Sulfhydryl-disulfide Interchange Research Articles

Heat treatment of quinoa (Chenopodium quinoa Willd.) albumin: Effect on structural, functional, and in vitro digestion properties.

Quinoa seeds are rich in protein, polyphenols, phytosterols, and flavonoid substances, and excellent amino acid balance that has been revisited recently as a new food material showing potential applied in fitness and disease prevention. Heat treatment is one of the most effective strategies for improving the physiochemical characteristics of a protein. However, research examining the effects of temperature on quinoa albumin (QA) properties is limited. In this study, QA was subjected to thermal treatment (50, 60, 70, 80, 90, 100, and 121°C). SDS−PAGE revealed that QA is composed of several polypeptides in the 10−40 kDa range. Amino acid (AA) analysis showed that the branched-chain amino acids (BCAAs), negatively charged amino acid residues (NCAAs), and positively charged amino acids (PCAAs) contents of QA were more than double that of the FAO/WHO reference standard. Additionally, heating induced structural changes, including sulfhydryl-disulfide interchange and the exposure of hydrophobic groups. Scanning electron microscopy demonstrated that the albumin underwent denaturation, dissociation, and aggregation during heating. Moreover, moderate heat treatment (60, 70, and 80°C) remarkably improved the functional properties of QA, enhancing its solubility, water (oil) holding capacity, and emulsification and foaming characteristics. However, heating also reduced the in vitro digestibility of QA. Together, these results indicate that heat treatment can improve the structural and functional properties of QA. This information has important implications for optimizing quinoa protein production, and various products related to quinoa protein could be developed. which provides the gist of commercial applications of quinoa seeds for spreading out in the marketplace.

Iso-seco-tanapartholide activates Nrf2 signaling pathway through Keap1 modification and oligomerization to exert anti-inflammatory effects

Covalent modification of Keap1 results in reducing ubiquitination and the accumulation of Nrf2, which subsequently initiates the transcription of cellular anti-oxidant and anti-inflammatory genes. Iso-seco-tanapartholide (IST), a sesquiterpene isolated from the traditional Chinese medicine Artemisia argyi, had been reported to possess NF-κB inhibitory activity. However, its deep anti-inflammatory effects and direct target have never been reported. Here we show that IST activated Nrf2 and increased its target gene expression. In particular, LPS-caused inflammation in vitro and in vivo was mitigated by IST-induced Nrf2 activation but aggravated by Nrf2 inhibition. Mechanically, IST targeted Keap1 proteins via alkylating its cysteine residues 151, 273, 288, and so on. Subsequently, the modifying agent IST was displaced by intermolecular sulfhydryl disulfide interchange to lead to a disulfide dimer of Keap1. The resulting conformational change of Keap1 liberated Nrf2 from sequestration and allowed it translocation to the nucleus to activate the transcriptional program. Further studies demonstrated that Keap1 dimer formation contributed to the anti-inflammatory effects of IST. Taken together, our findings reveal a new mechanism for Nrf2 activation and provide a potential lead compound to treat inflammatory diseases through targeting Keap1.

Combination of high-intensity ultrasound and hydrogen peroxide treatment suppresses thermal aggregation behaviour of myofibrillar protein in water

This study was aimed at evaluating the potential of high-intensity ultrasound (HIU, 450 W for 10 min) combined with hydrogen peroxide (H2O2) having various concentrations (0, 50, 100, 200, 400, 800 μmol/g protein) to inhibit the thermal aggregation behavior of myofibrillar proteins (MPs) in water. The results indicated that the addition of H2O2 interfered with the intermolecular sulfhydryl-disulfide interchange and inhibited the disulfide bond cross-linking. The H2O2-mediated conversion of cysteine to thiol derivatives appeared to be the primary mechanism of this effect. The HIU combined with H2O2, especially at the H2O2 concentration of 200 μmol/g, corresponded to a more significant inhibitory effect than that of only H2O2, which attributed to the dissociation of the filamentous myosin structure that led to an enhanced accessibility of the buried sulfhydryl groups. In conclusion, these findings provide direct evidence for the role of HIU combined with H2O2 in improving the thermal stability of MPs.

