From the Medical Nobel Institute for Biochemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden
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ABSTRACT |
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The immunostimulatory dinitrohalobenzene compound 1-chloro-2,4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arnér, E. S. J., Björnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479-3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.
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INTRODUCTION |
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TrxR1 catalyzes the NADPH-dependent reduction of the active site disulfide in oxidized thioredoxin to a dithiol in reduced thioredoxin. Thioredoxin is an ubiquitous 12-kDa protein with a large number of biological activities (1-6). Reduced thioredoxin is a powerful protein disulfide reductase catalyzing electron transport to ribonucleotide reductase and other reductive enzymes or redox regulation of enzymes and transcription factors. Secreted thioredoxin has cytokine-like effects on certain mammalian cells (2-7).
Thioredoxin reductase from Escherichia coli has been extensively characterized (1), and a high resolution x-ray structure shows surprisingly large differences to the other members of the pyridine nucleotide-disulfide oxidoreductase family (8, 9). Thus, the subunits of about 35 kDa are smaller than the about 50-kDa subunits present in glutathione reductase from all species. Furthermore the active site cysteine residues of E. coli TrxR are located in the central NADPH domain and separated by two amino acids (Cys-Ala-Thr-Cys), in comparison with the active site in glutathione reductase which is Cys-Val-Asn-Val-Gly-Cys and located in the NH2-terminal FAD domain, suggesting convergent evolution (9). The structural features of TrxR from E. coli with a high specificity for its homologous Trx are also typical for TrxR from prokaryotes, lower eukaryotes like yeast, or plants (1, 8-10).
It has long been known that mammalian TrxR has properties strikingly different to the enzyme from E. coli and lower organisms (2-4). The enzymes from calf liver and thymus and rat liver were first purified to homogeneity and showed 58-kDa subunits (11, 12). The mammalian thioredoxin reductases, including that of human placenta (13), are thereby larger than the E. coli enzyme and in contrast have a wide substrate specificity. Thus, the mammalian enzymes will reduce thioredoxins from different species (11), several low molecular weight disulfide substrates including DTNB used in assays (11, 12) or lipoic acid (14) as well as other nondisulfide substrates including selenodiglutathione (15), selenite (16), alloxan (17), or most surprising lipid hydroperoxides (18). The wide substrate specificity indicates an unusual structure of the active site, which is also demonstrated by the inhibition of mammalian TrxR by several drugs in clinical use including antitumor quinones (19, 20), nitrosoureas (21), or 13-cis-retinoic acid (22).
Recently, two proteins from transformed human cells having thioredoxin reductase activity, a 55-kDa subunit enzyme from a Jurkat T-cell line (23) and a 57-kDa subunit protein from a lung adenocarcinoma cell line (24), were shown to contain selenocysteine. A peptide sequence from the protein purified from the human T-cell line (23) agreed with a putative human placental TrxR cDNA sequence (25).
We have confirmed the sequence of the human enzyme and determined that of bovine TrxR peptides and a cDNA clone of rat TrxR (26). We have also shown that the selenocysteine residue, conserved between the mammalian species in the carboxyl-terminal motif Gly-Cys-Sec-Gly, is essential for catalytic activity (26). The mammalian TrxR amino acid sequence is highly homologous to GR and carries the identical sequence motif within the NH2-terminal FAD domain that contains the redox active cysteine residues of GR. The carboxyl-terminal motif with a penultimate Sec and a neighboring Cys residue is an elongation to the structure that is not present in GR, but is homologous to a Cys-Cys-containing elongation found in another related flavoprotein, mercuric reductase (26). The domain organization of TrxR is schematically depicted in Fig. 1.
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DNCB is an electrophilic compound used as a substrate in assays to determine glutathione S-transferase, which is involved in elimination of DNCB in vivo (27). DNCB is therefore also used in cell culture experiments as a GSH-depleting agent (28). Furthermore, DNCB has an established use as an immunomodulatory agent to provoke delayed-type hypersensitivity (29). Although proposed to function as a hapten, the mechanism of DNCB immunomodulation is not clear (30).
To summarize our earlier findings, we showed that DNCB irreversibly inhibited mammalian TrxR with second order kinetics by alkylating the enzyme, but that TrxR had to be reduced by preincubation with NADPH for the alkylation to occur (31). Upon alkylation, an NADPH oxidase activity could be detected, which was about 30-fold increased as compared with that of the native enzyme, and no consumption of NADPH was seen under anaerobic conditions (31). In the present study we demonstrate that the nitro groups of the dinitrophenyl-alkylated TrxR are necessary for the induction of the NADPH oxidase activity, and we identify the residues alkylated by DNCB.
