Thiol-disulfide Interchange Research Articles

Purification of Prealbumin from Human and Canine Serum Using a Two-Step Affinity Chromatographic Procedure

1. A two-step-method is described by which human and canine prealbumin have been isolated from serum in good yield and high purity. The first step involves binding and release of prealbumin by a thiol-disulfide interchange reaction with thiol-Sepharose 4B. The second step consists of reversible binding of prealbumin to retinol-binding protein covalently linked to Sepharose 4B. 2. The canine prealbumin was immunochemically identified as an alpha2-globulin. Like human prealbumin it is a tetramer with some 10% smaller subunits than human prealbumin. Both proteins are devoid of carbohydrate and both bind thyroxin.

Rates of thiol-disulfide interchange reactions between mono- and dithiols and Ellman's reagent

The rate constants for thiol-disulfide interchange between 21 mono- and dithiols and Ellman's reagent correlate with the pKu's of the thiol groups with a Bronsted coefficient of p = 0.36. The maximum rates ofreduction are observed for thiols having pK, values close to the pH of the solution in which the reactions were carried out. In the dilute solutions examined (10-4-10-6 M in each reagent), the rate of the second, intramolecular interchange step in reactions of dithiols was faster than that of the first, intermolecular interchange, regardless of the size of the cyclic disulfide formed. A convenient synthesis of a mixture of diastereomers of 1,4-dimercapto-2,3-butanediol (i.e., of a mixture of dithiothreitol, DTT, and dithioerythritol, DTE) has been developed from 1,2:,4-diepoxybutane and thiolacetic acid.

Preparative isolation of Band 3, the predominant polypeptides of the human erythrocyte membrane

Band 3 proteins are the predominant polypeptide components of the human erythrocyte membrane and have been implicated in various transport activities. Following extraction of membrane ghosts with dimethylmaleic anhydride to remove two polypeptides (Bands 4.2 and 6) associated with Band 3, Band 3 proteins were solubilized along with the other major membrane glycoproteins (PAS 1–3) with Triton X-100 under nondenaturing conditions. Band 3 proteins were then purified (>95%) on a large scale by chromatography via thioldisulfide interchange on activated thiol-Sepharose 4B [agarose-(glutathione-2-pyridyl disulfide) conjugate]. This procedure allows the preparation of 20 to 25 mg of purified Band 3 proteins in high yield (>80%) from ghosts in a soluble form suitable for physical, chemical, and functional characterization.

Thiol-disulfide interchange in the binding of bence jones proteins to alpha-antitrypsin, prealbumin, and albumin.

Native light Ig chains of kappa- but not of lambda-type form -S-S linked complexes with prealbumin, alpha1-AT and albumin in vivo. kappa-chains isolated from urines have cysteinyls which are more promptly reacting with dithionitrobenzoate (DTNB) than lambda-chains. Both are monomerized on this reaction. On addition to plasma mixed disulfides between both types of light chains and DTNB form larger amounts of complexes than the native chains. The lower reactivity of native lambda-chains to the plasma proteins can be explained by their higher dimer stability. From the light chain reactions obtained with isolated alpha1-AT and albumin it is concluded that alpha1-AT has a disulfide which efficiently interchanges with monomeric, light chain thiolate ions released from thionitrobenzoate derivates of light chains and that on interchange with the derivatized light chains albumin releases more free light chains into the solution than are bound to albumin. Addition of derivatized light chains to a mixture of alpha1-AT and albumin increases the yield of alpha1-AT complexes and decreases the amount of albumin complexes formed. The relative amount of the different complexes formed in the latter experiments corresponds to the findings in vivo in patients with Bence Jones proteinemia. Prealbumin and alpha1-AT in plasma have a roughly 10-fold stronger tendency to link the light chains than albumin. The complexes are formed through thiol-disulfide interchange though neither the disulfide of native alpha1-AT nor the thiols of prealbumin is available for reaction with DTNB. The three plasma proteins may together constitute a system for linkage and transport of peptides with reactive thiols or disulfides released into the extracellular fluids. The trypsin and elastase binding and inhibiting capacity of alpha1-AT remains after cleavage of the internal -S-S-bridge of alpha1-AT through interchange with a light chain thiol for which reason an intact internal -S-S-bridge of alpha1-AT is not necessary for inhibition and linkdage of the enzymes.

Disulfide reduction and sulfhydryl oxidation by microbial enzyme. III. Purification of thiol-disulfide interchange enzyme from Candida claussenii.

