Thiol-disulfide Interchange Reactions Research Articles

Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples.

To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S-S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S-S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S-S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity.

Solid-Phase Synthesis of an "Inaccessible" hGH-Derived Peptide Using a Pseudoproline Monomer and SIT-Protection for Cysteine.

The solid-phase peptide synthesis (SPPS) of the C-terminal sequence of hGH with one extra Tyr attached to its N-terminus (total of 16 residues with a disulfide bridge) has been accomplished for the first time by optimizing several synthetic parameters. First of all, the two Ser residues (positions 9 and 13 of the molecule) have been introduced as a single amino acid, Fmoc-Ser(ψMe,Mepro)-OH, demonstrating that the acylation of these hindered moieties is possible. This allows us to avoid the use of the corresponding dipeptides, Fmoc-AA-Ser(ψMe,Mepro)-OH, which are very often not commercially available or very costly. The second part of the sequence has been elongated via a double coupling approach using two of the most effective coupling methods (DIC-OxymaPure and HATU-DIEA). Finally, the disulfide bridging has been carried out very smoothly by a chemoselective thiol-disulfide interchange reaction between a SIT (sec-isoamyl mercaptan)-protected Cys residue and the free thiol of the second Cys. The synthesis of this short peptide has evidenced that SPPS is a multifactorial process which should be optimized in each case.

Physicochemical properties and interfacial dilatational rheological behavior at air-water interface of high intensity ultrasound modified ovalbumin: Effect of ionic strength

High intensity ultrasound (HIUS), as an efficient modification method, generally improves the interfacial properties of globular proteins. Herein, to better understand the intrinsic relationship between the foaming property and the interface behavior of HIUS-treated ovalbumin (OVA). The main purpose of this work was to investigate the correlation between the physicochemical properties of high intensity ultrasound (HIUS)-treated ovalbumin (OVA) and interfacial dilatational rheological behavior at air-water interface with different salt ionic strength. The results showed that HIUS treatment resulted in aggregation of OVA dominated by hydrophobic interaction. The size of HIUS-treated OVA gradually increased due to the charge shielding effect caused by the increase of salt ionic strength, while the surface net potential and free sulfhydryl content of the OVA particle were progressively reduced. As a result, the HIUS treated-OVA exhibited the highest diffusion rate and the amount of protein adsorbed at the air-water interface with salt ionic concentrations of 50 mM and 100 mM, respectively. On the other hand, the modified-OVA contact with the air-water interface led to an exposure of aromatic residues, the content of α–helix, β-helix, and β-turn decreased by 6.3%, increased by 10.3%, and decreased by 5.5%, respectively, and there was no salt ion strength dependence. Moreover, the conformational changes resulted in a thiol-disulfide interchange reaction between the OVA molecules at the interface, which was the main contribution to the viscoelastic properties of OVA absorbed layer. These findings provide better insights for an in-depth understanding of the foaming mechanism of ultrasound treated OVA.

Thermodynamic aspects of bis(3‑sulfopropyl) disulfide and 3‑mercapto‑1‑propanesulfonic acid in Cu electrodeposition

Organic additives play an important role in regulating film growth and properties in Cu electrodeposition. 3‑Mercapto‑1‑propane sulfonate (MPS) and its dimer bis(3‑sulfopropyl) disulfide (SPS) are representative accelerators that promote Cu electrodeposition rate. In this study, the formal reduction potential of SPS to MPS was determined by measuring the equilibrium constants of thiol–disulfide interchange reactions with the reference thiol/disulfide pairs by 1H nuclear magnetic resonance (1H NMR) spectroscopy. The calculated formal reduction potential of SPS to MPS was 0.153 V (vs. standard hydrogen electrode). Based on this calculation, the cell potentials of Cu(I)(thiolate) formation reaction with either SPS or MPS, which is a key reaction in accelerating mechanism, were also estimated by ultraviolet–visible absorption and 1H NMR spectroscopies: they were found to be 0.202 V and 0.214 V for SPS and MPS, respectively. The positive cell potential indicates that the formation of Cu(I)(thiolate) complexes is thermodynamically favorable in the presence of additives.

