Carbonate Anion Radical Generated by the Peroxidase Activity of Copper-Zinc Superoxide Dismutase:Scavenging of Radical and Protection of Enzyme by Hypotaurine and Cysteine Sulfinic Acid

Copper-zinc superoxide dismutase (SOD) is considered one of the most important mammalian antioxidant defenses and plays a relevant role due to its main function in catalyzing the dismutation of superoxide anion to oxygen and hydrogen peroxide. However, interaction between SOD and H2O2 produced a strong copper-bound oxidant (Cu(II)●OH) that seems able to contrast the self-inactivation of the enzyme or oxidize other molecules through its peroxidase activity. The bicarbonate presence enhances the peroxidase activity and produces the carbonate anion radical (CO3●–). CO3●– is a freely diffusible reactive species capable of oxidizing several molecules that are unwieldy to access into the reactive site of the enzyme. Cu(II)●OH oxidizes bicarbonate to the CO3●–, which spreads out of the binding site and oxidizes hypotaurine and cysteine sulfinic acid to the respective sulfonates through an efficient reaction. These findings suggest a defense role for sulfinates against the damage caused by CO3●–. The effect of hypotaurine and cysteine sulfinic acid on the CO3●–-mediated oxidation of the peroxidase probe ABTS to ABTS cation radical (ABTS●+) has been studied. Both sulfinates are able to inhibit the oxidation of ABTS mediated by CO3●–. The effect of hypotaurine and cysteine sulfinic acid against SOD inactivation by H2O2 (~42% protection of enzyme activity) has also been investigated. Interestingly, hypotaurine and cysteine sulfinic acid partially avoid the H2O2-mediated SOD inactivation, suggesting that the two sulfinates may have access to the SOD reactive site and preserve it by reacting with the copper-bound oxidant. In this way hypotaurine and cysteine sulfinic acid not only intercept CO3●–which could move out from the reactive site and cause oxidative damage, but also prevents the inactivation of SOD

Biradicals as ESR probes of conformations in model β-turn peptides

Electron spin-spin exchange interaction in biradicals has been examined for its potential usefulness in the conformational analysis of peptides in solution. Three peptides with high propensity to adopt β -turn conformations in solution, Boc-Cys-Pro-Xxx-Cys-NHMe (Xxx = Leu, 1; Xxx = Aib, 2; Xxx = Tyr, 3), have been modified into biradicals by spin labeling the thiols groups. Analysis of electron spin resonance spectra of these peptides in it variety of solvents and at different temperatures suggests that the population of folded conformations follows the order 2 > 1 > 3

Biradicals as ESR Probes of Conformations in Model \beta-turn Peptides

Electron spin-spin exchange interaction in biradicals has been examined for its potential usefulness in the conformational analysis of peptides in solution. Three peptides with high propensity to adopt \beta-turn conformations in solution, Boc-Cys-Pro-Xxx-Cys-NHMe (Xxx = Leu, 1; Xxx = Aib, 2; Xxx = Tyr, 3), have been modified into biradicals by spin labeling the thiols groups. Analysis of electron spin resonance spectra of these peptides in it variety of solvents and at different temperatures suggests that the population of folded conformations follows the order 2 > 1 > 3

Fast computation of dynamic EPR spectra of biradicals

A fast computation method for the simulation of an EPR spectrum of dynamically exchanging biradicals and a FORTRAN program are presented here. The lineshape has been developed in the Liouville representation of quantum mechanics following Heinzer's derivation described for broadened isotropic EPR spectra. Application of the Liouville density treatment to calculate the biradical EPR transitions permits simulation of the intramolecular exchange among many sites. The program DYNBIR can simulate the EPR spectrum in the complete range of slow- to fast-exchange limit involving a maximum of four conformations for the biradical. The program was tested by reproducing the results reported earlier by Parmon and Zhidamirov for nitroxyl biradical. Simulations of the spectrum under weak, intermediate, and strong spin-spin exchange in slow and fast dynamic situations are presented. Application to simulate the temperature-dependent spectrum of a long-chain biradical is also presented

Effect of bicarbonate on iron-mediated oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) may play an important role in atherosclerosis. We studied the effects of bicarbonate/CO(2) and phosphate buffer systems on metal ion-catalyzed oxidation of LDL to malondialdehyde (MDA) and to protein carbonyl and MetO derivatives. Our results revealed that LDL oxidation in mixtures containing free iron or heme derivatives was much greater in bicarbonate/CO(2) compared with phosphate buffer. However, when copper was substituted for iron in these mixtures, the rate of LDL oxidation in both buffers was similar. Iron-catalyzed oxidation of LDL was highly sensitive to inhibition by phosphate. Presence of 0.3-0.5 mM phosphate, characteristic of human serum, led to 30-40% inhibition of LDL oxidation in bicarbonate/CO(2) buffer. Iron-catalyzed oxidation of LDL to MDA in phosphate buffer was inhibited by increasing concentrations of albumin (10-200 μM), whereas MDA formation in bicarbonate/CO(2) buffer was stimulated by 10-50 μM albumin but inhibited by higher concentrations. However, albumin stimulated the oxidation of LDL proteins to carbonyl derivatives at all concentrations examined in both buffers. Conversion of LDL to MDA in bicarbonate/CO(2) buffer was greatly stimulated by ADP, ATP, and EDTA but only when EDTA was added at a concentration equal to that of iron. At higher than stoichiometric concentrations, EDTA prevented oxidation of LDL. Results of these studies suggest that interactions between bicarbonate and iron or heme derivatives leads to complexes with redox potentials that favor the generation of reactive oxygen species and/or to the generation of highly reactive CO(2) anion or bicarbonate radical that facilitates LDL oxidation