# Glutathione, Cysteine, and D-Penicillamine Role in Exchange of Silver Metal from the Albumin Metal Complex

Authors: Nahed S. Alharthi; Haroon Khan; Fahad Jibran Siyal; Zahid Ali Shaikh; Shumaila Parveen Arain; Lienda Bashier Eltayeb; Altaf Ali Mangi
Journal: BioMed Research International
 (2022)

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## Abstract

The purpose of this study is to investigate the exchange reaction taking place among the bovine serum albumin (BSA), 5,5′-dithiobis-(2-nitrobenzoic acid (ESSE), reduced glutathione, N-acetylcysteine, D-penicillamine (thiolates), and silver metal (AgI). For this purpose, stock solutions of BSA and Ellman’s reagent were prepared by dissolving 264 mg of BSA in 5 ml of reaction buffer (0.1 M KH2PO4 at pH 7.8) and 23.8 mg of ESSE in 1.0 ml of reaction buffer which were mixed together. Mixture of BSA-AgI was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. Blocking of Cys-34 of BSA with AgI was confirmed by treating different dilutions of BSA-AgI (500 μM) solutions with the solutions of ESSE (85 μM) and ES- (85 μM) and recording the spectra (300-450) with a UV-visible spectrophotometer. The chromatographed AgI-modified BSA ((BSA-S)AgI)) samples (typically 500 μM) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). AgI and modified BSA (typically 500 μM each) were treated with these low molecular weight thiolates and allowed to react overnight followed by chromatographic separation (Sephadex G25). The redox reactions of AgI-modified BSA with various low molecular weight thiols revealed a mechanically important phenomenon. In the case of reduced glutathione and N-acetylcysteine, we observed the rapid release of a commensurate amount of Ellman’s anion, indicating that an exchange has taken place and low molecular weight thiols (RSH) substituted AgI species at the Cys-34 of BSA eventually forming disulfide (BSA-SSR) at Cys-34. It can be anticipated from the phase of study involving bovine serum albumin that low molecular weight thiolates (reduced glutathione and N-acetylcysteine) take off AgI which are attached to proteins elsewhere in the physiological system, making these toxic metals free for toxic action.


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## Body

## 1. Introduction

Bovine serum albumin is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments (Peters, [1]). Cow’s milk contains around 30–35 g of proteins per litre and includes more than 25 different proteins, but only some of them are known to be allergenic. BSA is also commonly used to determine the quantity of other proteins, by comparing an unknown quantity of protein to known amounts of BSA. The ALB gene encodes the most abundant protein 5in human blood. Albumin has a high affinity for fatty acids, hematin, and bilirubin and a broad affinity for small negatively charged aromatic compounds [2–4]. It forms covalent adducts with pyridoxyl phosphate, cysteine, glutathione, and various metals [5]. Heavy metals compromise normal brain development and neurotransmitter function, leading to long-term deficits in learning and social behavior. Albumin, the most abundant protein in mammalian blood plasma, is involved in binding, transport, and delivery of a range of endogenous and exogenous small molecules or ions, such as fatty acids and metal ions [6]. It was used because of its easy availability in my research lab, and second thing, there are numerous advantages of silver metal like its medical uses as wound dressings, creams, and an antibiotic coating on medical devices and strong antioxidant effect [4]. Additionally, because of its abundance, human serum albumin plays a significant role in the pharmacokinetic behaviour of a variety of drugs, including drug half-life in the bloodstream, drug efficacy regulation, drug toxicity decrease, and drug targeting specificity improvement. Serum albumin has strong interactions with anionic and cationic ligands. Since the unbound form is being metabolized and/or excreted from the body, the bound fraction will be released in order to maintain equilibrium. Since albumin is alkalotic, acidic and neutral drugs will primarily bind to albumin. If albumin becomes saturated, then these drugs will bind to lipoprotein (Sadler et al., 1994; [7]), such as metal ions. The redox state of serum albumin’s Cys-34 has been proposed to be important for its biological function. In plasma, it is not supported by enzymes (glutaredoxin) and the GSH-regenerating system (hexose monophosphate shunt) [8], and the thiolation/dethiolation process is almost exclusively sustained by albumin. BSA is a serum albumin protein derived from bovine blood by a proprietary heat shock treatment. The plasma used for the process is collected as a byproduct of the meat industry. BSA meets and exceeds the exacting standards demanded by diagnostic, biopharmaceutical, and research customers worldwide. Plasma albumin has a theoretical concentration of 0.6 mM, only one -SH group, and several disulfide bridges. The cysteinyl residue, located in a well-conserved region in position 34 in all mammalian species, has a low pKa (about 5–7) [9–11], because of a salt bridge, with His 39 that stabilizes the thiolate anion. It is well known that albumin -SH is not well exposed, which impedes development of its high potential reactivity related to its low pKa. Indeed, the reaction rate of albumin towards -SH reagents (ESSE) is lower than that of thiol with higher pKa. Compounds such as fatty acids that change albumin conformation, improving Cys-34 exposure, lead to higher reactivity of albumin -SH (Simplicio et al., 1985; [12]). The difference of pKa of thiols involved in protein–thiol-mixed disulfides is an important feature to determine the kind of reaction (substitution or dethiolation) and consequently the end products. The premise is that the slow exchange of species bound to Cys-34 is the basis for a mechanism by which toxic species can become widely distributed around the body. In this study, we have sought to briefly investigate these issues. Since the method for the fractionation of human serum albumin on DEAE-Sephadex A-50, which had been worked out by Janatova [13], appeared to give somewhat better resolution of the albumin components than other published fractionation systems, therefore, this procedure was adopted for the fractionation of the bovine albumin preparations. The reaction of thiolates with excess Ellman’s reagent (ESSE, 5,5′-dithiobis(2-nitrobenzoic acid)) is used for quantitative estimation of thiol by measuring the absorption due to Ellman’s thiolate (ES-) at λ (412 nm). The reaction of thiolates with 5excess Ellman’s reagent is used for quantitative estimation of thiol by measuring the absorption at λ (412 nm) [14–16]. These metal-modified proteins have subsequently been challenged with thiolates (GSH, NAC, and Dpen) in an attempt to remove the metal and regenerate Cys-34. In the second phase of the study, we have metalated albumin with metals (silver nitrate (AgI species)). The disulfide exchange reactions occurring at Cys-34 of BSA with low molecular weight thiolates such as reduced glutathione (GSH), N-acetylcysteine (NAC), and D-penicillamine (Dpen) have been determined; in addition, the reduction of oxidized Cys-34 by these thiolates has also been studied in order to understand the reverse reaction. A reversible reaction is a chemical reaction where the reactants form products that, in turn, react together to give the reactants back. Reversible reactions will reach an equilibrium point where the concentrations of the reactants and products will no longer change. In this research, we have assessed the oxidative modification of and metal binding capacity of Cys-34 with heavy metals to investigate the ease with which it is possible to effect disulfide-thiol exchange at these sites or remove a metal bound at this position.


