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Is Ellman's reagent specific for low molecular weight proteins and thiols?

Is Ellman's reagent specific for low molecular weight proteins and thiols?



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Is it still possible to quantify cystein rich low molecular weight proteins such as Metallothionein in a given sample using Ellman's reagent if the sample is contaminated with some high molecular weight proteins?


It's specific for thiols in general. If the contaminating proteins contain solvent accessible thiols, DTNB will react with them.


Introduction

Thiols, also known as mercaptans, are a class of organic compounds that contain a sulfhydryl group (–SH) composed of a sulphur atom and a hydrogen atom attached to a carbon atom [1]. The plasma thiol pool is mainly formed by albumin thiols, protein thiols and slightly formed by low-molecular-weight thiols such as cysteine (Cys), cysteinylglycine, glutathione, homocysteine and γ-glutamylcysteine [2].

Thiols (RSH) can undergo oxidation reaction via oxidants and form disulphide (RSSR) bonds [3]. A disulphide bond is a covalent bond the linkage is also called a SS-bond or disulphide bridge. Under conditions of oxidative stress, the oxidation of Cys residues can lead to the reversible formation of mixed disulphides between protein thiol groups and low-molecular-mass thiols. The formed disulphide bonds can again be reduced to thiol groups thus, dynamic thiol–disulphide homeostasis is maintained [4].

Dynamic thiol disulphide homeostasis status has critical roles in antioxidant protection, detoxification, signal transduction, apoptosis, regulation of enzymatic activity and transcription factors and cellular signalling mechanisms [5], [6]. Moreover, dynamic thiol disulphide homeostasis is being increasingly implicated in many disorders. There is also a growing body of evidence demonstrating that an abnormal thiol disulphide homeostasis state is involved in the pathogenesis of a variety of diseases, including diabetes [7], cardiovascular disease [8], cancer [9], rheumatoid arthritis [10], chronic kidney disease [11], acquired immunodeficiency syndrome (AIDS) [12], Parkinson's disease, Alzheimer's disease, Friedreich's ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis [13], [14], [15] and liver disorder [16]. Therefore, determination of dynamic thiol disulphide homeostasis can provide valuable information on various normal or abnormal biochemical processes.

The plasma thiol level is most commonly measured using the classical Ellman reagent, 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB). This compound is stoichiometrically reduced by free thiols in an exchange reaction, forming a mixed disulphide and releasing one molecule of 5-thionitrobenzoic acid, which can be measured at 412 nm [17]. An alternative reagent to DTNB is 4,4′-dithiodipyridine (4-DPS) [18]. Reduction of 4-DPS leads to 4-thiopyridone tautomer, and this can be measured at 324 nm, which is a near ultraviolet wavelength. This wavelength cannot be used by automated analysers because the lowest wavelength is 340 nm in all automated analysers used in clinical chemistry laboratories.

To the best of our knowledge, there is no automated colourimetric measurement method for plasma/serum dynamic disulphide levels [19]. In a few recent studies, the disulphide and thiol levels of low-molecular-weight disulphide compounds of plasma have been determined using high-performance liquid chromatography (HPLC) [20], [21], fluorescence capillary electrophoresis [22] and bioluminescent systems [23]. In these sophisticated systems, separation processes such as the removal of the remaining reductants, which are NaBH4, Tris(2-carboxyethyl)phosphine (TCEP) and tributylphosphine, as well as precipitation of proteins, are also needed [19]. These pretreatment applications and measurement procedures are time-consuming, labour-intensive and costly, and require complicated techniques.

In this study, a novel and automated assay determining dynamic thiol/disulphide homeostasis is described and a new test cluster concept containing –S–S–, –SH, –S–S–/–SH, –S–S–/(–SH + –S–S–) and –SH/(–SH + –S–S–) is introduced.


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

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Note: In lieu of an abstract, this is the article's first page.


Abstract

Thiol groups in biological molecules play a significant role in various physiological functions and pathological conditions. Thiols are divided into two major groups: protein thiols and nonprotein thiols. Numerous methods have been reported for thiol assays. Most of these methods have been developed for glutathione, the principal nonprotein thiol, despite the fact that cellular protein thiols are more abundant than glutathione. Further, these methods usually involve a process of biological sample preparation followed by a separation method, and they are time-consuming. We reported previously a series of thiol-specific fluorogenic benzofurazan sulfides. These nonfluorescent benzofurazan sulfides react rapidly and specifically with a thiol to form a strong fluorescent thiol adduct. The rapid reaction, thiol-specific and fluorogenic nature of the sulfides successfully yielded an application of one of the sulfides for relative quantitation of total thiols in live cells through fluorescence microscopy. In this work, we employed the same compound to develop the first high-throughput method for simultaneous monitoring of protein thiols, nonprotein thiols, and total thiols in cells in a 96-well plate on a fluorescence microplate reader at λex = 430 nm and λem = 520 nm, respectively. The method is rapid and sensitive, and has been validated by an HPLC thiol assay method. The method can detect thiols with cell concentrations as low as 500 cells/well. We also demonstrated that the method can readily monitor changes in cellular thiol levels. Although the method cannot provide an absolute quantification for thiols because fluorescence intensity of different thiol adducts varies, it provides an accurate measurement of relative quantification, relative to the control. The method will be a valuable tool in thiol-related biomedical/pharmaceutical research.


