Thiol-disulphide Interchange Research Articles

Whey protein polymerisation and its applications in environmentally safe adhesives

Whey protein is a group of globular proteins from cheese whey. It is a safe, degradable, renewable and abundant natural material compared with synthetic polymers. Native whey proteins do not exhibit good adhesive properties due to their low molecular weights and compact globular structures; however, under thermal treatment, these globular structures can be unfolded to form polymers or aggregates through intermolecular disulphide bonds via a thiol–disulphide interchange. Heat‐induced whey protein polymer exhibits good adhesive properties. With the addition of a cross‐linker, stabiliser or co‐binder, different types of wood and paper adhesives can be formulated using polymerised whey protein.

Aggregation of proteins and its prevention by carbohydrate excipients: albumins and gamma-globulin.

Moisture-induced (2-10 microL added to 10 mg) aggregation of solid-state albumin and gamma-globulin was investigated by incubation at 37 degrees C for 24 h. The insoluble aggregates were centrifuged from a reconstituted solution, dissolved in a solution containing denaturant and reducing agent, and analysed by a Bio-Rad protein assay kit. Of the three albumins used, maximum aggregation (8.2%) was observed with bovine serum albumin that was essentially fatty-acid free. The maximum aggregation observed with gamma-globulin was 7.0%. A bell-shaped curve for percent aggregation was observed with increasing moisture content and was especially prominent for bovine serum albumin. When mixed with carbohydrate excipients in a 1:1 ratio, aggregation was reduced for both bovine serum albumin and gamma-globulin by all four of the following excipients used: Emdex, dextrose, trehalose and hydroxypropyl beta-cyclodextrin. For bovine serum albumin, the aggregation was reduced about sixfold, with Emdex being the most effective excipient. The likely mechanism of the resulting aggregation was covalent linkages formed due to intermolecular thiol disulphide interchange.

The putative thiol-disulphide interchange protein DsbG from Acidithiobacillus ferrooxidans has disulphide isomerase activity

The putative thiol-disulphide interchange protein, DsbG, is involved in the formation and rearrangement of disulphide bonds in Acidithiobacillus ferrooxidans but its exact role is so far unclear. The gene encoding DsbG from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli BL21 (DE3). The protein was purified by one- step affinity chromatography. The scRNaseA could be rescued to 89% of the native RNaseA activity by using the purified DsbG of A. ferrooxidans. This characteristic is unique and different from that of DsbG from E. coli, which does not catalyse the oxidative refolding of RNaseA in vitro. Site-directed mutagenesis of the DsbG protein revealed that Cys119A and Cys122A do not possess the disulphide isomerization activity, indicating Cys119 and Cys122 are catalytic residues and play a crucial role in disulphide isomerization.

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases

Background Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood. Methods We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay. Results We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol–disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme. Conclusion Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases. General significance The possible involvement of thiol–disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.

Conditions limiting the influence of thiol–disulphide interchange reactions on the heat-induced aggregation kinetics of β-lactoglobulin

The effects of calcium and N-ethylmaleimide addition on the heat-induced denaturation/aggregation of β-lactoglobulin at neutral pH were examined. Aggregation of unfolded β-lactoglobulin was found to be rate limiting on heating aqueous protein solutions, while stoichiometric additions of calcium indicated that the unfolding reaction was limiting. Increased binding of calcium reduced the zeta potential of β-lactoglobulin to a minimum of −5 mV, resulting in first-order denaturation/aggregation kinetics at pH 7.0. Thiol–disulphide interchange reactions had a positive effect on the aggregation of unfolded β-lactoglobulin at low protein concentrations and low ionic strength. In the presence of calcium, the absence of thiol–disulphide interchange reactions did not affect the protein aggregation kinetics. Thiol–disulphide interchange reactions had little effect on storage modulus development of heated β-lactoglobulin (3%, w/w) acid gels; however, they enhanced the strain stability. Results showed the importance of non-specific forces in controlling the heat-induced aggregation of unfolded β-lactoglobulin.

Factors affecting the acid gelation of sodium caseinate

Acid gelation, turbidity and particle size development of dispersions of sodium caseinate and other protein fractions were studied. Sodium caseinate dispersions became particulate prior to the onset of gelation. Casein particles had enhanced stability to gelation in the presence of sodium chloride. Removal of the hydrophilic part of the κ-casein molecule through renneting and acidification of the soluble sodium para-caseinate resulted in increased gelation pH. Removal of most of the κ-casein through ethanol fractionation of sodium caseinate resulted in an α s1- β-fraction, which was markedly destabilised at higher pH values during acidification in the presence of sodium chloride. Preheated β-lactoglobulin/sodium caseinate dispersions had similar acid gelation profiles in the presence of sodium chloride with or without N-ethylmaleimide, suggesting that secondary thiol-disulphide interchange reactions between κ-casein and pre-heated β-lactoglobulin aggregates did not effect the gel point and final storage modulus in the short time-frame (120 min) of acidification. It was found that κ-casein and sodium chloride played a significant role in both particle development and subsequent stability of sodium caseinate dispersions on acidification.

