Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Abstract

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K_M and V_max values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC₅₀ values, K_i constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag⁺; Cd²⁺, Cr²⁺ and Mg²⁺.

Keywords::

• Glutathione S-transferase
• rainbow trout
• erythrocytes
• metal ions

Introduction

Glutathione S-transferase plays a crucial role by detoxifying xenobiotics in combination with several mechanisms in the metabolism¹. This enzyme inactivates electrophilic xenobiotics by conjugating them with reduced glutathione (GSH) and eliminates them from the body². Glutathione S-transferase isoenzymes are homodimeric and heterodimeric proteins synthesized from a multitude of gene loci with a molecular mass of about 25 kDa. Each subunit has an active position involving two different functional sites, which are the G region that binds the physiological substrate and the hydrophobic H region that binds the structurally different electrophilic substrates. The amino acid composition of GST isozymes differs in the hydrophobic H region, which is the reason behind the substrate diversity of the enzyme family. Glutathione S-transferase is a member of the family of phase-II detoxification enzymes which catalyze the conjugation of glutathione with xenobiotics, to which living organisms are exposed voluntarily or involuntarily, and convert them into less toxic metabolites^3–5.

The effects of some metal ions on E. Coli GST⁶, human serum GST⁷, rainbow trout liver GR, human erythrocyte CA-I, CA-II and human erythrocyte GR activities have been investigated^8,9. However, no reports could be found in the literature about the effects of Ag⁺, Cd²⁺, Cr²⁺, and Mg²⁺ on rainbow trout erythrocytes GST. Dispersion and penetration properties of metals in the environment, which may cause vital danger in living organisms, are notable and very common. Metals are notable for their wide environmental dispersion from such activities as their tendency to accumulate in select tissues of the human body and their overall potential to be toxic even at relatively minor levels of exposure. On the other hand, some metals such as copper and iron are essential to life and play unavoidable roles in some critical enzyme systems. Other metals, xenobiotic, i.e., have no useful role in human physiology (and most other living organisms) and, even worse, as in the case of lead and mercury, may be toxic even at trace levels of exposure^8,9. The aim of this study was to investigate in vitro effects of some metal ions on purified and characterized rainbow trout erythrocytes GST activities.

Material and methods

Materials

Glutathione-agarose, Sephadex G-100, GSH, CDNB, protein assay reagents, chemicals for electrophoresis and all other chemicals used were obtained from either Sigma or Sigma-Aldrich Co. (Germany).

Preparation of the haemolysate

Fresh fish blood was sampled into heparinised (5 IU/mL) Vacuntainer^® tubes and centrifuged at 2.500g for 15 min. The plasmas were removed by drip. The erythrocytes were washed three times with isotonic NaCl solution (0.154 M) the samples being centrifuged at 2.500g each time and the supernatants removed. The erythrocytes were haemolysed with 5 vol. of ice-cold water and centrifuged (+4°C, 10.000g) for 30 min to remove ghosts and intact cells. Then, all haemolysate samples were pooled in a tube¹⁰.

Glutathione-agarose affinity chromatography

A 1 g lyophilized powder of glutathione-agarose was weighed for 10 mL of bed volume and washed several times with 200 mL distilled water to remove solid particles. Washing swelled the gel, which was degraded by using a water trompe and suspended by adding stabilization buffer (0.05 M K-phosphate (pH 7.4), 1 mM EDTA, and 1 mM DTT). The suspended gel was packed in a cooled column with a 1 × 10 cm closed system. After precipitation, the gel was washed using a peristaltic pump with stabilization buffer. Stabilization of the column was clear from the fact that the absorbance and pH of the eluate and buffer were identical. The affinity column was thus prepared. The prepared heamolysate was directly applied to the glutathione-agarose affinity column. Subsequently, the column was washed by passing through 10 mM KH₂PO₄ and 0.1 M KCl (pH 8.0). The washing procedure was monitored on a spectrophotometer through equal-to-blind absorbance values. After the column was stabilized, the enzyme was purified by gradient elution. Elution solvent was prepared from a solvent gradient containing 50 mM Tris–HCl and (1.25–10 mM GSH, pH 9.5). The eluates were taken into 1.5 mL tubes and their activity was measured by monitoring the increase in absorbance at 340 nm. The column flow rate was set to 35 mL/h using a peristaltic pump. The purification procedure was carried out at +4°C^2,11.

