Purification of carbonic anhydrase-II from sheep liver and inhibitory effects of some heavy metals on enzyme activity

Abstract

In this study; sheep carbonic anhydrase-II (SCA-II) (E.C: 4.2.1.1) was purified from sheep liver and in vitro effects of heavy metals on the enzyme was examined. SCA-II isozyme was purified with about 203 purification fold, a specific activity of 2320 EU/mg-protein and a yield of 72 by using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Purity of the SCA-II enzyme was verified by SDS-PAGE technique and subunit molecular mass of the enzyme was found as 29 kDa. In addition to this, inhibitory effects of some metal ions on the enzyme were examined. In this study, sheep liver tissue was chosen; because the liver is an organ in which metal wastes of air, water and food are collected and it is easy to obtain the liver tissue. Because of the very important duties of CA enzyme on living beings, the effect of metals on the CA enzyme was investigated.

Keywords::

• SCA-II
• purification
• metal
• inhibition

Introduction

CA enzyme, which exists commonly in living organisms, has various izoenzymes according to conditions and necessities of the medium. A new type of isoenzyme has been discovered almost for each year and now it has 16 isoenzymes. It is one of the most studied enzymes, and CA-I and CA-II are the most common isoenzymes. Carbonic anhydrases described in last 5 years has an increasing physiological and biochemical properties¹. Seven different CA isoenzymes have been characterized from vertebrates².

The carbonic anhydrases are wide family of zinc, including enzymes, which usually provide maintenance of pH homeostasis in the human body, catalyzing the reversible hydration of carbon dioxide in a two-step reaction to yield bicarbonate and protons^3–5,6. Sixteen isoenzymes have been defined up to now, that differ in their subcellular place, catalytic activity and emotional different types of inhibitors. Some of these enzymes are cytolosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII and CA XIV), two are mitochondrial (CA VA and CA VB), and one is secreted in saliva (CA VI). It has been clarified that there is no expression of CA XV in human and other primates, but it is plentiful in rodents and other higher vertebrates^7,8.

Carbonic anhydrase enzyme can be involved in metaloenzyme family encoded evolutionarily in at least five different gene families in prokaryotes and eukaryotes; α-class CAs (found in bacteria, algae, green plants and vertebrates), β-class CAs (mainly in bacteria, algae), γ-class CAs (in some arkeas and bacteria), δ-class CAs in marinated diatoms (thalassiosira weisflogii) and ϵ class CAs in cyano bacteria and bacteria. Until now, many carbonic anhydrase isoenzymes have been isolated from mammals. There are three different classes including α, β and γ class (in 1994) (and then ϵ class was isolated). These three classes do not have any significant similarity. Despite the structural differences, all three classes share one common property: zinc atom for catalysis in active site. Carbonic anhydrase is a member of zinc metaloenzyme family, and it has a structure that single peptide chain in the active site coordinated with Zn²⁺ ion⁹.

Heavy metals can change the activity of enzymes by binding to functional groups or displacing the metal associated with enzyme¹⁰. Rapid industrialization, poor emission control, unorganized urbanization, and increased motor traffic cause an increase of heavy metal concentration in the environment. Plants and other organisms in the habitat are affected by the contamination of soil, water, and atmosphere with heavy metals^11,12.

In the current study, we aimed at purifying the carbonic anhydrase from sheep liver and examining in vitro effects of heavy metals (Pb²⁺, Cu²⁺, Fe²⁺, Cr²⁺, Al³⁺, Ni²⁺, Mn²⁺, Cd²⁺, Zn²⁺, Mg²⁺) on the enzyme activity.

Materials and methods

Chemicals

Sepharose 4B, protein assay reagents and 4-nitrophenylacetate were obtained from Sigma-Aldrich Co (Germany). The other chemicals were of analytical grade and obtained from Merck (Germany).

Hydratase activity assay

Carbonic anhydrase hydratase activity was assayed by following the hydration of CO₂ according to the method described by Wilbur and Anderson¹³. CO₂-hydratase activity was calculated as an enzyme unit (EU) by using the equation (to-tc/tc) where t₀ and t_c are the times for pH change of the non-enzymatic and the enzymatic reactions, respectively.