Thiol–disulphide interchange in tubulin: kinetics and the effect on polymerization

All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] and 2,2'-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulphide products react further to form both intra- and intermonomer disulphide bonds that can be reversed by reducing agents. With MMTS (methyl methanethiosulphonate) or ODNB (n-octyl-dithio-2-nitrobenzoate), virtually no protein-protein disulphide bonds are formed and the ODNB reaction can be given as the sum of three exponential terms with pseudo-first-order rate constants of 0.206, 0.069 and 0.010 s(-1) at pH 6.5, suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of the helix content. MMTS-induced loss of SH groups leads to progressive increases in the critical concentration and loss of polymerization competence that can be reversed by assembly promoters such as higher protein concentration, taxol or high ionic strength. Under such conditions, the substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles.

Potential involvement of copper and thiol-disulphide interchange in prion proteins' conformational conversion

The prion protein PrP C in transmissible spongiform encephalopathy converts to the pathogenic isoform PrP Sc containing less α-helical structure and a greater β-pleated sheet content. The stability of PrP C protein is partly dependent on the disulphide bond between two α-helices designated B and C. Further stability could arise from ligand complexes of Cu(II) ions formed with carboxylic acid side chains in PrP C. Electron spin resonance (E.S.R.) spectra and atomic absorption measurements have shown for α-keratin that the formation of ligands by Cu(II) is 10 2 more rapid than interaction of Cu(II) with ionised thiols X–S − which form X–S–Cu +. X–S − destabilises disulphide bonds by thiol-disulphide interchange. When insufficient Cu(II) is present to form ligands with all available sites in PrP C then unblocked X–S − groups could potentially destabilise the disulphide bonds by thiol-disulphide interchange followed by reformation of the disulphide bond in the β form of PrP Sc and the release of X–S − to interact with other PrP C.

Mechanisms of protein modification during model anti-viral heat-treatment bioprocessing of beta-lactoglobulin variant A in the presence of sucrose.

To ensure the safety of plasma and recombinant therapeutic proteins, heat treatment is routinely applied to these biopharmaceuticals as a means of virus inactivation. However, to maintain protein integrity during heat treatment it is necessary to use high concentrations of thermostabilizing excipients, such as sucrose, in order to prevent protein damage. In this study we describe the covalent modifications inferred to a model protein, beta-lactoglobulin A, that occur during typical and extended anti-viral heat treatments. The chemical derivation and mechanisms by which these modifications arise are addressed. Heat treatment initiated hydrolysis of sucrose to glucose and fructose, which in turn were degraded to glyoxal. Glyoxal and the free reducing sugars reacted with free amino groups in beta-lactoglobulin A to yield Maillard glycation adducts and advanced glycation end products (AGEs). The major mechanism for AGE formation was via degradation of glucose-derived Schiff-base adducts. Heat treatment and glycation of beta-lactoglobulin A resulted in thiol-disulphide interchange reactions leading to protein oligomerization. A small population of beta-lactoglobulin A non-disulphide-linked dimers were also observed with increasingly harsh heat treatments. These findings have implications for (i) improvements in the safety and efficacy of heat-treated protein biopharmaceuticals and (ii) our understanding of the mechanisms of protein glycation and AGE adduct formation.

Quantitative determination of Cu-thionein from human fluids with application of solid-phase extraction on covalent affinity chromatography with thiol-disulphide interchange support.

The aim of our investigation was to carry out quantitative isolation of Cu-thionein (Cu-Th) in human body fluids (urine, plasma and breast milk) in order to determine the level of exposure to heavy metals. In the experiment covalent affinity chromatography with thiol-disulphide interchange gel (CAC-TDI) was used as a solid phase extraction (SPE) support for preconcentration of Cu-thionein (Cu-Th) protein and Cu bonded with MT from water and human fluids samples. The present paper is a continuation of early experiments on the quantitation of Hg-thionein (Hg-Th), Cd-thionein (Cd-Th) and Zn-thionein (Zn-Th) in human body fluids such as urine, plasma and breast milk.

Application of covalent affinity chromatography with thiol-disulphide interchange for determination of environmental exposition to heavy metals based on the quantitative determination of Zn-thionein from physiological human fluids by indirect method based on analysis of metal contents.

