Oxidative modification of ferritin induced by hydrogen peroxide

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. In this study, we assessed the modification of ferritin induced by H(2)O(2). When ferritin was incubated with H(2)O(2), the degradation of ferritin L-chain increased with the H(2)O(2) concentration whereas ferritin H-chain was remained. Free radical scavengers, azide, thiourea, and N-acetyl-(L)-cysteine suppressed the H(2)O(2)-mediated ferritin modification. The iron specific chelator, deferoxamine, effectively prevented H(2)O(2)-mediated ferritin degradation in modified ferritin. The release of iron ions from ferritin was increased in H(2)O(2) concentration-dependent manner. The present results suggest that free radicals may play a role in the modification and iron releasing of ferritin by H(2)O(2). It is assumed that oxidative damage of ferritin by H(2)O(2) may induce the increase of iron content in cells and subsequently lead to the deleterious condition.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Chemical modification of hyaluronic acid by carbodiimides.

Hyaluronic acid (HA) is a linear polysaccharide with repeating disaccharide units of glucuronic acid and N-acetylglucosamine and is found in the extracellular matrix of connective tissues. Reaction of high molecular weight sodium hyaluronate (NaHA, MW approximately 2 x 10(6] with EDC at pH 4.75, either in the presence or absence of a primary diamine, gave the N-acylurea and O-acylisourea as NaHA-carbodiimide adducts. None of the expected intermolecular coupling with the amine component was observed. On the basis of this new observation, this method for chemical modification of HA was used in conjunction with new synthetic carbodiimides to prepare HA derivatives bearing lipophilic, aromatic, cross-linked, and tethered functional groups. The degree of conversion to NaHA-acylurea products appears to depend upon both the characteristics of various carbodiimides and the conformational structure of NaHA.     

Inhibitory activity of berberine on DNA strand cleavage induced by hydrogen peroxide and cytochrome c

The inhibitory activity of berberine on the DNA single-strand cleavage induced by hydrogen peroxide and cytochrome c was measured. Berberine effectively inhibited single-strand cleavage of DNA and its effectiveness was concentration-dependent. As the berberine concentration increased, the inhibitory activity against the DNA single-strand cleavage increased. The treatments with 1, 5, 10, 50, and 100 microM berberine showed 7.7, 10.8, 32.2, 39.5, and 51.6% inhibition of DNA cleavage. This inhibitory activity of berberine against the DNA single-strand cleavage has never been reported previously. The inhibitory activity of berberine against DNA cleavage was stronger than caffeic acid and ascorbic acid. Berberine did not show strong hydroxyl radical scavenging activity, but showed strong superoxide anion radical quenching ability.     

Degradation of 2-deoxyaldoses by alkaline hydrogen peroxide

Reaction of 2-deoxy-D-arabino-hexose, 2-deoxy-D-lyxo-hexose, and 2-deoxy-D-erythro-pentose with alkaline hydrogen peroxide in the presence of magnesium hydroxide afforded the corresponding 2-deoxyaldonic acid, the 1,4-lactone, and the 1-O-formyl derivative of the next lower alditol. The 2-deoxyaldonic acids were separated in 60–80% yields, as new, crystalline lithium salts. The 1,4-lactones were obtained under conditions that precluded intermidiate formation of the free acids: presumably, the reaction proceeded by way of an intermediate, furanosyl hydroperoxide, which was converted into the lactone by elimination of water. With an excess of alkaline hydrogen peroxide, in the absence of magnesium hydroxide, the substrates were degraded to formic acid, with concurrent decomposition of hydrogen peroxide. It is shown that decomposition of hydrogen peroxide is catalyzed by hydroperoxide anion, and that it takes place by both a chain, and a non-chain, process. The decomposition reactions afford an abundant source of hydroxyl radical capable of oxidizing a wide variety of compounds.     

Oxidative modification of cytochrome c by hydrogen peroxide

Oxidative alteration of mitochondrial cytochrome c has been linked to disease and is one of the causes of pro-apoptotic events. We have investigated the modification of cytochrome c by H2O2. When cytochrome c was incubated with H2O2, oligomerization of the protein increased and the formation of carbonyl derivatives and dityrosine was stimulated. Radical scavengers prevented these effects suggesting that free radicals are implicated in the H2O2-mediated oligomerization. Oligomerization was significantly inhibited by the iron chelator, deferoxamine. During incubation of deoxyribose with cytochrome c and H2O2, damage to the deoxyribose occurred in parallel with the release of iron from cytochrome c. When cytochrome c that had been exposed to H2O2 was analyzed by amino acid analysis, the tyrosine, histidine and methionine residues proved to be particularly sensitive. These results suggest that H2O2-mediated cytochrome c oligomerization is due to oxidative damage resulting from free radicals generated by a combination of the peroxidase activity of cytochrome c and the Fenton reaction of free iron released from the oxidatively-damaged protein.     

Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging.

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.     

Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture.

The depression of proteoglycan synthesis in ten-day-old high density chondrocyte cultures was shown to be dependent on both the concentration and time of exposure of the cells to hyaluronic acid. Hyaluronic acid had no effect on the overall protein synthesis by the cultured cells. Using benzyl-beta-D-xyloside an exogenous acceptor, it was shown that glycosaminoglycan biosynthesis by the chondrocytes was not affected by hyaluronic acid. It was concluded that hyaluronic acid was effecting glycosaminoglycan chain initiation, hence proteoglycan biosynthesis, either by specifically depressing the synthesis of the core protein or by repressing the activity of the xylosyltransferase.     