Esterase activity assay

The esterase activity was assayed by following the change in absorbance of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm over a period of 3 min at 25°C using a spectrophotometer (BECKMAN COULTER UV-VIS) according to the method described by Verpoorte et al.¹⁴. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05 M Tris-SO₄ buffer (pH 7.4), 1.0 mL 3 mM 4-nitrophenylacetate, 0.5 mL H₂O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution.

Protein determination

During each purification step, protein determination was performed spectrophotometrically at 595 nm according to the Bradford method, using bovine serum albumin as the standard¹⁵.

Purification of carbonic anhydrase II from sheep liver by affinity chromatography

Sheep liver was obtained from Erzurum Ozbeyli Municipal Slaughterhouse and stored at −80°C until usage. Twenty grams of thawed liver was chopped into small pieces with a knife. The fragments were homogenized with liquid nitrogen and taken into 1.5–2 volumes of buffer solution (50 mM Tris-HCl, pH 7.4). The homogenate was filtered through four layers of cheesecloth and then centrifuged at 18,000 × g for 1 h. The pellet (mitochondria and cell debris) was discarded. The pH of the supernatant was adjusted to 8.7 with solid Tris. The homogenate was applied to the prepared Sepharose 4B L-tyrosine sulfanilamide affinity column equilibrated with 25 mM Tris-HCl/0.1M Na₂SO₄ (pH 8.7). The affinity gel was washed with 25 mM Tris-HCl/22 mM Na₂SO₄ (pH: 8.7). The SCA-II enzyme was eluted with 0.1 M NaCH₃COO/0.5 M NaClO₄ (pH 5.6). Purified SCA-II enzyme was dialyzed for 3 h against 0.05 M Tris-SO4/1 mM 2-mercaptoethanol (pH 7.4) (Table 1).

Table 1.  Summary of purification procedure for sheep liver CA II.

Purification steps	Activity (EU/ml)	Total volume (ml)	Protein (mg/ml)	Total protein (mg)	Total activity	Specific activity (EU/mg)	Yield %	Purification fold
Homogenate	145	20	12.7	254	2900	11.4	100.0	1.0
Affinity Chromatography After Dialyzed	348	6	0.15	0.9	2088	2320	72	203

SDS polyacrylamide gel electrophoresis

After the purification steps, SDS polyacrylamide gel electrophoresis was performed to verify enzyme purity. It was carried out in 10 and 3% acrylamide for the running and the stacking gel, respectively, containing 0.1% SDS according to Laemmli procedure. A 20 µg sample was applied to the electrophoresis medium. Gels were stained for 1.5 h in 0.1% Coommassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid, then detained with several changes of the same solvent without dye¹⁶ (Figure 1).

Figure 1.  SDS-PAGE analysis of purified sheep CA II. Lane (1) sheep- CA II. Lane (2) standard proteins (E. coli b-galactosidase (116 kDa), rabbit phosphorylase B (97.4 kDa), bovine serum albumin (66 kDa), chicken ovalbumin (45 kDa) and bovine carbonic anhydrase (29 kDa)).

In vitro inhibition effect of heavy metals on the enzyme activity

In this study, SCA-II was purified from sheep liver, and the activities of the effluents were defined by the hydratase method, in which CO₂ was used as substrate. Other kinetic studies were performed by using the esterase activity method and 4-nitrophenyl acetate (NPA) as substrate. For Zn²⁺, Cu²⁺, Al³⁺ and Cd²⁺, inhibition studies were performed by using esterase activity, whereas hydratase activity was used for Zn²⁺, Cu²⁺, Co²⁺, Cd²⁺ and Ni²⁺. All compounds were tested in triplicate at each concentration. Five different inhibitor concentrations were treated. In Table 2, we reported K_i values at three different inhibitor concentrations by using esterase activity method. In hydratase activity assay, inhibitor concentrations were calculated in Table 3 and IC₅₀ values were obtained from inhibitor concentration and Activity%—(Inhibitor) graphs.

