1 H NMR characterization of complexation of Glutathione with silver and aluminum metals in aqueous solution

Abstract

Metallo-elements have both pharmacological and toxic effects on plants, animals and humans. These are considered as a major public health issue worldwide. In particular, heavy metals such as silver (Ag) and aluminum (Al) are environmentally widespread, and their relative toxicity can lead to numerous pathologies such as nephropathy, cancers, vascular and skin diseases. The goal of this study was to examine the behavioral effects of Ag and Al salts (i.e. Silver nitrate and Aluminum sulfate) on glutathione (GSH), a potent oxidant in biological mixtures. We also aimed to suggest mechanisms of action of thiolate complexed to these metallo-elements in competitive studies with Ellman’s reagent (5,5’-dithiobis(2-nitrobenzoic acid aka ESSE). By proton nuclear magnetic resonance (NMR) spectroscopy, detailed titrations were carried out for these metal thiols interactions in the presence of ESSE in order to elucidate first equilibrium and possible second equilibrium. We found by ¹H NMR spectroscopy that GSH binds to Ag and Al, which highlighted possible in-vivo chelation mechanisms of GSH toward these toxic metallo-elements.

Keywords

Glutathione thiolates anions silver aluminium nuclear magnetic resonance spectroscopy

1 Introduction

For many years, Ellman’s reagent (5, 5’-dithiobis (2-nitrobenzoic acid aka ESSE) has been an important reagent for the spectrophotometric assay of sulfhydryl groups (-SH) in biology and so it is desirable that the Ellman’s reagent undergoes reaction with free -SH groups present in the solution system [1 –3]. The Ellman’s reagent (5,5^′-dithiobis-(2-nitrobenzoic acid) or DTNB) is a chemical used to quantify the number or concentration of thiol groups in a sample. It was developed by George L. Ellman. In the current study, to obtain accurate estimation of thiols by ESSE, we ensured that this reagent (i) bound to free -SH groups in aqueous solution system or biological mixtures, (ii) did not show any reactivity to the thiolate anions (RS^-) previously coordinated with metals (e.g. Ag, Al), (iii) reacts in such way that, at the end point, the reaction could terminate at the first equilibrium without shifting to the second equilibrium.

The accuracy of the analysis was then based on controlling the two related equilibria (equation 1), which are formed when ESSE is treated with RS-. Consequently the assay was conducted using an excess of ESSE, and experimental conditions were optimized to avoid the involvement of the second exchange reaction. Thereby, if ESSE was present at too high concentration during the analysis, the de-convolution of the bands (ESSEλ_max 325 nm; ES^–λ_max 412 nm) became difficult and the accuracy of the analysis was compromised. The solubility of ESSE and the pK_a of the sulfhydryl groups also required some attention. ESSE was only sparingly soluble in water at pH < 7 (acid). $\begin{matrix} K_{1} K_{2} \\ ESSE + 2 R S^{-} \Leftrightarrow ESSR + R S^{-} + E S^{-} \Leftrightarrow RSSR + 2 E S^{-} \end{matrix}$ (1)

The ESSE reaction is based on the reaction of 5,5^′-dithio-bis(2-nitrobenzoic acid (ESSE) with RS^–. The resulting Ellman’s anion (ES^–) exhibited intense light absorption at a wavelength (λ) of 410– 420 nm. The main limit of this method is linked to the intensity of the light absorption of ES^–, which is pH-independent when the pH of the medium is >7.3. Thus, this assay must be only employed above this pH value. However, if the pH rises above 8.0, the solution develops an intense and irreversible coloration [4]. Fortuitously, the important thiolate species typically studied (e.g. GSH) possess an appropriate pK_a (∼8.5) which makes the analysis possible at physiological pH.