Intoxication with heavy metals results in numerous poisonings and diseases. They disturb metabolism of the system, are the source of cancer, degeneration changes and others. As a result of kidney damage the urine of people exposed to heavy metals contains different low molecular weight proteins, oligopeptides and amino acids, indicating pathological changes. One of the proteins is a very specific metallopolythiopolypeptide--metallothionein (MT). Based on earlier investigations, a very good correlations has been found between the contents of metallothionein in urine and plasma and the concentration of heavy metals in the blood, urine, kidneys, liver and brain and general in level of exposition to heavy metals. The aim of our investigations was to carry out quantitative isolation of Zn-thionein (Zn-Th), in order to determine the level of exposition to heavy metals. For Zn-Th protein isolation by covalent affinity chromatography with thiol-disulphide interchange (CAC-TDI) was applied, which is a modern technique of separation of a high affinity, good repeatability and reproducibility, allowing specific isolation of the thiol-proteins CAC-TDI gel was used as a solid-phase extraction (SPE) support for preconcentration of Zn-Th protein and Zn bonded with Zn-Th from water, rine, plasma and breast milk samples. The investigations showed unfavourable effect of the support on separation of thiol proteins and good correlation between the concentration of MTs protein added to water, plasma and urine and the concentration of protein indirectly determined via atomic absorption spectrometric (AAS) method, by preconcentration on SPE support metals formerly bound with MT protein and absorbed on CAC-TDI gel and calculated from metals concentration. The present paper is a continuation of earlier experiments on quantitation of Hg-thionein and Cd-thionein in physiological fluids and homogenates.

Application of covalent affinity chromatography with thiol-disulphide interchange for determination of environmental exposure to heavy metals based on the quantitative isolation of Cd-thionein from human breast milk.

The aim of this study was to test a new chromatographic method for the quantitative determination of Cd-thionein (Cd-Th) in human breast milk, in order to determine the level of exposure to heavy metals. Cd-thionein was isolated by covalent affinity chromatography with thiol-disulphide interchange, which is a modern separation technique of high affinity, good repeatability and reproducibility, allowing specific isolation of the thiolproteins. The fundamentals of indirect determination of the contents of metallothionein protein from human milk were worked out through estimation of the quantities of cadmium bound with Cd-thionein and adsorbed on covalent affinity chromatography gel during a separation process.

Application of covalent affinity chromatography with thiol‐disulphide interchange for determination of environmental exposure to heavy metals based on the quantitative isolation of Cd-thionein from human breast milk

The aim of this study was to test a new chromatographic method for the quantitative determination of Cd-thionein (Cd-Th) in human breast milk, in order to determine the level of exposure to heavy metals. Cd-thionein was isolated by covalent affinity chromatography with thiol-disulphide interchange, which is a modern separation technique of high affinity, good repeatability and reproducibility, allowing specific isolation of the thiolproteins. The fundamentals of indirect determination of the contents of metallothionein protein from human milk were worked out through estimation of the quantities of cadmium bound with Cd-thionein and adsorbed on covalent affinity chromatography gel during a separation process. © 1998 John Wiley & Sons, Ltd.

The determination of environmental and industrial exposure to heavy metals based on the quantitative isolation of metallothionein from human fluids, with application of covalent affinity chromatography with thiol-disulphide interchange gel as a solid-phase extraction support

The aim of this study was to present a new analytical method for the quantitative determination of metallothionein (MT) proteins in human body fluids and tissues, in order to determine the level of environmental and industrial exposure to heavy metals. For MT isolation, covalent affinity chromatography with thiol-disulphide inter-change (CAC-TDI) was applied. Fundamentals of indirect determination of the contents of metallothionein proteins were worked out through estimation of the quantities of metals bound with metallothionein protein and adsorbed on covalent affinity chromatography gel as on solid-phase extraction support during separating process. The (CAC-TDI) gel, specially prepared, was used as a solid phase extraction support (SPE) for preconcentration of Hg–thionein (Hg-Th), Cd–thionein (Cd-Th), Zn–thionein (Zn-Th) and Cu–thionein (Cu-Th) proteins and Hg, Cd, Zn and Cu bonded with MTs from water, human fluids such as: urine, human plasma, breast milk and tissues homogenates.

Application of solid-phase extraction to the preconcentration of metallothionein and metals from physiological fluids

Solid-phase extraction (SPE) with covalent affinity chromatography with thiol–disulphide interchange (CAC–TDI) gels as sorbents were used for the preconcentration of Cd–thionein (Cd–Th), Zn–thionein (Zn–Th) and Cu–thionein (Cu–Th) proteins and Cd, Zn or Cu bonded with metallothioneins (MTs) from water, plasma and human urine samples. An indirect method of quantitation of MTs, based on the analysis of metal content, for the determination of MT in human body fluids is described. In experiments, different types of CAC–TDI gels were tested. The results showed a satisfactory correlation between the concentration of MT proteins added to water or physiological fluids and the concentration of protein indirectly determined via the concentration of metals by atomic absorption spectrometry.