Activity determination

Among its many other substrates, the aromatic electrophile 1-chloro-2,4-dinitrobenzene is one that is most often used to determine GST enzyme activity. As the product obtained by the use of this substrate, dinitrobenzene S-glutathione (DNB-SG) displays maximum absorbance at 340 nm. Activity measurements were thus carried out by using the absorbance increase at this wavelength. In order to measure the activity of enzyme firstly, 0.5 M 200 µL potassium phosphate buffer (K₂HPO₄/KH₂PO₄ pH 7.4) and 25 mM 20 µL 95% CDNB (in ethanol) were added in to the cuvettes. After incubation at 37°C for 3 min, 20 mM 50 µL GSH was added to cuvettes, and thoroughly stirred. After a quick addition of 50 µL enzyme sample, the cuvettes were placed in the spectrophotometer and reading was initiated¹².

Protein determination

Bradford’s method was followed in quantitative protein determination. This method is based on the binding of Coomassie Brilliant Blue G-250 to protein. The complex formed spectrophotometrically shows maximum absorbance at 595 nm. We used bovine serum albumin solution as standard¹³.

SDS-polyacrylamide gel electrophoresis

To control enzyme purity, Laemmli’s procedure was performed in 3% and 10% acrylamide concentrations for stacking and running gels, respectively. 1% SDS was added to this acrylamide solution. The obtained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel was first stabilized for 30 min in a solution containing 50% propanol, 10% TCA, and 40% distilled water. Then, the gel was stained for 2 h in a solution containing 0.1% Coomassie Brilliant Blue R-250, 50% methanol, and 10% acetic acid. Finally, washing was performed with a solvent containing 50% methanol, 10% acetic acid, and 40% distilled water until the protein bands were clear¹⁴.

Optimal pH determination

For the optimal pH determination, the enzyme activity was measured in 0.5 M phosphate buffers within the pH of 5.3 to 8.0.

Optimum ionic strength determination

For determination of optimum ionic strength, enzyme activity was determined using different concentrations of phosphate buffer, pH: 7.3, in the range from 0.01 M to 0.2 M.

Stable pH determination

For this purpose, the enzyme activity was determined in 1 M phosphate buffer at pH of 5.3, 5.8, 6.3, 6.8, 7.3, and 7.8. In each experiment, equal volumes of buffer and enzyme solutions were mixed and kept refrigerated (+4°C). Activity determinations were made within an interval of 1 day to 8 days.

Effect of temperature on GST activity

The enzyme activity was measured between 0 and 70°C at optimal pH and ionic strength for this purpose.

Molecular weight determination

Sephadex G-100 gel filtration

The molecular weight of the enzyme was determined according to Andrews¹⁵. At first, to calculate the void volume, Blue Dextran (2000 kDa) was passed through the column; then, cytochrome c (12 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), yeast alcohol dehydrogenase (150 kDa) and β-amylase (200 kDa) were used as standard proteins¹⁶.

SDS-PAGE

The subunit determination was made by SDS-PAGE¹⁰. Rabbit phosphorylase B (97.4 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa), and bovine carbonic anhydrase (29 kDa) were used as standards.

Kinetic studies

For K_M and V_max evaluation, we used Lineweaver-Burk curves obtained¹⁷ with five different concentrations of CDNB (0.0005, 0.001, 0.015, 0.002 and 0.0025 mM) and one constant concentration of GSH (1 mM). The same experiments were done with five different concentrations of GSH (0.001, 0.002, 0.003, 0.004 and 0.005 mM) and one fixed CDNB concentration (0.5 mM). All kinetic studies were performed at 25°C and at optimal pH (0.01 M K-phosphate, pH 7.3).

