Expression, S-Nitrosylation, and Measurement of S-Nitrosylation Ratio of Recombinant Galectin-2.

S-nitrosylation, which involves the coupling of an NO group to the reactive thiol of Cys residue(s) in a polypeptide, is an important posttranslational modification detected in a variety of proteins. Here, we present the S-nitrosylation of recombinant galectin-2 (Gal-2) using S-nitrosocysteine and the measurement of the molecular ratio of S-nitrosylation of Cys residues in the Gal-2 protein.

Cellulose-based biomaterials integrated with copper-cystine hybrid structures as catalysts for nitric oxide generation.

Bionanocomposite materials were developed from the assembly of polymer-coated copper-cystine high-aspect ratio structures (CuHARS) and cellulose fibers. The coating of the metal-organic materials with polyallylamine hydrochloride (PAH) allows their covalent linkage to TEMPO-oxidized cellulose by means of EDC/NHS. The resulting materials can be processed as films or macroporous foams by solvent casting and lyophilization, respectively. The films show good mechanical behavior with Young's moduli around 1.5 GPa as well as resistance in water, while the obtained foams show an open network of interconnected macropores with average diameters around 130 µm, depending on the concentration of the initial suspension, and compression modulus values around 450 kPa, similar to other reported freeze-dried nanocellulose-based aerogels. Based on these characteristics, the cellulose/PAH-CuHARS composites are promising for potential biomedical applications as implants or wound dressing materials. They have proved to be effective in the decomposition of low molecular weight S-nitrosothiols (RSNOs), similar to those existing in blood, releasing nitric oxide (NO). This effect is attributed to the presence of copper in the crystalline structure of the CuHARS building unit, which can be gradually released in the presence of redox species like ascorbic acid, typically found in blood. The resulting biomaterials can offer the interesting properties associated with NO, like antimicrobial activity as preliminary tests showed here with Escherichia coli and Staphylococcus epidermidis. In the presence of physiological concentration of RSNOs the amount of generated NO (around 360 nM) is not enough to show bactericidal effect on the studied bacteria, but it could provide other properties inherent to NO even at low concentration in the nM range like anti-inflammatory and anti-thrombotic effects. The cytotoxic effect recorded of the films on rat brain endothelial cells (BMVECs) is least significant and proves them to be friendly enough for further biological studies.

Role of Nitric Oxide Carried by Hemoglobin in Cardiovascular Physiology: Developments on a Three-Gas Respiratory Cycle.

A continuous supply of oxygen is essential for the survival of multicellular organisms. The understanding of how this supply is regulated in the microvasculature has evolved from viewing erythrocytes (red blood cells [RBCs]) as passive carriers of oxygen to recognizing the complex interplay between Hb (hemoglobin) and oxygen, carbon dioxide, and nitric oxide-the three-gas respiratory cycle-that insures adequate oxygen and nutrient delivery to meet local metabolic demand. In this context, it is blood flow and not blood oxygen content that is the main driver of tissue oxygenation by RBCs. Herein, we review the lines of experimentation that led to this understanding of RBC function; from the foundational understanding of allosteric regulation of oxygen binding in Hb in the stereochemical model of Perutz, to blood flow autoregulation (hypoxic vasodilation governing oxygen delivery) observed by Guyton, to current understanding that centers on S-nitrosylation of Hb (ie, S-nitrosohemoglobin; SNO-Hb) as a purveyor of oxygen-dependent vasodilatory activity. Notably, hypoxic vasodilation is recapitulated by native S-nitrosothiol (SNO)-replete RBCs and by SNO-Hb itself, whereby SNO is released from Hb and RBCs during deoxygenation, in proportion to the degree of Hb deoxygenation, to regulate vessels directly. In addition, we discuss how dysregulation of this system through genetic mutation in Hb or through disease is a common factor in oxygenation pathologies resulting from microcirculatory impairment, including sickle cell disease, ischemic heart disease, and heart failure. We then conclude by identifying potential therapeutic interventions to correct deficits in RBC-mediated vasodilation to improve oxygen delivery-steps toward effective microvasculature-targeted therapies. To the extent that diseases of the heart, lungs, and blood are associated with impaired tissue oxygenation, the development of new therapies based on the three-gas respiratory system have the potential to improve the well-being of millions of patients.