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EXPERIMENTAL PROCEDURES |
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Materials and Enzymes--
DNCB, DNFB, DNCB analogues, IAA, and
4-VP as well as epinephrine bitartrate salt and SOD were purchased from
Sigma. Mammalian TrxR was purified from human placenta and calf thymus
as described (12). Enzyme concentration was determined from
A280 nm measurements
(A280 A340 = 0.7 mg/ml) or by the activity with 5 mM DTNB assuming that 1200 A412 units/ml corresponded to 1 mg of protein/ml
and an Mr of 120,000, i.e, a specific
activity of 22 µmol of NADPH oxidized per min/mg of protein in the
DTNB assay (12).
Enzyme Activity Measurements--
All experiments were performed
in 0.5 ml of 50 mM Tris-Cl, 2 mM EDTA, pH 8.0, at 20 °C using a Zeiss PM Q3 or a Hitachi spectrophotometer with
semimicro quartz cuvettes. Details of activity determination using
selenite reduction have been given in a previous paper (16). DNCB or
analogues were used as stock solutions in 99.5% EtOH, and in all
samples the same final volume of EtOH (1%) was added, also to
controls. For analysis of inhibition and induction of NADPH oxidase
activity, 40-150 nM TrxR was used with 100 µM amounts of the dinitrobenzenes or DNCB analogues, 50 µM IAA or 1 mM 4-VP. For determination of
NADPH oxidase activity, oxidation of NADPH was followed at 340 nm using
a molar extinction coefficient of 6200 M1
cm
1 in calculations.
Isolation of dnp-alkylated TrxR with Sephadex G-25 Columns-- TrxR was incubated with 200 µM NADPH in 0.50 ml of 50 mM Tris-Cl, 2 mM EDTA, pH 8.0, containing 1 mg/ml bovine serum albumin. After incubation for 20-60 min with electrophilic compounds at 20 °C, each sample was applied to a Sephadex G-25 column (NAP-5, Amersham Pharmacia Biotech), equilibrated with 50 mM Tris-Cl, 2 mM EDTA, pH 8.0, and the enzyme was collected in 1.0 ml of buffer. The alkylated TrxR free from electrophilic compound in solution could then be further analyzed for enzyme activity. Nonalkylated TrxR treated in the same manner was used as control enzyme.
Detection of Superoxide by the Adrenochrome
Method--
Superoxide was followed by reaction with epinephrine (2 mM in both reference and sample cuvettes) under production
of adrenochrome, with an increase of absorbance at 480 nm using an
extinction coefficient of 4020 M1
cm
1 (32).
Identification of dnp-alkylated Amino Acid Residues--
TrxR
(75 µg, 1.3 nmol of subunit) was preincubated for 30 min at room
temperature with NADPH (250 nmol) in 15 µl of 50 mM Tris-Cl, 2 mM EDTA, pH 7.5. Then 0.5 µl of 260 mM DNCB in ethanol was added to give 130 nmol (TrxR
subunit:DNCB
1:100 molar ratio, 3% ethanol), and alkylation
was allowed to proceed for 20 min at room temperature. Then the sample
was put on ice and subsequently run on a Fast desalting PC 3.2/10
column equilibrated with 50 mM Tris-Cl, 2 mM
EDTA, pH 7.5, using the SMART HPLC system (Amersham Pharmacia Biotech,
Uppsala, Sweden). To the alkylated enzyme guanidine hydrochloride
(final concentration: 0.8 M) and 7.5 µg of Lys-C endoproteinase (Wako) was added, and the sample was incubated for
17 h at 37 °C, whereafter additional 2.5 µg of Lys-C
endoproteinase was added and incubation continued for 1.5 h at
37 °C. Then peptides were separated by automated fractionation using
the SMART HPLC system with a Sephasil µRPC C2/C18 2.1/10 column in a
gradient of 0.1% trifluoroacetic acid in water (buffer A) and 0.1%
trifluoroacetic acid in acetonitrile (buffer B) using 100% buffer A
for 6 min, a linear gradient to 50% buffer B in 120 min, followed by a
linear gradient to 100% buffer B in 3 min and finally 100% buffer B
for 15 min. Peptides were followed at 214 and 254 nm, while
dnp-alkylated peptides were identified by absorbance at 340 nm.
