Amount Of Free Thiol Groups Research Articles

Investigating sorghum protein solubility and in vitro digestibility during seed germination

In this work, we examined the impact of sorghum gain germination on kafirins solubility and digestibility. Two genotypes differing in their proteins and tannins contents were germinated under controlled conditions up to radicle emergence. Biochemical, physicochemical, and in vitro digestibility tests were applied on the germinated grains. Microscopic examination of grains endosperm revealed that germination resulted in pitted starch granules and protein matrix slackening. Apart cystine and the amount of free thiol groups which increased significantly, the overall amino acids composition remained rather unchanged, just as the kafirins solubility and size distribution. In contrast germination was demonstrated to improved significantly the in vitro protein digestibility, even after cooking and especially for the genotype poor in tannin. Without inducing major physicochemical changes, germination enhanced kafirins susceptibility to gastrointestinal proteases. Germination may be a way to improve the nutritional value of sorghum.

Synthesis and Characterization of a Cationic Thiomer Based on Ethyl Cellulose for Realization of Mucoadhesive Tablets and Nanoparticles.

PurposeEthyl cellulose (EC) based nanoparticles are being extensively studied for their ability to achieve prolonged drug release and improve drug stability. Within this study, the thiolation of unmodified EC using cysteamine as a ligand was carried out to design nanoparticles with mucoadhesive properties and comparatively strong lipophilic properties.MethodsThe thiolation was performed via oxidation and reductive amination, whereas the nanoparticles were generated via the ionic gelation followed by the precipitation method.ResultsThe number of free thiol groups on EC-cysteamine was in the range of 210–261 µmol per gram of polymer. Tablets based on EC-cysteamine demonstrated mucoadhesive properties 16.7-fold improved compared with those comprising unmodified EC. The mean diameter of the particles was in the range of 94–123 nm and the zeta potential was determined to be −7.97 to −14.70 mV. The nanoparticles remained attached to porcine intestinal mucosa for up to 36% after 3 h of incubation. The formation of nanoparticles improved the stability of EC-cysteamine conjugate against cellulase and provided a zero-order release. Moreover, both EC-cysteamine and the nanoparticles did not show any pronounced cytotoxicity.ConclusionAccordingly, EC-cysteamine nanoparticles could be a specific type of nanoparticulate delivery system with mucoadhesive properties. The amount of free thiol groups within EC-cysteamine nanoparticles together with their lipophilic properties could be further modified and modulated for a desired release behavior.

Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates.

This work describes N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential platform useful to formulate micro-(MP) and nano-(NP) particles via spray-drying techniques. These conjugates are mucoadhesive over the range of urine pH, 5.0-7.0, which makes them advantageous for intravesical drug delivery and treatment of local bladder diseases. NAC- and GSH-GC conjugates were generated with a synthetic approach optimizing reaction times and purification in order to minimize the oxidation of thiol groups. In this way, the resulting amount of free thiol groups immobilized per gram of NAC- and GSH-GC conjugates was 6.3 and 3.6mmol, respectively. These polymers were completely characterized by molecular weight, surface sulfur content, solubility at different pH values, substitution and swelling degree. Mucoadhesion properties were evaluated in artificial urine by turbidimetric and zeta (ζ)-potential measurements demonstrating good mucoadhesion properties, in particular for NAC-GC at pH 5.0. Starting from the thiolated polymers, MP and NP were prepared using both the Büchi B-191 and Nano Büchi B-90 spray dryers, respectively. The resulting two formulations were evaluated for yield, size, oxidation of thiol groups and ex-vivo mucoadhesion. The new spray drying technique provided NP of suitable size (<1μm) for catheter administration, low degree of oxidation, and sufficient mucoadhesion property with 9% and 18% of GSH- and NAC-GC based NP retained on pig mucosa bladder after 3h of exposure, respectively. The aim of the present study was first to optimize the synthesis of NAC-GC and GSH-GC, and preserve the oxidation state of the thiol moieties by introducing several optimizations of the already reported synthetic procedures that increase the mucoadhesive properties and avoid pH-dependent aggregation. Second, starting from these optimized thiomers, we studied the feasibility of manufacturing MP and NP by spray-drying techniques. The aim of this second step was to produce mucoadhesive drug delivery systems of adequate size for vesical administration by catheter, and comparable mucoadhesive properties with respect to the processed polymers, avoiding thiolic oxidation during the formulation. MP with acceptable size produced by spray-dryer Büchi B-191 were compared with NP made with the apparatus Nano Büchi B-90.

