Amount Of Free Sulfhydryl Groups Research Articles

Interfacial properties of protein nanofibrils with different morphology prepared using aqueous solvent with ethanol: Part I. preparation and characterization

β-lactoglobulin can self-assemble into nano-fibrils with high specific surface area and hydrophobic groups, resulting in excellent interfacial activity. In this paper, β-lactoglobulin fibrils with conformation from semi-flexible to worm-like were prepared by controlling the polarity of the self-assemble precursor solution. The effects of ethanol on the surface properties, microscopic interaction forces and secondary structure of the proteins in the precursor solution, and the morphology, secondary structure, mechanical properties, and surface properties of the formed fibrils were investigated, using techniques including atomic force microscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy and molecular dynamic simulation, etc. It was found that with the increase of ethanol concentration in the aqueous solvent, both the fibril length and diameter became smaller, the content of β-sheet in the fibrils gradually decreased whereas the content of random curl increased, resulting in decrease of the modulus of the fibrils (5.47 ± 0.54 GPa for semi-flexible fibrils and 0.52 ± 0.20 GPa for worm-like fibrils). With increasing ethanol concentration, the polarity of the precursor solution decreased, and both hydrophobic interactions and disulfide bonds of the proteins decreased whereas the electrostatic interactions with solvent were enhanced, which resulted in the increase of particle size of the precursor proteins and thus facilitated the final formation of fibrils with worm-like morphology. However the fibrillation process mainly occurs in the heating stage but not in the precursor solution. The surface hydrophobicity of the formed fibrils increased, whereas the amount of free sulfhydryl groups and surface charge decreased slightly with the increase of ethanol concentration. Their effects on the interfacial functional properties will be investigated further.

Effect of high intensity ultrasound on structure and foaming properties of pea protein isolate

The effects of high intensity ultrasound (HIUS, 20 kHz, at varying amplitude 30%, 60%, 90% for 30 min) on structure and foaming properties of pea protein isolate (PPI) were investigated. No significant change was observed from the electrophoresis profiles and circular dichroism (CD) spectrum. Analyses of fluorescence spectroscopy and the amount of free sulfhydryl groups showed that HIUS induced protein molecular partial unfolding. Furthermore, HIUS decreased particle size of PPI and increased exposed hydrophobicity, resulting in a reduction of the surface tension at the air−water interface. Therefore, the foaming ability of PPI increased from 145.6% to 200.0%. The foaming stability increased from 58.0% to 73.3% with the increasing amplitude after 10 min though all reduced to 50.0% with the extension of time. That suggested that HIUS treatment has a potential to be implemented to modify foaming properties of PPI.

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

BackgroundNitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions. MethodsBy means of UV–VIS and fluorescence spectroscopy methods we compared nitric oxide mediated changes of enzyme activities, amount of free sulfhydryl groups (-SH) and bis-ANS probe binding. Finally, we predicted potential cysteine residues modified by nitric oxide in studied proteins using GPS-SNO software. ResultsOur results indicated that nitric oxide reversibly inactivates GAPDH but does not affect the activity of LDH. Nitric oxide dependent GAPDH activity decline was accompanied by the reduction of the amount of free –SH groups and GAPDH-bound bis-ANS fluorescence. Reduction of the number of free –SH groups and protein-bound bis-ANS fluorescence was also observed in LDH treated with NO. Applied antioxidants increased inactivation of GAPDH and structural changes of GAPDH and LDH. ConclusionsNitric oxide modifies function and structure of thiol-dependent enzyme such as GAPDH and structure of LDH which function do not rely on cysteine thiols. Both resveratrol and melatonin exerted prooxidative properties in studied conditions. General significanceExtensively studied antioxidants: resveratrol and melatonin may function as a prooxidative species under in vitro nitrosative stress conditions.