Integrated microfluidic device for the separation, decomposition and detection of low molecular weight S-nitrosothiols.

S-nitrosothiols (RSNOs) are very important biomolecules that play crucial roles in many physiological and physiopathological processes. They act as NO-donors and are candidates for future medicines. Their identification and quantitation are therefore important for biomedical applications. One, two or more RSNOs can then be combined to design a drug and therefore, the quantification of each is important to establish an acceptable quality control process. Till date, miniaturized devices have been used to detect RSNOs based on their total quantitation without a preceding separation step. This study reports on an original and integrated microdevice allowing for the successive electrokinetic separation of low molecular weight RSNOs, their decomposition under metal catalysis, and their quantitation by amperometric detection of the produced nitrite in the end-channel arrangement, leading to their quantitation in a single run. For this purpose, a commercial SU-8/Pyrex microfluidic system was coupled to a portable and wireless potentiostat. Different operating and running parameters were optimized to achieve the best analytical data, allowing for an LOD equal to 20 µM. The simultaneous separation of S-nitrosoglutathione and S-nitrosocysteine was successfully obtained within 75 s. The proposed methodology using SU-8/Pyrex microfluidic devices opens new possibilities to investigate future drug candidates for NO-donors.

Direct Measurement of S-Nitrosothiols with an Orbitrap Fusion Mass Spectrometer: S-Nitrosoglutathione Reductase as a Model Protein.

Recent studies suggest cysteine S-nitrosation of S-nitrosoglutathione reductase (GSNOR) could regulate protein redox homeostasis. "Switch" assays enable discovery of putatively S-nitrosated proteins. However, with few exceptions, researchers have not examined the kinetics and biophysical consequences of S-nitrosation. Methods to quantify protein S-nitrosothiol (SNO) abundance and formation kinetics would bridge this mechanistic gap and allow interpretation of the consequences of specific modifications, as well as facilitate development of specific S-nitrosation inhibitors. Here, we describe a rapid assay to estimate protein SNO abundance with intact protein electrospray ionization mass spectrometry. Originally designed using recombinant GSNOR, these methods are applicable to any purified protein to test for or further study nitrosatable cysteines.

Chemical methods for mapping cysteine oxidation.

Cysteine residues in proteins are subject to diverse redox chemistry. Oxidation of cysteine to S-nitrosocysteine, cysteine sulfenic and sulfinic acids, disulfides and persulfides are a few prominent examples of these oxidative post-translational modifications. In living organisms, these modifications often play key roles in cell signalling and protein function, but a full account of this biochemistry is far from complete. It is therefore an important goal in chemical biology to identify what proteins are subjected to these modifications and understand their physiological function. This review provides an overview of these modifications, how they can be detected and quantified using chemical probes, and how this information provides insight into their role in biology. This survey also highlights future opportunities in the study of cysteine redox chemistry, the challenges that await chemists and biologists in this area of study, and how meeting such challenges might reveal valuable information for biomedical science.

Protein Cysteinyl-S-Nitrosylation: Analysis and Quantification.

The thiol moiety of cysteine residues can undergo a number of biologic modifications including oxidation, sulfenylation, nitrosylation, persulfidation, metalation, and other modifications. These modifications can control biological function, including gain as well as loss of function. Herein, we focus attention on the proteomic analysis of S-nitrosylation in health and disease. We describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. We present several studies where suitability of this approach for investigating endogenous S-nitrosylation is addressed.

Thiolate-based dinitrosyl iron complexes: Decomposition and detection and differentiation from S-nitrosothiols.

