Mixed Disulphide Research Articles

HPLC determination of biologically active thiols using pre-column derivatisation with 5,5′-dithio-(bis-2-nitrobenzoic acid)

5,5′-Dithio-(bis-2-nitrobenzoic acid), Ellmans reagent (ESSE), is used as a pre-column derivatisation reagent for the determination of biologically active thiols by HPLC. d-penicillamine, N-acetyl- d-penicillamine, N-acetylcysteine, cysteine, captopril and thiomalic acid all give well resolved derivatives. The calibration graph and reproducibility (%R.S.D.±1.3%) for the analysis of glutathione indicates that the method could be used for quantitative analysis. ESSE is widely used as a reagent in thiol determinations by electronic spectroscopy via the detection of the Ellmans anion (ES −) generated without any prior separation procedures. However, there are considerable reservations over its use for the spectrophotometric determination of thiols because of the possibility of side reactions which generate another Ellmans based species (ESO 2 −). The assay described determines the thiol as a derivatised mixed disulphide (ESSR) and since speciation between the anion ES − and the oxidation product ESO 2 − occurs it enables the process of oxidation to be monitored simultaneously.

Assignment of a single disulphide bridge in human α2-antiplasmin: implications for the structural and functional properties

Human alpha2-antiplasmin (alpha2AP) is a serpin involved in the regulation of blood coagulation. Most serpins, unlike smaller serine proteinase inhibitors, do not contain disulphide bridges. alpha2AP is an exception from this generalization and has previously been shown to contain four Cys residues organized into two disulphide bridges [Lijnen, Holmes, van Hoef, Wiman, Rodriguez and Collen (1987) Eur. J. Biochem. 166, 565-574]. However, we found that alpha2AP incorporates iodo[14C]acetic acid, suggesting that the protein contains reactive thiol groups. This observation prompted a re-examination of the state of the thiol groups, which revealed (i) a disulphide bridge between Cys43 and Cys116, (ii) that Cys76 is bound to a cysteinyl-glycine dipeptide, and (iii) and Cys125 exists as either a free thiol or in a mixed disulphide with another Cys residue. The disulphide identified between Cys43 and Cys116 appears to be conserved in orthologous proteins since the homologous Cys residues form disulphide bonds in bovine and possibly mouse alpha2AP. The conservation of this disulphide bridge suggests that it is important for functional aspects of alpha2AP. However, the structural and functional analysis described in this study does not support this conclusion.

Reduction of oxidative stress does not affect recovery of myocardial function: warm continuous versus cold intermittent blood cardioplegia.

OBJECTIVE: To compare oxidative stress after cardiac surgery in patients treated with two different methods of myocardial protection: warm continuous versus cold intermittent blood cardioplegia. To correlate oxidative stress with...

Effects of dough mixing and oxidising improvers on free reduced and free oxidised glutathione and protein-glutathione mixed disulphides of wheat flour

Changes in free reduced glutathione (GSH), free oxidised glutathione (GSSG) and protein-glutathione mixed disulphides (PSSG) during dough mixing were monitored. After a rapid decrease in the GSH content and an increase in the GSSG content, the contents of both GSH and GSSG decreased progressively, whereas the PSSG content increased, as a simple flour-water dough was mixed. Total glutathione (GSH plus GSSG plus PSSG) levels in simple flour-water doughs and doughs treated with ascorbic acid or potassium bromate remained essentially constant during dough mixing, indicating that the reactions in which glutathione is involved are simple oxidations of sulphydryl (SH) groups to disulphide (SS) bonds and SH/SS interchange reactions. Yeast contributed high levels of GSH and GSSG to doughs, but analysis of dough aqueous phases (liquors) and the similarity of the PSSG contents of simple and yeasted flour-water doughs suggested that the yeast GSH and GSSG were largely unavailable to react with flour proteins. The GSH content of ascorbic-acid-treated and yeasted dough decreased rapidly on wetting the flour, the magnitude of the decrease indicating that the ascorbate oxidation system oxidised the yeast intracellular GSH as well as the flour GSH. With further mixing, the GSH content of the ascorbate dough remained constant at low levels similar to those of the simple flour-water dough. The GSSG content of the ascorbate dough increased rapidly on wetting the flour, but declined as dough mixing continued. The PSSG content of the doughs increased markedly as dough mixing proceeded to the optimum and then stabilised. The increase in the PSSG content lagged behind the rapid oxidation of GSH to GSSG. Potassium bromate caused a pattern of changes similar to those observed for ascorbate, but the changes in GSH and PSSG contents were smaller in magnitude. The results indicate that the changes in the different glutathione pools and the effects of oxidising bread improvers are rather more complex than envisaged previously, particularly the effects on PSSG.