Fractions with absorbance at 340 nm were taken to amino acid sequence
determination using Edman degradation on a Procise Protein Sequencer
(Applied Biosystems) or to analysis using electrospray mass
spectrometry with an AutoSpec OATOFFPD (Micromass, Manchester, UK).
Yields at the individual cycles of the Edman degradation were as
follows (in picomoles): peptide in fraction B,
4.7-4.2-5.7-13-7.3-29-11-10-9.5-6.5-<0.5-<0.5-<0.5; peptide 1 in
fraction C,
>50-16-15-11-3.4-6.2-7.9-8.8-6.3-2.6-6.2-8.8-8.2-10-8.1-6.4-7.2-4.1-3.1-6.8-3.6-1.5-<0.5->3; peptide 2 in fraction C,
0.6-3.4-8.4-3.4-0.7-1.1-1.9-6.3-1.0-1.6-3.7-0.5-1.8-1.2-0.8-1.7-5.3-3.6-<0.5-1.1-<0.5-<0.5-<2. Mass spectra were recorded over an m/z range of
1500-300 at a resolution of 4000 (10% valley definition). Multiply
charged ions were observed corresponding to (M + 2H)2+, (M + 3H)3+, and (M + 4H)4+ protonated molecules
(see Fig. 5). The help with the mass spectrometry of Dr. William
Griffiths, Protein Analysis Center, Karolinska institutet, Sweden, is
acknowledged. Prior to the mass spectrometry, the fractions from the
HPLC had first been dried in a Speed-Vac and then peptides were
re-dissolved in 10 µl of 50% methanol.
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RESULTS |
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Reactivity of Dinitrohalobenzenes and Analogues with TrxR and Production of Superoxide-- Induction of an NADPH oxidase activity in mammalian TrxR upon alkylation with DNCB could be an effect due to the alkylation of the enzyme per se, due to specific properties of DNCB, or due to a combination of these factors. To analyze this, we compared the effects on mammalian TrxR of different DNCB analogues and of alkylating agents other than dinitrohalobenzenes. The well known alkylating agents IAA or 4-VP did not induce any NADPH oxidase activity, although TrxR was irreversibly inhibited, showing that only alkylation of the reduced enzyme was not sufficient to induce an NADPH oxidase activity (Table I). The DNCB analogues 3,4-DCNB, 2,5-DCNB, 4-chloronitrobenzene, and 1,4-dichlorobenzene neither inhibited TrxR nor induced an NADPH oxidase activity, whereas a second dinitrohalobenzene compound, DNFB, both irreversibly inhibited TrxR and induced an NADPH oxidase activity (Table I). The results indicated that specific properties of dinitrohalobenzenes were needed to concomitantly alkylate and induce an NADPH oxidase activity in TrxR.
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Identification of Alkylated Residues-- To determine the site(s) of alkylation, DNCB was in the presence of NADPH added to native TrxR at a 100-fold molar excess for 20 min at room temperature, i.e. conditions that yield a fully inactivated enzyme but concentrations and time of incubation sufficiently low to avoid extensive nonspecific alkylation. The dnp-alkylated protein was then desalted, digested with lysine-specific endoproteinase, and peptides were separated using reverse phase HPLC. Three fractions were found to exhibit absorbance at 340 nm, indicating the possible presence of dnp-alkylated peptides (Fig. 4, fractions A, B, and C). These fractions were subjected to Edman degradation, where fraction A gave no amino acid sequence, fraction B contained a peptide corresponding to the carboxyl-terminal end of the enzyme, and fraction C contained two peptides from the interface domain of the enzyme (Table III). When detection of phenylthiohydantoin-derived amino acids was changed to 340 nm at the Edman degradation of the peptide in fraction B, a peak of decreasing intensity was seen during the first 2-3 cycles (not shown), which indicated that the dnp group is lost from the alkylated amino acid(s) during Edman degradation, as has been suggested elsewhere in experiments with glutathione S-transferase (33). Therefore Edman degradation could not be utilized to positively identify the residue(s) alkylated with a dnp group.
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DISCUSSION |
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The NADPH oxidase activity of native TrxR was with 200 µM NADPH determined to be about 0.15 min1
(31), which is quite low compared with other flavoproteins (34). The
increase of NADPH oxidase activity in mammalian TrxR by DNCB was shown
here to be dependent not solely on the alkylation but on a combination
of this with a certain property of the dinitrohalobenzenes, no doubt
carried by the reactive nitro groups of these compounds.