Effects of thermal and mechanical treatments on the physicochemical properties of wheat flour

Planetary ball milling is a possible way to improve the technological properties of wheat flour by thermal and mechanical modifications. In the present study, roller milled common wheat flours (Triticum aestivum L.) were additionally modified in a ball mill by varying mill parameters such as rotation speed and grinding time. As a result of ball milling, the flours experienced temperatures of up to 100 °C, the range of particle sizes strongly increased as well as the content of damaged starch. The treated flours were also analyzed for the protein distribution by SDS-polyacrylamide-gel electrophoresis and proteins were quantitated by an extraction/high-performance liquid chromatography (Osborne fractionation) and an extraction/gel permeation chromatography procedure (SDSL/GMP fractionation). Intense mechanical/thermal treatment led to a decrease of the content of extractable protein and shifted ethanol-soluble gliadins to the glutenin fraction. Cysteine-containing alpha- and gamma-gliadins initially soluble in 60% ethanol were bound to ethanol-insoluble glutenins by thiol–disulfide interchange reactions. Linked to that, a high level of modification also significantly decreased the gliadin/glutenin ratio and increased the glutenin macropolymer content. Cysteine-free omega5- and omega1,2-gliadin types were only weakly affected by milling. Heating induced by mechanical treatment is supposed to be mainly responsible for these effects. Thiol–disulfide interchange can also explain the increase of the particle size after intense ball milling, because flour particles could stick together via disulfide bonds between proteins located on the surface of different particles.

Alkylation of Free Thiol Groups During Extraction: Effects on the Osborne Fractions of Wheat Flour

Wheat proteins are classified according to solubility into the so‐called Osborne fractions. Because wheat flour contains both free thiol and disulfide groups, thiol–disulfide interchange reactions are possible during extraction. Osborne fractionation of 12 different wheat flour samples was performed in the presence of N‐ethylmaleinimide (NEMI) to alkylate free thiol groups and without addition of NEMI (control). The addition of NEMI during extraction tended to decrease the content of gliadins (predominantly α‐gliadins) and caused an increase of the content of glutenins in most flour samples. Thus, alkylation of free thiol groups during extraction led to a decline of the gliadin/glutenin ratio from 2 (control) to approximately 1.5 (NEMI). NEMI and control gliadins were separated by gel‐permeation HPLC into an oligomeric subfraction (high‐molecular‐weight [HMW] gliadins) and two monomeric subfractions. In most flours (8 of 12), the addition of NEMI led to a significant increase of the content of HMW gliadins. HMW gliadins from cultivar Akteur wheat were preparatively isolated from NEMI and control gliadins and characterized by HPLC, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and N‐terminal sequencing. HMW gliadin isolated in the presence of NEMI had a significantly higher content of low‐molecular‐weight glutenin subunits and disulfide‐bound cysteine as well as a lower content of α‐gliadins and disulfide‐bound glutathione compared with the control.

Red/ox states of human protein disulfide isomerase regulate binding affinity of 17 beta-estradiol

Human protein disulfide isomerase (hPDI) is a key redox-regulated thiol-containing protein operating as both oxidoreductase and molecular chaperone in the endoplasmic reticulum of cells. hPDI thiol-disulfide interchange reactions lead to the adoption of two distinct red/ox conformations with different substrate preferences. hPDI also displays high binding capacity for some endogenous steroid hormones including 17 beta-estradiol (E2) and thus contributes to the regulation of their intracellular concentration, storage and actions. The primary focus of this study was to investigate the impact of E2 binding on functional activity of recombinant hPDI. Then, we examined the effect of E2 binding on structural alteration of hPDI red/ox conformations and its influence on affinity and position of interaction using experimental and computational analysis. Our results revealed that interaction of one E2 per each hPDI molecule led to the inhibition of hPDI reductase activity and conformational changes in both oxidation states. Mutually, E2-binding position were also redox-regulated with higher affinity in oxidized hPDI compare to the reduced form. The importance of histidine-256 protonation states in distinct binding preferences of E2 were also demonstrated in hPDI red/ox conformations. These findings might pave the way for better understanding of the mechanisms behind the redox-dependent hormone-binding activity of hPDI.

