Free SH Research Articles

Effect of free radical-induced aggregation on physicochemical and interface-related functionality of egg white protein

This study investigated the impact of free radical-induced aggregation by a redox pair (ascorbic acid + H2O2) on physicochemical characteristics, structure, and rheology as well as the emulsifying and foaming capacity of egg white protein (EWP). In general, this treatment led to oxidize the free SH groups, a little change in secondary structure, increasing surface hydrophobic (H0) and ζ-potential as well induce aggregation of egg white protein through disulfide cross-linking. The pH at which the oxidative aggregation reaction was performed had a significant effect on the functionality of the EWP. The EWP aggregated at pH 11 had a very high apparent viscosity, which justified by its greater H0, higher aggregation yield and larger aggregates with high effective volume, as confirmed by SDS-PAGE and AFM. Furthermore, emulsions prepared by aggregated EWP samples, particularly the EWP aggregated at pH 11, showed higher stability against droplet flocculation and phase separation during storage for 30 days at various pH (4–7) and salt concentration (0–200 mM) as compared to emulsions made with native EWP, presumably due to create a more viscoelastic film with promoted steric hindrance on oil droplets. Moreover, a dramatically enhanced foam stability was obtained by the EWP aggregated at pH 11. This study suggested that radical induced aggregation has a potential to improve the interface-related properties of egg white protein.

Comparative study of the antioxidant properties of monocarbonyl curcumin analogues C66 and B2BrBC in isoproteranol induced cardiac damage

AimTo test the antioxidant properties of the newly synthesized (2E,6E)-2,6-bis(2-bromobenzylidene)cyclohexanone (B2BrBC) in parallel with C66 in rats with cardiac hypertrophy. Materials and methodsThe protective effects of both C66 and B2BrBC against oxidative stress in rats with cardiac hypertrophy, was studied by evaluating the activity of antioxidant enzymes, the relationship between the ratio of the activities of the antioxidant enzymes R = SOD/(GPx + CAT) and levels of thiols and lipid peroxidation in the heart. In order to gain better understanding of the antioxidant properties of the studied compounds, computational methods were utilized. The properties of selected structurally related derivatives were obtained on optimized geometries for ground states, using semi-empirical PM3 quantum mechanical calculations. Key findingsThe ratio R shows disequilibrium in rats with induced hypertrophy (p < 0.001). Coextending changes were detected in total and free sulfhydryl group content (p = 0.011 for t-SH and p = 0.008, for free SH, respectively). The results with the B2BrBC, indicated strong thiol prevention reflected in the levels of both t-SH and f-SH. Taking into account the HOMO energies of B2BrBC (−9.398 eV) and C66 (−9.667), it can be concluded that B2BrBC has lower HOMO energy, which makes it a better electron donor and a better antioxidant. SignificanceThe obtained results indicated that the antioxidant ability of B2BrBC is positively associated with the catalytic SOD and GPx activities expressed through preserved t-SH levels. It seems plausible that for a compound to exhibit antioxidant activity, as most of the 2,6-bis(benzylidene)cyclohexanones do, they should be good electron donors. Impact statementUnderstanding the relationship between cardiac hypertrophy induced oxidative injuries and supporters of endogenous reparatory machinery will help in establishing the beneficial role of adequate antioxidant supplementation. In this study reliable data on the preventive effects of newly synthesized symmetric monocarbonyl curcumin analogue B2BrBC and its role in the prevention of oxidative injuries on three levels (enzymatic, protein and lipid), in the heart hypertrophic onset, were obtained.

ZnO Functionalization: Metal–Dithiol Superstructures on ZnO(0001) by Self-Assembly

We present a study of the functionalization of a monocrystalline ZnO surface using aromatic dithiols. The aim is to obtain a sulfur-terminated self-assembled monolayer (SAM), which is then used for creation of further molecular superstructures with intercalated metal atoms. These metal-molecule self-assembled structures are characterized by high-resolution X-ray photoelectron spectroscopy (XPS) and near edge X-ray adsorption fine structure (NEXAFS) measurements. Formation of a 5,5- bis(mercaptomethyl)-2,20- bipyridine (BPD) SAM on ZnO(0001) is demonstrated using a protocol developed by us earlier for dithiol assembly on gold, allowing production of standing-up SAMs with free SH groups. Thereafter the formation of a metal intercalated dithiol super lattice is achieved by first grafting metal atoms (Ag or Ni) and then attaching a second BPD molecular layer. Metal atoms bind to both sulfur and the pyridine nitrogens within the SAM. Clear changes in the valence band region near the Fermi level are observed, a...