Materials

All reagents and chemicals were of analytical or higher grade. Ellman reagent, (5,5&rsquo-dithio-bis-(2-nitrobenzoic acid), 2-mercaptoethanesulfonic acid, sodium salt (MES) and other chemicals used were obtained from VWR Chemicals (BDH Prolab) and Sigma Aldrich.

ODS AQ silica gel (product 12S50) was purchased from YMC Co. Ltd. Kyoto, Japan. Bio-Gel P2 from Bio-Rad Laboratories Inc.

All solvents used were of analytical grade supplied by Sigma Aldrich or Merck. Solvents for HPLC were Merck LiChroSolv grade. Europe GMBH.

Large glass columns were supplied by Soham Scientific, Fordham, Ely, Cambs., UK

LNCaP (androgen-sensitive human prostate adenocarcinoma cells, clone FGC-ECACC no. 89110211) and PNT2 cells (a normal human prostate epithelial cell line immortalized with SV40 virus) were purchased from the Public Health England Laboratories (ECACC-HPA) at Porton Down, Salisbury, England. Cells were grown to confluence in cell factories in a medium consisting of RPMI 1640 + 2mM glutamine + 1mM sodium pyruvate containing 10% Zone 2 FBS. The confluent cells were given a two-hour incubation in fresh medium before harvesting by trypsinization (Tryple Express). Cell counts were performed on a Nucleocounter NC3000 and the cells collected by centrifugation. The resultant cell pellets were snap frozen and stored at -80 0 C until required.


Results

In this report, we used N-acetylcysteine (NAC) and the thiol-containing redox protein, human thioredoxin-1 (Trx1), to test whether MB is thiol-reactive and covalently modifies protein thiols. Results ( Fig. 1 A) show that free thiol concentration decreased as a function of MB concentration with a complete loss when MB:NAC reached 2:1. A kinetic assay showed that the apparent 2nd order rate constant was 5.03 M -1 s -1 (data not shown). We next examined the reactivity of MB with protein thiol using recombinant Trx1 as a model. This protein contains thiol residues that are solvent accessible, have a spectrum of reactivity and are essential for its biological function [12]. Fig. 1 B shows a stoichiometric loss of protein thiols due to MB treatment. Trx1 contains 5 Cys residues and the biotin signal was lost when incubated with MB:Trx1 at a molar ratio of 5:1, indicating complete modification of thiols (either adduction or oxidation, see below). Reactivity of MB to primary amines was also investigated ( Fig. 1 C) using a procedure that is similar to visualizing protein thiols. Following MB incubation, the free amines were labeled and visualized using sulfo-NHS-biotinਊs demonstrated in  Fig. 1 B. These results show that MB does not react with amines under the conditions of the assay, preferentially modifying only protein thiols. The kinetics of the reaction was investigated with 5:1 concentration ratio using the mPEG2-biotin method ( Fig. 1 D). In agreement with the NAC reactivity data, results from this experiment show that the reaction between MB and Trx1 is relatively slow, approaching complete modification of all Trx1 thiols at 60 min.

Maneb (MB) is a thiol-reactive compound. (A) Addition of MB to N-acetylcysteine causes loss of thiol content as measured by Ellman's reagent. Maneb at 2:1 M ratio resulted in nearly complete loss of thiol. (B) Using thioredoxin-1 (Trx1) as a model protein, maneb stoichiometrically causes loss of protein thiols. Protein thiols labeled with mPEG2-biotin, a biotinylated N-ethylmaleimide, and visualized in Western blot analysis with fluorescently labeled streptavidin. (C) MB does not modify amines as detected by reaction of free amines with sulfo-NHS-biotin and subsequent Western blot analysis with fluorescently labeled streptavidin. (D) A time course demonstrates that maneb causes loss of most free thiols within 15�?min.

The activity of Trx1 was assessed to determine functional consequences of MB adduction. Trx1 was incubated for 1 h with MB (5:1, as above) and then desalted to remove any unreacted MB prior to the activity assay. Data show that MB modification of Trx1 slowed the Trx1-dependent oxidation of NADPH in the presence of Trx reductase by 43% ( Fig. 2 A and B). In contrast to these results, Trx1-catalyzed insulin reduction by DTT showed no effect on activity due to MB modification (data not shown). The incomplete inhibition of activity despite evidence for nearly complete modification of thiols indicated that MB-dependent thiol modification is likely to be reversible.

Maneb inhibits thioredoxin-1 (Trx1) activity. Trx1 was treated with 5 M equivalents of maneb and activity was measured in terms of insulin reduction in the presence of NADPH and thioredoxin reductase-1. Decreased activity was evident in the rate of NADPH oxidation (A) and the calculated activity (B). Assay with DTT as the reductant instead of the NADPH/thioredoxin reductase did not show inhibition of activity (not shown). Loss of protein thiols is associated with formation of a Trx1 dimer and reversed by addition of a reductant. Both the reductant DTT (C) and N-acetyl cysteine (D) can reverse maneb-mediated loss of protein thiols.

To examine reversibility, MB-modified Trx1 was incubated with 1 mM DTT, and thiol content was examined with mPEG2-biotinylation and Western blotting as above. Results showed that the band corresponding to the unmodified thiol form of Trx1 was restored ( Fig. 2 C). Titration with NAC (increasing the ratio of NAC:MB from 0 to 13.3) showed that greater than a 5-fold excess of NAC was required to restore all of the thiols ( Fig. 2 D). The results, therefore, show that modification of Trx1 by MB is completely reversible by treatment with thiols.

