Human Erythrocyte Carbonic Anhydrase Research Articles

Design, synthesis of antipyrine-based Schiff bases and investigation of their cholinesterase and carbonic anhydrase activities by in vitro and in silico approaches

In this study, a series of antipyrine-based Schiff bases (1-10) was designed, synthesized, and evaluated as potential inhibitors of various metabolic enzymes, including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human erythrocyte carbonic anhydrase I and II (hCA I and hCA II). The target molecules were characterized by UV-Vis, FT-IR, 1H NMR, 13CNMR, LC-HRMS, and elemental analysis. All tested derivatives demonstrated low nanomolar inhibition with Ki values of in the range of 20.58 ± 0.35 to 53.11 ± 1.02 nM against AChE, 21.84 ± 0.40 to 54.41 ± 1.05 nM against BChE, 27.45 ± 0.41 to 48.22 ± 0.91 nM against cytosolic hCA I isoform associated with epilepsy, and 6.02 ± 0.11 to 29.32 ± 0.54 nM against cytosolic hCA II isoform associated with glaucoma. In general, most these molecules, except for a few, inhibited these enzymes more than acetazolamide (AZA) and neostigmine. Among them, compounds 5, 7, 8, and 9 showed the best inhibitory activities against AChE, BChE, hCA I, and hCA II, respectively. Docking results were calculated for the compounds that showed the best inhibitory activity against these enzymes and for reference compounds. Based on the molecular docking results, they were determined to have high binding energies, including hydrogen bonds, electrostatic interactions, and hydrophobic interactions. Absorption, distribution, metabolism, and excretion (ADME) parameters determined that all the synthesized pyrazolone ring-bearing Schif base derivatives (1-10) have the expected physicochemical properties in terms of drug-likeness and can be evaluated as orally active potential. Properties such as the electrophilicity index and chemical hardness were also investigated by density functional theory (DFT) at the B3LYP/6-311G** level of theory.

Influence of Chirality of Benzimidazole Amine Hybrids on Inhibition of Human Erythrocytes Carbonic Anhydrase I, II and Acetylcholinesterase.

Novel chiral benzimidazole amine hybrids (4a-4d) were synthesized from commercially available amine [(R)- (+)-phenylethylamine, (-) (S)-(-)-phenylethylamine, (-) (R)-(-)-cyclohexylethylamine, (S)-(+)-cyclohexylethylamine] and 2-(chloromethyl)-N-tosyl-1H-benzimidazole. The synthesized compounds (4a-4d) were characterized by IR, NMR, and LC/MS analysis. The inhibitory effect of 4a-4d on human erythrocytes carbonic anhydrase I (hCA-I), II (hCA-II), and acetylcholinesterase (AChE) activity was investigated. For hCA-I, the IC50 values of 4a-4d were found to be 4.895 μM, 1.750 μM, 0.173 μM, and 0.620 μM, respectively, and for hCA-II, the IC50 values of 4a-4d were found to be 0.469 μM, 0.380 μM, 0.233 μM, 0.635 μM, respectively. Furthermore, IC50 values of 4a-4d on AChE were found as 87.5 nM, 100 nM, 26.92 nM, and 100 nM, respectively. In addition, molecular docking analysis was performed to evaluate the affinity of 4a-4d against hCA-I, hCA-II, and AChE and explain their binding interactions.

Vinyl functionalized 5,6-dimethylbenzimidazolium salts: Synthesis and biological activities.

A series of vinyl functionalized 5,6-dimethylbenzimidazolium salts are synthesized. All compounds were fully characterized by elemental analyses, MS, 1 H-NMR, 13 C-NMR, and IR spectroscopy techniques. Enzyme inhibition is a very active area of research in drug design and development. In this study, the synthesized novel benzimidazolium salts were evaluated toward the human erythrocyte carbonic anhydrase I (hCA I), and II (hCA II) isoenzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. They demonstrated highly potent inhibition ability against hCA I with Ki values of 484.8 ± 62.6-1389.7 ± 243.2 nM, hCA II with Ki values of 298.9 ± 55.7-926.1 ± 330.0 nM, α-glycosidase with Ki values of 170.3 ± 27-760.1 ± 269 μM, AChE with Ki values of 27.1 ± 3-77.6 ± 1.7 nM,and BChE with Ki values of 21.0 ± 5-61.3 ± 15 nM. As a result, novel vinyl functionalized 5,6-dimethylbenzimidazolium salts (1a-g) exhibited effective inhibition profiles toward studied metabolic enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly to treat some global disorders including glaucoma, Alzheimer's disease, and diabetes.