## 2. Experiment

All reagents were commercially obtained. Ellman’s reagent, bovine serum albumin (>98%; agarose gel electrophoresis lyophilised), and Sephadex (G25 coarse) were purchased from Sigma-Aldrich. UV-visible absorption spectra were recorded in a Unicam UV 300 spectrophotometer at room temperature. Thiols were made from haloalkanes by nucleophilic substitution of the halide ion by the sulfhydryl ion (HS−), which is an excellent nucleophile.


### 2.1. Preparation of Stock Solutions of BSA and Ellman’s Reagent

Different literature reviews, a handbook on pharmaceutical calculations, and some other stuff were followed before proceeding to lab work. BSA 264 mg was dissolved in 5 ml of reaction buffer (0.1 M KH2PO4 at pH 7.8). Dissolution of BSA was achieved by slow vortexing to avoid bubbles. The volume of BSA solution was made 20 ml with reaction buffer to get stock solution (200 μM) of BSA. Stock solution of ESSE (60 mM) was prepared by dissolving 23.8 mg of ESSE in 1.0 ml of reaction buffer.


### 2.2. Concentration of BSA Standard Solution

66.0 mg of BSA was dissolved in 20 ml of reaction buffer for preparation of standard solution (50μM) of commercially purchased BSA (unchromatographed). This standard solution was then serially diluted with reaction buffer to obtain 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM solutions of unchromatographed BSA. The UV spectra (250-350 nm) of the above five solutions of the BSA standard were recorded taking reaction buffer as a reference. Plotting the absorbance of the solution at λ (280 nm) gives a straight line (R2=0.999). The concentration of protein in solution was calculated using Beers’ law at λmax=280nm and ε=43,824cm−1M−1) (Peters, 1975). The absorbance of these known concentrations of the BSA standard was always used to adjust the working concentration of the column collected (chromatographed) albumin of unknown concentration.


### 2.3. Treatment of Ellman’s Modified Albumin (BSA-SSE) with Thiolates

The appearance of the protein in the eluent was identified by testing the liquors with trichloroacetic acid (which precipitates denatured protein) whereupon collection commenced. Periodic sampling identified when the eluent was protein free. The residual Ellman’s reagent and anion (identified as a yellow band) were eluted second and were from the column with further aliquots of reaction buffer. To study the reduction of Cys-34, initially, BSA was chemically modified at Cys-34 by Ellman’s reagent (ESSE) to give a bovine serum albumin-SE mixed disulfide (BSA-SSE). Solutions of BSA (200 mg, 0.5 ml) and Ellman’s reagent (ESSE) (1 mg, 0.5 ml) in reaction buffer (0.1 M KH2PO4, pH 7.4) were mixed (vol.ofthemixture=1ml) and allowed to react at room temperature overnight until absorbance at 412 nm was no longer changed. The solution was then carefully applied to a column (10cm×2cm) packed with swollen Sephadex (G25 coarse). The mixture was eluted with reaction buffer. Ellman’s assay is a useful tool that can be used to determine the sulfhydryl concentration of unknown solutions. It is done by following Beer’s law and the extinction coefficient of TNB.The BSA-SSE solution collected as above was diluted (1 ml of chromatographed protein solution was diluted to 5 ml) to generate a solution of known concentration (typically, 500μM, approximated from the working range of the known concentration of the BSA standard). The UV spectrophotometric spectrum (200–600 nm) was recorded. This BSA-SSE solution (typically, 500 μM) is separately titrated with 100, 200, 300, 400, and 500 μM of thiolates (reduced glutathione, N-acetyl cysteine, and D-penicillamine) and each time allowed to react overnight. The spectra were recorded UV spectrophotometrically. The release of Ellman’s anion is assessed at λmax=412nm (ε=14,150cm−1M−1).


### 2.4. Blocking of the Cys-34 in BSA

#### 2.4.1. Blocking the Cys-34 by AgI

Cysteines are unique among naturally occurring amino acids because of their thiol-containing side chain, which can undergo a variety of different nucleophilic reactions. The one-electron oxidation of a thiol(ate) group generates a thiyl radical, which gives rise to a diverse range of oxidation products, includingS-nitrosothiols. Mixture of BSA-AgI was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. The mole ratio of BSA with silver nitrate is 3 : 1 (BSA : AgI, 3 : 1). The BSA-AgI mixtures were allowed to react overnight. The BSA-AgI mixtures were carefully applied to a swollen Sephadex (G25 coarse) packed column. The mixtures were collected for metal bound proteins (BSA-AgI) by eluting with reaction buffer. The collected samples were approximated (diluted) with reaction buffer to BSA-AgI (125, 250, 375, and 500 μM) solutions by using the absorbance range of known concentrations of BSA standards. Blocking of Cys-34of BSA with AgI was confirmed by treating different dilutions of BSA-AgI (500 μM) solutions with the solutions of ESSE (85 μM) and ES- (85 μM) and recording the spectra (300-450) with a UV-visible spectrophotometer.


#### 2.4.2. Reaction of Thiolates with AgI-Capped BSA

The protein samples of BSA-AgI collected through swollen Sephadex (G25 coarse) packed column were mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) in separate procedures. Thiolates were added to BSA-AgI solutions in three equivalents to silver nitrate and allowed to react overnight. In separate procedures, the mixtures of BSA-AgI and solutions with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) were passed again through the clean Sephadex packed column and eluted with reaction buffer. The proteins (identified by testing the liquors with trichloroacetic acid) were eluted first and collected carefully to make sure that there are no free thiolate species in the collected protein samples. Since thiolates might be capable of taking off AgI previously bounded to BSA, rendering Cys-34 in BSA to be regenerated. The collected protein samples collected from the mixtures of BSA-AgI with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) were approximated (diluted) with reaction buffer to BSA-AgI/BSA-S (125, 250, 375, and 500 μM) by using the absorbance range of known concentrations of BSA standards. BSA-S- was then spectrophotometrically determined for free Cys-34 content by treating the protein dilutions with ESSE (85 μM). The release of Ellman’s anion is assessed at λmax=412nm (ε=14,150cm−1M−1).


## 2.1. Preparation of Stock Solutions of BSA and Ellman’s Reagent

Different literature reviews, a handbook on pharmaceutical calculations, and some other stuff were followed before proceeding to lab work. BSA 264 mg was dissolved in 5 ml of reaction buffer (0.1 M KH2PO4 at pH 7.8). Dissolution of BSA was achieved by slow vortexing to avoid bubbles. The volume of BSA solution was made 20 ml with reaction buffer to get stock solution (200 μM) of BSA. Stock solution of ESSE (60 mM) was prepared by dissolving 23.8 mg of ESSE in 1.0 ml of reaction buffer.


