Molecular mechanism for metal-independent production of hydroxyl radicals by hydrogen peroxide and halogenated quinones

We have shown previously that hydroxyl radicals (HO*) can be produced by H2O2 and halogenated quinones, independent of transition metal ions; however, the underlying molecular mechanism is still unclear. In the present study, using the electron spin resonance secondary radical spin-trapping method, we found that tetrachloro-1,4-benzoquinone (TCBQ), but not its corresponding semiquinone anion radical, the tetrachlorosemiquinone anion radical (TCSQ*-), is essential for HO* production. The major reaction product between TCBQ and H2O2 was identified by electrospray ionization quadrupole time-of-flight mass spectrometry to be the ionic form of trichlorohydroxy-1,4-benzoquinone (TrCBQ-OH), and H2O2 was found to be the source and origin of the oxygen atom inserted into the reaction product TrCBQ-OH. On the basis of these data, we propose that HO* production by H2O2 and TCBQ is not through a semiquinone-dependent organic Fenton reaction but rather through the following mechanism: a nucleophilic attack of H2O2 to TCBQ, forming a trichlorohydroperoxyl-1,4-benzoquinone (TrCBQ-OOH) intermediate, which decomposes homolytically to produce HO*. This represents a mechanism of HO* production that does not require redox-active transition metal ions.     

Angiotensin-converting enzyme inhibitors as neutralizers of hydroxyl radical]

Angiotensin converting enzyme inhibitors are utilized in the treatment of essential hypertension and of chronic cardiac failure. They are also employed in the treatment of the myocardial lesion of ischemia-reperfusion, which involves oxygen free radicals. In the present study we investigated the possibility of three angiotensin converting enzyme inhibitors (captopril, enalapril, lisinopril) to act as hydroxyl radical scavengers. The rate constants for reactions of those compounds with .OH were determined using the deoxyribose method. All there compounds proved to be good scavengers of .OH with rate constants of about 10(10)M-1s-1 and are iron chelators specially enalapril. The fact that captopril possesses a thiol group does not confer an higher antioxidative capacity. These results suggest that scavenging of oxygen free radicals may be a possible mechanism contributing to the therapeutic effect of angiotensin converting enzyme inhibitors.     

Effect of metal chelators and antiinflammatory drugs on the degradation of hyaluronic acid

Degradation of hyaluronic acid (measured viscometrically) by oxygen-derived free radicals (ODFR) generated 1) by autoxidation of ferrous EDTA chelates and 2) enzymatically by xanthine oxidase and hypoxanthine (XO/HX) was studied. Degradation of hyaluronic acid by XO/HX was strongly inhibited by superoxide dismutase and catalase, whereas degradation of hyaluronic acid by autoxidation of ferrous ions was weakly inhibited by catalase and unaffected by superoxide dismutase. Both ODFR-producing systems were inhibited by hydroxyl radical scavengers, suggesting that hydroxyl radical was the proximate damaging species in both systems. Penicillamine at concentrations of 1-5 mM stimulated hyaluronic acid degradation by ferrous EDTA chelates but inhibited degradation by the XO/HX system. Higher concentrations of penicillamine and all concentrations studied (1-100 mM) of other antiinflammatory drugs (chloroquine, gold sodium thiomalate, and salicylate) inhibited hyaluronic acid degradation by both the autoxidation and enzymatic ODFR-producing systems, with inhibitory potency similar to that seen with known hydroxyl radical scavengers. Both systems serve as in vitro models of ODFR-mediated tissue damage which may occur in vivo at sites of inflammation.     

The pH dependence of the mechanism of reaction of hydrogen peroxide with a nonaggregating, non-mu-oxo dimer-forming iron (III) porphyrin in water.

