Number Of Sulfhydryl Groups Research Articles

The effect of subzero temperatures on the properties and structure of soy protein isolate emulsions

Different freezing temperatures (−5, −20, −40 and −80 ℃) could change soy protein isolate (SPI) structure and emulsion properties. After freezing at −5 ℃ and −20 ℃, the structure of the SPI loosened, the fluorescence intensity was red shifted, and the proportion of Phe, Tyr and Trp exposed increased. With decreasing temperature, the surface hydrophobicity (H0 × 100), the number of sulfhydryl groups and the number of disulfide bonds all rose, then fell (−40 ℃), and rose again (−80 ℃). The β-sheet content in the protein secondary structure increased from 32.71% (control) to 50.66% (−40 ℃) and then decreased to 37.05% (−80 ℃), while the β-turn and random coil contents showed the opposite pattern, which also confirmed aggregation. The emulsification performance of SPI after freezing treatment was decreased. The results of this study provide theoretical support for future production of frozen foods with added SPI.

Exploring in vitro gastrointestinal digestion of myofibrillar proteins at different heating temperatures

The effects of different heating temperatures (40–115 °C) on the structure, oxidation, and digestibility of beef myofibrillar protein were investigated. Reductions in the number of sulfhydryl groups were observed, together with gradual increases in the number of carbonyl groups, indicating oxidation of the protein by the increased temperatures. At temperatures between 40 °C and 85 °C, β-sheets were converted to α-helices, and increased surface hydrophobicity showed that the protein expanded as the temperature approached 85 °C. These changes were reversed at temperatures over 85 °C, indicative of aggregation induced by thermal oxidation. Between 40 °C and 85 °C, the digestibility of the myofibrillar protein was increased, reaching a maximum of 59.5 % at 85 °C, after which it began to decrease. These results indicated that moderate heating and oxidation-induced protein expansion were beneficial to digestion while protein aggregation resulting from excessive heating is not conducive to digestion.

The Influence of Lactic Acid Bacteria Fermentation on the Bioactivity of Crayfish (Faxonius limosus) Meat

In recent years, new raw materials have been sought for use in processing. This category certainly includes invasive crayfish Faxonius limosus. One of the problems associated with their use is their short microbiological shelf life. Therefore, in the research presented here, an attempt was made to ferment crayfish meat with strains of Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, Lactobacillus casei, and yogurt culture. The analyses included an evaluation of changes in the microbial quality of the material, the content of free amino acids, reducing sugars, ascorbic acid, and the antioxidant properties of the fermented meat. Changes in the canthaxanthin content and the number of sulfhydryl groups and disulfide bridges were also evaluated. The study showed that carrying out lactic fermentation resulted in a decrease in meat pH (8.00 to 7.35–6.94, depending on the starter culture). Moreover, the meat was characterized by an increase in FRAP (2.99 to 3.60–4.06 mg AAE/g), ABTS (2.15 to 2.85–3.50 μmol Trolox/g), and reducing power (5.53 to 6.28–14.25 μmol Trolox/g). In addition, the study showed a favorable effect of fermentation on the content of sulfhydryl groups in the meat as well as for ascorbic acid content. The results obtained can serve as a starting point for the further development of fermented products based on crayfish meat.

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry ofchromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Protein aggregation and Ca2+-induced gelation of soymilk after heat treatment under slightly alkaline conditions

Alkaline heat treatment of soymilk is widely used in the production of soy products. This study aimed to clarify the effect of the thermal aggregation state of proteins on the coagulation characteristics of soymilk. Soymilk with different pH levels (6.6–8.0) was preheated, and the gelation processes induced by CaSO4·2H2O and the gel properties were investigated. Results showed that the aggregation and binding pattern of proteins changed after slightly alkaline heat treatment. The ability of 7S and the acidic (A) subunit of 11S to bind to the basic (B) subunit of 11S weakened. These subunits converted into non-particulate proteins, which decreased particulate protein content and particle size. The number of sulfhydryl groups on the protein surface reduced by 45%–62%, and more disulfide bonds formed in the protein subunits during recombination. During gelation, the soymilk after alkaline heat treatment exhibited a slow intermolecular binding in the “pre-aggregation” stage, which led to late gelation onset and a uniform gel network. With the increase in non-gelable protein content in the gel, the water holding capacity (WHC) enhanced and but the hardness decreased. When the pH was further increased (pH ≥ 7.8) to the isoelectric point of the B subunit, the B subunit excessively aggregated and formed large and dense aggregates, which increased gel hardness and produced a rough and uneven network.