## 2.2. Concentration of BSA Standard Solution

66.0 mg of BSA was dissolved in 20 ml of reaction buffer for preparation of standard solution (50μM) of commercially purchased BSA (unchromatographed). This standard solution was then serially diluted with reaction buffer to obtain 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM solutions of unchromatographed BSA. The UV spectra (250-350 nm) of the above five solutions of the BSA standard were recorded taking reaction buffer as a reference. Plotting the absorbance of the solution at λ (280 nm) gives a straight line (R2=0.999). The concentration of protein in solution was calculated using Beers’ law at λmax=280nm and ε=43,824cm−1M−1) (Peters, 1975). The absorbance of these known concentrations of the BSA standard was always used to adjust the working concentration of the column collected (chromatographed) albumin of unknown concentration.


## 2.3. Treatment of Ellman’s Modified Albumin (BSA-SSE) with Thiolates

The appearance of the protein in the eluent was identified by testing the liquors with trichloroacetic acid (which precipitates denatured protein) whereupon collection commenced. Periodic sampling identified when the eluent was protein free. The residual Ellman’s reagent and anion (identified as a yellow band) were eluted second and were from the column with further aliquots of reaction buffer. To study the reduction of Cys-34, initially, BSA was chemically modified at Cys-34 by Ellman’s reagent (ESSE) to give a bovine serum albumin-SE mixed disulfide (BSA-SSE). Solutions of BSA (200 mg, 0.5 ml) and Ellman’s reagent (ESSE) (1 mg, 0.5 ml) in reaction buffer (0.1 M KH2PO4, pH 7.4) were mixed (vol.ofthemixture=1ml) and allowed to react at room temperature overnight until absorbance at 412 nm was no longer changed. The solution was then carefully applied to a column (10cm×2cm) packed with swollen Sephadex (G25 coarse). The mixture was eluted with reaction buffer. Ellman’s assay is a useful tool that can be used to determine the sulfhydryl concentration of unknown solutions. It is done by following Beer’s law and the extinction coefficient of TNB.The BSA-SSE solution collected as above was diluted (1 ml of chromatographed protein solution was diluted to 5 ml) to generate a solution of known concentration (typically, 500μM, approximated from the working range of the known concentration of the BSA standard). The UV spectrophotometric spectrum (200–600 nm) was recorded. This BSA-SSE solution (typically, 500 μM) is separately titrated with 100, 200, 300, 400, and 500 μM of thiolates (reduced glutathione, N-acetyl cysteine, and D-penicillamine) and each time allowed to react overnight. The spectra were recorded UV spectrophotometrically. The release of Ellman’s anion is assessed at λmax=412nm (ε=14,150cm−1M−1).


## 2.4. Blocking of the Cys-34 in BSA

### 2.4.1. Blocking the Cys-34 by AgI

Cysteines are unique among naturally occurring amino acids because of their thiol-containing side chain, which can undergo a variety of different nucleophilic reactions. The one-electron oxidation of a thiol(ate) group generates a thiyl radical, which gives rise to a diverse range of oxidation products, includingS-nitrosothiols. Mixture of BSA-AgI was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. The mole ratio of BSA with silver nitrate is 3 : 1 (BSA : AgI, 3 : 1). The BSA-AgI mixtures were allowed to react overnight. The BSA-AgI mixtures were carefully applied to a swollen Sephadex (G25 coarse) packed column. The mixtures were collected for metal bound proteins (BSA-AgI) by eluting with reaction buffer. The collected samples were approximated (diluted) with reaction buffer to BSA-AgI (125, 250, 375, and 500 μM) solutions by using the absorbance range of known concentrations of BSA standards. Blocking of Cys-34of BSA with AgI was confirmed by treating different dilutions of BSA-AgI (500 μM) solutions with the solutions of ESSE (85 μM) and ES- (85 μM) and recording the spectra (300-450) with a UV-visible spectrophotometer.


### 2.4.2. Reaction of Thiolates with AgI-Capped BSA

The protein samples of BSA-AgI collected through swollen Sephadex (G25 coarse) packed column were mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) in separate procedures. Thiolates were added to BSA-AgI solutions in three equivalents to silver nitrate and allowed to react overnight. In separate procedures, the mixtures of BSA-AgI and solutions with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) were passed again through the clean Sephadex packed column and eluted with reaction buffer. The proteins (identified by testing the liquors with trichloroacetic acid) were eluted first and collected carefully to make sure that there are no free thiolate species in the collected protein samples. Since thiolates might be capable of taking off AgI previously bounded to BSA, rendering Cys-34 in BSA to be regenerated. The collected protein samples collected from the mixtures of BSA-AgI with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) were approximated (diluted) with reaction buffer to BSA-AgI/BSA-S (125, 250, 375, and 500 μM) by using the absorbance range of known concentrations of BSA standards. BSA-S- was then spectrophotometrically determined for free Cys-34 content by treating the protein dilutions with ESSE (85 μM). The release of Ellman’s anion is assessed at λmax=412nm (ε=14,150cm−1M−1).


## 2.4.1. Blocking the Cys-34 by AgI

Cysteines are unique among naturally occurring amino acids because of their thiol-containing side chain, which can undergo a variety of different nucleophilic reactions. The one-electron oxidation of a thiol(ate) group generates a thiyl radical, which gives rise to a diverse range of oxidation products, includingS-nitrosothiols. Mixture of BSA-AgI was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. The mole ratio of BSA with silver nitrate is 3 : 1 (BSA : AgI, 3 : 1). The BSA-AgI mixtures were allowed to react overnight. The BSA-AgI mixtures were carefully applied to a swollen Sephadex (G25 coarse) packed column. The mixtures were collected for metal bound proteins (BSA-AgI) by eluting with reaction buffer. The collected samples were approximated (diluted) with reaction buffer to BSA-AgI (125, 250, 375, and 500 μM) solutions by using the absorbance range of known concentrations of BSA standards. Blocking of Cys-34of BSA with AgI was confirmed by treating different dilutions of BSA-AgI (500 μM) solutions with the solutions of ESSE (85 μM) and ES- (85 μM) and recording the spectra (300-450) with a UV-visible spectrophotometer.