The reaction of hydrogen peroxide with 5, 10,15,20-tetrakis(2,6-dimethyl-3-sulfonatophenyl)porphinato- iron(III) hydrate [(P)FeIII(H2O)] has been investigated in water between pH 1 and pH 12. The water-soluble (P)FeIII(H2O) neither aggregates nor forms a mu-oxo dimer. The pH dependence and rate-limiting second-order rate constants (kly) for oxygen transfer from H2O2 and HO2- to the iron(III) porphyrin were determined by trapping of the resultant higher-valent iron-oxo porphyrin species with 2,2'-azinodi(3-ethylbenzthiazoline)-6-sulfonate (ABTS). Reactions were monitored spectrophometrically by following the appearance of the radical ABTS.+. From a plot of the logarithm of the determined second-order rate constants for reaction of hydrogen peroxide with iron(III) porphyrin vs. pH, the composition of the transition states can be assigned for the three reactions that result in oxygen transfer to yield a higher-valent iron-oxo porphyrin species. The latter not only reacts with ABTS to provide ABTS.+ in a peroxidase-type reaction but also reacts with hydrogen peroxide to provide O2 in a catalase-type reaction. The nitrogen base 2,4,6-collidine serves as a catalyst for oxygen transfer from hydrogen peroxide to the (P)FeIII-(H2O) and (P)FeIII(HO) species. The preferred mechanism involves a 1,2-proton shift concerted with heterolytic cleavage of the peroxide O-O bond. An analogous mechanism is believed to occur in the peroxidase enzymes.     

Dependence of the rates of dissolution of the Fe4S4 clusters of Chromatium vinosum high-potential iron protein and ferredoxin on cluster oxidation state

The influence of oxidation state on the pH dependence of the dissolution of the Fe(4)S(4) clusters of Chromatium vinosum ferredoxin and high-potential iron protein (HIPIP) has been studied. The first-order rate constants (k(obs)) for dissolution of both the Fe(4)S(4)(S-Cys)(4) (2-) and Fe(4)S(4)(S-Cys)(4) (3-) clusters of the ferredoxin follow the same overall kinetic equation but with differing specific rate and equilibrium constants. The dependence of rate and equilibrium constants upon oxidation state may be rationalized on the basis of the accompanying change in electrostatic affinity of a cluster toward H(+) and HO(-). A more drastic change in the pH dependence of the kinetics of dissolution of the Fe(4)S(4) cluster of the HIPIP accompanies its change in oxidation state. Whereas the values of k(obs) for dissolution of HIPIP containing the Fe(4)S(4)(S-Cys)(4) (2-) cluster are strictly second order to [H(+)] and [HO(-)], the pH dependence for dissolution of the HIPIP Fe(4)S(4)(S-Cys)(4) (1-) cluster indicates a first-order dependence upon [H(+)], a second-order dependence upon [HO(-)], and a spontaneous or water rate. These reactivity differences may be related to changes in cluster charge density. Mechanisms of dissolution involve preequilibrium protonation at acidic pH and preequilibrium ligand exchange at basic pH.     

Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibody-catalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into para-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a short-lived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen ((1)O*(2)) induced antibody-catalyzed water-oxidation pathways proceeds via the formation of dihydrogen trioxide (H(2)O(3)), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical HO(3)*, and we point to the remarkable potential of this either H(2)O(3)- or O(3)-derivable species to act as a masked hydroxyl radical HO* in a biological environment.     