Thiol-disulfide interchange enzyme which catalyzes the thiol-disulfide interchange was purified from cell-free extracts of Candida claussenii by acid treatment, ammonium sulfate fractionation, aqueous polymer two phase method (Dextran-PEG system), CM-Sephadex column chromatography, Sephadex G–100 and Sephadex G–200 gel filtrations. More than four active fractions were obtained on CM-Sephadex column. Further purification steps from one of these fractions resulted in two purified enzyme preparations D–l–1 and D–2 of which the increase in specific activities was 8150- and 8450-folds respectively, over the crude extract. Both purified enzymes were homogeneous in ultracentrifugal analysis.

Disulfide reduction and sulfhydryl oxidation by microbial enzyme. II. Screening for the producer of thiol-disulfide interchange enzyme in yeasts and culture conditions for the enzyme from Candida claussenii.

Screening was carried out to obtain microorganisms which produced the enzyme to reduce the disulfide bond, from our type cultures of yeast. Among many strains of yeast showing activity to reduce the disulfide bond, Candida claussenii, Candida brumptii and Candida rugosa were selected to have the highest activity. The enzyme activity was detected in the cell free extracts, but not in culture broth.The highest enzyme formation occured during the exponential growth phase, and rapid decrease of activity was observed in the stationary phase. Pantothenate and boron ion contributed to enzyme formation, and biotin and zinc ion to growth. The maximum enzyme activity was obtained in the following synthetic medium: 10% sucrose, 0.3% (NH4)2SO4, 0.5% KH2PO4, 0.15% MgCl2·6HO2 0.05% CaCl2, 0.015% MnCl2, 0.001% pantothenate, 0.0001% biotin, 0.0001% H3BO3, 0.00004% FeCl3·6H2O and 0.00008% ZnCl2. In addition, 30°C of the cultural temperature and vigorous aeration showed good results for enzyme formation.

An in Vitro System for Studying the Kinetics of Interchain Disulfide Bond Formation in Immunoglobulin G

Abstract An in vitro system for studying the order and kinetics of interchain disulfide bond formation (covalent assembly) in immunoglobulins has been developed using subclasses 1 and 4 of human immunoglobulin G. Noncovalent association of reduced, but not alkylated, heavy and light chains was achieved at pH 4.8 and air oxidation initiated by rapid transfer to aerated buffer at pH 8.2. Disulfide-bonded species in samples taken at various times were detected, following alkylation, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The principal intermediates in the covalent assembly of immunoglobulin G1, with either kappa or lambda light chains, were half-molecules (HL) and H2L. The half-time for the appearance of the completed molecule, H2L2, ranged from 70 to 95 min at 25°. Half-molecules were the predominant intermediate in the assembly of immunoglobulin G4. The disulfide bonds between the heavy chains formed slowly; H2L2 appeared with a half-time of 16 hours. The effects of temperature, pH, cupric ion concentration, and reducing agent on the air oxidation of reduced immunoglobulin were evaluated. Although all these parameters influenced the rate of H2L2 production, the order of disulfide bond formation was unaffected except in the presence of high concentrations of cupric ion. Covalent assembly utilizing a thiol-disulfide interchange system (oxidized and reduced glutathione) was also examined. Optimum rates of H2L2 production were observed with 10-2 m glutathione + 10-3 m oxidized glutathione. For immunoglobulin G1 assembly the half-time was reduced to 25 min at 25° and 10 min at 37° and to 30 min at 37° for subclass 4. These rates are comparable to those observed for covalent assembly in vivo.

Enzymatic catalysis of the reversible sulfitolysis of glutathione disulfide and the biological reduction of thiosulfate esters

Rat liver supernatants were shown to contain an enzymatic activity catalyzing in both forward and reverse directions the reversible sulfitolysis of glutathione disulfide. The enzymatic sulfitolysis has maximal activity at pH 7. S-Sulfoglutathione, which is a product of the sulfitolysis, was isolated by passage through an ion-exchange column. Three different assays were applied to determine S-sulfoglutathione, viz., methods based on the ninhydrin reaction, the formation of a thiazoline derivative in strong acid, and the use of radioactively labeled glutathione. The reversal of the sulfitolysis, i.e., the reaction of S-sulfoglutathione with glutathione, was studied directly by determination of sulfite with radioactive N-ethylmaleimide, or indirectly by coupling to the NADPH- and glutathione reductase-linked reduction of glutathione disulfide. Chromatographic analysis of rat liver supernatants demonstrated that all fractions catalyzing the reversible sulfitolysis did also catalyze the previously studied thiol-disulfide interchange of glutathione and the mixed disulfide of cysteine and glutathione. The reduction of thiosulfate esters, such as S-sulfocysteine and trimethylammonium-ethylthiosulfate, with glutathione was also catalyzed by the enzyme active in the sulfitolysis, which indicates an important biosynthetic role of the enzyme in microorganisms synthesizing cysteine via S-sulfocysteine. The enzyme is also capable of participating in the formation of the naturally occurring S-sulfoglutathione.

