How Does Thiourea Prevent Prevent Sn2 From Occurring Again
Biochim Biophys Acta. Author manuscript; available in PMC 2015 Feb ane.
Published in final edited form as:
PMCID: PMC3766385
NIHMSID: NIHMS464925
Quantification of Thiols and Disulfides
Jakob R. Winther
oneSection for Biomolecular Sciences, Section of Biology, University of Copenhagen, Copenhagen Biocenter, DK-2200 Copenhagen, Denmark
Colin Thorpe
2Section of Chemistry and Biochemistry, Academy of Delaware, Newark, Delaware 19716, Us
Abstract
Background
Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells become to great lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing claiming due, in function, to the facility of thiol/disulfide exchange reactions.
Scope of the review
In the present account, nosotros briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the fundamental chemical aspects of electric current methodology, together with identifying potential difficulties inherent in their experimental implementation.
Major conclusions
While many reagents accept been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox weather in living cells presents a standing challenge.
General Significance
Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while puddle sizes of redox pairs and their intracellular distribution are beingness uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important attribute of cellular metabolism.
Keywords: Detection, Modification, Redox, Substitution, Nucleophile
1. Introduction
ane.1 Thiol-disulfide commutation
Thiol-disulfide exchange reactions play critical roles in many aspects of cellular part. In these reactions, a nucleophilic thiolate attacks one of the 2 sulfur atoms of the target disulfide bail (Fig. one). Since the reactivity of the sulfhydryl group is dominated by that of its deprotonated form, we start briefly address aspects of the acidity (pKa) and nucleophilicity of thiolates. The protonated forms of simple alkyl thiols are practically unreactive as nucleophiles under normal weather; reacting some 1010–fold slower than their corresponding thiolates [1,2]. As the concentration of the thiolate is derived from the Henderson-Hasselbalch the equation, for the pH dependency of the reaction charge per unit for thiol-disulfide exchanges is governed by the following equation for reaction kinetic relationship [3]:
Thiol-disulfide commutation. The attack of a thiolate (the nucleophile, S-n) on a disulfide bond takes place through a linear transition state where the fundamental sulfur cantlet (Sc) will participate in a new disulfide bond and resolution of a new leaving grouping thiolate (Southward-lg). Which of the two sulfur atoms participating in the disulfide bail will eventually act as leaving group is dependent on steric, electrostatic and intrinsic acidity of the thiolate species involved.
Hither thouobs is the observed rate constant at a given pH, and k is the respective limiting rate constant for the thiolate at high pH values. Thus, thousandobs is one half of the limiting rate constant at the pKa, merely falls to 1/104 of the maximal reactivity at four pH units beneath the pKa.
Biological thiols show a very wide range of pKa values (from about 3 to 11, thus corresponding to an eight-guild of magnitude shift in the deprotonation equilibrium [4]). The factors contributing to this profound modulation of thiol pKa's are under, which and so profoundly modulate thiol pKa'southward, are nether active investigation, and include solvation, electrostatic effects with neighboring charges and dipoles, too as H-bonding interactions [v-7]. It is important to note that the pKa of thiols has two distinct effects on reactivity. Obviously, as noted in a higher place, a lower thiol pKa increases the fraction of thiol in its reactive thiolate form, however, the intrinsic reactivity of fully-formed thiolates (at the loftier pH limit) typically declines with decreasing thiol pKa for a series of structurally-related thiols [eight-10]. The ability of a thiol sulfur atom to retain a proton is to some extent a reflection of its intrinsic nucleophilicity, thus illustrating the correlation between nucleophilicity and pKa.
Although it might seem unnecessary in terms of populating the thiolate, some enzymes accept evolved to have pKa'south far below the predominant pH of a typical cellular environment. Such low pKa values might, however, suppress oxidative side reactions that would otherwise compromise catalysis. Another reason is that marked differences in acidity allow the equilibrium constant for thiol disulfide exchange to exist tuned by thiol pKa values. Thus, in the thiol-disulfide exchange reaction:
R1 − SH + R2 − S − S − R2 ⇌ R1 − S − South − R2 + R2 − SH
-- lowering the pKa of R2-SH with respect to R1-SH will amend the leaving-group backdrop of R2-SH and bias the equilibrium to the right [5,xi].
Two sequent thiol/disulfide exchange reactions accompany the overall redox reaction shown below:
Knowing the stability of one disulfide, together with the magnitude of Grandox, allows the stability of the other disulfide to exist direct calculated. Again the magnitude of Kox will be dependent on a combination of effects including steric, electrostatic and pKa values of the thiol species involved [12].
Finally, the rates of thiol-disulfide exchange reactions are influenced by the requirement for a linear organisation of the three sulfur atoms in the transition state [13,14]. In proteins, the two sulfur atoms of the disulfide bonds often differ markedly in their accessibility to an attacking thiolate nucleophile generating a single mixed disulfide intermediate. In the event that both disulfide sulfur atoms are exposed, the outcome of disulfide substitution may largely reflect discrimination based on pKa values (see above).
1.2 Overall principles for thiol-disulfide detection and quantifications
Thiols are typically detected straight past virtue of their relatively high reactivity compared to most other common species in biological systems. Disulfides, on the other hand, have no strong chemical signature, and are hence most commonly detected afterward reduction to their respective thiols. Thus, the most common methodologies for thiol and disulfide quantification involve determination of complimentary thiol concentration, followed by alkylation, reduction of disulfide bonds, and subsequent quantification of the additional exposed thiols. The processes of reduction and alkylation are thus pivotal for thiol quantification. In the conclusion of disulfides, the complete removal of the reducing species prior to detection is crucial and then that no cantankerous-reaction takes place between the reductant and the reagent used for thiol detection.
2. Quenching of thiol oxidation and exchange
2.1 Thiol alkylation
Alkylation of cysteine thiols with iodo, bromo or chloro substituted acetic acid or acetamide is a classic approach that has been exploited since the 1930′s. The relative reaction rates betwixt glutathione and these halogenated acetates are 100:60:i for iodo-, bromo- and chloro-acetates respectively [15]. Although iodoacetic acid or iodoacetamide are past far the most widely used haloalkanes for thiol alkylation, they can prove significant reactivity towards other nucleophilic side bondage. While such side-reactions may be fairly innocuous for most analytical applications, they become major problems in proteomic approaches involving the identification of reacted modified species past mass spectrometry. Thus iodoacetate treatment was shown to significantly modify lysine residues every bit demonstrated past mass spectroscopy [16]. The substitution of chloroacetic acid alleviated this problem only this solution cannot be adopted when thiol residues must be quenched apace.
Maleimides are very widely used reagents for the alkylation of thiols. The reaction represents a Michael addition of the thiolate on the electrophilic double bail of the maleimide (Fig. 2, reaction A). The enone functionality of North-ethylmaleimide (NEM) shows an extinction coefficient of 620 M−1cm−one at 302 nm allowing reactions with nucleophiles to exist conveniently followed spectrophotometrically [17]. A notable additional advantage of maleimides is that they react apace with thiols at neutral or slightly acidic pH values with rate constants that are some 3 to 4 orders of magnitude faster than iodoacetamide nether comparable weather condition [i,18,19]. Despite their utility, several reactions may complicate the apply of maleimides, specially at pH values above vii. Firstly, while maleimides are frequently characterized in the literature as irreversible thiol-modifying reagents, the adducts are subject to base-catalyzed reverse Michael reactions (Fig. ii, reaction B and C) leading to the possible migration of the maleimide betwixt thiol targets [20]. Farther maleimide adducts, specially those where N-R represents an aniline functionality, are prone to band-open by hydrolysis, yielding the isomeric products shown in Fig. 2, reaction D [21]. Such ring-opening reactions take been used to identify maleimide-labeled peptides, [22,23]. In amass, these secondary reactions may play an important office in modulating the stability of maleimide conjugates in vivo [xx,21].
