Disulphide Bonds Research Articles

Salivary biochemical assessments to determine smoking cessation under smoking cessation program

The study aim was to investigate the potential of salivary biochemical assessments to determine smoking cessation in patients under smoking cessation programs. The study validated smoking behavior and degree of nicotine dependence using Fagerström test for nicotine dependence in smokers and non-smokers. Protein components were examined using Raman spectroscopy. Salivary matrix metalloproteinases-8 (MMP-8) levels were analyzed using Quantikine human total MMP-8 immunoassay-kits. Thiocyanate (SCN−) concentrations, peroxidase and glutathione-peroxidase activities were determined using spectrophotometric methods. Human mononuclear cell line was examined for M1 polarization. The data were analyzed using Fisher’s exact test and two-tailed paired t-test at α = 0.05. Amide I peak in smoker group was significantly reduced as compared to non-smokers (p < 0.05). Sulfhydryl bond, dialkyl disulfides SS-stretch and corresponding disulphide bond at 500−545 cm-1 were significantly increased in smokers (p < 0.05). The CH2 wag showed higher intensity amongst smoker group representing a recoil of carbon atoms. Salivary MMP-8 levels of participants after smoking cessation was significantly lower than smoking salivary levels (p < 0.05). The SCN− and peroxidase concentrations were significantly higher in smoking group whereas an M1 polarization was seen in non-smokers. Salivary biochemical analysis can aid clinicians to verify smoking cessation in patients under smoking cessation programs.

A novel approach for characterisation of stabilising bonds in milk protein deposit layers on microfiltration membranes

The gel formation mechanism of low-pressure casein gels during the filtration of skim milk remains unknown this far. We investigated the prevailing bond types in this gel and correlated these findings with deposit layer properties and filtration performance. At high pH and low transmembrane pressure, calcium bridges predominated among the bond types stabilising the deposit layer gel. However, there were less calcium bridges present than within or between casein micelles in skim milk. With a decreasing amount of calcium bridges at low pH and higher transmembrane pressures, the deposit layer turned into a dense and mushy gel, which impaired solvent and solute permeability. Additionally, ionic and hydrogen bonds, as well as disulphide bonds, were formed between the highly concentrated deposited proteins during gelation. Hydrophobic interactions played a negligible role. We concluded that intramicellar calcium bridges are associated with a voluminous micelle and a stable gel backbone, which benefits filtration efficiency.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

Dissociation of the respiratory syncytial virus F protein-specific human IgG, IgA and IgM response

Human respiratory syncytial virus (RSV) is one of the most important causes of severe respiratory tract infections in early childhood. The only prophylactic protection is the neutralizing antibody, palivizumab, which targets a conformational epitope of the RSV fusion (F) protein. The F protein is generated as a F0 precursor containing two furin cleavage sites allowing excision of the P27 fragment and then gives rise to a fusion-competent version consisting of the N-terminal F2 subunit and the a C-terminal F1 subunits linked by two disulphide bonds. To investigate natural human F-specific antibody responses, F2 conferring the species-specificity of RSV, was expressed in Escherichia coli. Furthermore, the F0 protein, comprising both subunits F2 and F1, was expressed as palivizumab-reactive glycoprotein in baculovirus-infected insect cells. Six overlapping F2-derived peptides lacking secondary structure were synthesized. The analysis of IgG, IgA and IgM responses of adult subjects to native versions and denatured forms of F2 and F0 and to unfolded F2-derived peptides revealed that mainly non-conformational F epitopes, some of which represented cryptic epitopes which are not exposed on the proteins were recognized. Furthermore, we found a dissociation of IgG, IgA and IgM antibody responses to F epitopes with F2 being a major target for the F-specific IgM response. The scattered and dissociated immune response to F may explain why the natural RSV-specific antibody response is only partially protective underlining the need for vaccines focusing human antibody responses towards neutralizing RSV epitopes.

Influence of the Presence of Disulphide Bonds in Aromatic or Aliphatic Dicarboxylic Acid Hardeners Used to Produce Reprocessable Epoxidized Thermosets.

The design of polymers from renewable resources with recycling potential comes from economic and environmental problems. This work focused on the impact of disulphide bonds in the dicarboxylic acids reactions with three epoxidized vegetable oils (EVOs). For the first time, the comparison between aromatic vs. aliphatic dicarboxylic acids, containing or not S–S bonds with EVOs was discussed and evaluated by dynamic scanning calorimetry. The obtained thermosets showed reprocessability, by the dual dynamic exchange mechanism. The virgin and reprocessed materials were characterized and the thermomechanical properties were compared. The thermosets derived from EVOs with high epoxy content combined with aromatic diacids containing disulphide bridges showed high glass transition values (~111 °C), high crosslink densities and good solvent stability.

