Simple fluorescent reagents for monitoring disulfide reductase and S-nitroso reductase activities in vitro and in live cells in culture.

This study describes the theoretical basis and the methods for the facile synthesis and characterization of four fluorogenic probes, N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG), N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2) and N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2). In addition, the study describes the methodology for the application of these reagents for measuring and imaging of free thiols on cell surfaces as well as their use as pseudo substrates for the thiol reductase and S-nitrosothioldenitrosylase activities of protein disulfide isomerase (PDI) and S-nitrosothiol reductase activity of S-nitrosoglutathione reductase (GSNOR) in vitro and on live cells in culture.

The catalytic mechanism for NO production by the mitochondrial enzyme, sulfite oxidase.

Recently, Guenter Schwarz and colleagues published an elegant study in the Biochemical Journal (2019) 476, 1805-1815 which combines kinetic and spectroscopic studies with protein engineering to provide a mechanism for sulfite oxidase (SO)-catalyzed nitrite reduction that yields nitric oxide (NO). This work is noteworthy as it demonstrates that (i) for NO generation, both sulfite and nitrite must bind to the same molybdenum (Mo) center; (ii) upon sulfite reduction, Mo is reduced from +6 (MoVI) to +4 (MoIV) and MoIV reduces nitrite to NO yielding MoV; (iii) the heme moiety, linked to the Mo-center by an 11 amino acid residue tether, gets reduced by intramolecular electron transfer (IET) resulting in MoV being oxidized to MoVI; (iv) the reduced heme transfers its electron to a second nitrite molecule converting it to NO; (v) the authors demonstrate steady-state NO production in the presence of the natural electron acceptor cytochrome c; (vi) Finally, the authors use protein engineering to shorten the heme tether to reduce the heme-Mo-center distance with the aim of increasing NO production. Consequently, the rate of IET to cytochrome c is decreased but the enzymatic turnover rate for NO production is increased by ∼10-fold. This paper is unique as it provides strong evidence for a novel mechanism for steady-state NO production for human mitochondrial SO and serves as a potential template for studying NO production mechanisms in other enzymes by integrating the information gained from enzyme kinetics with EPR and UV/vis spectroscopy and protein engineering.

Greenhouse tomato plant roots/carboxymethyl cellulose method for the efficient removal and recovery of inorganic phosphate from agricultural wastewater.

Phosphate (P) is a biologically important compound that is commonly incorporated into fertilizers. Wastewater from agricultural processes results in excessive accumulation of P and eutrophication of lakes. We have developed a system for the remediation, recovery, and potential reuse of P from agricultural wastewater using tomato plant roots (roots) as a capture matrix and carboxymethyl cellulose (CMC) as an eluent and enhancer of P precipitation. Untreated roots can bind up to 55.2 ±â€¯15.2 grams of P per kilogram (g/kg) of roots in comparison to the maximum 8.2 ±â€¯1.5 g/kg bound by the previously used iron-chitosan (Fe-chito). The addition of CMC enhances the precipitation of P with a clearance of 97.2% as opposed to 33.3% without CMC. On site tests show an average removal of 226.5 µg/L per day or a total of ∼28 g of P removed after 23 days. This corresponds to a 71% P removal rate.

Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the αIIbß3 integrin.

The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbß3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn(2+)-treated platelets lacking ß3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbß3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbß3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats.

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress.

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells.

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.

Microplate-based colorimetric detection of free hydrogen sulfide.

Hydrogen sulfide (H2S) has recently been recognized as an important physiologically relevant gasotransmitter. Produced by the enzymes involved in the transsulfuration pathway, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), H2S has been implicated to control biological activity in virtually every organ system. In recent years it is being recognized that many commonly used H2S assays do not measure free H2S specifically and may be prone to artifacts. This has led to large variations in the reported H2S biological concentrations. In order to accurately study H2S's functions in biological systems accurate assays which measure free H2S specifically are required. In this work we present a simple microplate-based colorimetric assay for H2S gas. The underside of a 96-well microplate cover was coated with Nafion polymer doped with Ag(+) ions. H2S is a highly volatile gas, and as it is volatilized in the microplate well it reacts with Ag(+) to produce Ag2S nanoparticles, which have a strong absorbance in the low-UV range. By monitoring the absorbance change from formation of Ag2S nanoparticles, H2S production can be monitored in real time. The assay has a limit of detection (LOD) of 2.61 nmol (8.70 µM) and a liner range up to 30 nmol (100 µM). Using the assay, the KM and Vmax of recombinant CSE enzyme were determined to be 11.13 ± 0.57 mM and 0.45 ± 0.01 nmol min(-1), respectively. H2S production from mouse liver homogenate under aerobic conditions in the presence of cysteine was measured and determined to be 4.89 ± 0.19 nmol min(-1) mL(-1) homogenate. The assay is simple, low cost, and specific to free H2S gas.