The induced NADPH oxidase activity resembles that of GR incubated with TNBS studied in detail by Carlberg and Mannervik (35). This interaction was, however, reversible and was fully dependent on TNBS in solution. The kinetics of the TNBS induced NADPH oxidase activity in GR was most complex, sigmoidal in the absence and nonsigmoidal in the presence of NADP+, with the latter supporting one-electron transfer reactions (35). However, with TrxR alkylated by DNCB, addition of NADP+ did not change the velocity of NADPH oxidase activity (not shown) and the effects of DNCB in solution added to DNCB-alkylated TrxR (Fig. 3) did not show the complex kinetics that was found in the interaction between GR and TNBS.
Lipoamide dehydrogenase, which like GR is structurally related to mammalian TrxR (26), is known to carry diaphorase activity, i.e. having the capacity to transfer electrons to artificial acceptors such as dichlorophenolindophenol or ferricyanide (reviewed in Ref. 1). It is clear that the FAD of lipoamide dehydrogenase catalyzes this diaphorase activity directly without participation of the redox active disulfide, since both lipoamide dehydrogenase treated with -SH alkylators (36) point mutated lipoamide dehydrogenase with serines instead of cysteines at the redox active disulfide (37), or other chemical modifications inhibiting the normal redox reactions (1) can leave the electron transfer to alternate acceptors unaffected or even greatly increased.
The NAD(P)H-dependent aerobic redox cycling of aromatic nitro compounds with flavoenzymes under production of superoxide is a known phenomenon (36, 38-40). The overall mechanism is believed to be reduction of the flavin by the pyridine nucleotide, followed by one-electron transfers from the flavin to the nitro groups under production of nitro anion radicals, that in turn react with molecular oxygen under production of superoxide. We believe that this is the basic mechanism for the NADPH oxidase activity in mammalian TrxR induced by dinitrohalobenzenes and that the reacting nitro groups are either in solution or, alternatively, those positioned close to the active site by a dinitrobenzene alkyl group of the alkylated enzyme.
We found that three residues were dnp-alkylated in TrxR upon gentle incubation with DNCB. The fraction with the highest absorbance at 340 nm (fraction B, Fig. 4) contained the peptide corresponding to the carboxyl-terminal end of the enzyme (see Table III and Fig. 1) with both the Cys and the Sec alkylated. In addition, we found a peptide corresponding to a part of the interface region being alkylated with one dnp group, either at the His proposed to act as a base at the active site in GR and TrxR-like enzymes (1, 26) or at its neighboring Cys (peptide 2, fraction C, Table III). Alkylation at all three of these residues can in theory easily explain the inactivation of the normal enzymatic activity of the enzyme, all residues being envisioned to take part at the active site of the enzyme. It is not at this stage certain which of the nitro groups of the provided dnp groups that would give rise to superoxide formation. However, we may propose the following events. First, reduction of the enzyme with NADPH, via the FAD prosthetic group and probably the NH2-terminal GR-like redox active disulfide, must make the Cys and Sec at the carboxyl terminus, or possibly the whole active site, more accessible for alkylation, since oxidized TrxR is resistant to alkylation by DNCB (31) as well as digestion with carboxypeptidase Y (26). A combination of conformational change or reduction of a bridge between the carboxyl-terminal Cys and Sec residues2 could be proposed to account for the change in accessibility for alkylation. We then propose that the FAD of the dnp-alkylated enzyme still can be reduced by NADPH but instead of two-electron transfer of reducing equivalents to the NH2-terminal active site disulfide, as in the normal case, a nitro group of the dnp group is reduced in a one-electron transfer to form an anion radical, which in turn reacts with oxygen to form superoxide. A second consecutive step of a one-electron transfer to form another nitro radical with subsequent reaction with oxygen under formation of superoxide would regenerate fully oxidized FAD of the enzyme and NO2 groups in the participating dnp group. Adrenochrome formation is a good measure of superoxide formation formed by univalent reduction of oxygen by reduced flavins (32). The fact that 2 mol of adrenochrome were formed per mol of NADPH by the DNCB-alkylated TrxR and that SOD completely blocked this formation (Table II) supports the notion that two superoxides are formed per NADPH (or FADH2). Free DNCB in solution is, in addition, proposed to be able to react with the FAD prosthetic group of the alkylated TrxR, to form nitroradicals and superoxide. This is supported by the Michaeli-Menten type of kinetics of the increase in NADPH oxidase activity dependent on free DNCB added to already alkylated TrxR (Fig. 3).