## 2.4.2. Reaction of Thiolates with AgI-Capped BSA

The protein samples of BSA-AgI collected through swollen Sephadex (G25 coarse) packed column were mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) in separate procedures. Thiolates were added to BSA-AgI solutions in three equivalents to silver nitrate and allowed to react overnight. In separate procedures, the mixtures of BSA-AgI and solutions with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) were passed again through the clean Sephadex packed column and eluted with reaction buffer. The proteins (identified by testing the liquors with trichloroacetic acid) were eluted first and collected carefully to make sure that there are no free thiolate species in the collected protein samples. Since thiolates might be capable of taking off AgI previously bounded to BSA, rendering Cys-34 in BSA to be regenerated. The collected protein samples collected from the mixtures of BSA-AgI with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine) were approximated (diluted) with reaction buffer to BSA-AgI/BSA-S (125, 250, 375, and 500 μM) by using the absorbance range of known concentrations of BSA standards. BSA-S- was then spectrophotometrically determined for free Cys-34 content by treating the protein dilutions with ESSE (85 μM). The release of Ellman’s anion is assessed at λmax=412nm (ε=14,150cm−1M−1).


## 3. Results and Discussion

The subject of the thiol-disulfide interchange reaction is an important one in biochemistry and has been discussed extensively elsewhere. It has been known since long that glutathione also reacts with other thiol compounds, including proteins, and forms mixed disulfides. The physiological significance of this reaction has, however, been recognized only recently [17]. The subject of the thiol-disulfide interchange reaction is an important one in biochemistry and has been discussed extensively elsewhere. Oxidative stress causes the modification of proteins and impairs their biological functions. Among the amino acids found in albumin, cysteine-34 (Cys-34) is the most susceptible to modification by oxidants. Glutathione, present in the millimolar range in cells, prevents reactive sulfhydryls (Cys-34) of albumin from oxidative modification. It has been known since long that glutathione also reacts with other thiol compounds, including proteins, and forms mixed disulfides. The physiological significance of this reaction has, however, been recognized only recently [17]. Disulfide-reducing reagents are used in biochemistry for a number of purposes, especially in reduction of cysteine groups in albumin and in maintaining essential thiol groups in their reduced state [18]. In this research, we focus on the role of Cys-34 in albumin having a free SH group. A free thiol group in albumin (Cys-34) can be quantitatively determined by different methods, including the use of DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) ([14]; Jocelyn, 1972; [19]) also termed as Ellman’s reagent (ESSE). Ellman’s reagent (ESSE) was the earliest reagent widely used for estimating the number of thiol groups. The reaction between ESSE and the thiol group produces an equivalent amount of Ellman’s anion (ES-), the absorbance of which can be followed at 412 nm. Reaction rates of ESSE with serum albumins are much slower than those with small thiols of similar pKa; therefore, in the current study, overnight incubations have been provided to the mixtures involving the reaction of ESSE with BSA. Thiol groups are one of the most reactive groups in proteins and can participate in side reactions with reagents used to manipulate other functional groups in a protein and the other solution components.


### 3.1. Concentration of BSA Standard Solution

Five dilutions (10, 20, 30, 40, and 50μM) of the unchromatographed BSA sample were prepared and spectrophotometrically investigated for protein concentrations. The UV spectra of BSA were recorded at room temperature (Figure 1). In the wavelength range from 250 to 350 nm, the maximum absorption of BSA was at λ=278nm. Under the same conditions, the reaction buffer (0.1 M KH2PO4 at pH 7.4) was used as the blank solution. Concentrations of protein were calculated from the absorbance of the five standard dilutions of unchromatographed BSA at λ=278 using Beers’ law at λmax=278nm (ε=43,824cm−1M−1) (Peter, 1975). Concentrations of protein of the five standard BSA dilutions (10, 20, 30, 40, and 50 μM) were found to be 9.7, 19.4, 28.8, 38.2, and 44.13 μM, respectively.Figure 1
UV-vis spectra of 250-350 nm for five dilutions of unchromatographed BSA in reaction buffer.The protein concentration is unknown in the experiments which involve collection of free or complexed proteins through the Sephadex packed column, and they are required to be incorporated in this study of thiol disulfide exchange reactions with typically known concentrations. Hence, the absorbance range of the solutions of unchromatographed BSA and the subsequent calculation for protein concentration have been achieved which can be used in this piece of study for the adjustment of and approximating the working concentration of chromatographed protein samples of unknown concentration.


### 3.2. The Calculation of the Free Thiolate Content of BSA

The thiolate form of bovine serum albumin (BSA) typically comprises ~30-60% of the protein in the commercially available (unchromatographed) material. This value varies from batch to batch necessitating the calculation of the relative amount of thiolate present on the protein before the study can commence. The thiolate content of BSA is determined by the use of Ellman’s reagent (ESSE). The spectrophotometric assay of thiols with Ellman’s reagent (ESSE) depends on the cleavage of the disulfide bond of the almost colourless reagent, with the concomitant liberation of the colored anion (Ellman’s anion (ES-)) (Figure 2).Figure 2
Possible pathways for the reactions of BSA-SSE with RSH (J. JANATOVA et al. 1968).The pKa of the sulfhydryl group of Ellman’s anion is low enough so that the sulfhydryl group is essentially completely dissociated above pH 6.5. The magnitude of the absorbance at 412 nm is thus a measure of the sulfhydryl content of the added thiol.The thiolate status of the BSA was assessed initially by titrating each of the ten sequentially different dilutions of BSA (50-500μM) with Ellman’s reagent (85 μM) (Figure 3).Figure 3
UV-visible absorption (325-500 nm) of the titration of Ellman’s reagent with unchromatographed BSA (50-500μM) in reaction buffer.It is evident from the UV-visible spectra of unchromatographed BSA solutions (50-500μM) that the initial four solutions (up to 200 μM) of unchromatographed BSA exhibited linearity; hence, BSA was assessed subsequently by titrating each of the ten sequentially different dilutions of BSA (20-200 μM) with Ellman’s reagent (85 μM) (Figure 4).Figure 4
UV-visible absorption (325-500 nm) of the titration of Ellman’s reagent with unchromatographed BSA (50-200μM) in reaction buffer.Since ESSE reacts with thiols to give ES-, the amount of ES- released in solution represents the amount of thiol at the start of the reaction. Therefore, λ=412nm (ε=14,150cm−1M−1) was used for calculation of the thiolate content (Cys-34) of commercial BSA (unchromatographed). Rates of Cys-34 reduction were monitored by following the rise in absorbance at 412 nm for the release of ES-. Plotting the amount of Ellman’s anion released as the BSA concentration increases allows us to compensate for the natural absorbance of albumin at 412 nm (Figure 5) and hence gain an accurate value for its thiolate status. Using this approach, the BSA used here was found to be 53.9% in the thiolate form.
(1)BSA−S−+ESSE⟶⟵BSA−SSE+ES−Figure 5
The titration of BSA (20-200μM) with Ellman’s reagent (85 μM). The expected deviation from linear behaviour at high concentration of unchromatographed commercial BSA (>160 μM) is evident. An intercept is found which is consistent with residual absorbance by BSA at 412 nm.