Mechanism of action of enzymes catalyzing thiol—disulfide interchange. Thioltransferases rather than transhydrogenases

FEBS LettersVolume 38, Issue 3 p. 263-267 Full-length articleFree Access Mechanism of action of enzymes catalyzing thiol—disulfide interchange. Thioltransferases rather than transhydrogenases Per Askelöf, Per Askelöf Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this authorKent Axelsson, Kent Axelsson Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this authorStellan Eriksson, Stellan Eriksson Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this authorBengt Mannervik, Bengt Mannervik Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this author Per Askelöf, Per Askelöf Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this authorKent Axelsson, Kent Axelsson Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this authorStellan Eriksson, Stellan Eriksson Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this authorBengt Mannervik, Bengt Mannervik Department of Biochemistry, University of Stockholm, Stockholm, SwedenSearch for more papers by this author First published: January 15, 1974 https://doi.org/10.1016/0014-5793(74)80068-2Citations: 52AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume38, Issue3January 15, 1974Pages 263-267 ReferencesRelatedInformation

66] Covalent chromatography by thiol-disulfide interchange

The selective isolation and purification of enzymes and other biologically important macromolecules by “affinity chromatography” exploits the unique ability of proteins and polypeptides to bind ligands specifically and reversibly and is widely applied. Reversible binding in this context usually makes use of noncovalent bonds although covalent bonding may occur in some cases. A special type of affinity chromatography can be envisaged in which the material of the chromatography column reacts chemically with only one of the components of a mixture. After removal of the other components from the column by washing, the covalently bound component is released by reaction with a suitable reagent. The reaction that releases the component of interest should preferably leave the column in a form that can be readily “reactivated.” This type of chromatography, which relies upon the scission and formation of covalent bonds, has been called “covalent chromatography.”

Evidence for a glutathione-dependent interconversion of two forms of a thioltransferase from rat liver catalyzing thiol-disulfide interchange.

Evidence for a glutathione-dependent interconversion of two forms of a thioltransferase from rat liver catalyzing thiol-disulfide interchange.

Thiol-disulfide Transhydrogenase from Baker's Yeast and a New Method for the Direct Assay of an Enzyme-catalyzed Thiol-disulfide Interchange Activity

This paper presents a study on the enzyme reduction of the disulfide bond and the following results have been found.In enzyme preparation, antioxidants showed a stability effect and EDTA appeared to have both enzyme stabilization and solubilization. On the distribution of the enzyme activity in subcellular fractions, the water soluble fraction appeared to contain the major released enzyme activity. The enzyme was inhibited with several metals. Hg2+ and transition metals were the most toxic. The substrate specificity of this enzyme was wide for the low molecular substrates, but the protein disulfide reducing activity was not detected in this preparation. It was assumed that the thiol-disulfide transhydrogenase was coupled with glutathione reductase and the disulfide substrates were reduced by the system involving the two enzymes. A new method for the direct recording of an enzyme-catalyzed thiol-disulfide interchange using diphenyl disulfide and p,p-dinitro diphenyl disulfide was devised.

Glutathione VII. Differentiation among substrates by the thiol-oxidizing agent, diamide

1. 1. Glutathione reacts much more rapidly with the thiol-oxidizing agent, diamide, than do other small molecule cellular constituents like NADH, CoASH and lipoic acid. 2. 2. Combination of rate data and concentration data for important oxidizable cellular constituents leads to the conclusion that GSH is the major target for diamide within the cell. 3. 3. Conversion of GSH to GSSG is the most important immediate consequence of diamide treatment of a cell suspension. 4. 4. Consideration of the rate constants for thiol-disulfide interchange reactions leads to the idea that further chemical and physical changes may occur shortly after the initial GSH-GSSG equilibrium has been perturbed by diamide. The time scale of an experiment defines how extensive these sequellae might be. 5. 5. A new term, the thiol-disulphide status of the cell is defined. 6. 6. Perturbations of the thiol-disulfide status of cells and organisms by thiol-oxidizing agents have been demonstrated as important in many problems, including protein synthesis and neurotransmitter release.

The Creep Behaviour of Synthetic Flour Doughs

The complexity of rheological properties of wheat flour dough has prompted the present study of the viscoelasticity with synthetic flour dough, which was prepared by mixing three major components of wheat flour dough, i. e. the gluten protein, wheat starch, and water.The results obtained can be summarized as follows:(1) The source of the gluten protein fractionated from the commercially available wheat flours plays an important role in knowing the creep behaviour of synthetic flour Boughs of the same composition.(2) The values of the creep parameters derived from the creep data decrease with increasing measure of shear stresses, so that the synthetic system exhibits non-linear viscoelastic behaviour.(3) The introduction of a small amount of cystein into the system is pronouncedly effective to the softening, which may be due to the introduction of thiol-disulfide interchange reactions in the protein network.(4) The glutenin fraction of the gluten protein is a major component in the network structure of protein in the dough system.