The application of the spectrophotometric Ellman’s assay to more complex mixtures can be problematic, specifically when there is a potential for competition with -SH groups. This situation raised with the speciation of metals in biological mixtures, where there is a possibility of dynamic exchange between -SH groups and the parent ligand, solvent (i.e. water) or buffer [5]. In these instances, there is a great concern that ESSE can perturb the equilibrium, and compete either with thiolates previously coordinated with metals or with “free” -SH groups present in the solution. The consequences of this equilibrium alteration will be an over estimation of the amount of -SH groups present in the system [5 –8]. Thiophilic metal ions, such as silver and aluminum are present in virtually all organisms where they fulfil a multitude of functions and often bind to sulfur as their ligands. The resulting medley of metal binding and exchange on the one side and redox activity of some of these metals and sulfur ligands on the other leads to a truly complex chemistry that is reflected in their biochemistry [6, 7].

We were particularly interested in the behavior of heavy metals such as Ag and Al at -SH groups in biological mixtures, and so we decided to study thiolate complexes of monovalent Silver (i.e. Silver nitrate- AgNO₃) and trivalent Aluminum (i.e. Aluminum sulfate – Al₂(SO₄)₃) in competitive studies with ESSE.

In this manuscript, we report detailed ¹H NMR titrations of these metals-thiol interactions in the presence of ESSE, and provide insights about first and possible second equilibria, since such proton NMR studies allowed us to identify additional components of the reaction mixture and their relative abundance.

2 Materials and methods

2.1 Chemicals and reagents

All reagents were of analytical grade purity or equivalent unless otherwise stated, and included Silver nitrate (Sigma Aldrich) Aluminum sulphate (Across, Belgium), reduced L-Glutathione (Fluka AG), Deuterium oxide (Sigma Aldrich), distilled water (double refined), Ethanol 95% (Merck), Methanol HPLC grade (Merck), pH-Tablets (pH: 4 and pH 7) (Sigma Aldrich), Potassium dihydrogen phosphate (Merck), Sodium hydroxide (Fluka AG).

2.2 Equipments

The following instruments were used for this study, and included: Analytical weighing balance: Model AX 200 (Shimadzu, Japan), Magnetic stirrer (Chiltern, local market), Micropipettes (200μl, 500μl, 1000μl) (Socorex Swiss, Finland), NMR tubes 5 mm, Oven U-30,854: Model Memmert (Schwa Bach, Germany), pH meter: Model NOV-210 (Nova Scientific Company Ltd, Korea), Cary 300 Bio UV-Vis Spectrophotometer (Agilent), Varian Cary 1E UV-Vis Spectrophotometer (Agilent), Water Bath (Julabo, Germany), Bruker Avance-III 400 MHz NMR spectrometer (Brucker). The software used for NMR spectral data analysis was Bruker Topspin (Brucker).

2.3 Preparation of NMR samples

2.3.1 Stock solutions and reaction mixtures of silver nitrate with glutathione

Stock solution of GSH (C₁₀H₁₇N₃O₆S, FW: 307.32 g/mol) was prepared by dissolving 10 mg 32.53μmoles) of GSH in 1500μl buffer. Solutions of monovalent silver (Ag^I, here Silver nitrate, FWAgNO₃: 169.87 g/mol) were prepared in reaction buffer (0.1 M potassium phosphate (KH₂PO₄) in deuterium water (²H₂O) at pH 7.4) at four different molar equivalents: 0.215 mg (1.25μmoles), 0.43 mg (2.50μmoles), 0.645 mg (3.75μmoles) and 0.86 mg (5.0μmoles). In each of the four separate NMR tubes containing Ellman’s reagent, 300μl containing 2 mg (6.61μmoles) of GSH stock solution was mixed sequentially with 500μl of the respective concentration of Ag^I solution. An additional sample containing 2 mg GSH in a total volume of 800μL was prepared as a reference sample (i.e. negative control). The four following molar ratios between GSH and Ag^I were then used: 1:1.25; 1:2.50; 1:3.75; 1:50 these molar ratio are correct for GSH and silver while for GSH and silver the above mentioned ratios are correct Shown in the Table 1.