### 3.3. Treatment of BSA-SSE with Thiolates

Size exclusion chromatography (SEC) is used for separation of proteins (BSA) after reacting BSA with ESSE and subsequently with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). In size exclusion chromatography (SEC), the larger-sized molecules were essentially eluted first from the column. Size exclusion chromatography (SEC) separates polymer molecules according to their size in dilute solution, but what size to use has been a matter of debate for 35 years. In 1967, Benoit and coworkers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume VH, for a wide range of species and large-scale molecular architectures. However, both theory and simulations assume a thermodynamic separation principle. This assumption is based on experimental observations that elution volumes are independent of flow rates. Medium-sized molecules are relatively large compared to the pore size of the solid phase and therefore may find some pores in which they enter and spend some time. Smaller-sized molecules have more pores that are accessible to them and therefore spend more time inside the pores relative to larger-sized molecules. Therefore, smaller molecules are eluted last and larger molecules are eluted first in size exclusion chromatography.In order to completely access the -SH groups of the Cys-34 of the protein (BSA) by Ellman’s reagent, the BSA-SSE mixture was allowed for an overnight reaction. The BSA-SSE mixture was then eluted through the swollen Sephadex (G25 coarse) packed column. The assay method not only releases Ellman’s anion but generates a stoichiometric amount of BSA labelled with Ellman’s moiety at cysteine-34 (BSA-SSE, equation (1)). By taking advantage of this reaction, it is possible to synthesize significant amounts of BSA which has been capped with Ellman’s moiety. Thus, incubate albumin with a small excess of Ellman’s reagent overnight (based on the thiolate assay) followed by chromatographic separation (Sephadex G25); we obtained a solution of Ellman’s modified BSA in reaction buffer. The solution can be desalted and freeze-dried to give pure and concentrated BSA-SSE in reduced volume. Alternatively, the molar absorptivity of the solution at 280 nm can be used to give a suitable estimate of the protein concentration in the eluent sample. We are interested here in the ability of small thiolate species (glutathione, D-penicillamine, and N-acetylcysteine) to react with Cys-34 in its disulfide form, and as such, we opted to work with the protein solutions.The chromatographed BSA-SSE sample (typically, 500μM) is subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). BSA-SSE (typically, 500 μM) is treated with reduced glutathione (100, 200, 300, 400, and 500 μM) and allowed to react overnight followed by chromatographic separation (Sephadex G25). Reactions of small thiolates with BSA-SSE mixed disulfides were accompanied by rapid massive substitution of ES- (leaving group) by RS- (entering group), marked by an increase in absorption at 412 nm because of free chromophore and an increase in BSA-SSR concentration. The reduction reactions of BSA-SSE with various small thiols revealed a mechanically important phenomenon. We observed the rapid release of a commensurate amount of Ellman’s anion (Figure 6) indicating that an exchange has taken place leading us to hypothesize the following reaction adjustments, ultimately leading to the release of Ellman’s anion (ES-):
(2)BSA−SSE+RSH⟶⟵BSA−SSR+ES−(3)BSA−SSE+RSH⟶⟵BSA−SH+ESSR(4)ESSR+BSA−SH⟶⟵BSA−SSE+RSH(5)ESSR+BSA−SH⟶⟵BSA−SSR+ES−Figure 6
The titration of BSA-SSE (conc) with glutathione solutions (100-500μM). The formation of Ellman’s anion is evident from the appearance of a larger band at 412 nm.With excess thiols, two pathways for the reduction of BSA-SSE become apparent. When thiols (RSH) attack a disulfide, one sulfur atom in the disulfide bond is the electron acceptor and is incorporated into the new disulfide bond, while the other becomes a thiol. For example, the first step of the reaction can give either ES- and a BSA mixed disulfide adduct (BSA-SSR) or ESSR and the regenerated thiol form of BSA (equations (2) and (3)). The newly formed ESSR can then react further with BSA-SH (equations (4) and (5)). Ellman’s anion (ES-), the only product monitored, is the final product in each case and is reasonably stable due to its low pKa and the existence of several resonance forms.The chromatographed sample of BSA-SSE (conc., approximately 500μM) is treated with N-acetylcysteine (100, 200, 300, 400, and 500 μM) and allowed to react overnight followed by chromatographic separation (Sephadex G25). We observed the rapid release of a commensurate amount of Ellman’s anion (Figure 7) indicating that an exchange has taken place almost in the same fashion as observed with reduced glutathione.Figure 7
The titration of BSA-SSE (conc) with N-acetylcysteine solutions (100-500μM). The formation of Ellman’s anion is evident from the appearance of a larger band at 412 nm.These data, however, indicate that a significant portion of the GSH and NAC can form mixed disulfides with proteins in cells. Both the inhibition of glutathione biosynthesis and recycling would cause oxidative stress and elevated protein-GSH adducts. NAC is generally used to increase intracellular glutathione. Since GSH plays a major role in maintaining intracellular redox state, lowering GSH levels causes oxidative stress. Thus, the enhanced protein-GSH adducts might occur during the process of GSH depletion. Further examination would be required to clarify this.In contrast to reduced glutathione and N-acetylcysteine, the reaction of BSA-SSE (typically, 500μM) with D-penicillamine (100, 200, 300, 400, and 500 μM) produced no Ellman’s anion (Figure 8). D-Penicillamine is shown to be not able to substitute ES- in the mixed disulfide (BSA-SSE).Figure 8
The titration of BSA-SSE (conc) with D-penicillamine solutions (100-500μM). The reduced formation of Ellman’s anion is evident from the appearance of smaller bands at 412 nm.The absorbance peak produced for Ellman’s anion (ES-) is relatively higher if ES- of BSA-SSE is substituted by NAC compared to substitution by GSH. The peak produced for ES- of BSA-SSE by Dpen was lower than peaks of the first two thiols (NAC and GSH).When BSA-SSE reacted with the thiols, the substitution phase was identified by the release of ES- being dethiolated from BSA-SSE. The difference in BSA-SSE dethiolation made it possible to establish the rank of ES- substitution by NAC>GSH>DPen (Figure 9).Figure 9
Comparison of plots of the addition of a single aliquot (200μM) of reduced glutathione, N-acetylcysteine, and D-penicillamine to a BSA-SSE solution (200 μM).Studies reported elsewhere have shown that D-penicillamine does not exchange with its mixed disulfide of Ellman’s reagent to form penicillamine disulfide due to steric problems. This observation suggests that the nature of the protein pocket acts to prevent the release of entities bound at Cys-34. This observation, however, is also an important control for the reactions involving reduced glutathione and N-acetylcysteine. A result similar to that shown in Figures6 and 7 might be expected from an exchange of thiolate with Ellman’s reagent loosely bound (H-bonded or hydrophobically associated) to the protein which remains in solution as a result of poor chromatographic separation. If either of these situations were present, an exchange reaction with D-penicillamine would be expected.


