Free Sulphydryl Research Articles

Fabrication and in vitro evaluation of the collagen/hyaluronic acid PEM coating crosslinked with functionalized RGD peptide on titanium

Surface modification of titanium (Ti) using biomolecules has attracted much attention recently. In this study, a new strategy has been employed to construct a stable and bioactive coating on Ti. To this end, a derivative of hyaluronic acid (HA), i.e. HA-GRGDSPC-(SH), was synthesized. The disulfide-crosslinked Arg–Gly–Asp (RGD)-containing collagen/hyaluronic acid polyelectrolyte membrane (PEM) coating was then fabricated on Ti through the alternate deposition of collagen and HA-GRGDSPC-(SH) with five assembly cycles and subsequent crosslinking via converting free sulphydryl groups into disulfide linkages (RGD–CHC-Ti group). The assembly processes for PEM coating and the physicochemical properties of the coating were carefully characterized. The stability of PEM coating in phosphate-buffered saline solution could be adjusted by the crosslinking degree, while its degradation behaviors in the presence of glutathione were glutathione concentration dependent. The adhesion and proliferation of MC3T3-E1 cells were significantly enhanced in the RGD–CHC-Ti group. Up-regulated bone specific genes, enhanced alkaline phosphatase activity and osteocalcin production, the increased areas of mineralization were also observed in the RGD–CHC-Ti group. These results indicate that the strategy employed herein may function as an effective way to construct stable, RGD-containing bioactive coatings on Ti.

Characteristics of Enzymatic Hydrolysis of the Wheat Gluten Proteins Treated by Ultrasound Wave

Effects of sonication at 150W and 300W power output on hydrolysis of wheat gluten using two proteinases (Protamex and papain) were evaluated in the present study. Sonication resulted in the increase in amounts of free sulphydryl (SH) of wheat gluten. Significant (P < 0.05) increase in SH contents at 300W power output was found compared with the control and those samples sonicated at 150W power output. Degree of hydrolysis (DH) and protein recovery (PR) of the wheat gluten hydrolysates increased with sonication time and power output. Significant (P < 0.05) increase in DH and PR of the wheat gluten hydrolysate from the sample sonicated by 300W power output was found compared to those sonicated by 150W output. Sonication caused the decrease in the relative percent of the large peptide fractions and increase in the relative percent of the small peptide fractions in the hydrolysates compared to the control. Structure change in insoluble wheat gluten proteins was the principal reason of improvement in their characteristics of enzymatic hydrolysis.

Role of disulphide linkages between protein-coated lipid droplets and the protein matrix in the rheological properties of porcine myofibrillar protein–peanut oil emulsion composite gels

The objective of the study was to establish disulphide interaction between protein-coated oil droplets and the surrounding protein matrix in myofibrillar protein (MP)-emulsion composite gels. An MP-stabilized peanut oil emulsion was treated with 0, 1, 3, 5 and 10mM N-ethylmaleimide (NEM, a sulphydryl-blocking agent) and subsequently incorporated into a bulk MP sol to produce 5%-lipid, 2%-protein composites at pH 6.2. About 69% of sulphydryls in the emulsion (1% protein) were blocked by 1mM NEM, and almost all were bound at ≥3mM NEM. The loss of free sulphydryls resulted in a significant drop in the storage modulus (G′) and rupture force of the composite gels. Microstructural examination revealed pores and oil leakage from emulsion droplets by NEM treatments, corresponding to declining rheological properties of the MP-emulsion composites. The results supported the hypothesis that disulphide cross-linking between MP-coated oil droplets and protein matrix contributed to the stabilization and reinforcement of protein-emulsion composite gels formed in comminuted muscle foods.

Effects of Electric Fields on Protein Unfolding and Aggregation: Influence on Edible Films Formation

Electric fields application is receiving increased attention because of its uniform heating of liquids. The mechanisms of unfolding and aggregation of whey proteins during ohmic heating may influence properties of edible films made thereof. The aim of this work was to evaluate the effects of ohmic heating on physical and structural properties of whey protein edible films and compare them with those obtained by conventional heating. The results showed that ohmic heating determined less aggregation and lower concentration of free sulphydryls in film-forming solutions. Ohmic films were thinner, less permeable to water vapor and presented nearly the same mechanical properties of conventional films. Ohmic heating induced protein conformational changes by increasing the contents of β-sheet structures in the film network. This work emphasized the effects of ohmic heating in unfolding and aggregation mechanisms of whey proteins during heat denaturation, which determined the production of protein edible films with distinctive properties.

Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing

The successful construction of an immunosensor depends on having an effective procedure for immobilising the bio-recognition element to the transducer surface. In the present study, an amino-terminated 4-aminothiophenol (ATP) self-assembled monolayer (SAM) was modified with heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate to couple reduced anti-myoglobin half-antibody fragments. The disulphide groups present in the hinge region of IgG molecules were selectively cleaved by 2-mercaptoethylamine to produce reduced half-antibody fragments with free sulphydryl groups. The maleimide terminated 4-ATP SAM modified surface was coupled to these reduced antibody fragments to produce highly oriented immobilization of the half-antibody via its Fc domain and to allow free access to the Fv bindings sites. This represents an improvement by comparison with biotin/avidin mediated IgG attachment which is essentially randomly oriented. Functional immunosensors were able to detect myoglobin in both phosphate buffered saline and whole serum over the range of concentrations from 10 − 13 M to 10 − 6 M, and order of magnitude better than avidin/biotin linked immunosensors. In addition, atomic force microscopy (AFM) was carried out to elucidate the nanotopology of the immunosensor surface at different stages of fabrication; the images demonstrate that half antibodies bind as described and show structural changes on subsequent antigen binding.

Antibiotic therapy prevents, in part, the oxidative stress in the rat brain after meningitis induced by Streptococcus pneumoniae

Bacterial meningitis is associated with intense inflammation and also linked to the production of reactive oxygen species. To this aim, animals underwent a magna cistern tap and received either sterile saline as a placebo or an equivalent volume of a Streptococcus pneumoniae suspension. The animals began antibiotic therapy 16 h after induction. The animals were sacrificed at 24 or 48 h post-infection and the hippocampus and cortex were harvested. The activity of the enzymes superoxide dismutase, catalase, and thiobarbituric acid reactive species, protein carbonyls, and free sulphydryl groups were altered, but reversed, in part, by the antibiotic treatment. Our results support the hypothesis that antibiotic treatment prevents, in part, the oxidative stress in the bacterial meningitis induced by Streptococcus pneumoniae.

Effect of glucose oxidase, transglutaminase, and pentosanase on wheat proteins: Relationship with dough properties and bread-making quality

Glucose oxidase (Gox), transglutaminase (TG), and pentosanase (Pn) were investigated for their effect on bread quality. The changes introduced in wheat protein by the action of these enzymes were analysed to explain dough behaviour. Gox treatment decreased free sulphydryl groups ( SHf), increased glutenin macropolymer contents, and modified the electrophoretic pattern of protein fractions. Gox modified mainly albumin, globulin, and glutenin, forming large protein aggregates. These modifications explained the high strength of the dough and the low bread specific volume of samples with Gox. TG treatment modified solubility in SDS of protein and decreased glutenin macropolymer content. However, it formed large protein aggregates. The new cross-linking bonds introduced by this enzyme were different to S–S bonds and, consequently, the dough was less extensible and showed high resistance. Pn treatment increased water soluble pentosan content. Moreover, in these samples a tendency to increase SHf content was observed. In addition, Pn increased protein solubility in isopropanol, which indicates that the reduction of pentosans size decreases steric impediment of insoluble pentosans, thus increasing interaction among protein and making their extraction easier. These changes at the microscopic level allowed explaining the formation of softer dough and the production of higher specific volume in breads with Pn.

Characteristics of enzymatic hydrolysis of thermal-treated wheat gluten

Effect of thermal treatment at 50–90 °C on wheat gluten hydrolysis by papain was evaluated in this study. Thermal treatment decreased the amount of sodium dodecyl sulfate (SDS) extractable protein. The treatments at 80 and 90 °C had a strong impact on protein extractability. Thermal treatment for 30 min resulted in a significant reduction in SDS extractable glutenin level in wheat gluten. A significant drop in free sulphydryl level was found in wheat gluten treated at 70 °C for 30 min. It indicated that cross-linking of glutenin through S–S occurred during thermal treatment. The treatments at 70–90 °C led to significant decreases in soluble and nitrogen level, while significant increases in peptide nitrogen amount in the hydrolysates from treated gluten were found. A time-dependent effect was observed for the changes in soluble forms of nitrogen and PN. Thermal treatment resulted in molecular mass distribution change according to gel permeation chromatography analysis. Thermal treatment significantly increased the amount of fractions with molecular mass beyond 10 K (67.2%) in the hydrolysates and greatly decreased the amounts of fractions with MM of 10–5 K and below 5 K in hydrolysates.