An Azomethine-H-Based Fluorogenic Sensor for Formic Acid.

Formic acid (FA) is an important C1-containing feedstock that serves as a masked source of dihydrogen gas (H2). To encourage the adoption of cleaner (noncarbonaceous) energy sources, FA detection and sensing is thus of considerable interest. Here, we examine the use of a commercially available dye, azomethine-H (Az-H), for FA sensing. Solution studies confirm that FA quenches both the absorbance and the luminescence properties of Az-H. FA was additionally found to attenuate a known Az-H (E)-to-(Z) conformational change, suggesting an Az-H/FA interaction, possibly through hydrogen bonding; this phenomenon was probed using 1H NMR spectroscopy. Moving toward a solid-state sensor, the Az-H probe was incorporated into a gelatin-based matrix. On exposure to FA, the luminescence of this system was found to increase in a FA-dependent manner, attributed to the formation of stable hydrogen-bonded structures, facilitating a (Z)-to-(E) isomerization via imine protonation, allowing for production of the more luminescent (E)-isomer. This fluorogenic signal was used as a FA sensor with an estimated detection limit of ca. 0.4 ppb FA vapor. This work constitutes an important step toward a highly sensitive FA sensor in both the solution and solid state, opening new space for the detection of organic acids in differing chemical environments.

Polydimethylsiloxane permeability-based method for the continuous and specific detection of hydrogen sulfide.

Hydrogen sulfide (H(2)S) is known to play a physiological role in processes as diverse as vasodilation, maintenance of vascular tone, neurotransmission, and immune response. The multitude of physiological functions in which H(2)S is involved warrants the development of useful methods for its detection. Here, we introduce a simple and continuous H(2)S detection method that exploits the relatively high polydimethylsiloxane (PDMS) permeability of H(2)S in comparison to other thiols typically encountered in the cellular milieu. In this method, 96-well inserts constructed of PDMS act as an H(2)S-permeable membrane, eliminating nonspecific thiol detection. This design also makes it possible to use virtually any available thiol-specific probe such as Ellman's reagent which was used here to detect H(2)S once it crossed the PDMS membrane. Utilizing this method, a detection limit of 9.2 ± 1.9 ppb(m) (parts per billion (by mole) or ~0.51 µM in 1.6 mL of buffer) free H(2)S (detected as solution sulfide) was achieved. In addition, the assay was used to determine K(M) and V(max) for natural substrates of cystathionine γ-lyase (CSE), the main enzyme responsible for H(2)S production in peripheral tissues. The K(M) and V(max) of CSE for cysteine were 3.79 ± 2.07 mM and 0.37 ± 0.02 nmol H(2)S/min, respectively. K(M) and V(max) for homocysteine were 6.90 ± 1.78 mM and 1.10 ± 0.19 nmol H(2)S/min, respectively. In addition, the assay was used to examine the potential for a direct interaction of H(2)S and NO. The levels of detected H(2)S decreased in the presence of NO under normoxia but not under anoxia indicating that H(2)S does not react with NO but with N(2)O(3) likely formed in the hydrophobic environment of PDMS.

Platelet function and thymosin ß4.

Thymosin ß4 (Tß4) is a small, low-molecular-weight peptide ubiquitously expressed in all cells and extracellular fluids. It is a major actin sequestering protein present in the cells. In addition to this, Tß4 has also been shown to be involved in endothelial cell migration, angiogenesis, corneal wound healing, and stem cell differentiation. It is also released by platelets after activation. The amount of Tß4 increases at sites of injury and thus suggests an important role of this biopeptide in wound healing. Herein, we provide an overview of the role of Tß4 in thrombosis and platelet aggregation.

Fluorescein isothiocyanate, a platform for the selective and sensitive detection of S-Nitrosothiols and hydrogen sulfide.

