Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)₂Fe(NO)₂. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron–sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron–sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe–diethyldithiocarbamate (Fe–DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.     

过氧亚硝酸盐的化学发光反应与测定

过氧亚硝酸盐是一氧化氮衍生物,由一氧化氮和超氧阴离子反应生成,它是很强的生物氧化剂,具有很高的反应活性和不稳定性。本文详细介绍了ONOO-的化学发光分解、ONOO-与二氧化碳的化学发光反应、ONOO-化学发光测定和应用,并展望了其发展前景。引用参考文献54篇。     

过氧亚硝酸根对蛋白质损伤的研究进展*

过氧亚硝酸根--一氧化氮和超氧根阴离子快速结合的产物,是生物体内产生的一种强氧化剂和细胞毒性物质,它可以介导生物大分子包括DNA、蛋白质和脂质的修饰和损伤。近年来,由于许多疾病条件下都发现大量过氧亚硝酸根诱导的蛋白质损伤产物,因此过氧亚硝酸根与蛋白质的相互作用引起了人们的广泛关注。本文详尽地介绍了过氧亚硝酸根介导蛋白质损伤对人体的病理作用和过氧亚硝酸根与蛋白质作用机制等方面的最新研究进展,并对未来过氧亚硝酸根与蛋白质相互作用的研究作了展望。     

Formation of Nitrosyl Hemoglobin and Nitrotyrosine during Murine Leishmaniasis*

Peroxynitrite, the potent oxidant formed by the fast reaction between nitric oxide and superoxide anion, has been suggested to be the reactive intermediate responsible for some of the pathologies associated with an overproduction of nitric oxide. In this report, we demonstrate that both nitric oxide and peroxynitrite are formed during infection of the susceptible mouse strain, BALBk, with Leishmania amazonensis. Nitric oxide was detected as the nitrosyl hemoglobin complex by EPR analysis of blood drawn from mice at 35 , 64 and 148 days of infection. The levels of nitrosyl hemoglobin complex increased with disease evolution, which in the murine model used is characterized by skin lesions, ulceration and visceral-ization of the parasites. Peroxynitrite formation was inferred from immunoreaction of homogenates obtained from footpad lesions in the late stages of the infection with anti-nitrotyrosine antibody; homogenates from parasites drawn from the lesions were also immunoreactive, although to a lesser extent. Analysis of protein homogenates by gel electrophoresis and western blots suggests that peroxynitrite may degrade proteins in vivo, in addition to nitrating them. The results demonstrate that peroxynitrite is formed during murine leishmaniasis and may play a role in the aggravation of the disease.     

Preventing nitrite contamination in tetramethylammonium peroxynitrite solutions

Peroxynitrite prepared from superoxide and nitric oxide in liquid ammonia does not contain detectable levels of nitrite. However, the dissolution of nitrite salts can lead to variable levels of peroxynitrite depending on the conditions used to disolve the salt. Low levels of nitrite result when frozen peroxynitrite solutions are first brought to +1 degrees C and then to room temperature. These undergo only 2-3% decomposition after 1 h, in contrast with the findings of a recent report (Lymar, S. V.; Khairutdinov, R. F.; Hurst, J. K. Inorg. Chem. 2003, 42, 5259-5266), where high levels of nitrite ( approximately 20%) result from rapid thawing of these solutions to room temperature. Warming the frozen peroxynitrite solution directly to room temperature in 30 min leads to a nitrite level of 28%.     

低剂量铅对大鼠脑组织一氧化氮合酶表达及活性的影响

为探讨低水平铅损伤记忆功能的作用机理，采用ＮＡＤＰＨ－黄递酶组织化学方法和＾３Ｈ－瓜氨酸生成生物检测技术，观察了新生大鼠在４００ｍｇ／Ｌ醋酸铅染毒４０天后对脑组织内一氧化氮合酶表达及活性的影响。结果显示大脑皮质一氧化氮合酶阳性神经元数量明显减少（Ｐ〈０．０５），在海马区脑组织内一氧化氮合酶活性降低５４．４％。结论：低水平铅暴露后可抑制大鼠脑组织内一氧化氮合酶的表达及活性。     

Imaging of nitric oxide generation in the rat brain

Nitric oxide radicals produced in the rat brain subjected to ischemia-hypoxia were trapped by the systemically administered diethyldithiocarbamate and iron. The right hemisphere of the brain was then removed and frozen with liquid nitrogen. Three-dimensional spatial distribution of the nitric oxide radicals in this hemisphere was performed using electron paramagnetic resonance imaging techniques. The results suggest that nitric oxide radicals were produced and trapped in the areas which are known to have high nitric oxide synthase activity, such as piriform cortex, hippocampus, hypothalamus, amygdala, and substantia nigra. In this model, which did not interrupt the posterior circulation, the production and trapping of nitric oxide in the cerebellum was relatively weak.     

