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Year : 2018  |  Volume : 15  |  Issue : 4  |  Page : 349-356

Human carbonic anhydrase: Purification and characterization study in thalassemia major patients compared to healthy subjects

Department of Chemistry, College of Science, Kirkuk University, Kirkuk, Iraq

Date of Web Publication20-Dec-2018

Correspondence Address:
Israa Ghassan Zainal
Department of Chemistry, College of Science, Kirkuk University, Kirkuk
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/MJBL.MJBL_81_18

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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 61samples of blood(31males and 30females) 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

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

How to cite this URL:
Hussein SS, Zainal IG. Human carbonic anhydrase: Purification and characterization study in thalassemia major patients compared to healthy subjects. Med J Babylon [serial online] 2018 [cited 2020 Oct 22];15:349-56. Available from: https://www.medjbabylon.org/text.asp?2018/15/4/349/248049

  Introduction Top

Carbonic anhydrase(CA)(EC[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],[5],[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],[9],[10] CAs are involved in various physiological roles including “fluid secretion, acid/base balance thus pH regulation, gluconeogenesis, ureagenesis, gastric acid production, and transport of CO2 from the tissues to the lungs(in the form of HCO3)” through blood.[11],[12],[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+ (reaction 2).

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],[19],[20],[21],[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],[24],[25],[26],[27]

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.

  Materials and Methods Top

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(31males and 30females) from ß-thalassemia patients with age ranged 2–32years 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(13males and 27females) 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, 2–3ml of blood was collected by venipuncture in glass tubes within ethylenediaminetetraacetic acid-K3 as an anticoagulant. The tubes were centrifuged for 10min 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 660nm 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 6 μL of hemolysate placed in 1cm spectrometric cell containing 744 μL of 0.05 M Tris-HCl, pH7.4, and 750 μL of p-nitrophenyl acetate. The change in the absorbance at 348nm was measured over the period of 3min 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]

Purification of carbonic anhydrase

All the purification steps were carried out at a temperature of 4°C:

  1. Extraction with chloroform and ethanol: Solvent proportion (0.6ml of H2O, 0.4ml of ethanol, and 0.5ml chloroform) was added drop by drop to 1.5ml from red blood cells hemolysate(the sample set in an ice bath with stirring continuously) for 90min, then the sample was centrifuged at 784×g for 30min to remove excess of chloroform and ethanol.[33] The precipitate was dissolved in 1ml 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 14h at 4°C, the sample centrifuged at 948.64×g and 4°C for 30min, then redissolved in 1ml 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 pH7.4, overnight at 4°C with changing the buffer solution each 6h. 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, pH7.4. The sample containing about 1–5mg/ml of total protein(TP) was loaded on the Sepharose 4B column equilibrated as aforementioned. Fractions of 1ml/5min were collected, and the absorbance of protein was read at 280nm. 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.

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 Top

The native CA was isolated and purified to homogeneity at 4°C from the erythrocytes of ß-thalassemia patients and healthy individuals, thenCA activity, specific activity, and TP were determined and the results were mentioned as mean±SD as present in [Table1].
Table 1: Carbonic anhydrase activity, specific activity, and total protein in the sera of ß-thalassemia patients

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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[Table 2] and [Table 3].
Table 2: Purification steps for carbonic anhydrase of ß-thalassemia patients

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Table 3: Purification steps for carbonic anhydrase of healthy individuals

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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 [Figure 1] and [Figure 2].
Figure1: Protein absorption at 280nm of the elution fractions for patients and healthy individuals

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Figure2: Elution volume of the human carbonic anhydrase for patients and healthy individuals

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From [Figure2], 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. [Figure3] 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.
Figure3: Optimum carbonic anhydrase concentration(volume) for all the studied groups

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The effect of different substrate concentrations on the activity of CA in all studied groups is presented in [Figure4].
Figure4: Optimum substrate concentration of the carbonic anhydrase in all the studied groups

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From [Figure4], 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 [Table4] (calculated from Lineweaver–Burk plot) for all studied groups.
Table 4: Km and Vmax for all studied groups (calculated from Lineweaver-Burk plot)

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The results indicated that the affinity of CA to its substrate increased in the partially purified samples compared to crude samples[Table4]. The optimum(pH and temperature) for CA reaction of the human erythrocyte in all studied groups is represented in [Figure 5] and [Figure 6], respectively, and found equal to 7.4 and 37°C for patient group.
Figure5: Optimum pH of(crude and partially purified) carbonic anhydrase in all the studied groups

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Figure6: Optimum temperature of(crude and partially purified) carbonic anhydrase in all the studied groups

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[Figure7]a represents the PAGE(10%) for the CA of the crude sera of ß-thalassemia patients and healthy individual samples and [Figure7]b for the crude sera and partially purified CA from ß-thalassemia patient samples.
Figure7:(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

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[Figure 7]a 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 results confirmed those mentioned previously in [Table 1] for the CA activity. The results obtained in [Figure7]b indicated that the CA was partially purified by the steps used in this study.

