Steven P. A. Goss

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase ROLE OF CARBONATE ANION RADICAL

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-γ-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H2O2-mediated nitration of γ-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H2O2-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H2O2-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H2O2, suggest that SOD1/H2O2 oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H2O2 by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226–1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H2O2. However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.

The Effect of Nitric Oxide Release Rates on the Oxidation of Human Low Density Lipoprotein

1-Substituted diazen-1-ium-1,2-diolates, a class of nitric oxide (⋅NO) donor compounds that spontaneously release⋅NO at different rates, were used to investigate the effect of⋅NO release rate upon the oxidation of low density lipoprotein (LDL). All donor compounds conferred an inhibitory effect upon the oxidation of LDL; however, the effect exhibited a biphasic dependence upon the rate of ⋅NO release. The ⋅NO release rate that maximally inhibited oxidation was dependent upon the rate of oxidation. When LDL was rapidly oxidized by copper(II) sulfate, a faster release rate was more effective. In contrast, when LDL was oxidized slowly by 2,2′-azobis-2-amidinopropane hydrochloride, a slower release rate was most effective. This biphasic relationship between ⋅NO release rate and the duration of inhibition was also demonstrated when LDL oxidation was initiated with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium, a peroxynitrite generator. We conclude that the antioxidant ability of ⋅NO is dependent not only upon the rate of its release from ⋅NO donors, but also upon the rate of oxidation. This conclusion is supported by a kinetic model of LDL oxidation in the presence of ⋅NO.

Reactions of *NO, *NO2 and peroxynitrite in membranes: physiological implications.

Nitric oxide (*NO) and nitrogen dioxide (*NO2) are hydrophobic gases. Therefore, lipid membranes and hydrophobic regions of proteins are potential sinks for these species. In these hydrophobic environments, reactive nitrogen species will exhibit different chemistry than in aqueous environments due to higher local concentrations and the lack of hydrolysis reactions. The peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH) can freely pass through lipid membranes, making peroxynitrite-mediated reactions in a hydrophobic environment also of extreme relevance. The reactions observed by these reactive nitrogen species in a hydrophobic milieu include oxidation, nitration and even potent chain-breaking antioxidant reactions. The physiological and toxicological relevance of these reactions is discussed.

ANTIOXIDANT EFFECTS OF NITRIC OXIDE AND NITRIC OXIDE DONOR COMPOUNDS ON LOW-DENSITY LIPOPROTEIN OXIDATION

Nitric oxide, when slowly released from a donor compound, has a potent inhibitory effect on the oxidative modification of LDL. This can be studied by monitoring changes in the lipid, protein, and antioxidant components of the LDL particle. In addition, the kinetics of LDL oxidation provides an insight into the mechanistic basis of the nitric oxide-dependent inhibition of LDL oxidation.