Cyanylation of the thiol of the cysteine of flavodoxin from Anabaena PCC 7119

Flavodoxins are small microbial proteins that contain FMN as prosthetic group, that mediate electron transfer between other redox proteins at low potential and that have been shown to replace ferredoxin in certain reactions [ 11. Anubaena PCC 71 19 flavodoxin (molecular weight 20 m a ) is identical in sequence with flavodoxin from Anabaena PCC 7 120 [23 and it contains a single cysteine residue (cys-54). This residue is conserved in several flavodoxins including that from Megasphaera elsdenii [ 11. It is not dlrectly involved in FMN binding although it is adjacent to the flavinbinding site. Apoflavodoxin, prepared by trichloroacetic acid precipitation of the holoprotein [3], was reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form the mixed disulphide and then with K13CN to produce 13Ccyanylated apoflavodoxin [4-61. The apoflavodoxin derivatives formed by reaction with DTNB and by subsequent cyanylation both bind FMN, but the interactions are weaker than in unmodified flavodoxin (Kd values 0.1 nM, 7.89 _+ 2.63 nM (n = 11) and 4.16 k 2.61 nM (n = 13) for the apoflavodoxin [3], the TNBand the cyanylated derivatives respectively. The 13C NMR spectrum showed a signal at 114.3 ppm (Figure 1). This signal shifted to 115.5 ppm after addltion of FMN.

Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst of Escherichia coli.

DsbA, a periplasmic protein, catalyses the disulphide bond formation of other cell surface proteins in E. coli. Reoxidation of DsbA for catalytic turn over is assured by DsbB, a membrane protein with four essential cysteine residues facing the periplasm. We and others previously reported that the reactive Cys30 residue of DsbA forms a mixed disulphide with DsbB in the absence of its partner Cys33 residue. Under the medium condition in which the DsbA mutant lacking Cys33 forms a mixed disulphide only with DsbB, we examined cysteine mutants of epitope-tagged DsbB for their ability to form the complex. It was shown that Cys104 of DsbB is absolutely required while other three cysteines are also required for maximum interaction. Examination of the redox states of cysteines in wild-type and mutant DsbB suggested that Cys104 and Cys130 form a disulphide bond which will be transferred to DsbA. In agreement with this notion, DsbB mutants lacking one of the N-terminally located cysteines retain weak DsbB activity in vivo. The primary role of the N-terminally located thioredoxin-like motif of DsbB is probably to reoxidize Cys104 and Cys130. We propose the following reaction cycle. DsbB is initially oxidized (State A in Summary Figure). Disulphide interaction between Cys30 of DsbA and Cys104 of DsbB should then trigger the recycling reaction of DsbA (State B), allowing over all electron transfer from newly secreted protein via DsbA (Cys30/Cys33) to DsbB in which intrachain electron flow from Cys104/Cys130 (State C) to Cys41/Cys44 (State D) may occur.

Mixed disulphide formation in myoglobin of mouse ( Mus musculus)

The myoglobin, isolated from murine skeletal muscles by chromatofocusing, showed the three components, one major and two minor, with different electrophoretic mobilities. The major component with the isoelectric point (pl) of 7.55 had one reactive SH/mole, while the others with pl of 7.32 and 7.16 showed none, which could be rendered fully reactive by treating the proteins with β-mercaptoethanol. The three components were the same in their molecular weight (18 kDa), amino-acid composition with one Cys residue and oxygenation properties. By a sensitive high-performance liquid chromatography method, the occurrence of cysteine or glutathione mixed disulphide was verified in the two minor components. We conclude from these results and incubation experiments with low-molecular-weight thiols that the two minors were derived from the major by a mixed disulphide formation with either cysteine or glutathione of the cysteinyl SH at the 66th sequence.