Exploring enzyme inhibition profiles of novel halogenated chalcone derivatives on some metabolic enzymes: Synthesis, characterization and molecular modeling studies

Enzyme inhibition is a very active area of research in drug design and development. Chalcone derivatives have a broad enzyme inhibitory activity and function as potential molecules in the development of new drugs. In this study, the synthesized novel halogenated chalcones with bromobenzyl and methoxyphenyl moieties were evaluated toward the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes and human erythrocyte carbonic anhydrase I (hCA I), and II (hCA II) isoenzymes. They showed highly potent inhibition ability toward AChE with Ki values of 1.83 ± 0.21–11.19 ± 0.96 nM and BChE with Ki values of 3.35 ± 0.91–26.70 ± 4.26 nM; hCA I with Ki values of 29.41 ± 3.14–57.63 ± 4.95 nM, and hCA II with Ki values of 24.00 ± 5.39–54.74 ± 1.65 nM. Among the tested enzyme inhibitions, compounds 14 and 13 were the most active compounds against AChE and BChE. Docking studies were performed to the most active compounds against AChE, BChE, hCA I and hCA II to propose a binding mode in the active site and molecular dynamics simulations were studied to check the molecular interactions and the stability of the ligands in the active site. The results may contribute to the development of new drugs particularly to treat some global disorders including Alzheimer’s disease (AD), glaucoma, and diabetes.

Physical–chemical studies of new, versatile carbazole derivatives and zinc complexes: Their synthesis, investigation of in vitro inhibitory effects on α‐glucosidase and human erythrocyte carbonic anhydrase I and II isoenzymes

In this study, two novel zinc–phthalocyanine complexes (4a and 6a), containing carbazole rings, were investigated. The first phthalocyanine 4a has been synthesized from 4‐(2‐(3‐acetyl‐9H‐carbazol‐9‐yl)ethoxy at the peripheral position. Likewise, the second phthalocyanine 6a has been synthesized from 4‐(2‐(3‐cinnamoyl‐9H‐carbazol‐9‐yl)ethoxy at the same position. The new phthalocyanines (Pcs) and their ligands are soluble in organic solvents, such as dimethylformamide, dimethyl sulfoxide, dichloromethane, ethyl acetate, and methanol/ethanol. The Zn (II) complexes were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance (NMR) (except 13C NMR for complexes), elemental analysis, mass spectrometry, and UV–Vis spectroscopy. Also, photophysical and photochemical properties and cyclic voltammogram for the N‐substituted carbazole zinc–Pcs have been reported. The evaluation of carbazole Zn (II) complexes (4a and 6a) and ligands (3, 4, 5, and 6) for in vitro α‐glucosidase inhibitory activities was performed compared to standard drugs acarbose, and kinetic studies were also carried out to determine their inhibition modes. The inhibitory effects of these new Pcs (4a and 6a) and their ligands (3, 4, 5, and 6) on human erythrocytes carbonic anhydrase I (hCA I) and II (hCA II) isoenzymes were investigated. Moreover, in silico studies were also carried out to better investigate the interactions of N‐substituted carbazole containing zinc Pcs with the active sites of hCA I and II isoforms.

Synthesis of benzimidazole derivatives containing amide bond and biological evaluation as acetylcholinesterase, carbonic anhydrase I and II inhibitors

Acetylcholinesterase (AChE) and carbonic anhydrase I (CA-I) and II (CA-II) are two vital metabolic enzymes. AChE inhibitors are seen as target molecules in drug development studies for Alzheimer's treatment. CA inhibitors are target molecules for treating many diseases from glaucoma to cancer. For this reason, it is crucial to identify new AChE and CA inhibitors. In this study, four benzimidazole acetamide derivatives were synthesized and their inhibition effects were investigated against human erythrocyte carbonic anhydrase I (hCA-I), II (hCA-II), and AChE. IC50 values of 9a-10b were determined in the range of 0.936 to 17.07 µM for AChE. IC50 values of 9a–10b for hCA-I were found as 7.21 µM, 4.72 µM, 6.08 µM, 8.23 µM, respectively. On the other hand, IC50 values of 9a–9b for hCA-II were found as 8.64 µM, 7.07 µM, 4.12 µM, 5.93 µM, respectively. According to IC50 values, 9a–10b molecules exhibited strong inhibition effects for AChE and hCAI, II. Also, Molecular docking studies were carried out to explain the binding interaction of 9a–10b with AChE, hCA-I, and hCA-II.

