Human Carbonic Anhydrase: Purification and

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Original Article

Human Carbonic Anhydrase: Purification and Characterization Study in Thalassemia Major Patients Compared to Healthy Subjects Salwa Saleh Hussein, Israa Ghassan Zainal Department of Chemistry, College of Science, Kirkuk University, Kirkuk, Iraq

Abstract Background: Carbonic anhydrase (CA) catalyzes the reversible reaction of converting carbon dioxide to bicarbonate. Objective: This study was aimed to isolate and purify human erythrocytes CA and study its physicochemical properties of the enzyme reaction for ß‑thalassemia major patients. Materials and Methods: The blood samples included 61 samples of blood (31 males and 30 females) from ß‑thalassemia patients visited Azadi Hospital/Kirkuk city. Healthy individuals as control group included 40 participants. The separated fractions were obtained using four steps: extraction by ethanol and chloroform, ammonium sulfate precipitation, dialysis, and gel filtration chromatography; finally, the CA was analyzed by polyacrylamide gel electrophoresis. Results: The CA activity showed significant (P ≤ 0.05) decrease, total protein showed nonsignificant (P ≥ 0.05) increase, and specific activity significantly (P ≤ 0.05) increased in patients group compared to healthy individuals. CA was partially purified with a factor of 22.5 and 18 by extraction with ethanol and chloroform and 1.5,1.4 for Fraction I and 1,2 for Fraction II using gel filtration chromatography. The optimum conditions for the CA reaction in patients group were enzyme concentration (6 µl), substrate concentration (6 Mm), pH = 7.4, and temperature 37°C. The electrophoresis study indicated that the bands of CA in patients group showed bands with less intensity than the bands in healthy individuals. Conclusion: The best method to purify CA from human erythrocytes with high recovery and fold of purification was ethanol–chloroform extraction. Keywords: Carbonic anhydrase, electrophoresis, gel filtration, purification, ß‑thalassemia

Introduction Carbonic anhydrase (CA) (EC 4.2.1.1) [1] catalyzes the reversible reaction of converting carbon dioxide (CO2) to bicarbonate (HCO3−) as in the equation below:[2] CO2 + H2O ↔ H+ + HCO3− CA is a Zn‑metalloenzyme[3] present in plants, animals, and microorganisms, suggesting that the CA has many diverse metabolic roles in living organisms. [4‑6] CAs have seven evolutionarily unrelated CA‑gene families including α‑, β‑, γ‑, δ‑, ζ‑, η‑, and θ‑ CAs with no structural similarity; mammals have only α‑CAs with multiple isoforms of the enzyme.[7] The family of CA is known to contain 15 human α‑CA isoforms, all of which differ in their catalytic rates, inhibitor sensitivity and selectivity, cellular localization, and tissue distribution.[8‑10] CAs are involved in various physiological roles including “fluid secretion, acid/base balance thus pH regulation, Access this article online Quick Response Code:

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DOI: 10.4103/MJBL.MJBL_81_18

gluconeogenesis, ureagenesis, gastric acid production, and transport of CO2 from the tissues to the lungs (in the form of HCO3−)” through blood.[11‑13] CO2 released as a part of respiration by the tissues is not very soluble in blood and thus, to be transported, it is converted to HCO3  − by human CA II (HCA II).[14,15] Reduction in CA activity decreases the secretion of HCO3 − and aqueous humor, thereby reducing the pressure.[8,10,16,17] HCA II aids in the conversion of H2O and CO2 into HCO3 − and a proton through two steps called “ping‑pong mechanism” as in the equations below: E: Zn − OH‑+ CO2 ↔ E: Zn − H2O + HCO3− (reaction 1). E: Zn − H2O + B↔ E: ZnOH− + BH+

H2O

(reaction 2).

Address for correspondence: Dr. Israa Ghassan Zainal, Department of Chemistry, College of Science, Kirkuk University, Kirkuk, Iraq. E‑mail: [email protected]

This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: [email protected]

How to cite this article: Hussein SS, Zainal IG. Human carbonic anhydrase: Purification and characterization study in thalassemia major patients compared to healthy subjects. Med J Babylon 2018;15:349-56.

