Heavy metal ion inhibition studies of human, sheep and fish α-carbonic anhydrases

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) were purified from sheep kidney (sCA IV), from the liver of the teleost fish Dicentrarchus labrax (dCA) and from human erythrocytes (hCA I and hCA II). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The kinetic parameters of these enzymes were determined for their esterase activity with 4-nitrophenyl acetate as substrate. The following metal ions, Pb²⁺, Co²⁺, Hg²⁺, Cd²⁺, Zn²⁺, Se²⁺, Cu²⁺, Al³⁺ and Mn³⁺ showed inhibitory effects on these enzymes. The tested metal ions inhibited these CAs competitively in the low milimolar/submillimolar range. The susceptibility to various cations inhibitors differs significantly between these vertebrate α-CAs and is probably due to their binding to His64 or the histidine cluster.

Keywords::

• Carbonic anhydrase
• metal ion
• erythrocyte
• sheep kidney
• teleost fish

Introduction

Carbonic anhydrases (CAs; EC 4.2.1.1) are ubiquitous metalloenzymes present in all living organisms that are encoded by five different unrelated gene families. These are the α-CAs (present in vertebrates, bacteria, algae and cytoplasm of green plants), the β-CAs (predominantly in bacteria, algae, chloroplasts of monodicotyledons and dicotyledons), the γ-CAs (mainly in archaea and some bacteria), the δ- and ζ-CAs (present in marine diatoms^1–4). Up to date, 16 α-CA isozymes of have been described in mammals, which have various subcellular localizations, a different catalytic activity and susceptibility to different classes of inhibitors^1–4. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII, CA XIV and CA XV), two are mitochondrial (CA VA and CA VB), and one is secreted in saliva (CA VI). CA XV is an isoform not expressed in humans or other primates, but it is abundant in rodents and other higher vertebrates^5–8. These enzymes catalyze the reversible hydration of carbon dioxide in a two-step reaction to yield bicarbonate and protons^1,9. Many tissues contain CAs which have an important role in crucial biological processes, such as acid-base balance (pH homeostasis), respiration, carbon dioxide and ion transport, lipogenesis, gluconeogenesis, ureagenesis and body fluid secretion processes^{1–4,10–12}.

One of the most important problems of environmental toxicology is exposure to heavy metal ions. Most metal ions are toxic to humans, animals, plants and other living organisms. Because of bioaccumulation, man and other organisms are at great risk of health hazards associated with toxic metals^13–22. Several studies shown that people in different regions of the world are being exposed to metal ions by nutrition and respiration²². The meat, vegetables and bakery products may contain lead (Pb) and cadmium (Cd²²). Another study investigated the content in metal ions, such as copper (Cu) and zinc (Zn) in various fruits and vegetables sold in Egyptian markets, being found that the highest levels of Pb, Cd, Cu and Zn were in strawberries, cucumbers, dates and spinach²³. Furthermore, the concentration of heavy metals in commercially important species of fish, shellfish and fish products from the Cochin area showed that different metals (Cd, Pb, Hg, Cr, As, Zn, Cu, Co, Mn, Ni, and Se) were present in the samples at various, worrying levels²⁴. Determination of these substances in food is important in environmental monitoring for the prevention, control and reduction of pollution. Metal ions are known to act as inhibitors of many enzymes, including the CAs^22,25. Indeed, cations such as Pb²⁺, Co²⁺ and Hg²⁺ show inhibitory activity against the human cytosolic isoforms hCA I, II and the fish enzyme dCA^22,25. Sheep meat is consumed all over the world as an important protein source but few data are reported regarding the metalloenzymes present in it²².

Although there are studies regarding purification of CA from various tissues and different organisms, no reports are available in the literature on the purification and characterization of the enzyme from sheep kidney. In the present study, we purified and characterized one α-CA from sheep kidney, presumably a CA IV-type isoform (sCA IV) for the first time, and investigated hCA I, hCA II, dCA and sCA IV kinetic properties and inhibitory effects with metal ions recognized as pollutants, such as Pb²⁺, Co²⁺, Hg²⁺, Zn²⁺, Se²⁺, Cu²⁺, Al³⁺ and Mn³⁺ on these enzymes.