A versatile study of novel A3B-type unsymmetric zinc(II) phthalocyanines containing thiazolidin-4-one: Their, carbonic anhydrase I, II isoenzymes, and xanthine oxidase inhibitors evaluation

In this study, four novel phthalocyanine complexes containing zinc metal were synthesized. After preparing the starting compounds (2-(4-hydroxyphenyl)-3-phenylthiazolidin-4-one 1 and 2-(4-(2-hydroxyethoxy)phenyl)-3-phenylthiazolidin-4-one 3 by the conventional method, they were reacted with 4-nitrophthalonitrile separately using K2CO3 in DMF. Compounds 2 and 4, are well-documented compounds for obtaining phthalocyanines. Subsequently, synthesized phthalonitrile compounds 2 and 4 were reacted with Zn(II) salt at high temperature in the presence of DBU to convert them into targeted symmetrical (2a, 4a) and unsymmetrical (2b, 4b) phthalocyanines under suitable conditions. Their photochemical, photophysical, and electrochemical features were then examined. These metallophthalocyanines indicated good solubility in some organic solvents, such as DMSO, DMF, THF, DCM, and CHCl3. Furthermore, the structures of ligands (1, 2, 3, 4) were determined by 1H NMR, 13C NMR, and FT-IR spectrometry, while complexes (2a, 2b, 4a, 4b) were determined by FT-IR, UV–Vis, fluorescence, and MALDI-TOF spectrometry. Inhibitory effects of ligand and phthalocyanine compounds (1, 2, 3, 4, 2a, 2b, 4a, 4b) against human erythrocyte carbonic anhydrase I (hCA I) and II (hCA II) isoenzymes, as well as cow's milk xanthine oxidase (XO), were examined. It was found that 2a, 2b, 4a, and 4b had strong inhibition effects at micromolar levels against all three. The compounds 2b and 4b showed stronger inhibition effects for hCA I and II than 2a and 4a. In the case of XO, although the inhibition effects of these molecules (2b, 4a, 4b) were similar, 2a had the strongest inhibition effect. Since CA and XO inhibitors are the target molecules of drug development studies to be used in the treatment of many diseases, the results of this study will aid drug design studies in the development of new XO and CA inhibitors.

Some characteristics of new and innovative COX inhibitor derivatives: Potent hCA‐I and hCA‐II inhibitors supported by molecular docking studies

AbstractIn this study, two novel metallophthalocyanines (ZnPc and CoPc) were synthesized using the corresponding metal salts 4‐(4‐(4‐[4‐chlorophenyl]‐5‐methylisoxazol‐3‐yl)phenoxy)‐phthalonitrile (11), prepared from the reaction of 4‐nitrophthalonitrile and 4‐(4‐[4‐chlorophenyl]‐5‐methylisoxazol‐3‐yl)phenol (9). These metallophthalocyanines (MPcs) showed quite solubility in organic solvents such as dichloromethane (DCM), tetrahydrofuran (THF), dimethyl formamide (DMF), and dimethylsulfoxide (DMSO). The novel compounds 11a and 11b have been characterized using their UV–Vis, FT–IR, 1H NMR, 13C NMR, X‐Ray, and MALDI–TOF mass spectra. Supporting information cocerning with the study has been supplied. Photochemical, photophysical, and cyclic voltagram properties of these novel 4‐(4‐(4‐[4‐chlorophenyl]‐5‐methylisoxazol‐3‐yl)phenoxy substituted metallophthalocyanines (11a and 11b) were determined in DMF. DNA binding, metal chelating effect assay, and DPPH [2,2‐diphenyl‐1‐picrylhydrazyl hydrate] radical scavenging assay and electrochemical studies of MPcs were investigated. Further, the inhibitory effects of the COX‐inhibitor based novel metallophthalocyanines (11a and 11b) and their ligands (10 and 11) were examined on human erythrocyte carbonic anhydrase I (hCA‐I) and II (hCA‐II) isoenzymes, and the synthesized molecules exhibited very strong inhibitory effects on both isoforms. In addition, the hCA‐I and hCA‐II inhibition potential of Zn (II) and Co (II) Phthalocyanine complexes was supported by molecular docking studies. The binding interaction of metallophthalocyanines complexes 11a, 11b enzymes were analyzed in detail.