X-Ray crystallography showed that oxidized Trx1 is crystalized as a dimer formed by a disulfide between C73 residues [14]. We investigated the possibility that MB treatment caused formation of a Trx1 dimer by treating Trx1 with increasing concentrations of MB, separation via SDS-PAGE under non-reducing conditions and visualization with Coomassie blue ( Fig. 3 A). The data demonstrate that MB results in appearance of a band at 25 kD, corresponding to twice the molecular weight of Trx1, with as little as 1 M equivalent of MB. This result indicates that only one Cys residue is involved in the dimerization.

MB treatment causes Trx1 to form a dimer that is reversed by DTT (A). Mass spectral analysis of intact Trx1 (B) and MB-modified Trx1 shows (C) a single MB modification (addition of 210 mass units corresponding to the addition of the ethylene bis-dithiocarbamate (EBDTC)). The asterisk (*) denotes an m/z corresponding to a potential Mn-Trx1 adduct (+54.9).

Due to the observation of MB-mediated protein cross-linking, we conducted LC–MS studies of intact, MB-treated Trx1 using ESI in the positive ionization mode and detection with an LTQ-Orbitrap-Velos (Thermo). These experiments resulted in the detection of Trx1 and a single modified product ( Fig. 3 B and C). In the MB-modified sample, we observed a large decrease in intensity of the unmodified Trx1 peak (m/z 11,602) and a new peak (m/z 11,810) caused by the binding of ethylene bis-dithiocarbamate (EBDTC) to Trx1, resulting in a mass shift of 210 mass units from the unmodified peak. This result suggests that MB does not cause a simple oxidation of thiols to disulfides but rather participates in more complex reaction processes.


Abstract

Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme’s catalytic cysteine thiolate group. Importantly, low-molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively inactivated PTPs (PTP1B and SHP-2) by various low-molecular weight thiols and the enzyme thioredoxin. All monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. In general, molecules bearing low-pKa thiol groups were particularly effective. The biological thiol glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M –1 s –1 , while the dithiol dithiothreitol (DTT) displayed an apparent second-order rate constant of 0.325 ± 0.007 M –1 s –1 . The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 μM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10 –3 s –1 . The rates at which these agents regenerated oxidized PTP1B followed the order Trx > DTT > GSHand comparable values observed at 2 μM Trx, 4 mM DTT, and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1–20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M –1 s –1 . We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low-molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.


RESULT AND DISCUSSION

We strongly believe that only the development of chemical probes capable of selectively trapping SO2H will allow a clear elucidation of the role of protein sulfinylation. In this connection, we recently developed chemoselective S ulfinic acid N itroso L igation (SNL). 16 The addition of SO2H to C-nitroso compounds has been known for more than a century however, the resulting adduct is base-labile (Figure S1). In order to trap this unstable species, we have incorporated an electrophilic center ( Figure 1 ) in the ortho-position of a nitroso-benzene derivative (1). The transient oxyanion (2) reacts with the ester by intramolecular trans-esterification to form a stable benzisoxazolone (3). Basing our work upon this idea, we have synthesized a class of C-nitroso compounds that show fast reactivity with low molecular weight SO2H. These reagents do not react with other biologically relevant nucleophiles aside from thiols, which, however, do not form stable adducts (Figure S2).

Chemoselective labeling of sulfinic acid with aryl-nitroso compounds.

Using SNL for labeling protein sulfinic acids

Encouraged by these results, we employed SNL to develop chemical probes for detection of protein sulfinylation. First of all, we explored the ability of NO-Ph ( Figure 2 ), the C-nitroso derivative that has shown the best reactivity, to modify SO2H within the double mutant (C64,82S) of the thiol peroxidase Gpx3 from yeast. 17 In the presence of H2O2, Gpx3 forms an intramolecular disulfide bond through sulfenylation of catalytic C36, followed by condensation with the resolving C82. Mutation of C82 to serine stabilizes transient Cys36-SOH, allowing its further controlled oxidation to SO2H (see Supporting Information).

Nitroso probes for selective labeling of protein sulfinic acids.

