Toxicity of thiols and disulphides: Involvement of free-radical species

Sulphur is essential to life, and thiols and disulphides play essential roles in cellular biochemistry. Such compounds are also widely distributed in the food of man and his domestic animals, and they are extensively used in industry. However, many thiols and disulphides have been shown to be toxic. Aliphatic, aromatic, and heterocyclic compounds of this type are haemolytic agents in animals while aminothiols have been shown to induce many cytotoxic effects in vitro and the epidithiodioxopiperazine mycotoxin, sporidesmin, is a potent hepatotoxic agent. Structure-activity relationships among these compounds and factors which modulate their harmful effects are consistent with a toxic mechanism involving redox cycling between the thiol and the corresponding disulphide. Thiyl radicals and "active oxygen" species are formed in this process, and it is suggested that these substances are responsible for initiating the tissue damage provoked by thiols and disulphides.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

The histochemistry of thiols and disulphides. III. Staining patterns in rat tissues

Synopsis With the aid of new staining methods, thiol groups produced by the reduction of disulphide bonds were positively distinguished from pre-existing groups in paraffin sections of several organs of the rat. Good preservation of structures in which the natural thiol-disulphide balance had been maintained was sought by fixing the tissues in neutral formalin containing an organomercurial. After dissociation of the resulting mercaptide bonds that protected the native thiols, these were shown in one colour and then disulphide sites in another within the same sections. Intracellular granules and extracellular membranes rich in disulphides thereby stood out in red against the predominantly blue labelling of the cellular ground plasm. Intimate mixtures of the two forms in some places and the presumed transformation of thiols to disulphides in others, notably the keratinizing epithelium of the tongue, were readily seen. Supplemented by separate visualization of thiols and disulphides along with suitable controls for specificity of staining, the results obtained diverged in some major respects from those of previous investigations.     

The mechanism of disulphide reduction by mitochondria

1. Cystamine was reduced to the corresponding thiol by rat liver mitochondria, even in the presence of rotenone or antimycin A. 2. The reduction of disulphides was stimulated by the accumulation of NADH or by the addition of NADH to osmotically ;shocked' mitochondria. 3. Energy made available by oxidative phosphorylation was not essential for the reduction of disulphides. 4. Cystamine was not reduced during the oxidation of NADH by ultrasonically treated particles, which had lost their capacity for oxidation of alpha-oxo acids. 5. In intact mitochondria, arsenite and other inhibitors of vicinal dithiols caused a decrease in the capacity for reduction of disulphides concomitantly with an inhibition of the oxidation of alpha-oxo acids. 6. Isolated lipoamide dehydrogenase reduced cystamine at the expense of NADH, provided that lipoic acid was also present. 7. It is concluded that in mitochondria the reduction of cystamine and related disulphides is probably brought about by interaction with reduced lipoic acid, generated by the alpha-oxo acid dehydrogenase complexes during the oxidation of alpha-oxo acids or by reaction of lipoamide dehydrogenase with NADH.     

A method for the estimation of thiol esters.

1. A method is described for the estimation of thiol ester groups. The thiol ester is converted into the corresponding thiol by reaction with ammonia; the thiol is then titrated amperometrically with mercuric chloride. 2. The method may be used in the presence of SH and S.S groups. The SH groups are titrated at pH3 in the presence of excess of chloride; under these conditions thiol esters do not react with mercuric chloride. Thiol ester plus thiol is then estimated by titration after reaction with ammonia. Finally, titration after reaction with ammonia and sulphite gives the thiol ester plus thiol plus disulphide. 3. The procedure has been applied to glyceraldehyde phosphate dehydrogenase. The enzyme was found to contain 15-16 SH groups/mol. and no S.S groups. After reaction with acetyl phosphate 1.8-3.5 thiol ester groups were detected, the number depending on the conditions of acetylation. In the absence of bound NAD, the number of thiol ester groups formed was 1.8/mol., although a value of 2.9 labile acetyl groups/mol. was given by the method of Lipmann & Tuttle (1945). The presence of thiol ester groups in the S-(d-3-phosphoglyceryl)-enzyme was also demonstrated.     