High Homogenization Speeds for Preparing Unstable Myofibrillar Protein-Olive Oil Emulsions.

Natural protein-based oil-in-water emulsions have recently attracted a lot of attention because of their potential as a synthetic emulsifier replacer. It is, however, unclear how the emulsification process and protein concentration may affect the stability of such emulsions. Therefore, this study investigated the effects of homogenization speeds (4,000, 8,000, 12,000, and 16,000 rpm/min) and myofibrillar protein (MP) concentrations (10, 20, 30, 40, and 50 mg/mL) on the stability of MP-olive oil emulsion. The emulsifying creaming index, emulsifying activity index (EAI), droplet size, microstructure, free sulfhydryl content, and zeta potential of the emulsion were measured. The results showed that with the condition of sufficient emulsifier (at least 20 mg/mL), the EAI increased, and the droplet size and zeta potential of emulsions decreased with the increase of homogenization speed. Emulsions were stable at 4,000 and 8,000 rpm/min (20, 30, 40, and 50 mg/mL) within 48 hr, and they were unstable at 12,000 and 16,000 rpm/min (20, 30, 40, and 50 mg/mL) within 48 hr. This result is mainly attributed to the fact that sulfhydryl-disulfide interchange leads the excessive aggregate of MP at the oil-water interface. PRACTICAL APPLICATION: Myofibrillar protein-olive oil emulsions (oil-in-water) may be used to deliver nutrients into food products. In this study, myofibrillar protein-olive oil emulsions stabilized with the optimizing emulsification conditions. This study may have important implications to produce food-grade myofibrillar protein-olive oil emulsions to deliver nutrients.

Effect of oxidative modification on structural and foaming properties of egg white protein

Protein oxidation results in structural modification which affects its functionalities. In this study, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was selected as a representative of lipid peroxidation products to investigate the effect of oxidative modification on structural and foaming properties of egg white protein. Incubation of egg white protein with AAPH resulted in structural changes, regarding by sulfhydryl-disulfide interchange reaction and increase in dityrosine formation. Moderate oxidation led to exposure of hydrophobic groups of egg white protein, whereas, excessive oxidation induced a decrease in surface hydrophobicity. Molecular weight distribution and scanning electron microscopy showed that egg white protein underwent an aggregation after excessive oxidation. Furthermore, after treating with AAPH at 0.2 mM, the foaming ability of egg white protein increased from 85.6% to 91.4%, and the foaming stability increased from 80.6% to 85.3%. The excessive oxidation treatment (5 and 25 mmol/L AAPH) resulted in an enhanced foaming ability, while reduced its foaming stability. This study suggested that oxidation treatment has a potential to be implemented to modify foaming properties of egg white protein.

Effect of a multiple freeze-thaw process on structural and foaming properties of individual egg white proteins.

In this study, major albumen proteins (ovalbumin, ovomucoid, ovotransferrin, lysozyme and ovomucin) were singly subjected to a multiple freeze-thaw process, and the resulting changes in structural characteristics and foamability were investigated. Structural changes of proteins occurred during the process, regarding by sulfhydryl-disulfide interchange and exposure of hydrophobic groups. The differential scanning calorimetry and scanning electron microscopy showed that these albumen proteins underwent denaturation, dissociation and possibly aggregation. Correspondingly, the foaming ability of albumen proteins improved after the freeze-thaw treatment, except for ovalbumin. The foaming ability of whole egg white was higher than that of each albumen protein, and improved after the multiple freeze-thaw process. This study extended knowledge of the relative contribution of each albumen protein to foaming properties of whole egg white during a freeze-thaw process, and suggested that a multiple freeze-thaw process is a promising technique for improving foaming properties of egg white proteins.

Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions

Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H2O2 in the range of 0-0.144 H2O2 to protein ratios (HTPR) by the addition of the required quantity of H2O2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H2O2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H2O2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H2O2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA.

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.