Heat damage evaluation in ultra-high pressure homogenized milk

The evaluation of heat damage in ultra-high pressure homogenized (UHPH)-treated samples has been studied by analyzing different thermal indicators such as compounds that absorb at 340 nm, hydroxymethylfurfural, free sulphydryl content, furosine, lactulose and whey protein denaturation. Milk was treated at 200 and 300 MPa with inlet temperature of 30 and 40 °C and was compared to high-pasteurized milk (PA; 90 °C, 15 s) and to commercial UHT, in bottle-sterilized and pasteurized milks. UHPH samples were differentiated from PA milk, basically by the determination of absorbance at 340 nm and β-Lactoglobulin denaturation. Both parameters indicated a reduced thermal effect in UHPH samples over PA milk. Comparison of UHPH samples with UHT and sterilized milks showed an increase of all heating indicators when severe heat process was applied. Overall, in spite of the increase of temperature during UHPH treatments, heat damage was lower compared to PA milk, suggesting the potential of this technology as an alternative to pasteurization.

Physicochemical and functional properties of acidic and basic polypeptides of soy glycinin

The amino acid composition, some selected physicochemical and functional properties of acidic and basic polypeptides of soy glycinin were investigated and compared. Large amount of these polypeptides were obtained by DEAE-Sepharose fast flow column chromatography. Free sulphydryl contents, surface hydrophobicity, solubility and emulsifying activities (at different pH values) were evaluated. Different polypeptides had different patterns of amino acid composition, especially contents of acidic (and basic) and hydrophobic amino acids. The free sulphydryl contents (including total and exposed) and surface hydrophobicity considerably varied with the type of polypeptides. Compared with glycinin, isoelectric point (pI) of individual polypeptides shifted towards a more acidic pH. At a given pH value (e.g. above or below pI), the solubility and emulsifying ability index of these polypeptides were closely related to their relative contents of acidic (and basic) amino acids. The results indicated that glycinin polypeptides with different amino acid character have different physicochemical and functional properties, especially solubility and emulsifying ability.

Heat-induced modifications in the functional and structural properties of vicilin-rich protein isolate from kidney (Phaseolus vulgaris L.) bean

Abstract Heat-induced changes in the physico-chemical (and/or functional) and structural properties of protein isolate from kidney beans (KPI) were characterised. The extent of protein denaturation, free sulphydryl contents, surface hydrophobicity, as well as structural characteristics of the proteins were evaluated. Analyses of size-exclusion chromatography combined with laser scattering showed that the heating at 95 °C led to transformation of 7S-form vicilin to its 11S-form, and even higher molar mass (MW) oligomers or polymers. Moderate heating (for 15–30 min) significantly improved protein solubility, emulsifying and foaming activities (at neutral pH), whilst extensive heating (for 60–120 min) on the contrary decreased these properties. Spectral analyses of fluorescence and/or Raman spectroscopy showed that tertiary and secondary conformations of protein in KPI were remarkably affected to a varying extent by the heating. The results suggested a close relationship between functional properties of the vicilin from kidney bean and its conformational characteristics.

Thermal denaturation and gelation of vicilin-rich protein isolates from three Phaseolus legumes: A comparative study

The thermal denaturation and gelation properties of vicilin-rich protein isolates from red bean, red kidney bean and mung beans (further denoted as RPI, KPI and MPI) were investigated by differential scanning calorimetry (DSC) and dynamic oscillatory measurements. The relation between the properties of these proteins to their free sulphydryl (SH)/disulfide bond (SS) contents was also evaluated. DSC analyses showed that many DSC characteristics of major endothermic peak (corresponding to vicilin component), including its denaturation temperature ( T d), enthalpy changes (Δ H) and width at half-peak height (Δ T 1/2), significantly varied with the type of protein isolates. Furthermore, the heat-induced gelation, including onset of gelation and the development of mechanical moduli, was also dependent on the type of protein isolates. The thermal denaturation of these proteins was nearly unaffected by the presence of reducing agent dithiothreitol (DTT), while the presence of DTT weakened the gel formation. The T d of vicilin components and the mechanical moduli of corresponding formed gels were positively related to their SS contents. Additionally, the formed gels were thermo-irreversible, and heat pretreatment (carried out at temperatures higher than or close to the T d of these vicilins) could improve the gel network formation. These results confirm that vicilin-rich protein isolates from various legumes show different patterns of thermal denaturation and gelation, and these properties are to a great extent related to their SH and/or SS contents.