Preactivated thiolated pullulan as a versatile excipient for mucosal drug targeting

AimThe purpose of the present study was to generate a novel mucoadhesive thiolated pullulan with protected thiol moieties and to evaluate its suitability as mucosal drug delivery system. MethodsTwo different synthetic pathways: bromination-nucleophilic substitution and reductive amination including periodate cleavage were utilized to synthesize such thiolated pullulans. The thiomer (pullulan-cysteamine) with the highest amount of free thiol groups was further enrolled in a reaction with 6-mercaptonicotinamide and its presence in pullulan structure was confirmed via NMR analysis. Furthermore, unmodified, thiolated and preactivated thiolated pullulan were investigated in terms of mucoadhesion via rotating cylinder studies and rheological synergism method as well as their toxicity potential over Caco-2 cells. ResultsComparing both methods the reductive amination seems to be the method of choice resulting in comparatively higher coupling rates. Using this procedure pullulan-cysteamine conjugate displayed 1522±158μmol immobilized thiol groups and 280±70μmol free thiol groups per gram polymer. Furthermore, 82% of free thiol groups on this conjugate were linked with 6-mercaptonicotinamide (6-MNA). The adhesion time on the rotating cylinder was up to 46-fold prolonged in case of the thiolated polymer and up to 75-fold in case of the preactivated polymer. Rheological measurements of modified pullulan samples showed 98-fold and 160-fold increase in dynamic viscosity upon the addition of mucus within 60min, whereas unmodified pullulan did not show an increase in viscosity at all. Both conjugates had a minor effect on Caco-2 cell viability. ConclusionBecause of these features preactivated thiolated pullulan seems to represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems.

Thiolated α-Cyclodextrin: The Invisible Choice to Prolong Ocular Drug Residence Time

It was the aim of this study to develop cysteamine-conjugated α-cyclodextrin (α-CD) enabled to form disulfide bonds with cysteine-rich substructures of the ocular mucus layer to provide a prolonged residence time of incorporated drugs at the site of action. Cysteamine was covalently attached to oxidized α-CD via reductive amination. The resulting α-CD-cysteamine conjugates (α-CD-Cys) were characterized regarding the amount of free thiol groups attached to the oligomer backbone via Ellman's reagent; resazurin assay was conducted for cytotoxicity, and mucoadhesive properties were evaluated on porcine intestinal and ocular mucosal tissues. Furthermore, albino rabbits were used for assessing the irritation-masking effects of α-CD-Cys. Free thiol groups attached to the backbone were in the range of 558 ± 24-1143 ± 92 μmol/g. None of these α-CD-Cys unduly affected the viability of Caco-2 cells in a concentration of 0.5%. Mucoadhesive properties of α-CD-Cys were up to 32-fold improved compared to unmodified α-CD. Encapsulation of cetirizine into α-CD-Cys resulted in significantly reduced local ocular mucosal irritation of this model drug. According to these results, α-CD-Cys is a promising new tool to prolong drug residence time on the ocular mucosal surface.