Transamination of methionine after loading in patients with cirrhosis

Background/Aims: An impaired methionine degradation along the transsulfuration pathway has been widely described in cirrhosis. Evidence has been provided that methionine can also be degraded via a transamination pathway, leading to formation of methanethiol and its metabolites, protein-S-SCH 3 (a mixed disulphide of blood proteins and methanethiol), α-ketomethylthiobutyrate and X-S-SCH 3 (a mixed disulphide of a thiol with an unknown component X and methanethiol). This pathway seems to be of little importance in normal subjects, even after methionine loading, but its role in the presence of an acquired transsulfuration defect has never been tested. Methods: We measured the plasma concentration of methanethiol metabolites in six normal subjects and 11 patients with cirrhosis receiving a primed-continuous infusion of L-methionine, at rates able to increase plasma methionine to levels approximately 20 times basal concentrations. Results: Before methionine infusion, the sum of transamination metabolites was similar in the two groups (0.29±SD 0.07 μmol/l in controls and 0.45±SD 0.22 in patients with cirrhosis). During methionine infusion and after the end of infusion, there was a progressive increase of transamination metabolites, which reached values approximately 10 times basal concentrations, with no difference between groups. Conclusions: We conclude that transamination cannot represent a quantitatively important exit for excess methionine in subjects with cirrhosis, in the presence of an acquired block along the transsulfuration pathway.

Glutathionylspermidine metabolism in Escherichia coli

Intracellular levels of glutathione and glutathionylspermidine conjugates have been measured throughout the growth phases of Escherichia coli. Glutathionylspermidine was present in mid-log-phase cells, and under stationary and anaerobic growth conditions accounted for 80% of the total glutathione content. N1,N8-bis(glutathionyl)spermidine (trypanothione) was undetectable under all growth conditions. The catalytic constant kcat/Km of recombinant E. coli glutathione reductase for glutathionylspermidine disulphide was approx. 11,000-fold lower than that for glutathione disulphide. The much higher catalytic constant for the mixed disulphide of glutathione and glutathionylspermidine (11% that of GSSG), suggests a possible explanation for the low turnover of trypanothione disulphide by E. coli glutathione reductase, given the apparent lack of a specific glutathionylspermidine disulphide reductase in E. coli.

A kinetic explanation for the rearrangement pathway of BPTI folding.

Bovine pancreatic trypsin inhibitor (BPTI) does not fold by simple sequential formation of its native disulphide bonds. Instead, an initially formed intermediate, termed N', first rearranges to a more stable species in a slow process that requires substantial unfolding. We find that direct oxidation of N' is also inhibited by native structure which slows both the intermolecular step in oxidation--formation of a mixed disulphide bond with the oxidizing agent GSSG--as well as the subsequent intramolecular step. Folding does not occur appreciably by direct oxidation because the high GSSG concentrations required for efficient mixed disulphide formation cause N' to accumulate as a nonproductive, double-mixed disulphide species. The need to unfold previously acquired native structure, observed in the folding of BPTI, may be a common feature of disulphide-linked folding reactions.

Modelling molecular stability in gamma B crystallin

Molecular modelling and dynamics simulations of γ B crystallin and a number of its adducts were used to simulate effects of post-translational modifications. N-terminal modifications which disrupted the surface charge network of γ-crystallin did not produce significant unfolding in any part of the γ-crystallin molecule ( P > 0.05) consistent with the results of in vitro aggregation studies. There were no significant changes to the molecular volume of the protein, suggesting that adduct formation did not result in molten globule formation. Adduct formation did however increase the vulnerability of particular γ-crystallin cysteine residues to sulphydryl bond formation and so increased the possibility of mixed disulphide formation with glutathione. Modelling of such secondary modification products (containing both N-terminal modifications and glutathione adducts) demonstrated significant ( P < 0.05) structural changes. This suggests that a post-translational modification pathway leading to crystallin cross-linking and cataractogenesis proceeds via a number of co-operative steps involving both modification at the N-terminus and mixed glutathione formation.

Gliotoxin inactivates alcohol dehydrogenase by either covalent modification or free radical damage mediated by redox cycling

The fungal metabolite gliotoxin shows selective toxicity to cells of the immune system and has been implicated in the aetiology of invasive aspergillosis. The related toxin sporidesmin is the causative agent of facial eczema in sheep. The toxicity of these compounds has been related to their ability to redox cycle intracellularly and thus produce damaging free radicals. These toxins are also potentially capable of forming mixed disulphides with thiol groups on proteins by virtue of their bridged disulphide structure. We show here that gliotoxin can inactivate horse liver alcohol dehydrogenase by either oxidative damage or covalent modification of thiol groups on the enzyme. Either Cys-281 or Cys-282 is selectively modified. Neither of these residues are at the active site. Covalent modification occurs in the absence of reducing agents such as dithiothreitol. In the presence of dithiothreitol no protection is observed and the rate of inactivation is enhanced although as expected no covalent modification occurs. Gliotoxin can therefore inhibit alcohol dehydrogenase by either pathway and this will depend on the availability of reducing agents such as glutathione and/or how readily the reactive oxygen species generated are removed.