The effect of knockdown of insulin receptor substrates 1 and 2 on glucose and lipid metabolism in human hepatoblastoma cells

The European corn borer (Ostrinia nubilalis) is one of the most important insect pests of maize, and has a significant impact on the production of this crop. In this work, we examined the effects of different insecticides on the antioxidative defense system of O. nubilalis larvae. The experimental setup consisted of a completely randomized block design with 4 replicates. Four experimental groups were formed as follows: control (C), indoxacarb (250 mL ha-1), chlorantraniliprole (100 mL ha-1) and the chlorantraniliprole+lambda cyhalothrin (200 mL ha-1) group. Larvae from maize stems were collected 20 days after insecticide application and the whole larvae were homogenized. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), and the total amount of free SH groups were assessed. Comparison of the experimental groups showed that indoxacarb significantly affected the activities of GST, GPx and the total amount of free SH groups, while chlorantraniliprole significantly affected the activities of SOD, CAT, GST and the total amount of free SH groups, while chlorantraniliprole+lambda cyhalothrin significantly affected the activities of CAT, GST and the total amount of free SH groups. The results show that exposure to insecticides considerably affects the antioxidative defense components of the European corn borer larvae, especially chlorantraniliprole (T2).

Effects of different insecticides on the antioxidative defense system of the European Corn Borer (Ostrinia nubilalis Hübner) (Lepidoptera: Crambidae) larvae

The European corn borer (Ostrinia nubilalis) is one of the most important insect pests of maize, and has a significant impact on the production of this crop. In this work, we examined the effects of different insecticides on the antioxidative defense system of O. nubilalis larvae. The experimental setup consisted of a completely randomized block design with 4 replicates. Four experimental groups were formed as follows: control (C), indoxacarb (250 mL ha-1), chlorantraniliprole (100 mL ha-1) and the chlorantraniliprole+lambda cyhalothrin (200 mL ha-1) group. Larvae from maize stems were collected 20 days after insecticide application and the whole larvae were homogenized. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), and the total amount of free SH groups were assessed. Comparison of the experimental groups showed that indoxacarb significantly affected the activities of GST, GPx and the total amount of free SH groups, while chlorantraniliprole significantly affected the activities of SOD, CAT, GST and the total amount of free SH groups, while chlorantraniliprole+lambda cyhalothrin significantly affected the activities of CAT, GST and the total amount of free SH groups. The results show that exposure to insecticides considerably affects the antioxidative defense components of the European corn borer larvae, especially chlorantraniliprole (T2).

Immunogenic and structural properties of ovalbumin treated by pulsed electric fields

ABSTRACTIn this study, the immunogenic and structural properties of ovalbumin (OVA) induced by pulsed electric fields (PEF) treatment were investigated. The immunogenic properties were estimated by the IgG and IgE binding abilities, which were determined by enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibodies and egg-allergy patients’ sera, respectively. The structural changes were monitored by circular dichroism (CD), ultraviolet absorption, and fluorescence spectroscopy. When the OVA samples were treated at low electric field intensity (below 25 kV/cm, for 180 μs) or for short time (less than 60 μs, at 35 kV/cm), the IgG and IgE binding capacities gradually increased due to the partial unfolding of OVA. This was reflected in the increase in free SH content, surface hydrophobicity, and UV absorption. However, when the OVA samples were treated at high electric field intensity (exceeding 25 kV/cm, for 180 μs) or for long time (more than 60 μs, at 35 kV/cm), the IgG and IgE binding abilities significantly reduced due to the aggregation of OVA. This was also reflected in the decrease in free SH content, surface hydrophobicity, and UV absorption. Moreover, high-intensity PEF treatment caused a loss of the α-helix structure. The results showed that the largest decrease in the immunogenic properties was observed at 35 kV/cm for 180 μs. Therefore, PEF processing has the potential for utilization as a method for egg desensitization.

