Free SH Groups Research Articles

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).

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.

Research of indicators of the endogenous antioxidant system in rats affected with sodium nitrite on the background of tobacco intoxication

Under conditions of progression of environmental pollution, the one of priority directions of toxicology and medicine is the study of features and mechanisms of the combined effect of xenobiotics – risk factors of many ecologically depending diseases. In last years there is widely studied the influence of tobacco smoking on the human organism. The numerous researches proved that the wide use of mineral fertilizers in agriculture resulted in environmental pollution, especially pollution of atmospheric air, drinking water and food and created the real threat for life of both human and other living beings. All these anthropogenic toxicants penetrate the human organism and lead to the oxidative stress and activation of free radical processes in the organism and imbalance in functioning of pro- and antioxidant systems.The aim of the research is to establish the level of the oxidative stress and activity of indicators of the system of antioxidant defense in rats, injured with sodium nitrite on the background of 45 days tobacco intoxication for choosing adequate schemes of correction of detected disorders.Experiments were realized on non-linear white male rats of different age groups, who underwent the effect of tobacco smoke during 45 days. Two groups of animals, injured by smoke, received sodium nitrite in dose 45 mg / kg of body mass for 24 hours and 72 hours before the end of experiment. Euthanasia was realized under thiopental anesthesia. Blood, blood serum liver and kidneys were taken for the study.For determining AOF, there was realized extraction of blood neutrophils by the method of gradient centrifugation. For measuring AOF level in neutrophils, dichlofluorescein diacetate was used. The level of AOF production was analyzed by the intensity of dye stuff lighting on the current cytometer. Catalase activity in the reaction with ammonium molybdate, superoxide dismutase activity with nitrotetrazolium blue, the content of restored glutathione at interaction of Ellman reagent with free SH-groups were determined in blood serum and tissues. The content of ceruloplasmin in blood serum was determined by the reaction of para-phenylendiamine oxidation.The studies were realized observing general principles of experiments on animals, coordinated with statements of the European convention about protection of vertebral animals, used for experimental and other scientific aims.Rats of all age groups, injured by tobacco smoke demonstrated the increase of AOF level in blood that resulted in the oxidative process development in the organism. The synchronous intoxication of rats with tobacco smoke and sodium nitrite deepened even more all detected disorders that testify to the reliable (p≤0,05) increase of AOF that acquired the maximal values in immature animals. The oxidative stress, observed in the organism of rats of all age groups, caused inhibition of antioxidant enzymes activity: superoxide dismutase, catalase and decrease of the non-enzyme component of the antioxidant system – restored glutathione. The synchronous injure of animals by sodium nitrite and tobacco smoke resulted in the decrease of catalase activity in lungs of mature rats in 2,4times compared with intact animals in the final term of the study. After sodium nitrite intoxication of rats, toxicated by smoke, the content of restored glutathione was maximally decreased in the liver of mature rats (in 4,5 times). Most expressed changes of indicators of the antioxidant system were observed in blood serum and organs of mature rats

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.

Influence of pulsed light treatment on the aggregation of whey protein isolate

The effect of pulsed light (PL) on the aggregation of whey protein isolate (WPI) solutions was investigated. PL fluence values from 4 to 16J/cm2 were used to treat WPI (1% w/v) solutions in sodium phosphate buffer (pH=7.5). Whey protein structural modification and aggregation were assessed through the determination of free SH-groups and UV-absorption spectra. Additionally, covalent and non-covalently linked protein-protein interactions were identified through the measurement of turbidity, aggregation index, particle size distribution, and SDS-PAGE.WPI upon PL treatment showed structural changes as demonstrated by the immediate increase of free SH-group content (unfolding) and the subsequent formation of a small fraction of aggregation of unfolded proteins, due to both hydrophobic interactions and the formation of disulphide bonds. Turbidity, mean particle size, and aggregation index increased in samples treated at PL fluence from 4 to 16J/cm2. Furthermore, particle size distribution analysis of samples treated at higher fluence indicated that WPI dimer dissociation and formation of larger particles were likely to occur. The association of intermediate and larger protein molecules as well as the formation of soluble aggregates between β-lactoglobulin and α-lactalbumin were also observed in gel electrophoresis analysis. In conclusion, the results of this investigation demonstrated the potential of PL treatments to induce protein denaturation, with a minimal formation of soluble protein aggregates.

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.