Dinitrosyl iron complexes (DNIC) spontaneously form in aqueous solutions of Fe(II), nitric oxide (NO), and various anions. They exist as an equilibrium between diamagnetic, dimeric (bi-DNIC) and paramagnetic, monomeric (mono-DNIC) forms. Thiolate groups (e.g., on glutathione or protein cysteine residues) are the most biologically relevant anions to coordinate to Fe(II). Low molecular weight DNIC have previously been suggested to be important mediators of NO biology in cells, and emerging literature supports their role in the control of iron-dependent cellular processes. Recently, it was shown that DNIC may be one of the most abundant NO-derived products in cells and may serve as intermediates in the cellular formation of S-nitrosothiols. In this work, we examined the stability of low molecular weight DNIC and investigated issues with their detection in the presence of other NO-dependent metabolites such as S-nitrosothiols. By using spectrophotometric, Electron Paramagnetic Resonance, ozone-based chemiluminesence, and HPLC techniques we established that at neutral pH, bi-DNIC remain stable for hours, whereas excess thiol results in decomposition to form nitrite. NO was also detected during the decomposition, but no S-nitrosothiol formation was observed. Importantly, mercury chloride accelerated the degradation of DNIC; thus, the implications of this finding for the diagnostic use of mercury chloride in the detection of S-nitrosothiols were determined in simple and complex biological systems. We conclude S-nitrosothiol levels may have been substantially overestimated in all methods where mercury chloride has been used.

GC-ECNICI-MS analysis of S-nitrosothiols and nitroprusside after treatment with aqueous sulphide (S2-) and derivatization with pentafluorobenzyl bromide: Evidence of S-transnitrosylation and formation of nitrite and nitrate.

A GC-MS method is reported for the quantitative analysis of S-nitrosothiols (RSNO) derived from endogenous low- and high-molecular mass thiols (RSH) including hemoglobin, cysteine, glutathione, N-acetylcysteine, and the exogenous N-acetylcysteine ethyl ester. The method is based on the conversion of RSNO to nitrite by aqueous Na2S (S2-). 15N-Labelled analogs (RS15NO) or 15N-labelled nitrite and nitrate were used as internal standards. The nitrite (14NO2- and 15NO2-) and nitrate (O14NO2- and O15NO2- anions were derivatised by pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone and their PFB derivatives were separated by gas chromatography. After electron-capture negative-ion chemical ionization, the anions were separated by mass spectrometry and detected by selected-ion monitoring of m/z 46 for 14NO2-, m/z 47 for 15NO2-, m/z 62 for O14NO2-, and m/z 63 for O15NO2-. The expected thionitrites (-S14NO and -S15NO) were not detected, suggesting that they are intermediates and rapidly exchange their S by O from water, presumably prior to PFB-Br derivatization. The reaction of S2- with RSNO and sodium nitroprusside (SNP) resulted in the formation of nitrite and nitrate as the major and minor reaction products, respectively. The novel Na2S procedure was compared with established procedures based on the use of aqueous HgCl2 or cysteine/Cu2+ reagents to convert the S-nitroso group to nitrite. Our results provide evidence for an equilibrium S-transnitrosylation reaction between S2- with RSNO in buffered solutions of neutral pH. Use of Na2S in molar excess over RSNO shifts this reaction to the right, thus allowing almost complete conversion of RSNO to nitrite and nitrate. The Na2S procedure should be useful for the quantitative determination of RSNO as nitrite and nitrate after PFB-Br derivatization and GC-MS analysis. The Na2S procedure may also contribute to explore the complex reactions of S2- with RSNO, SNP and other NO-containing compounds.

Volatile S-nitrosothiols and the typical smell of cancer.

An unconventional approach to investigations into the identification of typical volatile emissions during illnesses gives rise to the proposal of a new class of cancer markers. Until now, cancer markers seem not to have been conclusively identified, though the obvious behavior of dogs points to their existence. The focus has been directed towards molecules containing sulfurous functionalities. Among such compounds, S-nitrosothiols (SNOs) are known to be involved in important physiological processes in living organisms and they are described as being typically elevated in cancer. Volatile SNOs (vSNOs) are proposed to be the source of the significant smell of cancer. Synthetic vSNOs are known to have lifetimes of between some minutes and several hours, which may be the main reason as to why they have been ignored until now, and also for the inability of analytics to detect them in vivo. Based on typical structures occurring in the volatile sulfur organics being emitted from human breath, four vSNOs have been synthesized and characterized by tandem mass spectrometry and gas chromatography/mass spectrometry. Simulating the relatively fatty consistency of cancer tissue by diluting the samples in n-decane, surprisingly reduces their tendency to decompose to lifetimes of weeks even at room temperature. A sniffer dog was trained with the synthetic vSNOs, and the results of the tests indicate that synthetic and cancer smells are very similar or even the same. The findings can be a clue for further target-oriented systematic optimization of existing sensitive measurement methods to prove vSNOs as cancer emissions and finally establish future methods for cancer diagnosis based on screening for this new class of volatile illness markers.

