Biomedical subjects

R J Singh

Publications and source records attributed to R J Singh.

At least 19 recordsLinked to original sources

PubMed1999

Mechanism of superoxide dismutase/H(2)O(2)-mediated nitric oxide release from S-nitrosoglutathione--role of glutamate.

S-Nitrosoglutathione (GSNO), a physiologically relevant nitric oxide ((*)NO) donor, exhibits antioxidant, anti-ischemic, and antiplatelet properties. The exact mechanism of (*)NO release from GSNO in biological systems has not been determined. Both copper ions and copper-containing enzymes have been shown to catalyze (*)NO release from GSNO. In this study we observed that copper-zinc superoxide dismutase (Cu,ZnSOD) in the presence of H(2)O(2) caused a rapid decomposition of GSNO, forming oxidized glutathione (GSSG) and (*)NO. The cupric ions (Cu(2+)) released from Cu,ZnSOD were bound to the glutamate moiety of GSNO, yielding a 2:1 (GSNO)(2)Cu(2+) complex. Strong chelators of cupric ions, such as histidine and diethylenetriaminepentaacetic acid, inhibited the formation of (GSNO)(2)Cu(2+) complex, GSSG, and (*)NO. GSSG alone inhibited Cu(2+)-induced decomposition of GSNO. This effect is attributed to complexation of copper by GSSG. We conclude that binding of copper to GSNO is obligatory for (*)NO release from GSNO; however, the rate of this reaction was considerably slowed due to binding of Cu(2+) by GSSG. The glutamate moiety in GSNO and GSSG controls copper-catalyzed (*)NO release from GSNO. Cu,ZnSOD and H(2)O(2) enhanced peroxidation of unsaturated lipid that was inhibited by GSNO. The antioxidant function of GSNO is related to the sequestering of copper by GSNO and its ability to slowly release (*)NO. Implications of these findings are discussed in relation to GSNO-induced cardioprotection and to neuropathological processes.

Animals↗

PubMed1999

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-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) 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 H(2)O(2). 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.

Animals↗

PubMed1999

The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors.

SIN-1 has been used, in vitro, to simultaneously generate nitric oxide (*NO) and superoxide (O*-2). However, the pharmacological activity of SIN-1 resembles that of a *NO donor. SIN-1 decays by a three-step mechanism. After initial isomerization to an open ring form, SIN-1A reduces oxygen by a one-electron transfer reaction to give O*-2 and the SIN-1 cation radical, which decomposes to form SIN-1C and *NO. Here we report that one-electron oxidizing agents, in addition to oxygen, can oxidize SIN-1A, resulting in the release of *NO without the concomitant formation of O*-2. We demonstrate that easily reducible nitroxides, such as the nitronyl and imino nitroxides, are able to oxidize SIN-1. Biological oxidizing agents such as ferricytochrome c also stimulate *NO production from SIN-1. In addition, decomposition of SIN-1 by human plasma or by the homogenate of rat liver, kidney, and heart tissues results in the formation of *NO. Our findings suggest that SIN-1 may react with heme proteins and other electron acceptors in biological systems to produce *NO. Thus, at the relatively low in vivo oxygen concentrations, SIN-1 is likely to behave more like an *NO donor than a peroxynitrite donor. The relevance of this reaction to myocardial protection afforded by SIN-1 in ischemia/reperfusion-induced injury is discussed.

Animals↗

PubMed1999

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.

Antioxidants↗

PubMed1999

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.

Animals↗

PubMed1998

Nitration of gamma-tocopherol and oxidation of alpha-tocopherol by copper-zinc superoxide dismutase/H2O2/NO2-: role of nitrogen dioxide free radical.

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O2*-) to O2 and H2O2. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H2O2, leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of L-alpha-lecithin liposomes was enhanced greatly during the SOD/H2O2 reaction in the presence of nitrite anion (NO2-) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO2- also greatly enhanced alpha-tocopherol (alpha-TH) oxidation by SOD/H2O2 in saturated 1, 2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was alpha-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing gamma-tocopherol (gamma-TH) were incubated with SOD/H2O2/NO2-, the major product identified was 5-NO2-gamma-TH. Nitrone spin traps significantly inhibited the formation of alpha-tocopheryl quinone and 5-NO2-gamma-TH. NO2- inhibited H2O2-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO2- by an SOD-bound oxidant to the nitrogen dioxide radical (*NO2). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO2- is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.