Photo-responsive liquid crystalline epoxy networks with exchangeable disulfide bonds

Disulfide exchange and thiol–disulfide interchange reactions allow for reprocessing and recycling of azobenzene-based liquid crystalline networks.

Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention

ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds theoxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes theessential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

Altered Maternal Serum Dynamic Thiol-Disulfide Interchange Reactions in Pregnant Women with Gestational Diabetes Mellitus

OBJECTIVE: We aimed to evaluate the alteration of dynamic thiol/disulfide homeostasis which basically shows the oxidative stress in body in pregnant women with gestational diabetes mellitus (GDM) by using a newly developed and automated analysis method. STUDY DESIGN: Study population consisted of body mass index and gestational age-matched pregnant women. Patients were subdivided into 3 groups based on their response to glucose challenge test (GCT) and oral glucose tolerance test (OGTT) results: (1) control group (normal GCT test results n= 87); (2) Impaired glucose tolerance (IGT) group ( n= 37); (3) GDM group (n=25). Maternal serum thiol and disulphide levels were compared among three groups. RESULTS: Maternal serum thiol values were found decreased and maternal serum disulphide levels were increased in GDM groups were compared to control and IGT groups (Thiols for controls: 398.19±30.49 µmol/L, for IGT group 395.92±35.52 µmol/L, and for GDM group 371.89±41.14 µmol/L p= 0.002). Disulphides for controls; 17.47±4.38 µmol/L, for IGT; 19.27±3.34 µmol/L, for GDM group; 25.46±4.21 µmol/L, (p=0.001). Disulphide/thiol ratio was found to be increased in GDM group comparing to other groups (Disulphide/thiol ratio: 0.044±0.012 for controls, 0.049±0.009 for IGT group, 0.068±0.0103 for GDM group p=0.001) CONCLUSION: Thiol- disulphide balance has shifted to the oxidative side in pregnant women with GDM. So blood glucose regulation is extremely crucial for reducing the oxidative stress which may lead to damages to vital organs of a mother or possibly to development of a fetus in women with GDM.

Reconstitution baking tests with defatted wheat flour are suitable for determining the functional effects of lipase-treated wheat lipids

A microscale reconstitution baking test, using wheat flour defatted with 2-propanol at 20°C, was established to determine the functional effects of lipids isolated from lipase-treated wheat dough. Proper selection of solvent and extraction temperature was of major importance to maintain the functionality of defatted flour. Dough and gluten from flour defatted with water-saturated 1-butanol (WSB; extracted at 20°C) and 2-propanol (extracted at 75°C) had inferior extensibility and loaf volume compared to control flour extracted with 2-propanol at 20°C. Quantitation of gluten proteins showed that defatting with WSB (20°C) or 2-propanol (75°C) decreased the gliadin and increased the glutenin content. Possible reasons were thiol-disulfide interchange reactions, caused either by heat (2-propanol, 75°C) or by the solvent WSB, which affected gluten proteins. Confocal laser scanning microscopy showed that regular, interconnected gluten structures were only present in dough from flour defatted with 2-propanol at 20°C.

Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

Switching the Biointerface of Displaceable Poly-p-xylylene Coatings.

A new class of functionalized poly-p-xylyene coating has been synthesized to provide switchable and displaceable surface properties for biomaterials. The switchability is achieved through a mechanism for detaching/attaching biomolecules and/or a mechanism through which the programmed restoration of functions or their replacement by other functions can be carried out. This advanced version of poly-p-xylylene comprises an integrated disulfide moiety within the functional side group, and the switching phenomenon between the immobilized functional molecules is triggered by the redox thiol-disulfide interchange reaction. These dynamically well-defined molecules on the surfaces respond simultaneously to altered biological properties and controlled biointerfacial functions, for example, switching wettability or reversibly altered cell adhesion activity. Poly-p-xylylenes are a key player in controlling surface properties for many important applications, such as medical implants, biosensors, bioMEMS devices, and microfluidics. The introduction of this new facet of poly-p-xylylenes enables the dynamic mimicry of biological functions relevant to the design of new biomaterials.