Table 1
Stock Solutions and Reaction Mixtures of Silver Nitrate with Glutathione

Amount of reactants Mole ratio

GSH Ag GSH: Ag GSH: AgNo₃

5.0μmoles 1.25μmoles 1: 1 1: 0.25

5.0μmoles 2.50μmoles 1: 1 1: 0.50

5.0μmoles 3.75μmoles 1: 1 1: 0.75

5.0μmoles 5.0μmoles 1: 1 1: 1

Amount of reactants	Mole ratio
GSH	Ag	GSH: Ag	GSH: AgNo₃
5.0μmoles	1.25μmoles	1: 1	1: 0.25
5.0μmoles	2.50μmoles	1: 1	1: 0.50
5.0μmoles	3.75μmoles	1: 1	1: 0.75
5.0μmoles	5.0μmoles	1: 1	1: 1

2.3.2 Stock solutions and reaction mixtures of aluminum sulfate with glutathione

In this case, stock solution of GSH (C₁₀H₁₇N₃O₆S, FW: 307.32 g/mol) was prepared by dissolving 15 mg (48.81μmoles) (This amount has been changed because Glutathione makes complex with silver in 1:1 ratio while glutathione with Aluminum makes with 3:1 ratio) of GSH in 1500μl buffer. Solutions of trivalent Aluminum (Al^III, here Aluminum sulfate, FWAl₂ (SO₄)₃: 342.15 g/mol) were prepared in reaction buffer (0.1 M KH₂PO₄ in ²H₂O at pH 7.4) at four different molar equivalents: 0.135 mg (0.40μmoles), 0.270 mg (0.80μmoles), 0.405 mg (1.20μmoles), 0.540 mg (1.60μmoles). In each of the four separate NMR tubes containing Ellman’s reagent, 300μl containing 3 mg 9.76μmoles) of GSH stock solution was mixed sequentially with 500μl of the respective concentration of Al^III solution. An additional sample containing 3 mg (this amount has been changed because Glutathione makes complex with silver in 1:1 ratio while glutathione with Aluminum makes with 3:1 ratio) GSH in a total volume of 800μl was prepared as a reference sample (i.e. negative control). The four following molar ratios between GSH and Al^III were then used: shown in Table 2

Table 2
¹H NMR analysis of thiolates complexation with of Ag^I and Al^III compounds

Amount of reactants Mole ratio

GSH Al^III * GSH: Al^III GSH: Al(SO₄)₃

1.2μmoles 0.4μmoles 3: 1 1: 0.25

2.4μmoles 0.8μmoles 3: 1 1: 0.50

3.6μmoles 1.2μmoles 3: 1 1: 0.75

4.8μmoles 1.6μmoles 3: 1 1: 1.00

Amount of reactants	Mole ratio
GSH	Al^III *	GSH: Al^III	GSH: Al(SO₄)₃
1.2μmoles	0.4μmoles	3: 1	1: 0.25
2.4μmoles	0.8μmoles	3: 1	1: 0.50
3.6μmoles	1.2μmoles	3: 1	1: 0.75
4.8μmoles	1.6μmoles	3: 1	1: 1.00

For NMR studies, inorganic salts of monovalent silver (Ag^I, here Silver nitrate) or trivalent Aluminum (Al^III, here Aluminum sulfate) were separately prepared in buffer (0.1 M KH₂PO₄ in ²H₂O at pH 7.4) and reacted with GSH. All the experiments were performed at room temperature (25°C) and the experimental steps involving solutions of thiols were maintained under argon atmosphere until the reaction mixture, in the NMR tube with tightly fitted cap, was analyzed by NMR. ¹H NMR spectra were eventually obtained at 400.12 MHz. The samples were maintained at 300 K during spectral acquisition. Free induction decay was generated by a 3.13μs pulse width corresponding to a 30° pulse with a 2 seconds delay between pulses. Each data set was collected in 32 K of memory. A 1 Hz line broadening function was applied before Fourier transformation to reduce the effect of the baseline noise.