Green synthesis with enhanced magnetization and life cycle assessment of Fe3O4 nanoparticles

Magnetic nanoparticles were synthesized by a novel eco-friendly method using glutathione as a reductant and stabilizer. A careful study of the life cycle in the synthesis process was performed to assess the environmental impact caused by the production of this material. From the results, it is possible to conclude that the environmental impact is much less significant than that of conventional routes, specifically the co-precipitation method with which it was compared. Nanoparticles with a saturation magnetization of 85.4 emu/g were obtained, which is similar to the maximum value obtained through the co-precipitation method. One of the main advantages of this method is the formation of magnetic nanoparticles with a coating of free and reactive SH groups, which offers interesting possibilities for its use in environmental remediation tasks, specifically in adsorbing heavy metals such as mercury.

Effects of high temperatures and drought during anthesis and grain filling period on wheat processing quality and underlying gluten structural changes.

Climate changes do not only affect wheat yield, but also its quality. Information on this topic gathered so far is somewhat contradictory and insufficient. Climate changes also affect wheat indirectly through their influence on the ecosystem, including insects and fungi that affect wheat technological quality. The aim of this study was to examine trends in structural and technological changes of wheat quality under conditions typical of climate changes. With this in mind, three groups of wheat varieties with the same Glu-score were examined in three production years, characterized by different production conditions. A production season characterized by climate change conditions results in lower activity of amylolytic enzymes. What is more, it results in lower content of gluten, higher gluten index value, its decrease after 1 h to 37 °C, lower number of free SH groups and higher content of free amino groups, which result in lower alveograph W, lower farinograph WA and higher extensograph dough resistance. Variability in wheat quality produced under different climatic conditions is mainly influenced by the production conditions, including their influence on ecosystem factors. The influence of wheat cultivar genetic predisposition is much less expressed. This indicates that differences among cultivars with different Glu-score might be diminished under the influence of altered production conditions, as a consequence of climate change. © 2017 Society of Chemical Industry.

Effects of alkali on protein polymerization and textural characteristics of textured wheat protein

The impact of alkali addition on the degree of gluten polymerization and textural characteristics of textured wheat protein was investigated. Results showed that the extrusion process increased the average molecular weight of gluten as evidenced by SDS-PAGE and SDS extractable protein. The addition of alkali not only promoted the degree of gluten polymerization, but also induced dehydroalanine-derived cross-linking. Alkali addition decreased the content of cystine and increased the contents of dehydroalanine and lanthionine. The obvious decrease of free SH showed that dehydroalanine-derived cross-linking was quantitatively less crucial than disulfide cross-linking. Furthermore, the protein cross-linking induced by alkali improved the texture properties of gluten extrudates. SEM analysis showed extrusion under alkaline condition conferred a more fibrous microstructure as a consequence of a compact gluten network.

In Ukrainian

Novel neutron capture agents for the boron and gadolinium neutron capture therapy magnetic nanostructures have been created. The magnetic nanocomposites obtained could be applied as carriers for targeted delivery of 157 Gd and 10 В in order to their retention in tissues. Such materials can find an application in neutron capture therapy of cancer and multimodal Т 1 /Т 2 MRI. The aim of research is the synthesis of B- and Gd-containing nanomaterials perspective for application in neutron capture therapy of cancer and MRI. All reagents were of analytical grade and used without further purification. Demineralized water was used for preparation of all sample solutions. Starch, sodium thiosulfate, 0.1 N iodine solution in water, Phosphate buffer (рН 7.2), DMSO, ethanol, FeCl 3 ×6H 2 O, FeSO 4 ×7H 2 O, Gd(NO 3 ) 3 ×6H 2 O, and aqueous NH 4 OH (25 %) were purchased from local supplier (Himlaborreaktiv, Ltd). Meso-2,3-dimercaptosuccinic acid (DMSA) was purchased from Sigma-Aldrich. Orto-thiocarborane was purchased from Merck Schuchardtohg. 2,2'-dipyridyldisulfide was purchased from Sigma. 2-Mercaptonicotinic acid ≥99.0 % (HPLC) was purchased from Sigma-Aldrich. UV-Vis spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray powder difraction (XRD) were used for analysis. In order to obtain novel neutron capture agents magnetic nanostructures have been developed for the boron and Gd neutron capture therapy based on Fe 3 O 4 with optional Gd content and molecules containing 10-boron atoms. Molecular formula of the composite is Fe 3 O 4 /Gd 2 O 3 -S-S-Н 11 В 10 С 2 . Ortho-thiocarborane immobilization was carried out by thiol-disulphide exchanging reaction. The surface of Fe 3 O 4 /Gd 2 O 3 composite was modified by meso-2,3 dimercaptosuccinic acid in order to create free SH groups on the surface. Dipyridildisulphide was used as @leaving@ molecule from the surface of DMSA-Fe 3 O 4 /Gd 2 O 3 composite. The XPS spectroscopy studies confirmed presence of boron about and S-S bonds on the surface. According to our estimations, 0.07 mmol/g of carborane was immobilized.