An Assay Study of Molecular Recognition of Amino Acids in Water: Covalent Imprinting of Cysteine

A novel synthetic N-(9-fluorenyl methoxy carbonyl)-L-Cysteine (Fmoc-Cys(SH)-OH) receptor was pre- pared by co-polymerizing (9-fluorenyl methoxy carbonyl)-S-(1-propene-2-thiol)-L-Cysteine (Fmoc-Cys(SCH2CHCH2)-OH) and a non-imprinted polymer prepared from 1-propene-1-thiol photo-chemically 15 h at room temperature and additional 3 h thermally at 80℃. Subsequently, disulfides were reduced with lithium aluminum hydride (LiAlH4) from imprinted polymers. The imprinted polymers selectively recognized Fmoc-Cys(SH)-OH with high binding constants in aqueous and protic solvents by thiol-disulfide exchange reactions. In order to estimate the covalent rebinding, particles were further extracted and disulfides reduced were estimated with the non-covalent recognized and covalently bounded analytes. From rebinding studies that were conducted, we observed that proved polymer particles could be reproducible and contain constant binding strengths and recognition properties. Furthermore, we proved that short incubation periods resulted in fast and efficient thiol-disulfide interchange reactions.

Effect of Cysteine on Lowering Protein Aggregation and Subsequent Hardening of Whey Protein Isolate (WPI) Protein Bars in WPI/Buffer Model Systems

Whey protein isolate (WPI) bar hardening without and with cysteine (Cys) or N-ethylmaleimide (NEM) was investigated in model systems (WPI/buffer = 6:4, by weight, pH 6.8, a(w) approximately 0.97) in an accelerated shelf-life test (ASLT) at 45 degrees C over a period of up to 35 days. The formation of insoluble aggregates as determined by solubility and the structural rearrangement of WPI protein aggregates as observed by SEM were responsible for the WPI bars' hardening. As corroborated by electrophoresis analysis, both beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) were involved in the formation of aggregates via the thiol-disulfide interchange reaction and/or noncovalent interactions. The former force dominated the bar hardening at an earlier stage, whereas the latter force played a role for the long-term hardening. In comparison with the control bar without Cys, the thiol-disulfide interchange reaction was significantly reduced by Cys (WPI/Cys = 0.05), increased by Cys (WPI/Cys = 0.25), and inhibited by NEM (WPI/NEM = 2). Therefore, bar hardening was significantly delayed by Cys (WPI/Cys = 0.05) and NEM but accelerated by Cys (WPI/Cys = 0.25).

Molecular docking studies of dithionitrobenzoic acid and its related compounds to protein disulfide isomerase: computational screening of inhibitors to HIV-1 entry

BackgroundEntry of HIV-1 into human lymphoid requires activities of viral envelope glycoproteins gp120 and gp41, and two host-cell proteins, the primary receptor CD4 and a chemokine co-receptor. In addition, a third cell-surface protein called protein disulfide isomerase (PDI) is found to play a major role in HIV-1 entry. PDI is capable of mediating thio-disulfide interchange reactions and could enable the reduction of gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. In this scenario, inhibition of HIV-1 entry can be brought about by introducing agents that can block thiol-disulfide interchange reaction of cell surface PDI. There have been studies with agents that inhibit PDI activity, but the exact mode of binding remains to be elucidated; this might provide insights to develop new drugs to target PDI. This study attempts to perceive the mode of binding of dithionitrobenzoic acid (DTNB), and its structurally related compounds on PDI enzyme.ResultsWe performed molecular docking simulation with six different inhibitors (ligand), which includes DTNB, NSC695265, thionitrobenzoic acid, 2-nitro-5-thiocyanobenzoic acid, 2-nitro-5-sulfo-sulfonyl-benzoic acid and NSC517871 into the redox-active site [C37-G38-H39-C40] of the PDI enzyme and the activity was inferred by redox inhibitory models. All ligands showed favorable interactions and most of them seemed to bind to hydrophobic amino acids Ala34, Trp36, Cys37, Cys40, His39, Thr68 and Phe80. The redox inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature.ConclusionWe demonstrated that in silico docking experiment can be effectively carried out to recognize the redox inhibitory models of PDI with inhibitor molecules. Interestingly we found that number of docked clusters with each ligand varies in the range of five to eight and conveys that the binding specificity of each inhibitor varies for PDI. We also identified that Cys37 of the enzyme plays an important role in hydrogen bonding with inhibitors. This residue can be considered to being an active site for anti-HIV drug design. Therefore, by inhibiting PDI, one can, not only prevent the viral entry but also circumvent the problem of viral resistance