Dialysis membrane sampler for on-line flow injection analysis/chemiluminescence-detection of peroxynitrite in biological samples

Peroxynitrite, as a derivative of nitric oxide, is a potent oxidant. It reacts with several biological molecules, makes cellular and tissue damages, and is related with many diseases; therefore, it is of major concern in current medical research works. In this work, a special perm-selective cellulose acetate membrane sampler is used to implement flow injection analysis (FIA)/chemiluminscence (CL)-detection method for the detection of peroxynitrite with Luminol CL-reagent. Optimum detection conditions were established, and the permeability of peroxynitrite through cellulose acetate (CA) membrane, as well as the interference from matrix constituents were studied. The proposed method has the high sensitivity of the CL-detection and the selectivity of perm-selective membrane sampler. The obtained detection limit of 1×10⁻¹¹ M (without dialysis membrane) and 1×10⁻¹⁰ M (with dialysis membrane), makes it possible to monitor the elusive peroxynitrite in biological samples. The mechanism of luminol CL-emission generated during oxidation by peroxynitrite and the kinetics of peroxynitrite decomposition were also studied using FIA/CL-detection set-up.     

Kinetic regularities of erythrocyte hemolysis and hemoglobin oxidation under the action of sulfur-nitrosyl iron complexes as nitric oxide donors

The kinetics of erythrocyte hemolysis and intra-erythrocyte hemoglobin oxidation under the action of synthetic sulfur-nitrosyl iron complexes was studied. The complexes capable of releasing nitric oxide due to spontaneous hydrolytic decomposition was studied. The addition of these complexes to a 0.2% suspension of mouse erythrocytes results in hemolysis. The kinetic curves of hemolysis exhibit an induction period, whose duration is different for each complex. The hemolysis is preceded by hemoglobin oxidation with nitric oxide penetrating into the cell. The oxidation of hemoglobin follows the first-order rate equation. The apparent first-order rate constants characterizing the NO-donating ability of each complex were determined. The hemolytic effect of the studied complexes is suggested to be related to the formation of peroxynitrite inside erythrocytes. Peroxynitrite is the cytotoxic product of interaction of nitric oxide and the superoxide radical anion.     

Angiogenin induces nitric oxide release independently from its RNase activity

Nitric oxide (NO), a biological mediator involved in vascular physiology, was sensed electrochemically using a microelectrode array. Angiogenin was shown to trigger nitric oxide synthase (NOS) activity in human umbilical vein endothelial cells and embryonic stem cell derived endothelial cells independently from its RNase activity.     

Indications for the Occurrence of Nitric Oxide Synthases in Fungi and Plants and the Involvement in Photoconidiation of Neurospora crassa*

Indications for the occurrence of nitric oxide synthases in Dictyostelium, Neurospora, Phycomyces and the leguminous plant Mucuna hassjoo as well as a physiological role of nitric oxide in Neurospora crassa are demonstrated. An exogenous nitric oxide donor, sodium nitroprus-side, inhibited light-stimulated conidiation in N. crassa. Specific inhibitors of nitric oxide synthase, like the arginine derivatives N^G-nitro-L-arginine (L-NA) and N^G-nitro-L-arginine-methyl ester (L-NAME), enhanced conidiation in darkness and in the light, whereas the stereo-isomer D-NAME was inactive. This communication reports to our knowledge the first time the presence of enzymatic activity of nitric oxide synthase in fungi and a higher plant and an effect of nitric oxide in fungal photophysiology.     

Temperature-dependent spin crossover in neuronal nitric oxide synthase bound with the heme-coordinating thioether inhibitors

A series of L-arginine analogue nitric oxide synthase inhibitors with a thioether tail have been shown to form an Fe-S thioether interaction as evidenced by continuous electron density between the Fe and S atoms. Even so, the Fe-S thioether interaction was found to be far less important for inhibitor binding than the hydrophobic interactions between the alkyl group in the thioether tail and surrounding protein (Martell et al. J. Am. Chem. Soc. 2010 , 132 , 798). However, among the few thioether inhibitors that showed Fe-S thioether interaction in crystal structures, variations in spin state (high-spin or low-spin) were observed dependent upon the heme iron oxidation state and temperature. Since modern synchrotron X-ray data collection is typically carried out at cryogenic temperatures, we reasoned that some of the discrepancies between cryo-crystal structures and room-temperature UV-visible spectroscopy could be the result of temperature-dependent spin-state changes. We, therefore, have characterized some of these neuronal nitric oxide synthase (nNOS)-thioether inhibitor complexes in both crystal and solution using EPR and UV-visible absorption spectrometry as a function of temperature and the heme iron redox state. We found that some thioether inhibitors switch from high to low spin at lower temperatures similar to the "spin crossover" phenomenon observed in many transition metal complexes.     