Probing the molecular forces involved in binding of selected volatile flavour compounds to salt-extracted pea proteins

Molecular interactions between heterologous classes of flavour compounds with salt-extracted pea protein isolates (PPIs) were determined using various bond disrupting agents followed by GC/MS analysis. Flavour bound by proteins decreased in the order: dibutyl disulfide>octanal>hexyl acetate>2-octanone=benzaldehyde. Benzaldehyde, 2-octanone and hexyl acetate interacted non-covalently with PPIs, whereas octanal bound PPIs via covalent and non-covalent forces. Dibutyl disulfide reacted with PPIs covalently, as its retention was not diminished by urea and guanidine hydrochloride. Using propylene glycol, H-bonding and ionic interactions were implicated for hexyl acetate, benzaldehyde, and 2-octanone. A protein-destabilising salt (Cl3CCOONa) reduced bindings for 2-octanone, hexyl acetate, and benzaldehyde; however, retention for octanal and dibutyl disulfide increased. Conversely, a protein-stabilising salt (Na2SO4) enhanced retention for benzaldehyde, 2-octanone, hexyl acetate and octanal. Formation of a volatile flavour by-product, 1-butanethiol, from dibutyl disulfide when PPIs were treated with dithiothreitol indicated occurrence of sulfhydryl–disulfide interchange reactions.

Effect of NaCl addition during diafiltration on the solubility, hydrophobicity, and disulfide bonds of 80% milk protein concentrate powder

We investigated the surface hydrophobicity index based on different fluorescence probes [1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-propionyl-2-(N,N-dimethylamino)-naphthalene (PRODAN)], free sulfhydryl and disulfide bond contents, and particle size of 80% milk protein concentrate (MPC80) powders prepared by adding various amounts of NaCl (0, 50, 100, and 150mM) during the diafiltration process. The solubility of MPC80 powder was not strictly related to surface hydrophobicity. The MPC80 powder obtained by addition of 150mM NaCl during diafiltration had the highest solubility but also the highest ANS-based surface hydrophobicity, the lowest PRODAN-based surface hydrophobicity, and the least aggregate formation. Intermolecular disulfide bonds caused by sulfhydryl-disulfide interchange reactions and hydrophobic interactions may be responsible for the lower solubility of the control MPC80 powder. The enhanced solubility of MPC80 powder with addition of NaCl during diafiltration may result from the modified surface hydrophobicity, the reduced intermolecular disulfide bonds, and the associated decrease in mean particle size. Addition of NaCl during the diafiltration process can modify the strength of hydrophobic interactions and sulfhydryl-disulfide interchange reactions and thereby affect protein aggregation and the solubility of MPC powders.

Investigation of the Protein−Protein Aggregation of Egg White Proteins under Pulsed Electric Fields

Egg whites were exposed to pulsed electric fields (PEFs) to investigate the protein-protein aggregation. No insoluble protein aggregate was found when egg whites were exposed to PEFs at 25, 30, and 35 kV/cm for 400 micros. However, atomic force microscopy showed that the sizes of the protein particles increased. Native polyacrylamide gel electrophoresis (PAGE) demonstrated the existence of aggregates under PEFs at 35 kV/cm for 400 micros. Sodium dodecyl sulfate (SDS)-PAGE in the presence and absence of 2-mercaptoethanol further indicated that sulfhydryl-disulfide interchange reactions occurred under PEFs. Differential scanning calorimetry scans showed 400 micros of PEF treatment at 35 kV/cm denatured 16.5% proteins. Insoluble egg white protein aggregates were induced by PEF (35 kV/cm, 800 micros) and heat (60 degrees C, 3.5 min) treatments. Disulfide bonds were the dominant binding forces in the formation of protein aggregates. However, the weakly noncovalent bonds play a much more important role in the protein aggregation forming in heat treatment (60 degrees C, 3.5 min) than that in PEF treatment (35 kV/cm, 800 micros).

A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.

Heat-induced gels of egg white/ovalbumins from five avian species: thermal aggregation, molecular forces involved, and rheological properties.