Here we show that the fluorescence of fluorescein isothiocyanate (FITC) is not altered by its reaction with primary amines. However, the fluorescence is rapidly quenched upon reaction with small molecular weight thiols including cysteine, glutathione, homocysteine, dithiothreitol, and sulfide. We have taken advantage of the thiol-dependent quenching of FITC to devise a sulfide specific assay by utilizing polydimethylsiloxane (PDMS) membranes that are permeable to hydrogen sulfide but not to larger charged thiols. In addition, we have discovered that the fluorescein dithiocarbamate (FDTC) formed by the reaction with sulfide can specifically react with S-nitrosothiols (RSNO) to regenerate FITC, thus serving as a specific, fluorogenic reagent to detect picomol levels of RSNO. FDTC was tested as an intracellular RSNO-sensor in germinating tomato seedlings (Solanum lycopersicum) via epifluorescence microscopy. Control plant roots exposed to FDTC showed low intracellular fluorescence which increased ∼3-fold upon exposure to extracellular S-nitrosoglutathione and ∼4-fold in the presence of N6022, a S-nitrosoglutathione reductase (GSNOR) inhibitor, demonstrating that FDTC can be used to visualize intracellular RSNO levels.

Conjugated Polymer Nanoparticles as a Universal High-Affinity Probe for the Selective Detection of Microplastics.

Microplastic (MP) pollution is a global challenge that requires immediate mitigation practices. Monitoring is crucial for quantifying MPs, but their mitigation remains very challenging due to several factors, including the lack of selective materials to specific polymers, and the low sensitivity of the current detection techniques. In this work, we introduce a novel design for the selective detection of MPs through fluorescence spectroscopy by exploiting conjugated polymer nanoparticles (CPNs). Fluorescent diketopyrrolopyrrole nanoparticles were prepared by nanoprecipitation to incorporate peripheral hyaluronic acid to increase their affinity for various plastics. The affinity of the new ligand for various types of MPs was examined through several characterization techniques, including fluorescence spectroscopy and microscopy, nanoparticle tracking analysis and computational studies. The new CPN were shown to be highly fluorescent in the presence of typically abundant MPs, achieving very strong binding constants in the picomolar range. This very strong affinity for a broad family of plastics was found to be the results of cooperative supramolecular effects and topographical affinity, as probed by advanced microscopy and in silico studies. Furthermore, the new affinity probes were shown to be highly selective for MPs, allowing for their detection in heterogeneous samples, including soil debris and other organic contaminants. The new materials design introduced in this work constitute a promising platform for the development of novel MP detection devices directly useable at the point of collection. Moreover, it opens new avenue for the mitigation of this environmental hazard through tailorable materials.

Sinapinic acid can replace ascorbate in the biotin switch assay.

BACKGROUND: Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins. METHODS: The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO(2)(-) released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells+/-S-nitrosocysteine (CysNO) exposure. RESULTS: We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges. CONCLUSIONS: Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay. GENERAL SIGNIFICANCE: The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.

Whole blood, flow-chamber studies in real-time indicate a biphasic role for thymosin ß-4 in platelet adhesion.

BACKGROUND: Thymosin beta 4 (Tß(4)) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing. METHODS: In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins-fibrinogen and collagen-were immobilized onto the middle ~25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tß(4), on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers. RESULTS: The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026±0.0015s(-1), corresponding to a half-life of 26.7s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tß(4) (0.2 µM to 0.5 µM) increased both the rate constant of platelet deposition by ~1.5-fold (i.e. half-life decreased from 26.7s to 17.6s) and the total number of deposited platelets by ~3-fold. However at higher concentrations (>1 µM) the Tß(4)-potentiating effect was diminished to near control levels. Tß(4) did interact with fibrinogen with an estimated K(D) of ~126±18nM or 66±20nM under equilibrium or flow, respectively. CONCLUSION: These results suggest that Tß(4) could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tß(4) could also bind to fibrinogen and as its concentration increased would prevent platelet-fibrinogen interactions resulting in the attenuation of platelet deposition. GENERAL SIGNIFICANCE: This work suggests that Tß(4) might have a dual role in platelet function.

Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins.

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.

Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia.

Nitrite (NO(2)(-)) recycling to nitric oxide (NO) is catalysed by a number of enzymes and induces a protective vasodilation effect under hypoxia/ischaemia. In the present work, we tested the in vitro ability of the three NOS (nitric oxide synthase) isoforms to release NO from nitrite under anoxia using electrochemical detection, chemiluminescence and absorption spectroscopy. The release of free NO from anoxic nitrite solutions at 15 muM was specific to the endothelial NOS isoform (eNOS) and did not occur with the neuronal (nNOS) or inducible (iNOS) isoforms. Unlike xanthine oxidase, the eNOS reductase domain did not recycle nitrite to NO, and wild-type eNOS did not reduce nitrate. Our data suggest that structural and, by inference, dynamic differences between nNOS and eNOS in the distal haem side account for eNOS being the only isoform capable of converting nitrite into NO at pH 7.6. In human dermal microvascular endothelial cells under careful control of oxygen tension, the rates of NO formation determined by chemiluminescence were enhanced approximately 3.6- and approximately 8.3-fold under hypoxia (2 p.p.m. O(2)) and anoxia (argon) respectively compared with normoxia ( approximately 22 p.p.m. O(2)) using 10 muM extracellular nitrite. NOS inhibitors inhibited this hypoxic NO release. Our data show that eNOS is unique in that it releases NO under all oxygen levels from normoxia to complete anoxia at physiological micromolar nitrite concentrations. The magnitude of the hypoxic NO release by the endothelial cells suggest that the endothelium could provide an appropriate response to acute episodic ischaemia and may explain the observed eNOS-expression-specific protective effect as a short-term response in animal models of acute hypoxia.