Inhibitory effect of microwave heating on cathepsin l-induced degradation of myofibrillar protein gel

This work was aimed to compare the effect of microwave (MW) heating on the cathepsin L (Cat L)-induced degradation of myofibrillar protein (MP) gels with that of water bath (WB) heating. First, Cat L from silver carp was purified and determined to be 45 kDa. The gel strength of the MW-heated MP gels were significantly higher than those of the WB-heated when Cat L was added (P < 0.05). The gel electrophoresis pattern and scanning electron microscopy analysis indicated that MW heating inhibited the Cat l-induced hydrolysis of MP gels. In addition, the number of sulfhydryl groups and surface hydrophobicity of MW-heated gels were lower than those of WB-heated gels when Cat L was added. These results indicated that MW heating could effectively weaken the degradation of Cat L on MP gels by manipulating disulfide bonds and hydrophobic amino acids, resulting in good gel properties and a compact protein network.

The Effect of Chromium and Boron on the Lipid Peroxidation and Antioxidant Status (in Experiment)

Current anthropogenic factors have a destructive effect on the environment. In this connection, it becomes necessary to study the negative effects of substances on living organisms and ways to reduce them. This article evaluated the impact of potassium dichromate (K2Cr2O7) and boric acid (H3BO3) on the status of lipid peroxidation (LPO) and antioxidant system. The work is performed on 24 male rats of Wistar weighing 180-220 g, were grown in the vivarium of the Central research laboratory of West Kazakhstan state medical University named after Ospanov. An experiment on Wistar rats: 1st - warning; 2nd - received K2Cr2O7; 3rd – H3BO3; 4th – K2Cr2O7 and H3BO. The introduction of K2Cr2O7 intensified LPO and reduced activity of catalase, glutathione peroxidase and superoxide dismutase, the number of sulfhydryl groups. The results of the study allow asserting that the H3BO3 in the conditions of joint use with K2Cr2O7 has an antioxidant effect, which leads to inhibition of LPO and activation of antioxidant system. Therefore, we first established the antioxidant effect of boric acid in the combined effects of potassium dichromate and boric acid.

Effect of Glycosylation on Gelling and Physicochemical Properties of Egg White Powder

Molecular characteristics of glycosylated egg white proteins(GEWP),including gelling properties,the number of sulfhydryl groups,hydrophobicity,molecular flexibility and Zeta potential were investigated.The results showed that gel strength and water holding capacity of glycosylated egg white proteins increased by 112.51% and 18.89%,respectively,when compared to control.After glycosylation,the protein molecules were partially unfolded,thus exposing the internal hydrophobic groups.At the same time,inter-and intra-protein disulfide bonds were formed,resulting in decreased total sulfhydryl content.In addition,glycosylation decreased the number of free e-NH2 on the surface of egg white protein molecules,reducing the positive charges,and thus,the isoelectric point of egg white proteins.

Complex Application of Preparation Containing Ultralow Doses of Antibodies for the Treatment of Anemia Caused by Pubertal Uterine Bleedings

Poetam and anaferon (pediatric formulation) administered in combination with iron preparation to patients with anemia induce pronounced positive shifts in metabolic and morphological status of mature erythrocytes: the number of sulfhydryl groups and lipoprotein complexes in cell membranes and dry weight of erythrocytes increased, while the number of forms at different stages of degeneration decreased.