Incubation of NO-Ph with C64,82S Gpx3-SO2H (22772 Da) yields the expected sulfonamide adduct (22949 Da) as confirmed by ESI-LC/MS analysis ( Figure 3A ). Our preliminary experiments with small molecules have shown that thiols react with C-nitroso compounds to yield an unstable sulfenamide adduct, which is cleaved by reaction with a second thiol (Figure S2). In order to confirm these results with protein-SH, we treated fully reduced C64,82S Gpx3 (22740 Da) with NO-Ph, followed by incubation with DTT. Surprisingly, ESI-LC/MS revealed the formation of a stable adduct with a mass of 22935 Da ( Figure 3B ). Alkylation of the Cys residue with N-ethylmaleimide (NEM), conversely, prevented adduct formation ( Figure 3C ). Even considering that DTT was unable to cleave the sulfenamide formed by the addition of NO-Ph to Cys36, the detected mass increase (Δm = 195) does not correspond to the expected adduct (Δm = 177). Generally, addition of thiol to C-nitroso aryl compound yields an unstable semimercaptale, which can react with a second thiol molecule or undergo rearrangement to form a more stable sulfinamide (Figure S3). 18 Spontaneous rearrangement occurs via dissociation of a hydroxyl anion and formation of a cationic nitrenium ion intermediate, which is later hydrolyzed by water. Following the same pathway, the addition of NO-Ph to C64,82S Gpx3-SH would form a sulfinamide adduct with a mass increase of 195 Da, which corresponds exactly to our observations. Acidic environment usually favors semimercaptale rearrangement. With NO-Ph, however, once the benzisoxazolone is generated, the rearrangement appears to occur even at neutral pH, probably because the carboxylate group is much more prone to dissociate from the nitrogen atom than is the hydroxyl anion (Figure S4). The formation of the rearranged sulfinamide would also explain why the adduct was not reduced by DTT. Since sulfinamide formation was never observed with low molecular weight thiols, 16 we wondered why the rearrangement occurred with protein-SH. We speculated that, in such cases, the attack of a second thiol molecule on the transient sulfenamide is generally faster than the rearrangement of the latter. Once the sulfenamide is formed, however, the attack of a second Cys-SH would be precluded with the use of C64,82S Gpx3-SH because of steric hindrance. To verify this hypothesis, we tested the reactivity of NO-Ph toward C64S Gpx3, which has both redox-active and resolving cysteines. As expected, the incubation of C64S Gpx3 with NO-Ph exclusively promoted the formation of the internal disulfide ( Figure 3D ). The presence of the resolving Cys, which can easily interact with the sulfenamide adduct formed with catalytic Cys, prevents the rearrangement of the latter. This result suggests that the formation of stable sulfinamide and disulfide reflects competitive reaction pathways influenced by kinetic factors (Figure S5). As additional proof, we incubated 2-methyl 2-propanethiol with an excess of NO-Ph. In this case, we speculated that, because the interaction of two molecules of thiols would be more hampered for steric hindrance, sulfenamide rearrangement would be facilitated. As anticipated, LC-MS analyses showed formation of the expected sulfinamide adduct (Figure S6).

ESI-LC/MS spectra of (A) C64,82S Gpx3-SO2H, (B) C64,82S Gpx3-SH, (C) C64,82S Gpx3-S-NEM and (D) C64S Gpx3-SH before and after treatment with NO-Ph. Each Gpx3 form was incubated with NO-Ph (40 equivalt.) for 1 h at room temperature in PBS pH 7.4.

Selective block of free cysteine residues

Sulfenamide rearrangement apparently limits the use of SNL for protein sulfinylation detection. As the experiment with NEM suggests, however, protection of the free cysteines can be employed to prevent formation of non-reducible adducts with hindered thiols. In fact, many chemical methods for detection of specific thiol modifications (e.g., S-nitrosylation) involve selective blocking of reduced thiols. 19 The success of these assays relies on the selectivity of the thiol-blocking step and the reagent’s efficiency in fully protecting free thiols without cross-reacting with SO2H. We evaluated the reactivity of several thiol-blocking reagents toward C64,82S Gpx3-SO2H. When we used a large excess of common alkylating agents such as NEM or iodoacetamide (IAM), ESI-LC/MS analyses detected small but significant amounts of alkylated sulfinic acid (Figures S7A and S7B). Although this result may seem unexpected, the reaction of low molecular weight SO2H with Michael acceptors and a-halo carbonyl compounds has been reported. 20 Conversely, sulfhydryl reactive compounds that promote mixed-disulfide formations, such as 2,2′-dipyridyl disulfide (DPS) and S-methyl methanethiosulfonate (MMTS), showed no cross-reactivity toward SO2H (Figures S7C and S7D). Next, we examined whether protection of free Cys residues as disulfides was sufficient to prevent cross-reactivity with NO-Ph. After C64S,82S Gpx3-SH was pre-incubated with DPS (Figure S8A) or MMTS (Figure S8B), the excess of thiol-blocking reagents was removed and the protein was incubated with NO-Ph. ESI-LC/MS analyses confirmed that both DPS and MMTS efficiently blocked formation of the sulfinamide adduct with the reduced protein.

Design and synthesis of NO-Bio

Having established that SNL can be efficiently employed for labeling protein SO2H, we designed and synthesized NO-Bio ( Figure 2 ). The new chemical probe combines the C-nitroso warhead (blue) with a biotin handle (violet), which allows detection of protein sulfinylation in biological samples. The synthesis of NO-Bio (Figure S9), which is described in detail in the Supporting Information, involved the coupling of commercially available Biotin-PEG4-NHS with a diamino-linker, N-tert-Butoxycarbonyl-1,6-hexanediamine. The protected amino group was then cleaved by TFA treatment, and the generated primary amine was coupled with the N-succimidyl ester of NO-Ph to yield NO-Bio, which was purified by reverse-phase HPLC.

