometric contractions, but provide no evidence that the species involved were derived from mitochondrial semiquinone radicals.

Nitrite determination in human plasma and synovial fluid using reactions of nitric oxide with 3, 5-dibromo-4-nitrosobenzenesulphonate (DBNBS).

DBNBS (3,5-dibromo-4-nitrosobenzenesulphonate) reacts with nitric oxide (NO) produced from nitrite ions in acid solution to give a radical with a characteristic electron spin resonance spectrum, attributable to a 'DBNBS-NO' product, and comprising a triplet with alphaN=0.96 mT. This is identical with the spectrum obtained when NO, introduced from the gas phase, reacts with DBNBS. Under certain conditions, an additional signal is observed, attributable to oxidation of DBNBS to the radical cation, DBNBS*+ (a triplet with alphaN=1.32 mT). Conditions are described for the determination of nitrite, which avoid this DBNBS oxidation. The height of the low-field signal from the DBNBS-NO product is directly proportional to the nitrite concentration up to about 0.08 mM nitrite. The method has been applied to the measurement of nitrite concentrations in whole blood, plasma and synovial fluid taken from rheumatoid arthritis patients. In order to avoid the oxidation of DBNBS when analysing biological samples of this type, it is necessary to treat the specimen by ultrafiltration as soon as possible after collection and before addition of DBNBS.

A novel, stable bioradical.

Several cellular systems have shown a novel EPR spectrum at 77 K, comprising a slightly anisotropic doublet centred on the free-spin g-value, with a hyperfine splitting of ca. 120 G. It is suggested that this species is an occluded protein radical centred on a tryptophan unit.

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity.

Nitric oxide (NO) synthesis is well-known to result from the oxidation of L-arginine by a family of NO synthases (NOS). However, under hypoxic conditions this mechanism of NO synthesis may be impaired and NO is formed by a NOS independent mechanism. This study was designed to examine the reduction of nitrite to NO by xanthine oxidase (XO) under hypoxia, because the bacterial nitrate/nitrite reductases have structural similarity to XO. We found that both purified and tissue containing XO catalyze the reduction of nitrite to NO, as demonstrated using a chemiluminescent NO meter. This redox reaction requires NADH as an electron donor, and is oxygen independent. The inhibitory profiles suggest that reduction of nitrite takes place at the molybdenum center of XO whilst NADH is oxidized at the FAD center. Heparin binding of XO caused an increase in the catalysis of nitrite reduction. The XO-catalyzed generation of NO may be important in redistribution of blood flow to ischaemic tissue as a supplement to NOS, since both nitrite and NADH have been shown to be elevated in hypoxic tissue.

A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor.

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.

Electron movement through proteins and DNA.

Nature utilizes the phenomenon of single electron transfer very widely, especially in metallo-proteins. In systems when the metal donor (D) is well separated from the acceptor (A) by polypeptide chains, the transferring electron is presumed to be bonded, in part, by these chains, which may influence the pathway taken. This situation can be probed by radiolytic injection of electrons into proteins at low temperatures. One aim of this brief review is to consider how information derived from such radiolysis studies, and followed by ESR spectroscopy, may possibly impinge on studies of D-A systems. Electrons can also be injected into duplex DNA in this way, and the results are compared with those for proteins. They are also considered in the light of recent studies of D-A electron-transfer via a polynucleotide strand. It seems that such transfers are very efficient, and it is tentatively suggested that Nature may also use this conductivity in some as yet undiscovered systems.