Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Abstract

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. K_M values were 1.59 and 0.53 mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. V_max values for CDNB and GSH were also determined as 5.58 and 1.88 EU/mL, respectively. In addition, inhibition effects of Ag⁺, Cu²⁺, Cd²⁺, Fe³⁺, Pb²⁺, Cr²⁺, Co²⁺ and Zn²⁺ metal ions were investigated on the enzyme activity and IC₅₀, K_i values were calculated for these metal ions.

• Glutathione S-transferase
• inhibition
• metal ions
• purification
• Van Lake fish

Introduction

The glutathione S-transferases (GSTs) belong to a multigene enzyme superfamily which play a significant role in the detoxification proses1. GSTs reduce the toxic effect of endogenous and environmental chemicals with phase II system. The reaction catalyzed by this enzyme is conjugation thiol group of reduced glutathione (GSH) with varieties of electrophiles. Conjugation with GSH renders toxic substance more soluble2. GSTs are divided in four classes including cytosolic, mitochondrial, microsomal and fosfomycin resistance proteins identified in bacteria3. Some GSTs use GSH not as substrate, but as cofactors. There are many functions of GSTs in metabolism. These functions are hormone biosynthesis, tyrosine degradation, peroxide breakdown, dehydroascorbate reduction and other functions4.

GSTs are classified according to the protein sequence and structure. Members of the same class of cytosolic GSTs match more than 40% amino acid sequence identity. Between classes, proteins have less than 25% sequence identity. Cytosolic GSTs are categorized as alpha5, beta6, delta7^,8, epsilon9, zeta10, theta11, mu12, nu13, pi14, sigma15^,16, tau17, phi18 and omega19.

GSTs in fish have not been characterized as much as mammalian GSTs. There is GST catalytic activity in all fish species examined to date. Similarity to the rodent GSTs, alpha, mu, pi class GSTs have been defined in brown bullhead (Ameriurus nebulosus) and largemouth bass (Micropterus salmoides)20, juvenile white sturgeon (Acipensert ransmontanus)21, chinook salmon (Oncorhynchus tshawytscha)22. Identified classes in fish species compose the major GST isoforms.

Up to now, fish have been proven to be useful experimental models for the evaluation of the health of aquatic ecosystems exposed to environmental pollution and the associated biochemical changes23–27. It is important to understand that the detoxification mechanism of aquatic species is contaminated with metals, pesticides, herbicides and persistent pollutants28.

Van Lake Fish (Chalcalburnus tarichii Pallas) is the only fish known to live in the Lake Van29. Rivers which breeding and feeding areas of C. tarichii Pallas are poured to Van Lake through areas where made intensive agricultural activities. On the other hand, it was seen that, fish getting in river to breed, enters fields through water supplies and poisoned by pesticides. Van Lake fish is called as Chalcalburnus tarichi by Pallas in 1811. Van Lake fish show endemic and anadromous behaviors in Van Lake basin belong to Cyprinidae family30.

To our understanding of toxicity mechanism and responses of C. tarichii Pallas muscle to some metal ions, a better understanding of GST is required. The present study was initiated for the purification and characterization of C. tarichii Pallas muscle GST. Here, we investigated effects of some metal ions on C. tarichii Pallas GST activity.

Materials and methods

Chemicals

GSH-agarose was obtained from Sigma-Aldrich (St. Louis, MO). Reduced GSH, 1-chloro-2,4-dinitrobenzene (CDNB), the protein assay reagent and the chemicals for electrophoresis were purchased from Sigma Chem. Company (St. Louis, MO). All other chemicals used were of analytical grade and purchased from either Sigma or Fluka (Munich, Germany).

Preparation of homogenates

All procedures were carried out at 4 °C. Muscle tissue of C. tarichii Pallas (10 g) was powdered with liquid nitrogen for 10 min to decompose cell membranes. The sample was homogenized using homogenizer for 2 min in 50 mM Tris/HCl buffer (pH 7.2) containing 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 13 000 g for 1  h and the supernatant was collected.

Purification of GST

Prepared homogenate was applied to GSH-agarose affinity column equilibrated with 10 mM K-phosphate buffer (pH 7.4) containing 150 mM NaCl. The column was washed with equilibrium buffer and then enzyme was eluted with gradient of 5–10 mM GSH in 50 mM Tris/HCl (pH 9.5). 1.5 mL fractions were collected.

Protein determinations

Protein content was measured according to the Bradford method31 using bovine serum albumin as a standard.

Enzyme assay

The activity of GST was determined in accordance with a previous procedure32 using CDNB as a model substrate. The assay system included a phosphate buffer (pH 6.5), GSH (20 mM) and CDNB (25 mM). A spectrophotometer (Shimadzu UV-1208, Japan) was used to estimate the changes in absorbance at 340 nm for 3 min.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

To determine the enzyme purity, SDS–PAGE was performed according to Laemmli’s method33. Gels were stained for appearance of protein band using a standard Coomassie Blue method. The electrophoretic pattern was photographed.