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The hydration step showed (first step) the zinc‑bound hydroxide which acts as a nucleophile, attacking the CO2, and ultimately forming HCO3−, this prompts H2O molecule bound to the Zn (reaction 1). The second step (reaction 2) recovers the Zn‑bound hydroxide through a proton transfer mechanism through “His64” in HCA II to solvent B.[18‑22] Erythrocytes CA has been investigated to link with various pathological conditions including diabetes mellitus, hypertension, lipid disorders, anemia, sickle‑cell disease, and leukemia.[23‑27]

6 μL of hemolysate placed in 1 cm spectrometric cell containing 744 μL of 0.05 M Tris‑HCl, pH 7.4, and 750 μL of p‑nitrophenyl acetate. The change in the absorbance at 348 nm was measured over the period of 3 min before and after adding the sample. The absorbance was measured by an ultraviolet (UV)‑Vis spectrophotometer (Shimadzu UV‑2600 Spectrophotometer). One unit of enzyme activity was expressed as μmol of p‑nitrophenol released/min/μL from hemolysate at room temperature 25°C.[29,32]

Based on the above facts, the present study estimates the CA activity in the erythrocytes of patients with ß‑thalassemia compared to healthy individuals to look at its use as a surrogate marker in patients with ß–thalassemia this study also including characterizing the physicochemical properties of the CA reaction after purified from the erythrocytes of patients with ß‑thalassemia and healthy individuals. This CA has been purified by extraction using ethanol and chloroform and ammonium sulfate (AS) precipitation, followed by gel‑filtration chromatography.

Purification of carbonic anhydrase

Materials and Methods Chemicals and subjects

All the chemicals were commercial products of the purest quality. Sepharose 4B and p‑nitrophenyl acetate were purchased from Solarbio Company. Sixty‑one sample of blood (31 males and 30 females) from ß‑thalassemia patients with age ranged 2–32 years were selected in this study. Those patients visited Azadi hospital/Kirkuk city/Iraq, during the period from April to October 2017. All patients were subjected to a personal interview using specially designed questionnaire format of full history with detailed information. Healthy individuals as control group included forty participants (13 males and 27 females) with the same age range as patients, any case may interfere with this study such as diabetes mellitus, hypertension, anemia, and liver diseases were discarded.

Blood collection

Using a disposable syringe, 2–3 ml of blood was collected by venipuncture in glass tubes within ethylenediaminetetraacetic acid‑K3 as an anticoagulant. The tubes were centrifuged for 10 min with 704 × g. The plasma was separated from the cells and buffy coat removed. The packed red cells were washed three times with normal saline (0.9% NaCl) and were then lysis with ice cold water, then stored at −20°C until analysis.

Determination of total protein

Quantitative protein determination was achieved by absorbance measurements at 660 nm according to the Lowry method 1951,[28] using bovine serum albumin as a standard.

Assay of carbonic anhydrase activity

CA activity was determined as mentioned by Verpoorte et  al.,[29] with the modification described by Parui et  al.[30] using a spectrophotometer. The esterase activity of CA was determined from the hydrolysis rate of 3 mM p‑nitrophenyl acetate to p‑Ntro phenol.[31] The assay system contained 350