In vitro metal ions effects

In order to determine the effects of some metal ions on human GST, different concentrations of Ag⁺ (0.005–0.06 mM), Cd²⁺ (0.025–0.5 mM), Cr²⁺ (0.25–1.5 mM), Mg²⁺ (20–68 mM), were added to the reaction medium. The enzyme activity was measured and an experiment in the absence of inhibitor was used as control (100% activity). The IC₅₀ values were obtained from activity (%) vs. metal ion concentration plots. In order to determine K_i constants in the media with inhibitor, the substrate (GSH) concentrations were 0.04, 0.1, 0.18, 0.25 and 0.4 mM. Inhibitors (metal ions) solutions were added to the reaction medium, resulting in 3 different fixed concentrations of inhibitors in 1 mL of total reaction volume. Lineweaver-Burk graphs were drawn by using 1/V vs. 1/[S] values and K_i constant were calculated from these graphs¹⁷.

Results

Purification of GST enzyme led to a specific activity of 16.54 EU/mg proteins, a yield of 59% and a purification coefficient of 11,026 (Table 1). SDS polyacrylamide gel electrophoresis was performed after the purification of the enzyme, and the electrophoretic pattern was photographed (Figure 1). For the standard proteins and GST, R_f values were calculated, and an R_f-Log MW graph (Figure 2) was obtained according to Laemmli’s procedure, showing a molecular weight of 23 kDa for GST. The molecular weight of the enzyme was also determined by gel filtration chromatography. A K_av-log MW graph was obtained (Figure 3), which showed a molecular weight of 47 kDa for GST. Optimal pH, optimal ionic strength, optimal temperature and stable pH for enzyme were found as 7.3 using 0.01 M K-phosphate (Figure 4), 10 mM K-phosphate (Figure 5) and 30°C (Figure 6) and 7.3 using 0.01 M K-phosphate (Figure 7), respectively. The Lineweaver-Burk graphs constructed for GSH and CDNB are shown in Figures 8 and 9, which were constructed for GSH and CDNB. A K_M of 0.0395 mM and a V_max of 0.0328 EU/mL were obtained for GSH, and 0.259 mM and 0.0655 EU/mL for CDNB. IC₅₀ values of Ag⁺, Cd²⁺, Cr²⁺, Mg²⁺ were 0.0348, 0.171, 1.042 and 46.33 mM, and the K_i constants 0.0243 ± 0.009, 0.0243 ± 0.009, 2.506 ± 1.1040, and 65.73 ± 17.378 mM, respectively (Figures 10 and 11 and Table 2). All inhibition types for metal ions were found non-competitive.

Table 1.  Purification scheme of GST from rainbow trout erythrocytes.

Purification step	Total volume (mL)	Activity (EU/mL)	Protein (mg/mL)	Total protein (mg)	Total activity (EU)	Specific activity (EU/mg)	Yield (%)	Purification factor
Haemolysate	35	0.073	48.86	1710	2.555	0.0015	100	1
Glutathione-Agarose affinity chromatography	7	0.215	0.013	0.091	1.505	16.54	59	11,026

Table 2.  K_i and IC₅₀ values obtained from regression analysis graphs for rainbow trout erythrocytes GST in the presence of different metal ion concentrations.

Metal ions	IC50 (mM)	Ki (mM)	Inhibition type
Ag^+	0.0348	0.0243 ± 0.009	Non-competitive
Cd^2+	0.1710	0.1117 ± 0.052	Non-competitive
Cr^2+	1.0420	2.506 ± 1.1040	Non-competitive
Mg^2+	46.330	65.73 ± 17.378	Non-competitive

Figure 1.  SDS-PAGE photograph. Lane 1: Rainbow trout erythrocytes GST. Lane 2: Standard proteins: rabbit phosphorylase B (97 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa), bovine erythrocyte carbonic anhydrase (29 kDa).

Figure 2.  Standard Rf-Log MW graph of GST using SDS-PAGE. (Standards: Rabbit phosphorylase B (97.4 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa), and bovine carbonic anhydrase (29 kDa).

Figure 3.  Standard Kav-Log MW graph of GST using gel filtration. (Standards: Cytochrome c (12 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), yeast alcohol dehydrogenase (150 kDa) and β-amylase (200 kDa).

Figure 4.  Activity-pH graph of GST.

Figure 5.  Activity – [K-phosphate] for determination optimal ionic strength of GST.

Figure 6.  The effect of the temperature on GST.

Figure 7.  Stable pH graph of GST in 0.01 M K-phosphate.

