Sulfhydryl-disulfide Interchange Reaction Research Articles

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 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.

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.

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.

The role of net charge on the renaturation of reduced lysozyme by the sulfhydryl-disulfide interchange reaction.

Reduced and acetylated lysozymes are basic proteins. When their amino groups were variously acetylated and then renatured by sulfhydryl-disulfide (SH-SS) interchange reaction at pH 8.0, the final folding yield decreased as the number of positive charges decreased. The final folding yield of native and Ac1 lysozyme, with one positive charge eliminated, was less sensitive to increasing protein concentration than that of Ac2 lysozyme, where two positive charges had been eliminated. The final folding yield of reduced Ac2 lysozyme increased in the presence of 1 M urea, which reduced the aggregation of unfolded lysozyme. Thus, the aggregation of unfolded lysozymes, which leads to a decrease in the final folding yield, was found to be heavily dependent on their net charges. Moreover, the final folding yield of reduced lysozyme was shown to be increased by use of cystamine as an oxidizing reagent in comparison with 2-hydroxyethyl disulfide or dithiodiglycolic acid. This may support the idea that the final folding yield is influenced by electrostatic interaction between unfolded lysozymes in the early stage of renaturation. In contrast, the concentration dependency of the final folding yield of Ac1 lysozyme was different from those of carboxymethylated His15 and Asp106 lysozymes whose positive net charges were similar to that of Ac1 lysozyme. On the basis of the observations, it is suggested that the formation of the aggregates in the renaturation process might also be affected by the structure of the unfolded state of lysozyme in solution.

Changes in electrophoretic patterns of acetic acid-insoluble wheat flour proteins during dough mixing

Acetic acid-insoluble (residue) protein fractions of wheat flours and respective doughs were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS—PAGE). Gel patterns of unreduced SDS buffer extracts from residue fractions showed that dough mixing caused systematic changes in the staining intensity of a group of relatively high molecular weight proteins comprising triplet band proteins and neighboring bands. Together, these proteins were named in this study the triplet zone proteins. Continued mixing of control doughs resulted in a progressive decrease in the intensity of the triplet zone bands, whereas mixing in the presence of potassium iodate (KIO 3 ) and N -ethylmaleimide (NEMI) caused an increase in staining intensity of these bands. These results appear to be related to sulfhydryl-disulfide interchange reactions that have been proposed to occur during dough mixing. Results for doughs from water-extracted flour suggested water soluble components of the flour interacted during mixing with the triplet zone proteins. SDS—PAGE patterns of reduced proteins that could not enter the separating gel under non-reducing conditions (slot protein) resembled the patterns of purified glutenin. Electrophoretic patterns of slot protein of SDS buffer extracts from acetic acid-insoluble residues showed the presence of subunits that corresponded in molecular weight to subunits of triplet band proteins. This suggested the possibility of strong interaction between triplet band proteins and SDS-buffer extractable glutenin in flour and dough residues.

Isolation, Purification, and Characterization of the Oligomeric Seed Globulin fromAmaranthus hypochondriacus

The main storage seed protein of Amaranthus hypochondriacus, the oligomeric salt-soluble globulin, was isolated and purified to homogeneity by Sephacryl S-300 gel filtration and Separon Hema-Bio 1000 DEAE anion-exchange chromatography. The globulin had two heterogeneous forms, a major and minor species with apparent molecular masses of 398,000 and 337,000 daltons, respectively. Each of the two species were analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (PAGE) and by native-PAGE (without prior reduction of disulfide bonds). Only slight differences were observed in their subunit patterns although appreciable differences in mobility and dissociation behaviour were noted on native-PAGE. The major protein fraction was oligomeric, consisting of a tetrameric and hexameric moiety held together by weak secondary forces. The minor fraction, a hexamer of slightly higher apparent molecular mass was a polymeric species derived in part from a sulfhydryl-disulfide interchange reaction.

Isolation, Purification, and Characterization of the Oligomeric Seed Globulin from Amaranthus hypochondriacus.