Add-on of thiols to maleimides together with selected exchange and band opening reactions. The reaction of thiol (R1-SH) with maleimides (in this instance NEM) is reversible, admitting shifted strongly toward the adduct formation (Reaction A). In the presence of excess thiols (R2-SH), however, reversal of the initial adduct leads to the accumulation of the alternative adduct (Reactions B and C). The adduct is susceptible to ring-opening by hydrolysis of the imide (Reaction D).
Mammalian cultured cells are permeable to NEM and this has encouraged its utilise for quenching thiols in intact cells. All the same, the inclusion of a denaturant, such every bit SDS, may be necessary to ensure rapid labeling of all gratuitous cysteine residues because about 20% of full cellular protein thiols are non susceptible to modification past NEM under native atmospheric condition [24].
Vinyl pyridine, like NEM, reacts with thiols at the double bond and was previously widely used. Since vinyl pyridine reacts more than 500-fold slower than NEM, both high concentration and long reaction times are required for complete reaction [25,26].
Cyanylation using 1-cyano-4-dimethylamino-pyridinium salts (CDAP) represents an efficient means of thiol blocking [27-30] (Fig. 3A). The reaction is rapid at pH four-v leading to quantitative derivatization of thiols using low mM concentrations of CDAP [thirty,31]. These properties are useful because they permit efficient alkylation at low pH where thiol commutation is minimal. An boosted characteristic of CDAP is that cyanylated peptide-thiol adducts are susceptible to specific cleavage in the presence of ammonia (Fig. 3B). Here, a cyclization involving the cyanylated side-concatenation results in cleavage of the peptide concatenation North-terminal to the target cysteine residue. In combination with mass spectroscopy this procedure allows for mapping of disulfide bond patterns in proteins [32]. On the other mitt, the cyanylated proteins are intrinsically unstable higher up pH 7. CDAP itself is stable in polar aprotic solvents, such as acetonitrile, merely is prone to hydrolysis in aqueous solutions above pH 5 [thirty].
Cyanylation using CDAP. Reaction (A) of CDAP with cysteinyl peptide results in formation of a cyanylated species. This species tin react further in ane.5 M NH4OH to carve the side by side N-proximal peptide bond (B).
Although rapid and indiscriminate alkylation of thiols is oftentimes the desired outcome of labeling protocols, less reactive reagents have been recently used very effectively in proteomic approaches for the identification of proteins containing hyper-reactive cysteine residues [33,34].
iii. Reduction of disulfide bonds
In the reduction of thiols for further assay there are three major concerns: a) that the disulfide reduction is quantitative and rapid, b) that the reducing agent is specific, and does non show significant side reactions, and c) that the reductant selected does non complicate down-stream reactions and processes. Disulfide reduction is accomplished primarily by thiol exchange type reagents (like dithiothreitol, DTT, or 2-mercaptoethanol, ME) or by various substituted phosphines such as tris(2-carboxyethyl)phosphine, TCEP [35].
3.i Phosphine- and Thiol-based reductants
Unlike thiol reagents, for all practical purposes phosphines are irreversible reductants of disulfide bonds in aqueous solutions (Fig. four). Here the phosphine performs a nucleophilic attack on one of the two sulfur atoms forming a phosphonium ion sulfur adduct which is subsequently hydrolyzed yielding the corresponding phosphine oxide. This irreversibility contrasts with thiol-based reductants, which are typically needed in large backlog and consequently must be removed, or quenched, prior to sample workup.
Reaction mechanism for the reduction of disulfide bonds by phosphines. Here, the reaction of tris-(two-hydroxyethyl) phosphine (THP) is shown. Several other water soluble phosphines are commercially available, or take been described in the literature.
TCEP is now a widely used substitute for DTT in a broad range of biochemical applications. Mono-, di- and tri-methyl esters of TCEP testify progressively lower pKa values at phosphorus [36]. Thus, while the pKa of the nucleophilic center in TCEP is 7.5, that of trimethyl-TCEP is 4.7 [36]. While lowering the pKa promotes the expected decreases in intrinsic nucleophilicity, these water-soluble phosphine methyl esters are more facile reductants than TCEP at lower pH values. For example, at pH 5, their effectiveness in the reduction of a disulfide-begetting model peptide was tri- > di- > mono-methyl-TCEP > TCEP ≫ DTT [36]. Nevertheless, none of these phosphine-based reductants are likely to exist useful at the low pH values needed to suppress H/D exchange in peptides liberated from peptic digests during the assessment of the surface accessibility and dynamics of proteins. (At pH values lower than iii, electrochemical or zinc metallic reductions may provide viable substitutes; encounter beneath). Finally, neutral h2o-soluble phosphines may prove useful alternatives to DTT in the modulation of cellular redox poise. For example, the tmTCEP analog penetrates model lipid bilayers much more rapidly than DTT [36]: a standard reductant used to employ reductive stress in cell culture studies. This permeability, combined with the irreversibility noted earlier, may exist advantageous for in vivo purposes when short pulses of reduction are desired.
Although TCEP is the virtually widely used of the water soluble phosphine reductants, the commercially-bachelor tris(2-hydroxyethyl) phosphine (THP) is peradventure more than useful. It is frequently more reactive as a disulfide reductant than TCEP [36,37]. Furthermore, THP tin can be added to solutions in mM amounts without the necessity for pH adjustment, and THP is less polar allowing improve access to hydrophobic environments than TCEP. Stock solutions of THP, as well as other phosphine reagents, can be standardized by any 1 of the methods described subsequently in this review.
In terms of the thiol-based reductants, while ME is very convenient to use, and may be adequate for e.k. reducing SDS-PAGE applications, ME is not more reducing than generic protein thiols. In large excess (for SDS-Folio typically >0.5 M) it will, withal, shift the equilibrium towards mixed ME-protein disulfides and possibly generate the reduced protein species. As the pKa of the ME thiol is 9.5 [38] it is essential that pH values ≥ 7 are used to obtain efficient reaction.
DTT, on the other mitt, has a low redox potential due to formation of a six-membered ring upon disulfide substitution. Indeed the equilibrium constant for the reaction between DTT and GSSG is effectually 200 [39]. Consequently, commercial preparations of GSH (the all-time of which often contain up to 1% GSSG) cannot typically accomplish whatsoever pregnant reduction of oxidized DTT; a property that must be kept in mind when establishing equilibria with very reducing disulfide bonds in proteins. While DTT is overwhelmingly the mostly widely used dithiol reductant for protein disulfides, a serial of newer alternatives, exhibiting a range of ring sizes, polarities and redox potentials, have been synthesized and characterized past Whitesides [40,41] and Raines [42] and their coworkers.
three.ii Other methods for disulfide reduction
A number of other reducing agents and methods take been described. These include reduction with sodium borohydride, metal zinc, and methods for electrochemical reduction. The advantage of the first two reductants is that excess reagent can be hands removed. Borohydride reductions take place at high pH, however, acidification of the reaction mixture completely discharges the reactive hydride, with the evolution of gaseous H2 [43,44]. A potential disadvantage of this procedure is that the elevated pH values required for disulfide reduction can generate undesirable side-reactions such equally peptide bond cleavage and glutamine/asparagine deamidation.