  Discussion Top

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 [Figure7] 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 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 [Figure7]a and [Figure7]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[Figure7]b. The results of CA purification indicated that for both patients and healthy individuals with 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 Top

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

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Conflicts of interest

There are no conflicts of interest.

  References Top

Del PreteS, VulloD, De LucaV, SupuranCT, CapassoC. Biochemical characterization of the δ-carbonic anhydrase from the marine diatom thalassiosira weissflogii, TweCA. JEnzyme Inhib Med Chem 2014;29:906-11.  Back to cited text no. 1
LiljasA. Carbonic anhydrase under pressure. IUCrJ 2018;5:4-5.  Back to cited text no. 2
SauzeJ, JonesSP, WingateL, WohlS, Ogée J. The role of soil pH on soil carbonic anhydrase activity. Biogeosciences 2018;15:597-612.  Back to cited text no. 3
Floryszak-WieczorekJ, Arasimowicz-JelonekM. The multifunctional face of plant carbonic anhydrase. Plant Physiol Biochem 2017;112:362-8.  Back to cited text no. 4
SupuranCT, CapassoC. An overview of the bacterial carbonic anhydrases. Metabolites 2017;7. pii:E56.  Back to cited text no. 5
SupuranCT. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 2008;7:168-81.  Back to cited text no. 6
KikutaniS, NakajimaK, NagasatoC, TsujiY, MiyatakeA, MatsudaY, etal. Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom phaeodactylum tricornutum. Proc Natl Acad Sci U S A 2016;113:9828-33.  Back to cited text no. 7
AggarwalM, BooneCD, KondetiB, McKennaR. Structural annotation of human carbonic anhydrases. JEnzyme Inhib Med Chem 2013;28:267-77.  Back to cited text no. 8
LindskogS. Structure and mechanism of carbonic anhydrase. Pharmacol Ther 1997;74:1-20.  Back to cited text no. 9
AlterioV, Di FioreA, D'AmbrosioK, SupuranCT, De SimoneG. Multiple binding modes of inhibitors to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem Rev 2012;112:4421-68.  Back to cited text no. 10
SupuranCT. Carbonic anhydrases–An overview. Curr Pharm Des 2008;14:603-14.  Back to cited text no. 11
SlyWS, HuPY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995;64:375-401.  Back to cited text no. 12
SupuranCT, ScozzafavaA. Carbonic anhydrases as targets for medicinal chemistry. Bioorg Med Chem 2007;15:4336-50.  Back to cited text no. 13
ParuiR, GambirKK, CruzI, HostenAO. Erythrocyte carbonic anhydrase: Amajor intracelluler enzyme to regulate cellular sodium metabolism in chronic renal failure patients with diabetes and hypertension. Int J Biochem 1992;26:809-20.  Back to cited text no. 14
ParuiR, GambirKK, MehrotraPP. Changes in carbonic anhydrase may be the initial step of altered metabolism in hypertension. Int J Biochem 1991;23:779-89.  Back to cited text no. 15
PastorekovaS, ParkkilaS, PastorekJ, SupuranCT. Carbonic anhydrases: Current state of the art, therapeutic applications and future prospects. JEnzyme Inhib Med Chem 2004;19:199-229.  Back to cited text no. 16
AggarwalM, McKennaR. Update on carbonic anhydrase inhibitors: Apatent review(2008–2011). Expert Opin Ther Pat 2012;22:903-15.  Back to cited text no. 17
TuCK, SilvermanDN, ForsmanC, JonssonBH, LindskogS. Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant. Biochemistry 1989;28:7913-8.  Back to cited text no. 18
MikulskiRL, SilvermanDN. Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase. Biochim Biophys Acta 2010;1804:422-6.  Back to cited text no. 19
SilvermanDN. Carbonic anhydrase: Oxygen-18 exchange catalyzed by an enzyme with rate-contributing proton-transfer steps. Methods Enzymol 1982;87:732-52.  Back to cited text no. 20
SilvermanDN, LindskogS. The catalytic mechanism of carbonic anhydrase: Implications of a rate-limiting protolysis of water. Acc Chem Res 1988;21:30-6.  Back to cited text no. 21