Development of a chemical approach for protein sulfinic acid detection

As Figure 4A shows, protein sulfinylation could be selectively detected by a two-step method. In the first, a sulfhydryl-reactive compound (DPS or MMTS) is introduced to selectively block free Cys residues. Thereafter the sample is treated with the biotin-tag probe, NO-Bio, to label sulfinic acids. We tested this approach using recombinant DJ-1 as model. The Parkinson’s associate protein has a conserved Cys residue, C106, which is extremely sensitive to oxidative stress and tends to form a stable SO2H. Many studies demonstrate that DJ-1 protects cells against oxidative stress-mediated apoptosis through the formation of C106-SO2H. 21,22 In addition, DJ-1 contains two other free Cys residues, C46 and C53, which are not redox-active. Though C53 is not modified by ROS, it is still very reactive toward electrophiles. 23 Accordingly, DJ-1 represents an excellent model for testing the selectivity of our strategy. Reduced or oxidized WT DJ-1 was incubated with DPS, following by treatment with NO-Bio. As shown in Figure 4B , mass analysis clearly confirmed the selective modification of the solely oxidized DJ-1. Interestingly, DPS promoted the formation of an internal disulfide between C46 and C53. We speculated that DPS would first react with the highly solvent-exposed C53 to yield a mixed-disulfide. Later, the relatively more deeply buried C46 would attack the active disulfide with consequent generation of an internal disulfide bond (Figure S10). Selective protection of the free Cys can be achieved using MMTS as well (Figure S11). However, our results indicate that MMTS reacts with thiols at a relatively slower rate than does DPS. In fact, small amounts of Cys-SO2H were detected even in the reduced sample, which indicates that C106 was partially oxidized during thiol blocking. Although addition of EDTA in the buffer prevented this unwanted oxidation, we opted to use the more efficient DPS in all subsequent experiments. The biotin handle of the probe allows visualization of the labeled proteins by streptavidin blotting. Therefore, the selectivity of NO-Bio labeling was also confirmed by Western blot analysis. Reduced or oxidized DJ-1 was treated with DPS, followed by incubation with NO-Bio. The reactions were then subjected to SDS-PAGE and analyzed by streptavidin blotting. Treatment of oxidized DJ-1 with NO-Bio afforded selective protein labeling, while DJ-1 was not detected at all by streptavidin blotting in the absence of the oxidant, demonstrating the specificity of our chemoselective approach (Figure S12). NO-Bio showed also higher sensitivity in comparison to a commercially available antibody against hyperoxidized DJ-1 (Figure S13), allowing detection of sulfinylated DJ-1 at relative low concentrations.

Labeling of DJ-1 sulfinic acid with NO-Bio.

DJ-1 possesses a highly conserved G18 residue, which facilitates the ionization of C106, reduces its pKa, and helps stabilize C106-SO2H. Small changes in this position can drastically influence the oxidative properties of C106. 24 For example, the E18D DJ-1 mutant has a lower propensity to form SO2H, but the structurally similar E18N mutant shows an increased oxidation propensity thanks to a strong stabilization of C106-SO2H. We evaluated the sensitivity of NO-Bio, probing the different oxidation propensities of various DJ-1 mutants including C106S DJ-1, which does not contain a redox-active cysteine. Each DJ-1 variant (WT, E18N, E18D and C106S) was exposed to H2O2, treated with DPS, and finally incubated with NO-Bio. Western blot analysis was consistent with expected results ( Figure 4c ). E18N DJ-1 showed a higher fraction of sulfinylation, even in the absence of H2O2. The sulfinylation level of the E18D mutant exposed to oxidative stress, in contrast, was almost negligible. It is worth noting that C106S DJ-1 treated with H2O2 was not detected by streptavidin blotting, confirming that only C106 is able to form SO2H under relatively mild oxidation conditions.

NO-Bio detects sulfinic acid-modified proteins in cell lysate

Having established the specificity and sensitivity of our two-step approach in homogenous protein solutions, we next investigated whether NO-Bio could detect protein-SO2H in a complex, unfractionated cell lysate. To this end, we tested our optimized chemistry in a whole human cervical cancer (HeLa) cell extract, which was obtained by lysing the cells in modified RIPA buffer containing Catalase and DTT. The reducing lysis buffer prevents further oxidation of SO2H and maintains free Cys in the reduced form, avoiding overestimation of protein sulfinylation. Figure 5A shows an HRP-streptavidin western blot, which indicates that robust levels of protein SO2H can be detected under normal conditions. To demonstrate that DPS efficiently trapped all free thiols and therefore that the streptavidin blot revealed only sulfinylated proteins, we employed iodoacetyl-PEG2-biotin (IAM-Bio). The biotinylated reagent is able to alkylate thiols such as free Cys residues. Pre-treatment of the sample with DPS completely abrogated the IAM-Bio-dependent signal ( Figure 5A , Lane 3), which indirectly proved that NO-Bio reacted only with protein SO2H. Next, we determined whether our chemical approach could detect increases in protein sulfinylation in human cell culture. HeLa cells were incubated with increasing amounts of H2O2 for 15 minutes (this time point was chosen after a preliminary time-dependent experiment – Figure S14A), lysed, and then labeled as described above. Western blot analysis showed that the level of protein sulfinylation was increased by H2O2 in a dose-dependent fashion (Figure S14B). Taken together, these results confirm that SO2H is stable enough to be successfully detected in cell lysates and does not require in vivo labeling.

Reactivity of No-Bio in cell lysates - 5 μg protein loaded per lane - (A). Analysis of protein sulfinylation in human lung tumor tissue lysates - 1 μg protein loaded per lane - (B). Legend: T1-001-T1 Papillary adenocarcinoma T1-001-N1 Matched normal tissue T1-005-T1 Adenocarcinoma T1-005-N1 Matched normal tissue T1-013-T1 Adeno-squamous cell carcinoma T1-013-N1 Matched normal tissue.

Protein sulfinylation levels in human lung cancer

In order to show that our method can be applied to more complex biological questions, we performed comparative sulfinic acid profiling in human lung tumor tissue. For these experiments, protein sulfinylation was characterized by western blot analysis of whole-cell lysates. Our results showed a highly variable presence of SO2H among the three patient tumor tissue samples ( Figure 5B ). All three tumor tissue (papillary adenocarcinoma, adenocarcinoma, and adeno-squamous cell carcinoma) exhibited significant increase in the extent of SO2H modifications vs. matched normal tissue. Although the number of samples was too small to draw broad conclusions, these initial observations suggest that elevated levels of SO2H could be used as a cancer marker.