Metallic zinc was widely used as a reductant for disulfides in the older literature and deserves renewed consideration. Reductions of peptides and certain proteins (using zinc dust in 1% TFA in aqueous acetonitrile mixtures; pH ∼ one) are consummate in minutes [45]. Moreover, millimolar levels of GSSG are speedily reduced by a modest backlog of metallic zinc in citrate buffer, pH 2.5 (CT, unpublished observations). Backlog zinc metal tin can be hands removed by centrifugation, or the reductant might be incorporated in a stationary phase for in-line processing. Since the reduction of disulfides by zinc is facilitated by low pH values, the process might show useful for high-throughput mass spectrometry applications. It should exist noted, even so, that zinc ions may be released into solution, both during reduction of disulfide bonds, and as an incidental consequence of exposure of the metal to acidic pH values.
Finally, studies on the reduction of small molecule and poly peptide disulfides at a dropping mercury electrode, or via stirred mercury surfaces, were initiated more than 50 years ago. In some instances small proteins could be reduced directly, in other cases chaotrophes were used with or without the presence of small mediating thiols [46-48]. Frequently, a big electrochemical over-potential was necessary to ensure efficient kinetic reduction, although more modest potentials may achieve controlled reduction of subsets of protein disulfides [46]. More contempo piece of work has explored a range of derivatized surfaces in the electrochemical reduction of disulfides [49-51].
4. Thiol detection
Thiols may be detected by a diversity of reagents and separation techniques. Thiol reaction may result in quantitative formation of a chromophore or fluorophore, just covalent thiol modification may also provide analyte bigotry during liquid chromatograpy and gel electrophoresis or mass spectrometry.
4.1 Colorimetric thiol detection
The classical chromogenic reagent for thiol detection is v,5′-dithiobis-(2-nitrobenzoic) acid (DTNB; as well known every bit Ellman'due south reagent [52]. This compound has a highly oxidizing disulfide bond, which is stoichiometrically reduced past free thiols in an commutation reaction, forming a mixed disulfide and releasing one molecule of five-thio-2-nitrobenzoic acid (TNB; Fig. 5). TNB is an excellent leaving grouping with a thiol pKa of 4.five [53]. If a second thiol R2-SH initially evades reaction with DTNB it may resolve the mixed analyte disulfide in Fig. v, thereby releasing the second TNB and generating a new mixed disulfide (R1-South-Southward-R2). In either example, one TNB is released for every thiol oxidized upon DTNB treatment.
Detection of thiols using Ellman's reagent (DTNB). Due to the depression pKa of the TNB it forms an extremely efficient leaving group. The net issue of this is that the analyte efficiently forms disulfide bonds if steric and concentration issues allow. Note that for each analyte thiol reacted, a TNB is formed.
While DTNB has weak absorption at 412 nm, the extinction coefficient for TNB is 14,100 G−1cm−i at pH 7.3 [53], but drops steeply at pH beneath reflecting protonation of the orange thiolate species.
The extinction coefficient of the thiolate is slightly dependent on ionic forcefulness. This is relevant when thiols are determined in half dozen 1000 guanidinium chloride where the extinction coefficient at 412 nm drops to xiii,700 M−onecm−one.
One should besides exist aware that DTNB is fairly sensitive to hydrolysis at elevated temperatures and, in particular, at pH values > 7. Decomposition is initiated via hydrolytic scission of the activated disulfide of DTNB to yield the sulfenate species shown in Fig. 6. A more than hydrolytically stable derivative of DTNB has been developed to address this instability [54].
Hydrolysis of DTNB. Hydroxyl ions volition attack the disulfide bond of DTNB forming a sulfenic acid and a thiolate. This reaction is particularly relevant for activated disulfides like that found in DTNB.
An emerging alternative to DTNB is 4,4′-dithiodipyridine (4-DPS). While reduction of DTNB generates the TNB thiolate, 4-DPS reduction leads to germination of the strongly absorbing resonance-stabilized four-thiopyridone tautomer (Fig. 7). The pH independent absorption of the pyridone (extinction coefficient 21,000 G−anecm−1 at 324 nm) over pH three – seven so allows quantification of thiols at relatively low pH values. Under these weather condition a number of unwanted side reactions are suppressed and hydrolytic scission of the disulfide bond is insignificant. While this is a conspicuous advantage of four-DPS, the longer wavelength maximum for TNB, over the pyridone production of 4-DPS, makes DTNB more than suitable for the quantification of thiols in solutions that strongly absorb in the almost UV. The much lower solubility of the electroneutral 4-DPS (∼ 3 mM in water) compared to the dianion grade of DTNB at pH vii is another cistron to consider. Consequently DTNB is considered membrane impermeant at neutral pH values [36], whereas four-DPS can admission thiols in hydrophobic environments and laissez passer through biological membranes.
Structure of iv-DPS showing subsequent reaction with thiols. For each thiol oxidized a molecule of thiopyridone is made. The latter is a favored tautomer of the thiol, which absorbs strongly at 324 nm. Note that as the thiopyridone is uncharged even at depression pH the absorbtion remains unaffected.
While these activated chromogenic reagents have proven extremely useful in the quantification of thiols, they must be used with an appreciation of their limitations. We mention one instance for illustration: the potential interference in the determination of thiol concentrations that is associated with the presence of sulfite in biological, technical or environmental samples. Sulfite attacks disulfide bonds effecting their net scission with the release of -SH and -Due south-Thenthree 2− functionalities. While generic disulfides are not particularly sensitive to sulfitolysis, the highly reactive disulfides of DTNB and DPS leads to a stoichiometric reaction with sulfite, and to a consequent overestimation of thiol concentration in the presence of sulfite [55].
4.ii Fluorescent adducts
Many reagents for the fluorimetric detection of thiols accept been adult and are the subject of extensive reviews e.one thousand. [56,57]. Here we identify a few highlights of selected reagent classes. Monobromobimane (mBBr) and benzofurazans proceeds fluorescence as they react with thiols. Thus mBBr shows a blue fluorescence only after thioether formation (heady at 380 nm and emitting at 480 nm [58]. The reagent has been used extensively both for detection of low molecular weight thiols besides equally proteins modified for in-gel detection.
There are ii commonly used varieties of benzofurazans: 7-fluorobenzo-two-oxa-one,iii-diazole-4-sulfonate (SBD-F) and 4-(aminosulfonyl)-7-fluoro-two,ane,iii-benzoxadiazole (ABD-F) Fig. viii [59,60]. Both reagents are non-fluorescent, but accomplish potent fluorescence at around 500 nm upon conjugation with thiols. Of particular interest is the ascertainment that these reagents do not significantly cantankerous-react with phosphines, allowing reduction of disulfides and labeling of total thiols to exist conducted in a single reaction mixture [61]. While SBD-F requires rather harsh conditions (sixty °C at pH 8.five for one h), modification with ABD-F is consummate at ambience temperatures over x min at pH 8. For this reason the use of ABD-F is probably preferable, although for some applications a certain lack of specificity may pose a problem [62]. ABD-F is furthermore fairly unstable in the presence of backlog thiols [63]. A specially useful application of benzofurazans is that several pocket-size-molecular weight thiol-adducts can be detected simultaneously following HPLC separation with fluorescence detection [64]. Recently a new grade of benzofurazans has been described which are more reactive and which have potential awarding in fluorescence microscopy [65].