Sensitive Detection of Sulfhydryl Groups in Surface-Confined Metallothioneins and Related Species via Ferrocene-Capped Gold Nanoparticle/Streptavidin Conjugates

Metallothionein (MT), a cysteine-rich metalloprotein that is purported to play an important role in heavy metal accumulation and detoxification, and its related peptidic species were attached onto dithiobissuccinimidyl propionate self-assembled monolayers. The spatially accessible sulfhydryl groups present in these immobilized biomolecules, tagged with N-biotinoyl-N'-[6-maleimidohexanoyl]hydrazide, were detected voltammetrically at a sensitive level via the use of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The method was established first by examining relatively simple peptides (e.g., glutathione). For the hexapeptidic species that resembles the N-terminus of MT with a sequence of Lys-Cys-Thr-Cys-Cys-Ala, concentration levels as low as 0.050 nM can be determined. Such a remarkable sensitivity is attributed to the presence of a large number of Fc caps present at each gold nanoparticle, which enhances the detection of a small number of surface-bound sulfhydryl groups. Microgravimetric measurements, performed with a quartz crystal microbalance, were used in tandem with voltammetry to quantify the number of tagged sulfhydryl groups. Through extraction of the metals present in MT adsorbate, it is demonstrated that this amplified voltammetric detection is also suitable for the investigation of the variation of the number of sulfhydryl groups present at an electrode and sensitive to the change of surface structure of an immobilized biomolecule. This work represents a new method for the determination of sulfhydryl groups inherent in surface-bound proteins or peptides and can facilitate the study on the environmental issues related to MTs.

Study of methyl bromide reactivity with human and mouse hemoglobin

A study has been carried out on in-vitro reactivity of human and mouse hemoglobin spectrophotometrically at physiological pH, using different protein to reagent ratios. Hemoglobin side chains were modified with different concentrations of methyl bromide on agro-soil fumigant. To ascertain if the site of alkylation was the reactive sulfhydryl group present at cystein – 93 on the b chain of hemoglobin (b-93 cys), a spectrophotometric measurement using 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was used to measure the free sulfhydryl groups before and after treatment of hemoglobin with various amounts of methyl bromide. The results show that the methylbromide reacted substantially with both human and mouse hemoglobin at b-93 cys. The decrease in the number of sulfhydryl groups (SH) per hemoglobin molecule on addition of various concentrations of methylbromide ranges from 5.10 to 2.35 + 0.01 and 5.01 to 0.93 + 0.01 for human and mouse hemoglobin, respectively. The results showed a dose-dependent decrease in the number of sulfhydryl group indicating that hemoglobin can serve as a biomarker of human occupational exposure to methy bromide fumigants. Keywords: Hemoglobin, sulfhydryl groups, methylbromide, Cysteine, histidine. (Global Journal of Pure and Applied Sciences: 2002 9(1): 91-100)

Effect of gamma irradiation on the microbiological quality and on the functional properties of proteins in dry red kidney beans (Phaseolus vulgaris)

Abstract Gamma-irradiation was found to affect the physicochemical properties of dry red kidney beans. The highest dose used (8 kGy) significantly ( P ⩽0.05) modified the extent of deamidation, the number of sulfhydryl groups, as well as the solubility and the hydrophobicity of the protein. Deamidation, protein solubility and hydrophobicity all increased with the irradiation dose while the number of sulfhydryl groups was reduced by the treatment. Furthermore, irradiation also affected the outgrowth of natural filamentous fungi contaminants present on the dry beans. A dose of 1.5 kGy reduced the number of filamentous fungi by 2 log cycles immediately after treatment. However, the highest dose used (3 kGy) did not eliminate the filamentous fungi completely. Moreover, the filamentous fungi population was a lot less diversified on the irradiated samples. Species of Aspergillus sp. and Penicillium sp. were more abundant on the unirradiated beans while the beans irradiated at 3 kGy contained were predominantly infected by species of Rhizopus sp. , Cladosporium sp. and Alternaria sp.

Physicochemical Properties of Dry Red Kidney Bean Proteins and Natural Microflora as Affected by Gamma Irradiation

ABSTRACTGammaγ‐irradiation affected (p≤0.05) the physicochemical properties of dry red kidney beans (Phaseolus vulgaris.) Protein deamidation, solubility and hydrophobicity increased and the number of sulfhydryl groups was reduced after irradiation. Irradiation also affected the outgrowth of natural mold contaminants. A dose of 1.5 kGy reduced the number of molds by 2 log cycles immediately after the treatment. However, incomplete inhibition was observed with the highest irradiation dose (3 kGy). Species of Aspergillus and Penicillium. were completely inhibited at 1.5 and 3 kGy respectively.