The reverse of the 'repair' reaction of thiols: H-abstraction at carbon by thiyl radicals

Thiyl radicals (RS) formed by the reaction of radiolytically generated OH radicals with thiols, e.g. 1,4-dithiothreitol (DTT), react with cis- and trans-2,5-dimethyltetrahydrofuran by abstracting an H atom in the alpha-position to the ether function (k approximately equal to 5 X 10(3) dm3 mol-1 s-1). The so-formed planar ether radical is 'repaired' by the thiol (k = 6 X 10(8) dm3 mol-1 s-1) thereby regenerating a cis- or trans-2,5-dimethyltetrahydrofuran molecule. In this reaction a thiyl radical is reproduced. Thus trans-2,5-Me2THF from cis-2,5-Me2THF and vice versa are formed in a chain reaction: at a dose rate of 2.8 X 10(-3) Gys-1 and a trans-2,5-Me2THF concentration of 1 X 10(-2) mol dm-3 using DTT as the thiol, G(cis-2,5-Me2THF) = 160 has been found. The chain reaction is very sensitive to impurities and also to disulphides such as those radiolytically formed. 2,5-Me2THF can be regarded as a model for the sugar moiety of DNA where the C(4')-radical is known to lead to DNA strand breakage. The possible role of cellular thiols in the repair of the C(4') DNA radical, and also the conceivable role of thiyl radicals inducing DNA strand breakage, are discussed.     

The histochemistry of thiols and disulphides. II. Methodology of differential staining

Synopsis The reduction of disulphide bonds by various mercaptans and tri-n-butylphosphine (TBP) has been examined in paraffin sections of rat tissues. A re-reduction procedure demonstrating any residual disulphides shows that nearly equivalent endpoints are reached by all of the reagents at pH 8.5 and room temperature, though at greatly differing rates. TBP is the reductant of choice in that it acts rapidly, cannot cause the thiolation which is more or less pronounced with certain mercaptans and least reverses the prior alkylation of native thiol groups by iodoacetate or N-substituted malemides. Supporting studies establish that, except in highly compact structures, native as well as generated thiol groups can be visualized with satisfactory completeness and specificity by N-(4-aminophenyl)maleimide followed by a diazotization and coupling sequence. These findings provide the basis for the selective staining of disulphides, either alone or differentiated from native thiols in the same section.     

Reaction of thiols with 7-methylbenzopentathiepin

Polysulfides typically react readily with thiols, thus, reactions of endogenous cellular thiols with the polysulfide linkage in naturally-occuring pentathiepin cytotoxins are likely to be an important aspect of their biological chemistry. Here, it is reported that the reaction of thiols with the pentathiepin ring system initially produces a complex mixture of polysulfides that further decomposes in the presence of excess thiol to yield the corresponding 1,2-benzenedithiol with concomitant production of H(2)S and dimerized thiol. In this reaction, a single molecule of the pentathiepin consumes approximately six equivalents of thiol. The reaction of thiols with the pentathiepin ring system is faster than the analogous reaction involving typical di- and trisulfides.     

Separation of urinary thiols as tributyltinmercaptides and determination using capillary isotachophoresis

The essential steps in the assay included electrolytic reduction of disulphides, neutralization, extraction of thiols with 0.1 M tributyltin hydroxide in octane, stripping of the extract with 2% acetic acid, fixing the washed-out amino thiols to a cation exchanger, elution with 2 M hydrochloric acid, oxidation with bromine and evaporation. The remaining octane extract was decomposed by dodecanethiol, the mercapto acids were washed out, oxidized with bromine and evaporated. Both residues were dissolved in water and analysed using capillary isotachophoresis at pH 3.0. Cysteamine was extracted from reduced urine at ca. pH 10, decomposed by dodecanethiol and re-extracted to boric acid followed by determination as a cation. The presence of the following thiols in urine has been confirmed: mercaptoacetic acid, 3-mercaptolactic acid, 2-mercaptopropionic acid, acetylcysteine, mercaptoethanol, cysteine, homocysteine and an un-identified amino thiol. Cysteamine and 3-mercaptopropionic acid could not be detected. Captopril, homocysteine and cysteine were determined quantitatively.     