### 3.4. Blocking by AgI Species

The exchange experiments were carried out to explain protein substitution and dethiolation and more exactly to estimate fate of the metals in thiol disulfide exchange reactions. Metal-modified BSA mixtures were subjected to nucleophilic attack by thiolate anions (as the entering group). The -SH group (Cys-34) of BSA was blocked with AgI by separately incubating BSA solution with a small excess of silver nitrate overnight, followed by chromatographic separation (Sephadex G25); we obtained a solution of AgI-modified BSA in reaction buffer. The chromatographed AgI-modified BSA was incubated overnight with the solution of ESSE. ESSE was found to be not able to react with the AgI-modified -SH groups of Cys-34 in BSA. Treatment with ESSE confirms that -SH groups of Cys-34 in BSA have been effectively blocked by AgI as there is no release of ES- in the UV-visible spectrum at 412 nm (Figure 10).Figure 10
UV-visible spectra (250-450 nm) of ESSE reactivity with AgI-modified BSA (125, 250, 375, and 500 μM). The spectrum shows no release of ES- at λ (412 nm).


### 3.5. Reaction of Thiolates with AgI-Modified BSA

The chromatographed AgI-modified BSA ((BSA-S)AgI)) samples (typically 500 μM) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). AgI and modified BSA (typically 500 μM each) were treated with these low molecular weight thiolates and allowed to react overnight followed by chromatographic separation (Sephadex G25). The redox reactions of AgI-modified BSA with various low molecular weight thiols revealed a mechanically important phenomenon. In the case of reduced glutathione and N-acetylcysteine, we observed the rapid release of a commensurate amount of Ellman’s anion (equations (6) and (7)) indicating that an exchange has taken place, and low molecular weight thiols (RSH) substituted AgI species at the Cys-34 of BSA eventually forming disulfide (BSA-SSR) at Cys-34. The obtained results lead us to elucidate the following reactions:
(6)BSA−S−Ag+RSH⟶⟵BSA−SSR+AgI(7)BSA−SSR+ESSE⟶⟵BSA−SSE+RS−+ES−With excess thiols, an exchange occurs at Cys-34 of BSA previously bound by AgI. It is apparent that when RSH (GSH (Figure 11) or NAC (Figure 12)) attack AgI-modified BSA, it binds to Cys-34 of BSA (forming BSA-SSR) and renders AgI previously bound to Cys-34 of BSA to be free. The subsequent treatment of ESSE with BSA-SSR produces larger bands (release reasonably stable ES-) in UV spectrophotometric spectra. Reduced Cys-34 was detected in the single-chain peptides, it was the byproduct, and overall the procedure was quite technical and comprehensive to handle.Figure 11
UV-visible spectra (250-500 nm) of GSH reactivity with AgI-modified BSA (125-500 μM). The spectrum shows sequential increase in ES- at λ (412 nm).Figure 12
UV-visible spectra (250-500 nm) of NAC reactivity with AgI-modified BSA (125-500 μM). The spectrum shows sequential increase in ES- at λ (412 nm).After an overnight incubation of D-penicillamine with AgI-modified BSA, the protein samples have been collected through chromatographic separation (Sephadex G25). In contrast to reduced glutathione and N-acetylcysteine, D-penicillamine was not able to take AgI from the -SH groups of Cys-34 in BSA (Figure 13).Figure 13
UV-visible spectra (250-500 nm) of DPen reactivity with AgI-modified BSA (125-500 μM). The spectrum shows no production of ES- at λ (412 nm).A study was carried out by Mukhtiar et al. (2017) on the role of glutathione, cysteine and D-penicillamine in exchanging palladium and vanadium metals from albumin metal complex, and that was in good agreement with the current study since it is bound with albumin strongly and cannot be displaced by antioxidant like GSH, cysteine, and D-penicillamine. So exposure of human to these metals may disturb their normal physiology. Another study carried out by Jalilehvand et al. (2009) on the cadmium(II) complex formation with cysteine and penicillamine is in disagreement with the current study that it only reveals differences between cysteine and penicillamine as ligands to the cadmium(II) ion that can explain why cysteine-rich metallothionines are capable of capturing cadmium(II) ions, while penicillamine, clinically useful for treating the toxic effects of mercury(II) and lead(II) exposure, is not efficient against cadmium(II) poisoning.


### 3.6. Statistical Analysis

The chi-square test was performed, and ap value of 0.002 showed significant results.


## 3.1. Concentration of BSA Standard Solution

Five dilutions (10, 20, 30, 40, and 50μM) of the unchromatographed BSA sample were prepared and spectrophotometrically investigated for protein concentrations. The UV spectra of BSA were recorded at room temperature (Figure 1). In the wavelength range from 250 to 350 nm, the maximum absorption of BSA was at λ=278nm. Under the same conditions, the reaction buffer (0.1 M KH2PO4 at pH 7.4) was used as the blank solution. Concentrations of protein were calculated from the absorbance of the five standard dilutions of unchromatographed BSA at λ=278 using Beers’ law at λmax=278nm (ε=43,824cm−1M−1) (Peter, 1975). Concentrations of protein of the five standard BSA dilutions (10, 20, 30, 40, and 50 μM) were found to be 9.7, 19.4, 28.8, 38.2, and 44.13 μM, respectively.Figure 1
UV-vis spectra of 250-350 nm for five dilutions of unchromatographed BSA in reaction buffer.The protein concentration is unknown in the experiments which involve collection of free or complexed proteins through the Sephadex packed column, and they are required to be incorporated in this study of thiol disulfide exchange reactions with typically known concentrations. Hence, the absorbance range of the solutions of unchromatographed BSA and the subsequent calculation for protein concentration have been achieved which can be used in this piece of study for the adjustment of and approximating the working concentration of chromatographed protein samples of unknown concentration.