A computer program for quantification of SH groups generated after reduction of monoclonal antibodies

Reduction of disulfide bonds to sulfhydryl (SH) groups for direct radiolabeling of antibodies for immunoscintigraphic studies of colorectal and other cancers continues to be of considerable research interest. We have developed a general strategy and a versatile computer program for the quantification of the number of SH per molecule of antibody (Ab) generated after the treatment of monoclonal antibodies (MAbs) with reducing agents such as 2-mercaptoethanol (2-ME), stannous chloride (SnCl 2), dithiothreitol (DTT), dithioerythritol (DTE), ascorbic acid (AA), and the like. The program we describe here performs an unweighted least-squares regression analysis of the cysteine standard curve and interpolates the cysteine concentration of the samples. The number of SH groups per molecule of antibody in the 2-mercaptoethanol and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. The linear least-squares method fit the standard data with a high degree of accuracy and with the correlation coefficient r of 0.999. A program has been written for the IBM PC compatible computer utilizing a friendly menu to interact with the users. The package allows the user to change parameters of the assay, to calculate regression coefficients slope, intercept and its standard errors, to perform statistical analysis, together with detailed analysis of variance, and to produce an output of the results in a printed format.

Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies

A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (μg/mL). After subtraction of the back-ground due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 μg, the lowest detection limit for cysteine quantification was 0.03 μg. An excellent linear correlation was found between both cysteine concentration and absorbance ( r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.

Diversity of nuclear protein fractions of hamster liver and hepatoma produced by DNasel

The structural and functional diversity between active and inactive chromatin is thought to depend, is part, open differences in DNA-bound protein composition, including changes in the number of sulfhydryl groups. The aim of the present study was to compare protein composition in untreated nuclei, DNaseI-released and resistant nuclear fractions of hamster liver and Kirkman-Robbins hepatoma cells. Electrophoretic analysis of nuclear proteins showed some evident quantitative and qualitative differences between normal and neoplastic cells. The most significant diversities were noticed in DNaseI solubilized fraction of both types of cells. Nuclease attack released a characteristic set of non-histones with mol. wt 37,000, 50,000, 74,000 and 130,000–160,000 from transformed cells, and polypeptides of mol. wt 45,000 and 76,080 from normal cells. Cell-specific distribution within examined nuclear polypeptides was revealed using selective staining of their protein-boundsulfhydryls. Immuneblot analysis demonstrated that a no -bistoae protein with mol. wt 48,000, overexpressed in rodent tumour cps, was exclusively concentrated in liver DNaseI-sensitive fraction, which amounted only to 8.3% ± 2.0% of total nuclear DNA. In hepatoma cells, however, this particular polypeptlde is distributed between nuclease-Sensitive and nuclease-resistant nuclear fractions. Non-histone protein of mol. wt 48,000 appeared to contain free sulthydryl groups. In summary, these results show molecular specificity of nuclear proteins from normal and tumour cells and differences in their distribution among nuclease-released and nuclease-resistant nuclear fractions. The diversity in molecular characteristics and sulfhydryl group patterns observed among the examined proteins of normal and neoplastic cells may suggest their involvement she some changes in the rearrangement of nuclei during neoplastic transformation.

Isolation and characterization of soluble β-galactoside-binding lectins from mammalian liver