Reaction of benzofurazanes with thiols. The reaction products are highly fluorescent.
iv.3 Formation of detectable thiol adducts using gel-shift assays
Numerous maleimide derivatives are available commercially, in which the ethyl group of NEM has been replaced by other substituents carrying fluorophores, affinity tags, solubility enhancing tags, or tags that introduce a large increment in molecular mass. Nosotros will apply the abridgement HMD to announce heavy maleimide derivatives when referring to this latter awarding. Hither the beefy substituent, post-obit thiol modification, results in a shift in mobility on SDS-PAGE detectable by staining or by immunoblotting. Nevertheless, the result of modification by HMDs may be unpredictable and methods must be optimized for each cysteine and poly peptide [66]. The near popular HMD reagents include 4-acetamido-4′-maleimidylstilbene-two,ii′-disulfonic acid (AMS; [67]) and polyethylene glycol maleimides (PEG-mal; [68]). The shift in mobility for AMS is typically equivalent to 0.5-1 kD. PEG-mal is commercially available in mono-disperse preparations with molecular weights of upward to 40 kD, however, 2 and v kD reagents are often the most relevant for gel shift assays since modification with PEG-mal results in shifts that exceed the bodily mass of the reagent. This is plain due to the interaction betwixt SDS and PEG and hence excess PEG-mal reagent must also be removed prior to SDS-Page [69].
4.4 Other reagents for thiol blocking
In improver to the thiol alkylating reagents mentioned earlier, Southward-methyl methanethiosulfonate (MMTS) has been widely employed to trap thiol groups. MMTS generates a methylthio-mixed disulfide with target thiols while simultaneously liberating the good leaving group methylsulfinic acid (Fig. ix). While MMTS has the advantage of introducing a very modest thiol substituent, this reagent grade needs to exist used with appropriate caution if the intent is to quench thiol/disulfide commutation reactions. Thus under insufficiently rigorous reaction conditions, MMTS tin promote intramolecular disulfides rather than quenching all thiols every bit their methyl disulfides [seventy]. Recently, a new thiourea-activated disulfide fluorescent reagent (FCAD) has been synthesized that reacts efficiently with thiols at pH three-4 [71]. FCAD thus reacts under conditions that minimize thiol/disulfide exchange and may discover uses in chromatography and gel-electrophoresis.
Reaction of MMTS with thiols. The formation of a methyldisulfide (Reaction A) is prone to shuffling with adjacent reactive thiols (Reaction B). This renders MMTS unreliable every bit a thiol quencher.
5. Practical considerations for determination of the thiol/disulfide redox country of proteins
Gel shift assays are oft used to determine the thiol/disulfide redox condition of a protein. In some cases the formation of a disulfide bond, particularly 1 from widely-spaced thiols, generates a gel-shift without the need for further derivatization using HMDs. Nonetheless, information technology is always essential to include an alkylation pace to block any costless thiols to preclude any potential thiol/disulfide shuffling during exposure to SDS. Indeed, shuffling can be quantitative when samples are heated for two minutes at 95 °C in grooming for SDS-PAGE analyses (JRW, unpublished data).
Quite often the formation of brusk-range disulfide bonds in proteins does non outcome in significant mobility shifts on SDS-Folio. Here, free thiols tin can be quenched with a small reagent, such as NEM, and, later removal of excess maleimide, the disulfide bonds are reduced so that the liberated thiols tin can be labeled with an HMD. If the redox status of only one pair of cysteines is investigated it may exist tempting to label the thiol fraction with the HMD directly and go out the disulfides unmodified. Information technology should be noted that maleimides are not rigorously specific, and can undergo side reactions with amines particularly at higher pH values [72-74]. Obviously alkylation of reactive amines with HMDs will innovate significant error into the interpretation of thiol/disulfide condition. If still, these amines are outset reacted with NEM, which does not significantly decrease the electrophoretic mobility of the protein, merely thiols liberated later subsequent reduction of the disulfide bonds will be modified by the HMD [75].
In practical terms, the initial alkylation with NEM leaves the sample containing a significant concentration of free NEM. This alkylating agent must be removed prior to the subsequent reduction step to avoid an underestimation of the disulfide-content of the poly peptide. Although the alkylating potential of NEM is discharged with either DTT or phosphine handling, this reaction may be slower than the de novo generation of thiols in the sample past these reductants. One fashion to remove NEM is to pre-treat the sample with a small excess of ME relative to the alkylating reagent prior to reduction [76]. ME is non a stiff plenty reductant to significantly shift the redox balance under denaturing conditions, but will still readily react with the alkylating reagent. NEM is relatively stable in 100% (w/v) aqueous solution of TCA, and can therefore conveniently be added to the TCA solution used for quenching (R.E. Hansen and JRW, unpublished information). Excess NEM tin can also exist removed prior to reduction following precipitation with TCA and washing the pelleted protein with acetone.
The quantification of GSH and GSSG has recently been reviewed extensively [77]. In brief, the classical assay adult by Tietze [78] remains very popular and can observe depression levels of both GSH and GSSG in both conventional cuvettes and using a 96-well microtiter plate format. The assay is based on the specific reduction of glutathione disulfide by glutathione reductase at the expense of the oxidation of NADPH. For the conclusion of total glutathione, the inclusion of DTNB oxidizes the GSH component, and allows the charge per unit of enzymatic reduction of the disulfide to be followed via release of the TNB anion. A primal characteristic of this analysis is that at low glutathione concentration (much lower than the 1000grand for glutathione reductase) the rate of TNB generation will be proportional to the glutathione concentration. This can be tested empirically by construction of a suitable standard curve leading to the conclusion of total glutathione. For the specific measurement of GSSG levels, the GSH component in the sample must exist initially alkylated so that it is not able to cycle in the assay. 2-vinylpridine has been used extensively for this purpose every bit it is capable of trapping GSH while it is ineffective at intercepting the thiol-based chemistry of the active site of glutathione reductase [79]. More recently an alternative vinylpyridine derivative has been suggested which more efficiently quenches of GSH without inhibiting glutathione reductase [eighty].
v.1 Electrochemical detection
In-line HPLC detection of thiols and disulfides by amperometric or coulometric methods represent interesting alternatives to conventional chemic conversion followed by chromogenic or fluorogenic detection. Here the redox active state of the thiols and disulfides species are directly detected either as a change in current or potential over suitable electrodes [81]. Thus, in that location is no demand for chemical modification of the sample. The external potential can be changed and then that it optimally matches the species of interest or array detectors can be employed [51,82,83].
v.2 Thiol-disulfide sensing using GFP-based probes
GFP-based sensors for thiol-disulfide reactions were developed about 10 years ago independently in two laboratories [84,85]. The expanding toolbox of bachelor sensors has recently been the subject field of an fantabulous review by Meyer and Dick [86]).