Electron Spin Resonance Spectroscopy↗

PubMed1998

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5, 5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/.OH formation from the SOD and SOD mutants. The relative ratios of DMPO/.17OH and DMPO/.16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/.17OH and 37% DMPO/.16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/.17OH (64%); DMPO/.16OH (36%) from A4V and DMPO/.17OH (62%); and DMPO/.16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/.OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/.OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/.OH or DEPMPO/.OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Amyotrophic Lateral Sclerosis↗

PubMed1998

Depletion of nitric oxide causes cell cycle alterations, apoptosis, and oxidative stress in pulmonary cells.

Nitric oxide (NO.) is important in the regulation of mitochondrial function, cell signaling, and gene expression. To elucidate how endogenous NO. regulates the function of airway epithelial cells, we used carboxy-PTIO, a hydrophilic, negatively charged NO. trap, to scavenge NO. from rat lung epithelial (RLE) and rat pleural mesothelial (RPM) cells and to determine the elicitation of cell cycle alterations, apoptosis, and oxidative stress. The reaction of NO. with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of NO. formation. ESR spectroscopy revealed the production of NO. in RLE or RPM cells over a period from 1 to 24 h of exposure, indicating scavenging of NO. by PTIO. Cycle analyses in confluent RLE or RPM cells revealed two- to threefold increases in S and G2/M phases after exposure to 100-200 microM PTIO as well as increases in the fraction of cells undergoing apoptosis. Direct addition of PTI to cells failed to elicit cell cycle perturbations or apoptosis. The guanylyl cyclase inhibitor ODQ mimicked the effects of PTIO. 8-Bromo-cGMP but not 8-bromo-cAMP ameliorated the PTIO- or ODQ-mediated cell cycle perturbations and apoptosis, suggesting that cGMP-dependent pathways are involved in these cell cycle perturbations. Treatment of log-phase cells with PTIO resulted in more dramatic cell cycle perturbations compared with cells treated at confluence. Assessment of 5-bromo-2'-deoxyuridine incorporation to measure DNA synthesis demonstrated decreases in PTIO-treated compared with sham cells in addition to a cell cycle arrest in late S or G2/M phase. Last, incubation with dichlorofluorescin diacetate revealed oxidative stress in PTIO- but not in PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous NO. induces oxidative stress, cell cycle perturbations, and apoptosis. Our findings illustrate the importance of endogenous NO. in the control of cell cycle progression and survival of pulmonary and pleural cells and that a critical balance between NO. and superoxide may be necessary for these physiological events.

Animals↗

PubMed1997

The mechanism of apolipoprotein B-100 thiol depletion during oxidative modification of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) is recognized to be a key step in atherogenesis. Previous studies show that LDL contains low-molecular-weight antioxidants such as vitamin E, beta-carotene, and ubiquinol, which can retard oxidative modification. In this report, we have evaluated the antioxidant potential of apolipoprotein B-100 (apo-B) thiols during LDL oxidation. Both apo-B thiols and vitamin E were depleted concomitantly during the lag phase of Cu(2+)-mediated LDL oxidation. The rate of thiol depletion was significantly inhibited by the lipophilic spin trap N-tert-butyl-alpha-phenylnitrone (PBN) but not by the water-soluble spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Blocking apo-B thiols with sulfhydryl modifying agents increased the oxidizability of LDL. As with Cu2+, peroxynitrite also caused depletion of apo-B thiols, and again thiol depletion was inhibited by PBN but not by POBN. A PBN/lipid-derived radical adduct was observed by the electron spin resonance technique during oxidation of LDL with peroxynitrite. We conclude that apo-B thiol depletion is mediated by lipid peroxidation, prior to the onset of the propagation phase of LDL oxidation. The implications of apo-B thiols an intrinsic antioxidants of LDL are discussed.

Antioxidants↗

PubMed1997

S-Nitrosoglutathione as a substrate for gamma-glutamyl transpeptidase.