3 Results and discussion

Reduced glutathione (GSH) is a likely candidate as a reducing agent because it is present in all living cells at high concentration (generally millimolar) and it exists primarily in its reduced form. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG), and under particular conditions, it may generate other oxidation products. Moreover, GSH plays an important role in the complexation and elimination of some toxic metals from organisms [9, 10] which is shared with more complex peptides and proteins such as metallothioneins in mammals or phytochelatins in plants [11]. Aluminum is the most abundant metal and the third most abundant element, after oxygen and silicon, representing about 8% of the earth's crust/surface layer. However, Aluminum is a very reactive element and is never found as the free metal in nature. Previously, using Ellman's method, we reported that different concentrations of Aluminium- acetyleacetonate as well as pH, temperature and time decrease the concentrations of GSH status in aqueous medium [10]. More recently, we hypothesize that these changes in glutathione level produced by aluminum could be due to conjugate (Al-(SG)₃) formation, endowing with information regarding mechanism of toxicity of aluminum inorganic and organic complexes [11].

The present investigation used ¹H NMR spectroscopy aimed to help detail binding reactions which occur in reaction buffer (0.1 M potassium phosphate (KH₂PO₄) in deuterium water (²H₂O) at pH 7.4) between silver (Ag^I) and Aluminum (Al^III) species and reduced gluthatione (GSH), a thiol of low molecular weight. For each body experiment, a total of sixty four ¹H NMR spectro-scans were obtained. Chemical shifts were referenced to D₂O (4.82 ppm). All ¹H NMR measurements were carried out at room temperature and at pH 7.4. GSH was chosen for study instead of other cellular sulfhydryl (-SH) compounds because of its potential involvement in the metabolism of silver. GSH (aka γ-L-glutamyl-L-cysteinylglycine) contains four ionizable protons [8 , 13], two in the carboxylic acids (-COOH) of the L-glutamyl and the glycyl groups, one in the L-cysteinyl sulfhydryl group and the other in the L-glutamyl ammonium group (Fig. 1).

Fig. 1

The 400 MHz ¹H NMR spectra of reduced Glutathione (1 mg/ml) in ²H₂O, giving proton resonance assignments at: (1) Glu-β-CH₂ (δ, 2.08 ppm); (2) Glu-γ-CH (δ, 2.46 ppm); (3) Cys-β-CH₂ (δ, 2.86 ppm); (4) Glu-α-CH (δ, 3.73 ppm); (5) Gly-α-CH₂ (δ, 3.89 ppm); (6) Cys- α-CH (δ, 4.48 ppm).

3.1 ¹H NMR spectral assignments of GSH

Since oxidation of GSH involves formation of the dimer GSSG via cysteinyl sulfhydryl groups, the greatest differences in the ¹H NMR spectra between GSH and GSSG were seen at the methylene group bonded to the sulfhydryl group of the cysteinyl residue (i.e. Cys-β-CH₂ group of GSSG and the protons attached to it) due to its chiral center. Indeed, the two cysteinyl Cys-β-CH₂ protons of GSH appeared as closely-spaced multiplets at δ, 2.86 ppm (Fig. 2), whereas two Cys-β-CH₂ protons of GSSG appeared as a multiplet at δ, 2.90 ppm and the other appeared as a closely-spaced multiplet at δ, 3.20 ppm (Fig. 2). The adjacent carbon atom (i.e. Cys-α-CH) and its methine proton were also shifted due to water-mediated oxidation, but to a reduced extent (Fig. 2).

Fig. 2

The 400 MHz ¹H NMR spectra of Glutathione disulfide (1 mg/ml) in 2H₂O, giving proton resonances at: (1) Glu-β-CH₂ (δ, 2.08 ppm); (2) Glu-γ-CH (δ, 2.46 ppm); (3) Cys-β-CH₂ (δ, 2.90 ppm, 3.20 ppm); (4) Glu-α-CH (δ, 3.73 ppm); (5) Gly-α-CH₂ (δ, 3.89 ppm); (6) Cys- α-CH (δ, 4.48 ppm).