Redox and Thiols in Archaea

This is a valuable review that brings together a range of information concerning the biochemistry of thiols and their distribution in prokaryotes, with particular emphasis on Archaea. The review is well organised and written, except for some very minor edits as suggested below

  • Be consistent with gamma-glutamylcysteine abbreviation throughout
  • p.11, l. 352 unclear sentence
  • p.12, l. 364 add UDP in Appendix A
  • p.12, l. 391 closing round bracket missing

Comments and Suggestions for Authors

This is a valuable review that brings together a range of information concerning the biochemistry of thiols and their distribution in prokaryotes, with particular emphasis on Archaea. The review is well organised and written, except for some very minor edits as suggested below

Be consistent with gamma-glutamylcysteine abbreviation throughout

Response: We now use &gammaGC consistently throughout the manuscript. We no longer alternate with &gamma-GC.

p.11, l. 352 unclear sentence

Response: We corrected the sentence with stating cysteine instead of CoA. This correction now clarifies the sentence.

p.12, l. 364 add UDP in Appendix A

Response: UDP is now defined in Appendix A.

p.12, l. 391 closing round bracket missing

Response: The closing round bracket is now added.

Manuscript antioxidants-775988 by Rawat and Maupin-Furlow

For the domains of Eukarya and Bacteria various studies of low molecular weight (LMW) thiols have been published in the past, leading to a better understanding of the role and biochemistry of LMW thiols in these organism groups. Therefore, it is known that LMW thiols play important roles such as regulating the redox homeostasis inside cells or as cofactors. However, for Archaea not much is known about the role and presence of LMW thiols until now. This manuscript gives an overview on the state of art of LMW thiols in Archaea. The authors summarize the distribution of currently known LMW thiol classes in archaea and the role and biochemistry of single molecules. The article is well structured and supported by well-designed figures.

The only issue I have in terms of content is that I missed somewhat a small paragraph on thioredoxins in Archaea. Specifically for the methanogens, some literature is available. The authors could easily integrate it into paragraph 3.2 or split this paragraph.

  1. 67 Transition between the two sentences is missing/not fluent: &lsquo&hellipare found in domestic animals or humans [25]. Variations of GSH exist&hellip&rsquo please rewrite
  2. 276-276 to me this sentence sounds like DTNB reduces the -SH groups of present LMW thiols: &lsquo&hellipusing DTNB, which would reduce all -SH groups&hellip&rsquo, the -SH groups represent the reduced form, to my knowledge DTNB itself gets reduced whereas the LMW thiols gets oxidized by forming a mixed disulfide please clarify what is meant here
  3. 311-312 not the protein is induced but the expression of ohrA
  4. 332-333 transition between the two paragraphs is missing/not fluent please rewrite
  5. 334 the Fiege and Frankenberg-Dinkel article showed that RdmS is not a histidine kinase but a tyrosine kinase
  6. 371-375 sentence difficult to follow and understand, or is there meant to be a period instead of a comma in l. 374 &lsquo&hellip[121,122], Mtr,&hellip&rsquoMtr please rewrite

Inconsistent usage of &lsquoArchaea&rsquo and &lsquoarchaea&rsquo, not clear to me why sometimes with capital letter and sometimes not, e.g. l.127 &lsquoArchaea and bacteria&rsquo. Might be used to differ between the domain Archaea and archaea as organisms in general.

  1. 30 such as
  2. 38 the canonical pathway
  3. 76 auto-oxidation
  4. 107 Either &lsquoThe InterPro database&rsquo or omit &lsquoThe&rsquo
  5. 109 Consistent naming of citations in running text, either &lsquo&&rsquo or &lsquoand&rsquo between names of authors
  6. 140 &lsquobased on&rsquo instead of &lsquobased in&rsquo
  7. 187 is there an &lsquoas&rsquo missing: &lsquocan also act as a&rsquo?
  8. 202 &lsquoinsulin&rsquo instead of &lsquoinulin&rsquo
  9. 210 InterPro
  10. 266 &lsquosequences&rsquo
  11. 275 introduction of the abbreviation &lsquoDTNB&rsquo is missing
  12. 281 introduction of the abbreviations &lsquoDHA&rsquo and &lsquoHED&rsquo are missing
  13. 322 introduction of the abbreviation &lsquoDTT&rsquo is missing
  14. 333 correct citation name
  15. 358 &lsquothan&rsquo instead of &lsquothat&rsquo
  16. 359 &lsquoeven at&rsquo instead of &lsquoat even&rsquo
  17. 387 &lsquoto involve to&rsquo?
  18. 390-391 &lsquoan archaeal&rsquo
  19. 411 better: &lsquosome archaeal species&rsquo

Fig. 2 grey boxes: shade of gray somewhat darker for better contrast

Fig. 3, legend: please add the explanation of FTR with iron-sulfur cluster

For the domains of Eukarya and Bacteria various studies of low molecular weight (LMW) thiols have been published in the past, leading to a better understanding of the role and biochemistry of LMW thiols in these organism groups. Therefore, it is known that LMW thiols play important roles such as regulating the redox homeostasis inside cells or as cofactors. However, for Archaea not much is known about the role and presence of LMW thiols until now. This manuscript gives an overview on the state of art of LMW thiols in Archaea. The authors summarize the distribution of currently known LMW thiol classes in archaea and the role and biochemistry of single molecules. The article is well structured and supported by well-designed figures.

