The relevance of tissue thiol histochemistry to diagnostic hematopathology

Expression analyses suggest that alterations of the antioxidant state of some diffuse large B-cell lymphomas can assist prognosis; reversibly oxidized thiols may serve as a surrogate marker for identifying such cases. Little is known about the distribution of free thiols and reversibly oxidized thiols in human tissues. We developed a staining technique that enables visualization of tissue thiols in situ using bright field microscopy and validated it using gastrointestinal tissue specimens. We used our thiol staining technique to assess benign tonsillectomy and diffuse large B-cell lymphoma specimens. The gastrointestinal series revealed the presence of free thiols within epithelial cells and cells of the lamina propria. Staining for reversibly oxidized thiols was robust in gastric foveolar cells, intestinal goblet cells and the mucus they produce. Tonsillectomy specimens exhibited diffuse presence of free thiols. Staining for reversibly oxidized thiols was confined to germinal center macrophages and sinus histiocytes. Among the diffuse large B-cell lymphoma specimens, we observed strong staining for free thiols within malignant cells. By contrast to benign B-cells, the malignant cells demonstrated pronounced and diffuse staining for reversibly oxidized thiols. We demonstrated intrinsic differences between benign and malignant cells.     

ortho- and meta-substituted aromatic thiols are efficient redox buffers that increase the folding rate of a disulfide-containing protein

Thiol based redox buffers are used to enhance the folding rates of disulfide-containing proteins in vitro. Traditionally, small molecule aliphatic thiols such as glutathione are employed. Recently, we have demonstrated that aromatic thiols can further enhance protein-folding rates. In the presence of para-substituted aromatic thiols the folding rate of a disulfide-containing protein was increased by 4-23 times over that measured for glutathione. However, several important practical issues remain to be addressed. Aromatic thiols have never been tested in the presence of denaturants such as guanidine hydrochloride. Only two of the para-substituted aromatic thiols previously examined are commercially available. To expand the number of aromatic thiols for protein folding, several commercially available meta- and ortho-substituted aromatic thiols were studied. Furthermore, an ortho-substituted aromatic thiol, easily obtained from inexpensive starting materials, was investigated. Folding rates of scrambled ribonuclease A at pH 6.0, 7.0 and 7.7, with ortho- and meta-substituted aromatic thiols, were up to 10 times greater than those with glutathione. In the presence of the common denaturant guanidine hydrochloride (0.5M) aromatic thiols provided 100% yield of active protein while maintaining equivalent folding rates.     

Ab initio studies of the properties of intracellular thiols ergothioneine and ovothiol

Intracellular naturally occurring aromatic thiols such as ergothioneine and the ovothiols have been shown to play a variety of roles in cellular function. A detailed ab initio electronic structure analysis of these thiols is reported evaluating the thermodynamics of the reactions of these intracellular thiols with alkyl thiols, HO*, H2O2, ascorbate and their disulfides.     

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.     

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis.

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30 degrees C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.     

Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures

An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.     

Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d⁷-ω-bromoacetonylquinolinium bromide (d⁷-BQB). BQB and d⁷-BQB both rapidly and selectively reacted with thiols in acidic medium within 3 min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d⁷-BQB, respectively. The BQB- and d⁷-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI–MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI–MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI–MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27 nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers.     

1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one hydrobromide, a sulfhydryl-specific compound which reacts irreversibly with protein thiols but reversibly with small molecular weight thiols

1-p-Chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one hydrobromide (CDDP) has been shown to react selectively with small molecular weight and protein thiols. The reaction of this compound with thiols can be monitored directly owing to the large decrease (approximately 21,000 M-1 cm-1 at 310 nm) in extinction coefficient subsequent to thiol addition. CDDP reacted stoichiometrically with large molecular weight (greater than 11,000) protein thiols. However, with small molecular weight thiols (less than 500) the reaction was less than stoichiometric, indicating a significant degree of back-reaction. The forward and reverse rate constants have been estimated. The fact that the reaction is reversible enables CDDP to be used for the direct monitoring of the oxidation of small molecular weight thiols.     

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30°C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.     

Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate

Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a γ-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes.     

Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase

Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.     

Interaction of glutathione and other low-molecular-weight thiols with DNA: evidence for counterion condensation and coion depletion near DNA

The interaction of low-molecular-weight thiols with sonicated DNA was examined using spin filtration to concentrate the DNA. Cationic thiols (WR 1065 and cysteamine) behaved as counterions and were found to have increased concentrations in the DNA retentate relative to the filtrate. Anionic thiols (GSH, 2-mercaptoethanesulfonate, mercaptosuccinate) behaved as coions and were decreased in concentration in the DNA fraction. Concentrations of the uncharged thiol 2-mercaptoethanol were little influenced by DNA. The results demonstrate the importance of counterion condensation and coion depletion in determining the concentrations of charged species near DNA. They provide a rationale for enhanced effectiveness of WR 1065 and cysteamine as radioprotectors compared to neutral and anionic thiols and suggest that anionic thiols such as GSH should be poor radioprotectors of DNA.     

Moderate physical exercise induces the oxidation of human blood protein thiols

Exercise is known to induce the oxidation of blood low-molecular-weight (LMW) thiols such as reduced glutathione (GSH). We previously reported that full-marathon running induced a decrease in human plasma levels of protein-bound sulfhydryl groups (p-SHs). Moderate exercise, a 30-min running at the intensity of the individual ventilatory threshold, performed by untrained healthy females caused a significant decrease in erythrocyte levels of p-SHs (mostly hemoglobin cysteine residues) and LMW thiols, but their levels returned to each baseline by 2 h. No significant change in plasma LMW thiols was observed. However, plasma levels of p-SHs significantly decreased after running and remained unchanged after 24 h. These results suggest that moderate exercise causes the oxidation of blood thiols, especially protein-bound thiols.     