Enzyme kinetics

The kinetic values of GST for GSH and CDNB were determined by the procedure as follows: enzyme activity was measured at five different concentrations of GSH or CDNB with fixed saturated concentrations of other substrate. K_M constant and V_max value were calculated from Lineweaver–Burk graphs.

Inhibition effect on enzyme activity

To determine the effects of metal ions on C. tarichii Pallas muscle GST activity, the different concentrations of Ag⁺, Cu²⁺, Cd²⁺, Fe²⁺, Pb²⁺, Cr²⁺, Co²⁺ and Zn²⁺ were added into the activity medium and activity of the enzyme was measured. The absence of metal ions was used as the control (100% activity). IC₅₀ values were calculated from activity (%)–metal ion concentration graph. In order to determine K_i constants, at five different fixed concentrations of substrate (GSH), three different inhibitor concentrations were added to the reaction medium. Lineweaver–Burk graphs were drawn by using 1/V versus 1/[GSH] values, and K_i constants and inhibition types were obtained from these graphs.

Results

Glutathione agarose affinity chromatography was used for the purification of C. tarichii Pallas GST. GST was purified with 301.5-fold and a total yield of 19.07%. The results of the purification of GST are summarized in Table 1. SDS–PAGE was used to check the purity of the enzyme. Two bands were observed after electrophoresis (Figure 1). The result indicates that the native enzyme is a heterodimer.

Figure 1. Lane 2: aliquots and lane 3: aliquots of purified GST from Chalcalburnus tarichii Pallas muscle; lane 1: standard proteins: β-galactosidase (120 kDa), albumin (85 kDa), ovalbumin (50 kDa), carbonic anhydrase (35 kDa), β-lactoglobulin (25 kDa) and lysozyme (20 kDa) were used as standards (peqGOLD Protein Marker III).

Table 1. Purification steps of Chalcalburnus tarichii Pallas muscle GST.

Purification step	Activity (EU/mL)	Total volume (mL)	Protein (mg/mL)	Total protein (mg)	Total activity (EU)	Specific activity (EU/mg)	Yield (%)	Purification fold
Homogenate	0.597	15	5.08	76.2	8.955	0.118	100	1
Glutathione agarose affinity chromatography	1.139	1.5	0.032	0.048	1.708	35.583	19.07	301.5

K_M constants and V_max values of GST for C. tarichii Pallas were calculated from the Lineweaver–Burk graphs and the results are shown in Table 2. These kinetic parameters were determined using GSH and CDNB as substrates. V_max values and K_M constant for each substrate were determined by linear regression analysis of 1/V versus 1/[S]. K_M constants were calculated as 1.59 and 0.53 mM, and also V_max values were found as 5.58 and 1.88 EU/mL for CDNB and GSH, respectively. According to K_M constants results, it can be said that the affinity of GSH is better than CDNB to enzyme as a substrate.

Table 2. V_max values, K_m constant of C. tarichii Pallas muscle GST.

Substrate	Vmax (EU/mL)	Km (mM)
CDNB	5.58	1.59
GSH	1.88	0.53

Inhibitory effects of Ag⁺, Cu²⁺, Cd²⁺, Fe²⁺, Pb²⁺, Ni²⁺, Co²⁺ and Zn²⁺ ions on C. tarichii Pallas GST activity were studied at various concentrations using GSH as the substrate. IC₅₀ values and K_i constants were determined by graphs. K_i values are shown in Figure 2. The results are summarized in Table 3.

Figure 2. Lineweaver–Burk graph with five different substrate (GSH) concentrations and three different metal ions (Ag⁺, Cu²⁺, Pb²⁺, Ni²⁺, Zn²⁺) concentrations for determination of K_i.

Table 3. K_i constants and IC₅₀ values obtained from regression analysis graphs for GST from C. tarichii Pallas muscle in the presence of different metal ions.

	IC50 (mM)	Ki (mM)	Inhibition type
Ag^+	0.011	0.014 ± 0.004	Noncompetitive
Cu^2+	0.103	0.088 ± 0.033	Competitive
Pb^2+	0.113	0.430 ± 0.092	Competitive
Ni^2+	2.5	7.00 ± 0.001	Noncompetitive
Zn^2+	0.253	0.897 ± 0.429	Noncompetitive
Cd^2+	Ineffective
Co^2+	Ineffective
Fe^2+	Ineffective

Cd²⁺, Co²⁺, Fe²⁺ had no inhibitory effect on C. tarichii Pallas GST activity. It is found that purified C. tarichii Pallas GST was inhibited, in a concentration dependent way, by Ag⁺, Cu²⁺, Pb²⁺, Ni²⁺ and Zn²⁺. The concentrations of metal ions needed to inhibit the enzyme with 50% are named as IC₅₀ values. The IC₅₀ values of Ag⁺, Cu²⁺, Pb²⁺, Ni²⁺ and Zn²⁺ on C. tarichii Pallas GST are 0.011, 0.103, 0.113, 2.5, 0.253 mM, and also K_i values of these metals are 0.014 ± 0.004, 0.088 ± 0.033, 0.430 ± 0.092, 7.00 ± 0.001, 0.897 ± 0.429, respectively. According to results, the best inhibitor for C. tarichii Pallas GST is Ag⁺.

