Characterization of carbonic anhydrase from Turkish native “Gerze” chicken and influences of metal ions on enzyme activity

Abstract

Carbonic anhydrase was purified and characterized from erythrocytes of the Turkish native chicken, Gerze, for the first time. The enzyme was purified 57.65-fold with a yield of 52%, and a specific activity of 954.08 U/mg proteins having optimum pH at 8.0; optimum temperature at 30 °C; optimum ionic strength at 10 mM and stable pH at 8.0. The purified enzyme had apparent K_M and V_max values of 0.73 mM and 0.236 μmol × min⁻¹, respectively. Al⁺³, Hg⁺², Cu⁺², Pb⁺², and Cd⁺² showed inhibitory effects on the enzyme. Pb⁺² exhibited the strongest inhibitory action. Cd⁺² and Hg⁺² were moderate inhibitor, whereas Al⁺³ and Cu⁺² showed weaker actions. All tested metals inhibited the enzyme in competitive manner. Our findings indicate that these metals inhibit the chicken enzyme in a similar manner to other α-CAs from mammals investigated earlier, but susceptibility to various metals differ between the native chicken and other mammalian enzymes.

• Carbonic anhydrase
• gerze
• inhibition
• metal

Introduction

Carbonic anhydrases (EC 4.2.1.1, CAs) are widespread zinc containing metalloenzyme family. The enzyme catalyzes the reversible inter-conversion of carbon dioxide and bicarbonate. This reaction is the main role of CA enzymes in physiological conditions1–5. Sixteen isozymes have been described up to now in mammals, the most active ones as catalysts for carbon dioxide hydration being CA II and CA IX1,2. The 16 isozymes differ in their subcellular localization, catalytic activity and susceptibility to different classes of inhibitors. Some of them are cytosolic (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 reported that CA XV isoform is not expressed in humans or in other primates, but it is abundant in rodents and other vertebrates1,3–7. CAs are produced in a variety of tissues where they participate in several important biological processes such as acid–base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation1,4–6. Up to now, CA has been purified from many different tissues including human erythrocytes8–10, fish liver5, fish erythrocytes6, rainbow trout gill and liver6,11, similarly some chemicals and metal ions inhibition effects on human and rainbow trout enzymes are investigated4–14.

Gerze chicken, also known as Hacı Kadı, is the one of two endemic chicken breeds of Turkey. It has been rearing in Sinop province of Northern part of Turkey, but unfortunately, nowadays Gerze chicken is threatened with extinction because of its disadvantages on economic productivity according to commercial hybrids. Today, a limited number of Gerze chicken enthusiasts and governmental institutes as Lalahan Livestock Central Research Institution and Agricultural District Directorate of Gerze affiliated to the Turkish Ministry of Food, Agriculture and Animal Husbandry, conserve this genetic resource15,16. Consequently, there is a deep need to define existing chicken populations and to investigate their biochemical features so as to benefit people living in rural areas and scientists interested in this area17.

In the present study, we purified and characterized carbonic anyhdrase from gerze chicken erythrocytes for the first time, and investigated inhibitory effects of some metal ions on enzyme activity.

Methods and materials

Chemicals

Al(NO₃)₃, Cu(NO₃)₂, Pb(NO₃)₂, Cd(NO₃)₂, Hg(NO₃)₂, Sepharose 4B, protein assay reagents, 4-nitrophenylacetate were obtained from Sigma-Aldrich Co., Munich, Germany. All other chemicals were of analytical grade and obtained from Merck, Darmstadt, Germany.

Preparation of the hemolysate

Blood was sampled from the caudal vein using a 10 ml plastic heparinized syringe (5 IU/ml) and transferred to tubes. Blood sample were centrifuged at 2500 × g for 15 min. The plasma was removed by drip. After the package of red cells was washed with KC1 solution (0.16 M) three times, the samples were centrifuged at 2500 × g each time and supernatants were removed. The erythrocytes were hemolyzed with five volumes of ice-cold water and centrifuged (+4 °C, 10 000× g) for 30 min to remove the ghosts and intact cells6.

Purification of carbonic anhydrase from chicken erythrocytes by affinity chromatography

The pH of the hemolysate was adjusted to 7.5 with solid Tris. The homogenate was applied to the prepared Sepharose 4BL-tyrosine-sulfanilamide affinity column equilibrated with 10 mM Tris-HCl/0.1 M Na₂SO₄ (pH 8.0). The affinity gel was washed with 10 mM Tris-HCl/22 mM Na₂SO₄ (pH 8.0). The gerze chicken erythrocytes carbonic anhydrase enzyme was eluted with 1.2 M NaCl/25 mM Na₂HPO₄ (pH 6.3). All procedures were performed at 4 °C.