Copper(II) (3,5-diisopropylsalicylate)2 oxidizes thiols to symmetrical disulfides and oxidatively converts mixtures of 5-thio-2-nitrobenzoic acid and nonsymmetrical 5-thio-2-nitrobenzoic acid disulfides to symmetrical disulfides

L-cysteine, D-penicillamine, and L-glutathione were oxidized to symmetrical disulfides in the presence of Cu(II)(3,5-DIPS)2 and air-oxygen at physiologic pH, 7.3. Air-oxygen caused the oxidation of thiol reduced copper, Cu(I), to Cu(II), as evidenced by expected spectrophotometric changes in these reaction mixtures. L-cysteine, D-penicillamine, and L-glutathione formed mixed disulfides and TNB with the addition of DTNB to solutions of these thiols. The observed order of reactivity for these thiols with DTNB was: L-cysteine greater than D-penicillamine greater than L-glutathione. Surprisingly, Cu(II)(3,5-DIPS)2 converted these mixed disulfides to their symmetrical disulfides and DTNB, and although the initial conversion rate was rapid, complete conversion required more than two hours. These observations suggest caution with regard to the spectrophotometric determination of thiols immediately after the addition of Ellman's reagent. These results also clarify an earlier report concerning the oxidation of thiols by Cu(II)(o-phenanthroline)2 and offer caution with regard to the determination of thiols using DTNB in the presence of copper complexes. Spectrophotometric data are provided in support of the suggestion that analysis of plasma or cellular samples for thiols be done in the absence of copper(II) complexes to avoid false negative results.     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary The fungus Fusarium aquaeductuum synthesizes large amounts of carotenoids only after illumination or in the presence of mercuribenzoate (HMB) in the dark. The effect of HMB is abolished entirely by the addition of excess thiols such as cysteine or mercaptoethanol; evidently active HMB must be present continuously within the cell. In contrast, photoinduction needs only a short exposure to light to set off pigment production in a subsequent dark period. Illumination of mycelia preincubated with HMB in the dark induces an additional proportion of carotenoid synthesis which shows the same kinetics and quantitative yields as that observed in untreated controls. This demonstrates that HMB, though inhibiting O₂-uptake by 50%, does not interfere with light-induced carotenoid production as such. The effects of light and HMB are additive under various experimental conditions tested. After addition of thiols to mycelia treated with HMB in the light, carotenoid production decreased to an extent equal to the amount mediated by HMB in the dark controls. These results provide strong evidence that the mechanisms and the action sites of light and HMB in carotenoid synthesis are different. Incubation with HMB after exposure to light reduces the agent's effectiveness, which declines gradually to about 30% during the first two hours.These findings are interpreted to mean that in Fusarium there are two isoenzyme systems at work in carotenoid production: a constitutive one showing very low net activity is subject to direct stimulation by HMB; light somehow eliminates this enzyme system and, at the same time, induces an isoenzyme set which exhibits high carotenogenic activity and is insensitive to HMB treatment.

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Folgende Abkürzungen werden im Laufe dieser Arbeit benutzt HMB p-Hydroxymercuribenzoat - CMB p-Chloromercuribenzoat - CMS p-Chloromercuriphenylsulfonsäure     

Thiol detection, derivatization and tagging at micromole to nanomole levels using propiolates

Thiols, simple and complex, including polypeptide and protein thiols, react rapidly and selectively with esters of propiolic acid under very mild conditions (aqueous buffer, room temperature, pH 7) to give thioacrylates. These stable derivatives exhibit strong, characteristic ultraviolet spectra, maximal at 280–290 nm, molar absorbance ca 12,500. In a single, simple experiment the thiol is detected, its amount is estimated, it is stabilized against oxidation and disulfide scrambling, it is converted into a derivative amenable to isolation and structure elucidation procedures, and it is tagged with recognizable ultraviolet and NMR characteristics generated de novo. The reagents are stable and inexpensive and the procedures are quick, easy, sensitive and selective.     