Free radical scavenger activity of rutosides

BACKGROUND: The O-(beta-hydroxyethyl)-rutosides (HRs) are a standard mixture of flavonoid-derivatives that have a clinico-pharmacological activity on peripheral circulation, particularly on the endothelial cells of veins and lymphatics. Flavonoids are believed to prevent the oxidative damage derived from radical oxidative species (ROS), like hydroxyl radicals (HO.) and hypochlorite (-OCl). The aim of the study was to investigate the stability and capability of HRs in toto and of their single components (7-mo-nohydroxy ethyl rutoside; 7,4'-dihydroxyethyl rutoside; 7,3',4'-trihydroxyethyl rutoside and the 7,5,3',4'-tetrahydroxyethyl rutoside) of scavenging ROS and other radicals generated by different oxidative systems, and also their anti-lipoperoxidative activity at mM concentrations (1.0-10.0 mM). METHODS: The following oxidative systems have been employed: Fenton reaction for the hydro-xylation of l-tyrosine to l-DOPA and the peroxidation of arachidonic acid; photo-Fenton type reaction for the oxidation of toluene in the aqueous UV irradiated TiO2 system; the azocompound 2.2'-azobis(2, 4-dimethylvaleronitrile (AMVN) to produce peroxy radicals and the daily autoxidation of arachidonic acid. Analyses were performed by HPLC, HPLC-MS, GC-MS, and spectrophotometry. RESULTS: At 5.0 mM concentration, HRs produced the following inhibitions: 63+/-5% of the overall formation of cresols, benzaldehyde, benzyl alcohol, and biphenyl induced by photo-Fenton reaction; 91.6+/-5% and 59+/-8% of the synthesis of l-DOPA induced by HO. generated by Fenton reaction; 45+/-7% and 52+/-6% of the oxidation of arachidonic acid induced by Fenton reaction and AMVN; 60+/-4% of the autoxidation of arachidonic acid. These effects were strictly concentration dependent. CONCLUSIONS: At mM concentrations, HRs display a significant antilipoperoxidative activity due to their notable scanvenging activity against HO.; moreover these actions are concentration-dependent.     

Carbon monoxide binding kinetics in "capped" porphyrin compounds.

The rate constants for CO binding to the five-coordinate ferrous iron complexes of 5,10,15,20-[pyromellitoyl(tetrakis-o-oxyoxyphenyl)]porphyrin and 5,10,15,20-[pyromellitoyl(tetrakis-o-oxypropoxyphenyl)]porphyrin have been measured and compared with the corresponding rate constants for other hemes and hemoproteins. The second-order rate constant is independent of cap size and is comparable to that of high-affinity state hemoglobin (k5 approximately 4 X 10(6) M-1s-1). Therefore, these capped porphyrins provide no steric hindrance to CO binding. In addition, a kinetic scheme involving an unusual seven-coordinate porphyrin species is described.     

Reactivity of free and coordinated radicals in biology and chemical carcinogenesis. II. Electron transfer from 3,4-benzopyrene to molecular oxygen and to peroxides, and interpretation of ESR signals of the intermediate radicals of oxidation

Already a trace of oxygen mediates the one-electron transfer from 3,4-benzopyrene (benzo(a)pyrene, BP) to hydrogen peroxide or to tert. butyl hydroperoxide (ROOH), leading in this way to generation of highly reactive HO X, HO2 X or RO X and RO2 X radicals in nonpolar solvents at biological temperatures. At slightly higher concentration of O2 in solution and at moderately elevated temperature (40-60 degrees C) a stable radical pair (HO-BPO X)2 in equilibrium with its diamagnetic dimer of quinone-hydroquinone type is formed. The paradiamagnetic equilibrium of this redox system is reversibly shifted with temperature. At low temperature (up to -40 degrees C) the paramagnetism disappears. The precursor of the radical pair, which can be decomposed, applying a polar solvent, is the keto form of the hydroxy derivative (6-HO-BP) at ambient temperature. According to the study of highly resolved ESR spectra of the primary temporarily formed ion radical pair [BP+ X O-2 X] of BP oxidation in the dark, of the secondary radical pair (HO-BPO X)2 and of the coordinated unhindered phenoxy radicals of hydroxy derivatives BPO X CoIII, the mechanisms of one-electron or of hydrogen-atom transfer from radical intermediates of BP to potential biological targets is discussed.     

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.

Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.     

Oxygen free radical scavengers and reperfusion injury in dog lung preserved in cold ischemia.