Effect of heat on rheology, surface hydrophobicity and molecular weight distribution of glutens extracted from flours with different bread-making quality

Gluten was extracted from flours of several different wheat varieties of varying baking quality. Creep compliance was measured at room temperature and tan δ was measured over a range of temperatures from 25 to 95 °C. The extracted glutens were heat-treated for 20 min at 25, 40, 50, 60, 70 and 90 °C in a water bath, freeze-dried and ground to a fine powder. Tests were carried out for extractability in sodium dodecyl sulphate, free sulphydryl (SH) groups using Ellman's method, surface hydrophobicity and molecular weight (MW) distribution (MWD) using field-flow fractionation and multi-angle laser light scattering. With increasing temperature, the glutens showed a decrease in extractability, with the most rapid decreases occurring between 70 and 90 °C, a major transition in tan δ at around 60 °C and a minor transition at 40 °C for most varieties, a decrease in free SH groups and surface hydrophobicity and a shift in the MWD towards higher MW. The poor bread-making variety Riband showed the highest values of tan δ and Newtonian compliance, the lowest content of free SH groups and the largest increase of HMW/LMW with increasing temperature. No significant correlations with baking volume were found between any of the measured parameters.

Effects of high pressure on functional properties of soy protein

Simultaneous measurement of the effects of low soy protein concentration, pH and high pressure treatment at room temperature on solubility, emulsifying properties and rheological properties (loss modulus, G″) of soy protein isolate (SPI) were evaluated. Central composite rotatable designs (2 3) were employed over two pH ranges (2.66–4.34 and 5.16–6.84) with SPI concentration (0.32–3.68%) and pressure (198–702 MPa) as the other independent variables. The surface responses were obtained for protein solubility, emulsifying activity index (EAI) and G″. The samples with the highest effect on protein solubility, EAI and G″ values were evaluated, as well, by electrophoresis and free sulphydryl determination. The pH was the main factor that affected protein solubility, with solubility at a maximum at pH < 3 or pH > 6. Increasing SPI concentration and decreasing/increasing the pH away from the isoelectric point both caused a reduction in EAI. Loss modulus ( G″) was found to increase with SPI concentration in both pH ranges.

Purine Catabolism in Advanced Carotid Artery Plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.

Impact of redox agents on the physico-chemistry of wheat gluten proteins during hydrothermal treatment

The impact of the oxidants potassium bromate and potassium iodate and the reducing agent dithiothreitol (DTT) on the rheological behaviour of 20% (w/v) gluten-in-water suspensions during thermal treatment was monitored with the rapid visco analyser (RVA). The suspensions were subjected to a linear temperature increase from 40 to 95 °C in 14 min, a holding step of 40 min at 95 °C, a cooling step (7 min) with a linear temperature decrease to 50 °C, and a final holding step at 50 °C (13 min). Potassium iodate (1.18 and 1.77 μmol/g protein) and potassium bromate (1.52 and 15.2 μmol/g protein) decreased RVA viscosities in the holding step and increased sodium dodecyl sulphate (SDS) protein extractabilities suggesting a greater heat resistance and decreased gliadin–glutenin cross-linking. In contrast, in the presence of DTT (1.65 and 3.30 μmol/g protein) RVA viscosity increased at lower temperatures and lowered SDS extractabilities. It is postulated that low concentrations of reducing agent facilitate gliadin–glutenin cross-linking during heating while oxidants hinder gluten polymerization due to decreased levels of free sulphydryl groups and less flexibility of the glutenin chains.