Materials and methods

Pb(NO₃)₂, CoCl₂.6H₂O, HgCl₂, ZnCl₂, SeCl₂, CuSO₄.5H₂O, Al(NO₃)₃, MnCl₃, CNBr-activated Sepharose 4B, protein assay reagents, p-aminobenzene sulfonamide, L-tyrosine, 4-nitrophenyl acetate (NPA) and chemicals for electrophoresis were purchased from Sigma-Aldrich Co, Germany. All other chemicals were of analytical grade and obtained from either Sigma or Merck.

Purification of CA isozymes I, II, Dicentrarchus labrax liver and sheep kidney by affinity chromatography

The purification of hCA I and II isozymes were performed with a simple one step method by a Sepharose-4B-aniline-sulfanilamide affinity column chromatoghrapy^8–10. hCA I was purified, 102-fold with a specific activity of 839.6 EU × mg⁻¹ and overall yield of 59.7%; hCA II was purified, 580-fold with a specific activity of 4773.3 EU × mg⁻¹ and overall yield of 53%^8–10. The purification of dCA isozyme was performed with a simple one step method by a Sepharose-4B-aniline-sulfanilamide affinity column chromatoghrapy. dCA was purified, 78.8-fold with a specific activity of 751.72 EU·mg⁻¹ and overall yield of 46%²². Fresh sheep kidneys were taken from Erzurum Slaughterhouse under cold conditions (4°C). After the kidney was obtained, it was washed in isotonic saline containing 1 mM EDTA and stored at −20°C before use. One hundred grams of kidney was first cut into small pieces which were thereafter homogenized with 200 mL of buffer A (25 mM triethanolamine sulfate, 60 mM sodium sulfate and 1 mM benzamidine buffer, pH 8.0). The homogenate was centrifuged at 18,000 g for 60 min and the precipitate was removed. Supernatant was centrifuged at 45,000 rpm for 60 min and the precipitate containing sCA IV was separated. This was washed three times, and centrifuged at 45,000 rpm for 60 min. It has been weighted and suspended in 10 mL per g of buffer A and was then dialyzed at 4°C in 50 mM Tris-Sulfate (pH 7.4), for 1 h. The pH of the supernatant was adjusted to 8.7 with solid Tris. The homogenate was applied to the prepared Sepharose 4B-L-tyrosine-sulfanylamide affinity column equilibrated with 25 mM Tris-HCl/0.1 M Na₂SO₄ (pH 8.7). The affinity gel was washed with 25 mM Tris-HCl/22 mM Na₂SO₄ (pH 8.7). The sCA IV isozyme was eluted with 0.1 M CH₃COONa/0.5 M NaClO₄ (pH 5.6). All procedures were performed at 4°C.

Hydratase activity assay

CA activity was assayed by following the hydration of CO₂ according to the method described by Wilbur and Anderson²⁶. CO₂-hydratase activity as an enzyme unit (EU) was calculated by using the equation (t_c − t_c) where t₀ and t_c are the times for pH change of the nonenzymatic and the enzymatic reactions, respectively.

Esterase activity and in vitro inhibition assay

CA activity was assayed by following the change in absorbance at 348 nm of NPA to 4-nitrophenoxide ion over a period of 3 min at 25°C using a spectrophotometer according to the method described by Verpoorte et al.²⁷. 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 mL 3 mM NPA, 0.5 mL H₂O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of lead, cobalt, mercury, iron, aluminium, zinc, manganese and selenium were examined. All compounds were tested in triplicate at each concentration used. Different inhibitor concentrations were used. hCA I enzyme activities were measured in the presence of cadmium (1–3 mM), zinc (4–6 mM), selenium (0.5–3 mM), copper (0.5–1.5 mM), aluminium (0.5–3 mM) and manganese salts (3–7 mM). hCA II enzyme activities were measured for cadmium (0.5–1 mM), zinc (4–6 mM), selenium (0.5–2 mM), copper (0.05–0.5 mM), aluminium (0.1–1 mM) and manganese salts (3–10 mM); dCA enzyme activities were measured for cadmium (0.05–1 mM) and selenium salts (0.1–2 mM) and sCA IV enzyme activities were measured in the presence of lead (0.5–2 mM), cadmium (0.5–2 mM), zinc (0.5–2 mM), copper (0.1–0.5 mM) and aluminium (1–2.5 mM) salts. Control activity in the absence of inhibitor was taken as 100%. For each inhibitor, an Activity% − [Inhibitor] graph was obtained. To determine K_i values, several different inhibitor concentrations were tested. In these experiments, NPA was used as substrate at five different concentrations (0.15–0.75 mM). The Lineweaver–Burk curves were used to determine kinetic parameters and inhibition constants²⁸.