S-nitroso-proteome in poplar leaves in response to acute ozone stress.

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = -3.6) and caffeic acid O-methyltransferase (-3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.

Proteomic approaches to evaluate protein S-nitrosylation in disease.

Many of nitric oxide (NO) actions are mediated through the coupling of a nitroso moiety to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process known as S-nitrosylation or S-nitrosation. In many cases this reversible post-translational modification is accompanied by altered protein function and aberrant S-nitrosylation of proteins, caused by altered production of NO and/or impaired SNO homeostasis, has been repeatedly reported in a variety of pathophysiological settings. A growing number of studies are directed to the identification and characterization of those proteins that undergo S-nitrosylation and the analysis of S-nitrosoproteomes under pathological conditions is beginning to be reported. The study of these S-nitrosoproteomes has been fueled by advances in proteomic technologies that are providing researchers with improved tools for exploring this post-translational modification. Here we review novel refinements and improvements to these methods, and some recent studies of the S-nitrosoproteome in disease.

Mechanism-based triarylphosphine-ester probes for capture of endogenous RSNOs.

Nitrosothiols (RSNOs) have been proposed as important intermediates in nitric oxide (NO(•)) metabolism, storage, and transport as well as mediators in numerous NO-signaling pathways. RSNO levels are finely regulated, and dysregulation is associated with the etiology of several pathologies. Current methods for RSNO quantification depend on indirect assays that limit their overall specificity and reliability. Recent developments of phosphine-based chemical probes constitute a promising approach for the direct detection of RSNOs. We report here results from a detailed mechanistic and kinetic study for trapping RSNOs by three distinct phosphine probes, including structural identification of novel intermediates and stability studies under physiological conditions. We further show that a triarylphosphine-thiophenyl ester can be used in the absolute quantification of endogenous GSNO in several cancer cell lines, while retaining the elements of the SNO functional group, using an LC-MS-based assay. Finally, we demonstrate that a common product ion (m/z = 309.0), derived from phosphine-RSNO adducts, can be used for the detection of other low-molecular weight nitrosothiols (LMW-RSNOs) in biological samples. Collectively, these findings establish a platform for the phosphine ligation-based, specific and direct detection of RSNOs in biological samples, a powerful tool for expanding the knowledge of the biology and chemistry of NO(•)-mediated phenomena.

Protein s-nitrosylation measurement.

G protein-coupled receptors (GPCRs) are the most abundant and diverse type of cell surface receptors. GPCR signal duration and amplitude are both controlled by posttranslational modifications, principally phosphorylation. Emerging evidence demonstrates that the GCPRs and their effectors are also subject to S-nitrosylation modification. Protein S-nitrosylation involves the covalent attachment of a nitric oxide (NO) group to the thiol side chain of select cysteine residues (S-NO) that impacts the protein function. Progress in this area of research has been hampered by technical limitations to measure biologic S-nitrosylation, but obstacles have been substantially alleviated over the past few years. The two most commonly used methods to detect S-nitrosylation require decomposition of the S-NO covalent bond and consequent detection of reduced thiol or released NO groups. In this review, we summarize current methods for detection of protein S-nitrosylation with a focus on the biotin switch technique and related methods.

Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine.

Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (>90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.

Visible photolysis and amperometric detection of S-nitrosothiols.

