Ravinder Jit Singh

Mechanism of Nitric Oxide Release from S-Nitrosothiols

S-Nitrosothiols have many biological activities and have been suggested to be intermediates in signal transduction. The mechanism and products of S-nitrosothiol decomposition are of great significance to the understanding of nitric oxide (·NO) biochemistry. S-Nitrosothiols are stable compounds at 37°C and pH 7.4 in the presence of transition metal ion chelators. The presence of trace transition metal ions (present in all buffers) stimulates the catalytic breakdown of S-nitrosothiols to ·NO and disulfide. Thiyl radicals are not formed as intermediates in this process. Photolysis of S-nitrosothiols results in the formation of ·NO and disulfide via the intermediacy of thiyl radicals. Reduced metal ion (e.g. Cu+) decomposes S-nitrosothiols more rapidly than oxidized metal ion (e.g. Cu2+) indicating that reducing agents such as glutathione and ascorbate can stimulate decomposition of S-nitrosothiol by chemical reduction of contaminating transition metal ions. Transnitrosation can also stimulate S-nitrosothiol decomposition if the product S-nitrosothiol is more susceptible to transition metal ion-catalyzed decomposition than the parent S-nitrosothiol. Equilibrium constants for the transnitrosation reactions of reduced glutathione, either with S-nitroso-N-acetyl-DL-penicillamine or with S-nitroso-L-cysteine indicate that S-nitrosoglutathione formation is favored. The biological relevance of S-nitrosothiol decomposition is discussed.

The role of glutathione in the transport and catabolism of nitric oxide.

Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5–10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S‐nitrosoglutathione (GSNO). GSNO reacts with glutathione (k = 8.3 × 10−3 M−1 · s−1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG (k = 4.8 × 10−4 s−1 at 5 mM GSH). In both aerobic and anaerobic situations the nitroxyl anion may be an intermediate in the synthesis of nitrous oxide and, under aerobic conditions, nitroxyl anion may generate peroxynitrite. We present a hypothesis for the intracellular interaction between nitric oxide and glutathione.

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.

Photosensitized decomposition of S‐nitrosothiols and 2‐methyl‐2‐nitrosopropane Possible use for site‐directed nitric oxide production

Irradiation of S‐nitrosoglutathione (GSNO) with light (λ = 550 nm) resulted in the homolytic decomposition of GSNO to generate glutathionyl radical (GS ·) and nitric oxide (·NO), which were monitored by ESR spectrometry. Inclusion of Rose Bengal (RB) resulted in a 9‐fold increase in the quantum yield for ·NO production and also an increase in the rate of thiyl radical formation. The bimolecular rate constant for the interaction of triplet RB with GSNO has been estimated to be approximately 1.2 × 109 M−1s−1 by competition with oxygen. Hematoporphyrin (HP) also enhanced the rate of ·NO and tert‐butyl radical. Aluminum 2‐Methyl‐2‐nitrosopropane (MNP) decomposed on irradiation (λ = 660 nm) to form ·NO and tert‐butyl radical. Aluminum phthalocyanine tetrasulphonate enhanced the rate of decomposition of MNP by 10‐fold. These studies show that photosensitizers enhance the release of ·NO from donor compounds.

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (.NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-332, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and .NO is 0.63 +/- 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between .NO and O2 is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of .NO, the reaction between .NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose mechanism for the reaction between PTIO and .NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and .NO is dependent on the rate of generation of .NO and is 1:1 only at low rates of .NO generation (i.e., 10(-13) M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of .NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of .NO. The model agrees with previous experimental observations that the aqueous oxidation of .NO in air saturated solutions will exclusively form nitrite and predicts that .NO will only generate substantial amounts of nitrate if it is released at a rate less than 10(-17) M/s. This may have important consequences in cellular systems where the concentration of .NO is typically measured from nitrite production.

TRAPPING OF NITRIC OXIDE FORMED DURING PHOTOLYSIS OF SODIUM NITROPRUSSIDE IN AQUEOUS AND LIPID PHASES: AN ELECTRON SPIN RESONANCE STUDY

Abstract— Photolytic decomposition of sodium nitroprusside (SNP), a widely used nitrovasodilator, produced nitric oxide (NO), which was continuously monitored by electron spin resonance (ESR) spectroscopy. The NO present in the aqueous or the lipid phase was trapped by either a hydrophilic or a hydrophobic nitronyl nitroxide, respectively, to form the corresponding imino nitroxide. The conversion of nitronyl nitroxide to imino nitroxide was monitored by ESR spectrometry. The quantum yield for the generation of NO from SNP, measured from the rate of decay of nitronyl nitroxide, was 0.201 ± 0.007 and 0.324 ± 0.01 (¯± SD, n = 3) at 420 nm and 320 nm, respectively. The action spectrum for NO generation was found to overlap the optical absorption spectrum of SNP closely. A mechanism for the reaction between SNP and nitronyl nitroxide in the presence of light is proposed and computer‐aided simulation of this mechanism using published rate constants agreed well with experimental data. The methodology described here may be used to assay NO production continuously during photoactivation of NO donors in aqueous and lipid environments. Biological implications of this methodology are discussed.

PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully‐saturated dimyristoylphosphatidylcholine vesicles, mono‐unsaturated 1‐palmitoyl‐2‐oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane‐bound MC540 in aqueous buffer was determined to be 0.065 ± 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer‐dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.

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.

Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein: antioxidant implications

Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2(+)-mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.

Physical and chemical interactions between nitric oxide and nitroxides

The physical and chemical interaction of nitric oxide (NO) with stable nitroxides have been studied in both aqueous and membrane environments. The ESR spectrum of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1- yloxy (CTPO) was observed to broaden upon exposure to NO. This effect can be explained by invoking Heisenberg spin exchange as has been previously reported for molecular oxygen. No loss of total spin was observed negating the possibility of a chemical reaction between NO and CTPO. The extent of signal broadening was proportional to the concentration of NO and can thus be used to monitor NO concentration. We have used this method to observe the partitioning of NO into model membranes. We also report the use of multiquantum ESR to detect directly the effects of NO on the membrane bound spin label 12-doxylstearic acid. This methodology may prove useful for detecting NO in both aqueous and lipid environments and for examining the physical properties of NO within biological membranes.