Protein determination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method, using bovine serum albumin as the standard²⁹. SDS-PAGE was performed after purification of the enzymes. It was carried out in 10% and 3% acrylamide for the running and the stacking gel, respectively, containing 0.1% SDS according to Laemmli³⁰.

Molecular weight determination

Sephadex G-200 gel filtration

The molecular weight of the enzyme was determined according to Andrews’s method³¹. At first, for establishing the void volume, Blue Dextran (2000 kDa) was passed through the column; then, bovine erythrocyte CA (29 kDa), bovine serum albumin (66 kDa) and alcohol dehyrogenase (150 kDa) were used as standard proteins (Sigma MW-GF-200).

Results and discussion

At the respiratory epithelium, erythrocytic CA catalyses the rapid dehydration of bicarbonate to molecular carbon dioxide. Moreover, the CO₂/HCO₃⁻ system constitutes one of the most important physiological buffers for acid-base regulation^32–34. Studies regarding influences of various substances on mammalian or organisms CAs have gained a great attention in the recent years^35–38. For instance, in vitro effect of some heavy metals on enzymes, such as the gill, intestinal and liver ones from various fish species showed that these enzymes were significantly inhibited by heavy metal ions^22,39–41.

In this study, hCA I, hCA II, dCA and sCA IV were purified from human erythrocytes, D. labrax liver and sheep kidney by a simple one step procedure using Sepharose 4B-tyrosine-sulfanilamide affinity column. The activity of the eluents were determined by the hydratase method, with CO₂ as substrate²⁶ and further kinetic studies were performed using the esterase method, with NPA as substrate³⁰.

We report here the first study on the inhibitory effects of metal ions on the sCA IV esterase activity. The previous report by Ekinci et al.²⁵ investigated metal ions (including Hg²⁺, Co²⁺ and Pb²⁺) by using esterase activity method, NPA hydrolysis assay for monitoring CA inhibition. Data of Table 1 show the following regarding inhibition of hCA I, hCA II, dCA and sCA IV with some metal ions:

Table 1.  K_i values for the inhibition of hCA I, hCA II, dCA and sCA IV with metal ions by an esterase method with 4-NPA as substrate²⁷.

Inhibitor	Ki hCA I (mM)	Ki hCA II (mM)	Ki dCA (mM)	Ki sCA IV (mM)
Pb^2+	0.99^a	0.056^a	0.24^b	1.13
Co^2+	3.91^a	1.70^a	0.53^b	NI
Hg^2+	1.42^a	0.31^a	0.77^b	NI
Cd^2+	2.18	0.24	0.21	1.04
Zn^2+	4.41	2.78	0.72^b	0.96
Se^2+	2.34	1.97	0.98	NT
Cu^2+	0.98	0.16	0.17^b	4.70
Al^3+	1.47	0.58	0.15^b	3.45
Mn^3+	10.78	8.17	3.46	NI

Mean from at least three determinations. Errors in the range of 1–3% of the reported value (data not shown).

NI, no inhibition, less than 30% inhibition at the concentration of 100 mM; NT, no tested.

^aEkinci et al. (2007)²⁵.

^bCeyhun et al. (2011)²².

1. Against the first cytosolic isozyme hCA I, Co²⁺, Zn²⁺ and Mn³⁺ ions behave as weak, millimolar inhibitors, with K_i-s in the range of 3.91–10.78 mM. A second group of these metal ions, including Cd²⁺ and Se²⁺, show better inhibitory activity as compared to the previously mentioned metal ions, with K_i-s in the range of 2.18 and 2.34 mM (Table 1). Data of Table 1 also show that similarly to organic nitrates and inorganic anions^2,9 most of the investigated metal ions act as non-competitive inhibitors with 4-NPA as substrate, i.e. they bind in different regions of the active site cavity as compared to the substrate. However, the binding site of 4-NPA itself is unknown, but it is presumed to be in the same region as that of CO₂, the physiological substrate of this enzyme⁷. It is rather probable that these metal ions interact with the His64 present in many CA II-type enzymes or with the histidine cluster of which His64 is part^42,43.