Impact of microfluidization on colloidal properties of insoluble pea protein fractions

Microfluidization is a technique commonly used to disrupt and homogenize dispersions such as oil-in-water emulsions or cellular suspensions. In this study, we investigated its ability to alter the physicochemical properties of plant-derived insoluble protein aggregates such as those found in pea protein extracts. Insoluble pea protein dispersions (5% w/w, pH 7) were homogenized at 25–150 MPa for 1–5 cycles. Increasing the homogenization pressure and cycles decreased the particle size (d43) of the unhomogenized insoluble pea proteins from 180 ± 40 μm to 0.2 ± 0.0 μm (at ≥ 125 MPa), leading to more transparent dispersions. Furthermore, the solubility of the insoluble pea proteins increased from 23 ± 1% to 86 ± 4%. Treatments with chaotropic agents, dithiothreitol and urea, revealed that insoluble pea protein aggregates were stabilized not only by disulphide bonds but also by hydrogen bonds and hydrophobic interactions. These molecular interactions were disrupted by microfluidization. The study provides insights into the disruption mechanism of insoluble pea proteins by applying microfluidization and offers a mean to improve their technofunctional properties to facilitate further use in food manufacture.

Physicochemical, textural and structural characteristics of wheat gluten-soy protein composited meat analogues prepared with the mechanical elongation method

• Meat analogues were prepared by the mechanical elongation (ME) method. • Striated texture was formed by highly compacted ultrathin filaments. • Wheat gluten demonstrated potential in structuring meat analogues using ME method. This study investigated the effect of mechanical elongation method by combining wheat gluten (WG) and soy protein isolate (SPI) (at the ratio of 100:0, 80:20, 60:40 and 40:60 % w/w dry protein basis), and studied the physicochemical, textural and structural characteristics of meat analogues (∼52 % moisture). The meat analogues were analysed and compared against commercial control samples of firm tofu, mock chicken, and steamed chicken. Meat analogues made with 100 %WG showed the lowest hardness and chewiness. Analysis of meat analogues by scanning electron microscopy (SEM) showed their unique compact ultrastructure which was distinct from the commercial control samples. However, the proportional concentration of WG had an insignificant impact on the ultrastructure. Increasing SPI concentrations improved lysine contents of meat analogues but were still significantly lower ( p< 0.05) than steamed chicken. The results from protein solubility analysis suggested that hydrogen bonds, disulphide bonds and hydrophobic interactions were the forces responsible for the formation, stabilisation and retention of the structures within the meat analogues. It was concluded that the use of WG and WG-SPI produced acceptable doughs that could be used for the development of meat analogues using the mechanical elongation method.

Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress

BackgroundVon Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C‐domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined. MethodsVWF free thiols were blocked with N‐ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF‐C4 domain. ResultsBlockade of VWF free thiols reduced VWF‐mediated platelet capture to collagen in a shear‐dependent manner, with platelet capture virtually abolished above 5000 s−1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen‐bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol‐blockade reduced the lifetime and strength of the VWF‐collagen bond. Pulling simulations of the VWF‐C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free‐thiol exchange. ConclusionsWe conclude that free thiols in the C‐domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.

Understanding the hydration of alkali-induced duck egg white gel at high temperature

In this paper, prepared alkali-induced egg white gel (EWG) was treated at different temperatures (100–121 °C) to explore the hydration mechanism under high temperatures (≥100 °C). The physicochemical, mechanical and rheological properties and intermolecular interactions of the EWG were investigated. Results showed that with increasing temperature the pH, free alkalinity and surface hydrophobicity of EWG increased initially but then decreased, together with gel hydration, and the browning intensity increased markedly. The mechanical properties, including hardness, puncture strength and springiness, rheological characteristics―determined via apparent viscosity and frequency sweep results―and water-holding capacity significantly decreased as temperature increased. Fourier-transform infrared (FTIR) spectroscopy revealed that the β-sheet contents decreased significantly, and the α-helical and β-turn contents improved markedly with increasing temperature. The protein fraction results showed that hydrogen bonds, hydrophobic interactions and disulphide bonds were destroyed in the heating process. This study provides deep insights into EWG destruction under high temperature and the relationship with intermolecular interactions, enabling control over EWG quality.