The purification and properties of glutathione reductase from the cestode Moniezia expansa

Glutathione reductase has a central role in glutathione metabolism and as such is a potential target for chemotherapy. The aim of the work was to purify and characterise glutathione reductase from the cestode Moniezia expansa and to compare the properties of the helminth enzyme with its mammalian counterpart. The enzyme was purified by a combination of anion exchange and affinity chromatography and further characterized by chromatofocusing and gel electrophoresis. Analysis revealed a single isoenzyme of glutathione reductase in Moniezia expansa, with a pI of 5.8. The enzyme was a homodimer of native molecular weight 114 kDa, subunit weight 63 kDa. Enzyme activity was affected by buffer concentration and the presence of monovalent sodium salts. The pH optimum was 7.4 with NADPH as cofactor and 5 with NADH. The K m a for oxidized glutathione was 76 μM and for NADPH and NADH, 21 and 350 μM respectively. In addition to oxidized glutathione only the mixed disulphide between CoA and glutathione (CoASSG) showed any significant activity as substrate. The cestode enzyme was inhibited by a variety of compounds including arsonic derivatives, 2,4,6 trinitrobenzene sulfonate 1,3-bis (2-chlorethyl)-1-nitrosourea and oxidized glutathione. In conclusion the glutathione reductase of M. expansa resembles the mammalian enzyme in its general physical properties and its substrate and inhibitor profile. However, the parasite enzyme shows an unusually high activity with the mixed disulphide of coenzyme A and glutathione (CoASSG) and appears to be more sensitive to inhibition by sodium ions.

Thiol groups in proteins as endogenous reductants to determine glutathione-protein mixed disulphides in biological systems

A novel method for glutathione-protein mixed disulphide (GSSP) determination, based on the use of protein sulphydryl groups as endogenous reductant and on the spectrophotometric determination of reduced glutathione, is described. The procedure is based on the observation that acid-precipitated proteins from different rat tissues rapidly release GSH from GSSP when brought to neutral pH. The basal GSSP content determined in rat liver, heart, lung, testis, spleen and brain corresponded to that reported in the literature and determined by more complex sample preparation or labor-intensive analytical procedures.

Refolding of the mixed disulphide of RNase T1 and glutathione

Refolding of the mixed disulphide of RNase T1 and glutathione

Analysis of RNase A refolding intermediates by electrospray/mass spectrometry

Electrospray/mass spectrometry (ESIMS) was extensively used to obtain information on disulphide-containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular SS are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides. The results indicate that ES/MS is a suitable technique to detect and characterize refolding intermediates.

Glutathione Dependent Modification of Bovine Lens Aldose Reductase

Bovine lens aldose reductase (ALR2) is readily modified by glutathione disulphide (GSSG) to an enzyme form (GS-ALR2) exhibiting a reduced catalytic efficiency with all the substrates tested and a reduced susceptibility to inhibition. The modification, which is completely reversed by reduced glutathione (GSH) or dithiothreitol occurs by a pseudo-first-order process with respect to the enzyme and a second order rate constant of 30 ± 0·1 mol -1 min -1 at 25°C was determined. By measuring the residual activity of ALR2 incubated in different glutathione redox buffers at 25°C, an apparent redox equilibrium constant of 1·4 ± 0·1 was evaluated. Thus the rate and the maximal extent of ALR2 inactivation are proportional to the redox ratio of the thiol used as modifying agent (i.e. [GSH]/[GSSG]). The stoichiometric reversibility of the enzyme modification might be impaired by a reduced solubility of GS-ALR2 with respect to ALR2 and by an increased susceptibility of the modified enzyme to proteolysis. While the native enzyme form is rather insensitive to proteolytic breakdown, GS-ALR2 is easily degraded by chymotrypsin with the generation of a peptide of 26 kDa with an aminoacid sequence at the aminoterminal side compatible with proteolysis at level of Tyr 7 of aldose reductase. A reduced efficiency in the enzyme-cofactor binding following the GSSG dependent modification of ALR2, appears to be associated to the thiol accessibility of GS-ALR2 measured at different temperatures. GS-ALR2 is characterized by the presence of one glutathione residue, linked through a mixed disulphide bond. This is sustained by: (i) the isoelectric point for the modified enzyme of 4·75, which is 0·1 pH units lower than that observed for the native enzyme, which indicates the contribution of an acidic residue to the pI of GS-ALR2; (ii) the incorporation of radioactivity coming from [ 3H] labelled GSSG accounting for the presence of one equivalent of glutathione per mole of enzyme. Besides being a general feature of protein reactivity in oxidative conditions, the glutathione-mediated ALR2 modification might be part of a cell strategy to preserve reducing power in conditions of oxidative stress.