The only issue I have in terms of content is that I missed somewhat a small paragraph on thioredoxins in Archaea. Specifically for the methanogens, some literature is available. The authors could easily integrate it into paragraph 3.2 or split this paragraph.

Response: Thanks, we have altered this section of the manuscript to enhance organization and highlight the insight provided on thioredoxins in Archaea including reference to methanogens.

67 Transition between the two sentences is missing/not fluent: &lsquo&hellipare found in domestic animals or humans [25]. Variations of GSH exist&hellip&rsquo please rewrite

Response: Thanks, we have rewritten these sentences to emphasis the theme of this section is focused on GSH synthesis.

276-276 to me this sentence sounds like DTNB reduces the -SH groups of present LMW thiols: &lsquo&hellipusing DTNB, which would reduce all -SH groups&hellip&rsquo, the -SH groups represent the reduced form, to my knowledge DTNB itself gets reduced whereas the LMW thiols gets oxidized by forming a mixed disulfide please clarify what is meant here

Response: we now state that DTNB would presumably oxidize all of the &ndashSH groups.

311-312 not the protein is induced but the expression of ohrA

Response: we now state that it [leads to the induction of expression of the gene encoding OhrA]

332-333 transition between the two paragraphs is missing/not fluent please rewrite

Response: transition now added.

334 the Fiege and Frankenberg-Dinkel article showed that RdmS is not a histidine kinase but a tyrosine kinase

Response: this point is now corrected.

371-375 sentence difficult to follow and understand, or is there meant to be a period instead of a comma in l. 374 &lsquo&hellip[121,122], Mtr,&hellip&rsquoMtr please rewrite

Response: we have reorganized this paragraph to more clearly explain this section of the manuscript.

Inconsistent usage of &lsquoArchaea&rsquo and &lsquoarchaea&rsquo, not clear to me why sometimes with capital letter and sometimes not, e.g. l.127 &lsquoArchaea and bacteria&rsquo. Might be used to differ between the domain Archaea and archaea as organisms in general.

Response: we now consistently use archaea in a general manner - since we do not specifically state Archaea domain in the text.

Response: corrected to autoxidation.

107 Either &lsquoThe InterPro database&rsquo or omit &lsquoThe&rsquo

109 Consistent naming of citations in running text, either &lsquo&&rsquo or &lsquoand&rsquo between names of authors

Response: corrected to all &lsquoand&rsquo.

140 &lsquobased on&rsquo instead of &lsquobased in&rsquo

187 is there an &lsquoas&rsquo missing: &lsquocan also act as a&rsquo?

Response: corrected all Interpro to InterPro.

275 introduction of the abbreviation &lsquoDTNB&rsquo is missing

Response: Ellman's reagent [(5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB)] is now defined.

281 introduction of the abbreviations &lsquoDHA&rsquo and &lsquoHED&rsquo are missing

Response: Docosahexaenoic acid (DHA) and bis(2‐hydroxyethyl) disulfide (HED) are now defined.

322 introduction of the abbreviation &lsquoDTT&rsquo is missing

Response: dithiothreitol (DTT) is now defined.

333 correct citation name

Response: We were unclear what is meant by this point but have added that the information is related to the binding of MsvR to its own promoter to clarify which promoter is under discussion if this is what the reviewer meant.

line 333 is related to the following: Incubation of oxidized MsvR with the M. acetivorans thioredoxin system, consisting of NADPH, thioredoxin reductase and one of the 7 thioredoxins, leads to reduction of the cysteines and binding to its own promoter [105].

The citation references the work by the following which appears appropriate for the information presented:

Sheehan, R. McCarver, A.C. Isom, C.E. Karr, E.A. Lessner, D.J. The Methanosarcina acetivorans thioredoxin system activates DNA binding of the redox-sensitive transcriptional regulator MsvR. J Ind Microbiol Biotechnol 2015, 42, 965-969, doi:10.1007/s10295-015-1592-y.

359 &lsquoeven at&rsquo instead of &lsquoat even&rsquo

411 better: &lsquosome archaeal species&rsquo

Fig. 2 grey boxes: shade of gray somewhat darker for better contrast

Response: the shade of gray is now darker for better contrast.

Fig. 3, legend: please add the explanation of FTR with iron-sulfur cluster

Response: FTR/FDR added to legend with explanation and citation.

The review &ldquoRedox and thiol in Archaea&rdquo by Mamta Rawat and Julie A. Maupin- Furlow provides a clear and extensive panorama on a topic regarding Low molecular weigh (LMW) thiols of which up to now there has not been an updated overview. The detailed analysis of the different LMWs in the different kingdoms of life is well written and deepens biochemical pathways of the different LMWs. In the Archaea the role of LMW is still little known but this review has analyzed the different distribution of LMW thiols in this kingdom and has highlighted their possible function. Therefore, this manuscript could be worthy of publication. However, I have some points that, if addressed, would go a long way towards reducing my reservations and making this publication even better.