3.2 ¹H NMR characterizations of Ag^I complexation with GSH

¹H NMR spectra of mixtures of Ag^I and GSH were found to be different from those of either GSSG or GSH (Fig. 3). Solutions containing four different molar ratios of GSH and Ag^I (i.e. 1:1.25; 1:2.50; 1:3.75; 1:5.0) were used (Fig. 3). As the amount of Ag^I was increased, all peak positions were shifted towards higher resonance frequencies, the most affected signals being those of the Cys-β-CH₂ methylene protons. Indeed, one of the two Cys-β-CH₂ methylene protons appeared as a multiplet at δ, 3.15 ppm, and the other gave a multiplet at δ, 3.28 ppm. This change in the peak positions of the Cys-β-CH₂ methylene protons indicates that GSH binds to Ag via its sulfhydryl group – SH group. Then, this is to say that Cys-β-CH₂ methylene protons shifted downfield upon forming the Ag-SG complex. Relatively to NMR spectra previously obtained with free specific ligands (GSH and AgI, Figs. 1 and 2, respectively), the other groups of GSH (i.e. Glu-α-CH and Gly-α-CH₂) were not coordinated to Ag since no changes in the proton resonances were observed. These findings are in close agreement with several studies [13 –15] and confirm the structures of these compounds. When the molar ratio, GSH:Ag^I was changed from 1.00:0.25 to 1.00:1.00 (These are more likely equivalent molar ratios Answer: as we already discussed that GSH and silver makes 1:1 ratio complex. So to further clear the complex peak we used four different concentrations of silver and metal and kept the glutathione concentration constant

Fig. 3

The 400 MHz ¹H NMR spectra of Ag^I-GSH complexes. Spectra were obtained by titrating a solution of silver nitrate (Ag^I) with either 0.215 mg (1.25μmoles), 0.43 mg (2.50μmoles), 0.645 mg (3.75μmoles) and 0.86 mg (5.0μmoles), in each of the four separate NMR tubes containing 3 mg (9.76μmoles) glutathione (GSH) in reaction buffer (i.e. 0.1 M KH₂PO₄ in 2H₂O at pH 7.4) ratio (bottom to top); (a) GSH:Ag^I (1:1) (1:0.75),(1:0.50), (1:0.25).

GSH: AgNO₃ = 1: 0.25

GSH: AgNO₃ = 1: 0.50

GSH: AgNO₃ = 1: 0.75

GSH: AgNO₃ = 1: 1

The presence of unchanged GSH was first evidenced at a ratio of 1:1 (GSH was taken in slight excess and peak for Glu-α-CH (δ, 3.73 ppm) were observed to be restored. This means that as we increase the concentrations of metal the complex peaks became more visible. These data indicate that the stoichiometry of the complex of Ag^I with GSH is 1:1. Hence the complex can be postulated to be Ag-SG and the reaction can be represented as: $1 H^{+} + AgN O_{3} + GSH \leftrightarrow Ag - SG + H_{2} O$ (2)

Eventually, our findings propose a model for detoxification and clearance of silver as GSH conjugate at the cellular level. Indeed, we were able to demonstrate Ag^I conjugation with GSH at pH 7.4; hence, our results can explain those of Baldi et al. who identified the presence of free and GSH-conjugated silver metabolites in the bile and urine of rats. The roles of Ag^I and GSH complexes in transport and metabolism vivo are unknown and require further studies [16].

3.3 ¹H NMR characterizations of Al^III complexation with GSH

The related experiments were carried out in identical lab conditions than those performed to detect the binding reactions of Ag^I with GSH. Chemical shifts were referenced to the acetate peak present in the Al^III (δ, 1.86 ppm). The chemical shifts of the Cys-β-CH₂ methylene protons present in GSH were found to be good markers of complex formation between Al^III and GSH by ¹H NMR (Fig. 4). Solutions containing four different molar ratios of Al^III and GSH were employed (Fig. 4). The ¹H NMR spectra of the different solutions showed changes in the chemical shift of the Cys-β-CH₂ (Fig. 4) (Table 3) peak for Cys-β-CH₂ of coordinated GSH splits into multiples (δ, 2.90 ppm) and can be noticed (Fig. 4) with respect to the spectral analysis with free GSH (Fig. 1). These changes in the proton resonances of Cys-β-CH₂ indicate that, at pH 7.4, the cysteinyl thiolate group was coordinated to the Al (III) center, which is in agreement with previous studies [16, 17]. However, in our experimental conditions, the Glu-α-CH and Gly-α-CH₂ groups of GSH were not coordinated (Fig. 4), since no changes in the proton resonances were observed for Glu-α-CH and Gly-α-CH₂ groups when compared to that of the free ligand (i.e. GSH) (Fig. 1). Taken together, our data shows that GSH is capable to bind to the Al3 + ion.