Cholinesterases, carbonic anhydrase inhibitory properties and in silico studies of novel substituted benzylamines derived from dihydrochalcones

A series of novel urea, sulfamide and N,N-dipropargyl substituted benzylamines were synthesized from dihydrochalcones. The synthesized compounds were evaluated for their cholinesterases and carbonic anhydrase inhibitory actions. The known dihydrochalcones were converted into four new benzylamines via reductive amination. N,N-Dipropargylamines, ureas and sulfamides were synthesized following the reactions of benzylamines with propargyl bromide, N,N-dimethyl sulfamoyl chloride and N,N-dimethyl carbamoyl chloride. The novel substituted benzylamines derived from dihydrochalcones were evaluated against some enzymes such as human erythrocyte carbonic anhydrase I and II isoenzymes (hCA I and hCA II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The novel substituted benzylamines derived from dihydrochalcones exhibited Ki values in the range of 0.121–1.007 nM on hCA I, and 0.077–0.487 nM on hCA II closely related to several pathological processes. On the other hand, Ki values were found in the range of 0.112–0.558 nM on AChE, 0.061–0.388 nM on BChE. As a result, novel substituted benzylamines derived from dihydrochalcones showed potent inhibitory profiles against indicated metabolic enzymes. In addition, Induced-Fit Docking (IFD) simulations and ADME prediction studies have also been carried out to elucidate the inhibition mechanisms and drug-likeness of the synthesized compounds. Therefore, these results can make significant contributions to the treatment of some global diseases, especially Alzheimer's diseases and glaucoma, and the development of new drugs.

ADME properties, bioactivity and molecular docking studies of 4-amino-chalcone derivatives: new analogues for the treatment of Alzheimer, glaucoma and epileptic diseases.

The online version contains supplementary material available at 10.1007/s40203-021-00094-x.

Synthesis of New Schiff Bases and Assessment of Their in vitro Biological Effects on Acetylcholinesterase and Carbonic Anhydrase Isoenzymes Activities

In this study, three new Schiff bases have been synthesized by the reactions of commercially available phenylglycinol, phenylalaninol, and leuicinol with 4-{[2-(4-formylphenoxy)ethyl](methyl)amin}benzaldehyde and characterized by 1H and 13C NMR, FTIR, and UV-Vis spectroscopy and LCMS/MS. In vitro effects of synthesized new Schiff bases on human erythrocyte carbonic anhydrase I (hCA I) and II (hCA II) isoenzymes and acetylcholinesterase (AChE) activity were investigated. Schiff base synthesized from phenylglycinol showed no meaningful effect on hCAI and hCAII. Schiff bases synthesized from phenylalaninol and leuicinol exhibited a strong activation effect on hCAI and hCAII. On the other hand, all of the synthesized three Schiff bases exhibited a strong inhibitory effect on AChE activity.

Synthesis and Biological Evaluation of Novel Dihydro [2,3D] Pyridine Substituted Enaminosulfonamide Compounds as Potent Human Erythrocyte Carbonic Anhydrase II (hCAII)

Dihydro [2,3D] pyridine substituted enaminosulfonamide compounds have been synthesized and their effects on carbonic anhydrase II (hCAII) have been evaluated. Pyrido [2,3 d] pyrimidines were synthesized from barbituric acid derivatives, malonanitrile, aldehyde derivatives in basic condition and then hydrolyzed with hydrochloric acid. The targeted compounds were syn-thesized from amino sulfanilamide, dihydro [2,3D] pyridine compounds, and triethylorthoformate. 1H NMR, 13C NMR, FT-IR and elemental analysis were used for the structural analysis of the compounds. The half maximal inhibitory concentration (IC50) values of the compounds were determined to be between 27.03 and 104.39 μM for hCA II and 19.85-76.64 μM for Ki.

Synthesis of nitrogen, phosphorus, selenium and sulfur-containing heterocyclic compounds – Determination of their carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase and α-glycosidase inhibition properties

Sulfur-containing pyrroles (1–3), tris(2-pyridyl)phosphine(selenide) sulfide (4–5) and 4-benzyl-6-(thiophen-2-yl)pyrimidin-2-amine (6) were synthesized and characterized by elemental analysis, IR and NMR spectra. In this study, the synthesized compounds of nitrogen, phosphorus, selenium and sulfur-containing heterocyclic compounds (1–6) were evaluated against the human erythrocyte carbonic anhydrase I, and II isoenzymes, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase enzymes. The synthesized heterocyclic compounds showed IC50 values in range of 33.32–60.79 nM against hCA I, and 37.05–66.64 nM against hCA II closely associated with various physiological and pathological processes. On the other hand, IC50 values were found in range of 13.13–22.21 nM against AChE, 0.54–31.22 nM against BChE, and 13.51–26.55 nM against α-glycosidase as a hydrolytic enzyme. As a result, nitrogen, phosphorus, selenium and sulfur-containing heterocyclic compounds (1–6) demonstrated potent inhibition profiles against indicated metabolic enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly in the treatment of some global disorders including glaucoma, Alzheimer’s disease and diabetes.