Metabolic fate of S,S,S-tributyl phosphorotrithioate (DEF) in the lactating goat.

1. Metabolism of [1-14C] DEF (S,S,S-1-14C-tributyl phosphorotrithioate, 1) in the lactating goat has been investigated. A goat was dosed orally by capsule on 3 consecutive days at a rate of 0.82 mg/kg body weight/day based on 25 times the maximum DEF residue anticipated in animal feed. 2. Urine and milk were collected throughout the study. The goat was killed 21 h following the last treatment, and kidney, liver and composite samples of muscle and fat were collected. The radioactive residue levels (following the three doses) were 3.45 ppm in liver, 0.35 ppm in kidney, 0.19 ppm in fat, 0.06 ppm in muscle and 0.12 ppm in milk collected at the final 16 h and prior to killing. 3. Urinary metabolic profile indicated that DEF was efficiently metabolized to many metabolites. Tissue and milk extracts also indicated that DEF was extensively metabolized. 4. DEF comprised 31 and 5% of the total radioactive residue in fat and milk, respectively. The amount of DEF in liver, kidney and muscle represented < 1% of the total radioactive residue. 5. A major metabolite, 3-hydroxybutylmethyl sulphone (HBM sulphone, UP3), was found in tissue, milk and urine. The identification of this metabolite was accomplished by a combination of MS, nmr and comparison with an authentic standard. The glucuronide (UP1) and sulphate (UP2) conjugates of HBM sulphone were found in urine, and the sulphate conjugate was a major metabolite in kidney. 6. The hydrolytic products of DEF, S,S-dibutyl phosphorodithioate (Dibufos, U16) and S-butyl phosphorothiate (Bufos, U8), were identified as minor components in urine, comprising 5 and 4% of the total radioactive residue, respectively. Butyl mercaptan was not found, but mixed disulphides of butyl mercaptan with either glutathione (U10, 3%) or N-acetyl cysteine (U13, 2%) were found. 7. Direct evidence for the incorporation of DEF residue into natural constituents was also established. Fatty acids from milk and fat were isolated and shown to be radioactive.

Inactivation of mouse liver glutathione S-transferase YfYf (Pi class) by ethacrynic acid and 5,5′-dithiobis-(2-nitrobenzoic acid)

Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.

The long-term use of D-penicillamine for treating rheumatoid arthritis: is continuous therapy necessary?

Patients with RA often fail to comply with their therapy. We investigated the extent of non-compliance with D-penicillamine therapy and also whether patients needed continuous treatment after they had shown a therapeutic response. We developed a simple urinary test, measuring cysteine-penicillamine mixed disulphide by a high performance liquid chromatography technique, as an indicator of compliance. Using this method we evaluated compliance in 59 consecutive RA patients attending rheumatology outpatients for monitoring of D-penicillamine therapy. Evidence of poor compliance was shown in 39%. There was no relationship between poor compliance and disease activity. A possible explanation for this paradox emerged from a therapeutic study of the efficacy of intermittent treatment in patients in partial clinical remission on long-term D-penicillamine. In 14 randomly selected patients the daily dose of D-penicillamine was reduced in frequency over 6 months from 250-750 mg daily to the same dose taken 1 week out of every 4. The patients were compared to matched controls who remained on continuous therapy. After 30 months both the intermittent and continuous therapy groups had similar clinical and laboratory scores for disease activity. Our results suggest that many patients may not comply with their prescribed D-penicillamine regime, but such variation in compliance may not be clinically important since intermittent treatment may sustain response in the longer term.