The main storage seed protein of Amaranthus hypochondriacus, the oligomeric salt-soluble globulin, was isolated and purified to homogeneity by Sephacryl S-300 gel filtration and Separon Hema-Bio 1000 DEAE anion-exchange chromatography. The globulin had two heterogeneous forms, a major and minor species with apparent molecular masses of 398,000 and 337,000 daltons, respectively. Each of the two species were analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (PAGE) and by native-PAGE (without prior reduction of disulfide bonds). Only slight differences were observed in their subunit patterns although appreciable differences in mobility and dissociation behaviour were noted on native-PAGE. The major protein fraction was oligomeric, consisting of a tetrameric and hexameric moiety held together by weak secondary forces. The minor fraction, a hexamer of slightly higher apparent molecular mass was a polymeric species derived in part from a sulfhydryl-disulfide interchange reaction.

Effect of Chemical Modifications of Tryptophan Residues on the Folding of Reduced Hen Egg-White Lysozyme1

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.

Sulfhydryl groups and food safety.

Sulfhydryl (thiol) compounds such as cysteine, N-acetylcysteine, and reduced glutathione interact with disulfide bonds of plant protease inhibitors via. sulfhydryl-disulfide interchange and oxidation reactions. Such interactions with inhibitors from soy and lima beans facilitate their heat inactivation of inhibitors, resulting in beneficial nutritional effects. Since thiols are potent nucleophiles, they have a strong avidity for unsaturated electrophilic centers of several dietary toxicants, including cyclopropene fatty acids, aflatoxins, sesquiterpene lactones, and trichothecenes. Such interactions may be used in vitro to lower the toxic potential of our food supply, and in vivo for prophylactic and therapeutic effects against antinutrients and food toxicants. A number of examples are cited to illustrate the concept of applying site-specific nucleophilic reagents such as thiols to reduce antinutritional and toxic manifestations of food ingredients by modifying pharmacophoric and toxicophoric electrophilic sites. Several related interactions between sulfhydryl groups and toxic compounds are also briefly mentioned to illustrate the generality of this approach.KeywordsDisulfide BondFood SafetySulfhydryl GroupSesquiterpene LactoneLima BeanThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

On the role of disulfide bonds in polymerization of soybean 7S globulin during storage.

Modification of soybean 7S globulin with N-ethylmaleimide (NEM) was effective for preventing humidity-induced insolubilization during storage. The sulfhydryl-disulfide interchange reaction and/or oxidation of the sulfhydryl groups to form disulfide bonds closely related to the polymerization of the 7S globulin. A dimer, consisting of α′ and α subunits linked with disulfide bonds, was observed in the polymerized 7S globulin fractions, but this dimer was not readily apparent in the NEM-7S globulin. The formation of the dimer certainly initiates the insolubilization of the 7S globulin during storage. Although the β subunit had sulfhydryl groups, it did not take a part in the formation of the dimer.

Clianges in rheological and chemical properties of soy protein dope solution through increase of aging time.

Experiments were carried out to examine the effects of aging time on rheological and chemical properties of soy protein dope solution. The results obtained were as follows: 1) Viscosity and normal force of soy protein dope solution decreased with the increase in aging time. 2) Electrophoretic patterns of the dope solution became obscure as the aging time increased, however, the sedimentation coefficients were constant and 2_??_3 S. 3) Amino acid analysis showed the remarkable decrease of cystin and the increase of cystein and lysino-alanine with the increase in aging time. 4) Viscosity of the dope solution with added mercapto-ethanol was low and did not change with the aging time. Moreover, the power law of 3.5 existed between the viscosity and the concentration of disulfide bonds. From these results, it was speculated that the decrease of viscosity and normal force in the soy protein dope solution with the increase in aging time might be caused mainly by the disruption of the intermolecular disulfide bonds due to the sulfhydryl-disulfide interchange reaction and the cleavage of C-S bonds to form lysinoalanine by alkaline hydrolysis.

About some common biochemical and pharmacological properties of bile salts and non steroidal anti-inflammatory drugs.

Glyco- and tauro-deoxycholate, -chenodeoxycholate, -dehydrocholate and -cholate uncouple the mitochondrial oxidative phosphorylation in this order of potency. They also induce pseudo-energized mitochondrial swelling and lymphocyte swelling in the same order of potency. Similar swelling properties were observed for glyco- and tauro-lithocholate. Glyco- and tauro-deoxycholate induce alkalinization of the intra-mitochondrial space as determined on bromothymol blue pre-loaded mitochondria. These two bile salts conjugates also increase the amount of -SH groups available on lymphocyte membranes for sulfhydryl-disulfide interchange reactions. All these biochemical properties have been described for non-steroidal anti-inflammatory drugs. They could thus be implicated in the jaundice remission observed in various inflammatory states.