Characterization of physicochemical and structural properties of atmospheric cold plasma (ACP) modified zein

Atmospheric cold plasma (ACP) has been widely reported as an efficient approach in material surface modification. The effects of dielectric barrier discharge (DBD) ACP technology on the physicochemical and structural properties of powdered zein were investigated. The results indicated that the average diameter of zein particles decreased significantly indicating a depolymerization of aggregations after ACP treatment. There was an increase of free SH group concentration and decrease of pH in zein solutions. Significant improvement of zein solubility in both neutral and acidic solutions could be observed after treatment with max solubility at 75V. Moreover, Differential Scanning Calorimetry (DSC) and X-ray diffraction (XRD) results indicated that ACP treatment rearranged the conformation. FTIR spectrum confirmed the change of secondary structure for amide I region (1600–1700cm−1) after ACP treatment. Furthermore, the film forming properties of zein were also studied. It was found that the increase of ACP treatment voltage level could reinforce the tensile strength (TS) and surface hydrophilicity of zein films. It is suggested that the DBD ACP technology can effectively modify the physicochemical properties for protein powders.

Oxygen binding and nitric oxide dioxygenase activity of cytoglobin are altered to different extents by cysteine modification.

Cytoglobin (Cygb), like other members of the globin family, is a nitric oxide (NO) dioxygenase, metabolizing NO in an oxygen (O2)‐dependent manner. We examined the effect of modification of cysteine sulfhydryl groups of Cygb on its O2 binding and NO dioxygenase activity. The two cysteine sulfhydryls of Cygb were modified to form either an intramolecular disulfide bond (Cygb_SS), thioether bonds to N‐ethylmaleimide (NEM; Cygb_SC), or were maintained as free SH groups (Cygb_SH). It was observed that the NO dioxygenase activity of Cygb only slightly changed (~ 25%) while the P50 of O2 binding to Cygb changed over four‐fold with these modifications. Our results suggest that it is possible to separately regulate one Cygb function (such as O2 binding) without largely affecting the other Cygb functions (such as its NO dioxygenase activity).

Improved Performance of Recombinant Protein A Immobilized on Agarose Beads by Site-Specific Conjugation.

Protein A affinity adsorbent with high antibody-binding capacity plays a prominent part in the purification of biopharmaceuticals to decrease the manufacturing costs. We describe a site-specific covalent conjugation strategy for protein A to immobilize on agarose beads. Recombinant protein A, which has one cysteine introduced at the C terminus through genetic engineering technology, was immobilized site-specifically on maleimide-functionalized agarose beads by the thiol–maleimide reaction. As a comparison, the recombinant protein A was randomly immobilized on the aldehyde-functionalized agarose beads via free amino groups on the protein surface. The site-specific conjugation of recombinant protein A on the agarose beads was validated through the assay of free SH groups on the adsorbents using the Ellman’s reagent. Adsorbents containing various amounts of protein A were used to adsorb antibody from human plasma. Analysis of immunoturbidimetry showed that the adsorbed fractions contained the 90.1% IgG, 4.2% IgA, and 5.7% IgM. The maximal antibodies-binding capacities with static adsorption and dynamic adsorption were approximately 64 and 50 mg, respectively, per swollen gram for site-specifically conjugated adsorbent and 31 and 26 mg for randomly conjugated adsorbent. Remarkably, the high antibody-binding capacity for site-specifically conjugated adsorbent outperformed the existing commercial protein A Sepharose (approximately 30 mg/g). The orientation of a protein is crucial for its activity after immobilization, and these results demonstrate that the site-specifically conjugated protein molecule is in a functionally active form to interact with the antibody with weak steric hindrance. The proposed approach may be an attractive strategy to synthesize affinity adsorbents with high-binding capacity.