All these sensors feature a pair of engineered cysteines at the surface of the protein in close proximity to the fluorophore. The formation of a disulfide results in a slight movement of adjacent β-strands, with an associated modulation of the fluorescence of the probe. For whatsoever sensor the useful dynamic range must cover relevant practical conditions, in this case redox potential. The other prerequisite is the power to equilibrate with the species to be adamant. Somewhat serendipitously, the initial prepare of probes all proved to be highly reducing and somewhat oxidized nether steady-state conditions in the eukaryotic cytosol. On should note that whatsoever thiol-disulfide redox sensor is initially synthezised on the ribosome in its reduced country. Thus, any equilibration with the surrounding medium necessitates an oxidation to establish equilibrium. A crucial, and maybe somewhat surprising discovery, was that equilibration with the cytosolic glutathione buffer is catalyzed by glutaredoxin, whereas the steric backdrop of the sensing thiols render them inaccessible to reaction with thioredoxin [87]. It was demonstrated that cellular depletion of glutaredoxin activity slowed downwardly oxidation of the sensor and thus prolonged the time required to for newly synthesized redox sensor reach equilibrium in vivo. This glutathione-sensing ability can be farther enhanced past covalent linkage of glutaredoxin [88,89] allowing for GFP-based sensors to be used under steady-state weather. Since these sensors employ a disulfide-sensing mechanism they cannot be used to make up one's mind absolute concentrations of GSH and GSSG, however, they answer to the redox potential of the glutathione buffer (that is to the [GSH]two/[GSSG] ratio) according to the relationship:
The kinetic separation from the thioredoxin pathway ensures that the sensing is specific for the glutathione pool [90].
Interestingly, the redox potentials determined in this mode [85,87] are dramatically lower than those determined from classical chemic approaches. Likely, the challenges inherent in quenching thiol/disulfide exchange reactions, and the possible contamination of samples with the contents of cellular compartments with widely differing redox potentials, contribute to this discrepancy [91].
6. Challenges and pitfalls
6.1 Quench by acidification
A primal issue in the study of thiol/disulfide equilibria remains the adequacy of the methods used for quenching. Because the thiolate represents the reactive species in these exchange reactions, it is common practice to quench redox reaction by lowering the pH. However, some cysteine residues have very low pKa values (e.one thousand. 3.2 for DsbA [three,92]) and their parent proteins may themselves exist fairly stable at low pH [93]. Thus, acidification may perturb thiol/disulfide equilibria between thiols of widely unlike pKa values (equally shown for DsbA [94]) earlier the reaction is finer quenched by denaturation of the protein. Information technology may thus be necessary to combine quenching with the inclusion of chaotrophes (e.thou. the guanidinium ion at low pH, or 10-fifteen% trichloroacetic acrid). Flash freezing before quenching with acid may testify advantageous.
6.2 Quench by alkylation
In place of acidification, NEM is often used for in vivo quenching in situations where a certain degree of protein integrity is required (e.g. during co-immunoprecipitation). Nonetheless, to our knowledge, the effectiveness of NEM penetration into cells and organelles has never been quantitatively evaluated. Indeed the high intracellular concentrations of reduced glutathione must surely filibuster the effectiveness of NEM as a trapping agent in vivo and this may even bias the equilibria that NEM is intended to quench.
six.3 Oxygen and metal ions
A further claiming to the interpretation of thiol/disulfide equilibria is the artefactual generation of disulfide by oxidation past ambient molecular oxygen. This reaction is stimulated past traces of metals (Cu2+ > Fe3+ > Ni2+ ≫ Co2+ [95]) and is particularly prevalent with copper and iron [96] which are common contaminants of reagents and surfaces. Chelators, such as EDTA, attenuate, simply exercise non entirely suppress, thiol oxidation. While lowering the pH significantly slows unwanted thiol oxidation, such conditions are non always compatible with alkylation reactions. Information technology is therefore prudent to purge solutions with nitrogen or argon and, wherever possible, to manipulate solutions nether a blanket of argon or within an anaerobic glove box.
7. Concluding remarks and perspectives
The depression kinetic barriers associated with many thiol/disulfide commutation reactions pose potential pitfalls for scientists who wish to determine the concentrations of these critical partners in redox homeostasis. In addressing these challenges, it is important to appreciate the chemical fundamentals of these deceptively simple commutation reactions. In item, information technology is critical to ensure that the methods selected to quantify the redox status of thiols and disulfides do not themselves adjy the very arrangement under investigation. Thiol/disulfide interconversions, which are in rapid equilibrium, present particular difficulties in this regard; not to the lowest degree when relevant enzymes are around. Hither, the outcome may be biased past the kinetic reactivity profiles of the trapping agent. Information technology is thus appropriate to verify that quenching procedures practise not modify the outcome of the analysis by spiking samples with relevant thiols or disulfides or in other means introducing stringent controls.
The methods summarized in this review have focused heavily on the determination of thiol/disulfide concentrations without regard to how these levels are maintained (e.one thousand. whether these concentrations stand for to a system in rapid equilibrium, or to i reflecting the kinetics of the steady country). While but determining cellular thiol/disulfide levels will go on to pose meaning technical difficulties, these measurements simply accost one aspect of redox homeostasis. Thus, virtually nothing is known about the flux in thiol/disulfide redox pathways. For instance, how fast is the GSH pool turned over intracellularly by redox cycling? What percentage of the maximal capacity of glutathione reductase is employed in the typical exponentially growing yeast cell? Or what flux disulfides accompanies oxidative protein folding in the endoplasmic reticulum of a typical jail cell? The exploration of the fluxes accompanying thiol/disulfide homeostasis is an emerging frontier that will require new experimental approaches and a new toolbox of reagents and probes.
Acknowledgments
Work from the authors' laboratories was supported in part by the Danish Council for Independent Inquiry (JRW) and past NIH GM26643 (CT).
