ascorbic-acid and 3-5-dibromo-4-nitrosobenzenesulfonate

Exposure of human blood plasma to peroxynitrite in the presence of 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) resulted in the trapping of a strongly immobilized nitroxide radical adduct. The adduct was due to protein-centred radicals derived not only from serum albumin but also from other major plasma proteins (fibrinogen, IgG, alpha1-antitrypsin and transferrin). Urate significantly protected plasma from the peroxynitrite-induced DBNBS-plasma protein adduct, whereas ascorbate and glutathione were protective at concentrations exceeding those usually found in plasma. Alkylation of plasma -SH groups did not affect the intensity of DBNBS-plasma protein adduct, whereas bicarbonate increased its formation, thus showing a pro-oxidant effect. The DBNBS-plasma protein adduct provided little structural information, but subsequent non-specific-protease treatment resulted in the detection of an isotropic three-line spectrum, indicating the trapping of radicals centred on a tertiary carbon. The nitrogen hyperfine coupling constant of this adduct and its superhyperfine structure were similar to those of DBNBS-tryptophan peptides with the alpha-amino group of tryptophan linked in the amide bond, consistent with a radical adduct formed at C-3 of the indole ring of tryptophan-containing peptides. DBNBS was unable to trap radicals derived from peroxynitrite-treated tyrosine or tyrosine-containing peptides. Methionine treated with peroxynitrite resulted in the trapping of at least two DBNBS-methionine adducts with hyperfine structures different from that of protease-treated DBNBS-plasma proteins. These results demonstrate that peroxynitrite induced in blood plasma the formation of protein radicals centred on tryptophan residues and underline the relevance of the one-electron oxidation pathway of peroxynitrite decomposition in biological fluids.

Topics: Ascorbic Acid; Benzenesulfonates; Bicarbonates; Blood Proteins; Chromatography, Gel; Electron Spin Resonance Spectroscopy; Electron Transport; Free Radicals; Glutathione; Humans; Methionine; Nitrates; Nitroso Compounds; Peptides; Pronase; Spin Trapping; Sulfhydryl Compounds; Tryptophan; Uric Acid

Degradation of collagen by oxidant species may play an important role in the progression of rheumatoid arthritis. Whilst the overall effects of this process are reasonably well defined, little is known about the sites of attack, the nature of the intermediates, or the mechanism(s) of degradation. In this study electron paramagnetic resonance spectroscopy with spin trapping has been used to identify radicals formed on collagen and related materials by metal ion-H2O2 mixtures. Attack of the hydroxyl radical, from a Fe(II)-H2O2 redox couple, on collagen peptides gave signals from both side chain (.CHR'R"), and alpha-carbon[.C(R)(NH-)CO-,R = side-chain]radicals. Reaction with collagen gave both broad anisotropic signals, from high-molecular-weight protein-derived radicals, and isotropic signals from mobile species. The latter may be low-molecular-weight fragments, or mobile side-chain species; these signals are similar to those from the alpha-carbon site of peptides and the side-chain of lysine. Enzymatic digestion of the large, protein-derived, species releases similar low-molecular-weight adducts. The metal ion employed has a dramatic effect on the species observed. With Cu(I)-H2O2 or Cu(II)-H2O2 instead of Fe(II)-H2O2, evidence has been obtained for: i) altered sites of attack and fragmentation, ii) C-terminal decarboxylation, and iii) hydrogen abstraction at N-terminal alpha-carbon sites. This altered behaviour is believed to be due to the binding of copper ions to some substrates and hence site-specific damage. This has been confirmed in some cases by electron paramagnetic resonance studies of the Cu(II) ions.