Regulatory and essential light-chain interactions in scallop myosin. I. Protection of essential light-chain thiol groups by regulatory light-chains

The reactivity of the thiol groups of the essential light-chains of scallop myosin is greatly reduced by the presence of regulatory light-chains on myosin. The thiol groups of the essential light-chains react with iodoacetate only if the regulatory light-chains have been removed by treatment with EDTA. No alkylation of the essential light-chains could be detected in myosins containing regulatory light-chains (untreated or reconstituted myosins) after an overnight incubation with excess iodoacetate at 4 °C. In contrast, similar treatment alkylated two to three thiol groups of essential light-chains in desensitized myosins from which the regulatory light-chains had been removed. In addition, up to seven of the 20 heavy-chain thiols were also alkylated; however, the reactivity of the heavy-chain thiols did not depend on the presence of the regulatory light-chains. ATPase activities were not inhibited by alkylation with iodoacetate. Regulatory light-chains also protected essential light-chain thiols against reaction with N-iodoacetyl-N^′-(l-sulfo-5-naphthyl) ethylenediamine and against dansylation at pH 6.7, although treatment with these reagents caused a considerable loss of ATPase activities. Rebinding of the regulatory light-chains was impaired by alkylation. The results indicate an extensive interaction between the regulatory and the essential light-chains in scallop myosin.     

Long-chain acyl thioesters prepared by solvent-free thioesterification and transthioesterification catalysed by microbial lipases

Long-chain acyl thioesters (thio wax esters) have been prepared in high (80% to more than 90%) yields by solvent-free esterification of fatty acids (lauric, myristic, palmitic and stearic acids) with long-chain thiols, such as decane thiol, dodecane thiol, tetradecane thiol and hexadecane thiol, catalysed by lipases from Candida antarctica (Novozym) and Rhizomucor miehei (Lipozyme) in the presence of a 0.4-nm molecular sieve. In the thioesterification reaction Novozym was a more effective biocatalyst than Lipozyme. The extent of thioesterification increased with increasing molar ratio of fatty acid to alkane thiol (1:1 to 3:1) and with temperature (40 °C compared to 60 °C), as well as with the amount of the enzyme preparation and the amount of 0.4-nm molecular sieve. Decreasing the chain length of the alkane thiol from C₁₆ to C₁₀ also increased the extent of thioesterification. Lipase-catalysed solvent-free transthioesterification of fatty acid methyl esters with alkane thiols was less effective for the preparation of acyl thioesters than was thioesterification of fatty acids with alkane thiols. In transthioesterification, Lipozyme was slightly more effective as a biocatalyst than Novozym. Received: 3 September 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

Kinetics of inhibition of aldehyde dehydrogenase isoenzymes of rat liver by fluorine-containing disulfides

New disulphides synthesized on the basis of dithiocarboxylic acid derivatives and heterocyclic thiols containing the fluorine atoms were studied as applied to inhibit aldehyde dehydrogenase (ALDH) isozymes of the rat liver mitochondria. The most effective rat liver inhibitors of ALDH isozymes were revealed. Inhibition of the rat liver isozymes by disulphides I, II, IV, VI-VIII and fluorinated pyridine disulphide was found to be irreversible. The values of isozyme inactivation rate constants are reported. The ALDH inhibition by disulphides I, IV, VI-VIII was competitive both for the cofactor and for the substrate of the reaction. The protective effect of the NAD+ against ALDH I and II inactivation by disulfiram and disulphides I, IV, VI-VIII and X is shown. NADP+ protects isozyme II against inactivation by disulfiram and also disulphides I, VI-VIII.     

Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols

Problems inherent in the isolation of thiols from natural sources, such as oxidation, undesirable addition reactions, and low concentration of thiol species in cell-free extracts, can be circumvented by reversible derivatization to a less labile form which can be concentrated selectively. These objectives are realized by converting thiols to heterodisulfides in which the thiol partner is an apolar thiol with strong affinity for hydrophobic stationary phases. When reacted with 2-S-(2(')-thiopyridyl)-6-hydroxynaphthyldisulfide at pH<5, where most thiol species are relatively stable to atmospheric oxidation, mixed disulfides with 2-mercapto-6-hydroxynaphthalene as the apolar partner are obtained in good yield and can be concentrated onto a hydrophobic stationary phase. Such heterodisulfides exhibit excellent chromatographic properties when separated on reversed-phase media and the derivatization reaction can, therefore, be conveniently monitored. Following their isolation as the heterodisulfides the thiol species of interest are recovered by reduction and facile separation from the apolar 2-mercapto-6-hydroxynaphthalene partner.     