Effects of added fruit polyphenols and pectin on the properties of finished breads revealed by HPLC/LC-MS and Size-Exclusion HPLC

The positive roles of fruit polyphenols (PPs) and dietary fibres (DFs) in promoting human health and well-being are now well recognised. This study investigates the effects of added fruit PPs and pectin on the composition and extractability of PPs and proteins from finished breads, and complements our recently published papers that focus on the rheological and physicochemical properties of breads containing added PPs and pectin. Breads were prepared in the absence or presence of a high methoxyl (HM) apple pectin and kiwifruit, blackcurrant or apple PP extract, and subjected to texture measurements (crumb firmness), informal sensory evaluations and chemical analyses. An epicatechin standard was also used in place of the fruit PP extract to estimate the extractability and recovery of PPs from bread. Results show that adding a PP extract and pectin to bread modified bread firmness, specific volume, recovery of added PPs, amount of free thiol groups of bread proteins, and also the extractability of PPs and proteins from bread. The recovery of added PPs was the lowest for the APE + HM pectin bread which had the highest bread firmness. High performance liquid chromatography (HPLC)-mass spectrometry (MS) PP profiling revealed that baking caused oxidation of some added PPs such as quercetin and myricetin. Size-Exclusion HPLC analysis showed that adding a combination of PP extract and HM pectin decreased the S–H group concentration and increased the percentage of the Unextractable High Molecular Weight (UHMW) proteins, compared to the control bread. The added pectin and PPs, and possibly the oxidation products of the added PPs, affected the interactions between water and bread components (such as gluten proteins) during dough development and bread baking, causing differences in bread cross-linking microstructure and textural properties, which ultimately influenced the extractability and/or stability of added PPs.

Mucoadhesive interaction of cysteine grafted poly(2-hydroxyethyl aspartamide) with pig mucin layer of surface plasmon resonance biosensor

A new type of thiolated polyaspartamide was synthesized and characterized with a surface plasmon resonance (SPR) biosensor. l-Cysteine grafted poly(2-hydroxyethyl aspartamide) (PHEA)s was obtained by grafting S-(Trityl)- l-cysteine to poly(succinimide) and subsequently deprotecting Trityl groups. The degree of substitution was confirmed by 1H NMR, and elemental analysis. The amount of free thiol groups immobilized on the PHEA was determined by colorimetric reaction of thiols with Ellman's reagent. The stability of thiol groups at different pH levels was also reported. Mucoadhesive interaction was determined by pig mucin immobilized SPR biosensor. It is concluded that cysteine grafted PHEA enhances the interaction with mucin but that the amount of free thiol groups among the grafted thiols determines the mucoadhesive properties.

Development and In Vitro Characterization of Liposomes Coated with Thiolated Poly(Acrylic Acid) for Oral Drug Delivery

Mucoadhesive drug delivery systems offer promising opportunities for oral drug delivery. The aim of this study was to investigate the feasibility of preparing liposomes that are coated with the multifunctional polymer poly(acrylic acid)-cysteine (PAA-Cys). Cationic multilamellar vesicles (MLV) as well as cationic submicron-sized liposomes (ssLip) were prepared and coated with PAA-Cys. Size, zeta potential, amount of free thiol groups, aggregation behavior, drug-loading, and drug release of these novel carriers were evaluated. A switch of the initial positive zeta potential to a negative value after coating indicated the successful coating procedure. In both size ranges, MLV and ssLip, the amount of free thiol groups was comparable to that in a PAA-Cys solution of the same concentration. Drug loading of the hydrophilic marker fluorescence-isothiocyanate 4 kDa (FD4) was higher in PAA-Cys liposomes in comparison to noncoated liposomes, but lower in comparison to liposomes coated with unmodified poly(acrylic acid) (PAA). Only a minor ssLip or no increase MLV of the drug-loading was observed when using carboxyfluorescein (CF). These effects were attributed to interactions between the markers and the poly(acrylates). Coating of liposomes with PAA-Cys and PAA did not influence the release profile of FD4 and CF, whereas the release profile was affected by the molecular mass of the marker and the liposome size. In conclusion, the feasibility of coating liposomes with PAA-Cys was demonstrated, and it could be shown that this novel carrier system fulfills the basic requirements for an intended use in oral drug delivery.