2. A rather similar activity of these metal ions has been observed also for the inhibition of the hCA II (Table 1). Thus, a first groups of these metal ions, including Co²⁺, Zn²⁺ and Se²⁺ showed modest hCA II inhibitory activity with K_i-s in the range of 1.70–2.77 mM, whereas the remaining four metal ions, i.e. the same acting as efficient hCA I inhibitors, showed K_i-s in the range of 0.98–1.42 mM (Table 1). Again most of these metal ions act as non-competitive inhibitors with 4-NPA as substrate, except for Pb²⁺ and Hg²⁺ which are uncompetitive inhibitors (Table 1).

3. Pb²⁺, Cd²⁺, Cu²⁺ and Al³⁺ were also strong inhibitors of dCA, with K_i-s in the range of 0.15–0.24 mM. However, again Se²⁺ and Mn³⁺ are weak inhibitors (K_i-s of 0.98–3.46 mM). Other metal ions were determined to be weaker inhibitors (K_i-s of 0.53–0.77 mM) (Table 1).

4. Cu²⁺ and Al³⁺ were weak inhibitors of sCA IV, with K_i-s of 3.45–4.70 mM. A second group of these metal ions, including Pb²⁺, Cd²⁺ and Zn²⁺, show better inhibitory activity as compared to the previously mentioned metal ions, with K_i-s in the range of 0.963–1.132 mM (Table 1).

We used a single step chromatographic technique for purifying this new enzyme, sCA IV, employing Sepharose 4B-tyrosine-sulfanilamide affinity chromatography which strongly binds most α-CAs^9,10. The optimum pH for the purification of the enzyme was determined to be 7.5. To determine the native molecular weight of the enzyme, gel filtration was carried out. For this purpose, K_av values for the enzyme and standard proteins were calculated, and a K_av − LogM_W graph was obtained. The molecular weight was determined to be 29 kDa. Similar results have been observed for the enzyme from different sources. For example, human erythrocyte CA is of 29 kDa⁹, the teleost fish D. labrax (European seabass) liver CA (dCA) is also of 29 kDa²². The molecular weight was proved to be 28.9 kDa by SDS-PAGE (Figure 1). We have investigated the esterase activity of sheep CA IV with NPA as substrate (Figure 2). The K_M and V_max values were calculated for NPA hydrolysis catalyzed by the sheep enzyme by means Lineweaver–Burk graphs (Figure 2). The Michaelis–Menten constant K_M constant was calculated to be of 0.203 mM, and V_max was 0.128 mmol × min⁻¹ for NPA.

Figure 1.  SDS-PAGE photograph. Lane 1: Sheep liver CA IV. Lane 2: Standard proteins: rabbit phosphorylase B (97.4 kDa), truncated β-galactosidase (83 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa) and bovine carbonic anhydrase (29 kDa).

Figure 2.  Lineweaver–Burk curves for 4-NPA hydrolysis catalyzed by sheep CA IV enzyme, at five different concentrations of substrate.

Conclusions

Some metal ions used in this study inhibit the activity of hCA I, hCA II, dCA and sCA IV isozymes. Most of the cations we have investigated here show activity in the low milimolar range, probably due to their binding to His64 or the His cluster of these enzymes^42,43. According to K_i values, the best inhibitor for human CA I was the copper(II) cation, for human CA II was the lead(II) one, for the fish liver dCA was aluminum(III) whereas, for sheep kidney sCA was zinc(II). Metal-ligand interactions are stronger especially in aqueous media. The inhibition mechanism of these metal ions against CAs is probably due to the interactions with histidine residues present at the entrance of the active site cavity of these enzymes, which thereafter perturbs the proton shuttling effects in which these amino acid participate and which are essential for the catalytic cycle. Although the inhibitory effects we describe here are weak (submilimolar range), considering the abundance of polluting cations in many environmental niches, they may be significant in such systems due to the bioaacumulation processes which concentrate heavy metal ions in many exposed organisms⁴⁴.

Declaration of interest

This study was financed by Turkish Republic Prime Ministry State Planning Organization (DPT), (Project no: 2010K120440) for (MS).