Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for α-globin over that for β-globin and intact HbA in an adenosine 5′-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the α-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in α-globin compared with intact haemoglobin, HbA and the β-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the α-globin chain over that of the β-globin in the haemoglobin disorder β-thalassaemia.     

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.     

Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.     

Pretreatment of lignocellulosic materials with hydrogen peroxide in presence of manganese compounds

Pretreatment of lignocellulosic materials such as newspaper, rice straw, pulp waste, and municipal solid waste with hydrogen peroxide in the presence of manganese compounds greatly enhances their susceptibility to enzymatic saccharification. This pretreatment can be achieved using rather mild conditions with only a minimal decrease in the recovery and little change in composition. Manganese salts in this hydrogen peroxide pretreatment works effectively in particular when the concentration of hydrogen peroxide is relatively low. The susceptibility of hydrogen-peroxide-pretreated substrate to enzymatic saccharification increases with increasing the molar ratio of manganes to hydrogen peroxide up to 1 : 100.     

Peroxynitrite decay in the presence of hydrogen peroxide,mannitol and ethanol: A reappraisal

We have reported previously that the apparent rate of peroxynitrite (ONOO^-) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331–1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing peroxynitrate (O₂NOO^-) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458–3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695–10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxide dismutase and, in the case of mannitol and ethanol, by reducing the dioxygen concentration of the reaction solutions. Thus, superoxide formation is necessary for the decrease in the rate of absorbance decay. In addition, by simulations using known rate constants and absorption coefficients, we show that the slowing effect can be quantitatively accounted for by the formation of peroxynitrate.     

Peroxynitrite decay in the presence of hydrogen peroxide, mannitol and ethanol: a reappraisal

We have reported previously that the apparent rate of peroxynitrite (ONOO(-) ) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331-1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing peroxynitrate (O(2) NOO(-) ) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458-3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695-10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxide dismutase and, in the case of mannitol and ethanol, by reducing the dioxygen concentration of the reaction solutions. Thus, superoxide formation is necessary for the decrease in the rate of absorbance decay. In addition, by simulations using known rate constants and absorption coefficients, we show that the slowing effect can be quantitatively accounted for by the formation of peroxynitrate.     

Killing of schistosomes by elastase and hydrogen peroxide: implications for leukocyte-mediated schistosome killing

Activated leukocytes participate in immunity to infection by the parasitic blood fluke Schistosoma mansoni. They attach to the surface of schistosomes and secrete schistosomicidal substances. Cationic proteins, hydrolytic enzymes, and oxidants, produced by the leukocytes, have been implicated in the damage to the schistosomes. To examine the possible involvement of elastase in the killing of schistosomes by leukocytes, young and adult stages of S. mansoni were treated in vitro with pancreatic elastase (PE) and neutrophil elastase (NE). Schistosomula, lung-stage schistosomula (LSS), and adult worms (AW) have been found to be sensitive to both PE and NE. Male AW were more sensitive to PE than female AW. The enzymatic activity of elastase is essential for its toxic effect because heat-inactivation and specific elastase inhibitors prevented elastase-mediated schistosome killing. Thus, alpha1-antitrypsin and the chloromethyl ketone (CMK)-derived tetrapeptides Ala-Ala-Pro-Val-CMK and Ala-Ala-Pro-Ala-CMK but not Ala-Ala-Pro-Phe-CMK and Ala-Ala-Pro-Leu-CMK blocked PE caseinolytic and schistosomulicidal activities. As shown previously, schistosomes are also efficiently killed by hydrogen peroxide. LSS appear to be more resistant than AW and early-stage schistosomula to the lytic effects of hydrogen peroxide. Cotreatment experiments with both elastase and hydrogen peroxide indicated that they exert an additive toxic effect and that hydrogen peroxide sensitizes schistosomula to the toxic effect of elastase but not vice versa. These results demonstrate, for the first time, that elastases may be toxic molecules used by neutrophils, eosinophils, and macrophages to kill various developmental stages of S. mansoni.     

The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution

Background  

The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire.     

Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.     