β-D-Glukan, Lektin, Linoleik Asit ve β-Karoten Bileşiklerinin Karbonik Anhidraz Enzimleri Üzerindeki Etkilerinin Araştırılması

β-D-glukan, lektin, linoleik asit ve β-karoten; hayvansal, bitkisel ve bakteriyel kaynaklarda bulunan, biyolojik olarak aktif bileşiklerdir. Glokom, dünya genelinde körlüğe neden olma bakımından katarakttan sonra ikinci sırada gelen bir hastalıktır. Karbonik anhidraz (CA) inhibitörleri uzun yıllardır glokom tedavisinde kullanılmaktadır. Ancak, inhibitör ajanların yan etkileri azımsanmayacak derecede çoktur. Yeni CA inhibitörlerinin geliştirilmesi üzerine çeşitli araştırma grupları çalışmalar yapmaktadırlar. Doğal kaynaklı bileşiklerin CA inhibitörü olarak kullanımının araştırılması da son yıllarda popülerlik kazanmıştır. Bu çalışmada, β-D-glukan, lektin, linoleik asit ve β-karoten bileşiklerinin, insan eritrosit CA izoenzimleri olan hCA I ve hCA II üzerine etkileri in vitro koşullarda araştırılmıştır. Bileşiklerin Ki değerleri hCA I için 0.45±0.09 µM − 37.02±17.85 µM aralığında, hCA II için 3.12±1.38 µM − 61.23±25.46 µM aralığındadır.

Evaluation of some thiophene-based sulfonamides as potent inhibitors of carbonic anhydrase I and II isoenzymes isolated from human erythrocytes by kinetic and molecular modelling studies.

Thiophene(s) are an important group in therapeutic applications, and sulfonamides are the most important class of carbonic anhydrase (CA) inhibitors. In this study, inhibition effects of some thiophene-based sulfonamides on human erythrocytes carbonic anhydrase I and II isoenzymes (hCA-I and hCA-II) were investigated. Thiophene-based sulfonamides used in this study showed potent inhibition effect on both isoenzymes at very small concentrations. We report on the purification of the carbonic anhydrase I and II isoenzymes (hCA-I and hCA-II) using affinity chromatography method. The inhibition effect of the thiophene-based sulfonamides was determined by IC50 and Ki parameters. A molecular docking study was performed for each molecule. Thiophene-based sulfonamides showed IC50 values of in the range of 69nM to 70µM against hCA-I, 23.4nM to 1.405µM against hCA-II. Ki values were in the range of 66.49 ± 17.15nM to 234.99 ± 15.44µM against hCA-I, 74.88 ± 20.65nM to 38.04 ± 12.97µM against hCA-II. Thiophene-based sulfonamides studied in this research showed noncompetitive inhibitory properties on both isoenzymes. To elucidate the mechanism of inhibition, a molecular docking study was performed for molecules 1 and 4 exhibiting a strong inhibitory effect on hCA-I and hCA-II. The compounds inhibit the enzymes by interacting out of catalytic active site. The sulfonamide and thiophene moiety played a significant role in the inhibition of the enzymes. We hope that this study will contribute to the design of novel thiophene-based sulfonamide derived therapeutic agents that may be carbonic anhydrase inhibitors in inhibitor design studies.