Protein thiyl free radicals produced by nitric oxide and nitric oxide donors

The spin-trapping technique was used to study the radical intermediates produced by reaction of nitric oxide (^*NO) and peroxynitrite with serum albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Our results show that the major radical product induced by ^*NO and by peroxynitrite with serum albumin and GAPDH was a thiyl radical. The same radical can be detected in the ^*NO-transfer from S-nitroso albumin to low molecular weight thiols. Moreover, ^*NO or peroxynitrite treatment of GAPDH was able to induce NAD-dependent covalent modification of the enzyme in erythrocyte ghosts.     

Effect of CO2 Laser Radiation on Physiological Tolerance of Wheat Seedlings Exposed to Chilling Stress

To determine the effect of CO₂ laser pretreatment of wheat seeds on the physiological tolerance of seedlings to chilling stress, wheat seeds were exposed to CO₂ laser radiation for 300 s. After being cultivated for 48 h at 25°C, the wheat seedlings were subjected to chilling stress for 24 h. Selected physiological and biochemical parameters were measured in 6-day-old seedlings. We observed that chilling stress enhanced the concentrations of malondialdehyde and oxidized glutathione while decreasing the activities of nitric oxide synthase, catalase, peroxidase, superoxide dismutase and the concentrations of nitric oxide and glutathione in the wheat leaves compared with controls. When the chilling stress was preceded by CO₂ laser irradiation, the concentrations of malondialdehyde and oxidized glutathione were decreased while the activities of nitric oxide synthase, catalase, peroxidase, superoxide dismutase and the concentrations of nitric oxide and glutathione increased. Furthermore, chilling stress decreased the biomass, biophoton intensity and GHS/GSSG ratios of seedlings while these parameters increased when the seedlings were treated with CO₂ laser irradiation prior to the chilling stress. The results suggest that a suitable dose of CO₂ laser stimulation can enhance the physiological tolerance of wheat seedlings to chilling stress.     

A novel mass spectrometry‐based method for simultaneous determination of asymmetric and symmetric dimethylarginine,l‐arginine and l‐citrulline optimized for LC‐MS‐TOF and LC‐MS/MS

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l‐ arginine to l‐ citrulline. l‐ Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l‐ arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l‐ arginine, l‐ citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6‐pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse‐phase column. Serum aliquots (100 μL) were spiked with isotope‐labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R ² = 0.9966–0.9986) for all derivatized compounds. Good accuracy (94.67–99.91%) and precision (1.92–11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l‐ arginine and l‐ citrulline in angiologic patients. This robust and fast LC‐ESI‐MS method may be a useful tool in quantitative analysis of l‐ arginine, ADMA, SDMA and l‐ citrulline.     

The reaction of oximes with 4-phenyl-1,2,4-triazoline-3,5-dione to produce nitric oxide – Model compounds for nitric oxide synthase

Certain oximes form nitric oxide upon reaction with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD). The oximes appear to undergo an Alder-ene reaction with the PTAD enophile to form a nitroso intermediate capable of dimerization and/or nitric oxide formation. Upon exposure to oxygen, the nitroso compounds eventually form ketones. This reaction may serve as a model for the study of nitric oxide synthase (NOS), the enzyme responsible for physiological production of nitric oxide. NOS is known to produce an oxime intermediate which reacts with oxygen to produce nitric oxide and citrulline.     

Nitric Oxide-Mediated Resistance to Antitumor Photodynamic Therapy

As an antitumor modality based on sensitizer photoexcitation by tumor-directed light, photodynamic therapy (PDT) has the advantage of being site-specific compared with conventional chemotherapy or radiotherapy. Like these other therapies, however, PDT is often limited by pre-existing or acquired resistance. One type of resistance, discovered in the author’s laboratory, involves nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) in tumor cells. Using human breast, prostate and brain cancer cell lines, we have shown that iNOS is dramatically upregulated after a moderate PDT challenge sensitized by 5-aminolevulinic acid-induced protoporphyrin IX. The elevated NO not only elicited a greater resistance to cell photokilling, but also an increase in the growth and migration/invasion rate of surviving cells. Greater iNOS/NO-based resistance was also demonstrated at the in vivo level using a breast tumor xenograft model. More recent studies have shown that NO from PDT-targeted cells can stimulate a progrowth/promigration response in non-targeted bystander cells. These novel effects of NO, their negative impact on PDT efficacy and possible mitigation thereof by anti-iNOS/NO pharmacologic agents will be discussed.     

EPC-K1 protects neuronal cells from peroxynitrite-mediated oxidative damage

Protective effects of EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyldecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt, a difunctional derivative of vitamin C and vitamin E) on neuronal cell damage mediated by peroxynitrite were studied. Primary cultures of cerebellar granule cells were exposed to peroxynitrite by treatment with 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), which generated nitric oxide and superoxide anion simultaneously upon decomposition. The results showed that SIN-1 treatment triggered time-dependent cell death, which was accompanied by the decrease in the cellular GSH level, the increase in the lipid peroxidation level, and the alteration of cell membrane biophysical characteristics. EPC-K1 showed only moderate scavenging effect on peroxynitrite, but could effectively protect neuronal cells from oxidative damage mediated by peroxynitrite.     

Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase

Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.