## 3.2. The Calculation of the Free Thiolate Content of BSA

The thiolate form of bovine serum albumin (BSA) typically comprises ~30-60% of the protein in the commercially available (unchromatographed) material. This value varies from batch to batch necessitating the calculation of the relative amount of thiolate present on the protein before the study can commence. The thiolate content of BSA is determined by the use of Ellman’s reagent (ESSE). The spectrophotometric assay of thiols with Ellman’s reagent (ESSE) depends on the cleavage of the disulfide bond of the almost colourless reagent, with the concomitant liberation of the colored anion (Ellman’s anion (ES-)) (Figure 2).Figure 2
Possible pathways for the reactions of BSA-SSE with RSH (J. JANATOVA et al. 1968).The pKa of the sulfhydryl group of Ellman’s anion is low enough so that the sulfhydryl group is essentially completely dissociated above pH 6.5. The magnitude of the absorbance at 412 nm is thus a measure of the sulfhydryl content of the added thiol.The thiolate status of the BSA was assessed initially by titrating each of the ten sequentially different dilutions of BSA (50-500μM) with Ellman’s reagent (85 μM) (Figure 3).Figure 3
UV-visible absorption (325-500 nm) of the titration of Ellman’s reagent with unchromatographed BSA (50-500μM) in reaction buffer.It is evident from the UV-visible spectra of unchromatographed BSA solutions (50-500μM) that the initial four solutions (up to 200 μM) of unchromatographed BSA exhibited linearity; hence, BSA was assessed subsequently by titrating each of the ten sequentially different dilutions of BSA (20-200 μM) with Ellman’s reagent (85 μM) (Figure 4).Figure 4
UV-visible absorption (325-500 nm) of the titration of Ellman’s reagent with unchromatographed BSA (50-200μM) in reaction buffer.Since ESSE reacts with thiols to give ES-, the amount of ES- released in solution represents the amount of thiol at the start of the reaction. Therefore, λ=412nm (ε=14,150cm−1M−1) was used for calculation of the thiolate content (Cys-34) of commercial BSA (unchromatographed). Rates of Cys-34 reduction were monitored by following the rise in absorbance at 412 nm for the release of ES-. Plotting the amount of Ellman’s anion released as the BSA concentration increases allows us to compensate for the natural absorbance of albumin at 412 nm (Figure 5) and hence gain an accurate value for its thiolate status. Using this approach, the BSA used here was found to be 53.9% in the thiolate form.
(1)BSA−S−+ESSE⟶⟵BSA−SSE+ES−Figure 5
The titration of BSA (20-200μM) with Ellman’s reagent (85 μM). The expected deviation from linear behaviour at high concentration of unchromatographed commercial BSA (>160 μM) is evident. An intercept is found which is consistent with residual absorbance by BSA at 412 nm.


## 3.3. Treatment of BSA-SSE with Thiolates

Size exclusion chromatography (SEC) is used for separation of proteins (BSA) after reacting BSA with ESSE and subsequently with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). In size exclusion chromatography (SEC), the larger-sized molecules were essentially eluted first from the column. Size exclusion chromatography (SEC) separates polymer molecules according to their size in dilute solution, but what size to use has been a matter of debate for 35 years. In 1967, Benoit and coworkers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume VH, for a wide range of species and large-scale molecular architectures. However, both theory and simulations assume a thermodynamic separation principle. This assumption is based on experimental observations that elution volumes are independent of flow rates. Medium-sized molecules are relatively large compared to the pore size of the solid phase and therefore may find some pores in which they enter and spend some time. Smaller-sized molecules have more pores that are accessible to them and therefore spend more time inside the pores relative to larger-sized molecules. Therefore, smaller molecules are eluted last and larger molecules are eluted first in size exclusion chromatography.In order to completely access the -SH groups of the Cys-34 of the protein (BSA) by Ellman’s reagent, the BSA-SSE mixture was allowed for an overnight reaction. The BSA-SSE mixture was then eluted through the swollen Sephadex (G25 coarse) packed column. The assay method not only releases Ellman’s anion but generates a stoichiometric amount of BSA labelled with Ellman’s moiety at cysteine-34 (BSA-SSE, equation (1)). By taking advantage of this reaction, it is possible to synthesize significant amounts of BSA which has been capped with Ellman’s moiety. Thus, incubate albumin with a small excess of Ellman’s reagent overnight (based on the thiolate assay) followed by chromatographic separation (Sephadex G25); we obtained a solution of Ellman’s modified BSA in reaction buffer. The solution can be desalted and freeze-dried to give pure and concentrated BSA-SSE in reduced volume. Alternatively, the molar absorptivity of the solution at 280 nm can be used to give a suitable estimate of the protein concentration in the eluent sample. We are interested here in the ability of small thiolate species (glutathione, D-penicillamine, and N-acetylcysteine) to react with Cys-34 in its disulfide form, and as such, we opted to work with the protein solutions.The chromatographed BSA-SSE sample (typically, 500μM) is subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). BSA-SSE (typically, 500 μM) is treated with reduced glutathione (100, 200, 300, 400, and 500 μM) and allowed to react overnight followed by chromatographic separation (Sephadex G25). Reactions of small thiolates with BSA-SSE mixed disulfides were accompanied by rapid massive substitution of ES- (leaving group) by RS- (entering group), marked by an increase in absorption at 412 nm because of free chromophore and an increase in BSA-SSR concentration. The reduction reactions of BSA-SSE with various small thiols revealed a mechanically important phenomenon. We observed the rapid release of a commensurate amount of Ellman’s anion (Figure 6) indicating that an exchange has taken place leading us to hypothesize the following reaction adjustments, ultimately leading to the release of Ellman’s anion (ES-):
(2)BSA−SSE+RSH⟶⟵BSA−SSR+ES−(3)BSA−SSE+RSH⟶⟵BSA−SH+ESSR(4)ESSR+BSA−SH⟶⟵BSA−SSE+RSH(5)ESSR+BSA−SH⟶⟵BSA−SSR+ES−Figure 6
The titration of BSA-SSE (conc) with glutathione solutions (100-500μM). The formation of Ellman’s anion is evident from the appearance of a larger band at 412 nm.With excess thiols, two pathways for the reduction of BSA-SSE become apparent. When thiols (RSH) attack a disulfide, one sulfur atom in the disulfide bond is the electron acceptor and is incorporated into the new disulfide bond, while the other becomes a thiol. For example, the first step of the reaction can give either ES- and a BSA mixed disulfide adduct (BSA-SSR) or ESSR and the regenerated thiol form of BSA (equations (2) and (3)). The newly formed ESSR can then react further with BSA-SH (equations (4) and (5)). Ellman’s anion (ES-), the only product monitored, is the final product in each case and is reasonably stable due to its low pKa and the existence of several resonance forms.The chromatographed sample of BSA-SSE (conc., approximately 500μM) is treated with N-acetylcysteine (100, 200, 300, 400, and 500 μM) and allowed to react overnight followed by chromatographic separation (Sephadex G25). We observed the rapid release of a commensurate amount of Ellman’s anion (Figure 7) indicating that an exchange has taken place almost in the same fashion as observed with reduced glutathione.Figure 7
The titration of BSA-SSE (conc) with N-acetylcysteine solutions (100-500μM). The formation of Ellman’s anion is evident from the appearance of a larger band at 412 nm.These data, however, indicate that a significant portion of the GSH and NAC can form mixed disulfides with proteins in cells. Both the inhibition of glutathione biosynthesis and recycling would cause oxidative stress and elevated protein-GSH adducts. NAC is generally used to increase intracellular glutathione. Since GSH plays a major role in maintaining intracellular redox state, lowering GSH levels causes oxidative stress. Thus, the enhanced protein-GSH adducts might occur during the process of GSH depletion. Further examination would be required to clarify this.In contrast to reduced glutathione and N-acetylcysteine, the reaction of BSA-SSE (typically, 500μM) with D-penicillamine (100, 200, 300, 400, and 500 μM) produced no Ellman’s anion (Figure 8). D-Penicillamine is shown to be not able to substitute ES- in the mixed disulfide (BSA-SSE).Figure 8
The titration of BSA-SSE (conc) with D-penicillamine solutions (100-500μM). The reduced formation of Ellman’s anion is evident from the appearance of smaller bands at 412 nm.The absorbance peak produced for Ellman’s anion (ES-) is relatively higher if ES- of BSA-SSE is substituted by NAC compared to substitution by GSH. The peak produced for ES- of BSA-SSE by Dpen was lower than peaks of the first two thiols (NAC and GSH).When BSA-SSE reacted with the thiols, the substitution phase was identified by the release of ES- being dethiolated from BSA-SSE. The difference in BSA-SSE dethiolation made it possible to establish the rank of ES- substitution by NAC>GSH>DPen (Figure 9).Figure 9
Comparison of plots of the addition of a single aliquot (200μM) of reduced glutathione, N-acetylcysteine, and D-penicillamine to a BSA-SSE solution (200 μM).Studies reported elsewhere have shown that D-penicillamine does not exchange with its mixed disulfide of Ellman’s reagent to form penicillamine disulfide due to steric problems. This observation suggests that the nature of the protein pocket acts to prevent the release of entities bound at Cys-34. This observation, however, is also an important control for the reactions involving reduced glutathione and N-acetylcysteine. A result similar to that shown in Figures6 and 7 might be expected from an exchange of thiolate with Ellman’s reagent loosely bound (H-bonded or hydrophobically associated) to the protein which remains in solution as a result of poor chromatographic separation. If either of these situations were present, an exchange reaction with D-penicillamine would be expected.


