Partial Purification of Glutathione S-transferase Enzyme From the Seed of Mallow (Malva Slyvestris L.) and Investigation of the Inhibition Kinetics of Some Heavy Metals

Glutathione S-Transferase (GST) enzyme is abundant in mammals, insects, fish and microorganisms, as well as in various tissues of these species, particularly in tissues exposed to xenobiotics from the environment. As a result, the enzyme execute detoxifying function by scavenging a diverse range of xenobiotics, such as chemotherapeutic medicines, environmental carcinogens and endogenous compounds. In this study, GST enzyme was partially purified from mallow (Malva slyvestris L.) seed for the first time and the kinetic parameters were determined. The optimum ionic intensity was found in 400 mM Tris-Buffer, optimum pH: 7.0, and optimum substrate concentration was determined as 0.2 mM. One of the biggest reasons for deterioration of ecological balance in nature is heavy metal accumulation in soil, air and water which becomes a major threat to the vital activities of living things. In this study, inhibitory effects of Cd+ 2, Ag+, Zn+ 2 and Fe+ 3 heavy metals, which are common in nature, on mallow seed glutathione S-transferase enzyme were investigated. Each heavy metal showed micromolar inhibitory effects on enzyme activity. IC50 values of the metals were calculated as 60.93, 74.602, 178.22 and 369 µM, respectively.


Introduction
Mallow (Malva sylvestris L.) a plant native to North Africa, Europe and Southwest Asia, is primarily found in Mediterranean area of Turkey. Some components of the plant have been utilized to produce medication due to their medicinal properties. Furthermore, the leaves exhibit anti-inflammatory, antioxidant, anti-complementary, anti-cancer, and skin tissue integrity action. Mallow has positive interactions with other organisms since it has a host characteristic. Another noteworthy feature of mallow is its sensitivite ozone response. Ozone enters the apoplastic fluid surrounding the cells of mallow leaves and is quickly transformed to reactive oxygen. Reactive oxygen builds up along the veins, structure of stressed tissues and organs [3]. Knowledge of the adaptation mechanisms of plants is very important for their reactions to stressful conditions. To know the tolerance limits for heavy metal toxicity in plants, the availability of types and amounts of metals, the severity and type of damage, and the duration of damage must be taken into account. Knowing these properties is very important for the development and vitality of plants [5].
Oxygen molecules, necessary for the life of living beings, form extremely reactive intermediates, the so-called free radicals, in metabolism. These molecules, known as reactive oxygen species, damage cellular components such as lipids, proteins, DNA and RNA. Free radicals of oxygen and nitrogen; those derived from oxygen are called reactive oxygen species (ROS), and those derived from nitrogen are called reactive nitrogen species (RNS) [6,7].
Free radicals damage living cells by changing the structure of proteins, lipids, carbohydrates and cell components such as RNA and DNA. Our body provides an antioxidant defense mechanism by producing antioxidants against these damages caused by free radicals [8]. Antioxidants form endogenous and exogenous defense mechanisms against reactive oxygen species. Endogenous antioxidants are grouped as enzymatic and nonenzymatic. Exogenous antioxidants are grouped as vitamins, exogenous antioxidants and exogenous antioxidants used as drugs.
Enzymes are biological catalysts that are produced by living cells and accelerate processes in live metabolism providing 100% product yield [9]. Glutathione (GSH) is an antioxidant that functions as a substrate for Glutathione S-transferase (GST) and plays a vital role in the transport of amino acids, protein, and DNA synthesis [10][11][12]. The glutathione S-transferase enzyme catalyzes the binding of endogenous and external hydrophobic electrophiles to GSH [13]. GST's primary function is to protect the cell from oxidative damage. By covalently binding xenobiotics and endogenous electrophiles with GSH, the GST enzyme assists in detoxification. It facilitates the elimination process of the nutrients eaten without losing their nutritional properties during the metabolic process of hazardous chemicals taken with meals. One of its most essential roles is to attach to GSH's thiol (-SH) groups and catalyze processes that detoxify reactive electrophilic sites in xenobiotics [14].
Our environment is currently contaminated by metal waste, agricultural operations, ordinary human activities and industrial and commercial trash. Heavy metals are present in the environment, and living creatures are constantly exposed to heavy metals in nature. As a result, the harmful effects of heavy metals on living organisms have been extensively investigated by researchers, and it is now a key study topic in the toxicological field [15][16][17].
So far, we have not encountered any studies on the purification and characterization of GST from mallow. Therefore, we characterized GST enzyme from the mallow seed for the first time. In addition we investigated the effects of heavy metals Cd + 2 , Ag + , Zn + 2 and Fe + 3 . This is also the first investigation of these metals on mallow GST enzyme.