Fig. 4

The 400 MHz ¹H NMR spectra of Al^III -GSH complexes. Spectra were obtained by titrating a solution of Aluminum sulfate (Al^III) with either 0.135 mg (0.40μmoles), 0.270 mg (0.80μmoles), 0.405 mg (1.20μmoles), 0.540 mg (1.60μmoles), in each of the four separate NMR tubes containing 2 mg (6.51μmoles) glutathione (GSH) in reaction buffer (i.e. 0.1 M KH₂PO₄ in 2H₂O at pH 7.4).

Table 3

¹H NMR assignments of chemical shifts

Assign peaks	Chemical shift ppm (δ)	# Protons	Species Assignments
(1)	2.08	2	Glu-β-CH₂
(2)	2.46	2	Glu-γ-CH
(3)	2.86	2	Cys-β-CH₂
(4)	3.73	2	Glu-α-CH
(5)	3.89	2	Gly-α-CH₂
(6)	4.48	2	Cys- α-CH

Table 4

Chemical shift observed after complexation of glutathione with silver and aluminum

Two multiplet of each complex	Ag-GSH complex (Cys-α-CH2)	Al-GSH complex
H_a Quartet J1 = 2 Hz	δ, 2.85 ppm	δ, 2.90 ppm
H_b Quartet J1 = 2 Hz	δ, 3.0 ppm	δ, 3.25 ppm

It is worth to note that, in comparison to Ag^I, little information is available on the structure of Al^III-thiolate ligands (e.g. GSH) complexes, in spite of their common presence within cells or their exogenous administration as heavy-metal chelating agents Al^III ions displayed a large range of coordination numbers often in irregular geometric coordination. The toxic effects of Al^III are frequently attributed to the displacement of essential metals (e.g. Ca^II and Zn^II) by Al^III ions which, along with simultaneous binding to structural and catalytic protein sites, disturbs normal biological activity [17, 18]. Toxicological studies have shown that patients exposed to Al^III occupationally, obtain decreased levels of GSH in their blood, strongly suggesting that Al^III-GSH complexes are formed in-vivo. Also, animals exposed to aluminum, registered a significant decrease in the levels of reduced glutathione, and oxidized glutathione as well as in the activity of glutathione reductase in all the different brain regions studied when compared to normal control animals [19]. Eventually, it has been shown that Aluminum causes oxidative damage on cellular biological processes by inhibiting glutathione regeneration through the inhibition of NADPH supply in mitochondria, but only a little inhibitory effect on the glutathione generation in cytosol [21].

4 Conclusion

Our findings suggest that the metals silver and aluminum conjugated with glutathione in aqueous medium. The chances of such assumed conjugation reactions further increases with time elapse we have also used UV/visible spectrophotometer to study the complex formation. In our research work, we used different parameters i.e. concentration effect, time dependent effect and pH effect and observed that these complex are more pronounced as concentration increases, time elapsed and at pH 7.6), increasing concentration of the metallo-elements and/or suitable pH 7.4. These conjugation reactions have at least a biological importance taking into account that silver and aluminum have xenobiotic nature of causing oxidative stress and glutathione is known to exert detoxification and biotransformation actions of these metallo-elements. Therefore, this study performed in-situ can be used as a model for in-vivo assays.

Declarations

Conflict of interest

There is no conflict of interest among the authors

Funding

No funding received from any agency or institution for conducting this study.

Availability of data

The data can be obtained from corresponding author on request.

References

Ellman

G.L.

, Tissue sulfhydryl groups, Arch Biochem Biophys 82 (1959), 70–77.

Riddles

P.W.

, Blakeley

R.L.

and Zerner

, Ellman’s reagent: 5,5^′-dithiobis(2-nitrobenzoic acid)-a reexamination, Anal Biochem 94 (1979), 75–81.

Danehy

J.P.

, Elia

V.J.

and Lavelle

C.J.

