Surface Thiol Research Articles

Properties of the Protein and Carbohydrate Fractions in Immature Wheat Kernels

The time course of compositional changes in the polysaccharide and protein fractions, and of changes in protein structural properties, was investigated in grains of two Italian durum wheat cultivars, Ofanto and Duilio, and the pattern of changes was found to be specific to each cultivar. The fructo-oligosaccharide content in ripening kernels was different between cultivars, as was the pattern of starch accumulation. Evaluation of gluten proteins by Western-blotting using broad-specificity anti-gliadin antibodies showed the sudden appearance of immunoreactive proteins during ripening. Protein surface hydrophobicity and thiol content and accessibility were evaluated in conditions apt at assessing changes in protein interaction and/or in aggregative patterns. Proteins in the two cultivars showed differences in their surface hydrophobicity and in their content of accessible thiols. The simultaneous evaluation of changes in the individual components indicates that use of immature grains for the production of functional foods will require accurate monitoring of the rapidly evolving biopolymer patterns, and careful choice of the cultivar.

Ultrathin gradient films using thiol‐ene polymerizations

AbstractThe application of surface‐attached, thiol‐ene polymer films for controlling material properties in a gradient fashion across a surface was investigated. Thiol‐ene films were attached to the surface by first depositing a thiol‐terminated self‐assembled monolayer and performing a thiol‐ene photopolymerization reaction on the surface. Property gradients were created either by creating and modifying a gradient in the surface thiol density in the SAM or by changing the polymerization conditions or both. Film thickness was modified across the substrate by changing either the density of the anchoring thiol functional groups or by changing the reaction conditions such as exposure time. Thicker films (1–11 nm) were obtained by polymerizing acrylate polymer brushes from the surface with varying exposure time (0–60 s). The two factors, that is, the surface thiol density and the exposure time, were combined in orthogonal directions to obtain thiol‐ene films with a two‐dimensional thickness gradient with the maximum thickness being 4 nm. Finally, a thiol‐acrylate Michael type addition reaction was used to modify the surface thiol density gradient with the cell‐adhesive ligand, Arg‐Gly‐Asp‐Ser (RGDS), which subsequently yielded a gradient in osteoblast density on the surface. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 7027–7039, 2006

Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes

CO 2 changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO 3 – / NO 2 radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO 3 – and NO 2 are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO 2 peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO 2 peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.

Formation of Single Pt Atoms on Thiolated Carbon Nanotubes Using a Moderate and Large‐Scale Chemical Approach

Single Pt atoms are formed on thiolated carbon nanotubes (see figure) by using a moderate and large-scale chemical approach instead of conventional physical methods that require severe conditions. The surface modification results from the formation of bonds between Pt and surface thiol groups by weak physisorption. Furthermore, the electronic state of single Pt atoms is close to that of bulk Pt rather than the precursor ion.

Density functional study of a typical thiol tethered on a gold surface: ruptures undernormal or parallel stretch

The mechanical and dynamical properties of a model Au(111)/thiol surface system wereinvestigated by using localized atomic-type orbital density functional theory in the localdensity approximation. Relaxing the system gives a configuration where the sulfur atomforms covalent bonds to two adjacent gold atoms as the lowest energy structure.Investigations based on ab initio molecular dynamics simulations at 300, 350 and 370 Kshow that this tethering system is stable. The rupture behaviour between the thiol and thesurface was studied by displacing the free end of the thiol. Calculated energy profilesshow a process of multiple successive ruptures that account for experimentalobservations. The process features successive ruptures of the two Au–S bonds followed bythe extraction of one S-bonded Au atom from the surface. The force required torupture the thiol from the surface was found to be dependent on the direction inwhich the thiol was displaced, with values comparable with AFM measurements.These results aid the understanding of failure dynamics of Au(111)-thiol-tetheredbiosurfaces in microfluidic devices where fluidic shear and normal forces are ofconcern.

A Novel and Simplified Procedure for Patterning Hydrophobic and Hydrophilic SAMs for Microfluidic Devices by Using UV Photolithography