Effect of pulsed electric field on functional and structural properties of canola protein by pretreating seeds to elevate oil yield

Pulsed electric field (PEF) was applied to moisture-preconditioned canola seeds to evaluate the effect on oil yield. The effect of PEF on functional and structural properties of canola protein left over after oil extraction were also examined in albumin and globulin fractions obtained at optimal conditions for oil yield. Generally, PEF pretreatment significantly improved (P < 0.01) the following functional properties of canola protein and its fractions: solubility, water-holding capacity, emulsibility, emulsion stability, oil-holding capacity, foamability, and foam stability. Infrared spectrometry revealed that the secondary structure of the protein changed upon PEF, as shown by changes in the proportions of α-helices, β-sheets, and β-turns in the amide I region. An increased amount of free sulfhydryl groups and surface hydrophobicity suggested alterations in tertiary structure as well. PEF caused the formation of new protein aggregates with low molecular mass, demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and changes in the number of total sulfhydryl groups.

Evaluation of physicochemical properties of gluten modified by Glucose oxidase and Xylanase

The effect of Glucose oxidase (GOX) and Xylanase (XYL) on gluten-starch separation and physicochemical properties of gluten was studied. The gluten yield and Rmax increased and the extensibility of gluten and the amount of free sulfhydryl groups (SHf) decreased in comparison to the control with the addition of GOX up to 80 ppm. These observations indicated the high strength of gluten network by 80 ppm of enzyme. GOX caused a shift to larger particle size distribution of glutenin macropolymer (GMP) gel. Here, GMP gel at low enzyme level was more elastic and stiffer. Generally, an improvement of GMP gel quality can be obtained by the addition of GOX, although inverse effects were obtained when excessive enzyme levels were added. The results indicated that XYL leads to increase the tendency to aggregate by degradation of pentosane and decreasing the viscosity. The enzyme led to decrease the particle size distribution in GMP gel. In these samples, a tendency to increase the amount of SHf content was observed and it seems that the incorporation of pentosan and protein prevented the disulfide crosslinking. GMP gel separated with XYL had higher delta value and lower storage modulus in comparison to GMP gel separated without XYL.

High intensity ultrasound modified ovalbumin: Structure, interface and gelation properties

Influence of high intensity ultrasound (HIUS) on the structure and properties of ovalbumin (OVA) were investigated. It was found that the subunits and secondary structure of OVA did not change significantly with HIUS treatment from the electrophoretic patterns and circular dichroism (CD) spectrum. The amount of free sulfhydryl groups increased and intrinsic fluorescence spectra analysis indicated changes in the tertiary structure and partial unfold of OVA after sonication increased. Compared with the untreated OVA, HIUS treatment increased the emulsifying activity and foaming ability, and decreased interface tension (oil–water and air–water interface), which due to the increased surface hydrophobicity and decreased the surface net charge in OVA, while the emulsifying and foaming stability had no remarkable differences. The increased particle size may be attributed to formation of protein aggregates. Moreover, the gelation temperatures of HIUS-treated samples were higher than the untreated OVA according to the temperature sweep model rheology, and this effect was consistent with the increased in surface hydrophobicity for ultrasound treated OVA. These changes in functional properties of OVA would promote its application in food industry.

Understanding the differences in gelling properties between lupin protein isolate and soy protein isolate

The gelling properties of lupin protein isolate (LPI) were compared with those of soy protein isolate (SPI). It was found that LPI behaves fundamentally different than SPI, evidenced by the formation of weaker and deformable gels. Further investigation shows that both protein isolates can be considered particle gels and that LPI particles do not swell as much as SPI particles inside the network. Besides, heating hardly affects LPI particles while SPI particles show additional swelling. To explain the differences, the sulfhydryl reactivity of LPI was tested. The amount of free sulfhydryl groups on LPI was higher than the amount of free sulfhydryl groups on SPI. Upon heating the amount of free sulfhydryl groups on LPI increases. We hypothesize that the compact, heat stable structure of the protein particles suppresses the intermolecular bonding through disulphide bridge formation and favours intramolecular crosslinking. The small sulphur-rich proteins that are not incorporated within the particles but are present in the surrounding solution cannot strengthen the particle network, due to their low concentration. LPI did not form gels of similar consistency as SPI and may therefore be less useful for solid food products. The thermal stability of LPI could offer opportunities for high-protein foods that require low viscosity after heating.