Esterase activity assay

Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25 °C using a spectrophotometer according to the method described by Verpoorte et al.18. 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 4-nitrophenylacetate, 0.5 ml H₂O and 0.1 ml enzyme solution18. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of Al⁺³, Cu⁺², Cd⁺², Pb⁺² and Hg⁺² were examined. All compounds were tested in triplicate at each concentration used. Different inhibitor concentrations were used. Control cuvette activity in the absence of inhibitor was considered as 100%. For each inhibitor, an Activity (%)−[Inhibitor] graph was drawn. To determine K_i values, three different inhibitor concentrations were tested. In these experiments, 4-nitrophenylacetate was used as substrate at five different concentrations (0.10–0.60 mM). The Lineweaver–Burk curves were drawn19.

Protein determination

Protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method, using bovine serum albumin as the standard20.

SDS polyacrylamide gel electrophoresis

SDS polyacrylamide gel electrophoresis 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 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 destained with several changes of the same solvent without the dye21.

Stable pH determination

For this aim, equal volumes of the buffers K-phosphate at pH of 6.0, 6.5, 7.0, 7.5 and 8.0, Tris-HCl at pH of 7.5, 8.0, 8.5 and 9.0, and purified enzyme were mixed and kept in a refrigerator (+4 °C). The enzyme activity was assayed at 5-day intervals22.

Optimum pH determination

In order to determine the optimum pH, Tris-HCl and K-phosphate buffers were used in the pH range of 5.5–8.0 and 7.5–9.0, respectively22.

Optimum temperature determination

For determination of the optimum temperature, enzyme activity was assayed at different temperatures in the range from 5 °C to 70 °C. The desired temperature was provided by using a Grant bath22.

Optimum ıonic strength determination

For determination of optimum ionic strength, enzyme activity was determined using different concentrations of K-phosphate buffer, pH: 7.5, in the range from 10 mM to 1000 mM22.

Molecular weight determination with SDS-PAGE

The subunit of the enzyme was determined by SDS-PAGE (Figure 1). E. coli β-galactosidase (116 kDa), Rabbit phosphorylase B (97.4 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa) and bovin carbonic anhdrase (29 kDa) were used as standards (Sigma MW-SDS-200).

Figure 1. SDS-PAGE analysis of purified gerze chicken erythrocytes CA (gcCA); Lane (1, 2): gcCA and Lane (3): standard proteins (Chicken ovalbumin (45 kDa), bovine carbonic anhydrase (29 kDa) and chicken egg white lysozyme (16.5 kDa)).

In vitro effects of metal ıons

In order to determine the effects of the metal ions on chicken erythrocytes CA, different concentrations of metal ions 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 (%) versus metal ion concentration plots.

In order to determine K_i constants in the media with inhibitor, the substrate (NPA) concentrations were 0.01, 0.02, 0.035, 0.05, and 0.07 mM. Inhibitor solutions (metal salts) were added to the reaction medium, resulting in three different fixed concentrations of inhibitors in 1 ml of total reaction volume. Lineweaver–Burk graphs were drawn by using 1/V versus 1/[S] values and K_i constant were calculated from these graphs19. Regression analysis graphs were drawn for IC₅₀ using inhibition % values by a statistical package (SPSS-for windows; version 10.0) on a computer (student t test; n = 3).

Results and discussion

Here, we isolated the CA enzyme from the Turkish native Gerze chicken erythrocytes for the first time and characterized. We achieved to purify the enzyme in a single step using affinity chromatography on Sepharose 4B tyrosine-sulfanilamide. The enzyme was purified 57.65-fold with a recovery ratio of 52.82% compared to the homogenate (Table 1). After the sample had completely passed through, the column was washed with 10 mM Tris-HCl/0.1 M Na₂SO₄ buffer whose pH was 8.0. During washing, absorbencies of fractions were spectrophotometerically measured at 280 nm and 348 nm. These values of the absorbance showed that some proteins, bound to the affinity material, have been removed from the column by the washing solutions. Then, the enzyme was eluted with 1 M NaCl/25 mM Na₂HPO₄ pH 6.3. At the end of the last step, a highly purified enzyme was obtained exhibiting a single band on SDS-PAGE (Figure 1). We used only one chromatographic technique, Sepharose 4B tyrosine-sulfanilamide affinity chromatography by modification of washing and elution conditions.