Plesae change &hellip&hellipdifferent LMW thiol, bacillithiol&hellip. in &hellip&hellip different LMW thiols, such as bacillithiol

I would like that the authors describe and comment the role of PDO not only in relationship to Sur but also as part of redox system involved in the regeneration of prx in S. sofataricus . PDO activity should be commented

The authors should include the following papers among references :

- Limauro D, D'Ambrosio K, Langella E, De Simone G, Galdi I, Pedone C, Pedone E, Bartolucci S. Exploring the catalytic mechanism of the first dimeric Bcp: Functional, structural and docking analyses of Bcp4 from Sulfolobus solfataricus. Biochimie. 2010 92(10):1435-44. doi: 10.1016/j.biochi.2010.07.006

- D'Ambrosio K, Limauro D, Pedone E, Galdi I, Pedone C, Bartolucci S, De Simone G.Insights into the catalytic mechanism of the Bcp family: functional and structural analysis of Bcp1 from Sulfolobus solfataricus. Proteins. 2009 76(4):995-1006. doi: 10.1002/prot.22408.

In these papers a new disulfide redox system that reduces peroxiredoxins in Saccharolobus solfataricus is described. The canonical system NADPH / Tr / Trx, generally used to reduce Prxs, in S. solfataricus is replaced by the NADPH / Tr / PDO to regenerate the bacterioferritin (Bcps) comigratory proteins.

Line 147 change glutaredoxin and thioredoxin to Grx and Trx respectively

Line 149 changes glutaredoxin to Grx

In Fig. 3 NTR could also be connected to Prx by Protein Disulfide Oxidoreductase (PDO) as reported in the previous comment. Also the legend must be completed inserting PDO of S. solfataricus

Line 163 Please change Halobacterium salinarum to H. salinarum

Line 167 Please change Pyrococcus furiosus to P. furiosus

Line 195: Sulfolobus solfataricus must be changed to Saccharolobus solfataricus, based on the update of the taxonomy within the Sulfolobaceae family

Lane 205: Please change Pyrococcus horikoshii in P. horikoshii

Lane 288 Edit S. solataricus in S. solfataricus

Lane 298 Edit S. sulfotaricus in S. solfataricus

Lane 320 Insert the follow reference: Lipscomb, G.L. , Schut, G.J. Scott RA, Adams, M.W.W. SurR is a master regulator of the primary electron flow pathways in the order Thermococcales. Mol Microbiol 2017, 104, 869-881, doi: 10.1111/mmi.13668

Lane 347 Please Change Pyrococcus furiosus in P. furiosus and Sulfolobus solfataricus in S. solfataricus

Comments and Suggestions for Authors

The review &ldquoRedox and thiol in Archaea&rdquo by Mamta Rawat and Julie A. Maupin- Furlow provides a clear and extensive panorama on a topic regarding Low molecular weigh (LMW) thiols of which up to now there has not been an updated overview. The detailed analysis of the different LMWs in the different kingdoms of life is well written and deepens biochemical pathways of the different LMWs. In the Archaea the role of LMW is still little known but this review has analyzed the different distribution of LMW thiols in this kingdom and has highlighted their possible function. Therefore, this manuscript could be worthy of publication. However, I have some points that, if addressed, would go a long way towards reducing my reservations and making this publication even better.

Plesae change &hellip&hellipdifferent LMW thiol, bacillithiol&hellip. in &hellip&hellip different LMW thiols, such as bacillithiol

Response: we replaced the , with : to avoid confusion and clarify that bacillithiol is one example of a LMW thiol that differs from GSH.

I would like that the authors describe and comment the role of PDO not only in relationship to Sur but also as part of redox system involved in the regeneration of prx in S. sofataricus . PDO activity should be commented

The authors should include the following papers among references :

- Limauro D, D'Ambrosio K, Langella E, De Simone G, Galdi I, Pedone C, Pedone E, Bartolucci S. Exploring the catalytic mechanism of the first dimeric Bcp: Functional, structural and docking analyses of Bcp4 from Sulfolobus solfataricus. Biochimie. 2010 92(10):1435-44. doi: 10.1016/j.biochi.2010.07.006

- D'Ambrosio K, Limauro D, Pedone E, Galdi I, Pedone C, Bartolucci S, De Simone G.Insights into the catalytic mechanism of the Bcp family: functional and structural analysis of Bcp1 from Sulfolobus solfataricus. Proteins. 2009 76(4):995-1006. doi: 10.1002/prot.22408.

In these papers a new disulfide redox system that reduces peroxiredoxins in Saccharolobus solfataricus is described. The canonical system NADPH / Tr / Trx, generally used to reduce Prxs, in S. solfataricus is replaced by the NADPH / Tr / PDO to regenerate the bacterioferritin (Bcps) comigratory proteins.

In Fig. 3 NTR could also be connected to Prx by Protein Disulfide Oxidoreductase (PDO) as reported in the previous comment. Also the legend must be completed inserting PDO of S. solfataricus

Response: Thanks, we now include all of this information in the manuscript text in the section which discusses Prxs and the work performed in archaea. The references listed above and citations are now in the main body of the text.

Line 147 change glutaredoxin and thioredoxin to Grx and Trx respectively

Line 149 changes glutaredoxin to Grx

Response: We now use GRX and TRX throughout the manuscript.

Line 163 Please change Halobacterium salinarum to H. salinarum

Line 167 Please change Pyrococcus furiosus to P. furiosus

Line 195: Sulfolobus solfataricus must be changed to Saccharolobus solfataricus, based on the update of the taxonomy within the Sulfolobaceae family

Lane 205: Please change Pyrococcus horikoshii in P. horikoshii

Lane 288 Edit S. solataricus in S. solfataricus

Lane 298 Edit S. sulfotaricus in S. solfataricus

Lane 347 Please Change Pyrococcus furiosus in P. furiosus and Sulfolobus solfataricus in S. solfataricus

Response: We have made all of these taxonomic changes and now use appropriate abbreviations.