Discussion

It is well-known that the aquatic environment is contaminated with several types of organic and non-organic pollutants34. These pollutants influence the integrity of ecosystems and physiological functions of animals34 as well as of people as consumers35. Pollutants water can be evaluated with chemical analysis; however, chemical monitoring is not indicated their generally negative impact on organisms. To detect and determine the effect of contamination on aquatic organisms, biochemical and molecular markers are required36. Biochemical and molecular markers may be defined as eliminating the toxic effects of chemical substances in the organism, such as GSTs37.

Fish have been used as aquatic contamination indicators for many years. One of the intensely investigated biochemical markers in fish is the GST conjugation enzyme. The GST activity assay as a response of the fish organism to aquatic contamination has been described in many papers from all over the world36^,38.

Metabolism of xenobiotics occurs in two stages. The first stage of metabolism, unmasking or adding reactive functional groups, involves oxidation, reduction or hydrolysis39. The second stage is conjugation reactions which add water-soluble groups such as reduced GSH, glucuronic acid linked uridine diphosphate-glucuronic acid or glycine. The resulting conjugate is usually a very polar substance that cannot be resorbed, and it is therefore excreted from the organism18.

In fish, GSTs are used primarily as a biomarker indicating aquatic environment pollution with wastewater of industrial and agricultural origin. GST activity has been studied in various tissues of different fish species both under laboratory and under natural conditions. To date, GSTs are purified from some fish species such as Monopterus albus40, rainbow trout (Comaklı), bonito liver (Guller). In this study, C. tarichii Pallas muscle GST was purified and characterized. In this study, GST was first purified from C. tarichii Pallas muscle. The purification steps included preparation of homogenate and GSH-agarose affinity chromatography. Table 1 shows a purification with a specific activity of 35 583 EU/mg protein, a yield of 19.07%, and a purification coefficient of 301.7. Huang et al. purified GST from M. albus liver using Sepharose 6B affinity column and achieved 300-fold purification with 14% recovery40. Comaklı et al. purified GST from rainbow trouterythrocytes, and they achieved 11.026-fold purification with 59% recovery. Akkemik et al. purified GST from turkey liver, and they found 252.7-fold with a yield of 45%, with a specific activity of 164.31 U/mg41. K_M constants and V_max values for CDNB and GSH substrates were calculated via Lineaweaver–Burk graphs. K_M constant and V_max value for CDNB (at saturate concentration of GSH) were estimated as1.59 mM and 5.58 EU/mL, respectively. Similarly, K_M constant as 0.53 mM and V_max value as 1.88 EU/mL were obtained for GSH (at saturate concentration of CDNB). According to our result, it can be said that the affinity of GSH is better than CDNB to enzyme as a substrate. To understand toxic impacts of some metal ions on C. tarichii Pallas muscle GST, inhibition effect of them was examined. We determined that Ag⁺, Cu²⁺, Pb²⁺, Ni²⁺ and Zn²⁺ inhibited the GST from C. tarichii Pallas muscle, while Cd²⁺, Co²⁺and Fe²⁺ did not affect the enzyme activity. Since Ag⁺, Ni²⁺ and Zn²⁺ exhibit a non-competitive inhibition effect on the enzyme activity, V_max values are decreased and K_M values are not changed. Since Cu²⁺ and Pb²⁺ have competitive inhibition effect on the enzyme activity, these inhibitors have no effect on V_max but raises K_M. According to IC₅₀ and K_i results, Ag⁺ was the most effective inhibitor with the IC₅₀ value of 0.011 mM and K_i constants of 0.014 ± 0.004 mM. The inhibition order of metal was Ag⁺ > Cu²⁺ > Pb²⁺ > Zn²⁺ > Ni²⁺, and IC₅₀ values and K_i constants are given in Table 3.

Cytosolic GSTs are dimeric proteins made up of two identical subunits or two different subunits, i.e. homodimers or heterodimers42, the molecular weight of which is approximately 25 kDa. In our study, cytosolic C. tarichii Pallas muscle GST was purified. Our SDS–PAGE result is indicating that the enzyme is a heterodimer, because of exhibiting two protein bands. Similar results have showed other eukaryotic species as follows: rat liver43, bovine brain44, turkey liver41, human liver45 and kidney46. These results support our findings.

Conclusions

Consequently, we purified GST from C. tarichii Pallas muscle for the first time. Here, we determined K_M constants and V_max values of C. tarichii Pallas GST using CDNB and GSH as substrates. Also, inhibitory effects of metals on enzyme activity were reported. The results are important to understand of toxic effects metal ions on C. tarichii Pallas.

Declarations of interest

The authors report no declarations of interest.