Changes in the Chemical Composition of Wool During Dyeing and Finishing and Their Effects on Fabric Properties

The chemical composition of a wool fabric is determined after each of a variety of dyeing and finishing processes. The main concern is to observe the effect of dye-fiber interactions and dyeing recipe differences. Dyeing liquor pH and decatizing have a large influence on chemical composition. Reactive dyes react at histidine and cysteine sites, and affect the behavior of cystine during dyeing and both cysteine and cystine during decatizing. Low stress fabric mechanical properties are related to cysteine levels. It is clear that cysteine plays a role in fabric property changes during processing, possibly due to its importance in the thiol-disulphide interchange reaction, which is thought to be important in stress relaxation processes.

The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin.

1. The selectivity observed when the potentially general technique for the isolation of fully active forms of cysteine proteinases, covalent chromatography by thiol-disulphide interchange, is applied to chymopapain M and to actinidin was investigated by a combination of experimentation and computer modelling. Neither of these enzymes is able to react with the original Sepharose-GSH-2-dipyridyl disulphide gel, but fully active forms of both enzymes are obtained by using Sepharose-2-hydroxypropyl-2'-dipyridyl disulphide gel, which is both electrically neutral and sterically less demanding than the GSH gel. Electrostatic potential calculations, minimization and molecular-dynamics simulations provide explanations for the unusual, but different, specificities exhibited by actinidin and chymopapain M in the interactions of their active centres with ligands. 2. The unique behaviour of chymopapain M in exerting an almost absolute specificity for substrates with glycine at the P1 position and in resisting inhibition by cystatin was examined by the computer-modelling techniques. A new, modelled, structure of the complete chicken egg-white cystatin molecule based on the crystal structure of a short form of cystatin was deduced as a necessary prerequisite. The results suggest that electrostatic repulsion prevents reaction of actinidin with the GSH gel, whereas a steric 'cap' resulting from a unique arginine-65-glutamic acid-23 interaction in chymopapain M prevents reaction of the gel with this enzyme and accounts for the lack of its inhibition by cystatin and its specificity in catalysis. 3. Use of chymopapain M as a structural variant of papain demonstrates the validity of the predictions of Lowe and Yuthavong [Biochem. J. (1971) 124, 107-115] relating to the structural requirements and binding characteristics of the S1 subsite of papain.

Reactions of β-ethyl sulphone crosslinking agent with wool: Part III: Wool fractionation and amino acid analysis

A study of the effect of the application to wool of the new substantive crosslinking agent, 2-chloro-4, 6-di(-aminobenzene-4-β-sulphato-ethylsulphone)-1,3,5-s-triazine (XLC) is reported, with special reference to determining crosslinking at individual amino acid sites. Amino acid analysis was used to detect reactions of this compound at specific amino acid residues. Lysine and histidine residues were shown to be important sites of reaction, and indirect evidence was obtained that the compound restricts the degree of thiol-disulphide interchange which occurs when boiling wool under mildly acidic aqueous conditions (pH values 5–6).

The active adenovirus protease is the intact L3 23K protein.

The L3 23K protein was isolated from adenovirus type 2 and shown to cleave purified substrates, confirming that this protein is the adenovirus protease. Separate antisera, prepared against the amino- and carboxy-terminal regions of the 23K protein react with active protease, demonstrating that, contrary to previous reports, zymogen activation is not involved in the regulation of this enzyme. Molecular exclusion chromatography indicated that the protease is active as a monomer. Purified protease was shown to be inhibited by Zn2+ and Cu2+ and by some, but not all, recognized cysteine protease inhibitors, indicating participation of a thiol group and providing additional support to the suggestion that regulation of the enzyme involves a form of thiol-disulphide interchange.

The mechanism of separation of polythiolpeptides and metallopolythiolpeptides by covalent affinity chromatography

Covalent affinity chromatography with thiol-disulphide interchange (CAD-TDI) is a method for the separation of thiolproteins based on the interaction between disulphide bridges immobilised on a insoluble support and thiol groups in the protein undergoing separation. Mercury-thionein, cadmium-thionein and apothionein have been used as low molcular weight thiolproteins rich in cysteine residue. The proposed separation mechanism is more complex than those in the literature and can have anionic, cationic or a free radical character depending on the protein, the ligand and the conditions during separation.