S-Nitrosoglutathione (GSNO) has been used as a nitric oxide (.NO) donor compound and has also been postulated to be involved in the transport of .NO in vivo. In this study we have examined the possibility that GSNO is a substrate for gamma-glutamyl transpeptidase (gamma-GT), an enzyme that hydrolyses the gamma-glutamyl moiety of glutathione to give glutamate and cysteinylglycine. gamma-GT accelerated the decomposition of GSNO, forming S-nitrosocysteinylglycine (CG-SNO) by a mechanism inhibitable by the gamma-GT inhibitors acivicin and S-methylglutathione. The Km of gamma-GT for GSNO was found to be 28 microM. In the presence of contaminating transition metal ions, gamma-GT accelerated the release of ;NO from GSNO, as CG-SNO is more susceptible to transition metal ion-dependent decomposition than GSNO. However, in the presence of the transition metal ion chelator diethylenetriaminepentaacetic acid, neither GSNO nor CG-SNO decomposed to generate .NO. Neither S-methylglutathione nor acivicin affected the vasodilatory response to GSNO in an isolated perfused rat heart. However, rat kidney homogenate stimulated the decomposition of GSNO by an acivicin-inhibitable mechanism. It is likely therefore that gamma-GT is involved in the decomposition of GSNO in the kidney but not in the heart.

Animals↗

PubMed1997

Phylogenetic and genomic relationships in the genus Glycine Willd. based on sequences from the ITS region of nuclear rDNA.

Phylogenetic relationships among all 18 species of the genus Glycine were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Pairwise sequence divergence values ranged from 0.2% (a single nucleotide) between Glycine max and Glycine soja to 8.6% between Glycine hirticaulis and Glycine falcata. The length of the ITS1 and ITS2 sequences ranged from 215 to 238 nucleotides and from 205 to 222 nucleotides, respectively, and that of 5.8S was 168 nucleotides across all the species. Phylogenetic analyses of the ITS region clearly resolved all the genomic groups that were established previously based on cytogenetic and biochemical studies. Based on this study, we assign new genome symbols: HH to Glycine arenaria, H1H1 to Glycine hirticaulis, H2H2 to Glycine pindanica, II to Glycine albicans, and I1I1 to Glycine lactovirens. Parsimony analysis of the entire ITS region, using subgenus Soja as outgroup, resulted in a trichotomy consisting of the clades: G. falcata (F genome), Glycine cyrtoloba and Glycine curvata (C genome), and all other species (A, B, D, E, H, and I genomes) of the subgenus Glycine.

Base Sequence↗

PubMed1996

Detection of thiyl radical adducts formed during hydroxyl radical- and peroxynitrite-mediated oxidation of thiols--a high resolution ESR spin-trapping study at Q-band (35 GHz).

Thiyl radicals (RS.) formed during peroxynitrite- or hydroxyl radical-dependent oxidation of thiols, i.e., glutathione (GSH) and L-cysteine (CySH) were trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and analyzed by X-band and Q-band electron spin resonance (ESR) spectroscopy. At X-band, the ESR parameters of DMPO-glutathionyl radical adduct (DMPO/.SG) and DMPO-hydroxyl radical adduct (DMPO/.OH) are nearly similar in aqueous solutions and as a result, except for the lowfield spectral line, the remaining spectral lines of DMPO/ .SG virtually over-lap with those of the DMPO/.OH adduct. In contrast, at Q-band, most of the spectral lines due to the DMPO/.SG were separated from the DMPO/ .OH. Inclusion of a superoxide dismutase (SOD) mimic completely abolished the formation of the DMPO/.OH adduct and not the DMPO/.SG adduct during ONOO(-)-mediated oxidation of GSH and DMPO. In the presence of formate, the DMPO/.SG spectrum was replaced by the DMPO/.CO2- spectrum which was monitored by Q-band ESR spectroscopy. Thus, spin-trapping at Q-band provides unambiguous proof for the glutathionyl radical-dependent oxidation of formate by peroxynitrite. High resolution Q-band ESR spectra of DMPO/.Scys were also obtained. Biological applications of the Q-band spin-trapping technique to detect thiyl radicals in cellular systems are discussed.

Biological Assay↗

PubMed1996

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 degrees 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.

Cyclic N-Oxides↗

PubMed1996

The reaction between nitric oxide and alpha-tocopherol: a reappraisal.