Redox regulation of cell cycle through nitric oxide

This paper investigates the effects of sodium nitroprusside as NO donor on two cell lines in culture: transformed cells of mice fibroblasts (L929) and malignant cells of human eritroleukemia (K562). Low concentrations of NO have stimulative effect, while high concentrations have inhibitive effects on proliferation of K562 cells in a dose-dependent manner. In our experiments, by using sodium nitroprusside (SNP) as NO donor and two kinds of superoxide dismutase, Cu,Zn-SOD and Mn-SOD, we created conditions to generate several kinds of signal molecules and investigated reaction of transformed (L929) and malignant (K562) cells to dose. Results of experiments are showing that chosen parameters (amount of free thiol groups and glutathione) may be relevant in monitoring the effect of exogenous nitrate oxide and its redox descendants in different, both transformed and malignant cell lines.

DNA-binding Activity of the ERp57 C-terminal Domain Is Related to a Redox-dependent Conformational Change

ERp57, a member of the protein-disulfide isomerase family, although mainly localized in the endoplasmic reticulum is here shown to have a nuclear distribution. We previously showed the DNA-binding properties of ERp57, its association with the internal nuclear matrix, and identified the C-terminal region, containing the a' domain, as being directly involved in the DNA-binding activity. In this work, we demonstrate that its DNA-binding properties are strongly dependent on the redox state of the a' domain active site. Site-directed mutagenesis experiments on the first cysteine residue of the -CGHC-thioredoxin-like active site lead to a mutant domain (C406S) lacking DNA-binding activity. Biochemical studies on the recombinant domain revealed a conformational change associated with the redox-dependent formation of a homodimer, having two disulfide bridges between the cysteine residues of two a' domain active sites. The formation of intermolecular disulfide bridges rather than intramolecular oxidation of active site cysteines is important to generate species with DNA-binding properties. Thus, in the absence of any dedicated motif within the protein sequence, this structural rearrangement might be responsible for the DNA-binding properties of the C-terminal domain. Moreover, NADH-dependent thioredoxin reductase is active on intermolecular disulfides of the a' domain, allowing the control of dimeric protein content as well as its DNA-binding activity. A similar behavior was also observed for whole ERp57.

In vitro evaluation of calcium binding capacity of chitosan and thiolated chitosan poly(isobutyl cyanoacrylate) core–shell nanoparticles

The ability of chitosan and its derivatives to bind cations is well known. Chitosan and thiolated chitosan were recently associated with poly(isobutyl cyanoacrylate) (PIBCA) nanoparticles leading to very promising results in terms of bioadhesion and permeation enhancement properties. Taking into account the influence that cations concentration have in the maintenance of both the permeation and the enzymatic barrier of the oral route, the possible cation binding capacity of these colloidal systems might be interesting in the use of these nanocarriers for the oral administration of pharmacologically active peptides. The aim of the present work was to in vitro evaluate the capacity of these colloidal systems to bind calcium, a model cation of physiological interest in the intestinal tract. The presence of chitosan on the nanoparticle surface importantly increased the calcium binding ability, in comparison to non-coated PIBCA nanoparticles. In addition, its presentation in the gel layer surrounding the nanoparticles, also beneficiated its binding capacity, obtaining 2–3 folds higher values when the polymer coated the nanoparticles than when it was in solution. The cross-linked structure observed for thiolated chitosan, due to the formation of inter- and intra-chain disulphide bonds, diminished the accessibility of cation to active sites of the polymer, decreasing the binding capacity of the calcium ion. However, when the amount of free thiol groups on the nanoparticle surface was high enough, the binding behaviour observed was higher than for nanoparticles elaborated with non-modified polymer.

Microbial Transglutaminase as a Tool to Restore the Functionality of Gluten from Insect‐Damaged Wheat

ABSTRACTIn some wheat‐growing countries, considerable quantities of commercial wheat are rendered unusable in standard baking because of preharvest damage of the grain by protease‐injecting bugs. In the present study, we studied the ability of transglutaminase (TG) treatment of damaged wheat flour to return the functionality of the gluten network. To confirm the TG cross‐linking, the degree of protein hydrolysis, the amount of free thiol groups, and the electrophoresis properties of glutenin subunits were determined. The effectiveness of the TG treatment on insect‐damaged wheat was analyzed by measuring the dough mixing behavior and the gluten quality. A decrease in the degree of hydrolysis (or free amino groups), a reduction in thiol group concentration, and a decrease of extractable high molecular weight glutenin subunits (HMW‐GS) (measured by high‐performance capillary electrophoresis) confirmed the protein cross‐linking catalyzed by TG, the simultaneous formation of disulfide bonds by the proximity of the cross‐linked polypeptide chains, and the formation of aggregates of high molecular weight. The TG treatment of the damaged wheat flour led to a recovery of the consistograph parameters and gluten index value, and the covalent nature of the bonds ensured the stability of the protein changes.