Chemicals
All chemicals used for purification were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were obtained from Merck (Darmstadt, Germany).

Preparation of the Homogenate and in vitro Enzyme Assay
The mallow seeds were crushed in a mortar with liquid nitrogen. The resulting content was collected in a 50 ml centrifuge tube and made up to 30 ml with 100 mM KH 2 PO 4 buffer (pH: 7.7) + 0.5 mM EDTA + 1% PVP buffer. After centrifugation for 30 min at + 4 °C at 15,000 xg, the supernatant and the precipitate were filtered and the measurement was performed with the supernatant part. Activity measurement was performed at 340 nm with the help of Shimadzu UV-1800 spectrophotometer for 3 min [14].

Ammonium Sulphate Precipitation and Dialysis
Different levels of ammonium sulfate (NH 4 ) 2 SO 4 precipitation were used as follows: 0-20%, 20-40%, and 40-60%. After precipitation, the precipitate was dissolved in the optimum buffer solution (400 mM Tris) and prepared for the next step. According to the activity measurements, the maximum activity value was discovered at 40-60% ammonium sulfate saturation. The enzyme activity was then measured using dialysis. It was dialyzed for 2 h at 4 °C in 1 mM Tris buffer (pH 7.0). The enzyme solution was stored at 4 °C [18, 19].

Characterization of the Enzyme with Kinetic Parameters
In order to determine optimum pH, enzyme activity was measured in 400 mM Tris-HCl over the pH ranges of 6.5-8.0. Optimum ionic strength was determined in 10-600 mM Tris (pH 7) buffer concentrations. The optimum substrate concentration was determined between 0.1 and 0.3 mM CDNB intervals [18]. The optimal ionic strength was found to be 400 mM Tris buffer, the optimum pH 7.0, and the optimum substrate value 0.2 mM CDNB, respectively.

In vitro Effects of Heavy Metals
Several concentrations of heavy metals were added to the reaction medium to assess the effects of the heavy metals on mallow seed GST. The enzyme activity was evaluated, and an experiment without heavy metals served as a control (100% activity).

Results
In this study, we isolated the GST enzyme from mallow for the first time. We used a very simple and cost-effective purification method including homogenate preparation, ammonium sulphate precipitation and dialysis. Following homogenate preparation, precipitate saturation of the enzyme was determined to be 40-60% with solid (NH 4 ) 2 SO 4 . This precipitation interval is comparable to others derived from other sources [18][19][20]. These findings show that the purification process employed is adequate and beneficial for future research. This purification method also has the benefit of being quite quick, lasting only one day.
After purification, GST enzyme was tested with metals Cd + 2 , Ag + , Zn + 2 and Fe + 3 . The IC 50 values for Cd + 2 , Ag + , Zn + 2 and Fe + 3 were determined to be 60.93, 74.602, 178.22 and 369 µM, respectively. Table 1 shows the inhibition findings. As seen, metals exhibited micromolar inhibitions. ( Table 1     ). In that study, it was found that Cd 2+ , Ni 2+ , Cu 2+ , and Mg 2+ have an inhibition effect on Cherry Laurel GST enzyme. The IC 50 values of these heavy metals were between 0,33 and 1.79 mM, and the most strongly inhibiting heavy metal was found to be Mg 2+ [13]. Another study reported by Halušková et al. investigated heavy metal application for barley [30]. Balcı et al. investigated the inhibition of some metal ions in Vaccinium arctostapylous L. The IC 50 values for Cd 2+ , Cu 2+ , Ni 2+ , and Mg 2+ inhibition values used in the study were found as 0.502, 0.701, 0.789, and 0.127 mM, respectively. Mg 2+ was found to be the most effective inhibitor [31].
Heavy metals affect not only plants but also animals. Ozaslan et al. 2017 reported inhibition analysis of GST enzyme with metal ions from the Van Lake fish gills (Chalcalburnus tarichii Pallas). In the study, it was found that Ag + , Cu 2+ , Cd 2+ and Zn 2+ ions had inhibitory effects on the enzyme. The IC 50 values were found to be 16.43, 320.25, 450.32 and 1510.13 µM, respectively. Ag + showed better inhibitory effect compared with other metal ions [32]. In another study, inhibition effects of Al + 3 , B + 3 , Ba + 2 , and Se − 2 metal ions on the GST enzyme of gill tissue of Van Lake Pearl Mullet (Chalcalburnus tarichi) were investigated by Zaric. The IC 50 values of these metal ions were calculated as 0.072 mM, 0.100 mM, 0.082 mM and 0.083 mM, respectively [33]. Taysi and Temel 2021 investigated the inhibition effects of Ag + , Cd 2+ , Ni 2+ , Zn 2+ and Al 3+ metals on quail liver GST enzyme. The IC 50 values of these metals were 239, 258, 265, 322, 594 µM, respectively. The most strongly inhibiting heavy metal was found to be Ag + [34]. In another study Ahmed et al. reported the inhibition of metal ions on GST enzyme characterized from quail heart tissue. In the study, it was determined that Cu 2+ , Ag + and Cd 2+ metals caused significant decrease in enzyme activity. The IC 50 values of these metals were 0.0028, 0.382 and 3.127 mM, respectively. The strongest inhibitory metal was found to be Cu 2+ [35]. In the study of Akkemik et al. 2012, GST enzyme was purified from turkey liver and heavy metal inhibition was investigated. As a result of the research, inhibition of Ag + , Cu 2+ and Hg 2+ metals was observed. The IC 50 values were found to be 0.378, 245 and 257, respectively. Ag + was found as the metal with the strongest inhibition [18]. Aksoy et al. investigated the inhibition effect of Ag + , Cu 2+ , Pb 2+ , Ni 2+ and Zn 2+ metals on GST from the muscle tissue of Lake Van fish (Chalcalburnus tarichii Pallas). The IC 50 values of these metals were found as 0.011, 0.103, 0.113, 2.5, 0.253 mM, respectively. Ag was found to be the metal with the strongest inhibitory effect [36]. As can be seen from the provided literature data, our findings are in good agreement