Abbreviations
| ABD-F | 4-(aminosulfonyl)-7-fluoro-2,one,3-benzoxadiazole |
| AMS | four-acetamido-iv′-maleimidylstilbene-2,two′-disulfonic acrid |
| CDAP | 1-cyano-four-dimethylamino-pyridinium |
| 4-DPS | iv,4′-dithiodipyridine |
| DTNB | five,5′-dithiobis-(2-nitrobenzoic) acrid |
| EDTA | ethylenediamine tetraacetic acrid |
| ER | endoplasmic reticulum |
| GSH | glutathione |
| GSSG | glutathione disulfide |
| HMD | heavy maleimide derivative |
| MBBr | Monobromobimane |
| ME | 2-mercato ethanol |
| MMTS | S-methyl methanethiosulfonate |
| PAGE | polyacrylaminde gel electrophoresis |
| PEG | polyethyleneglycol |
| SDS | sodium dodecylsulfate |
| TCEP | tris(2-carboxyethyl) phosphine |
| SBD-F | 7-fluorobenzo-2-oxa-1,three-diazole-4-sulfonate |
| THP | tris(2-hydroxyethyl) phosphine |
| TNB | 5-thio-2-nitrobenzoic acrid |
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early on version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its concluding citable course. Please note that during the product process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
1. Bednar RA. Reactivity and pH-dependence of thiol conjugation to N-ethylmaleimide -Detection of a conformational change in chalcone isomerase. Biochemistry. 1990;29:3684–3690. [PubMed] [Google Scholar]
two. Nagy P. Kinetics and mechanisms of thiol-disulfide substitution roofing direct commutation and thiol oxidation-mediated pathways. Antioxid Redox Signal. 2013 [PMC free commodity] [PubMed] [Google Scholar]
iii. Nelson JW, Creighton TE. Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bail germination in vivo. Biochemistry. 1994;33:5974–5983. [PubMed] [Google Scholar]
4. Pinitglang South, Watts AB, Patel Yard, Reid JD, Noble MA, Gul South, Bokth A, Naeem A, Patel H, Thomas EW, Sreedharan SK, Verma C, Brocklehurst K. A classical enzyme active center motif lacks catalytic competence until modulated electrostatically. Biochemistry. 1997;36:9968–9982. [PubMed] [Google Scholar]
5. Gilbert HF. Molecular and cellular aspects of thiol-disulfide exchange. Adv Enzymol Relat Areas Mol Biol. 1990;63:69–172. [PubMed] [Google Scholar]
6. Hansen RE, Ostergaard H, Winther JR. Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu. Biochemistry. 2005;44:5899–5906. [PubMed] [Google Scholar]
vii. Roos Yard, Foloppe Due north, Messens J. Agreement the pKa of redox cysteines: The key office of hydrogen bonding. Antioxid Redox Point. 2013;18:94–127. [PubMed] [Google Scholar]
8. Bulaj G, Kortemme T, Goldenberg DP. Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry. 1998;37:8965–8972. [PubMed] [Google Scholar]
ix. March J. Avant-garde Organic Chemistry. John Wiley; New York: 1985. [Google Scholar]
10. Shaked Z, Szajewski RP, Whitesides GM. Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values. Biochemistry. 1980;19:4156–4166. [PubMed] [Google Scholar]
11. Iversen R, Andersen PA, Jensen KS, Winther JR, Sigurskjold BW. Thiol-disulfide exchange betwixt glutaredoxin and glutathione. Biochemistry. 2010;49:810–820. [PubMed] [Google Scholar]
12. Houk J, Whitesides GM. Structure reactivity relations for thiol disulfide interchange. J Am Chem Soc. 1987;109:6825–6836. [Google Scholar]
13. Rosenfield RE, Parthasarathy R, Dunitz JD. Directional preferences of nonbonded diminutive contacts with divalent sulfur. 1. Electrophiles and nucleophiles. J Am Chem Soc. 1977;99:4860–4862. [Google Scholar]
14. Bach RD, Dmitrenko O, Thorpe C. Mechanism of thiolate-disulfide interchange reactions in biochemistry. J Org Chem. 2008;73:12–21. [PubMed] [Google Scholar]
15. Dickens F. Interaction of halogenacetates and SH compounds. The reaction of halogenacetic acids with glutathione and cysteine. The machinery of iodoacetate poisoning of glyoxalase. Biochem J. 1933;27:1141–1151. [PMC complimentary article] [PubMed] [Google Scholar]
16. Nielsen ML, Vermeulen G, Bonaldi T, Cox J, Moroder 50, Mann Thousand. Iodoacetamide-induced antiquity mimics ubiquitination in mass spectrometry. Nat Methods. 2008;five:459–460. [PubMed] [Google Scholar]
17. Riordan JF, Vallee BL. Reactions with N-ethylmaleimide and p-mercuribenzoate. Methods Enzymol. 1972;25:449–456. [PubMed] [Google Scholar]
xviii. Gilbert HF. Thiol/disulfide exchange equilibria and disulfide bond stability. Methods Enzymol. 1995;251:8–28. [PubMed] [Google Scholar]
19. MacQuarrie R, Bernhard SA. Mechanism of alkylation of rabbit musculus glyceraldehyde iii-phosphate dehydrogenase. Biochemistry. 1971;x:2456–2466. [PubMed] [Google Scholar]
twenty. Baldwin AD, Kiick KL. Tunable deposition of maleimide-thiol adducts in reducing environments. Bioconjugate Chem. 2011;22:1946–1953. [PMC free article] [PubMed] [Google Scholar]
21. Lin D, Saleh S, Liebler DC. Reversibility of covalent electrophile - Poly peptide adducts and chemical toxicity. Chem Res Toxicol. 2008;21:2361–2369. [PMC free article] [PubMed] [Google Scholar]
22. Borges CR, Watson JT. Recognition of cysteine-containing peptides through prompt fragmentation of the 4-dimethylaminophenylazophenyl-4′-maleimide derivative during assay by MALDI-MS. Poly peptide Sci. 2003;12:1567–1572. [PMC free commodity] [PubMed] [Google Scholar]
23. Gehring H, Christen P. A diagonal procedure for isolating sulfhydryl peptides alkylated with Due north-ethylmaleimide. Anal Biochem. 1980;107:358–361. [PubMed] [Google Scholar]
24. Lind C, Gerdes R, Hamnell Y, Schuppe-Koistinen I, von Lowenhielm HB, Holmgren A, Cotgreave IA. Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Curvation Biochem Biophys. 2002;406:229–240. [PubMed] [Google Scholar]
25. Gorin Thousand, Matic PA, Doughty Yard. Kinetics of reaction of Due north-ethylmaleimide with cysteine and some congeners. Arch Biochem Biophys. 1966;115:593–597. [PubMed] [Google Scholar]
26. Lindorff-Larsen Grand, Winther JR. Thiol alkylation below neutral pH. Anal Biochem. 2000;286:308–310. [PubMed] [Google Scholar]
27. Wakselman Grand, Guibejampel East, Raoult A, Busse WD. ane-cyano-four-dimethylamino-pyridinium salts - New water-soluble reagents for cyanylation of protein sulfhydryl groups. J Chem Soc Chem Com. 1976:21–22. [Google Scholar]
28. Wu J, Watson JT. A novel methodology for consignment of disulfide bail pairings in proteins. Poly peptide Sci. 1997;vi:391–398. [PMC free article] [PubMed] [Google Scholar]
29. Li Ten, Chou YT, Husain R, Watson JT. Integration of hydrogen/deuterium commutation and cyanylation-based methodology for conformational studies of cystinyl proteins. Anal Biochem. 2004;331:130–137. [PubMed] [Google Scholar]
30. Pipes GD, Kosky AA, Abel J, Zhang Y, Treuheit MJ, Kleemann GR. Optimization and applications of CDAP labeling for the assignment of cysteines. Pharmaceut Res. 2005;22:1059–1068. [PubMed] [Google Scholar]