The cycle of events proposed above would explain the inactivation of TrxR with DNCB and concomitant induction of an NADPH oxidase activity. Since only three residues of the enzyme were found to be alkylated, the importance of these amino acids for the catalytic activity is illustrated. It should be pointed out that the active site structure of mammalian TrxR most likely is composed of the combination of the GR-like NH2-terminal redox active disulfide motif of one subunit and the carboxyl-terminal selenocysteine containing redox active motif of the other subunit (26), in analogy with mercuric reductase (1, 41). This implies that the nitro groups of the dnp groups at the carboxyl-terminal motif could be proposed to become positioned in the vicinity of the NH2-terminal redox active motif, which normally contains the cysteine residues that accept reducing equivalents from FADH2.
In this study we could not detect dnp derivatization of the NH2-terminal GR-like active site dithiol/disulfide motif, which we previously found to be alkylated by 4-VP in a similar experiment with NADPH-dependent alkylation and identification of redox active residues of the enzyme (26). The explanation could be that, specifically, the nitro groups of DNCB sterically hinders alkylation of the NH2-terminal redox active motif, or, more likely, that the carboxyl-terminal motif generally is more accessible and easily alkylated. It should be noted that the conditions for alkylation in this study were milder than in that with 4-VP (26).
It is of interest to note that the Cys as well as the Sec residue of the carboxyl-terminal peptide had been alkylated. The selenol group of Sec usually has a high nucleophilic reactivity and a low pKa value, which makes this residue a natural target for alkylation with electrophilic compounds, providing that it is sterically accessible. The finding that the neighboring Cys residue was alkylated as well indicates that in mammalian TrxR, its sulfhydryl group has an unusual reactivity indicating a low pKa value.
Does the specific and high reactivity with mammalian TrxR of dinitrohalobenzenes like DNCB play a role in the mechanism of the immunomodulating properties of these compounds? In this context, it is of importance to note that all of the DNCB analogues that failed to inhibit TrxR or to induce any NADPH oxidase activity (Table I) previously had been tested in vivo for induction of hypersensitivity reactions and shown to provoke no reaction (30). In the same study, O2 utilization, H2O2 production, and NADPH consumption in skin or liver microsomes was also measured upon addition of dinitrohalobenzenes or the DNCB analogues. All of these properties correlated well to mouse ear swelling upon application of the compounds, whereas changes in levels of GSH or GSSG did not (30). The enzyme(s) responsible for the NADPH consumption and superoxide (or H2O2) production were not identified, but based upon our study it is safe to conclude that TrxR is a strong candidate. How would the interaction with TrxR by dinitrohalobenzenes take part in the mechanism of immunostimulation by these compounds? Two mechanisms would be the most probable. First, Trx plays a central role in redox regulation of cell function (3, 5, 42-44), and an irreversible inhibition of TrxR would therefore with certainty affect Trx-related functions in the immune system. Second, the induced NADPH oxidase activity and increased production of superoxide (and H2O2 in the presence of SOD) by TrxR alkylated by dinitrohalobenzenes in combination with inhibition of the many anti-oxidant functions of the mammalian Trx system should give rise to a significant oxidative stress, which in itself is immunostimulatory (45).
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FOOTNOTES |
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* This work was supported by the Swedish Cancer Society (961, 3628, and 3775), the Swedish Medical Research Council (13X-3529), the Inga-Britt and Arne Lundbergs Stiftelse, the Knut and Alice Wallenbergs Stiftelse, Stiftelsen Sigurd och Elsa Goljes minne, and the Karolinska Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 46-8-728-69-83;
Fax: 46-8-31-15-51; E-mail: elias.arner{at}mbb.ki.se.
1 The abbreviations used are: TrxR, thioredoxin reductase; Trx, thioredoxin, Trx-S2, oxidized Trx; Trx-(SH)2, reduced Trx; SOD, superoxide dismutase; GR, glutathione reductase; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); DNCB, 1-chloro-2,4-dinitrobenzene; DNFB, 1-fluoro-2,4-dinitrobenzene; dnp, dinitrophenyl; 2,5-DCNB, 2,5-dichloronitrobenzene; 3,4-DCNB, 3,4-dichloronitrobenzene; 4-VP, 4-vinylpyridine; IAA, iodoacetic acid; Sec or U, selenocysteine; TNBS, 2,4,6-trinitrobenzenesulfonic acid; HPLC, high performance liquid chromatography.
2 A redox active selenyl sulfide between the carboxyl-terminal Cys and Sec residues was recently demonstrated (L. Zhong, E. S. J. Arnér, and A. Holmgren, manuscript in preparation).
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REFERENCES |
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