, The alkaline decomposition of organic disulfides. IV. A limitation on the use of Ellman’s Reagent, 2,2^′ Dinitro-5,5^′-dithiodibenzoic acid, J Org Chem 36 (1971), 1003–1005.

Dixon

D.P.

, Cummins

, Cole

D.J.

and Edwards

, Purification, regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs, Curr Opin Plant Biol 1 (1998), 258–266.

Reglinski

, Hoey

, Smith

W.E.

and Sturrock

R.D.

, Cellular response to oxidative stress at sulfhydryl group, Anal Chem 34 (1998), 345–360.

Mulay

, Kim

, Choi

, Lee

D.Y.

, Choi

, Lee

, Jon

and Churchill

D.G.

, Enhanced doubly activated dual emission fluorescent probes for selective imaging of glutathione or cysteine in living systems, Anal Chem 90 (2014), 2648–2654.

Tang

, Gounaris

, Griffiths

and Selkirk

M.E.

, Heterologous expression and enzymatic properties of a selenium-independent glutathione peroxidase from the parasitic nematode Brugia pahangi, J Biol Chem 270 (1995), 18313–18318.

Abdillah

, Sonawane

P.M.

, Kim

, Mametov

, Shimodaira

, Park

and Churchill

D.G.

, Discussions of Fluorescence in Selenium Chemistry: Recently Reported Probes, Particles, and a Clearer Biological Knowledge, Molec 26 (2021), 692–.

Raab

, Meharg

A.A.

, Jaspars

, Genney

D.R.

and Feldmann

, Silver-glutathione complexes: their stability in solution and during separation by different HPLC modes, J Anal Atom Spec 19 (2004), 183–190.

10.

Yoshinari

, Hirai

, Kawamoto

, Igashira-Kamiyama

, Tsuge

and Konno

, Reversible conversion of tetranuclear, trinuclear, and mononuclear palladium (ii) complexes with D-penicillaminate, Chem Lett 38 (2009), 1056–1057.

11.

Freeman

H.C.

, Stevens

G.N.

and Taylor

I.F.

, Inorganic Reactions and Methods, the Formation of Bonds to C, Si, Ge, Sn, Pb Chem Commun, J Chem 39 (1974), 366–367.

12.

Vicky

and Farideh

, Lead (II) Complex Formation with Glutathione, Inorg Chem 51 (2012), 6285–6298.

13.

Delnomdedieu

, Basti

M.M.

, Otvos

J.D.

and Thomas

D.J.

, Transfer of arsenite from glutathione to dithiols: A model of interaction, Res Toxicol 6 (1993), 598–602.

14.

Haroon

, Farid

M.K.

, Barkat

A.K.

, Ghulam

and Naheed

, Evaluation of the Interaction of Aluminium Metal with Glutathione in Human Blood Components, Biomed Res 23 (2012), 237–240.

15.

Haroon

, Khan

M.F

, Jan

S.U.

and Ullah

, Effect of aluminium metal on glutathione (GSH) level in plasma and cytosolic fraction of human blood, Pak J Pharm Sci 24 (2011), 13–18.

16.

Baldi

, Minoia

and Nucci

, Effects of silver in isolated rat hepatocytes, Toxicol Lett 41 (1988), 261–268.

17.

Dallas

and Rabenstein

, Nuclear magnetic resonance studies ofthe acid-base chemistry of amino acids and peptides. I. Microscopicionization constants of glutathione and methylmercury-complexedglutathione, J Am Chem Soc, 95 (1973), 2797–2803.

18.

Bryan

and Rabenstein

D.L.

, Nuclear magnetic resonance studies of the solution chemistry of metal complexes. IX. Binding of cadmium, zinc, lead, and mercury by glutathione, Int J Chem Soc 14 (2016), 302–311.

19.

Murakami

and Yoshino

, Aluminum decreases the glutathione regeneration by the inhibition of NADP-isocitrate dehydrogenase in mitochondria, J Cell Biochem 93 (2004), 1267–1271.

20.

Bimla

and Punita

, Reversal of an aluminium induced alteration in redox status in different regions of rat brain by administration of centrophenoxine, Mol Cell Biochem 290 (2006), 185–191.