The Physiological Implications of S-Nitrosoglutathione Reductase (GSNOR) Activity Mediating NO Signalling in Plant Root Structures.

Nitrogen remains an important macronutrient in plant root growth due to its application in amino acid production, in addition to its more elusive role in cellular signalling through nitric oxide (NO). NO is widely accepted as an important signalling oxidative radical across all organisms, leading to its study in a wide range of biological pathways. Along with its more stable NO donor, S-nitrosoglutathione (GSNO), formed by NO non-enzymatically in the presence of glutathione (GSH), NO is a redox-active molecule capable of mediating target protein cysteine thiols through the post translational modification, S-nitrosation. S-nitrosoglutathione reductase (GSNOR) thereby acts as a mediator to pathways regulated by NO due to its activity in the irreversible reduction of GSNO to oxidized glutathione (GSSG) and ammonia. GSNOR is thought to be pleiotropic and often acts by mediating the cellular environment in response to stress conditions. Under optimal conditions its activity leads to growth by transcriptional upregulation of the nitrate transporter, NRT2.1, and through its interaction with phytohormones like auxin and strigolactones associated with root development. However, in response to highly nitrosative and oxidative conditions its activity is often downregulated, possibly through an S-nitrosation site on GSNOR at cys271, Though GSNOR knockout mutated plants often display a stunted growth phenotype in all structures, they also tend to exhibit a pre-induced protective effect against oxidative stressors, as well as an improved immune response associated with NO accumulation in roots.

Platelet microparticle-associated protein disulfide isomerase promotes platelet aggregation and inactivates insulin.

Platelet-derived microparticles (pMP) have been shown to be pro-aggregatory and retain most of their platelet membrane markers. Recent studies have correlated elevated pMP levels with pathogenesis of diabetes mellitus and cardiovascular disease. The pro-aggregatory effect of pMP has been largely attributed to their negatively charged outer surface and activation of factor X by membrane associated Tissue factor (TF). Here we sought to investigate whether, like platelets, protein disulfide isomerase (PDI) is present on the surface of pMP and, if so, to analyze its contribution to platelet hyperaggregability and insulin degradation. Using a fluorescent assay based upon a novel pseudo-substrate of PDI, flow cytometry and immunological techniques, we have demonstrated the presence of PDI on the surface of pMP (termed msPDI) and its ability to influence insulin-mediated Akt phosphorylation (Thr308) in 3T3-L1 fibroblasts. Moreover, pMP are shown to contain catalytically active PDI, capable of both promoting platelet aggregation and disrupting insulin signaling. pMP increased initial rates of aggregation by 4-fold and the pro-aggregatory activity of pMPs could be attenuated with an anti-PDI antibody. The pMP insulin-reductase activity was further attributed to PDI based on the ability of anti-PDI antibodies to block the degradation of insulin, thereby restoring insulin signaling. Plasma pMP counts were also obtained from diabetic (n=10) and non-diabetic individuals (n=10) and found to be elevated in the diabetic state. Detection of increased levels of PDI-containing microparticles in patients with T2D raises the possibility that platelet hypersensitivity and insulin desensitization observed in diabetes can partially be attributed to msPDI activity.

Disulfide-linked, gold nanoparticle based reagent for detecting small molecular weight thiols.

Disulfide-linked gold nanoparticles (AuNP) were synthesized by reacting dithiobis[succinimidylpropionate] (DSP) coated nanoparticles with glutathione disulfide. AuNP-cross-linking was monitored by the red shift and broadening of the AuNP's localized surface plasmon absorption resonance (LSPR) spectrum. The exposure of the disulfide-linked AuNPs to a variety of free thiols with systematically varying molecular weight revealed a AuNP-disulfide stability to reduction by thiols up to a critical molecular weight, M(c), of >300 Da thus making the disulfide-linked AuNP the first reagent that can discriminate thiols based on their size.

Evidence for an Allosteric S-Nitrosoglutathione Binding Site in S-Nitrosoglutathione Reductase (GSNOR).

Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill-Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen-deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme.