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1.
Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.  相似文献   

2.
Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70 degrees C, and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducing reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the beta-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine; that is, a free sulfhydryl residue was formed via beta-elimination of a disulfide bond. Intermolecular disulfide bonds were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.  相似文献   

3.
Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.  相似文献   

4.
Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.  相似文献   

5.
The reactions of oxidized glutathione generated from endogenous glutathione by the addition of ascorbic acid (AA) prior to dough mixing on free thiol groups of gluten proteins have been investigated. A small amount of (35)S-labeled glutathione was added as a tracer to identify the reaction products of GSSG and free protein thiols by radioactivity measurement. First, gluten was isolated from the dough, then the gliadins were extracted, and residual glutenin was partially hydrolyzed with thermolysin. After preseparation by gel permeation chromatography, the fractions with the highest radioactivity were separated by high-performance liquid chromatography. Radioactive peptides were identified, isolated, sequenced, and assigned to amino acid sequences of gluten protein components. The isolated peptides contained exclusively the cysteine residues C(b) and C(x) of low molecular weight subunits of glutenin, which are supposed to be highly reactive in forming intermolecular disulfide bonds. From these results it can be assumed that the cysteine residues C(b) and C(x) of the low molecular weight subunits of glutenin are at least partly present in the thiol form in flour. During dough mixing they are converted to protein-protein disulfides or glutathione-protein mixed disulfides by thiol/disulfide interchange reactions. Oxidized glutathione necessary for this reaction is generated from glutathione by the action of AA. These results are in accordance with the major hypothesis about the mechanism of action of AA.  相似文献   

6.
The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.  相似文献   

7.
For a number of applications, gluten protein polymer structures are of the highest importance in determining end‐use properties. The present article focuses on gluten protein structures in the wheat grain, genotype‐ and environment‐related changes, protein structures in various applications, and their impact on quality. Protein structures in mature wheat grain or flour are strongly related to end‐use properties, although influenced by genetic and environment interactions. Nitrogen availability during wheat development and genetically determined plant development rhythm are the most important parameters determining the gluten protein polymer structure, although temperature during plant development interacts with the impact of the mentioned parameters. Glutenin subunits are the main proteins incorporated in the gluten protein polymer in extracted wheat flour. During dough mixing, gliadins are also incorporated through disulfide‐sulfhydryl exchange reactions. Gluten protein polymer size and complexity in the mature grain and changes during dough formation are important for breadmaking quality. When using the gluten proteins to produce plastics, additional proteins are incorporated in the polymer through disulfide‐sulfhydryl exchange, sulfhydryl oxidation, β‐eliminations with lanthionine formation, and isopeptide formation. In promising materials, the protein polymer structure is changed toward β‐sheet structures of both intermolecular and extended type and a hexagonal close‐packed structure is found. Increased understanding of gluten protein polymer structures is extremely important to improve functionality and end‐use quality of wheat‐ and gluten‐based products.  相似文献   

8.
The kinetics of heat-induced polymerization of gliadin, that is, a mixture of monomeric wheat storage proteins, was studied using a model system. Samples were heated at pH 6.0 and 8.0 at 110, 120, and 130 °C for up to 240 min, and their extractabilities were compared under nonreducing and reducing (with 1% dithiothreitol) conditions. Extraction media were sodium dodecyl sulfate (SDS) containing buffer (pH 6.8, SDS buffer) and/or 70% ethanol. Gliadin cross-linking mainly resulted from intermolecular disulfide (SS) bond formation. At higher temperatures and, preferably, alkaline pH, intramolecular SS bonds in gliadin underwent β-elimination reactions, leading to the formation of dehydroalanine (DHA) and free sulfhydryl (SH) groups. The latter interchanged rapidly with SS bonds, leading to intermolecular SS bonds and gliadin extractability loss. When free SH groups had been formed, gliadin extractability in SDS buffer decreased following first-order reaction kinetics, the reaction rate constant of which increased with temperature and pH. Furthermore, the extractabilities of α- and γ-gliadin in 70% ethanol decreased according to first-order reaction kinetics. ω-Gliadin extractability was much less affected. Under the experimental conditions, gliadin polymerization through SH-SS interchange occurred much more rapidly than β-elimination of cystine.  相似文献   

9.
Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.  相似文献   

10.
Interactions between alpha-lactalbumin (alpha-La) and ovalbumin (OVA) in mixed systems (1:1 ratios; 2, 4, and 8% w/w total protein, respectively) heated at pH 7 and 80 degrees C for 15 min were studied using sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), gel filtration chromatography (GFC), and competitive enzyme-linked immunosorbent assay (ELISA). Although alpha-La alone did not form aggregates upon heating, it formed large aggregates when heated with OVA. The aggregated molecules eluted at the void volume had a molecular mass >300 kDa. The aggregation process was quantitatively affected by different avian OVAs from five species, possessing different numbers of free sulfhydryl groups. The amount of aggregates (M(w) > 300 kDa) increased in proportion to total protein concentration, and the amount of intermediate components (M(w) < 300 kDa) and monomeric OVA and alpha-La also changed, correlating with total protein concentration during heating. The results also indicated that the aggregates and intermediates, which contained dimeric and trimeric alpha-La, were mainly formed by the intermolecular disulfide bonds. The different interactions observed in several avian OVAs may explain heat-induced gelation of various avian OVAs as well as the enhancement of heat-induced gelation of OVA by alpha-La.  相似文献   

11.
Variation in power and time of sonication of gluten and glutenin suspensions was used to gain information about the mechanism of molecular breakdown. At low power, an increase in sulfhydryl (SH) content of solubilized gluten could be ascribed to additional glutenin brought into the solution. Higher power sonication of gluten suspensions, at constant protein concentration and with increasing times, progressively shifted the molecular weight distribution (measured by size exclusion HPLC) to lower molecular weights. It was accompanied by a parallel decrease in SH content. This suggested that the freed cysteine residues formed by scission of disulfide (SS) bonds, caused by sonication, reacted to form new intramolecular SS bonds with free cysteines on the same molecular fragments. Circular dichroism measurements appeared to support this conclusion. High‐power ultrasound produced SE‐HPLC profiles with diffuse peaks corresponding to HMW‐GS and LMW‐GS. SDS‐PAGE patterns of protein fractions obtained in different elution ranges of SEHPLC confirmed that individual subunits had been produced by sonication. A fraction of the polymeric protein eluting at the void volume of SE‐HPLC appeared to be resistant to breakdown by sonication.  相似文献   

12.
The structural and antimicrobial functions of lysozyme reduced with food-compatible reducing agents-cysteine (Cys) and glutathione (GSH)-were investigated. The disulfide bonds were partially reduced by thiol-disulfide exchange reactions under heat-induced denaturing conditions from 55 to 90 degrees C. The results showed that treatment of lysozyme with Cys and GSH resulted in the introduction of new half-cystine residues (2-3 residues/mol of protein). The released SH groups, in turn, rendered the lysozyme molecule more flexible, being accompanied by a dramatic increase in the surface hydrophobicity and exposure of tryptophan residues. As a consequence, the resulting reduced lysozymes were more capable of binding to lipopolysaccharides (LPS) and permeabilizing the bacterial outer membrane, as evidenced by the liposome leakage experiment, than were native or heated lysozyme. Both reduced lysozymes displayed significantly higher antimicrobial activity than native or heated lysozyme against Salmonella enteritidis (SE) in sodium phosphate buffer (10 mM, pH 7.2) at 30 degrees C for 1 h. Their minimal inhibitory concentrations (MICs) against the tested bacteria were about 150- and 25-fold lower than their respective MICs of native or heated lysozyme. The results suggest that partially reduced lysozyme could be used as a potential antimicrobial agent for prevention of SE attack.  相似文献   

13.
The aim of this work is to evaluate the impact of sulfhydryl groups on ovalbumin aggregation and gelation. Ovalbumin was chemically modified to add sulfhydryl groups in various degrees. The rate of aggregation was not affected by the introduction of sulfhydryl groups, and disulfide bond formation was preceded by physical interactions. Hence, disulfide interactions may not be the driving force for the aggregation of ovalbumin. Investigation of the aggregates and gels by electron microscopy and rheology suggested that a critical number of sulfhydryl groups can be introduced beyond which the microstructure of the aggregates transforms from fibrillar into amorphous. Rheological studies further suggested that covalent networks, once formed, do not have the possibility to rearrange, reducing the possibility to attain a stronger network. These results show that, even though aggregation of ovalbumin may be primarily driven by physical interactions, formed disulfide bonds are important to determine the resulting aggregate morphology and rheological properties.  相似文献   

14.
Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.  相似文献   

15.
In order to study the functional properties of glutenin subunits added to a dough, they must be incorporated into the glutenin polymer. This requires partial reduction to open up the polymer, followed by oxidation to incorporate the added monomer into the polymer. Existing methods for incorporating glutenin subunits were suitable only for studies on mixing properties and needed to be modified for use in studies on extension and baking. A range of concentrations and of reaction times was therefore tested for both the reductant and the oxidant. In addition, mixing time as well as relaxation time before extension were varied. Extension curves and loaf heights were used to evaluate the treatments. Optimum conditions were developed that provided extension curves of normal dimensions but with altered shape. The conditions were reduction with 0.2 mg/mL of dithiothreitol (DTT) solution for 1 min followed by oxidation with 5 mg/mL of KIO3 solution, then mixing the dough to 70% of the peak dough development time. For microbaking, the conditions of 2 mg/mL of DTT for 1 min, 2.5 mg/mL of KIO3 for 5 min, and mixing the dough to peak development time allowed loaf height to be retained. The size distribution of the glutenin polymer was analyzed using size‐exclusion HPLC and field‐flow fractionation methods. This showed that the monomers were incorporated into the polymer and that polymer size was restored to control levels following reduction and oxidation.  相似文献   

16.
Electron paramagnetic resonance (EPR) spectroscopy was used to study free radical production in hard red wheat flours extruded according to a two-level fractional factorial experimental design (11 and 14% protein content, 160 and 185°C, 16 and 20% moisture, 300 and 500 rpm screw speed, and mass flow rate of 225 and 400 g/min). All spectra showed dominant broad singlets (g = 2.0053–2.0059) from nitrogen-centered radicals originating from heat-induced peptide scission and reactions of lipid radicals with side-chain amino groups. At 77 K, sulfur-oxyl or peroxyl radicals (g = 2.008–2.018), thiyl radicals (g = 2.025), and disulfide radical species (g = 2.032–2.035 and 2.05–2.06), resulting from intra- and intermolecular electron migration and shear-induced scission of disulfides, sometimes were present. The strongest EPR signals occurred under conditions of maximum free radical production and minimum opportunity for radical recombination: high protein flour (14%), high die temperature (180°C), and low moisture (16%). EPR signals correlated with sulfhydryl and disulfide (SH-SS) levels and physical properties of extrudates, indicating that free radicals are integrally involved in molecular changes that occur during extrusion.  相似文献   

17.
Feather keratins were extracted from chicken feathers with an aqueous solution of urea and 2-mercaptoethanol. The keratin solution obtained was dialyzed to remove the reagents. Upon dialysis, extensive protein aggregation occurred. To obtain stable solutions or dispersions in water, cysteine residues were modified prior to dialysis with iodoacetamide, iodoacetic acid, or bromosuccinic acid, thereby blocking free thiol groups and introducing hydrophilic groups. For the development of biodegradable materials with good mechanical properties from these biopolymers, disulfide bonds between the keratin molecules are needed. Therefore, cysteine residues were only partially modified by using different reagent/cysteine molar ratios. The reaction rate constants of iodoacetate with glutathione and 2-mercaptoethanol were successfully used to predict the degree of modification of keratin cysteine. It was shown that, for carboxymethylated keratin, fewer aggregates were formed for higher degrees of cysteine modification, while more protein was present as oligomers. Aggregates and oligomers were stabilized through intermolecular disulfide bonds.  相似文献   

18.
In plant-based food, phenolic compounds usually do not exist in their native form, but as esters, glycosides, or polymers. The native forms, however, require deglycosylation for their intestinal absorption, and aglycone has been considered to be the potential health-protecting/promoting form. The binding of the aglycones of phenolic compounds to bovine and reindeer beta-lactoglobulins (betaLG) using fluorescence quenching was studied. The effects of pH and storage were also studied. Of the compounds investigated, the majority of flavones, flavonols, flavanones, and isoflavones were bound to betaLG. In the pH studies, no significant effects were found. The fact that the phenolic compounds were not released at pH 2 might indicate that they bind to an external part rather than to the central cavity. Studies implicated that betaLG could act as a binder or carrier for phenolic compounds in acidic, basic, or neutral conditions and that the ligand/betaLG complex can remain stable during storage.  相似文献   

19.
为研究贮藏过程中蛋白质氧化对蛋白质分子间作用力的影响机理,通过建立羟基自由基(·OH)氧化体系体外模拟乌贼肉在冻藏过程中蛋白质氧化的过程,从而探究蛋白质分子间作用力与结构的变化情况。结果表明,随着·OH氧化体系中H2O2浓度的增加,乌贼肌原纤维蛋白分子间作用力平衡被打破,离子键和氢键含量逐渐降低,疏水作用力逐渐增强,二硫键和非二硫共价键含量逐渐增加;肌原纤维蛋白表面疏水性逐渐增加;巯基与活性巯基含量逐渐降低。应用红外光谱(FTIR)分析氧化过程中蛋白质二级结构的变化规律,结果表明,自由基对氨基酸侧链和蛋白肽链进行了攻击,随着H2O2浓度的增加,光谱带向不同波数方向有规律地移动,蛋白质二级结构发生变化,α-螺旋和β-折叠含量降低,β-转角和无规则卷曲含量增加。石蜡切片显示,随着H2O2浓度的增加,肌纤维蛋白结构趋于疏松、间隙持续增大、肌丝变细且断裂卷曲量增多。本研究为探究乌贼肉蛋白质氧化的机理提供了理论依据,为延长乌贼贮藏期、提高经济效益与食用品质等相关研究奠定了理论基础。  相似文献   

20.
Free sulfhydryl groups in sulfur compounds have been reported to act directly on natural toxins to reduce toxicity. The objective of this study was to reduce protease inhibitors and glycoalkaloids in simulated snack foods by the addition of sulfur-containing compounds prior to extrusion. Thiamine, methionine, and benzyl disulfide were added to potato flakes at levels of 0.5% or 1.0% prior to twin-screw extrusion. Total and free thiols and protease inhibitors were monitored before and after extrusion by colorimetric assays. Potato glycoalkaloids were analyzed by HPLC and by immunoassay. Extrusion reduced potato flake disulfide bonds; disulfide bonds were higher in samples containing added sulfur compounds. Trypsin inhibitor activity was reduced by as much as 79% by extrusion plus methionine. Extrusion significantly reduced carboxypeptidase inhibitor, but only when benzyl disulfide and 0.5% methionine were not added. One percent methionine and thiamine resulted in 60% reductions in glycoalkaloids.  相似文献   

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