Product processing (heating, pH change, etc.) usually alters protein structure, improves rheological properties, and gives a unique texture to foods. The thermal aggregation and structural properties of ovalbumins from five avian species were studied at different pH values by polyacrylamide gel electrophoresis (PAGE) and determinations of sulfhydryl group content and surface hydrophobicity. The results showed that sulfhydryl group content changed insignificantly in heat-denatured ovalbumins other than hen ovalbumin (pH-independent), and surface hydrophobicity markedly increased (pH-dependent) after heating, with a significant difference among species. Furthermore, it was demonstrated that the hydrophobic interaction and sulfhydryl-disulfide interchange reaction were necessary in the aggregation and cross-linking of gel networks. Creep tests were also used to characterize the gel network structures of various egg white/ovalbumins upon heating. The viscoelastic behavior of the ovalbumins of all species was dependent on pH values, and changed significantly with the phylogeny of these species. With increases in pH value (7.0-8.5), the heat-induced gels of ovalbumins gradually changed from turbid to translucent, the instantaneous modulus (E(0)) increased slightly and reached a nearly constant value, and the Newtonian modulus (etaN) increased significantly in each sample. The heated egg white from these five avian species also formed highly viscoelastic gels, with a good correlation of viscoelastic behavior between ovalbumin and egg white in corresponding species.

Inactivation Kinetics Study of the Kunitz Soybean Trypsin Inhibitor and the Bowman-Birk Inhibitor.

The inactivation of trypsin inhibitors (TIs) in soy flour exhibits a two-phase inactivation behavior. It is sometimes assumed that this behavior is caused by a difference in the heat stabilities of the Kunitz soybean trypsin inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). Kinetics studies with KSTI and BBI in soy flour showed that this two-phase inactivation behavior of TIs could not be explained by the difference in the heat stabilities of KSTI and BBI. Inactivation of KSTI and BBI in an aqueous solution and in a starch matrix followed a first-order reaction. KSTI and BBI in a starch matrix with added cysteine showed a two-phase inactivation behavior. The existence of thiols in soy flour seems to be responsible for the two-phase inactivation of TIs in soy flour. It is suggested that TIs in soy flour are inactivated by sulfhydryl-disulfide interchange during the first inactivation phase and by heat during the second phase.

SULFHYDRYL AND DISULFIDE GROUPS OF THE OLIGOMERIC SEED GLOBULIN FROM AMARANTHUS HYPOCHONDRIACUS K343

The hetero-oligomeric seed globulin from amaranth was found to contain high levels of sulfhydryl and disulfide groups. Results suggested that the majority of sulfhydryl groups were buried deep within subunits of this hetero-oligomer and were found to have varying degrees of reactivities. In the presence of Nethylmaieimide (NEM), total alkylation of sulfhydryl groups was not possible and was the probable reason for the inability to completely inhibit polymerization of the globulin via a sulfhydryl-disulfide interchange mechanism. Disulfide linkages were important in stabilizing the protein and for the spontaneous dimerization (following reduction) of certain subunits after elimination of the reductant. In addition, these disulfide linkages are thought to be important for maintenance of the quaternary structure of the globulin. Little change in the secondary structure of the globulin was shown to occur upon reduction.

Effects of N-ethylmaleimide and CaCl 2 on cold gelation of whey protein isolate

The combined effect of CaCl 2 (10–120 mM) and N-ethylmaleimide [NEM] (0–5 mM) on colour and textural properties of Ca 2+-induced cold-set whey protein isolate (WPI) gels was investigated. Raising [CaCl 2] resulted in increasing gel opacity and hardness while reducing cohesiveness. In gels prepared by dialyzing against a 10 mM CaCl 2 solution, increasing [NEM] to 2 mM resulted in a slight increase in opacity and hardness. At > 2 mM NEM, both parameters were reduced due to the lower degree of sulfhydryl-disulfide interchange reaction. Gels prepared by dialyzing against ≥ 30 mM CaCl 2 showed an increase in opacity and a decrease in hardness as [NEM] was raised from 0 to 5 mM. These changes were likely to be related to the degree of WPI polymerization during the pre-heating stage, i.e. prior to calcium addition and gelation.