## 3.4. Blocking by AgI Species

The exchange experiments were carried out to explain protein substitution and dethiolation and more exactly to estimate fate of the metals in thiol disulfide exchange reactions. Metal-modified BSA mixtures were subjected to nucleophilic attack by thiolate anions (as the entering group). The -SH group (Cys-34) of BSA was blocked with AgI by separately incubating BSA solution with a small excess of silver nitrate overnight, followed by chromatographic separation (Sephadex G25); we obtained a solution of AgI-modified BSA in reaction buffer. The chromatographed AgI-modified BSA was incubated overnight with the solution of ESSE. ESSE was found to be not able to react with the AgI-modified -SH groups of Cys-34 in BSA. Treatment with ESSE confirms that -SH groups of Cys-34 in BSA have been effectively blocked by AgI as there is no release of ES- in the UV-visible spectrum at 412 nm (Figure 10).Figure 10
UV-visible spectra (250-450 nm) of ESSE reactivity with AgI-modified BSA (125, 250, 375, and 500 μM). The spectrum shows no release of ES- at λ (412 nm).


## 3.5. Reaction of Thiolates with AgI-Modified BSA

The chromatographed AgI-modified BSA ((BSA-S)AgI)) samples (typically 500 μM) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). AgI and modified BSA (typically 500 μM each) were treated with these low molecular weight thiolates and allowed to react overnight followed by chromatographic separation (Sephadex G25). The redox reactions of AgI-modified BSA with various low molecular weight thiols revealed a mechanically important phenomenon. In the case of reduced glutathione and N-acetylcysteine, we observed the rapid release of a commensurate amount of Ellman’s anion (equations (6) and (7)) indicating that an exchange has taken place, and low molecular weight thiols (RSH) substituted AgI species at the Cys-34 of BSA eventually forming disulfide (BSA-SSR) at Cys-34. The obtained results lead us to elucidate the following reactions:
(6)BSA−S−Ag+RSH⟶⟵BSA−SSR+AgI(7)BSA−SSR+ESSE⟶⟵BSA−SSE+RS−+ES−With excess thiols, an exchange occurs at Cys-34 of BSA previously bound by AgI. It is apparent that when RSH (GSH (Figure 11) or NAC (Figure 12)) attack AgI-modified BSA, it binds to Cys-34 of BSA (forming BSA-SSR) and renders AgI previously bound to Cys-34 of BSA to be free. The subsequent treatment of ESSE with BSA-SSR produces larger bands (release reasonably stable ES-) in UV spectrophotometric spectra. Reduced Cys-34 was detected in the single-chain peptides, it was the byproduct, and overall the procedure was quite technical and comprehensive to handle.Figure 11
UV-visible spectra (250-500 nm) of GSH reactivity with AgI-modified BSA (125-500 μM). The spectrum shows sequential increase in ES- at λ (412 nm).Figure 12
UV-visible spectra (250-500 nm) of NAC reactivity with AgI-modified BSA (125-500 μM). The spectrum shows sequential increase in ES- at λ (412 nm).After an overnight incubation of D-penicillamine with AgI-modified BSA, the protein samples have been collected through chromatographic separation (Sephadex G25). In contrast to reduced glutathione and N-acetylcysteine, D-penicillamine was not able to take AgI from the -SH groups of Cys-34 in BSA (Figure 13).Figure 13
UV-visible spectra (250-500 nm) of DPen reactivity with AgI-modified BSA (125-500 μM). The spectrum shows no production of ES- at λ (412 nm).A study was carried out by Mukhtiar et al. (2017) on the role of glutathione, cysteine and D-penicillamine in exchanging palladium and vanadium metals from albumin metal complex, and that was in good agreement with the current study since it is bound with albumin strongly and cannot be displaced by antioxidant like GSH, cysteine, and D-penicillamine. So exposure of human to these metals may disturb their normal physiology. Another study carried out by Jalilehvand et al. (2009) on the cadmium(II) complex formation with cysteine and penicillamine is in disagreement with the current study that it only reveals differences between cysteine and penicillamine as ligands to the cadmium(II) ion that can explain why cysteine-rich metallothionines are capable of capturing cadmium(II) ions, while penicillamine, clinically useful for treating the toxic effects of mercury(II) and lead(II) exposure, is not efficient against cadmium(II) poisoning.


## 3.6. Statistical Analysis

The chi-square test was performed, and ap value of 0.002 showed significant results.


## 4. Conclusion

It can be anticipated from the phase of the study involving bovine serum albumin that low molecular weight thiolates (reduced glutathione and N-acetylcysteine) take off AgI which are attached to proteins elsewhere in the physiological system, making these toxic metals free for toxic action. The low molecular weight thiolates seem to be engaged in regeneration of AgI-bound sulfhydryls of protein across the living systems.



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*Source: 3619308-2022-08-08.xml*