Table 2.  K_i values and inhibition types for four metal ions of SCA II.

Type of metals	Average values of Ki	Type of inhibition
Zn2+	3.91	Uncompetitive
Cu2+	1.51	Uncompetitive
Fe2+	–	–
Cd2+	6.7	Non-competitive
Pb2+	–	–
Co2+	–	–
Cr2+	–	–
Al3+	1.34	Uncompetitive
Ni2+	–	–
Mn2+	–	–

Table 3.  IC₅₀ values of inhibitors for SCA-II hydratase activity.

Type of metals	IC50 mM values for hydratase activity
Zn2+	0.058
Cu2+	0.00041
Fe2+	–
Cd2+	0.66
Pb2+	–
Co2+	0.75
Cr2+	–
Al3+	–
Ni2+	1.44
Mn2+	–

Results and discussion

In the current study, we examined the effects of some metals on liver CA II enzyme activity of sheep. And as we know, sheep liver is an important source of nutrient for people. The aim of this study is purifying SCA-II from unstudied tissue of sheep liver and examining the effects of heavy metals on enzyme activity by using both hydratase and esterase activity. In this study, the liver tissue was chosen; because the liver is an organ in which metal wastes of air, water and food are collected and it is easy to obtain the liver tissue. Because of the very important duties of CA enzyme on living beings, the effect of metals on the CA enzyme was investigated.

The enzyme has esterase activity besides its hydratase activity, but physiologically hydratase activity is very important. For instance, it plays an important role in the regulation of acid—base balance in the organisms. Where this balance is impaired, for example, in intraocular tension, therapeutic intervention to carbonic anhydrase enzyme activity is a common applied method. In this respect, compounds of carbonic anhydrase inhibitors gain a clinical importance¹⁷.

Carbonic anhydrase which is a widespread metaloenzyme has previously been purified and characterized from many living organisms including animals^18–21. The isozymes of CA play important roles in different tissues^22,23. The similarities of CAs in various sources have been determined from their crystal structures²⁴. It is known that carbonic anhydrase has been purified many times from different organisms and the effects of various chemicals, pesticides and drugs on its activity have been investigated^25–27. Hundreds of pollutants in the form of metals, acids, bases and other toxic compounds are being added to rivers, seas and the atmosphere, and this causes a situation which results in destruction of the natural balance²⁵.

Inhibition and activation effects of many chemicals and drugs on carbonic anhydrase activity have been investigated by scientists and reported in the literature^8,28,29. Today, all living things can inevitably be exposed to the effects of different chemical substances. This situation can be arisen from people’s environment like their work environment. Although there is no connection with the atmosphere of such a work environment, people and other organisms are primarily affected by the waste created by people. In particular, the elimination of waste materials from factories in various countries has been made by burying into the ground or releasing to the air. As a result, many toxic substances such as heavy metals, have been passing into the soil, water, plants, and then from there to animals and people. So today, especially enzyme activity studies made by using these substances remains its popularity^25,30–32.

Christensen and Tucker³³ found that Ag¹⁺, Cd²⁺, Cu²⁺ and Zn²⁺ ions have inhibitory effect on fish CA enzyme. In another study about enzyme inhibition reported by Ozdemir and colleagues³⁵, the effects of some active cations on the activities of bovine CA and human (HCA-I and HCA-II) enzymes were investigated in vitro. According to this study, Al³⁺ inhibited the enzymes competitively, and Mn²⁺, Sr²⁺, Hg²⁺, Ni²⁺, Ca²⁺ and Cd²⁺ ions showed non-competitive inhibition.

In another study with fish liver CA, Al³⁺, Cu²⁺, Pb²⁺, Co³⁺, Ag¹⁺, Zn²⁺ and Hg²⁺ were found to show inhibitory action³³. In addition, Co²⁺ and Hg²⁺ ions showed inhibition (K_i 3.91 and 1.42, respectively) effects on human CA enzymes³².