31. Barbirz S, Happersberger HP, Przybylski Grand, Glocker MO. Selective cyanylation of cysteinyl residues as an approach for the mass spectrometric determination of protein structures. Eur Mass Spec. 1999;5:123–131. [Google Scholar]
32. Wu J, Yang Y, Watson JT. Trapping of intermediates during the refolding of recombinant human epidermal growth factor (hEGF) past cyanylation, and subsequent structural elucidation by mass spectrometry. Protein Sci. 1998;seven:1017–1028. [PMC free commodity] [PubMed] [Google Scholar]
33. Weerapana E, Wang C, Simon GM, Richter F, Khare S, Dillon MBD, Bachovchin DA, Mowen K, Baker D, Cravatt BF. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature. 2010;468:790–795. [PMC free commodity] [PubMed] [Google Scholar]
34. Serafimova IM, Pufall MA, Krishnan Due south, Duda K, Cohen MS, Maglathlin RL, McFarland JM, Miller RM, Frodin M, Taunton J. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat Chem Biol. 2012;8:471–476. [PMC free commodity] [PubMed] [Google Scholar]
35. Getz EB, Xiao M, Chakrabarty T, Cooke R, Selvin PR. A comparison between the sulfhydryl reductants tris(two- carboxyethyl)phosphine and dithiothreitol for use in poly peptide biochemistry. Anal Biochem. 1999;273:73–fourscore. [PubMed] [Google Scholar]
36. Cline DJ, Redding SE, Brohawn SG, Psathas JN, Schneider JP, Thorpe C. New water-soluble phosphines as reductants of peptide and protein disulfide bonds: Reactivity and membrane permeability. Biochemistry. 2004;43:15195–15203. [PubMed] [Google Scholar]
37. Daithankar VN, Wang WZ, Trujillo JR, Thorpe C. Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover. Biochemistry. 2012;51:265–272. [PMC free article] [PubMed] [Google Scholar]
38. Jocelyn PC. Standard redox potential of cysteine-cystine from thiol-disulphide exchange reaction with glutathione and lipoic acrid. Eur J Biochem. 1967;two:327–331. [PubMed] [Google Scholar]
39. Chau MH, Nelson JW. Straight measurement of the equilibrium between glutathione and dithiothreitol by high-performance liquid-chromatography. Febs Lett. 1991;291:296–298. [PubMed] [Google Scholar]
40. Singh R, Lamoureux GV, Lees WJ, Whitesides GM. Reagents for rapid reduction of disulfide bonds. Methods Enzymol. 1995;251:167–173. [PubMed] [Google Scholar]
41. Lees WJ, Whitesides GM. Equilibrium-constants for thiol disulfide interchange reactions - A coherent, corrected set. J Org Chem. 1993;58:642–647. [Google Scholar]
42. Lukesh JC, Palte MJ, Raines RT. A potent, versatile disulfide-reducing agent from aspartic Acid. J Am Chem Soc. 2012;134:4057–4059. [PMC free article] [PubMed] [Google Scholar]
43. Chocolate-brown WD. Reduction of poly peptide disulfide bonds by sodium borohydride. Biochim Biophys Acta. 1960;44:365–367. [Google Scholar]
44. Hansen RE, Ostergaard H, Norgaard P, Winther JR. Quantification of poly peptide thiols and dithiols in the picomolar range using sodium borohydride and 4,four′-dithiodipyridine. Anal Biochem. 2007;363:77–82. [PubMed] [Google Scholar]
45. Erlandsson M, Hallbrink M. Metal zinc reduction of disulfide bonds between cysteine residues in peptides and proteins. Int J Pept Res Ther. 2005;xi:261–265. [Google Scholar]
46. Cecil R, Weitzman PD. The electroreduction of the disulphide bonds of insulin and other proteins. Biochem J. 1964;93:1–10. [PMC costless commodity] [PubMed] [Google Scholar]
47. Leach SJ, Meschers A, Swanepoel OA. Electrolytic reduction of proteins. Biochemistry. 1965;4:23–27. [PubMed] [Google Scholar]
48. Torchinskii IM, Metzler DE. Sulfur in Proteins. Pergamon. 1981 [Google Scholar]
49. Kruusma J, Benham AM, Williams JAG, Kataky R. An introduction to thiol redox proteins in the endoplasmic reticulum and a review of current electrochemical methods of detection of thiols. Analyst. 2006;131:459–473. [PubMed] [Google Scholar]
fifty. Zhang Y, Cui WD, Zhang H, Dewald Hard disk, Chen H. Electrochemistry-assisted top-down label of disulfide-containing proteins. Anal Chem. 2012;84:3838–3842. [PMC free article] [PubMed] [Google Scholar]
51. Harfield JC, Batchelor-McAuley C, Compton RG. Electrochemical determination of glutathione: a review. Analyst. 2012;137:2285–2296. [PubMed] [Google Scholar]
52. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70–77. [PubMed] [Google Scholar]
53. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman's reagent. Methods Enzymol. 1983;91:49–60. [PubMed] [Google Scholar]
54. Zhu JG, Dhimitruka I, Pei D. 5-(2-aminoethyl)dithio-two-nitrobenzoate as a more than base-stable alternative to Ellman'south reagent. Org Lett. 2004;vi:3809–3812. [PubMed] [Google Scholar]
55. Humphrey RE, Ward MH, Hinze Due west. Spectrophotometric determination of sulfite with 4,4′-dithiodipyridine and 5,5′-dithiobis-(2-nitrobenzoic acid) Anal Chem. 1970;42:698–702. [Google Scholar]
56. Tyagarajan K, Pretzer Eastward, Wiktorowicz JE. Thiol-reactive dyes for fluorescence labeling of proteomic samples. Electrophoresis. 2003;24:2348–2358. [PubMed] [Google Scholar]
57. Chen 10, Zhou Y, Peng XJ, Yoon J. Fluorescent and colorimetric probes for detection of thiols. Chem Soc Rev. 2010;39:2120–2135. [PubMed] [Google Scholar]
58. Kosower NS, Kosower EM, Newton GL, Ranney HM. Bimane fluorescent labels: Labeling of normal human being red cells under physiological weather condition. Proc Nat Acad Sci U Due south A. 1979;76:3382–3386. [PMC gratuitous commodity] [PubMed] [Google Scholar]
59. Imai K, Toyooka T, Watanabe Y. A novel fluorogenic reagent for thiols - ammonium 7-fluorobenzo-two-oxa-1,3-diazole-four-sulfonate. Anal Biochem. 1983;128:471–473. [PubMed] [Google Scholar]
60. Toyo'oka T, Imai Thousand. Isolation and label of cysteine-containing regions of proteins using four-(aminosulfonyl)-7-fluoro-ii,1,3-benzoxadiazole and high-performance liquid chromatography. Anal Chem. 1985;57:1931–1937. [PubMed] [Google Scholar]
61. Mentum CC, Wold F. The utilise of tributylphosphine and four-(aminosulfonyl)-seven-fluoro-2,one,three-benzoxadiazole in the report of protein sulfhydryls and disulfides. Anal Biochem. 1993;214:128–134. [PubMed] [Google Scholar]
62. Husted LB, Sorensen ES, Sottrup-Jensen L. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole is non specific for labeling of sulfhydryl groups in proteins as it may also react with phenolic hydroxyl groups and amino groups. Anal Biochem. 2003;314:166–168. [PubMed] [Google Scholar]
63. Treuheit MJ, Kirley TL. Reversibility of cysteine labeling past 4-(aminosulfonyl)-seven-fluoro-2,1,3-benzoxadiazole. Anal Biochem. 1993;212:138–142. [PubMed] [Google Scholar]