Figure 8.  Lineweaver-Burk graph in 5 different GSH concentrations and in constant CDNB concentration.

Figure 9.  Lineweaver-Burk graph in 5 different CDNB concentrations and in constant GSH concentration.

Figure 10.  Activity % vs [metal ions] regression analysis graphs for rainbow trout GST in the presence of 5 different (a) [Ag⁺] (b) [Cd²⁺] (c) [Cr²⁺] (d) [Mg²⁺] concentrations.

Figure 11.  Lineweaver-Burk graph with 5 different substrate (GSH) concentrations and 3 different (a) [Ag⁺] (b) [Cd²⁺] (c) [Cr²⁺] (d) [Mg²⁺] concentrations for determination of Ki.

Discussion

As is well known, medications used for treatment of different diseases have numerous adverse effects for metabolism. The metabolism contains various enzymatic and non-enzymatic defense systems to eliminate or minimize such side effects. Enzymatic defense is provided by several enzyme systems such as glutathione S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase¹⁸. With a specific role in the metabolism of a wide group of electrophilic substrate that include antibiotic, analgesic, and anticancer medications, glutathione S-transferases^10–12 constitute a group of multifunctional enzymes which are involved in conjugating both endogenous and exogenous electrophilic compounds with reduced glutathione (GSH) and thus catalyzing their conversion into less toxic metabolites^19–21.

Rainbow trout erythrocyte GST was purified in this study by heamolysate preparation and glutathione-agarose affinity chromatography. Table 1 shows a purification characterized with a specific activity of 16.54 EU/mg proteins, a yield of 59% and a purification coefficient of 11,026. These figures mean that the procedure used in the study is good enough to be used by investigators. This purification procedure also has an advantage of short experimental duration of about 6 to 7 h. Figure 1 exhibits the SDS-PAGE done for the determination of purity and molecular weight of the enzyme. A high purity for the enzyme has been obtained. Also to perform kinetic studies; optimal pH, optimal ionic strength, optimal temperature and stable pH (Figures 4–7) and K_M and V_max for substrate’s (Figures 8 and 9) were found for enzyme.

Additionally, molecular weights of GST were found 23 and 47 kDa with SDS-PAGE and gel filtration respectively (Figures 2 and 3). Since the molecular weight determined by gel filtration chromatography was approximately two-fold greater than that of SDS-PAGE electrophoresis, native GST may be a homodimer.

Heavy metals have various toxicological effects on living organisms. For example, it has been reported that heavy metals such as mercury and cadmium exist exert toxic action in a synergistic fashion with salinity^22,23. To the best of our knowledge, although the toxic effects of the metal ions have been described, their effects on rainbow trout erythrocyte GST have not been studied yet.

In order to show inhibitory effects, while the most suitable parameter is the K_i constant, some researchers use the IC₅₀ value. Therefore, in this study, both the K_i and IC₅₀ parameters of these metal ions for GST were determined²⁴. As shown in Table 2, the K_i values were 0.0243 ± 0.009, 0.0243 ± 0.009, 2.506 ± 1.1040, and 65.73 ± 17.378 mM for Ag⁺, Cd²⁺, Cr²⁺, and Mg²⁺, respectively and the corresponding IC₅₀ values were 0.0348, 0.1710, 1.0420 and 46.330 mM, respectively. K_i values and IC₅₀ values show that Ag⁺ was the most potent inhibitor followed by Cd²⁺, Cr²⁺ and Mg²⁺ respectively.

An examination of the K_i constants and IC₅₀ values of the medications with inhibitory effect on the activity of rainbow trout erythrocyte GST enzyme (Table 2) demonstrates that all of them are strong inhibitors of GST, but Ag⁺ is the medication displaying the highest inhibitory effect. Both the results of our study and those of others in the literature we have mentioned have revealed that the metal ions used in the present research are potential inhibitors for metabolic enzymes. Such medications inhibit not only GST enzyme, but also other enzymes that produce glutathione as a GST substrate (glutathione reductase), which will result in a serious failure of detoxification reactions. Therefore, the people and especially the industrialists and administrators need to be extremely careful about the metal and industrial residues.

Declaration of interest

The authors declare no conflicts of interest.