The effects of the thiolation with thioglycolic acid and l-cysteine on the mucoadhesion properties of the starch-graft-poly(acrylic acid)

The aim of this study is to investigate the effects of the thiolation on the mucoadhesion characteristics of the gelatinized and crosslinked wheat starch-graft-poly(acrylic acid) [(WS-g-PAA)gc] for potential use in drug delivery via vaginal route. Thiolation of (WS-g-PAA)gc was first time realized using l-cysteine hydrochloride monohydrate (CyS) and thioglycolic acid (TGA). These conjugates [(WS-g-PAA)gcth] were characterized using FTIR. The free SH group, mucoadhesion, cytotoxicity characteristics and the mechanism of the thiolation were also evaluated. To obtain fundamental data for possible application such as drug carrier, in vitro and in vivo progesterone release profiles from the mucoadhesive tablet formulations were also determined. The results showed that, vaginal tablet containing (WS-g-PAA)gc-TGA, which has not contain free SH groups in its structure, displays higher mucoadhesion than (WS-g-PAA)gc and (WS-g-PAA)gc-CyS. This tablet formulation can also be used as a drug carrier in vaginal applications.

Effect of transglutaminase cross-linking on the conformational and emulsifying properties of peanut arachin and conarachin fractions

Peanut protein is widely consumed in many countries of the world. The effects of transglutaminase (TGase)-induced cross-linking on the functional properties of peanut protein fractions of arachin and conarachin were investigated. TGase promoted protein cross-linking and caused lower protein solubility. SDS-PAGE and high-performance size exclusion chromatography displayed that larger polymers or aggregates were formed during TGase treatment and the conarachin fraction was more susceptible to cross-linking than arachin. Conarachin has higher surface hydrophobicity (H0) and emulsifying properties than arachin. TGase incubation led to decrease in total free SH content and increase in exposed SH and H0 before 90 min treatment, suggesting unfolding of the protein molecules. The emulsifying properties for conarachin were also progressively improved by TGase treatment before 90 min, which was consistent with the change of H0. This study presented the variance in structure and emulsifying properties changes between arachin and conarachin fractions and their functional properties change caused by TGase cross-linking.

Ball-milling changed the physicochemical properties of SPI and its cold-set gels

In this study, the effect of ball-milling (BM, Mixer Mill MM 400) treatments on the physicochemical properties of soybean protein isolate (SPI) and its cold-set gels induced by glucono-δ-lactone was studied. BM of SPI increased the gel strength and water holding capacity of cold-set gels significantly after 4 min of BM treatments. BM treatments did not alter the primary structure of SPI, but caused minor changes of the secondary structure. Furthermore, surface hydrophobicity of SPI increased gradually from 2058 to 5051 after 10 min of BM, while free SH groups reduced from 3.92 to 2.65 μmol/g protein. Moreover, near-UV CD spectra indicated that the tertiary conformation stability was increased and the hydrophobic groups might shift to a more hydrophobic microenvironment. In conclusion, appropriate BM treatments could change the physicochemical properties of SPI and increased the gelation properties of SPI cold-set gels.