This work describes how selective patterning of hydrophobic and hydrophilic areas inside microchannels of microfluidic devices can be achieved by combining well-known chemical protocols and standard photolithography equipment (365 nm). Two techniques have been performed and compared. The first technique is based on the preparation of self-assembled monolayers of photocleavable organosilane and the second one on photoassisted grafting (365 nm) of self-assembled monolayers (SAMs) on a silicon or glass substrate. In the first case, we begin with monolayers carrying an o-nitrobenzyl function (hydrophobic area) that is photochemically cleaved, revealing a carboxylic acid group (hydrophilic area). The problem is that the energy necessary to cleave this monolayer is too high and the reaction time is more than 1 h with 50 mW/cm(2) irradiation flux. To overcome this practical disadvantage, we propose another approach that is based on the thiol-ene reaction with benzophenone as photoinitiator. In this approach, a monolayer of mercaptopropyltrimethoxysilane (MPTS) is prepared first. Subsequently, a hydrocarbon chain is photografted locally onto the thiol layer, forming a hydrophobic surface while the reminding unmodified thiol surface is oxidized into sulfonic acid (hydrophilic area). We demonstrated the feasibility of this approach and synthesized high-quality self-assembled monolayers by UV grafting with an irradiation time of 30 s at 365 nm (50 mW/cm(2)). The modified surfaces have been characterized by contact angle measurements, X-ray photoelectron spectroscopy (XPS), AFM, and multiple internal reflection infrared spectroscopy (MIR-FTIR). The difference in the contact angles on the hydrophilic and hydrophobic surfaces reached a remarkable 77 degrees. We have also demonstrated that this method is compatible with selective surface grafting inside microfluidic channels.

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that the lower capacity to produce ROS is associated with an increased number of reduced thiol groups (-SH) on T cell membrane surfaces. Artificially increasing the number of reduced thiols on T cells from animals with arthritis-protective Ncf1 alleles by glutathione treatment lowered the threshold for T cell reactivity and enhanced proliferative responses in vitro and in vivo. Importantly, T cells from immunized congenic rats with an E3-derived Ncf1 allele (DA.Ncf1E3 rats) that cannot transfer arthritis to rats with an arthritis-associated Dark Agouti (DA)-derived mutated Ncf1 allele (DA.Ncf1DA rats) became arthritogenic after increasing cell surface thiol levels. This finding was confirmed by the reverse experiment, in which oxidized T cells from DA.Ncf1DA rats induced less severe arthritis compared with controls. Therefore, we conclude that ROS production as controlled by Ncf1 is important in regulating surface redox levels of T cells and thereby suppresses autoreactivity and arthritis development.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 μM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) (100 μM) or inhibiting PDI with bacitracin (5 mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 μM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 μM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-( N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Ultrathin Patterned Polymer Films on Surfaces Using Thiol−Ene Polymerizations

A simple and straightforward method for growing ultrathin, micropatterned polymer films on surfaces with precise thickness and spatial control is presented. A difunctional ene monomer and a difunctional thiol were photopolymerized on a surface terminated with thiol groups. The surface thiols participate in the polymerization, and a linear polymer is formed on the surface. Brush growth was spatially controlled by selectively polymerizing the monomers through a photomask, while the brush thickness was controlled by changing the ratio of thiol and ene monomers or by changing the monomer functionality. Further, the surface was passivated by attaching a patterned, cross-linked polymer film, which was then backfilled with another monomer, demonstrating the attachment of multiple functionalities on the surface in a controlled, photolithographically patternable manner. Thiol−ene polymerizations were carried out without an initiator and used to graft patterned, polymer films. The films were observed using scanning...

Pyrococcus furiosus 4Fe-ferredoxin, chemisorbed on gold, exhibits gated reduction and ionic strength dependent dimerization

Pyrococcus furiosus ferredoxin is a small metalloprotein that shuttles electrons between redox enzymes. In its native 4Fe-4S form the protein is highly thermostable. In addition to three cluster-ligating cysteines, two surface cysteine residues (C21 and C48) are present. We used the reactivity of these surface thiols to directly immobilize ferredoxin on a bare gold electrode, with an orientation in which the cluster is exposed to solution. Voltammetry, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) studies established the immobilization of the 4Fe form. Native and recombinant wild-type ferredoxins were compared with the C48S, C21S, and C21S/C48S mutants. The variants with one and two surface cysteines can be directly chemisorbed on bare gold. Cyclic voltammetry demonstrated that the reduction potentials are similar to those in solution. The interfacial electron transfer kinetics revealed that the reduction is gated by the interconversion between two oxidized species. AFM images showed that dimers are chemisorbed at low ionic strength, while monomers are present at high ionic strength. XPS spectra revealed the presence of S, Fe, C, N, and O at the surface, which are assigned to the corresponding atoms in the peptide and the cofactor. Analysis of the sulfur spectrum corroborates that both C21 and C48 form gold-thiolate bonds. Moreover, two inorganic sulfide and two iron species were identified, suggesting an inhomogeneous charge distribution in the 4Fe-4S cluster. In conclusion, P. furiosus ferredoxin can be directly and vectorially chemisorbed on gold with retention of its properties. This may provide a biocompatible electrode surface with docking sites for redox enzymes.

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST-GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2 degrees. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.