Oxygen-derived free radicals are now considered important contributors to tissue injury associated with ischemia and reperfusion. The purpose of this study was to determine the influence of oxygen free radical scavengers on reperfusion injury. The left lower lobes of 15 canine lungs were isolated, preserved, and then reperfused for 120 minutes. Three groups of lobes were studied: Group 1 (n = 5), without ischemia, group 2 (n = 5) four hours of cold ischemia in Euro-Collins solution, group 3 (n = 5) four hours cold ischemia+oxygen free radical scavenger glutathione (0.1 nmol/L) given at the moment of perfusion. Extravascular lung water (grams per gram of blood-free dry lobe weight) after reperfusion was 2.82 +/- 0.32, 5.06 +/- 0.45, 4.21 +/- 0.33 for groups 1 through 3 respectively (p less than 0.001 group 1 versus group 2, p less than 0.001 group 2 versus group 3). Lung tissue lipid peroxidation, measured as thiobarbituric acid reactive material was 125 +/- 11, 270 +/- 30, and 185 +/- 17 nmol/g dry lobe weight for groups 1, 2 and 3 respectively (p less than 0.05 group 2 versus 1 and group 3 versus group 2). The data suggest that oxygen free radical scavengers attenuate reperfusion injury.     

Hydroxyl radical scavengers inhibit lymphocyte mitogenesis.

Agents that are known to be scavengers of hydroxyl radicals inhibit lymphocyte mitogenesis induced by phorbol myristate acetate (PMA) to a greater extent than they inhibit mitogenesis induced by concanavalin A or phytohemagglutinin. These agents include dimethyl sulfoxide, benzoate, thiourea, dimethylurea, tetramethylurea, L-tryptophan, mannitol, and several other alcohols. Their inhibitory effect is not associated with cytotoxicity. The hydroxyl radical scavengers do not inhibit PMA-dependent amino acid transport in T cells or PMA-induced superoxide production by monocytes. Thus, they do not inhibit the primary interaction of PMA with responding cells. Treatment of peripheral blood mononuclear cells with PMA increased cellular guanylate cyclase in most experiments, and dimethyl sulfoxide tended to inhibit this increase. In addition to inhibition of PMA-induced mitogenesis, hydroxyl radical scavengers markedly inhibited the activity of lymphocyte activating factor (interleukin 1). The differential inhibition of lymphocyte mitogenesis induced by different mitogens appears to be related to the differential macrophage requirements of the mitogens. The data suggest that hydroxyl radicals may be involved in mediating the triggering signal for lymphocyte activation. Some of the hydroxyl radical scavengers are inducers of cellular differentiation,. nd it is possible that their differentiating activity is related to their ability to scavenge free radicals.     

Role of hydrogen peroxide and hydroxyl radical formation in the killing of Ehrlich tumor cells by anticancer quinones.

The cytotoxicity of the clinically important antineoplastic quinones doxorubicin, mitomycin C, and diaziridinylbenzoquinone for the Ehrlich ascites carcinoma was significantly reduced or abolished by the antioxidant enzymes catalase and superoxide dismutase, the hydroxyl radical scavengers dimethyl sulfoxide, diethylurea, and thiourea, and the iron chelators deferoxamine, 2,2-bipyridine, and diethylenetriaminepentaacetic acid. However, tumor cell killing by 5-iminodaunorubicin, a doxorubicin analog with a modified quinone function that prohibits oxidation-reduction cycling, was not ameliorated by any of the free radical scavengers tested. Furthermore, treatment of intact tumor cells with doxorubicin, mitomycin C, and diaziridinylbenzoquinone but not 5-iminodaunorubicin generated the hydroxyl radical, or a related chemical oxidant, in vitro in a process that required hydrogen peroxide, iron, and intact tumor cells. These results suggest that drug-induced hydrogen peroxide and hydroxyl radical production may play a role in the antineoplastic action of redox active anticancer quinones.     

Mechanisms for the oxidation of reduced gluthathione by stimulated granulocytes

We have reported previously that human granulocytes have an irreversible fall in their endogenous reduced soluble sulfhydryls following zymosan stimulation. In the present study, we demonstrate that stimulated granulocytes release one or more reactive oxygen species (ROS) with the capacity to oxidize reduced glutathione (GSH). One or more of these compounds is stable enough to be detected in the supernatant. The formation of these stable oxidants appears to require H2O2 and heme or a heme-containing enzyme. However, once formed, the compound reacts with GSH without these factors. The ROS is not superoxide or hydroxyl radical, since neither superoxide dismutase nor the hydroxyl scavengers, mannitol and benzoic acid, change the rate of the reaction. Methionine has recently been demonstrated to be oxidized to a sulfoxide by a reactive oxygen species that is dependent on H2O2 and heme for its production. We found that methionine could directly react with the same ROS that degrades GSH. The ROS also has the capacity to oxidize iodide and fix halogen to proteins. Our data indicate that stimulated granulocytes release a ROS with the capacity to oxidize GSH, react with methionine, and oxidize and fix I- to protein. The compound, therefore, appears dependent on H2O2 and the myeloperoxidase system for its production, and is either hypochlorous acid (HOCI) or a compound derived from HOCI, such as a chloramine. The capacity of GSH to react with this ROS suggests an additional role for this tripeptide in cellular protection against oxidant injury.     

Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene

L-[U-¹⁴C]Methionine fed to apple tissue was efficiently converted to ethylene when the tissue was incubated in air. In nitrogen, however, it was not metabolized to ethylene but was instead converted to 1-aminocyclopropane-1-carboxylic acid (ACC). When apple tissues were fed with L-[methyl-¹⁴C]methionine or L-[³⁵S]methionine and incubated in nitrogen, radioactivity was found subsequently in methylthioribose. This suggests that methionine is first converted to S-adenosylmethionine which is in turn fragmented to ACC and methylthioadenosine. Methylthioadenosine is then hydrolyzed to methylthioribose. The conclusion that ACC is an intermediate in the conversion of methionine to ethylene is based on the following observations: Labeled ACC was efficiently converted to ethylene by apple tissue incubated in air; the conversion of labeled methionine to ethylene was greatly decreased in the presence of unlabeled ACC, but the conversion of labeled ACC to ethylene was little affected by the presence of unlabeled methionine; and 2-amino-4-(2′-aminoethoxy)trans-3-butenoic acid, a potent inhibitor of pyridoxal phosphate-mediated enzyme reactions, greatly inhibited the conversion of methionine to ethylene but did not inhibit conversion of ACC to ethylene. These data indicate the following sequence for the pathway of ethylene biosynthesis in apple tissue: methionine → S-adenosylmethionine → ACC → ethylene. A possible mechanism accounting for these reactions is presented.     

NBD-5-acylcholine: fluorescent analog of acetylcholine and agonist at the neuromuscular junction.

We have studied the properties of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-omega-aminohexanoic acid beta-(N-trimethylammonium)ethyl ester, a fluorescent analog of acetylcholine at the cellular level by using pharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetylcholine receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is a powerful agonist of acetylcholine at the neuromuscular junction and also strongly desensitizes muscle fibers. Interaction with acetylcholine receptor is accompanied by large changes in the drug's fluorescence. From the kinetics of interaction studied by means of a stopped-flow fluorimeter with laser light source, we obtained a second-order forward rate constant in excess of 1 X 10(8) M-1 sec-1 and an initial dissociation rate constant (k1) of 0.5 sec-1 for receptor from Electrophorous electricus. Interaction of this analog with acetylcholine esterase from E. electricus is accompanied by a transient decrease in fluorescence followed by an increase leading to a stable plateau value at a level near the original one. The initial decrease in fluorescence followed second-order kinetics with k2 of the order of 10(9) M-1 sec-1. The slower consecutive reaction which could be blocked by phosphorylation of the esteratic site, was of first order with k1 = 0.05 sec-1.     

Singlet oxygen interaction with Ca(2+)-ATPase of cardiac sarcoplasmic reticulum.