Dependence of Vitamin B1 Metabolism and the State of Astroglia in the Rat Brain on the Supply with this Vitamin

Vitamin В1 deficiency was modeled in rats; their daily diet during 6 weeks contained about 20% of the necessary amount of thiamine. Under these conditions, the total content of vitamin В1 (thiamine) in the liver was, on average, 18.3% of the control, while the content of thiamine diphosphate (ТDP) in the blood and cerebral tissues was 40%. General indices of the redox balance (levels of reactive oxygen species, ROSs, and of free SH groups) were also smaller. The activity of thiamine pyrophosphokinase (TPK) in the brain homogenate of animals with thiamine deficiency (TD) was 35% higher than that in the control. In three cerebral structures, the cortex, cerebellum, and hippocampus, we measured the contents of proteins involved in thiamine metabolism, namely thiamine transporter (ТHTR) and TPK. In TD, directions of alterations of these indices for the above-mentioned proteins were opposite; the TPK content in all cerebral structures increased, while the THTR content in the cerebellum and hippocampus decreased. After single injections of 2.0 mg/kg thiamine into animals with TD 24 h before the completion of the experiment, the content of TPK in all cerebral structures increased. As to ТHТR, such effect was observed only in the cortex, while the reactions to thiamine injections in the cerebellum and hippocampus were not clearly manifested. We also estimated the content of glial fibrillary acidic protein (GFAP), an astrocyte marker, in all examined cerebral structures. In TD, this index was stably lower in all structures but returned to nearly control levels after single thiamine injection. Possible relations between alterations in the content of key proteins involved in thiamine metabolism and the supply of the organism with this vitamin are discussed.

Effects of ultrasound pre-treatment on the structure of β-conglycinin and glycinin and the antioxidant activity of their hydrolysates

The effect of power, time and temperature of ultrasound on the structure of β-conglycinin (7S) and glycinin (11S), and on the antioxidant activity of their hydrolysates were investigated. All ultrasound treated 7S and 11S fractions showed an increase in the α-helix and β-turn proportions, and a decrease in β-sheet and random coil proportions. The polarity of 7S and 11S microenvironment increased after ultrasound treatment. Ultrasound treatment significantly increased the reduction capacity and iron chelating capacity of 7S and 11S hydrolysates. The degree of hydrolysis and free SH groups of 7S and 11S hydrolysates increased after ultrasound pre-treatment. The relative content of high molecular weight peptides reduced, and the relative content of low molecular weight peptides increased in ultrasound treated 7S and 11S hydrolysates. The ultrasonication exposed certain groups of 7S and 11S fractions, improved contact with enzymes, and increased the content of highly active soybean antioxidant peptides.

Mechanistic investigation of capability of enzymatically synthesized polycysteine to cross-link proteins

BackgroundPreviously, we had reported that α-chymotrypsin–catalyzed polymerization of l-cysteine ethyl ester in a frozen buffer provided poly-l-cysteine (PLCys) in good yield, of which degree of polymerization had been determined to be 6–11. Almost all of SH groups in PLCys were in free forms. Such a multi-thiol peptide may cross-link proteins through thiol/disulfide (SH/SS) exchange reactions, considering the knowledge that other synthetic multi-thiol additives changes properties of protein materials. MethodsThis study explored the capability of PLCys to cross-link proteins using lysozyme as a model protein which has four disulfide bonds but no free SH group. The protein was incubated with PLCys at neutral pH and at below 70°C to avoid PLCys-independent, β-elimination-mediated cross-linkings. Protein polymerization was analyzed by SDS-PAGE and SEC. PLCys peptides involved in the protein polymer, which were released by reduction with dithiothreitol, were analyzed by RP-HPLC. ConclusionsAddition of urea and thermal treatment at 60°C caused PLCys-induced lysozyme polymerization. Compared with free cysteine, a higher level of PLCys was required for the polymerization probably due to its low water solubility. RP-HPLC analyses suggested that PLCys played a role in the protein polymerization as a cross-linker. General significanceEnzymatically synthesized PLCys shows promise as a peptidic cross-linker for the production of protein polymers with novel physiochemical properties and functionalities.

Maleimide-functionalized closo-dodecaborate albumin conjugates (MID-AC): Unique ligation at cysteine and lysine residues enables efficient boron delivery to tumor for neutron capture therapy

Maleimide-conjugating closo-dodecaborate sodium form 5c (MID) synthesized by the nucleophilic ring-opening reaction of closo-dodecaborate-1,4-dioxane complex 2 with tetrabutylammonium (TBA) azide was found to conjugate to free SH of cysteine and lysine residues in BSA under physiological conditions, forming highly boronated BSA that showed high and selective accumulation in tumor and significant tumor growth inhibition in colon 26 tumor-bearing mice subjected to thermal neutron irradiation.