Arachidonic acid metabolites, hydrogen peroxide, and EDHF in cerebral arteries

We tested the hypotheses that EDHF in rat middle cerebral arteries (MCAs) involves 1) metabolism of arachidonic acid through the epoxygenase pathway, 2) metabolism of arachidonic acid through the lipoxygenase pathway, or 3) reactive oxygen species. EDHF-mediated dilations were elicited in isolated and pressurized rat MCAs by activation of endothelial P2Y(2) receptors with either UTP or ATP. All studies were conducted after the inhibition of nitric oxide synthase and cyclooxygenase with N(omega)-nitro-l-arginine methyl ester (10 microM) and indomethacin (10 microM), respectively. The inhibition of epoxygenase with miconazole (30 microM) did not alter EDHF dilations to UTP, whereas the structurally different epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanoic acid (20 or 40 microM) only modestly inhibited EDHF at the highest concentration of UTP. An antagonist of epoxyeicosatrienoic acids, 14,15-epoxyeicosa-5(Z)-enoic acid, had no effect on EDHF dilations to UTP. Chronic inhibition of epoxygenase in the rat with 1-aminobenzotriazol (50 mg/kg twice daily for 5 days) did not alter EDHF dilations. The inhibition of the lipoxygenase pathway with either 10 microM baicalein or 10 microM nordihydroguaiaretic acid produced no major inhibitory effects on EDHF dilations. The combination of superoxide dismutase (200 U/ml) and catalase (140 U/ml) had no effect on EDHF dilations. Neither tiron (10 mM), a cell-permeable scavenger of reactive oxygen species, nor deferoxamine (1 or 10 mM), an iron chelator that blocks the formation of hydroxyl radicals, altered EDHF dilations in rat MCAs. We conclude that EDHF dilations in the rat MCA do not involve the epoxygenase pathway, lipoxygenase pathway, or reactive oxygen species including H(2)O(2).     

N-dealkylation of aminopyrine catalyzed by soybean lipoxygenase in the presence of hydrogen peroxide

A hypothesis that lipoxygenase may mediate N-dealkylation of xenobiotics was investigated using the prototype drug aminopyrine and soybean lipoxygenase as a model enzyme in the presence of hydrogen peroxide. Formaldehyde production as a result of N-demethylation of aminopyrine exhibited pH optimum of 6.5. The reaction was dependent on the incubation time, amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. Under the experimental conditions employed, the specific activity for N-demethylation of aminopyrine was found to be 823 ± 93 nmoles per min/mg protein or 89 ± 10 nmoles per min/nmole of enzyme. The reaction was significantly inhibited by nordihydroguaiaretic acid and gossypol, the classical inhibitors of lipoxygenase. Spectrophotometric analyses indicated the generation of a nitrogen-centered free-radical cation as the initial oxidation product of aminopyrine. The rate of accumulation of this radical species was also dependent on pH, the amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. The radical production was markedly suppressed by ascorbate, glutathione, and dithiothreitol in a concentration-dependent manner. Preliminary data gathered for the oxidation of other chemicals indicated that the lipoxygenase exhibits a unique substrate specificity. Collectively, the evidence presented suggests for the first time that lipoxygenase pathway may be involved in N-demethylation of aminopyrine and other chemicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 175–183, 1998     

Degradation of high-molecular-weight hyaluronan by hydrogen peroxide in the presence of cupric ions

Dynamic viscosity (eta) of the high-molecular-weight hyaluronan (HA) solution was measured by a Brookfield rotational viscometer equipped with a Teflon cup and spindle of coaxial cylindrical geometry. The decrease of eta of the HA solution, indicating degradation of the biopolymer, was induced by a system containing H2O2 alone or H2O2 plus CuCl2. The reaction system H2O2 plus CuCl2 as investigated by EPR spin-trapping technique revealed the formation of a four-line EPR signal characteristic of a *DMPO-OH spin adduct. Thus, hydroxyl radicals are implicated in degradation of high-molecular-weight HA by the system containing H2O2 and CuCl2.     

Consequences of MnSOD interactions with nitric oxide: nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^- ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^-  species initiate the formation of peroxynitrite (ONOO^- ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^-  decomposition and ONOO^- -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^-  is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Light-stimulated formation of hydrogen peroxide and hydroxyl radical in the presence of uroporphyrin and ascorbate

Blue light irradiation of 2-deoxyribose (DOR) in the presence of uroporphyrin I (UP), ascorbate (AH-), trace iron, and phosphate buffer resulted in a strong stimulation of hydroxyl radical (OH.)-dependent oxidation of DOR. Photostimulated generation of H2O2 was monitored after removal of residual AH- (i) by ascorbate oxidase treatment, or (ii) by anion exchange on mini-columns of DEAE-Sephadex. Irradiation of the above mixture produced a strong burst of H2O2 which was intensified by desferrioxamine and suppressed by catalase or EDTA. The mechanism suggested by these observations is one in which photoreduction of UP to the radical anion initiates the formation of H2O2, which gives rise to OH. via Fenton chemistry. This is the first known investigation of H2O2 fluxes in a Type I (free radical) photoreaction involving AH- as the electron donor.     

High-performance liquid chromatography of globin chains in the identification of human globin gene abnormalities

This article summarizes experience and data obtained using a previously developed reverse-phase high-performance liquid chromatography method (J.B. Shelton, J.R. Shelton and W.A. Schroeder, J. Liq. Chromatogr. 7 (1984) 1969) in the study of a number of hemoglobinopathies in the Sardinian population. The occurrence and incidence of several abnormal hemoglobins are described, as well as aspects of the expression of abnormal gamma-globin gene arrangements and thalassemic genes.