All the purification steps were carried out at a temperature of 4°C: 1. Extraction with chloroform and ethanol: Solvent proportion (0.6 ml of H2O, 0.4 ml of ethanol, and 0.5 ml chloroform) was added drop by drop to 1.5 ml from red blood cells hemolysate (the sample set in an ice bath with stirring continuously) for 90 min, then the sample was centrifuged at 784 × g for 30 min to remove excess of chloroform and ethanol.[33] The precipitate was dissolved in 1 ml of 0.05M Tris‑HCl buffer (pH‑7.4). Finally, CA activity and protein concentration were determined for each separated fraction 2. By AS precipitation: Three milliliters of the obtained sample from Step 1 above was first brought to 50% saturation with solid AS (the sample set in ice bath with stirring slowly and continuously).[33] After resting for 14 h at 4°C, the sample centrifuged at 948.64  ×  g and 4°C for 30 min, then redissolved in 1 ml of 0.05M Tris‑HCl buffer (pH‑7.4). Both enzyme activity and protein concentration were determined for each separated fraction 3. Dialysis against buffer: The obtained AS precipitate (enzyme solution) was dialyzed in the presence of 0.05M Tris‑HCl buffer at pH 7.4, overnight at 4°C with changing the buffer solution each 6 h. Fractions were checked in terms of both protein concentration and CA activity 4. Gel‑filtration chromatography: The sample from the Step 3 above was further purified by gel‑filtration chromatography on Sepharose 4B resin. Sepharose 4B resin was applied to an empty column (38 × 0.7) cm and equilibrated with 0.05M Tris‑HCl buffer, pH 7.4. The sample containing about 1–5 mg/ml of total protein (TP) was loaded on the Sepharose 4B column equilibrated as aforementioned. Fractions of 1 ml/5 min were collected, and the absorbance of protein was read at 280 nm. Enzyme activity and protein concentration of the partially purified samples were checked at 348 and 660 nm, respectively; tubes with CA activity were collected for physicochemical properties of the enzyme reaction studies.

Discontinuous polyacrylamide gel electrophoresis (Laemmli method) Polyacrylamide gel electrophoresis (PAGE) was carried out according to the Laemmli method[34] for the crude and partially purified samples to locate the position of CA bands. Gel was stained with Coomassie Brillant Blue G‑250.

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Statistical analysis

Statistical analysis was performed with GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA), values were expressed as mean ± standard deviation [SD]) and P ≤ 0.05 was considered as statistically significant. The comparison of mean ± SD was performed using the Student’s t‑test. Statistical significance was defined as P ≤ 0.05.

Results The native CA was isolated and purified to homogeneity at 4°C from the erythrocytes of ß‑thalassemia patients and healthy individuals, then CA activity, specific activity, and TP were determined and the results were mentioned as mean ± SD as present in Table 1. The results indicated that there was nonsignificant increase (P ≥ 0.05) in the TP concentration, significant (P ≤ 0.05) decrease and increase in the activity and specific activity of CA, respectively, in the patients group compared to healthy individuals. The results of CA activity disagreed with Midiwo et  al.,[27] they studied the activity of CA in children with sickle‑cell anemia and found that there was significant elevation in the activity of CA. Osterman et al.[35] found an increase in serum CA activity in all patients with muscular dystrophy,

chronic polymyositis, and amyotrophic lateral sclerosis and in many with myasthenia gravis. To the best of our knowledge, there is no study to evaluate the activity and specific activity of erythrocytes CA in the patients with ß‑thalassemia. This study also examined the purification and characterization of crude and partially purified CA reaction for the ß‑thalassemia patients compared to healthy individuals [Tables 2 and 3]. The results indicated that most of the CA activity was recovered in the soluble fraction of cell extract after using ethanol–chloroform method; the specific activity of the CA in ß‑thalassemia patients sample increased from 0.762 to 16.4 U/mg and from 0.762 to 13.58 U/mg in healthy individuals after extraction with ethanol and chloroform, then decreased after using another steps of purification. The fold of purification and the yield % were also increased after extraction with chloroform and ethanol, then decreased for the two studied groups. Contaminants of proteins were precipitated by the addition of solid AS (50% saturation). Following the AS precipitation, the CA activity was detected in the supernatant fraction, which was fractioned by gel‑filtration chromatography for both healthy individuals and ß‑thalassemia patients samples [Figures 1 and 2].

