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The effect of bicarbonate anion (HCO 3 ؊ ) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5,5-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2,2-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO 3 Ϫ enhanced SOD/H 2 O 2 -dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UVvisible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H 2 O and not from H 2 O 2 . This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO 3 Ϫ enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO 3 Ϫ -mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis. Bicarbonate anion (HCO 3Ϫ ) is abundantly present in biological systems (1). In vitro studies have shown that HCO 3 Ϫ can dramatically alter the nitrating and oxidizing ability of reactive nitrogen and oxygen species (2-4). Recently, HCO 3 Ϫ was shown to exacerbate the peroxidase activity of the enzyme, copper, zinc superoxide dismutase (SOD1) (5).1 HCO 3 Ϫ enhanced both the hydroxylation of 5,5Ј-dimethyl-1-pyrroline N-oxide (DMPO) to DMPO-OH and oxidation of ABTS chromophore to the ABTS radical cation in the presence of SOD1 and H 2 O 2 (5). HCO 3 Ϫ was reported to increase the peroxidase activity of SOD1 by facilitating the redox cleavage of H 2 O 2 (5).Stadtman, Yim, and co-workers (6) originally proposed that SOD1 reacts with H 2 O 2 to form hydroxyl radicals as evidenced by increased hydroxylation of nitrone traps and peroxidation of ABTS chromophore. Subsequently, it was suggested that familial amyotrophic lateral sclerosis-associated SOD1 mutants increased formation of hydroxyl radical upon reaction with H 2 O 2 (7, 8). HCO 3 Ϫ stimulated the peroxidase activity of the extracellular SOD activity, which was attributed to an increased formation of hydroxyl radicals (9). Hydroxyl radicals formed from the reaction between SOD1 and H 2 O 2 were suggested to be responsible for the increased cytotoxicity of SOD1 (10).In the present work, we show that HCO 3 Ϫ enhances (i) the hydroxylation of nitrone spin trap, DMPO, (ii) oxidation and nitration of tyrosine, and (iii) oxidation of the peroxidase probe ABTS to the ABTS radical cation in the presence of SOD1 and H 2 O 2 . We propose that the carbonate anion radical (CO 3 . )formed from oxidation of HCO 3 Ϫ at the active site of SOD1 by the copper, zinc SOD-bound hydroxyl radical (SOD1-Cu 2ϩ -⅐ OH) is responsible for hydroxylati...

Synthetic dopa melanin and cysteinyldopa melanin have different electron spin resonance spectra. Data are reported for mixtures of these melanins and for dopa-cysteinyldopa copolymers, which are spectroscopically similar. A simple parameterization of the spectra allows estimation of the relative amounts of (i) dopa melanin and cysteinyldopa melanin in mixtures and of (ii) dopa and cysteinyldopa incorporated into copolymers. Several natural eumelanins and pheomelanins have been characterized and shown to be copolymers.

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Synthetic pheomelanins from enzymic oxidation of the 3,4-dihydroxyphenylalanine (dopa) derivative 5-S-cysteinyldopa have been examined by ESR spectroscopy. These alkalisoluble polymers contain a novel kind of free radical that is spectroscopically distinct from that found in eumelanins. Delocalization of the unpaired electron onto a nitrogen atom and the ability of the radical to chelate complexing metal ions strongly suggest an o-semiquinonimine structure. The synthetic pheomelanin was compared with natural red pigments extracted from human red hair and from red chicken feathers. Spectroscopically, the chicken feather pheomelanin is almost identical to synthetic cysteinyldopa pheomelanin. In contrast, the pigment from red hair has a major spectral component very similar to that found in dopa melanin, with a smaller component corresponding to that found in cysteinyldopa melanin.Melanin pigmentation ofhair, skin, and eye is thought to result from two separate but biogenetically interrelated classes ofpigments: eumelanins (black-brown, insoluble in dilute alkali) and pheomelanins (yellow to reddish-brown, alkali soluble) (1). Eumelanins are generally considered to be derived from the enzymic oxidation of tyrosine through 3,4-dihydroxyphenylalanine (dopa) (structure I) (2), whereas pheomelanins arise by a diversion of the eumelanin pathway through the intervention of cysteine or related sulfhydryl compounds (3). A major intermediate in the biosynthesis of pheomelanins is the dopa metabolite 5-S-cysteinyldopa (structure II).

The characterization and identification of semiquinone radicals from catechol(amine)s and catechol estrogens by electron spin resonance spectroscopy is addressed. The use of diamagnetic metal ions, especially Mg2+ and Zn2+ ions, to detect transient semiquinone radicals in biological systems and to monitor their reactions, is discussed. A brief account of the identification and reactions of quinones is also presented.

Electron spin resonance spectroscopy has recently been used by others to detect directly radical species in isolated perfused hearts. Sample processing prior to spectroscopy in this study involved pulverization of tissue, which can artifactually generate radical species. We assessed in isolated perfused hearts the influence of tissue pulverization on the identity of radical species detected by spectroscopy and then, using a processing technique less likely to induce artifacts, whether myocardial ischemia and reperfusion generate radical species. Rat and rabbit hearts (n = 8) were perfused aerobically for 10 min and freeze-clamped to -196C. Frozen tissue was processed at -196°C for spectroscopic analysis by pulverization vs. chopping. Spectra of pulverized tissue consisted of three components: a semiquinone (g = 2.004), a lipid peroxy radical (g11 = 2.04 and gL = 2.006), and a carboncentered radical that is possibly a lipid radical (gl,, = 2.002 and A' 50 G). Chopped tissue consisted of a single component, a semiquinone (g = 2.004). Rat hearts (n = 8 per group) also underwent 10-min global no-flow normothermic ischemia followed by 5-60 sec of either aerobic or anaerobic reperfusion, with frozen tissue chopped prior to spectroscopy. Spectra of ischemic tissue consisted of an iron-sulfur center and a semiquinone. Aerobic reperfusion resulted in a spectrum similar to the control but with increased amplitude that peaked after 10-15 sec of reflow. Anaerobic reperfusion yielded a spectrum identical to that of ischemic tissue. We conclude that pulverization of frozen myocardial tissue artifactually generates radical species. Using a nonpulverization technique for tissue processing, we found that myocardial ischemia and reperfusion produce radical species but that molecular oxygen is necessary for the burst of radical production during reflow.Myocardial ischemia occurs when myocardial oxygen demand exceeds oxygen supply. Unless reversed, this situation results in cell injury. The clinical event is myocardial infarction. Reperfusion of the ischemic myocardium during thrombolysis, coronary artery bypass surgery, or angioplasty can restore oxygen and energy substrates to the ischemic myocardial cell, but this process may create another form of myocardial damage, termed "reperfusion injury." This is characterized by tissue disruption, enzyme leakage, and development of contracture (1). Damage to the myocardial cell induced by a cycle of ischemia and reperfusion has been proposed to be due in part to the generation of toxic oxygen-derived free radicals (2, 3). Until recently, evidence to support the role of free radicals in myocardial cell injury has been of an indirect nature. Their production during ischemia and reperfusion has been implied through the use either of inhibitors of free radical generation or of scavengers of radical production (4-7).Nearly 2 decades ago, Beinert and coworkers (8-10) used low-temperature ESR spectroscopy to study the mechanism of electron transport in the cardiac mitochondrial respirat...