Table 1. Summary of purification procedure for gerze chicken erythrocytes carbonic anhydrase enzyme by a Sepharose-4B-tyrosine-sulfanilamide affinity column chromatography.

Purification steps	Activity (EU/ml)	Total volume (ml)	Protein (mg/ml)	Total protein (mg)	Total activity (EU)	Specific activity (EU/mg)	Yield (%)	Purification factor
Hemolysate	118	18	7.13	128.34	2124	16.55	100	1
Sepharose-4B-tyrosine-sulfanilamide affinity column chromatography	187	6	0.196	1.176	1122	954.08	52.82	57.65

The optimum pH, optimum temperature, optimum ionic strength and stable pH were determined to be 8.0, 30 °C, 10 mM and 8.0 for the enzyme, respectively. The stable pH profile of the enzyme was determined at four different pHs in 10 mM Tris-HCl and five different pHs in 10 mM K-phosphate buffer. The enzyme was able to protect 92% of maximum activity at the end of 14 days in 10 mM Tris-HCl buffer (pH: 8.0). The optimum pH was found to be 8.0. The result is similar to CAs obtained from human erythrocytes23,24. The enzyme was seen to exhibit the highest activity at 25 °C in a study of temperatures between 5 and 70 °C. The optimum ionic strength of the enzyme was estimated to be 10 mM Tris-HCl buffer. However, about 85% of the maximum activity was present in the broad range from 10 mM to 1000 mM.

The molecular weight was determined to be 30 kDa. Similar results have been observed for the enzyme from different sources. For example, human erythrocyte CA is 29 kDa, bovine erythrocyte CA is 29 kDa and European seabass liver CA is 30.2 kDa3–6. In addition to establishment of molecular weight of the enzyme, Rf values were calculated for the enzyme and standard proteins, and Rf-LogMW graph was obtained according to Laemmli’ method. The molecular weight of the enzyme was determined to be 30 kDa. K_M and V_max values were calculated for NPA by Lineweaver–Burk graph (Figure 2). K_M constants were calculated as 0.73 mM, V_max values as 0.236 μmol × min⁻¹ for NPA.

Figure 2. Lineweaver–Burk curves for p-NPA hydrolysis catalyzed by the gcCA enzyme, at five different concentrations of substrate.

Moreover, effects of substrates on enzyme activity were studied. Measurements showed that, as NPA concentration became higher, the activity became less and less. This result shows that aggregation occurs and activity decreases similarly at high NPA concentrations.

In addition to purification and characterization of the enzyme, Al ⁺³, Cu⁺², Cd⁺², Pb⁺² and Hg⁺² were chosen to investigate their inhibitory effects on gerze chicken erythrocytes CA (gcCA) and K_i parameters of these metals were determined. Metal ions inhibited the enzyme activity at low concentrations. It is clear that Pb²⁺ and Hg²⁺ are the most potent inhibitors for gcCA enzyme (Table 2). According to values, the best inhibitor for gcCA is lead. The mechanism of toxicological effects of the metals is probably due to the interactions between the histidines and the metals or the interaction of the metals with other aminoacids around the active site.

Table 2. K_i values for the inhibition of hCA I, hCA II, sCA IV, dCA and gcCA (gerze chicken CA) with metal ions (mM).

Metal ion	hCA I	hCA II	sCA IV	dCA	gcCA
Pb^2+	0.99*	0.056*	1.13‡	0.24†	0.97
Hg^2+	1.42*	0.31*	–	0.77†	1.26
Co^2+	3.91*	1.70*	–	0.53†	–
Al^3+	1.47‡	0.58‡	3.45‡	0.15†	2.24
Cu^2+	0.98‡	0.16‡	4.70‡	0.17†	2.78
Cd^2+	2.18‡	0.24‡	1.04‡	0.21‡	1.35

*Ekinci et al4.

†Ceyhun et al5.

‡Demirdag et al6.

Conclusions

Consequently, we purified carbonic anhydrase from Turkish native chicken, Gerze for the first time, and analyzed characteristic features. Our results are in good agreement with others reported in literature. Also, inhibitory effects of metals on enzyme activity were reported. This study is supportive for medicinal chemists who are interested in enzyme inhibitors. It is also useful for paultry researchers because economically efficient hybrids have begun to replace the indigenous breeds with lower potential due to spread of intensive production. This endemic Gerze chicken is under the risk of extinction and therefore, the studies about its specific physiological responses to environmental conditions, especially chemical toxicity has great importance.

Declaration of interest

The authors report no conflicts of interests. The authors alone are responsible for the content and writing of this article.