Structural and functional changes in ultrasonicated bovine serum albumin solutions

Effects of high-intensity ultrasonication on functional and structural properties of aqueous bovine serum albumin (BSA) solutions were investigated. The functional properties of BSA were altered by ultrasonication. Surface activity of BSA increased. Minimal changes were observed in the global structure of BSA but surface charge increased particularly at basic pH values (e.g. pH > 9). While dynamic light scattering measurements indicated that the particle size increased up to 3.4 times after 90 min of sonication, no significant increase in the oligomeric state of BSA using blue native PAGE was observed. The amount of free sulfhydryl groups in BSA after 90 min of sonication decreased. The increased particle size and decreased number of free sylfhydryl groups may be attributed to formation of protein aggregates. Surface hydrophobicity increased and circular dichroism spectroscopy and FTIR analysis indicated changes in the secondary structure of BSA. We hypothesize that mechanical, thermal and chemical effects of ultrasonication resulted in structural changes in BSA that altered the functional properties of the macromolecule which may be attributed to the formation of an ultrasonically induced state that differs from a thermally, mechanically or solvent induced state.

Generation of oligomeric insulin receptor forms by intramolecular sulfhydryl-disulfide exchange. Involvement of masked sulfhydryl groups.

Insulin receptors from rat liver membranes were labelled with a 125I-labelled photoreactive insulin analogue or by iodination using lactoperoxidase and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under nonreducing conditions different receptor forms with Mr 400,000 (alpha 2 beta 2), 360,000 (alpha 2 beta beta'), 330,000 (alpha 2 beta' beta'), 320,000 (alpha 2 beta), 280,000 (alpha 2 beta'), 240,000 (alpha 2), 210,000 (alpha beta), 165,000 (alpha beta') and 115,000 (alpha) were detected. The subunit composition of these receptor forms was determined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence and presence of dithioerythritol. During denaturation in sodium dodecyl sulfate in the absence of reductants, the Mr 400,000 receptor form (alpha 2 beta 2) was converted into the Mr 320,000 (alpha 2 beta) and Mr 240,000 (alpha 2) receptor form. This conversion was prevented either by N-ethylmaleimide, oxidants, or low pH. In contrast, alkylation of the receptor with N-ethylmaleimide under non-denaturing conditions did not prevent the appearance of intermediate-sized receptor forms. Furthermore, the inhibition of receptor cleavage by N-ethylmaleimide during denaturation was also observed when the amount of free sulfhydryl groups was reconstituted by the addition of an unlabelled and non-alkylated receptor sample to the alkylated and photoaffinity-labelled receptor. These results suggest, that the generation of different oligomeric receptor forms detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis is due at least in part to the cleavage of one or both beta-subunits from the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Omeprazole, a specific inhibitor of gastric (H+-K+)-ATPase, is a H+-activated oxidizing agent of sulfhydryl groups.