Inhibition effects of many substances such as medical drugs, various metals, anions and pesticides have been reported in the literature^20,22,35,36. Many chemicals affect metabolism by changing normal enzyme activity, particularly inhibition of a specific enzyme^36,37 and the effects can be dramatic and systemic³⁸. Hence, heavy metals have various toxicological effects on living organisms. For example, it has been reported that heavy metals, such as mercury and cadmium, exert a toxic action in a synergistic fashion with salinity^26,38,39. Also, it has been expressed that the binding of heavy metals with membrane transport ligands can alter their catalytic function^26,40. Pb²⁺, Cu²⁺, Fe²⁺, Cr²⁺, Al³⁺, Ni²⁺, Mn²⁺, Cd²⁺, Zn²⁺, Mg²⁺ were chosen for investigation of their inhibitory effects on CA in this study, and it was important that heavy metals inhibited the enzyme activity at low concentrations. K_i parameters of these metals were determined, and it was found that the metals were potent inhibitors of CA. Lineweaver-Burk⁴¹ plots showed that Zn²⁺, Cu²⁺, Al³⁺ inhibited the enzyme uncompetitively and Cd²⁺ non-competitively.

Soyut et al. examined the inhibitory effects of cobalt, copper, zinc, silver and cadmium on carbonic anhydrase purified from brains of rainbow trout. They founded that cobalt, silver and cadmium inhibited the enzyme competitively, copper inhibited non-competitively, whereas zinc inhibited the enzyme uncompetitively⁴² In another study, Soyut and Beydemir examined the effects of some metals (cobalt, copper, zinc, silver) on the CA activity of rainbow trout liver. Their results showed that cobalt and zinc inhibited the enzyme in a competitive manner and copper and silver inhibited the enzyme in an uncompetitive manner⁴³. Besides, Soyut and Beydemir examined the effects of metals (Co²⁺, Zn²⁺, Cu²⁺, Cd²⁺) on the activity of CA from rainbow trout kidney. They found in vitro inhibition of the metals in the order of Co²⁺> Zn²⁺> Cu²⁺> Cd²⁺> Ag²⁺⁴⁴.

Air, water, soil and other natural sources can be contaminated with metal ions because of technological reasons. These metal ions entering the metabolism through respiration, water and foods show toxic effects in the body. In addition, the metal ions thrown as industrial waste dirt streams and give harm to the plants and animals. Received metal ions affect all living organs in the body, especially in developing children shows itself as mental retardation, learning disabilities and physical growth delay.

In this study, Zn²⁺, Cu²⁺, Cd²⁺ and Al³⁺ were chosen to investigate their inhibitory effects on SCA II. K_i parameters of these metals were determined. Metal ions inhibited the enzyme activity at low concentrations. It is clear that Cu²⁺ and Al³⁺ are the most potent inhibitors for SCA-II enzyme. K_i shows that metals inhibit SCA-II in uncompetitive manner (Table 2). According to K_i values, the best inhibitor for SCA-II is aluminium. Metal-ligand interactions are stronger especially in aqueous media. The mechanism of toxicological effects of the metals is probably due to the interactions between negative charged amino acids and the metals or the interaction of the metals with other amino acids around the active site^45,46. We investigated the effects of metals on the SCA-II isoenzyme by measuring hydratase activity occurring in physiological conditions and esterase activity in vitro conditions. We saw that Zn²⁺, Cu²⁺, Cd²⁺ ions inhibited the enzyme either in vitro, or physiological environment. Co²⁺ and Ni²⁺ ions inhibited the enzyme only in the physiological environment, Al³⁺ ion found to inhibit the enzyme in vitro. The majority of heavy metals in this study showed inhibitory effect in both physiological and in vitro conditions.

According to these results, SCA-II enzyme, which is available nearly at all living beings and very important at metabolic events, was purified from sheep liver. And then the effects of some metals were examined, and it was found that the metals inhibited the enzyme even at very low concentrations. As many harmful materials, some metals entrance to the body and their accumulation in it may be the reason of stress factor. The people, especially who work and live at the industrial regions and other living beings, are under the threat of toxic. Because of this, it is possible to see problems at the metabolism of living beings which are exposed to these harmful materials.

Declaration of interest

The authors report no conflicts of interest.