Inhibition Effect of Eosin Y on Carbonic Anhydrase (CA) I and II Isoenzymes Purified from Human Erythrocytes

All cells produce carbon dioxide (CO2), which is released as a result of metabolism and must be removed from the body. A large part of this CO2 is converted to bicarbonate by the carbonic anhydrase (CA) enzyme in erythrocytes and is discarded from the body. So, CA has a vital role in red blood cells. In addition to, CA involved in many other pathological and physiological processes and it was determined that the inhibitors of CA were effective in the treatment and diagnosis of many diseases particularly glaucoma. Considering the importance of the CA's inhibitors, in this study it was intended to research the inhibition effects of Eosin Y on CA I and CA II isoenzymes activity purified from human erythrocytes. Eosin Y is a dye molecule commonly used in histological and medical applications. For this purpose, firstly CA I and CA II isoenzymes were purified from human erythrocytes by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography. Then the inhibitory effect of Eosin Y on the activity of these human erythrocyte CA I (hCA I) and CA II (hCA II) isoenzymes was investigated. It was determined that hCA I and hCA II were inhibited by Eosin Y in the millimolar range. IC50 values were found to be 3.78 mM for hCA I and 2.04 mM for hCA II and Ki values were determined as 9.65±0.968 mM and 7.52±2.88 mM for hCA I and hCA II, respectively. In conclusion, it is hoped that the results obtained in this study may be beneficial in the development of new CA inhibitors which may be drug candidates.

The effects of cardiac drugs on human erythrocyte carbonic anhydrase I and II isozymes

Cardiovascular diseases are the leading cause of mortality worldwide. In recent years, the relationship between carbonic anhydrase inhibitors and atherosclerosis has attracted attention. In this study, we aimed to determine the in vitro effects of 35 frequently used cardiac drugs on human carbonic anhydrase I (hCA I) and II (hCA II). The inhibitory effects of the drugs on hCA I and hCA II were determined with both the hydratase and esterase methods. The most potent inhibitors observed were propafenone (hCA I: 2.8 µM and hCA II: 3.02 µM) and captopril (hCA I: 1.58 µM and hCA II: 6.25 µM). Isosorbide mononitrate, propranolol, furosemide, and atorvastatin were also potent inhibitors. The inhibitor constant, K i, value from the Lineweaver–Burk plot for propafenone was 2.38 µM for hCA I and 2.97 µM for hCA II. The tested cardiac drugs showed potent in vitro inhibition of the hCA I and II isozymes. Especially, in patients with atherosclerotic heart disease, these drugs may be preferred primarily due to the beneficial effects of carbonic anhydrase inhibition on atherosclerosis.

Evaluation of Inhibition Effects of Some Cardiovascular Therapeutics on Human Erythrocyte Carbonic Anhydrase Isoenzymes

Abstract Carbonic anhydrase enzyme plays a vital role in metabolic events such as acid-base regulation and respiration. In our research, it is tried to determine the inhibitory influences of the cardiovascular therapeutics esmolol hydrochloride, amiodarone hydrochloride and lidocaine hydrochloride on human erythrocytes carbonic anhydrases (hCAI and II). In accordance with this purpose, carbonic anhydrase isoenzymes were purified from human erythrocytes by using affinity chromatography method. Enzyme purity was checked by SDS-PAGE electrophoresis method. After the aldose reductase enzymes were purified, the inhibitory affects of cardiovascular therapeutics on these enzymes, using esterase activity, which is the method of measuring in vitro activity, were examined. The three cardiovascular therapeutics dose-dependently decreased activity of hCAs. IC 50 values of amiodarone hydrochloride, esmolol hydrochloride and lidocaine hydrochloride were found to be, respectively, 0.91 mM, 5 mM, 5.8 mM for hCA-I and 0.41 mM, 3.5 mM, and 6.36 mM for hCA-II. Our results proved that, under in vitro conditions, cardiovascular therapeutics significantly inhibit human CA-I and II activities. So, irregular use of these medicines may cause serious adverse effects in terms of human health.

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

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 s-thalassemia major patients. Materials and Methods: The blood samples included 61 samples of blood (31 males and 30 females) from s-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.

The effects of ex vivo ozone treatment on human erythrocyte carbonic anhydrase enzyme

Ozone autohemotherapy is used in the treatment of some diseases. Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes and play a role in homeostatic mechanisms. The aim of this study was to investigate the effects of ozone on human red blood cell CA (hCA) enzyme activity. Blood samples were treated with different doses of ozone (10, 20, 30 µg/ml) and the erythrocyte total CA activities were determined. Also, purified hCAI and hCAII isozymes were treated with the same doses of ozone and the enzyme activities were measured. About 30 µg/ml ozone treatment decreased the purified hCAI and hCAII activity and increased the total CA activity compared to the control. Because the implication of CAs on many physiological and biochemical processes is linked to pathologies, it can be suggested that the ozone at a concentration of 30 µg/ml is safely used by autohaemotherapy in a well-designed clinical trial.