Role of thiols in degradation of proteins by cathepsins.

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.     

Reduction of a disulfide bond of thrombospondin in the supernatant solution of activated platelets

Incubation of the material secreted by activated platelets leads to the formation of disulfide-linked dimers and multimers of one of the proteins, thrombospondin. To determine whether these complexes formed as a result of thiol-disulfide exchange (no change in the number of thiols) or of oxidation of thiols (a decrease in the number of thiols), the number of thiols in TSP was measured during formation of multimers. The number of thiols increased from about 3/mol to 4.8/mol. The half-time for the disappearance of monomers of thrombospondin was fourfold greater than the half-time for appearance of new thiols. The appearance of new thiols, as well as the formation of multimers, was inhibited by Ca2+. The appearance of new thiols was reversible; addition of Ca2+ reversed the process, and at pH 8, but not at pH 6 or 7, the appearance of new thiols spontaneously reversed. No new thiols formed during incubation of partially purified thrombospondin or after the supernatant solution had been treated with activated thiol-Sepharose to remove reactive thiol compounds. It is concluded that thrombospondin has a disulfide bond that is unstable in the absence of Ca2+. It can be attacked by a thiol of another molecule of thrombospondin to form disulfide-linked multimers, by a thiol of the same molecule of thrombospondin to generate isomerization of disulfide bonds or, as observed in this study, by another secreted thiol compound to give a mixed disulfide and a new thiol.     

Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: proteomic identification of target proteins

Protein thiols are targets of oxidative stress. Their modification was analysed in gill extracts of the mussel Mytilus edulis, exposed to menadione. Diagonal gel electrophoresis revealed two clusters of carbonylated proteins involved in interchain disulphide linkages. Immunoblotting identified these as being associated with protein disulphide isomerase (PDI) and actin and this was confirmed by immunoprecipitation. Protein free thiols (-SH) were identified in 2-DE separations by labelling with 5-iodoacetamidofluorescein (IAF). Cysteines involved in disulphide bridges were identified by blocking free -SH with N-ethylmaleimide, reducing disulphides with DTT and IAF labelling. Several protein spots containing free thiols disappeared on exposure to menadione. Conversely, new protein spots containing disulphides appeared in response to menadione which may be protective against oxidative stress. In-gel tryptic digestion followed by LC/MS-MS and database searching identified some of the free thiol targets: PDI; hsp gp96; calreticulin; heavy metal binding protein. Tubulin, PDI, enolase and gelsolin contained new disulphide bridges in response to menadione. Our findings indicate a protein level response to oxidative stress principally involving PDI, chaperone-like and cytoskeletal proteins. Since many environmental pollutants cause oxidative stress, studies on PDI and structural proteins may be particularly relevant to understanding toxicity in this popular sentinel species.     

The histochemistry of thiols and disulphides. IV. Protective fixation by organomercurial-formalin mixtures

Summary Formation of mercaptides as the result of adding organomercuric salts to neutral formalin used for fixation was found to protect protein thiols from autoxidation, provided the tissues were washed in distilled and not tap water. Such bloking, in contrast to that given by HgCl₂, could be reversed quantitatively by mercaptoethanol made strongly acid to keep it from reducing disulphides. However, some cleavage of disulphides by the mercurials themselves caused slight arbfactual thiol staining in a limited number of sites. Three of the nine compounds tested are sufficiently soluble to penetrate tissues with reasonable speed, stable enough to preclude more than incidental mercurial deposits and currently available commercially. Of them, the diuretic mercurial Mersalyl is at present the protecting, agent of choice since methyl- and ethylmercuric chlorides are too toxic to recommend for routine use.