We investigated the role of singlet oxygen (generated from photoactivation of rose bengal) on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum (SR). Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at 560 nm resulted in significant inhibition of Ca2+ uptake (from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min [mean +/- SEM], p less than 0.01) and Ca(2+)-ATPase activity (from 2.08 +/- 0.05 to 0.28 +/- 0.04 mumol Pi/min.mg [mean +/- SEM], p less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal-derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. Singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal-derived activated oxygen species, but superoxide dismutase and catalase did not attenuate the inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal for up to 14 minutes demonstrated complete loss of the Ca(2+)-ATPase monomer band, which was significantly protected by histidine. The addition of dithiothreitol (5 mM) had a slight protective effect, showing that new disulfide bond formation was not a major cause of aggregation. The results were also confirmed by high-performance liquid chromatography of the SR exposed to irradiated rose bengal. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide radical (generated from xanthine oxidase action on xanthine) and hydroxyl radical (in the presence of Fe(3+)-EDTA or 0.5 mM H2O2 plus Fe(2+)-EDTA) as well as H2O2 (0.25-12 mM) were without any effect on the 97,000-d Ca(2+)-ATPase band of SR. Generation of radical species (superoxide and hydroxyl radical) from rose bengal was studied by electron paramagnetic resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results showed that irradiation of rose bengal formed a 1:2:2:1 quartet, characteristic of the DMPO-OH adduct, which was scavenged by ethanol but not by superoxide dismutase, catalase, or histidine. No radical species could be detected from irradiated rose bengal or irradiated DMPO under the assay conditions used. Peroxy adducts of DMPO might be produced but would be observed only at very low temperatures. Similarly, we could not detect any measurable.O2- anion from irradiation of rose bengal as indicated by either cytochrome c reduction at 550 nm or nitro blue tetrazolium reduction at 560 nm. These results show that SR is damaged most likely by singlet oxygen derived from rose bengal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction with melatonin and with other hydroxyl radical scavengers

Barbituric acid (2,4,6-pyrimidinetrione) can be transformed by a non-enzymatic hydroxylation into alloxan (2,4,5,6-pyrimidinetetrone). This transformation can be used as a reaction indicating the formation of hydroxyl radicals (.OH). This conversion was detected using HPLC. Formation of .OH was demonstrated by electron spin resonance (ESR) spectroscopy combined with spin-trapping techniques. It was shown that .OH generated via the Fenton reaction abstracts first a hydrogen atom from barbituric acid (BA) and forms intermediately a paramagnetic derivative of BA. After a second attack by another .OH, the BA radical is transformed into dialuric acid (DA), which autoxidizes via the alloxan radical (.ALX) to ALX. Superoxide radicals (.O2-) are formed during autoxidation of DA and.ALX. They are able to regenerate ferrous ions. As a result, traces of iron salts are capable of catalyzing the conversion of large amounts of BA into ALX. Several scavengers of .OH were tested with regard to their efficiency in preventing the transformation of BA into ALX. Of all the scavengers analyzed, melatonin was shown to be one of the most potent compounds.     

Corticosteroid receptors in the kidney of chick embryo. III. Nature, properties, and ontogeny of aldosterone receptor

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4 degrees revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association (ka) and dissociation (kd) rate constants were, respectively, 4.94 X 10(5) M-1 sec-1 and 8.33 X 10(-6) sec-1. From their ratio a KA value of 5.9 X 10(10) M-1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the KA at equilibrium, obtained from the Scatchard plot, was 3.1 +/- 1.2 X 10(8) M-1, whereas the Nmax was 172 +/- 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.     

Phagocyte-derived free radicals stimulated by ingestion of iron-rich Staphylococcus aureus: a spin-trapping study.

Phagocytic cells generate superoxide (O2-) and hydrogen peroxide (H2O2), creating the substrates for hydroxyl radical (HO.) in the presence of redox active metals. Previously it was shown that HO. is not a physiologic product of human neutrophils or monocytes but can be generated in the presence of high concentrations of iron. This study was undertaken to determine whether bacterial iron could be used for the generation of HO. The growth of Staphylococcus aureus under iron-rich conditions increased bacterial iron concentration and phagocytosis of iron-rich bacteria allowed neutrophils to accumulate threefold more iron than ingestion of iron-starved organisms. Neither neutrophils nor monocytes ingesting iron-rich S. aureus generated iron-catalyzed HO. at levels detectable by spin-trapping techniques. No differences in the killing of iron-rich organisms by neutrophils was noted. The results suggest that HO. does not play a role in the killing of S. aureus by human neutrophils, regardless of their ability to deliver iron to the cell.