Soluble β-galactoside-binding lectins were isolated by chromatography on asialofetuin-Sepharose-4B column in 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 5 mM CaCl 2 and 1 mM 2-mercaptoethanol. The three lectins moved essentially as single polypeptide bands, of 18, 22 and 24 kDa, respectively, for sheep, goat and buffalo hepatic lectins. Sheep and goat lectins each contained 4 mol of hexose, whereas the hexose content of the buffalo lectin was 7 mol. The number of sulfhydryl groups in sheep, goat and buffalo lectins were determined to be 3.2, 4.3 and 4.8, respectively. The optical properties of the three lectins were similar to those of tryptophan-containing proteins. Lectin mediated hemagglutination of trypsinized rabbit erythrocytes was most effectively inhibited by lactose, followed by o-nitrophenyl β-galactopyranoside and galactose, but remained unaffected by glucose, mannose, fucose and fructose. Calcium ions substantially enhanced their hemagglutinating activity. Goat and buffalo lectins, but not sheep lectin, were also stimulated by Mg 2+, Mn 2+, Sr 2+ and Ni 2+ ions. The lectins lost activity after treatment with para-hydroxy-mercuribenzoate and N-ethylmaleimide. However, iodoacetamide treatment had no effect on the activity. The results show that the three lectins are different from the soluble β-galactoside-binding lectins studied thus far.

Purification and Characterization of Alpha-Macroglobulin and Ovo macroglobulin of the Green Turtle (Chelonia my das japonica)1

The plasma alpha-macroglobulin and egg white ovomacroglobulin were purified from the sea turtle, Chelonia mydas japonica, and their structural and functional properties were studied with the aim of clarifying the degree of evolutional divergence of two homologous proteins specific to different tissues of the same animal. The concentration of alpha-macroglobulin in green turtle plasma was about 4 mg/ml. The protein was purified from the plasma by precipitation with polyethylene glycol 6000, followed by zinc chelate chromatography and gel chromatography on Sepharose CL-6B. The concentration of ovomacroglobulin in green turtle egg white was about 0.4 mg/ml. Ovomacroglobulin was purified by gel chromatography on Sepharose CL-6B. The two proteins had similar molecular weights and amino acid compositions, and both inhibited proteinases such as trypsin, chymotrypsin, papain, and thermolysin. The amino terminal sequences of the two proteins were homologous to each other but higher homologies were found between the ovomacroglobulin of turtle and chicken, and between the serum macroglobulins of the same animals. The functional difference between turtle alpha-macroglobulin and ovomacroglobulin became clear when they were treated with methylamine, which is known to destroy the inhibitory activity of human alpha 2-macroglobulin by splitting internal thiolester bonds. The inhibitory activity of the turtle plasma protein was completely destroyed by methylamine but that of ovomacroglobulin was only partially affected. The number of sulfhydryl groups as titrated with 5,5'-dithiobis(2-nitrobenzoate) before and after treatment with proteinases or methylamine was different for the two proteins. The amount of radioactive methylamine that was incorporated was also different between the two proteins. The two proteins purified in this study had no immunological cross-reactivity.

Thiol-group modification of Torpedo californica acetylcholine receptor: subunit localization and effects on function.

The effects of thiol-group modifications on acetylcholine receptor (ACHR) function were measured with purified ACHR reconstituted into asolectin vesicles. N-Phenylmaleimide (NPM) was used to modify sulfhydryl groups on ACHR in the absence of any prior reduction of dithiothreitol, so that only the functional relevance of free sulfhydryls was examined. Modification by NPM led to the inhibition of ion-channel activity without a detectable effect on ligand binding. The ion flux inhibition by NPM primarily affected channel activation, since the initial rates of activation were decreased over a wide range of carbamylcholine concentrations. The [3H]NPM subunit labeling pattern of ACHR (a multisubunit membrane protein with alpha 2 beta gamma delta stoichiometry) revealed that there was preferential labeling of the gamma subunit. At high NPM concentrations, the number of sulfhydryl groups on the gamma subunit that could be modified with NPM was approximately two. Detergent was required during labeling for functionally relevant thiol-group modifications, and most of the label was protected from protease digestion in the reconstituted membranes. These results are consistent with the presence of the NPM modification in a bilayer and/or cytoplasmic domain.

Subunit distribution of the sulfhydryl groups of the F 1 adenosine triphosphatase of Salmonella typhimurium

The number of sulfhydryl groups in each subunit of the F 1 adenosine triphosphatase of Salmonella typhimurium was measured by the method of T. E. Creighton [1980, Nature (London) 284, 487–489]. The α, β, γ, δ, and ϵ subunits of this enzyme contained 4, 1, 2, 2, and 0 sulfhydryl groups per molecule of subunit, respectively.