Surface thiolation of carbon nanotubes as supports: A promising route for the high dispersion of Pt nanoparticles for electrocatalysts

This paper introduces a unique method for achieving high dispersion of precious metal nanoparticles on carbon nanotube supports for electrocatalysts: the surface thiolation of multiwall carbon nanotubes (MWNT). The high dispersion of Pt nanoparticles [i.e., small average particle size (1.5 nm) and narrow size distribution] was obtained by the surface thiolation of MWNT as supports, despite a considerably large Pt load (40 wt% Pt to MWNT) and the use of MWNT with a relatively small surface area as supports to common activated carbon. In particular, EXAFS and XPS data revealed that the interaction between Pt nanoparticles and surface thiol groups on MWNT was the reason for the high dispersion. These highly dispersed Pt nanoparticles showed enhanced and stable electrocatalytic activity in methanol oxidation and oxygen reduction reactions. This result indicates that the surface thiolation of supports is an effective way to obtain highly dispersed precious metal nanoparticles to enhance electrocatalytic activity.

Fine Size Control of Platinum on Carbon Nanotubes: From Single Atoms to Clusters

Bond formation between single Pt atoms and surface thiol groups is the result of the reduction of a Pt precursor in the presence of surface-thiolated carbon nanotubes. By setting the temperature of the following heat treatment, finely size-controlled and monodispersed Pt clusters were obtained from the dispersed single atoms (see picture).

Regulation of redox-sensitive exofacial protein thiols in CHO cells

Thiols affect a variety of cell functions, an effect known as redox regulation, largely attributed to modification of transcription factors and intracellular signaling mechanisms. Since exofacial protein thiols are more exposed to redox-acting molecules used in cell culture and may represent sensors of the redox state of the environment, we investigated their susceptibility to redox regulation. Exofacial protein thiols were measured using cell-impermeable Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB]. For quantification, we also set up an ELISA assay based on the cell-impermeable biotinylated SH reagent, N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM). Exposure of CHO cells to H(2)O(2) induces oxidation of surface thiols at concentrations not affecting intracellular GSH. Depletion (50%) of GSH decreases surface thiols by 88%. Surface thiols are also highly sensitive to thiol antioxidants, since exposure to 5 mM N-acetyl-L-cysteine (NAC) for 2 h augmented their expression without increasing GSH levels. Using BIAM labeling and two-dimensional gel electrophoresis, we show that this increase in surface thiols is due to the reduction of specific membrane proteins. Peptide mass fingerprinting by MALDI mass spectrometry allowed us to identify two of these proteins as Erp57 and vimentin.

Surface PEGylation and Ligand Exchange Chemistry of FePt Nanoparticles for Biological Applications

FePt magnetic nanoparticles (MNPs) were functionalized with a mixed monolayer of poly(ethylene glycol)-terminated thiol and dopamine ligands. The resulting nanoparticles were soluble and stable in aqueous media, including water, ionic solutions, and cell culture medium. The surface thiol ligands are readily exchanged with other thiols bearing chain-end functionalities. MNPs featuring either a cationic or an anionic surface were synthesized by ligand exchange chemistry to afford ligand peripheries capable of binding biomolecules. Surface binding of cationic MNPs to DNA and anionic MNPs to chymotrypsin was enabled by incorporation of a charged functionality on the nanoparticle surface. This approach represents a general strategy to synthesize functionalized FePt nanoparticles that form stable solutions in water and facilitates the use of these magnetic FePt nanoparticles in biological applications.

Covalent modification of actin by 4‐hydroxy‐trans‐2‐nonenal (HNE): LC‐ESI‐MS/MS evidence for Cys374 Michael adduction

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.

The Kinetics of Heat-Induced Structural Changes of β-Lactoglobulin

Heat-induced structural changes of β-lactoglobulin were studied at temperatures ranging from 67.5 to 82.5°C, and at pH 7.5. These changes were monitored by measurement of surface hydrophobicity, thiol availability, and protein solubility. Kinetic studies were conducted to quantitatively describe the contribution of hydrophobic and SH/SS interchange reactions to the thermal structural changes of β-lactoglobulin. Results indicate that β-lactoglobulin is sensitive to heat-induced interchange reactions with consequences for protein solubility. The extent of changes measured by the increase in surface hydrophobicity and the decrease in slow-reacting SH groups content could be described by a first-order fractional conversion model and were characterized by activation energy values of 233.9±8.6 and 148.2±6.7kJ/mol, respectively. The break in the Arrhenius plot suggested in literature for β-lactoglobulin denaturation was confirmed in this study only for the kinetics of exposed SH groups.

Controlled self-assembly of thiol-terminated poly(amidoamine) dendrimer and gold nanoparticles

A dendrimer-mediated “bricks and mortar” strategy for the ordering of gold nanoparticles into structured assemblies was described. The poly(amidoamine) (PAMAM) dendrimer used were generation 2.0–5.0 having surface thiol group (G2.0–5.0 SH). By using thiol-terminated PAMAM dendrimer, stable self-assembled gold clusters in water were obtained as characterized by UV–vis spectroscopy and transmission electron microscopy. In this approach, direct control of interparticle space is provided through choice of dendrimer generation.

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K- ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin–SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K- ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.