Table 1: Carbonic anhydrase activity, specific activity, and total protein in the sera of ß‑thalassemia patients Groups

n

Mean±SD Carbonic anhydrase, U/ml

Protein concentration, mg/ml

Specific activity, U/mg

0.1168±0.02455 0.1112±0.004582 0.8544**

19.02±1.523 14.9±0.9821 0.0467*

Patients 61 1.327±0.05061 Healthy 40 1.524±0.06268 0.0157* P *P≤0.05=Significant, **P≥0.05=Nonsignificant. SD: Standard deviation

Table 2: Purification steps for carbonic anhydrase of ß‑thalassemia patients Steps

CA activity (U/ml)

Total volume (ml)

Protein (mg/ml)

1. Crude sera 0.375 2 0.489 2. Ethanol chloroform 0.704 1.5 0.043 3. AS 0.142 4 0.049 4. Dialysis 0.052 3 0.078 5. Gel filtration a 0.035 1 0.032 6. Gel filtration b 0.025 3 0.028 CA: Carbonic anhydrase, AS: Ammonium sulfate, TP: Total protein

TP (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Fold of purification

0.978 0.0644 0.197 0.235 0.032 0.084

0.714 1.056 0.568 0.157 0.035 0.075

0.73 16.4 2.886 0.67 1.104 0.886

100 148 80 22 5 10.5

1 22.5 4 1 1.5 1.4

TP (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Fold of purification

0.923 0.123 0.148 0.113 0.02 0.0242

0.704 1.666 0.769 0.228 0.017 0.0374

0.762 13.58 5.203 2.015 0.857 1.54

100 237 109 32.4 2.5 5.3

1 18 7 3 1 2

Table 3: Purification steps for carbonic anhydrase of healthy individuals Steps

CA activity (U/ml)

Total volume (ml)

Protein (mg/ml)

1. Crude sera 0.235 3 0.308 2. Ethanol chloroform 0.833 2 0.061 3. AS 0.22 3.5 0.042 4. Dialysis 0.152 1.5 0.075 5. Gel filtration a 0.017 1 0.02 6. Gel filtration b 0.025 1.5 0.016 CA: Carbonic anhydrase, AS: Ammonium sulfate, TP: Total protein

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From Figure 2, the CA appeared with two Fractions I and II. The second part of this study was aimed to evaluate the physicochemical properties (optimum conditions) of CA

reaction in the crude and Fractions (I and II) of patients and healthy individuals. Figure 3 represents the optimum CA concentration in all studied groups, and it is clear that CA activity elevated with increasing the volume of the added serum to the CA reaction; the volume of CA used was 6 µl as the optimum concentration of CA. The effect of different substrate concentrations on the activity of CA in all studied groups is presented in Figure 4.

Figure 1: Protein absorption at 280 nm of the elution fractions for patients and healthy individuals

From Figure 4, the results of the crude and partially purified CA fractions (I and II) samples appeared with hyperbolic figure, while the results appeared with S shape after partially purified, the optimum substrate concentration was 6 mM for all studied groups. The values of Km and Vmax are represented in Table 4 (calculated from Lineweaver–Burk plot) for all studied groups. The results indicated that the affinity of CA to its substrate increased in the partially purified samples compared to crude samples [Table 4]. The optimum (pH and temperature) for CA reaction of the human erythrocyte in all studied groups is represented in Figures 5 and 6, respectively, and found equal to 7.4 and 37°C for patient group. Figure 7a represents the PAGE (10%) for the CA of the crude sera of ß‑thalassemia patients and healthy individual samples and Figure 7b for the crude sera and partially purified CA from ß‑thalassemia patient samples.

Figure 2: Elution volume of the human carbonic anhydrase for patients and healthy individuals

Figure 7a indicates that the band which cleared in this figure represents the CA and the intensity of the band was less clear in patients samples compared to healthy individuals. These

Figure 3: Optimum carbonic anhydrase concentration (volume) for all the studied groups 352

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Figure 4: Optimum substrate concentration of the carbonic anhydrase in all the studied groups

Table 4: Km and Vmax for all studied groups (calculated from Lineweaver‑Burk plot) Groups Crude sample of thalassemia patient Crude sample of healthy subject Partially purified of thalassemia patient ‑ Fraction I Partially purified of thalassemia patient ‑ Fraction II Partially purified of healthy subject ‑ Fraction I Partially purified of healthy subject ‑ Fraction II

Km Vmax (µmol/ (mM) min/µl) 10 6.7 0.5 0.22 0.29 0.25

1.25 1 1.7×102 1×102 1.33×102 2×102

results confirmed those mentioned previously in Table 1 for the CA activity. The results obtained in Figure 7b indicated that the CA was partially purified by the steps used in this study.