Discussion
Metal pollution pose one of the most serious threats to the environment and human health. Metals enter the biosphere through natural processes as well as through human activities such as mining, fuel burning, and a variety of other industrial operations [21]. Interactions of specific enzymes with metals and other chemicals should be well characterized [22].
Metals can interfere with the intracellular detoxification processes of organic contaminants which may cause amplification of their toxicity. Glutathione transferases (GSTs) are important proteins in the detoxification of both organic xenobiotics and endobiotic metabolites. When metals enter an organism, the defensive system against oxidative stress and other negative consequences increases GST activity. However, there is increasing evidence that metals at hazardous quantities have an effect on GSTs, either directly by inhibiting enzyme activity or indirectly by lowering the concentration of reduced glutathione [23].
Glutathione (GSH) is a tripeptide that is produced in the liver and present in cytosol, nucleus and mitochondria of cells. The GSH/GSSG ratio indicates the cell's oxidative status [24]. One of the two primary roles of glutathione, which is plentiful in the cell cytosol, is to eliminate harmful metabolites from the cell, and the other is to keep the sulfhydryl groups stable due to its reduced form [25]. GSH has reduced thiol groups which enable it to serve as antioxidant [26,27].
All living species in nature are in risk of extinction as a result of the world's rising environmental degradation. It is critical to consume nutrients that boost the body's resilience against this critical threat. Enzymes roles in living organisms are to keep critical functions healthy and sustained. GST enzyme, which is one of the most essential enzymes that will give our bodies with resistance, detoxifies xenobiotics and is a well-known enzyme in the detoxification of xenobiotic chemicals [28]. It is responsible for the conjugation of xenobiotics with reduced glutathione. As a result, GSTs offer phase II transformation of endogenous chemicals and xenobiotics such as anticancer medicines and environmental carcinogens produced as a result of oxidative stress, rendering xenobiotics soluble and eliminated from the body via bile and urine [29].  with the previous investigations on GST enzymes from different sources.

Conclusion
Foods, plants and aquatic organisms can be damaged by microbial or environmental reasons. Heavy metals, pesticides, industrial wastes and other chemical agents are examples of environmental pollutants. Metal ions are often found in vegetables, dairy and meat products, which are eaten everyday by humans and other species as part of their diet. Findings of our study may provide important data to make people aware of the impacts of heavy metals on specific enzyme systems. The another important part of our study is that GST enzyme has been first characterized from mallow which is a frequently consumed plant. Therefore, our data will help further researchers who are interested in antioxidant enzymes of this plant. According to our characterization findings, the optimal ionic strength was determined as 400 mM Tris buffer, the optimum pH was 7.0, and the optimum substrate value was 0.2 mM CDNB.
Our results showed that metal ions inhibited GST activity at micromolar concentrations in vitro with the ordering Cd + 2 >Ag + >Zn + 2 >Fe + 3 . Therefore, the findings show that metal ions are potent inhibitors of the GST enzyme purified from mallow seed and may cause undesirable results by impairing detoxification and antioxidant defense, although the inhibition mechanisms are currently unknown.