Activation energy determinations suggest that thiols reverse autooxidation of tetrahydrobiopterin by a different mechanism than ascorbate

In neutral aqueous solutions tetrahydrobiopterin is oxidized by dioxygen in a reaction that is succinctly described as autooxidation. Ascorbate and thiols moderate this reaction by reversing the oxidative process. In the present study the effect of various thiols on the apparent Arrhenius activation energy of tetrahydrobiopterin autooxidation was characterized and compared to that of ascorbate determined previously. We observed that - in sharp contrast to ascorbate - the efficiency of thiols to protect tetrahydrobiopterin decreased with the elevation of temperature from 22 to 37 degrees C. Accordingly, the apparent Arrhenius activation energies (in kJ/mol) measured in the presence of thiols were consistently greater than the value determined with tetrahydrobiopterin alone (59.6 +/- 1.4) or in the presence of ascorbate (59.9 +/- 2.8). Thus, the energy values were 88.8+/-1.1 with glutathione, 87.6 +/- 2.1 with N-acetylcysteine, 79.2 +/- 1.6 with cysteine, 75.1 +/- 2.4 with dithiotreitol and 70.3 +/- 0.9 with homocysteine. Since thiols are as potent reducing agents as ascorbate, these findings suggest that thiols and ascorbate protect tetrahydrobiopterin from oxidation acting at different steps of the oxidation process. It is likely that thiols reduce quinoidal dihydrobiopterin, whereas ascorbate scavenges the trihydrobiopterin radical to tetrahydrobiopterin. Furthermore, the results indicate that thiols are excellent tools to protect tetrahydrobiopterin from autooxidative decomposition in laboratory experiments conducted at relatively low temperatures, whereas the protective effect diminishes at 37 degrees C, i.e. under physiological conditions.     

Isotope-coded affinity tag approach to identify and quantify oxidant-sensitive protein thiols

An approach is described for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT reagent, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. To validate our approach, creatine kinase with four cysteine residues, one of which is oxidant-sensitive, was chosen as an experimental model. ICAT-labeled peptides derived from creatine kinase were used to evaluate the relative abundance of the free thiols in samples subjected (or not) to treatment with hydrogen peroxide. As predicted, hydrogen peroxide decreased the relative abundance of the unmodified oxidant-sensitive thiol residue of cysteine-283 in creatine kinase, providing proof of principle that an ICAT-based quantitative mass spectrometry approach can be used to identify and quantify oxidation of cysteine thiols. This approach opens an avenue for proteomics studies of the redox state of protein thiols.     

Therapeutic efficacy of green tea polyphenols on cellular thiols in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis

In cancer, a high flux of oxidants not only depletes the cellular thiols, but damages the whole cell as well. Epidemiological studies suggest green tea may mitigate cancers in human and animal models for which several mechanisms have been proposed. In the present investigation, the levels of cellular thiols such as reduced glutathione (GSH), oxidised glutathione (GSSG), protein thiols (PSH), total thiols, lipid peroxidation product conjugated dienes and the activity of gamma glutamyl transferase (GGT) were assessed in tongue and oral cavity. In 4-Nitroquinoline 1-oxide- (4-NQO) induced rats, there was a decrease in the levels of GSH, PSH and total thiols and an increase in the levels of GSSG, conjugated dienes and the activity of GGT. On supplementation of green tea polyphenols (GTP) for 30 days (200 mg/kg) for the oral cancer-induced rats, there was a moderate increase in the levels of GSH, PSH and total thiols and a decrease in the levels of GSSG, conjugated dienes and the activity of GGT. Thus, GTP reduces the oxidant production thereby maintains the endogenous low molecular weight cellular thiols in oral cancer-induced rats. From the results, it can be concluded that GTP supplementation enhances the cellular thiol status thereby mitigate oral cancer.     

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides

 Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is ɛ₅₂₀=34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells. Accepted: 17 December 1996     

Role of thiol/disulfide exchange in newcastle disease virus entry

Newcastle disease virus (NDV) entry into host cells is mediated by the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. We previously showed that production of free thiols in F protein is required for membrane fusion directed by F protein (S. Jain et al., J. Virol. 81:2328-2339, 2007). In the present study we evaluated the oxidation state of F protein in virions and virus-like particles and its relationship to activation of F protein by HN protein, F protein conformational intermediates, and virus-cell fusion. F protein, in particles, does not have free thiols, but free thiols were produced upon binding of particles to target cells. Free thiols were produced at 16°C in F protein in virions bound to the target cells. They also appeared in different fusion defective mutant F proteins. Free thiols were produced in the presence of mutant HN proteins that are defective in F protein activation but are attachment competent. These results suggest that free thiols appear prior to any of the proposed major conformational changes in F protein which accompany fusion activation. These results also indicate that HN protein binding to its receptor likely facilitates the interaction between F protein and host cell isomerases, leading to reduction of disulfide bonds in F protein. Taken together, these results show that free thiols are produced in F protein at a very early stage during the onset of fusion and that the production of free thiols is required for fusion in addition to activation by HN protein.     

Synthesis of stable cysteine-heterodisulphides

The reaction of Fmoc-Cys(Npys)-OH under acidic conditions with several thiols has been studied. Results show that aromatic thiols give stable heterodisulphides.