Maltol inhibits apoptosis of human neuroblastoma cells induced by hydrogen peroxide

To analyze the effect of Maltol on the apoptosis of Human Neuroblastoma Cells (SH-SY5Y) treated by free radical which was generated from Hydrogen Peroxide (H2O2), flow cytometry analysis on Phosphatidylserine (PS) inverting percentage was applied to determine the apoptosis. MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was employed to analyze the cell viability. DNA electrophoresis was used to detect DNA fragmentation. Moreover intracellular calcium of concentration ([Ca2+]i) was measured by fluorescence emission. Flow cytometry analysis on the function of mitochondria and Western blot analysis of NF-kappaB. The results showed that the pretreatment with maltol for 2 hours could prevent the H2O2-induced apoptosis. Maltol could reduce the inverting percentage of PS, DNA fragmentation and [Ca2+]i, and enhance the cellular function of mitochondria. NF-kappaB activated by H2O2 is reduced. The experiments suggest that maltol could effectively inhibit the apoptosis induced by H2O2. As a novel anti-oxidant, maltol is a new promising drug in protecting the neurological cells from the damage by free radical.     

Complete amino acid sequence of a human platelet proteoglycan

The primary structure of a human platelet proteoglycan (P.PG) core was established by a combination of amino acid sequence analysis and cDNA cloning. The deduced 131 amino acid long protein contains eight Ser-Gly repeats. The significance of homologies observed between P.PG and promyelocytic leukemia cell line proteoglycans is discussed.     

Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.     

Abscisic acid mediates hydrogen peroxide production in peanut induced by water stress

Peanut plants exposed to water stress induced by polyethylene glycol (PEG) accumulated abscisic acid (ABA) and hydrogen peroxide (H₂O₂), the increase being significant at 12 and 24 h after addition, respectively. To address the question whether the increase in H₂O₂ production was related to ABA accumulation, the peanut leaves were pretreated with ABA biosynthesis inhibitor (sodium tungstate) and then exposed to water stress. Under these conditions, a decrease of ABA and H₂O₂ content were found after 12 h. The addition of 100 μM ABA restored H₂O₂ content reaching values similar to those under water stress at 12 h. We concluded that ABA accumulation is the first signal that triggers the H₂O₂ generation in peanut during first 12 h but its subsequent production is partially ABA-independent.     

A small proteoglycan isolated from human cartilage containing a nonfunctional hyaluronic acid binding region.

A low buoyant density fraction (A4) was isolated from human cartilage by CsCl density gradient ultracentrifugation. This fraction contained a hydrodynamically small proteoglycan (Kav, 0.74 on Sepharose CL-2B) that reacted with monoclonal antibody 12/20/1C6 specific for the hyaluronic acid binding region (G1 globe) of the large aggregating high-density proteoglycan isolated from many animal cartilages. Despite the presence of the hyaluronic acid binding region, this small proteoglycan did not form proteoglycan aggregates with hyaluronan, not even in the presence of link protein.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage

Oxygen-derived reactive species, generated enzymatically by the action of xanthine oxidase upon hypoxanthine, significantly inhibit proteoglycan synthesis by cultured bovine articular cartilage (Bates, E.J., Lowther, D.A. and Handley, C.J. (1984) Ann. Rheum. Dis. 43, 462-469). Here we extend these investigations and show, through the use of catalase and the specific iron chelator diethylenetriaminepentaacetic acid, that the active species involved is H2O2 and not the hydroxyl radical. Incubations of cartilage with H2O2 at concentrations of 1 X 10(-4) M and above are also inhibitory to proteoglycan synthesis. Subsequent recovery of the tissue is dependent upon the initial dose of xanthine oxidase or H2O2. Xanthine oxidase at 84 mU per incubation results in a prolonged inhibition of proteoglycan synthesis which is still apparent after 14 days in culture. Lower concentrations of xanthine oxidase (21-66 mU) are inhibitory to proteoglycan synthesis, but the tissue is able to synthesise proteoglycans at near normal rates after 3 days in culture. The inhibition of proteoglycan synthesis by 1 X 10(-4) M H2O2 is completely reversed after 5 days in culture, whereas 1 X 10(-3) M H2O2 results in a more prolonged inhibition. The synthesis of the proteoglycan core protein is inhibited, but the ability of the newly formed proteoglycans to aggregate with hyaluronic acid is unimpaired.     

Studies on the generation of hydrogen peroxide during some non-enzymic reactions changing the hyaluronic acid molecule.

The effect of catalase on non-enzymic-induced changes in the conformation of hyaluronic acid in a vitreous humour preparation was measured using viscometry. Ascorbate, heavy metal ions, riboflavin or EDTA all lowered the viscosity of hyaluronic acid solutions. These effects could be prevented by the addition of catalase. This suggested that H2 O2 is produced by these compounds and that the resulting change in conformation of hyaluronic acid may be due to peroxyl and hydroxyl attack by the free radicals thus generated.