КОРИГУВАЛЬНИЙ ВПЛИВ ТІОТРИАЗОЛІНУ НА ТОКСИЧНІСТЬ І СТАН ПРО- ТА АНТИОКСИДАНТНОГО ГОМЕОСТАЗУ В ЩУРІВ ЗА УМОВ ТОКСИЧНОЇ ДІЇ НАНОХРОМУ ЦИТРАТУ

Мета роботи. Вивчити коригувальний вплив тіотриазоліну на загальну токсичність та параметри біохімічної детоксикації за дії токсичних доз нанохрому цитрату.Матеріали і методи. У гострому досліді на щурах досліджено вплив тіотриазоліну (100 мг/кг) на токсичність нанохрому цитрату (НХЦ) і стан про- та антиоксидантного гомеостазу на тлі одноразового введення сублетальних (DL0) та летальних (DL50, DL100) доз сполуки (3 мг/кг, 4,5 мг/кг, 5 мг/кг). В еритроцитах крові визначали вміст малонового альдегіду (МА), відновленого глутатіону (Г-SH) та активність каталази (КТ). У плазмі крові – загальну антиоксидантну активність (АОА), вміст окисно-модифікованих білків (ОМБ) та вільних SН-груп. У постядерних супернатантах гомогенатів печінки визначали вміст МА, Г-SH, ОМБ та активність КТ. Результати й обговорення. Введення тіотриазоліну з лікувальною метою на тлі сублетальних і летальних доз НХЦ значно зменшило загальні прояви отруєння, подовжило життя тварин. Якщо неліковані тварини гинули на 2–3 добу, то при дії тіотриазоліну – на 6–8 добу. При введенні токсичних доз НХЦ посилюються процеси вільнорадикального окиснення ліпідів і променів, знижується активність КТ, АОА плазми крові, вміст вільних SН-груп та відновленого Г-SH. Ефективнішою виявилася дія тіотріазоліну на тлі мінімально токсичної дози НХЦ (3 мг/кг) – порівняно з нелікованими тваринами вірогідно зросла АОА в плазмі крові без помітних змін вмісту SН-груп.Висновки. У гострому досліді на щурах на тлі токсичних доз нанохрому цитрату тривале введення тіотриазоліну зменшує прояви інтоксикації та летальність тварин. Тіотріазолін запобігає розвитку порушень оксидантно-антиоксидантного гомеостазу в організмі інтоксикованих тварин, однак на тлі летальних доз нанохрому цитрату одноразове введення тіотриазоліну малоефективне.

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.

Modification of albumin with different degrees of the oxidation of SH-groups in the reaction with glucose

A molecule of the major blood protein albumin contains 34 cysteine residues involved in disulfide bonds and one unpaired SH-group of residue Cys34. Normally, 20–30% of these SH-groups are oxidized and form disulfide bonds or the derivatives of sulfenic, sulfinic, and sulfonic acids. The goal of the present work was to study the influence of the degree of oxidation of sulfhydryl groups on the capacity of albumin for glycation. Commercially available human albumin containing 0.4 moles of sulfhydryl groups per 1 mole of the protein (nonmercaptalbumin) was used. Disulfide bonds in this preparation were reduced with dithiothreitol to 0.7 mole/mole to give mercaptalbumin. The preparations were incubated for three weeks with glucose at a concentration of 5 and 50 mM. The content of ketoamine, a glycation product, was determined by the colorimetric method, the content of pentosidine (glycation end product) was analyzed by fluorescence, and the content of SH-groups was determined using the Ellman’s reagent. Changes in the structure and properties of the protein during glycation were studied by fluorescence and HPLC. During the incubation of both albumin preparations with 5 mM glucose, no significant increase in the ketoamine content was observed, whereas the incubation with 50 mM glucose was accompanied by a considerable accumulation of ketoamine. It was found that the greatest amount of ketoamine under these conditions forms in nonmercaptalbumin; in this case, the intensity of tryptophan fluorescence decreases. The intensity of pentosidine fluorescence increases with increasing content of ketoamine. The results obtained enable the conclusion that the oxidation of free SH-groups of the protein changes its conformation; as a result, the glycation of earlier hidden sites becomes possible, and the degree of protein glycation increases.

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.