Identification of monoclonal antibodies that recognize different disulfide bonded forms of thrombospondin 1

Thrombospondin 1 (TSP1) is a multidomain glycoprotein from platelets and cells which functions in cell-cell and cell-matrix interactions. The structure of TSPI is regulated by sulfhydryl-disulfide interchange in the carboxy-terminal Ca 2+-binding loops and globular domain which markedly influence its interaction with cell surface integrins and its inhibition of neutrophil enzymes. We have identified murine monoclonal antibodies that recognized different disulfide-bonded forms of TSP1, made by preparing TSPI in buffers containing either 0.1 mM or 2 mM Ca 2+. Antibody HB8432 recognizes TSPI prepared in buffers containing either 0.1 or 2 mM Ca 2+, while antibodies D4.6 and A65M recognized only TSP1 prepared in buffers containing 0.1 mM Ca 2+. The antibodies recognize these different TSP1 preparations either adsorbed to plastic or extracellular matrix. Immunohistochemistry of human rheumatoid synovial tissue using HB8432 resulted in staining of numerous blood vessel walls and matrix cells, while D4.6 and A65M stained a subset of the HB8432 positive blood vessels and only occasionally stained matrix cells. These results suggested that different disulfide-bonded forms of TSP1 were being expressed in different areas of inflamed tissue.

Effect of pH during the dry heating on the gelling properties of egg white proteins

Gel strength and elasticity of dried egg white proteins greatly increased by heating in the dry state at alkaline pH region (under 9.5) for a short period of time (3–5 days) without losing water solubility. Circular dichroism and differential scanning calorimetry spectrum revealed that the increased degree of denaturation found during preheating was accelerated in alkaline pH region. The polymerization of the proteins was also enhanced by alkaline dry heating through sulfhydryl-disulfide interchange. Alkaline dry heating resulted in high molecular weight polymer of partially unfolded egg white proteins which, in turn, contribute in the formation of low molecular weight and narrow molecular distribution of the aggregate found during the subsequent heat for gelation. Thus, heating of dried egg white proteins in the dry state at alkaline pH (under 9.5) is an effective method to obtain firm and elastic gels. The degree of unfolding of the proteins upon dry heating may play a crucial role in the gelling process of the proteins.

Kinetically trapped structure in the renaturation of reduced oxindolealanine 62 lysozyme.

The refolded products of reduced native lysozyme and reduced OX62 lysozyme, in which Trp62 is converted to oxindolealanine (OX62) during the renaturation of sulfhydryl-disulfide interchange reactions at pH 8 and 37 degrees C, were investigated. On gel-chromatography eluted with 10% aqueous acetic acid containing 4 M urea, two peaks appeared in the refolded product of reduced OX62 lysozyme while a single peak appeared in the refolded product of reduced native lysozyme. From the analyses of the activity and primary and the tertiary structures of the derivative, the structure of the derivative from reduced native lysozyme was confirmed to be identical to that of the untreated one. On the other hand, the refolded product from reduced OX62 lysozyme had the same primary structure but a different tertiary structure compared to the untreated one. The tertiary structure of the refolded product from the reduced OX62 lysozyme was changed to that of the untreated one by the denaturation-renaturation treatment under nonreduced conditions. However, the refolded species was barely changed to that of the untreated one by incubation under physiological conditions. Therefore, the refolded product from reduced OX62 lysozyme was suggested to be a metastable and kinetically trapped product in the renaturation process of reduced OX62 lysozyme. In addition, an interaction involving the folding process of reduced lysozyme was discussed on the basis of the NMR analyses of the metastable structure.

The Stress/Strain Curve of α-Keratin Fibers and the Structure of the Intermediate Filament

Staying in the tradition of Astbury's hypothesis about the role of the α ⇔ β trans formation for the stress/ strain-curve of wool fibers and of Feughelman's X/Y-zones model, the interrelation between the morphological structure of keratin fibers and the shape of their stress/strain-curve in water is reevaluated. The yield and post-yield regions can be attributed to the opening up of two distinctly different and well defined portions of the monomer of the intermediate filament; the increased slope in the post- yield region can be attributed to the influence of the sulfur bonds in one of the segments. The role of the sulfhydryl-disulfide interchange reaction for the appearance of the post-yield region is pointed out and the molecular mechanisms for achieving the max imum possible strain are discussed.