Topics: Amino Acids; Ascorbic Acid; Benzenesulfonates; Collagen; Collagenases; Copper; Electron Spin Resonance Spectroscopy; Hydrogen Peroxide; Hydroxyl Radical; Iron; Metals; Nitroso Compounds; Oxidants; Oxidation-Reduction; Peptides; Phenylhydrazines; Protein Binding; Spin Trapping

Depletion of antioxidants and the presence of products of free radical damage in plasma suggest that oxidative stress is increased in uremia. We have developed an application of electron spin resonance spectroscopy, and used this method to show that a stable oxidizing component or components of plasma accumulate in uremia. No oxidizing activity was detectable in plasma from subjects with normal renal function. The oxidant was detected by its capacity to oxidize the spin trap 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS). The oxidant was dialyzable from plasma, had an upper molecular weight limit of about 3,000 Daltons and was stable over many months. Physiological plasma concentrations of vitamin C, a water soluble congener of vitamin E and reduced glutathione were unable to inhibit the oxidizing capacity of uremic plasma. Thus, uremia is associated with accumulation of an endogenous oxidizing activity at much higher concentrations than in subjects with normal renal function.

Topics: Adult; Antioxidants; Ascorbic Acid; Azides; Benzenesulfonates; Chromans; Creatinine; Electron Spin Resonance Spectroscopy; Endopeptidases; Female; Glutathione; Humans; Hydrolysis; Leukocyte Count; Male; Middle Aged; Neutrophils; Nitroso Compounds; Oxidants; Oxidation-Reduction; Renal Dialysis; Spin Labels; Uremia; Vitamin E

Extracellular fluids contain low-molecular-weight antioxidants that are actively involved in the defense against reactive oxygen species. The antioxidant activity of these compounds is largely due to their ability to trap oxygen radicals. Less known is the ability of extracellular antioxidants to scavenge carbon-centered free radicals (C-radicals). These radicals can be involved in the damage under hypoxic/anoxic conditions as well as in ischemia/reperfusion injury. We studied the reactivity of some plasma antioxidants toward a water-soluble C-radical generated by the azocompound 2,2'-azobis(2-amidinopropane) hydrochloride (AAP) under anaerobic conditions. The AAP C-radical in plasma was trapped by the spin trap 3,5-dibromo-4-nitrosobenzene-sulfonic acid (DBNBS) and produced a DBNBS radical. The scavenging properties of urate, cysteine, glutathione, natural amino acids, and serum albumin were assessed by the inhibition of the intensity of DBNBS radical. The antioxidant activity of ascorbate and that of vitamin E was measured directly by the formation of their free radicals. Urate, vitamin E and non-SH amino acids were ineffective and ascorbate was a poor scavenger of AAP C-radical. At variance, cysteine and glutathione (0.1-1.0 mM) were effective scavengers of AAP C-radicals and, importantly, protected plasma ascorbate from oxidation under both aerobic or anaerobic conditions. Our data show that ascorbate in aerobic plasma can reduce vitamin E radical and the oxidized ascorbate may be recycled by a thiol antioxidant cycle. Low-molecular-weight antioxidants accounted only partially for plasma scavenging activity of C-radicals. Plasma strongly reduced the intensity of DBNBS radical and, after dialysis, its activity was reduced by approximately 10%. Serum albumin showed an antioxidant activity comparable to dialyzed plasma. Also the cysteine residue of serum albumin was an efficient scavenger of C-radicals as shown by approximately 20% decrease in the protein scavenging activity after thiol alkylation. These results suggest that elevation in the concentration of total reduced thiols in plasma may improve its antioxidant activity under hypoxic/anoxic conditions. This may be particularly useful since other important antioxidant mechanisms such as urate, ascorbate, and vitamin E appear to be inefficient.

Topics: Aerobiosis; Amidines; Anaerobiosis; Antioxidants; Ascorbic Acid; Benzenesulfonates; Blood Proteins; Carbon; Cysteine; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Glutathione; Humans; Molecular Weight; Nitroso Compounds; Serum Albumin; Spin Labels; Sulfhydryl Compounds; Uric Acid; Vitamin E