64. Abukhalaf IK, Silvestrov NA, Menter JM, von Deutsch DA, Bayorh MA, Socci RR, Ganafa AA. High performance liquid chromatographic assay for the quantitation of total glutathione in plasma. J Pharm Biomed Anal. 2002;28:637–643. [PubMed] [Google Scholar]
65. Li YH, Yang Y, Guan XM. Benzofurazan sulfides for thiol imaging and quantification in alive cells through fluorescence microscopy. Anal Chem. 2012;84:6877–6883. [PMC free commodity] [PubMed] [Google Scholar]
66. Appenzeller-Herzog C, Ellgaard L. In vivo reduction-oxidation state of protein disulfide isomerase: The 2 active sites independently occur in the reduced and oxidized forms. Antioxid Redox Indicate. 2008;10:55–64. [PubMed] [Google Scholar]
67. Joly JC, Swartz J. In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC. Biochemistry. 1997;36:10067–10072. [PubMed] [Google Scholar]
68. Goodson RJ, Katre NV. Site-directed pegylation of recombinant interleukin-two at its glycosylation site. Bio-Technol. 1990;eight:343–346. [PubMed] [Google Scholar]
69. Odom OW, Kudlicki West, Kramer Chiliad, Hardesty B. An effect of polyethylene glycol 8000 on protein mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a method for eliminating this effect. Anal Biochem. 1997;245:249–252. [PubMed] [Google Scholar]
seventy. Karala AR, Ruddock LW. Does S-methyl methanethiosulfonate trap the thiol-disulfide country of proteins? Antioxid Redox Betoken. 2007;nine:527–531. [PubMed] [Google Scholar]
71. Nielsen JW, Jensen KS, Hansen RE, Gotfredsen CH, Winther JR. A fluorescent probe which allows highly specific thiol labeling at low pH. Anal Biochem. 2012;421:115–120. [PubMed] [Google Scholar]
72. Smyth DG, Konigsberg W, Blumenfeld OO. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. 1964;91:589–595. [PMC gratuitous commodity] [PubMed] [Google Scholar]
73. Sharpless NE, Flavin K. The reactions of amines and amino acids with maleimides. Structure of the reaction products deduced from infrared and nuclear magnetic resonance spectroscopy. Biochemistry. 1966;5:2963–2971. [PubMed] [Google Scholar]
74. Brewer CF, Riehm JP. Prove for possible nonspecific reactions betwixt North-ethylmaleimide and proteins. Anal Biochem. 1967;18:248–255. [Google Scholar]
75. Chakravarthi S, Jessop CE, Bulleid NJ. The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep. 2006;vii:271–275. [PMC free commodity] [PubMed] [Google Scholar]
76. Hansen RE, Roth D, Winther JR. Quantifying the global cellular thiol-disulfide status. Proc Natl Acad Sci U S A. 2009;106:422–427. [PMC free commodity] [PubMed] [Google Scholar]
77. Monostori P, Wittmann G, Karg E, Turi S. Determination of glutathione and glutathione disulfide in biological samples: An in-depth review. J Chromatogr B. 2009;877:3331–3346. [PubMed] [Google Scholar]
78. Tietze F. Enzymic method for quantitative determination of nanogram amounts of full and oxidized glutathione - Applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502–522. [PubMed] [Google Scholar]
79. Griffith OW. Conclusion of glutathione and glutathione disulfide using glutathione-reductase and ii-vinylpyridine. Anal Biochem. 1980;106:207–212. [PubMed] [Google Scholar]
lxxx. Shaik IH, Mehvar R. Rapid conclusion of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples. Anal Bioana Chem. 2006;385:105–113. [PMC free article] [PubMed] [Google Scholar]
81. Melnyk Due south, Pogribna M, Pogribny I, Hine RJ, James SJ. A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection. J Nutr Biochem. 1999;10:490–497. [PubMed] [Google Scholar]
82. Diopan V, Shestivska V, Zitka O, Galiova M, Adam V, Kaiser J, Horna A, Novotny K, Liska M, Havel L, Zehnalek J, Kizek R. Determination of plant thiols by liquid chromatography coupled with coulometric and amperometric detection in lettuce treated by pb(II) Ions. Electroanal. 2010;22:1248–1259. [Google Scholar]
83. Sun YP, Smith DL, Shoup RE. Simultaneous detection of thiol-containing and disulfide-containing peptides by electrochemical high-operation liquid-chromatography with identification by mass-spectrometry. Anal Biochem. 1991;197:69–76. [PubMed] [Google Scholar]
84. Ostergaard H, Henriksen A, Hansen FG, Winther JR. Shedding calorie-free on disulfide bond formation: technology a redox switch in green fluorescent protein. EMBO J. 2001;20:5853–5862. [PMC complimentary article] [PubMed] [Google Scholar]
85. Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY. Imaging dynamic redox changes in mammalian cells with dark-green fluorescent protein indicators. J Biol Chem. 2004;279:22284–22293. [PubMed] [Google Scholar]
86. Meyer AJ, Dick TP. Fluorescent protein-based redox probes. Antioxid Redox Indicate. 2010;xiii:621–650. [PubMed] [Google Scholar]
87. Ostergaard H, Tachibana C, Winther JR. Monitoring disulfide bail formation in the eukaryotic cytosol. J Jail cell Biol. 2004;166:337–345. [PMC costless article] [PubMed] [Google Scholar]
88. Bjornberg O, Ostergaard H, Winther JR. Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent poly peptide. Biochemistry. 2006;45:2362–2371. [PubMed] [Google Scholar]
89. Gutsche Grand, Sobotta MC, Wabnitz GH, Ballikaya S, Meyer AJ, Samstag Y, Dick TP. Proximity-based Protein Thiol Oxidation by H2O2-scavenging Peroxidases. J Biol Chem. 2009;284:31532–31540. [PMC free article] [PubMed] [Google Scholar]
90. Bjornberg O, Ostergaard H, Winther JR. Measuring Intracellular Redox Conditions Using GFP-Based Sensors. Antioxid Redox Indicate. 2006;8:354–361. [PubMed] [Google Scholar]
91. Morgan B, Ezerina D, Amoako TN, Riemer J, Seedorf K, Dick TP. Multiple glutathione disulfide removal pathways mediate cytosolic redox homeostasis. Nat Chem Biol. 2012;9:119–125. [PubMed] [Google Scholar]
92. Grauschopf U, Winther JR, Korber P, Zander T, Dallinger P, Bardwell JC. Why is DsbA such an oxidizing disulfide catalyst? Cell. 1995;83:947–955. [PubMed] [Google Scholar]
93. Hatahet F, Ruddock LW. Protein disulfide isomerase: A disquisitional evaluation of its function in disulfide bond formation. Antioxid Redox Point. 2009;11:2807–2850. [PubMed] [Google Scholar]
94. Wunderlich M, Glockshuber R. Redox properties of poly peptide disulfide isomerase (DsbA) from Escherichia coli. Protein Sci. 1993;2:717–726. [PMC gratuitous article] [PubMed] [Google Scholar]
95. Bagiyan GA, Koroleva IK, Soroka NV, Ufimtsev AV. Oxidation of thiol compounds by molecular oxygen in aqueous solutions. Russ Chem Bull. 2003;52:1135–1141. [Google Scholar]
96. Munday R, Munday CM, Winterbourn CC. Inhibition of copper-catalyzed cysteine oxidation by nanomolar concentrations of atomic number 26 salts. Free Radical Bio Med. 2004;36:757–764. [PubMed] [Google Scholar]
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766385/
0 Response to "How Does Thiourea Prevent Prevent Sn2 From Occurring Again"
Post a Comment