SilvermanDN, McKennaR. Solvent-mediated proton transfer in catalysis by carbonic anhydrase. Acc Chem Res 2007;40:669-75.  Back to cited text no. 22
DemirC, DemirH, EsenR, AtmacaM, TagdemirE. Erythrocyte catalase and carbonic anhydrase activities in acute leukemias. Asian Pac J Cancer Prev 2010;11:247-50.  Back to cited text no. 23
RussellBJ, LoesebrinkB, ChernickV. Enhanced fetal erythrocyte carbonic anhydrase activity by hydrocortisone. Pediatr Res 1976;10:779-82.  Back to cited text no. 24
AlvarezBV, JohnsonDE, SowahD, SolimanD, LightPE, XiaY, etal. Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy. JPhysiol 2007;579:127-45.  Back to cited text no. 25
KuoWH, YangSF, HsiehYS, TsaiCS, HwangWL, ChuSC, etal. Differential expression of carbonic anhydrase isoenzymes in various types of anemia. Clin Chim Acta 2005;351:79-86.  Back to cited text no. 26
MidiwoC, OkunD, GwerS, OgwenoG. Plasma carbonic anhydrase II level is increased in children with sickle cell anaemia compared to healthy controls. Arch Paediatr Dev Pathol 2017;1:1013.  Back to cited text no. 27
LowryOH, RosebroughNJ, FarrAL, RandallRJ. Protein measurement with the folin phenol reagent. JBiol Chem 1951;193:265-75.  Back to cited text no. 28
VerpoorteJA, MehtaS, EdsallJT. Esterase activities of human carbonic anhydrases B and C. JBiol Chem 1967;242:4221-9.  Back to cited text no. 29
ParuiR, GambhirKK, CruzI, HostenAO. Erythrocyte carbonic anhydrase: Amajor intracellular enzyme to regulate cellular sodium metabolism in chronic renal failure patients with diabetes and hypertension. Biochem Int 1992;26:809-20.  Back to cited text no. 30
Ibrahim SI, Amodu AD, Ene-Ojo AS, Ismaila UA, Fakhrudeen M. Effect of hyperglycemia on erythrocyte carbonic anhydrase and lactic acid in type II diabetic subjects. J Diabetes Mellitus 2016;6:158-65.  Back to cited text no. 31
GambhirKK, OatesP, VermaM, TemamS, CheathamW. High fructose feeding enhances erythrocyte carbonic anhydrase 1 mRNA levels in rat. Ann N Y Acad Sci 1997;827:163-9.  Back to cited text no. 32
da Costa OresJ, SalaL, CerveiraGP, KalilSJ. Purification of carbonic anhydrase from bovine erythrocytes and its application in the enzymic capture of carbon dioxide. Chemosphere 2012;88:255-9.  Back to cited text no. 33
Amersham Biosciences. Protein Electrophoresis, Technical Manual. Hand Book. U.S.A.: Amersham Biosciences Inc.; 1999.  Back to cited text no. 34
OstermanPO, AskmarkH, WistrandPJ. Serum carbonic anhydrase III in neuromuscular disorders and in healthy persons after a long-distance run. JNeurol Sci 1985;70:347-57.  Back to cited text no. 35
BooneCD, HabibzadeganA, GillS, McKennaR. Carbonic anhydrases and their biotechnological applications. Biomolecules 2013;3:553-62.  Back to cited text no. 36
KeilinD, MannT. Carbonic anhydrase. Purification and nature of the enzyme. Biochem J 1940;34:1163-76.  Back to cited text no. 37
NymanPO. Purification and properties of carbonic anhydrases from human erythrocytes. Biochim Biophys Acta 1961;62:1-12.  Back to cited text no. 38
RickliEE, EdsallJT. Zinc binding and the sulfhydryl group of human carbonic anhydrase. JBiol Chem 1962;237:PC258-60.  Back to cited text no. 39
LaurentG, MarriqC, NahonD, CharrelM, Der-RienY. On the proteins accompanying human hemoglobin in its preparations. I. Isolation and complexity of the non-heme protein. Compt Rend Sot Biol 1962;166:1456.  Back to cited text no. 40
LaurentG, CharrelM, MarriqC, DerrienY. Identification des protéines érythrocytaires Y, X1 et X2 aux anhydrases carboniques humaines. Bull Sot Chim Biol 1962;44:419.  Back to cited text no. 41
TasginE, NadarogluH, DemirY, DemirN. Purification and properties of carbonic anhydrase from bone marrow. Asian J Chem 2009;21:5117-22.  Back to cited text no. 42
Demür N, Demür Y, Coþkun F. Purification and characterization of carbonic anhydrase from human erythrocyte plasma membrane. Turk J Med Sci 2001;31:477-82.  Back to cited text no. 43


  [Figure 1], [Figure 2], [Figure3], [Figure4], [Figure 5], [Figure 6], [Figure7]

  [Table1], [Table 2], [Table 3], [Table4]


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