Omeprazole (5-methoxy-2-[[(4-methoxy-3,5- dimethylpyridinyl)methyl]sulfinyl]-1H-benzimidazole) appeared to inhibit gastric (H+-K+)-ATPase by oxidizing its essential sulfhydryl groups, since the gastric ATPase inactivated by the drug in vivo or in vitro recovered its K+-dependent ATP hydrolyzing activity upon incubation with mercaptoethanol. Biological reducing agents like cysteine or glutathione, however, were unable to reverse the inhibitory effect of omeprazole. Moreover, acidic environments enhanced the potency of omeprazole. For example, in vivo pretreatment of rats with carbachol, a secretagogue, enhanced the activity of omeprazole to inhibit gastric (H+-K+)-ATPase, while pretreatment with cimetidine, an antisecretory agent, reduced its potency. In vitro, lowering pH of incubation media from 7.4 to 5.0 improved the ability of omeprazole to inhibit hog gastric (H+-K+)-ATPase almost 60-fold. The inhibitory effect of the drug was accompanied by a dose-dependently decreased amount of free sulfhydryl groups in the isolated hog gastric membranes. The chemical reactivity of omeprazole with mercaptans is also consistent with the biological action of omeprazole. The drug, only under acidic conditions, reacted with a stoichiometric amount of ethyl mercaptan (or beta-mercaptoethanol) to produce regio-isomers of N-sulfenylated omeprazole sulfide (5-methoxy-2[[(4-methoxy-3,5- dimethyl-2-pyridinyl)methyl]thio]-1- or 3-(ethylthio)benzimidazole). The N-sulfenylated compound reacted at neutral pH with another stoichiometric amount of ethyl mercaptan to produce omeprazole sulfide quantitatively. The gastric polypeptides of 100 kilodaltons representing (H+-K+)-ATPase in the rat gastric mucosa or isolated hog gastric membranes were covalently labeled with [14C]omeprazole. The radioactive label bound to the ATPase, however, could not be displaced by mercaptoethanol under the identical conditions where the ATPase activity was fully restored. These observations suggest that the essential sulfhydryl groups which reacted with omeprazole did not form a stable covalent bond with the drug, but rather that they further reacted with adjacent sulfhydryl groups to form disulfides which could be reduced by mercaptoethanol.

Thermal coagulation of bovine serum albumin.

The effects of salts were investigated on thermo-coagulation of bovine serum albumin (BSA). Salts affected the formation of coagulum, and a “minimum” (a col) on the turbidity-pH curve was observed at about pH 7.5. When the time course of turbidity was measured at more acidic pH (6.5) or alkaline pH (9.5), an induction period was observed at the more alkaline pH (9.5). The turbidity- pH curve for carboxyamidomethylated BSA was similar to that of original BSA. When salts were added to reduced, carboxyamidomethylated BSA solution, no “minimum” appeared on the turbidity-pH curve. The amount of free sulfhydryl groups, either in the presence or absence of salts, increased with heating time at more alkaline pH (pH 9.5). Intermolecular protein interactions occurred easily when salts were present. The endothermic peak on differential scanning calorimetry thermograms did not appear when salts were absent, but appeared when they were present.

Tubulin sulfhydryl groups and polymerization in vitro: Effects of di- and trivalent cations

The cations, Hg 2+, Cu 2+, Cd 2+, Ni 2+, Mn 2+, Co 2+, Zn 2+, Cr 3+ and Al 3+ have been investigated with respect to their effect on the polymerization and on the amount of free sulfhydryl groups of a brain tubulin preparation. Tubulin polymerization was completely inhibited by Hg 2+, Cu 2+, Cd 2+ and Cr 3+ at 10 −3 M but only slightly by Ni 2+ and Al 3+. In the presence of 10 −4 M Ni 2+ a stimulatory effect was observed. Co 2+ at 10 −3 M caused formation of sheet polymers of tubulin. The sheets were stable against colchicine and Ca 2+ depolymerization. Mn 2+ did not affect the polymerization. The amount of free sulfhydryl groups was markedly decreased by Hg 2+, Cu 2+ and Cd 2+ at 10 −3 M but less by Co 2+, Mn 2+ and Zn 2+. In the presence of Ni 2+, Cr 3+ and Al 3+ at the same concentration no effects were observed. The results indicate that some but not all free sulfhydryl groups of tubulin are directly involved in the polymerization process, and also that other metal binding groups could be of importance.

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.

Effects of Heat on Sulfhydryl and Disulfide Groups of Milk Proteins as Measured by the Spectrofluorometric Method

A spectrofluorometric method for the determination of sulfhydryl and disulfide groups in proteins, adapted by our laboratory, has been applied to determine the effects of heat on these groups in milk proteins.These studies revealed that no measurable free sulfhydryl groups are present in native caseins and only limited amounts are generated by high heat treatment. Serum proteins in native form contain a significant amount of free sulfhydryl groups which increased by heat-denaturation at the expense of disulfide in closed systems. In open systems sulfhydryl and disulfide decreased due to volatilization. Heating skimmilk revealed a behavior parallel to serum protein systems.