Synthesis of a novel poly-thiolated magnetic nano-platform for heavy metal adsorption. Role of thiol and carboxyl functions

We report a novel strategy for the synthesis of magnetic nano-platforms containing free thiol groups. It first involves the synthesis of a poly(acrylic acid) copolymer containing disulfide bridges between the linear chains through di-ester linkages, followed by the anchoring of this new ligand to magnetite nanoparticles using a ligand exchange reaction. Finally, free SH groups are obtained by treating the resulting disulfide-functionalized magnetic nano-system with tributyl phosphine as reducing agent. The characterization of the resulting 17nm nanoparticles (Fe3O4@PAA-HEDred) by FTIR and TGA confirms the attachment of the copolymer through iron carboxylates. XRD, TEM and magnetic measurements indicate an increase in the inorganic core diameter and the occurrence of strong magnetic inter-particle interactions during the exchange reaction, although coercitivity and remanence drop to near zero at room temperature. Afterwards, Fe3O4@PAA-HEDred nanoparticles were tested as sorbent for Pb2+ and Cd2+ cations in aqueous media. XPS measurements were performed in order to unravel the role of both carboxyl and thiol functions in the adsorption process. For the sake of comparison, the same study was performed using bare Fe3O4 nanoparticles and a nanosystem with disulfide groups (Fe3O4@DMSA). The joint analysis of the Pb 4f, Cd 3d, Fe 2p and S 2p high resolution spectra for the nanostructured materials indicates that metal-sulfur interactions are dominant if free SH groups are present, but if not, the main adsorption route entails metal-carboxyl interactions. Even in presence of unbound thiol moieties, carboxyl groups participate due to favoured steric availability.

Modification of interactions between selected volatile flavour compounds and salt-extracted pea protein isolates using chemical and enzymatic approaches

Effects of chemical (acetylation and succinylation) and enzymatic (Alcalase) modifications on the binding properties of salt-extracted pea protein isolates (PPIs) to 2-octanone, octanal, hexyl acetate and dibutyl disulfide were monitored using GC/MS and related to changes in protein structure, based on differential scanning calorimetry and native-PAGE analyses. Addition of acetic and succinic anhydrides from zero to one gram per gram of protein gradually released the bound octanal and hexyl acetate mainly due to the loss of reactive NH2 and OH groups and dissociation of protein inherent structure. Initial addition of these dicarboxylic acid anhydrides (<0.1 g) resulted in partial denaturation of PPIs and increased binding for 2-octanone and dibutyl disulfide; however, further addition of anhydrides reduced retention of these flavour compounds probably due to extensive protein denaturation and masking of free SH groups on the protein. The binding of dibutyl disulfide was found to be positively correlated with the number of free sulfhydryl groups on acylated PPIs inferring potential sulfhydryl-disulfide interchange reactions. Enzymatic hydrolysis of PPIs by Alcalase released bound ketone and ester flavours; however, the retention levels for aldehyde and disulfide flavours were enhanced. A strong relationship was found between protein-flavour binding affinities, the type of flavour and the structure of the protein as affected by different protein modification methods.

Albumin-based nanoparticle trehalose lyophilisation stress-down to preserve structure/function and enhanced binding

The aim of this study was to preserve albumin nanoparticle structure/function during the lyophilisation process. Bovine serum albumin nanoparticles were obtained by γ-irradiation. Nanoparticles were lyophilised in buffer, miliQ water or in trehalose/miliQ solution. The size and charge of the nanoparticles were tested after lyophilisation by light scattering and Z potential. The most relevant results in size of BSA nanoparticle were those lyophilised in PBS between 20 and 350nm, assembled in different aggregates, and negative Z potential obtained was 37±8mV in all, and those nanoparticles lyophilised with trehalose had a size range of 70±2nm and a negative Z potential of 20±5mV.Structure determination of surface aminoacids SH groups in the BSA NP lyophilised in PBS showed an increase in the free SH groups. Different aggregates had different amount of SH groups exposure from 55 to 938 (from smaller to bigger aggregates), whereas BSA NP lyophilised with trehalose showed no significant difference if compared with BSA NP.The binding properties of the BSA nanoparticle with a theragnostic probe (merocyanine 540) were studied after lyophilisation. Results showed more affinity between the BSA NP lyophilised with trehalose than that observed with non lyophilised BSA NP.As a result, the lyophilisation condition in trehalose 100μM solution is the best one to preserve the BSA NP structure/function and the one with the enhance binding affinity of the BSA NP.