Site-Specific Dense Immobilization of Antibody Fragments on Polymer Brushes Supported by Silicone Nanofilaments

For site-specific dense immobilization of antibodies on a solid support, we prepared phosphorylcholine copolymer brushes on silicone nanofilaments. The nanofilaments were prepared on a silicon wafer by treatment with trichloromethylsilane (MeSiCl 3). To generate Si-OH groups on the nanofilaments, O 2 plasma was irradiated on the surface. Initiators for atom transfer radical polymerization (ATRP) were then coupled on the filaments. Phosphorylcholine copolymer brushes were prepared by a "grafting from" process, and pyridyl disulfide groups were introduced into the polymer chains. F(ab') fragments were then specifically immobilized onto these surfaces via a thiol-disulfide interchange reaction. The amount of antibodies immobilized on the nanofilament-supported copolymer brushes was approximately 65 times greater than that on smooth wafer-supported copolymer brushes.

Covalent immobilization of antibody fragments on well-defined polymer brushes via site-directed method

Well-defined polymer brushes and block copolymer brushes consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC) and glycidyl methacrylate (GMA) were prepared by surface-initiated atom transfer radical polymerization (ATRP). The polymer brushes were used for the immobilization of antibody fragments in a defined orientation. Pyridyl disulfide moieties were introduced to the polymer brushes via a reaction of epoxy groups in GMA units. Fab’ fragments were then immobilized onto these surfaces via a thiol-disulfide interchange reaction and the reactivity of antibodies with antigens was investigated. Antigen/antibody binding on the polymer brushes was more preferable than that on epoxysilane films as a control surface. Furthermore, the activity of the antibodies immobilized on the block copolymer brushes having biocompatible PMPC was greater than that on other surfaces that did not have PMPC in their structures.

Polyrotaxane and Polyrotaxane Network: Supramolecular Architectures Based on the Concept of Dynamic Covalent Bond Chemistry

This review article concerns with the syntheses of polyrotaxanes and polyrotaxane networks that can be constructed mainly on the basis of the concept of dynamic covalent bond chemistry. At the beginning of the review, synthetic methods of rotaxanes are briefly summarized along with several high yielding preparations. Synthesis of poly[3]rotaxane by the reaction of homoditopic monomers utilizing the thiol-disulfide interchange reaction was discussed in detail, among polyrotaxanes possessing topological bonds for the monomer linking in the main chain. Polyrotaxane network was prepared by a similar protocol from a polyfunctional crown ether and a dumbbell-shaped homoditopic axle containing two sec-ammonium salt moieties and centrally located disulfide bond. Some characteristics of the polyrotaxane network were demonstrated, including the recyclable property as the crosslinked polymer. The meaning and applicability of the reversible crosslinking/decrosslinking system developed in the polyrotaxane network are specially emphasized for unique and potential application.

Synthesis and characterization of three-dimensional crosslinked networks based on self-assemly of α-cyclodextrins with thiolated 4-arm PEG using a three-step oxidation.

A kind of novel three-dimensional crosslinked networks based on the self-assemly of α-CDs with thiolated four-arm PEG was prepared by thiol-disulfide interchange reaction using a three-step oxidation. The supramolecular structure of the resulting hydrogels was characterized FTIR, H NMR, XRD, DSC and TGA measurements. The results demonstrated that the channel-type crystalline structure of inclusion complexes is still maintained after the multi-step oxidation. According to the swelling and degradation tests, the swelling behaviors and degradation properties can be readily regulated by tuning the feed compositions of α-CDs and PEG-thiolated prepolymer. Furthermore, the threading α-CDs clearly protects disulfide bonds from an attack of a reduced form of glutathione as a result of prolonging the degradation time of the hydrogels in water.