The impact of heating and cooling on the physico-chemical properties of wheat gluten–water suspensions

The rapid visco analysis (RVA) system was used to measure rheological behaviour in 20% (w/v) gluten-in-water suspensions upon applying temperature profiles. The temperature profiles included a linear temperature increase, a holding step, a cooling step with a linear temperature decrease to 50 °C, and a final holding step at 50 °C. Temperature and duration of the holding phase both affected RVA viscosity and protein extractability. Size-exclusion and reversed-phase HPLC showed that increasing the temperature (up to 95 °C) mainly decreased glutenin extractability. Holding at 95 °C resulted in polymerisation of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily increased with longer holding times while the gliadin and glutenin extractabilities decreased. Their reduced extractability in 60% ethanol showed that γ-gliadins were more affected after heating than α-gliadins and ω-gliadins. Enrichment of wheat gluten in either gliadin or glutenin showed that both gliadin and glutenin are necessary for the initial viscosity in the RVA profile. The formation of polymers through disulphide bonding caused a viscosity rise in the RVA profile. The amounts of free sulphydryl groups markedly decreased between 70 and 80 °C and when holding the temperature at 95 °C.

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a β‐barrel structure

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.

Glutathione and free sulphydryl content of seminal plasma in healthy medical students during and after exam stress

It has been reported that there is a relationship between stress and infertility. The mechanisms of stress-related semen quality alterations have not been fully elucidated. In the present study, we investigated the effect of examination stress on seminal glutathione and free sulphydryl content and sperm quality. Semen samples were collected from 34 healthy volunteers who were students of medical school in the fourth semester just before (stress period) and 3 months after (non-stress period) their final examinations. Their psychological examination stress was measured by the State Trait Anxiety Inventory (STAI) questionnaire. After standard semen analysis, semen samples were centrifuged at 10 000g for 15 min. Glutathione and free sulphydryl concentration of seminal plasma were measured. During the period of examination stress, the glutathione and free sulphydryl content of seminal plasma and the motility index of spermatozoa were significantly lower, whereas the percentage of morphologically abnormal spermatozoa was higher, than during the non-stress period (P < 0.001, for all). An association between seminal plasma glutathione and motility index was observed at both periods (P < 0.05 and P < 0.01, respectively). This study demonstrated that glutathione and free sulphydryl levels in seminal plasma decreased in subjects undergoing examination stress. Furthermore, poor sperm quality may be due to loss of glutathione and free sulphydryl content of seminal plasma.

Protective Effects of N-Acetylcysteine and Vitamin E Derivative U83836E on Proteins Modifications Induced by Methanol Intoxication

Methanol is oxidized into the formaldehyde and formate and these processes are accompanied by free radicals' generation. Formaldehyde and free radicals induce chemical modifications of proteins, leading to changes in their structure and function. The aim of this paper has been to evaluate the effect of N-acetylcysteine and vitamin E derivative U83836E on free radicals' generation and protein modifications induced during acute methanol intoxication. U83836E is an analog of α-tocopherol and similarly protects cells against oxidative damage. Moreover, this compound has hydrophilic properties and can be dissolved in an aqueous phase of blood and interstitial fluid, and next, membranes readily take it up. This compound belonging to the benzopyran family contains the reactive trolox ring and possesses antioxidant properties. The ESR determination indicates the increase in free radicals' signal 6 and 12 h after intoxication. Methanol ingestion causes a significant decrease in GSH level (by about 35%) and a significant increase in the lipid peroxidation product malondialdehyde (by about 25%). During methanol metabolism the aromatic amino acids of proteins are modified—the amount of carbonyl groups is increased (by about 42%) and fluorescence intensity of tryptophan is statistically decreased (by about 30%). The increase (by about 200%) in bityrosine fluorescence is also observed. Moreover, a significant decrease in free sulphydryl (by about 40%) and amino groups (by about 30%) in liver proteins is observed during intoxication. This is accompanied by the loss of lysosomal protease-cathepsin B activity (by about 25%). N-acetylcysteine (in dose 150 mg/kg body weight) and U83836E (in dose 10 mg/kg body weight) prevent free radicals' generation to a similar degree. U83836E protects membrane phospholipids against peroxidation a little stronger than N-acetylcysteine (concentration of MDA is decreased by 9 to 20% in the U83836 group and by 7 to 14% in the N-acetylcysteine group compared to methanol group). However after treating methanol-intoxicated rats with N-acetylcysteine, the changes in protein modification parameters are significantly smaller than in the group receiving methanol alone and they are a little smaller than after U83836E application.These findings suggest that N-acetylcysteine and to a smaller degree U83836E protect protein from modification in methanol intoxication, which can prevent liver pathologies.