Recently Gorbunov et al. reported that nitric oxide (.NO) can directly oxidize alpha-tocopherol to alpha-tocopheroxyl radical (Gorbunov et al., Biochem. Biophys. Res. Commun., 219, 835-841, 1996). We have reinvestigated this reaction and report that a direct reaction between .NO and alpha-tocopherol does not occur. However, the reaction between .NO and oxygen generates an oxidant which oxidizes alpha-tocopherol to alpha-tocopheryl quinone. Exposure of alpha-tocopherol to a low flux of .NO generated from spermine NONOate (100 microM) results in no consumption of alpha-tocopherol under either aerobic or anaerobic conditions. A higher flux of .NO, generated from 1 mM spermine NONOate, oxidizes alpha-tocopherol only under aerobic conditions. Artifactual oxidation of alpha-tocopherol can be observed when using commercial .NO that is contaminated with higher oxides of nitrogen, such as dinitrogen trioxide and dinitrogen tetraoxide.

Free Radicals↗

PubMed1996

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 X 10(-3) M-1 X s-1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG (k= 4.8 X 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.

Aerobiosis↗

PubMed1995

Interaction of nitric oxide with photoexcited rose bengal: evidence for one-electron reduction of nitric oxide to nitroxyl anion.

The interaction of nitric oxide (.NO) with Rose Bengal (RB) in the presence of electron donors was investigated. Upon illumination of a mixture of RB and .NO with visible light, an enhancement in the rate of .NO consumption was observed that increased with increasing RB concentration. In the presence of electron donors (NADH, glutathione, or ascorbate), the rates of .NO depletion increased further. NADH enhanced .NO depletion to a greater extent than either glutathione or ascorbate. Photoactivated RB under anaerobic conditions reacts with NADH to form the RB anion radical (RB.-), which has a characteristic visible absorption band centered at 418 nm. Rose Bengal anion radical disporportionates to give RB and a colorless reduced form of RB, RBH-. The net result of this process is the photobleaching of RB. The presence of .NO during irradiation of RB and NADH introduced a lag time into the kinetics of RB photobleaching. The length of this lag time was proportional to the concentration of .NO. A similar lag time, which was also dependent on the .NO concentration, was observed in the kinetics of formation of RB.-. The three-line electron spin resonance (ESR) spectrum of RB.-, with an intensity ratio 1:2:1, was obtained during irradiation of RB and NADH under anaerobic conditions. .NO introduced a concentration-dependent lag time into the kinetics of the appearance of this ESR signal. We propose that .NO oxidizes RB.- to regenerate RB and thus inhibit photobleaching until .NO is consumed. This reaction predicts the formation of NO-, the one-electron reduced form of .NO. Nitrous oxide, a characteristic dimerization product of NO-, was detected by gas chromatography. This evidence indicates the occurrence of a Type I mechanism between photoactivated RB and .NO.

Anions↗

PubMed1995

Spin-labeling study of the oxidative damage to low-density lipoprotein.

In this study, we have spin-labeled the lysine and cysteine residues of low-density lipoprotein (LDL) using N-4-(2,2,6,6-tetramethylpiperidinyl-1-oxyl-4-yl) maleimide (MAL-6) and succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate (SSL), respectively. The electron spin resonance (ESR) spectrum of SSL bound to LDL indicated that the nitroxide moiety was relatively mobile. In contrast, the ESR spectrum of MAL-6 bound to LDL showed that the nitroxide moiety was rotationally restricted. Using the continuous-wave power saturation technique in the presence of hydrophobic and hydrophilic paramagnetic relaxing agents, we have determined that (i) approximately 60-70% of lysine-bound SSL is exposed to the aqueous phase, (ii) approximately 30-40% of SSL-LDL is buried in a hydrophobic region, and (iii) MAL-6 bound to LDL is localized predominantly in the hydrophobic region. During Cu(2+)-initiated oxidation of spin-labeled LDL, nitroxide labels located in a hydrophobic environment were predominantly degraded. Nitroxide destruction was inhibited by butylated hydroxytoluene, indicating the role of lipid peroxidation in this process. ESR data also showed that Cu2+ binding to lysine is essential for LDL oxidation. The spin label methodology may be useful for the investigation of site-specific radical reactions in LDL.

Apolipoproteins B↗

PubMed1995

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 (lambda = 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 x 10(9) M-1s-1 by competition with oxygen. Hematoporphyrin (HP) also enhanced the rate of .NO production by 2-3-fold. 2-Methyl-2-nitrosopropane (MNP) decomposed on irradiation (lambda = 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.

Electron Spin Resonance Spectroscopy↗