A method for the direct recording of an enzyme-catalyzed thiol-disulfide interchange using a mixed disulfide of glutathione and 3-carboxy-4-nitrobenzenethiol

A method for the direct recording of an enzyme-catalyzed thiol-disulfide interchange using a mixed disulfide of glutathione and 3-carboxy-4-nitrobenzenethiol

The reduction of the L-cysteine-glutathione mixed disulfide in rat liver. involvement of an enzyme catalyzing thiol-disulfide interchange

The reduction of the L-cysteine-glutathione mixed disulfide in rat liver. involvement of an enzyme catalyzing thiol-disulfide interchange

Structural Characterization of Vulcanizates Part X. Thiol-Disulfide Interchange for Cleaving Disulfide Crosslinks in Natural Rubber Vulcanizates

Abstract The thiol-disulfide exchange reaction is shown to be applicable to cleavage of disulfide crosslinks in accelerated sulfur vulcanizates of natural rubber. The reaction, in conjunction with the previously reported selective cleavage of polysulfide crosslinks, is used to determine the distribution of crosslink types for several accelerated sulfur vulcanization systems as a function of cure time. Discrepancies between the results and published results obtained using the reagent sodium di-n-butyl phosphite for disulfide crosslink cleavage are discussed.

Thiol-disulfide interchange reactions between serum albumin and disulfides

Thiol-disulfide interchange between serum albumins and l-cystine or lysine vasopressin was studied by p- chloromercuribenzoate (PCMB) titration and by incubation with radioactive disulfides followed by separation of reaction products using Sephadex G-15 gel filtration. Good agreement between these two techniques was obtained. Serum albumins were purified by Sephadex G-150 gel filtration, and lysine vasopressin was purified by CM-cellulose ion-exchange chromatography and Sephadex G-25 gel filtration. [ 3H]Lysine vasopressin was prepared by reduction of the iodinated hormone with 3H and purified by ion-exchange chromatography and gel filtration. The PCMB technique was primarily used to confirm the gel filtration technique. The gel filtration technique was used to measure the kinetic and equilibrium parameters of reactions between l-[ 14C]cystine and human serum albumin and between [ 3H]lysine vasopressin and bovine mercaptalbumin. The pH profiles for these reactions were also determined. Kinetic experiments indicated that these thiol-disulfide interchange reactions proceeded at two distinct rates, and equilibrium experiments were measured only with respect to the fast-reacting component. Mg 2+ was demonstrated to inhibit the rate and extent of reaction between [ 3H]lysine vasopressin and bovine serum albumin, while Ca 2+ had no significant effect at similar concentrations.

Structural characterization of vulcanizates part X. Thiol‐disulfide interchange for cleaving disulfide crosslinks in natural rubber vulcanizates

AbstractThe thiol‐disulfide exchange reaction is shown to be applicable to cleavage of disulfide crosslinks in accelerated sulfur vulcanizates of natural rubber. The reaction, in conjunction with the previously reported selective cleavage of polysulfide crosslinks, is used to determine the distribution of crosslink types for several accelerated sulfur vulcanization systems as a function of cure time. Discrepancies between the results and published results obtained using the reagent sodium di‐n‐butyl phosphite for disulfide crosslink cleavage are discussed.

Studies related to the concept that a thiol disulfide interchange is involved in the action of insulin on muscle cells.

Abstract The accumulation and retention of [ 131 I]insulin were studied in isolated frog sartorius muscles that were first incubated with [ 131 I]insuline and then washed with Ringer's solution. Most of the radioactive material that was retained in the tissue after thorough washing with ice-cold Ringer's solution could be extracted later with 8 M urea. A portion of the labeled material that remained in the tissue after extraction with 8 M urea could be removed by additional washing with solutions that contained cysteine. These findings agree with the earlier results of other investigators who concluded that the action of insulin may involve formation of a disulfide bond linking the hormone to the tissue. However, the present extension of these studies has revealed that the amount of radioactive material (derived from labeled insulin) that could be extracted from frog sartorius muscles with cysteine solutions was not directly related to the magnitude of the effect of insulin on permeability to sugar. Therefore, demonstration of a release of radioactive material in the presence of cystein does not necessarily indicate that the biological action of insulin in skeletal muscle involves the formation of a disulfide bond between insulin and a specific receptor site in the tissue.