The concentration of S-nitrosothiols (RSNOs), endogenous transporters of the signaling molecule nitric oxide (NO), fluctuate greatly in physiology often as a function of disease state. RSNOs may be measured indirectly by cleaving the S-N bond and monitoring the liberated NO. While ultraviolet photolysis and reductive-based cleavage both decompose RSNOs to NO, poor selectivity and the need for additional reagents preclude their utility clinically. Herein, we report the coupling of visible photolysis (i.e., 500-550 nm) and amperometric NO detection to quantify RSNOs with greater selectivity and sensitivity. Enhanced sensitivity (up to 1.56 nA µM(-1)) and lowered theoretical detection limits (down to 30 nM) were achieved for low molecular weight RSNOs (i.e., S-nitrosoglutathione, S-nitrosocysteine) by tuning the irradiation exposure. Detection of nitrosated proteins (i.e., S-nitrosoalbumin) was also possible, albeit at a decreased sensitivity (0.11 nA µM(-1)). This detection scheme was used to measure RSNOs in plasma and illustrate the potential of this method for future physiological studies.

Identification of protein nitrosothiols using phosphine-mediated selective reduction.

Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.

Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence.

S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.

Direct vasoactive properties of thienopyridine-derived nitrosothiols.

Thienopyridines (ticlopidine, clopidogrel, and prasugrel) require in vivo metabolism to exhibit a critical thiol group in the active form that binds to the P2Y12 platelet receptor to inhibit platelet activation. We hypothesized that formation of thienopyridine-derived nitrosothiols (ticlopidine-SNO, clopidogrel-SNO, and prasugrel-SNO) occurs directly from the respective parent drug. Pharmaceutical-grade thienopyridine (ticlopidine, clopidogrel chloride, clopidogrel sulfate, clopidogrel besylate, or prasugrel) was added to nitrite in aqueous solution to form the respective thienopyridine-SNO (Th-SNO). An isolated aortic ring preparation was used to test vasoactivity of the Th-SNO derivatives. Increasing nitrite availability resulted in increased Th-SNO formation for all drugs (other than ticlopidine). Th-SNO induced significant endothelium-independent relaxation of preconstricted aortic rings. Clopidogrel-chloride-SNO displayed rapid-release kinetics in a chemical environment, which was reflected by immediate and transient vasorelaxation when compared with the SNO derivatives of the other thienopyridines. Accounting for differences in yield, clopidogrel-chloride-SNO exhibited the greatest propensity to immediately relax vascular tissue. Th-SNO derivatives exhibit nitrovasodilator properties by supplying NO that can directly activate vascular soluble guanylate cyclase to induce vasorelaxation. Differences in SNO yield and vasoactivity exist between thienopyridine preparations that might be important to our understanding of the direct pharmacological effectiveness of thienopyridines on vascular and platelet function.

Formation of vascular S-nitrosothiols and plasma nitrates/nitrites following inhalation of diesel emissions.

Epidemiological studies have associated traffic-related airborne pollution with adverse cardiovascular outcomes. Nitric oxide (NO) is a common component of fresh diesel and gasoline engine emissions that rapidly transforms both in the atmosphere and once inhaled. Because of this rapid transformation, limited information is available in terms of potential human exposures and adverse health effects. Young rats were exposed to whole diesel emissions (DE) adjusted to 300 µg/m(3) of particulate matter (containing 3.5 ppm NO) or 0, 3, or 10 ppm NO as a positive control. Animals were also pre-injected (ip) with either saline or N-acetylcysteine (NAC), a precursor of glutathione. Predictably, pure NO exposures led to a concentration-dependent increase in plasma nitrates compared to controls, which lasted for roughly 4 h postexposure. Whole DE exposure for 1 h also led to a doubling of plasma NOx. NAC injection increased the levels of plasma nitrates and nitrites (NOx) in the DE exposure group. Inhibition of nitric oxide symthase (NOS) by N(G)-nitro-L-arginine (L-NNA) did not block the rise in plasma NOx, demonstrating that the increase was entirely due to exogenous sources. Both DE and pure NO exposures paradoxically led to elevated eNOS expression in aortic tissue. Furthermore, coronary arterioles from NO-exposed animals exhibited greater constriction to endothelin-1 compared to controls, consistent with a derangement of the NOS system. Thus, NO may be an important contributor to traffic-related cardiovascular morbidity, although further research is necessary for proper hazard identification.