Discussion The CAs are mostly zinc‑containing metalloenzymes which catalyze the reversible hydration/dehydration of CO2/HCO3−;[36] the CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. The high catalytic rate, relatively simple procedure of expression and purification,[36] relative stability, and extensive biophysical studies of HCA II have made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors, and CO2 sequestration systems, among others. However, there had been few studies on the chemical characterization of CA from the human erythrocytes, Keilin and Mann,[37] partially

purified CA; in this study, CAs from healthy individuals and ß‑thalassemia patients were partially purified using four sequential steps including extraction with ethanol and chloroform, AS precipitation, dialysis, and finally, using gel‑filtration chromatography. Different researchers had reported that CA in human erythrocytes may be fractionated to two and/or three or more fractions. Kyman[38] separated three active fractions by column electrophoresis. Rickli and Edsall[39] separated two active fractions by hydrophobic chromatography with phosphate buffers. Laurent et al.[40] have separated three components by chromatography on Amberlite CG50 and have found them to contain CA activity,[41] one being much more active than the other two. This study separated two fractions from CA erythrocytes using Sepharose 4B gel filtration, Fractions (I and II) from all studied groups. Tasgin et  al.[42] purified CA from bovine bone marrow using affinity chromatography using Sepharose 4B‑L‑tyrosine sulfanilamide, then investigated its kinetic properties. The present study also aimed to investigate the kinetic properties of CA, therefore, the CA which was purified from erythrocytes and then determined its kinetic properties. To check the purity of the partially purified CA from previously purified steps, conventional electrophoresis analysis was carried out on the crude and partially purified CA in healthy and patient samples. It is obvious from Figure 7 that the comparison between protein profile of the crude and the partially purified CA for the studied groups, the proteins in the crude separated into several protein bands and the purified samples showed

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Figure 5: Optimum pH of (crude and partially purified) carbonic anhydrase in all the studied groups

Figure 6: Optimum temperature of (crude and partially purified) carbonic anhydrase in all the studied groups

less number and intensity bands which reflected that there were other proteins present in the crude sample which removed when the sample partially purified. It is clear from Figure 7a and b that the blue bands were demonstrated in each of the crude and the purified CA of the studied groups, which could be concluded that the purified CAs appeared as a single band [Figure 7b]. The results of CA purification indicated that for both patients and healthy individuals with 354

the first step (ethanol–chloroform extraction), best recovery and fold of purification were obtained. Demür et al.[43] studied CA in the human erythrocyte membrane and using affinity chromatography to purify the enzyme.

Conclusion The results of this study indicated that the CA was isolated from

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b

Figure 7: (a) Analysis of the carbonic anhydrase enzyme in healthy individuals and thalassemia patients by polyacrylamide gel electrophoresis 10% gel was stained for: (1) Crude sample of thalassemia patients. (2) Crude sample for healthy individuals. (b) (1) Crude sera from thalassemia patients. (2) Partially purified by ethanol and chloroform from thalassemia patients. (3) Partially purified by ammonium sulfate from thalassemia patient. (4) Dialysis from thalassemia patients. (5) Partially purified by gel filtration first band from thalassemia patients. (6) Partially purified by gel filtration second band from thalassemia patients. (7) Partially purified by gel filtration first band from thalassemia patients. (8) Partially purified by gel filtration second band from thalassemia patients

the human erythrocytes and can conclude that the best method to purify CA from the human erythrocytes with high recovery and fold of purification was ethanol–chloroform extraction.

Financial support and sponsorship Nil.

Conflicts of interest

There are no conflicts of interest.

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Medical Journal of Babylon  ¦  Volume 15  ¦  Issue 4  ¦  October-December 2018

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