Effect of atrazine on glutathione levels, glutathione s-transferase and glutathione reductase activities in pea and wheat plants

Changes were studied in the endogenous level of glutathione (total and oxidised), and in the amount of free thiol groups as caused by the herbicide atrazine on two species of plants with different sensitivity to it. The activities of two enzymes related to glutathione metabolism (glutathione reductase and glutathione S-transferase) were also determined. The application of the herbicide on leaf increased the levels of total and oxidised glutathione in pea and wheat plants. Increased activity glutathione S-transferase in wheat plants was found.

Phospholipid hydroperoxide cysteine peroxidase activity of human serum albumin

Human serum albumin (HSA) reduced the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-l-3-phosphatidylcholine (PLPC-OOH) to the corresponding hydroxy-derivative with a high apparent affinity (Km=9. 23+/-0.95 microM). Removal of bound lipid during purification increased this activity. At physiological concentration, HSA reduced the phospholipid hydroperoxide in the absence of a cofactor. However, in the presence of a cofactor (reductant), the rate of the reaction was increased. All of the major aminothiols in plasma could act as reductants, the best being the most abundant, cysteine (Km=600+/-80 microM). For every nanomole of PLPC-OOH reduced by HSA, 1.26 nmol of cystine was formed, indicating a reaction stoichiometry of 1 mol PLPC-OOH to 2 mol cysteine. We used chemical modification to determine which amino acid residues on HSA were responsible for the activity. Oxidation of thiol group(s) by N-ethylmaleimide led to a reduction in the rate of activity, whereas reduction of thiols by either dithiothreitol or the angiotensin-converting enzyme inhibitor, captopril, increased the activity. Both N-ethylmaleimide-modified HSA and dithiothreitol-treated HSA exhibited increased apparent affinities for PLPC-OOH. For a range of preparations of albumin with different modifications, the activity on PLPC-OOH was dependent on the amount of free thiol groups on the albumin (correlation coefficient=0.91). Patients with lowered albumin concentrations after septic shock showed lowered total plasma thiol concentrations and decreased phospholipid hydroperoxide cysteine peroxidase (PHCPx) activities. These results therefore show for the first time that HSA exhibits PHCPx activity, and that the majority of the activity depends on the presence of reduced thiol group(s) on the albumin.

Binding of secretory component to dimers of immunoglobulin A in vitro. Mechanism of the covalent bond formation.

A complex between secretory component and an immunoglobulin A (IgA) myeloma dimer has been studied in vitro as a model to elucidate the mechanism of the formation of disulfide bonds during assembly in vivo of secretory immunoglobin A. A small amount of free thiol groups, totally about 0.4 groups per mole of protein, were shown to be present on both the heavy and light chains of the IgA dimer, but not on its J-chain, while no such groups could be demonstrated on free secretory component. The SH-groups on IgA most likely exist as a result of incomplete oxidation of some intra-or interchain disulfide bonds of the molecule, analogous to what has been suggested for IgG. Several types of evidence indicated that the disulfide bonds between secretory component and IgA are formed after the noncovalent association of the two proteins by a sulfhydryl group-disulfide bond exchange reaction, in which the small amount of free sulfhydryl groups on the IgA dimer initiate the reaction by reducing a reactive disulfide bond on secretory component. This exchange reaction, which thus proceeds by the mechanism of so-called disulfide interchange reactions, requires certain conformational features of one or both of the proteins and leads to the formation of presumably two new interchain disulfide bonds between secretory component and IgA. The reaction does not progress to completion, however, but ends in an equilibrium so that a small proportion of the secretory component molecules always are unattached by disulfide bonds.