Trans-gauche-trans disulphide conformers measured by means of FT-Raman may be predictors of apparent digestibility of crude protein in feather meal fed to rainbow trout (Oncorhynchus mykiss)

The present study assessed the suitability of an FT-Raman method previously developed in our laboratory to predict the digestibility of crude protein (CP) in feather meals (FeMs) fed to rainbow trout (Oncorhynchus mykiss). Since we have already demonstrated that this FT-Raman method could efficiently predict the digestibility of CP in FeMs fed to broiler chickens, we decided to assess the suitability of this method in rainbow trout nutrition. A digestibility trial was conducted to assess the apparent digestibility coefficient (ADC) of CP in 7 FeMs of different origins in rainbow trout. Levels of disulphide conformers trans-gauche-trans (SStgt), gauche-gauche-trans (SSggt) and gauche-gauche-gauche (SSggg) in the 7 FeMs were studied by means of FT-Raman spectroscopy. Values of ADC were regressed against disulphide conformers to determine the presence of linear and quadratic correlations. The ADC of CP in the FeMs ranged from 0.64 to 0.71. Significant (P < 0.05) positive linear correlation existed between total disulphide bonds (P < 0.05) and ADC of CP in FeMs. Moreover, we observed significant linear and quadratic correlations between SStgt and ADC of CP in FeMs. The slope equations generated by the linear and quadratic regressions of SStgt against ADC of CP provided predicted values of ADC of CP that correlated linearly with observed values of ADC of CP in statistically significant fashions (P < 0.001). This study showed correlations between the digestibility of CP in FeM and SStgt levels, which may be used as predictors of ADC of CP in FeMs fed to rainbow trout.This study indicated that the ileal digestibility of CP in FeM may be affected by SStgt levels, which can be used as predictors of ADC of CP in FeMs fed to rainbow trout.

Identification of the Primary Factors Determining theSpecificity of Human VKORC1 Recognition by Thioredoxin-Fold Proteins.

Redox (reduction–oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol–disulphide exchange reactions between PDI and hVKORC1.

Brief descriptions of the principles of prominent methods used to study the penetration of materials into human hair and a review of examples of their use.

Consumers are attracted to the latest fashion trends and different looks. This drives the search for novel hair treatments. Some chemicals present in hair treatment products can penetrate the hair shaft. These materials can either nourish or injure the hair cortex. Different techniques have been used to investigate the mechanism of molecule penetration and the conditions under which penetration occurs. This article reviews the techniques applied for this purpose. Various microscopy techniques are used to capture clear and colourful images to determine the diffusion pathways and the exact location of the molecules under study. However, the laborious sample preparation often leads to sample destruction since cross-sectioning is often required. While various other techniques have been successfully used for investigating the penetration methods, most of these require different amounts of work to be put in for sample preparation and instrumentation. Several spectroscopic techniques have been used to study the penetration of the molecules because of the high levels of accuracy and the quick response time of these techniques. Moreover, the samples are not damaged during the investigation.

Variability in linear polypeptide stabilizes proteoglycan than zinc finger protein in vascular smooth muscle cells: An In Silico approach

Background: Structural and physicochemical topologies of proteins play a considerable role in differentiating the functional properties of the biological system. We aimed to study the physicochemical similarities, structural and functional differences of versican (VCAN) and early growth response (Egr) proteins involved in vascular injuries. Methods: For the primary structure prediction, the proteomic tools Expasy's Protparam is used, likewise, for secondary structure and content prediction SOPM and SOPMA tool is used. The transmembrane regions in VCAN and EGR proteins are predicted through SOSUI (Classification and Secondary Structure Prediction of Membrane Proteins) server. The CYSREC tool is used to identify the presence of disulphide bonds in all the VCAN and EGR proteins, additionally through homology modelling the disulphide bonds are visualized and structure of the modelled proteins are validated through Rampage (Ramachandran plot), ProQ (Protein Quality Server) and ProSA (Protein Structure Analysis) server. Results: VCAN and Egr proteins resemble hydrophilic in nature, similarly negative score of the grand average of hydropathicity index confirms hydrophilic nature. The maximum molecular weight for VCAN is observed as 39265 and 61623 Dalton for EGR protein. VCAN proteins showed a higher level of basic residues except Q86W61, while all the Egr proteins were acidic residues. The extinction coefficient (EC) has unique absorbance at 280 nm wavelength. Based on the aliphatic index (AI ≥ 45) and instability index (II ≥ 40) most of the VCAN and Egr proteins were unstable. The Classification and Secondary Structure Prediction of Membrane Proteins server classifies all Egr and few VCAN and proteins are soluble nature. Secondary structure content prediction and SOPM server show most of the VCAN proteins are beta sheets and many Egr proteins are alpha-helical, while few with mixed structures. Besides these differences, the VCAN protein stability was identified by most probable disulfide (SS) bridges using CYS_REC tool and confirmed by homology modeling in tertiary structure. Whereas the probable disulfide bonds in Egr proteins were not identified. Conclusion: The findings with these functional and structural properties will add an extra room in understanding their dual role.

Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins.

Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins) with multiple intramolecular disulphide bonds which are typically difficult to express in a soluble and folded state in the reducing environments found inside a cell. In some cases, the inclusion of a recombinant disulphide isomerase DsbC further enhances the expression levels of correctly folded hydrophobins. Using this protocol, we can achieve milligram levels of protein expression per ml of reaction. While our target proteins are the fungal hydrophobins, it is likely that this protocol with some minor variations can be used to express other proteins with multiple intramolecular disulphide bonds in a natively folded state. Graphic abstract: Figure 1.Workflow for cell-free protein expression and single-step purification using affinity chromatography. A. E. coli S30 lysate prepared as described in Apponyi et al. (2008) can be stored for up to several years at -80°C without any loss of activity in our experience. B. The S30 lysate, plasmid DNA that encodes for the protein of interest along with an affinity tag and components required for transcription and translation are added to the reaction mix. Following a single-step protein purification, the protein of interest can be isolated for further use.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

A proteolytic nanobiocatalyst with built-in disulphide reducing properties.

We report a method to equip proteolytic nanobiocatalysts with intrinsic disulphide bond reducing properties. After immobilisation onto silica particles, selected protease enzymes are partially shielded in a nanometre-thick mercaptosilica layer acting not only as a protective system but also as a substrate reducing agent. The biocatalysts produced efficiently perform simultaneous disulphide bond reduction and protein digestion. Besides a significant simplification of the proteolysis process, this strategy allows for a drastic increase of the enzyme stability.

Complex formation of copper(II), nickel(II) and γ-L-glutamyl-L-cysteinyl-glycine (GSH)

The formation of Cu(II) and Ni(II) complexes of GSH in conditions of 2-fold GSH excess over Ni(II) was studied by MALDI MS and MS/MS. The instrumental data demonstrated that S and N of GSH are involved in the coordination metal spheres, while COOH did not participate to complexation. NiL2 is a square-planar Ni(II) specie able to form one electron redox couple with Ni-(III) complex. It was found to dimerize rapidly via disulphide bond formation in the presence of air.

Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2-casein

Bovine milk αS2-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala81 to Lys113 is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of αS2-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of αS2-casein is a functional, yet amyloidogenic, structure.

Impact of heat treatment on the acid induced gelation of brewers’ spent grain protein isolate

Brewers’ spent grain (BSG) is a major side stream of the beer-brewing sector, which comprises a plethora of food macromolecules and micronutrients. In the present work, BSG protein isolates were assessed for their ability to form gels via delta-gluconolactone induced acidification. The impact of the thermal pre-treatment (native, heating at 72, 85 or 92 °C for 20 min, or autoclaving) of the BSG-PI dispersions (5% w/w) on the acid-induced gelation kinetics and the microstructural, rheological and water holding capacity characteristics of the acid gels was investigated. The gelation lag phase and the maximal gelation rate values were reduced proportionally to the intensity of the heat treatment allowing a better-controlled aggregation of the protein protofibrils into clusters, which in turn resulted in a percolated network formed by the protein clusters supramolecular interactions. The thermal treatment of the BSG-PI dispersions favoured the occurrence of segregative phase separation between glutelins and alkali-soluble cell wall dietary fibres. The arrestment of the segregative phase separation during the acidification process resulted in the development of composite acid gels of enhanced elasticity, stiffness and deformation resilience. All gel systems had high water holding capacity (>86%), which postulates the occurrence of covalent (disulphide bonds) interactions between the protein inter-flocs.

&lt;p&gt;Berberine-Loaded Thiolated Pluronic F127 Polymeric Micelles for Improving Skin Permeation and Retention&lt;/p&gt;

BackgroundChallenges associated with local antibacterial and anti-inflammatory drugs include low penetration and retention of drugs at the expected action site. Additionally, improving these challenges allows for the prevention of side effects that are caused by drug absorption into the systemic circulation and helps to safely treat local skin diseases.MethodsIn the current study, we successfully prepared a thiolated pluronic F127 polymer micelles (BTFM), which binds to keratin through a disulphide bond, to produce skin retention. In addition, the small particle size of polymer micelles promotes the penetration of carriers into the skin. The current study was divided into two experiments: an in vitro experiment; an in vivo experiment that involved the penetration of the micelle-loaded drugs into the skin of rats, the skin irritation test and the anti-inflammatory activity of the drug-loaded micelles on dimethyl benzene-induced ear edema in mice.ResultsResults from our in vitro transdermal experiment revealed that the amount of drug absorbed through the skin was decreased after the drug was loaded in the BTFM. Further, results from the vivo study, which used fluorescence microscopy to identify the location of the BTFM after penetration, revealed that there was strong fluorescence in the epidermis layer, but there was no strong fluorescence in the deep skin layer. In addition, the BTFM had a very good safety profile with no potentially hazardous skin irritation and transdermal administration of BTFM could significantly suppress ear edema induced by dimethyl benzene. Therefore, these findings indicated that BTFM reduced the amount of drug that entered the systemic circulation. Our results also demonstrated that the BTFM had a certain affinity for keratin.ConclusionOur experimental results suggest that the BTFM may be an effective drug carrier for local skin therapy with good safety profile.